**Part 2**

**Etiopathogenesis** 

24 Challenges in Rheumatology

[23] Zhu T, Tam L, Lee V, Lee K and Li E. The Impact of Flare on Disease Costs of Patients

[24] Quintana G, Coral-Alvarado P, Díaz J. Direct costs of health care of lupus nephritis in

[25] Weening JJ, D'Agati VD, Schwartz MM, Seshan SV, Alpers CE, Appel GB, et al. The

[26] Wilson E, Jayne D, Dellow D and Fordham R. The cost-effectiveness of mycophenolate

Research); 2009:1159–1167

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Colombia. Ann Rheum Dis 2011;70(Suppl3):694

Am Soc Nephrol. 2004 Feb;15(2):241-50.

With Systemic Lupus Erythematosus. Arthritis & Rheumatism (Arthritis Care &

classification of glomerulonephritis in systemic lupus erythematosus revisited. J

mofetil as first line therapy in active lupus nephritis. Rheumatology 2007;46;1096–

**3** 

*Japan*

Shunsei Hirohata

**Role of Bone Marrow in the** 

*Kitasato University School of Medicine* 

**Pathogenesis of Rheumatoid Arthritis** 

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent synovial proliferation. Thus, joints in RA consist of massive proliferating synovium, forming an invading tissue termed pannus, which results in the destruction of cartilage and bone. One of the most important histologic characteristics of the synovium in RA includes cellular proliferation in the lining layer as well as in the sublining layer (Tak, 2004). In the lining layer, both type A and type B synoviocytes, alternatively called intimal macrophages and fibroblast-like synoviocytes, respectively, are found to proliferate (Tak, 2004). In the sublining layer, there is infiltration of a variety of cells, including dendritis cells (DC), lymphocytes, plasma cells, and polymorphnuclear leukocytes. Notably, lymphoid cluster in RA synovium sometimes forms pseudo-germinal center, consisting of CD20+ B cells in the center surrounded by CD4+ T cells (Tak, 2004; Hirohata, 2004). In the synovium of RA, neovascularization is usually accompanied by lining cell proliferation and inflammatory cell infiltration (Firestein, 1999). In fact, lining cells and inflammatory cells have been found to produce angiogenic growth factors (Koch, 1998). It should be noted, however, that the synovium of RA also showed neovascularization in the areas without either lining cell proliferation or inflammatory cell infiltration, suggesting that the neovascularization might be one of the primary abnormal features that are most proximal to the etiology of RA

A number of studies have suggested that abnormal activation of normal joint constituents, such as synovial lining cells, play a pivotal role in the synovial hyperplasia in RA (Shiozawa & Tokuhisa, 1992). However, increasing attention has emerged to the role of bone marrow in the pathogenesis of RA. The present article overviews an update on the role of bone marrow

We previously showed that peripheral blood monocytes in patients with active RA are already activated to express higher densities of CD14 (Shinohara et al.,1992). It is also suggested that peripheral blood monocytes in patients with RA may have intrinsic abnormalities as evidenced by the enhanced expression of FcγR, which is repeatedly observed regardless of the disease activity of RA (Shinohara et al.,1992). It has been also demonstrated that CD14, FcγRI and FcγRII are involved in the regulation of various

**2. Bone marrow and type A synoviocytes (intimal macrophages) in RA** 

**2.1 Abnormalities in peripheral blood monocytes in RA** 

**1. Introduction** 

(Hirohata & Sakakibara, 1999).

in the pathogenesis of RA.

## **Role of Bone Marrow in the Pathogenesis of Rheumatoid Arthritis**

## Shunsei Hirohata

*Kitasato University School of Medicine Japan*

## **1. Introduction**

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent synovial proliferation. Thus, joints in RA consist of massive proliferating synovium, forming an invading tissue termed pannus, which results in the destruction of cartilage and bone. One of the most important histologic characteristics of the synovium in RA includes cellular proliferation in the lining layer as well as in the sublining layer (Tak, 2004). In the lining layer, both type A and type B synoviocytes, alternatively called intimal macrophages and fibroblast-like synoviocytes, respectively, are found to proliferate (Tak, 2004). In the sublining layer, there is infiltration of a variety of cells, including dendritis cells (DC), lymphocytes, plasma cells, and polymorphnuclear leukocytes. Notably, lymphoid cluster in RA synovium sometimes forms pseudo-germinal center, consisting of CD20+ B cells in the center surrounded by CD4+ T cells (Tak, 2004; Hirohata, 2004). In the synovium of RA, neovascularization is usually accompanied by lining cell proliferation and inflammatory cell infiltration (Firestein, 1999). In fact, lining cells and inflammatory cells have been found to produce angiogenic growth factors (Koch, 1998). It should be noted, however, that the synovium of RA also showed neovascularization in the areas without either lining cell proliferation or inflammatory cell infiltration, suggesting that the neovascularization might be one of the primary abnormal features that are most proximal to the etiology of RA (Hirohata & Sakakibara, 1999).

A number of studies have suggested that abnormal activation of normal joint constituents, such as synovial lining cells, play a pivotal role in the synovial hyperplasia in RA (Shiozawa & Tokuhisa, 1992). However, increasing attention has emerged to the role of bone marrow in the pathogenesis of RA. The present article overviews an update on the role of bone marrow in the pathogenesis of RA.

## **2. Bone marrow and type A synoviocytes (intimal macrophages) in RA**

### **2.1 Abnormalities in peripheral blood monocytes in RA**

We previously showed that peripheral blood monocytes in patients with active RA are already activated to express higher densities of CD14 (Shinohara et al.,1992). It is also suggested that peripheral blood monocytes in patients with RA may have intrinsic abnormalities as evidenced by the enhanced expression of FcγR, which is repeatedly observed regardless of the disease activity of RA (Shinohara et al.,1992). It has been also demonstrated that CD14, FcγRI and FcγRII are involved in the regulation of various

Role of Bone Marrow in the Pathogenesis of Rheumatoid Arthritis 29

Monocyte-like cells are shown by arrow heads.

(Hematoxylin and eosin, original magnification x25)

Fig. 2. Synovium-like tissue at pericardium lesion in an RA patient

including proteoglycans, cytokines, arachidonic acid metabolites, and matrix metalloproteinases (MMPs), that lead to the destruction of joints (Firestein,1996). Unlike intimal macrophages, the precise origin of type B synoviocytes remains unclear, although they are thought to arise from the sublining tissue or other support structures of a joint (Firestein,1996). On the other hand, a number of studies have shown that peripheral blood dendritic cells (DC) accumulate in the synovium, where they undergo phenotypic and functional differentiation in situ (Zvaifler et al.,1985; Thomas et al.,1994). It has been also shown that synovial DC gradually lose their dinstinct morphologic appearance and become indistinguishable from fibroblasts in vitro (Hendler et al.,1985). Moreover, Kyogoku et al. identified the presence of DC-like cells that strongly express major histocompatibility

(Electron microscopy, the scale bar at the right-bottom corner indicates 5 μm) Fig. 1. Transendothelial migration of monocyte into RA synovium.

functions of monocytes, including the production of cytokines (Krutmann et al.,1990) and the expression of adhesion molecules (Lauener et al.,1990). Therefore, the observed abnormalities in our studies suggest that the recruitment of RA peripheral blood monocytes may result in further activation and adhesion of these cells in the synovial tissues, thus contributing to extending the rheumatoid disease process.

## **2.2 Accelerated generation of CD14+ monocyte-lineage cells from the bone marrow**

Although previous studies have suggested a role of dysregulated proliferation of synoviocytes in synovial hyperplasia (Lafyatis et al.,1989), it was found that rheumatoid synovium had rarely evidence of mitosis, and that only 4% of rheumatoid synovial cells showed uptake of thymidine (Harris Jr.,1993). Thus, there has been no evidence for accelerated or dysregulated in situ proliferation of synoviocytes in rheumatoid synovium. We have disclosed that the spontaneous generation of CD14+ cells from bone marrow CD14 progenitor cells was accelerated in RA patients compared with control patients (Hirohata et al.,1996). Moreover, the expression of HLA-DR on the bone marrow-derived CD14+ cells was also accelerated in RA patients compared with controls, confirming the accelerated maturation of macrophages in RA bone marrow. Consistently, CD14+ CD16+ blood monocytes with high expression of chemokine receptors and CD54 were found to be increased in active RA (Kawanaka et al., 2002). It should be also pointed out that the expression of a variety of chemokines and adhesion molecules is enhanced in vascular endothelium and fibroblast-like synoviocytes in the RA synovium (Oppenheimer-Marks & Lipsky,1998; Patel et al.,2001; Kanbe et al.,2002 ), possibly facilitating the entry of such CD14+ CD16+ blood monocytes into the synovium. These observations strongly support the hypothesis that the accelerated generation of CD14+ cells from bone marrow progenitor cells and the accelerated maturation of such CD14+ cells into tissue-infiltrative CD16+ monocytes before entry into the joint might play an important role in the pathogenesis of RA.

## **2.3 Evidence for recruitment of cells from systemic circulation in RA**

It is noteworthy that accelerated angiogenesis has been demonstrated in RA synovium (Harris Jr.,1993), which might facilitate the recruitment of bone marrow-derived monocytes as well as lymphocytes into the synovium. In fact, the transendothelial migration of monocytes in RA synovium can be frequently observed under electron microscopy (Fig. 1).

Of interest, the formation of synovium-like tissue was also observed at the site of non-union formed after bone fracture as well as in the pericardial lesions in an RA patient (Fig. 2). Since such formation of the synovium-like tissue took place in the place without original synovial tissues, it is suggested that all the constituents of the newly formed tissue might be recruited from the systemic circulation. Finally, proliferative synovial tissues usually disappear at the site of bony ankylosis and total immobility. These observations strongly support the hypothesis that the accelerated generation and continuous recruitment of bone marrowderived cells might play a critical role in the synovial hyperplasia in RA, thus accounting for the discrepancy between the marked synovial hyperplasia and the lack of evidence for accelerated proliferation of synoviocytes.

## **3. Origin of type B synoviocyte**

## **3.1 Origin of type B synoviocytes**

Type B synoviocytes, which are called fibroblast-like synoviocytes, have the morphologic appearance of fibroblasts as well as the capacity to produce and secrete a variety of factors,

functions of monocytes, including the production of cytokines (Krutmann et al.,1990) and the expression of adhesion molecules (Lauener et al.,1990). Therefore, the observed abnormalities in our studies suggest that the recruitment of RA peripheral blood monocytes may result in further activation and adhesion of these cells in the synovial tissues, thus

**2.2 Accelerated generation of CD14+ monocyte-lineage cells from the bone marrow**  Although previous studies have suggested a role of dysregulated proliferation of synoviocytes in synovial hyperplasia (Lafyatis et al.,1989), it was found that rheumatoid synovium had rarely evidence of mitosis, and that only 4% of rheumatoid synovial cells showed uptake of thymidine (Harris Jr.,1993). Thus, there has been no evidence for accelerated or dysregulated in situ proliferation of synoviocytes in rheumatoid synovium. We have disclosed that the spontaneous generation of CD14+ cells from bone marrow CD14 progenitor cells was accelerated in RA patients compared with control patients (Hirohata et al.,1996). Moreover, the expression of HLA-DR on the bone marrow-derived CD14+ cells was also accelerated in RA patients compared with controls, confirming the accelerated maturation of macrophages in RA bone marrow. Consistently, CD14+ CD16+ blood monocytes with high expression of chemokine receptors and CD54 were found to be increased in active RA (Kawanaka et al., 2002). It should be also pointed out that the expression of a variety of chemokines and adhesion molecules is enhanced in vascular endothelium and fibroblast-like synoviocytes in the RA synovium (Oppenheimer-Marks & Lipsky,1998; Patel et al.,2001; Kanbe et al.,2002 ), possibly facilitating the entry of such CD14+ CD16+ blood monocytes into the synovium. These observations strongly support the hypothesis that the accelerated generation of CD14+ cells from bone marrow progenitor cells and the accelerated maturation of such CD14+ cells into tissue-infiltrative CD16+ monocytes before entry into the joint might

contributing to extending the rheumatoid disease process.

play an important role in the pathogenesis of RA.

accelerated proliferation of synoviocytes.

**3. Origin of type B synoviocyte 3.1 Origin of type B synoviocytes** 

**2.3 Evidence for recruitment of cells from systemic circulation in RA** 

synovium can be frequently observed under electron microscopy (Fig. 1).

It is noteworthy that accelerated angiogenesis has been demonstrated in RA synovium (Harris Jr.,1993), which might facilitate the recruitment of bone marrow-derived monocytes as well as lymphocytes into the synovium. In fact, the transendothelial migration of monocytes in RA

Of interest, the formation of synovium-like tissue was also observed at the site of non-union formed after bone fracture as well as in the pericardial lesions in an RA patient (Fig. 2). Since such formation of the synovium-like tissue took place in the place without original synovial tissues, it is suggested that all the constituents of the newly formed tissue might be recruited from the systemic circulation. Finally, proliferative synovial tissues usually disappear at the site of bony ankylosis and total immobility. These observations strongly support the hypothesis that the accelerated generation and continuous recruitment of bone marrowderived cells might play a critical role in the synovial hyperplasia in RA, thus accounting for the discrepancy between the marked synovial hyperplasia and the lack of evidence for

Type B synoviocytes, which are called fibroblast-like synoviocytes, have the morphologic appearance of fibroblasts as well as the capacity to produce and secrete a variety of factors,

Monocyte-like cells are shown by arrow heads. (Electron microscopy, the scale bar at the right-bottom corner indicates 5 μm)

Fig. 1. Transendothelial migration of monocyte into RA synovium.

(Hematoxylin and eosin, original magnification x25)

Fig. 2. Synovium-like tissue at pericardium lesion in an RA patient

including proteoglycans, cytokines, arachidonic acid metabolites, and matrix metalloproteinases (MMPs), that lead to the destruction of joints (Firestein,1996). Unlike intimal macrophages, the precise origin of type B synoviocytes remains unclear, although they are thought to arise from the sublining tissue or other support structures of a joint (Firestein,1996). On the other hand, a number of studies have shown that peripheral blood dendritic cells (DC) accumulate in the synovium, where they undergo phenotypic and functional differentiation in situ (Zvaifler et al.,1985; Thomas et al.,1994). It has been also shown that synovial DC gradually lose their dinstinct morphologic appearance and become indistinguishable from fibroblasts in vitro (Hendler et al.,1985). Moreover, Kyogoku et al. identified the presence of DC-like cells that strongly express major histocompatibility

Role of Bone Marrow in the Pathogenesis of Rheumatoid Arthritis 31

synovial fluid from RA (Jongbloed et al.,2006). In fact, previous study showed that pDC are recruited to RA synovial tissues and contribute into the local inflammatory environment (Lande et al,2004; Cavanagh et al.,2005). Recent studies have disclosed that the characteristic clinical phenomenon of destructive arthritis spreading between joints is mediated, at least in part, by the transmigration of activated RA synovial fibroblasts (Lefèvre et al.,2009). Thus, RA synovial fibroblasts showed an active movement from human RA synovial tissue or human cartilage-sponge complex containing RA synovial fibroblasts implanted into the SCID mouse to the naive cartilage implanted at the contralateral flank via the vasculature, leading to a marked destruction of the target cartilage (Lefèvre et al.,2009). The movement of DC-like cells from the sublining layer to the lining layer in RA synovium (Kyogoku et al.,1992) and the presence of DC in synovial fluid (Jongbloed et al.,2006; Lande et al,2004) strongly suggest that DC might be also released from the joint to the systemic circulation via draining veins. Since bone marrow pDC as well as mDC have capacities to differentiate into type B synoviocyte-like cells, it is possible that DC, as precursors for synovial fibroblasts,

A number of studies have demonstrated that RA is strongly associated with HLA-DR4 or DR1 (Nepom,2001), which are involved in the presentation of antigens to T cells. These results suggest that the antigen presentation involving HLA-DR4 or the shared epitope might play a critical role in the development of synovitis in human RA. In fact, the interactions between DC and T cells, possibly through MHC class II antigens, have been disclosed in the sublining layers of the RA synovium (Kyogoku et al.,1992). If the antigens presented by APC to T cells are perpetuating antigens, such as autoantigens or antigens of persistently infected virus, that are presented through MHC class II molecules, continuing activation of APC might take place. Further studies to explore such antigens that involve persistent interactions between APC and T cells would be still important for the complete

As highlighted above, bone marrow derived monocytes and DC have been shown to be the precursors of type A synoviocytes and type B synoviocytes, respectively. It is therefore suggested that RA might be a disease of dysregulated activation of antigen-presenting cells (APC), leading to synovial proliferation. Lymphocytes activation in the synovium can also be triggered by the activation of APC, accounting for activation of T cells and B cells in the synovium. Triggering with arthritogenic antigens, followed by dysregulated generation of APC from the bone marrow might result in persistent recruitment of APC into the synovium.

A number of studies indicated that neovascularization is crucial to the synovial hyperplasia in RA (Koch,1998; Hirohata & Sakakibara,1999). Postnatal neovascularization has been attributed to so-called angiogenesis, a process characterized by the sprouting of new capillaries from preexisting blood vessels (Folkman & Shing,1992). However, recent studies have demonstrated that endothelial progenitor cells of bone marrow origin play a significant role in the de novo formation of capillaries without preexisting blood vessels, socalled vasculogenesis (Asahara et al.,1997; Gehling et al.,2000; Bhattacharya et al.,2000; Lin et

also contribute to the spread of destructive arthritis between joints in RA.

**4. RA as a disease of antigen-presenting cells** 

understanding of the pathogenesis of RA.

al.,2000).

**5. Bone marrow abnormalities and angiogenesis in RA** 

complex (MHC) class II antigens and interact with T lymphocytes, in the sublining layers of the RA synovium (Kyogoku et al.,1992). They also showed that the sublining DC-like cells proliferate and differentiate into type A as well as type B synoviocytes to replace the lining layers (Kyogoku et al.,1992).

## **3.2 Generation of type B synoviocytes from bone marrow CD34+ cells in RA**

Since it was shown that DC are derived from bone marrow CD34+ cells (Reid et al.,1992; Szabolcs et al.,1995; Chen et al.,2004), it was also likely that type B synoviocytes might be induced from bone marrow progenitors. In this regard, we previously demonstrated that bone marrow CD34+ cells from RA patients have abnormal capacities to respond to tumor necrosis factor-α (TNF-α) and to differentiate into fibroblast-like cells (FLC) producing MMP-1, suggesting that bone marrow CD34+ cells might generate type B synoviocytes and thus could play an important role in the pathogenesis of RA (Hirohata et al.,2001). Thus, CD34+ cells from the bone marrow of RA patients differentiated into cells with fibroblastlike morphology, which expressed prolyl 4-hydroxylase, in the presence of stem cell factor (SCF), GM-CSF, and TNF-α, much more effectively than CD34+ cells from the bone marrow of control subjects (Hirohata et al.,2001).

## **3.3 Capacity of bone marrow DC to differentiate into type B synoviocytes**

We have recently demonstrated that bone marrow plasmacytoid DC (pDC) as well as myeloid DC (mDC), irrespective of their origin from RA bone marrow or osteoarthritis (OA) bone marrow, have prominent capacity to differentiate into FLC producing MMP-1 especially under influences of TNF-α (Hirohata et al.,2011). Of note, depletion of pDC from RA bone marrow CD34+ cells significantly diminished their capacities to differentiate into FLC, which were restored by addition of pDC in a dose-response manner (Hirohata et al.,2011).

It should be pointed out that generation of FLC from RA bone marrow CD34+ cells or pDC was correlated with MMP-1 levels in culture supernatants (Hirohata et al.,2001; Hirohata et al.,2011). On the other hand, it has been demonstrated that cadherin-11 is abundantly expressed in type B synoviocytes (Chang et al.,2010) compared with lung or dermal fibroblasts (Vandooren et al.,2008). Accordingly, the FLC induced from RA and OA bone marrow pDC expressed comparable amounts of cadherin-11 mRNA to RA and OA synovial FLC (Hirohata et al.,2011**)**. These results indicate that DC are one of the progenitors of type B synoviocytes irrespective of RA or OA and suggest that bone marrow CD34+ cells might differentiate into type B synoviocyte-like cells via DC, since DC have been demonstrated to originate from CD34+ cells (Reid et al.,1992; Szabolcs et al.,1995; Chen et al.,2004). It is also likely that expansion of immature DC from bone marrow CD34+ cells might be upregulated in RA compared with in OA, accounting for the enhanced capacity of RA bone marrow CD34+ cells to differentiate into FLC upon stimulation with TNF-α, although further studies are required to confirm this point.

## **3.4 Recruitment of type B synoviocytes and their precursors into RA joints: Role of DC**

It is thus suggested that the presence of abnormal precursors within the bone marrow progenitor cells might play an important role in the pathogenesis of RA by providing a repopulating reservoir of type B synoviocytes, as has been also suggested in other recent studies (Sen et al.,2000). Notably, the numbers of mDC and pDC have been found to be significantly decreased in RA peripheral blood, whereas both mDC and pDC are present in

complex (MHC) class II antigens and interact with T lymphocytes, in the sublining layers of the RA synovium (Kyogoku et al.,1992). They also showed that the sublining DC-like cells proliferate and differentiate into type A as well as type B synoviocytes to replace the lining

Since it was shown that DC are derived from bone marrow CD34+ cells (Reid et al.,1992; Szabolcs et al.,1995; Chen et al.,2004), it was also likely that type B synoviocytes might be induced from bone marrow progenitors. In this regard, we previously demonstrated that bone marrow CD34+ cells from RA patients have abnormal capacities to respond to tumor necrosis factor-α (TNF-α) and to differentiate into fibroblast-like cells (FLC) producing MMP-1, suggesting that bone marrow CD34+ cells might generate type B synoviocytes and thus could play an important role in the pathogenesis of RA (Hirohata et al.,2001). Thus, CD34+ cells from the bone marrow of RA patients differentiated into cells with fibroblastlike morphology, which expressed prolyl 4-hydroxylase, in the presence of stem cell factor (SCF), GM-CSF, and TNF-α, much more effectively than CD34+ cells from the bone marrow

We have recently demonstrated that bone marrow plasmacytoid DC (pDC) as well as myeloid DC (mDC), irrespective of their origin from RA bone marrow or osteoarthritis (OA) bone marrow, have prominent capacity to differentiate into FLC producing MMP-1 especially under influences of TNF-α (Hirohata et al.,2011). Of note, depletion of pDC from RA bone marrow CD34+ cells significantly diminished their capacities to differentiate into FLC, which were

It should be pointed out that generation of FLC from RA bone marrow CD34+ cells or pDC was correlated with MMP-1 levels in culture supernatants (Hirohata et al.,2001; Hirohata et al.,2011). On the other hand, it has been demonstrated that cadherin-11 is abundantly expressed in type B synoviocytes (Chang et al.,2010) compared with lung or dermal fibroblasts (Vandooren et al.,2008). Accordingly, the FLC induced from RA and OA bone marrow pDC expressed comparable amounts of cadherin-11 mRNA to RA and OA synovial FLC (Hirohata et al.,2011**)**. These results indicate that DC are one of the progenitors of type B synoviocytes irrespective of RA or OA and suggest that bone marrow CD34+ cells might differentiate into type B synoviocyte-like cells via DC, since DC have been demonstrated to originate from CD34+ cells (Reid et al.,1992; Szabolcs et al.,1995; Chen et al.,2004). It is also likely that expansion of immature DC from bone marrow CD34+ cells might be upregulated in RA compared with in OA, accounting for the enhanced capacity of RA bone marrow CD34+ cells to differentiate into FLC upon stimulation with TNF-α, although further studies

**3.4 Recruitment of type B synoviocytes and their precursors into RA joints: Role of** 

It is thus suggested that the presence of abnormal precursors within the bone marrow progenitor cells might play an important role in the pathogenesis of RA by providing a repopulating reservoir of type B synoviocytes, as has been also suggested in other recent studies (Sen et al.,2000). Notably, the numbers of mDC and pDC have been found to be significantly decreased in RA peripheral blood, whereas both mDC and pDC are present in

**3.2 Generation of type B synoviocytes from bone marrow CD34+ cells in RA** 

**3.3 Capacity of bone marrow DC to differentiate into type B synoviocytes** 

restored by addition of pDC in a dose-response manner (Hirohata et al.,2011).

layers (Kyogoku et al.,1992).

of control subjects (Hirohata et al.,2001).

are required to confirm this point.

**DC** 

synovial fluid from RA (Jongbloed et al.,2006). In fact, previous study showed that pDC are recruited to RA synovial tissues and contribute into the local inflammatory environment (Lande et al,2004; Cavanagh et al.,2005). Recent studies have disclosed that the characteristic clinical phenomenon of destructive arthritis spreading between joints is mediated, at least in part, by the transmigration of activated RA synovial fibroblasts (Lefèvre et al.,2009). Thus, RA synovial fibroblasts showed an active movement from human RA synovial tissue or human cartilage-sponge complex containing RA synovial fibroblasts implanted into the SCID mouse to the naive cartilage implanted at the contralateral flank via the vasculature, leading to a marked destruction of the target cartilage (Lefèvre et al.,2009). The movement of DC-like cells from the sublining layer to the lining layer in RA synovium (Kyogoku et al.,1992) and the presence of DC in synovial fluid (Jongbloed et al.,2006; Lande et al,2004) strongly suggest that DC might be also released from the joint to the systemic circulation via draining veins. Since bone marrow pDC as well as mDC have capacities to differentiate into type B synoviocyte-like cells, it is possible that DC, as precursors for synovial fibroblasts, also contribute to the spread of destructive arthritis between joints in RA.

## **4. RA as a disease of antigen-presenting cells**

A number of studies have demonstrated that RA is strongly associated with HLA-DR4 or DR1 (Nepom,2001), which are involved in the presentation of antigens to T cells. These results suggest that the antigen presentation involving HLA-DR4 or the shared epitope might play a critical role in the development of synovitis in human RA. In fact, the interactions between DC and T cells, possibly through MHC class II antigens, have been disclosed in the sublining layers of the RA synovium (Kyogoku et al.,1992). If the antigens presented by APC to T cells are perpetuating antigens, such as autoantigens or antigens of persistently infected virus, that are presented through MHC class II molecules, continuing activation of APC might take place. Further studies to explore such antigens that involve persistent interactions between APC and T cells would be still important for the complete understanding of the pathogenesis of RA.

As highlighted above, bone marrow derived monocytes and DC have been shown to be the precursors of type A synoviocytes and type B synoviocytes, respectively. It is therefore suggested that RA might be a disease of dysregulated activation of antigen-presenting cells (APC), leading to synovial proliferation. Lymphocytes activation in the synovium can also be triggered by the activation of APC, accounting for activation of T cells and B cells in the synovium. Triggering with arthritogenic antigens, followed by dysregulated generation of APC from the bone marrow might result in persistent recruitment of APC into the synovium.

## **5. Bone marrow abnormalities and angiogenesis in RA**

A number of studies indicated that neovascularization is crucial to the synovial hyperplasia in RA (Koch,1998; Hirohata & Sakakibara,1999). Postnatal neovascularization has been attributed to so-called angiogenesis, a process characterized by the sprouting of new capillaries from preexisting blood vessels (Folkman & Shing,1992). However, recent studies have demonstrated that endothelial progenitor cells of bone marrow origin play a significant role in the de novo formation of capillaries without preexisting blood vessels, socalled vasculogenesis (Asahara et al.,1997; Gehling et al.,2000; Bhattacharya et al.,2000; Lin et al.,2000).

Role of Bone Marrow in the Pathogenesis of Rheumatoid Arthritis 33

therefore likely that intrinsic abnormalities that were not secondary to the influences of TNF-α might be present in bone marrow progenitor cells, leading to recurrence of RA. In fact, although abnormal regulatory networks in the immune response and cell cycle categories were identified in bone marrow mononuclear cells from RA patients (Lee et al.,2011), it is possible that such changes might be secondary to systemic inflammation, presumably due to proinflammatory cytokines. In this regard, beyond its role in angiogenesis, the demonstration of the abnormal expression of VEGFR-2/KDR mRNA in RA bone marrow CD34+ cells (Hirohata & Yanagida et al.,2004) have brought an impact as

Mesenchymal stem cells (MSCs) have been shown to have potent anti-inflammatory and immunomodulatory properties through suppression of Th1/Th17 response and induction of Treg response (Macdonald et al., 2011). However, it remains unclear whether MSC therapy

As mentioned above, bone marrow CD34+ cells from RA patients have abnormal capacities to respond to TNF-α and to differentiate into FLC producing MMP-1, suggesting that abnormalities in bone marrow CD34+ cells might play a role in the pathogenesis in RA (Hirohata et al.,2001). TNF-α is one of the first triggers to be found effective for the activation of NFκ B (Müller-Ladner et al.,2002). Of note, we have recently demonstrated that RA bone marrow CD34+ cells showed enhanced expression of NFκ B1 (p50), silencing of

Nakamura et al. recently disclosed that the expression of several genes including amphiregulin (AREG), chemokine receptor 4 (CXCR4), and FK506-binding protein 5 (FKBP5), was augmented in bone marrow mononuclear cells from RA patients compared with those from OA patients (Nakamura et al.,2006). Interestingly, FKBP5 was found to be involved in nuclear translocation and activation of NFκB by degradation of inhibitor of NFκB alpha (IκBα) in a human megakaryoblastic leukemia cell line (Bouwmeester et al.,2004; Komura et al.,2005). It is therefore suggested that the up-regulated expression of both NFκB1 and FKBP5 mRNAs in bone marrow CD34+ cells from RA patients might be involved cooperatively in their abnormal responses to TNF-α to differentiate into type B

Although the expression of mRNAs for AREG, CXCR4 and FKBP5 has been shown to be augmented in RA bone marrow mononuclear cells (Nakamura et al.,2006), only FKBP5 mRNA expression was significantly upregulated in bone marrow CD34+ cells from RA (Matsushita et al.,2010). Therefore, it is suggested that the up-regulation of the expression of mRNAs for AREG and CXCR4 in RA bone marrow mononuclear cells might be sequelae of systemic inflammation of RA. By contrast, the up-regulation of FKBP5 mRNA expression in RA bone marrow CD34+ cells might not be secondary to systemic inflammation, but a primary abnormality in bone marrow CD34+ cells (Matsushita et al.,2010). It has been previously shown that TNF-α enhanced NFκB1 mRNA expression in bone marrow CD34+ cells from healthy individuals (Hirohata et al.,2006). However, TNF-α did not enhance FKBP5 mRNA expression in bone marrow CD34+ cells from healthy individuals (Matsushita et al.,2010). It is therefore confirmed that apart from NFkB1, the enhanced

which resulted in prevention of their differentiation into FLC (Hirohata et al.,2006).

the fist evidence for the intrinsic abnormality in RA bone marrow.

is beneficial for treatment of RA.

**6.2 Nuclear factor kappa B1 (NFkB1)** 

**6.3 FK506-binding protein5 (FKBP5)** 

synoviocyte-like cells.

We also showed that RA bone marrow CD34+ cells have enhanced capacities to differentiate into endothelial cells in relation to synovial vascularization (Hirohata & Yanagida et al.,2004). Therefore, bone marrow CD34+ cells might contribute to synovial neovascularization by supplying endothelial precursor cells and, thus, play an important role in the pathogenesis of RA.

Neovascularization of the synovium is not unique to RA. It has also been observed in OA synovium and has been shown to play an important role in the development of new cartilage and mineralization (Brown et al.,1980; Giatromanolaki et al.,2003; Haywood et al.,2003). Of note, recent studies have revealed that levels of expression of the angiogenic factors VEGF and platelet-derived endothelial cell growth factor are increased in RA as well as in OA, relative to normal subjects, whereas the presence of an activated synovial vasculature was high only in RA (Giatromanolaki et al.,2003). Moreover, the vascular activation by VEGF/KDR was significantly lower in OA than in RA patients, although the activation of the hypoxia inducing factor α (HIFα) pathway was comparable in OA and RA patients (Giatromanolaki et al.,2003). These observations suggest the presence of intrinsic abnormalities in synovial endothelial cells in RA patients. Of note, we have disclosed that the expression of VEGFR-2/KDR mRNA in RA bone marrow CD34+ cells was significantly higher than that in OA bone marrow CD34+ cells (Hirohata & Yanagida et al.,2004). It is therefore likely that the differences in VEGF/KDR vascular activation in bone marrow CD34+ cells might result in differences in their capacity to generate endothelial progenitor cells between RA and OA patients (Koch et al.,1994; Giatromanolaki et al.,2001).

It has been shown that decreased numbers and impaired function of endothelial progenitor cells (EPCs) resulting in defective vasculogenesis are associated with RA, leading to premature atherosclerosis (Herbrig et al., 2006; Pakozdi et al., 2009). On the other hand, it has been recently disclosed that EPCs can be differentiated into 2 subpopulations, EPCs of monocytic versus hemangioblastic origin, which have been denoted as early-outgrowth and late-outgrowth EPCs, respectively (Jodon de Villeroché et al, 2010). More importantly, lateoutgrowth EPCs have been found to be increased and have higher colony formation capacity in the active stage of RA. It is therefore likely that hemangioblastic EPC-dependent vasculogenesis might be associated with active inflammation and accelerated atherosclerosis in RA.

## **6. Abnormal gene expression in bone marrow CD34+ cells in RA**

## **6.1 RA and hematopoietic stem cell transplantation**

Although autologous hematopoietic stem cell transplantation (HSCT) has been used to treat severe RA in limited case reports (Joske,1997; Durez et al.,1998), a study with greater numbers of patients have disclosed that recurrence of RA is frequent after the autologous HSCT (Snowden et al.,2004; Bingham & Moore, 2004). Such frequent recurrence after autologous HSCT clearly indicates that abnormalities in bone marrow stem cells persist after the treatment. It has been proposed that bone marrow CD34+ progenitor cell reserve and function are defective in RA probably due, at least in part, to a TNF-α mediated effect, because significant restoration of the disturbed hematopoiesis was obtained following anti-TNF-α treatment (Papadaki et al.,2002; Porta et al.,2004). It should be noted, however, that blockade of TNF-α is not curative for RA in spite of its epoch-making impact on treatment of RA (Feldmann & Maini,2001). Thus, recurrence of RA is noted after discontinuation of blockade of TNF-α or even during anti-TNF-α therapy (Feldmann & Maini,2001). It is therefore likely that intrinsic abnormalities that were not secondary to the influences of TNF-α might be present in bone marrow progenitor cells, leading to recurrence of RA. In fact, although abnormal regulatory networks in the immune response and cell cycle categories were identified in bone marrow mononuclear cells from RA patients (Lee et al.,2011), it is possible that such changes might be secondary to systemic inflammation, presumably due to proinflammatory cytokines. In this regard, beyond its role in angiogenesis, the demonstration of the abnormal expression of VEGFR-2/KDR mRNA in RA bone marrow CD34+ cells (Hirohata & Yanagida et al.,2004) have brought an impact as the fist evidence for the intrinsic abnormality in RA bone marrow.

Mesenchymal stem cells (MSCs) have been shown to have potent anti-inflammatory and immunomodulatory properties through suppression of Th1/Th17 response and induction of Treg response (Macdonald et al., 2011). However, it remains unclear whether MSC therapy is beneficial for treatment of RA.

## **6.2 Nuclear factor kappa B1 (NFkB1)**

32 Challenges in Rheumatology

We also showed that RA bone marrow CD34+ cells have enhanced capacities to differentiate into endothelial cells in relation to synovial vascularization (Hirohata & Yanagida et al.,2004). Therefore, bone marrow CD34+ cells might contribute to synovial neovascularization by supplying endothelial precursor cells and, thus, play an important

Neovascularization of the synovium is not unique to RA. It has also been observed in OA synovium and has been shown to play an important role in the development of new cartilage and mineralization (Brown et al.,1980; Giatromanolaki et al.,2003; Haywood et al.,2003). Of note, recent studies have revealed that levels of expression of the angiogenic factors VEGF and platelet-derived endothelial cell growth factor are increased in RA as well as in OA, relative to normal subjects, whereas the presence of an activated synovial vasculature was high only in RA (Giatromanolaki et al.,2003). Moreover, the vascular activation by VEGF/KDR was significantly lower in OA than in RA patients, although the activation of the hypoxia inducing factor α (HIFα) pathway was comparable in OA and RA patients (Giatromanolaki et al.,2003). These observations suggest the presence of intrinsic abnormalities in synovial endothelial cells in RA patients. Of note, we have disclosed that the expression of VEGFR-2/KDR mRNA in RA bone marrow CD34+ cells was significantly higher than that in OA bone marrow CD34+ cells (Hirohata & Yanagida et al.,2004). It is therefore likely that the differences in VEGF/KDR vascular activation in bone marrow CD34+ cells might result in differences in their capacity to generate endothelial progenitor

cells between RA and OA patients (Koch et al.,1994; Giatromanolaki et al.,2001).

**6. Abnormal gene expression in bone marrow CD34+ cells in RA** 

**6.1 RA and hematopoietic stem cell transplantation** 

It has been shown that decreased numbers and impaired function of endothelial progenitor cells (EPCs) resulting in defective vasculogenesis are associated with RA, leading to premature atherosclerosis (Herbrig et al., 2006; Pakozdi et al., 2009). On the other hand, it has been recently disclosed that EPCs can be differentiated into 2 subpopulations, EPCs of monocytic versus hemangioblastic origin, which have been denoted as early-outgrowth and late-outgrowth EPCs, respectively (Jodon de Villeroché et al, 2010). More importantly, lateoutgrowth EPCs have been found to be increased and have higher colony formation capacity in the active stage of RA. It is therefore likely that hemangioblastic EPC-dependent vasculogenesis might be associated with active inflammation and accelerated atherosclerosis

Although autologous hematopoietic stem cell transplantation (HSCT) has been used to treat severe RA in limited case reports (Joske,1997; Durez et al.,1998), a study with greater numbers of patients have disclosed that recurrence of RA is frequent after the autologous HSCT (Snowden et al.,2004; Bingham & Moore, 2004). Such frequent recurrence after autologous HSCT clearly indicates that abnormalities in bone marrow stem cells persist after the treatment. It has been proposed that bone marrow CD34+ progenitor cell reserve and function are defective in RA probably due, at least in part, to a TNF-α mediated effect, because significant restoration of the disturbed hematopoiesis was obtained following anti-TNF-α treatment (Papadaki et al.,2002; Porta et al.,2004). It should be noted, however, that blockade of TNF-α is not curative for RA in spite of its epoch-making impact on treatment of RA (Feldmann & Maini,2001). Thus, recurrence of RA is noted after discontinuation of blockade of TNF-α or even during anti-TNF-α therapy (Feldmann & Maini,2001). It is

role in the pathogenesis of RA.

in RA.

As mentioned above, bone marrow CD34+ cells from RA patients have abnormal capacities to respond to TNF-α and to differentiate into FLC producing MMP-1, suggesting that abnormalities in bone marrow CD34+ cells might play a role in the pathogenesis in RA (Hirohata et al.,2001). TNF-α is one of the first triggers to be found effective for the activation of NFκ B (Müller-Ladner et al.,2002). Of note, we have recently demonstrated that RA bone marrow CD34+ cells showed enhanced expression of NFκ B1 (p50), silencing of which resulted in prevention of their differentiation into FLC (Hirohata et al.,2006).

## **6.3 FK506-binding protein5 (FKBP5)**

Nakamura et al. recently disclosed that the expression of several genes including amphiregulin (AREG), chemokine receptor 4 (CXCR4), and FK506-binding protein 5 (FKBP5), was augmented in bone marrow mononuclear cells from RA patients compared with those from OA patients (Nakamura et al.,2006). Interestingly, FKBP5 was found to be involved in nuclear translocation and activation of NFκB by degradation of inhibitor of NFκB alpha (IκBα) in a human megakaryoblastic leukemia cell line (Bouwmeester et al.,2004; Komura et al.,2005). It is therefore suggested that the up-regulated expression of both NFκB1 and FKBP5 mRNAs in bone marrow CD34+ cells from RA patients might be involved cooperatively in their abnormal responses to TNF-α to differentiate into type B synoviocyte-like cells.

Although the expression of mRNAs for AREG, CXCR4 and FKBP5 has been shown to be augmented in RA bone marrow mononuclear cells (Nakamura et al.,2006), only FKBP5 mRNA expression was significantly upregulated in bone marrow CD34+ cells from RA (Matsushita et al.,2010). Therefore, it is suggested that the up-regulation of the expression of mRNAs for AREG and CXCR4 in RA bone marrow mononuclear cells might be sequelae of systemic inflammation of RA. By contrast, the up-regulation of FKBP5 mRNA expression in RA bone marrow CD34+ cells might not be secondary to systemic inflammation, but a primary abnormality in bone marrow CD34+ cells (Matsushita et al.,2010). It has been previously shown that TNF-α enhanced NFκB1 mRNA expression in bone marrow CD34+ cells from healthy individuals (Hirohata et al.,2006). However, TNF-α did not enhance FKBP5 mRNA expression in bone marrow CD34+ cells from healthy individuals (Matsushita et al.,2010). It is therefore confirmed that apart from NFkB1, the enhanced

Role of Bone Marrow in the Pathogenesis of Rheumatoid Arthritis 35

As summarized in Fig.4, accumulating evidence has been provided for the involvement of bone marrow in the pathogenesis of RA. Thus, all the constituents in the proliferating synovial tissues might be supplied from bone marrow CD34+ cells. Apparently, RA bone marrow CD34+ cells have abnormal mRNA expression for several genes, possibly resulting in abnormal differentiation of monocytes and DC. Moreover, it is strongly suggested that DC, as precursors for synovial fibroblasts, might also contribute to the spread of destructive

arthritis between joints in RA (Hirohata et al.,2011; Lefèvre et al.,2009).

Fig. 4. Schema for the suggested role of bone marrow in the pathogenesis of RA

In the past decade, the importance of TNF-α in the pathogenesis of RA has come to be increasingly appreciated (Feldmann et al.,1996). We have revealed that CD34+ cells from bone marrow of RA patients have abnormal responsiveness to TNF-α (Hirohata et al.,2001). However, the precise sequelae of abnormal responses of CD34+ cells from bone marrow of RA patients to TNF-α remain unclear. KLF-5 might upregulate the expression of mRNAs for NFkB1 and VEGFR-2/KDR, whereas FKBP5 might enhance activation of NFkB, resulting in further upregulation of NFkB1 mRNA expression. Further studies that explore in detail the mechanism of abnormal expression of the genes, especially KLF-5 and FKBP5, in CD34+ cells from bone marrow of RA patients would be helpful in gaining a complete understanding of the etiology as well as the pathogenesis of RA. In this regard, we showed previously that GM-CSF-stimulated bone marrow CD34+ cells from 3 of 8 RA patients, but none from 7 OA patients, gave rise to spontaneous transformation of highly purified B cells of Epstein-Barr virus (EBV)-seronegative healthy donors, whereas neither GM-CSFstimulated bone marrow CD34+ cells alone nor highly purified B cells alone gave rise to spontaneously transformed B cell lines (Hirohata et al.,2000). All the transformed B cell lines

**7. Conclusion** 

FKBP5 mRNA expression in RA bone marrow CD34+ cells is not secondary to systemic inflammation of RA.

#### **6.4 Krüppel like factor 5 (KLF-5)**

Krüppel like factor 5 (KLF-5), a zinc finger-containing transcription factor, activates many genes, including platelet-derived growth factor (PDGF) A/B, plasminogen activator inhibitor-1, inducible nitric oxide synthase and VEGF receptors (Shindo et al.,2002; Nagai et al.,2005). KLF-5 has been shown to cooperate with NFkB1 to activate PDGF-A gene expression (Nagai et al.,2005; Aizawa et al.,2004), which might be involved in synovial fibroblast-like cell proliferation (Ohba et al.,1996). KLF-5 mRNA expression in bone marrow CD34+ cells was significantly higher in RA patients than in OA patients (Hirohata et al.,2009). It is thus likely that the upregulation of VEGFR-2/KDR mRNA expression might be secondary to the enhanced KLF-5 mRNA expression in RA bone marrow CD34+ cells. Of note, TNF-α enhanced NFkB1 mRNA expression, but not KLF-5 mRNA expression, in bone marrow CD34+ cells from normal individuals (Fig.3) (Hirohata et al.,2009).

Fig. 3. Effect of TNF-α on the expression of mRNAs for NFkB1 and KLF-5 in bone marrow CD34+ cells from a healthy donor

Previous studies also demonstrated that the suppression of KLF-5 by silencing RNA resulted in a reduction of NFkB1 mRNA in IEC6 cells stimulated with lipopolysaccharide, indicating that KLF-5 is an upstream regulator for NFkB1 mRNA expression in IEC6 cells (Chanchevalap et al.,2006). Taken together, it is most likely that the upregulation of KLF-5 mRNA expression might lead to the enhanced expression of NFkB1 mRNA in bone marrow CD34+ cells, but not vice versa, in RA. In addition, the upregulation of KLF-5 mRNA as well as NFkB1 mRNA in RA bone marrow CD34+ cells might result in their abnormal capacities to differentiate into FLC. Although it is strongly suggested that KLF-5 might be an upstream regulator of NFkB1 mRNA in bone marrow CD34+ cells, further studies to explore the mechanism of abnormal expression of KLF-5 mRNA in BM CD34+ cells and its relation with FKBP5 would be important.

## **7. Conclusion**

34 Challenges in Rheumatology

FKBP5 mRNA expression in RA bone marrow CD34+ cells is not secondary to systemic

Krüppel like factor 5 (KLF-5), a zinc finger-containing transcription factor, activates many genes, including platelet-derived growth factor (PDGF) A/B, plasminogen activator inhibitor-1, inducible nitric oxide synthase and VEGF receptors (Shindo et al.,2002; Nagai et al.,2005). KLF-5 has been shown to cooperate with NFkB1 to activate PDGF-A gene expression (Nagai et al.,2005; Aizawa et al.,2004), which might be involved in synovial fibroblast-like cell proliferation (Ohba et al.,1996). KLF-5 mRNA expression in bone marrow CD34+ cells was significantly higher in RA patients than in OA patients (Hirohata et al.,2009). It is thus likely that the upregulation of VEGFR-2/KDR mRNA expression might be secondary to the enhanced KLF-5 mRNA expression in RA bone marrow CD34+ cells. Of note, TNF-α enhanced NFkB1 mRNA expression, but not KLF-5 mRNA expression, in bone marrow CD34+ cells from normal individuals (Fig.3)

Fig. 3. Effect of TNF-α on the expression of mRNAs for NFkB1 and KLF-5 in bone marrow

Previous studies also demonstrated that the suppression of KLF-5 by silencing RNA resulted in a reduction of NFkB1 mRNA in IEC6 cells stimulated with lipopolysaccharide, indicating that KLF-5 is an upstream regulator for NFkB1 mRNA expression in IEC6 cells (Chanchevalap et al.,2006). Taken together, it is most likely that the upregulation of KLF-5 mRNA expression might lead to the enhanced expression of NFkB1 mRNA in bone marrow CD34+ cells, but not vice versa, in RA. In addition, the upregulation of KLF-5 mRNA as well as NFkB1 mRNA in RA bone marrow CD34+ cells might result in their abnormal capacities to differentiate into FLC. Although it is strongly suggested that KLF-5 might be an upstream regulator of NFkB1 mRNA in bone marrow CD34+ cells, further studies to explore the mechanism of abnormal expression of KLF-5 mRNA in BM CD34+ cells and its relation with

inflammation of RA.

(Hirohata et al.,2009).

CD34+ cells from a healthy donor

FKBP5 would be important.

**6.4 Krüppel like factor 5 (KLF-5)** 

As summarized in Fig.4, accumulating evidence has been provided for the involvement of bone marrow in the pathogenesis of RA. Thus, all the constituents in the proliferating synovial tissues might be supplied from bone marrow CD34+ cells. Apparently, RA bone marrow CD34+ cells have abnormal mRNA expression for several genes, possibly resulting in abnormal differentiation of monocytes and DC. Moreover, it is strongly suggested that DC, as precursors for synovial fibroblasts, might also contribute to the spread of destructive arthritis between joints in RA (Hirohata et al.,2011; Lefèvre et al.,2009).

Fig. 4. Schema for the suggested role of bone marrow in the pathogenesis of RA

In the past decade, the importance of TNF-α in the pathogenesis of RA has come to be increasingly appreciated (Feldmann et al.,1996). We have revealed that CD34+ cells from bone marrow of RA patients have abnormal responsiveness to TNF-α (Hirohata et al.,2001). However, the precise sequelae of abnormal responses of CD34+ cells from bone marrow of RA patients to TNF-α remain unclear. KLF-5 might upregulate the expression of mRNAs for NFkB1 and VEGFR-2/KDR, whereas FKBP5 might enhance activation of NFkB, resulting in further upregulation of NFkB1 mRNA expression. Further studies that explore in detail the mechanism of abnormal expression of the genes, especially KLF-5 and FKBP5, in CD34+ cells from bone marrow of RA patients would be helpful in gaining a complete understanding of the etiology as well as the pathogenesis of RA. In this regard, we showed previously that GM-CSF-stimulated bone marrow CD34+ cells from 3 of 8 RA patients, but none from 7 OA patients, gave rise to spontaneous transformation of highly purified B cells of Epstein-Barr virus (EBV)-seronegative healthy donors, whereas neither GM-CSFstimulated bone marrow CD34+ cells alone nor highly purified B cells alone gave rise to spontaneously transformed B cell lines (Hirohata et al.,2000). All the transformed B cell lines

Role of Bone Marrow in the Pathogenesis of Rheumatoid Arthritis 37

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[Epub ahead of print] PMID:21647863

inflammation in rheumatoid arthritis. *Arthritis Rheum.* 46: 2578-86.

chemokine receptor 4. *Arthritis Rheum.* 46: 130-7.

*Arthritis Res. Ther.* 8: R15.

350: 337-8.

4149-56.

951-62.

*Cancer Res.* 65: 3281-9.

*Nippon Rinsho* 50: 483-89.

*Arthritis Res. Ther.* 13:R89.

145: 1337-42.

distinct dendritic cell subsets in psoriatic arthritis and rheumatoid arthritis.

human chondrocytes by the interaction of stromal cell-derived factor 1 and CXC

cytokine modulating endothelial function in rheumatoid arthritis. *J. Immunol.* 152:

pathway in transforming growth factor-beta1 production in idiopathic myelofibrosis: possible relationship with FK506 binding protein 51 overexpression.

triggers IL-6 production. Role in anti-CD3-induced T cell activation. *J. Immunol.* 

rheumatoid arthritis ―as a clue to elucidate its pathogenesis― (in Japanese).

independent growth of synoviocytes from arthritic and normal joints. Stimulation by exogenous platelet-derived growth factor and inhibition by transforming

plasmacytoid dendritic cells in synovial fluid and tissue of patients with chronic

induces lymphocyte function-associated antigen-1/intercellular adhesion

Abnormal networks of immune response-related molecules in bone marrow cells from patients with rheumatoid arthritis as revealed by DNA microarray analysis.


**4** 

*France* 

**Estrogens Involvement in the** 

Philippe Galera and Laure Maneix

*University of Caen/Lower-Normandy,* 

*Faculty of Medicine, Caen* 

*Laboratory of Extracellular Matrix and Pathology,* 

Safa Moslemi, Magali Demoor, Karim Boumediene,

**Physiopathology of Articular Cartilage** 

Life expectancy increases in developed countries and results in a high prevalence of agerelated diseases namely in women. Elderly subjects generate a huge demand on the social and health services linked to their disability and dependence. Besides the own effects of aging, the estrogenic deficiency, especially during menopause, is the major cause of degenerative diseases such as osteoporosis, skin aging, Alzheimer's disease and osteoarthritis (OA) (Antonicelli et al., 2008; Candore et al., 2006; Elders, 2000; Felson & Nevitt, 1998; Pietschmann et al., 2008; Stovall & Pinkerton, 2008). OA is a worldwide public health problem which may affect different sites of the skeleton. In the Western world, at least 10% of the population has OA symptoms and 80% of the population will be potentially affected after the age of 70 years. Knee OA represents one of the major causes of morbidity and disability in relation to the worsening of quality of life (Elders, 2000). Pathogenesis of knee OA involves multiple factors including gender, weight, and genetics. Although agedependent degenerative pathologies, especially OA, involve complex mechanisms in which the imbalance of the cytokines/growth factors plays a crucial role, it is more and more obvious that estrogens participate, with their receptors and modulators, to all of these diseases affecting connective tissues (Calleja-Agius & Brincat, 2009). Estrogens are steroidal hormones irreversibly synthesized from androgens *via* a crucial enzyme of steroidogenesis called aromatase, a member of cytochrome P450 superfamily, encoded by the *CYP19* gene

Although non genomic action of estrogens is now recognized but these sexual hormones act often as signaling molecules that exert their effects by binding to estrogen receptors (ER)

The estrogen-receptor complex interacts then with DNA to change the expression of estrogen-responsive genes. The two known estrogen receptors, ER and ER, are present in numerous tissues other than those associated with reproduction, including bone, liver, heart, brain and cartilage and being selectively expressed in some targets; for instance ER is selectively expressed in lung and intestine. The presence of ER and ER was detected in articular chondrocytes of different species, especially in human, rat, pig, cow and rabbit

and being expressed in both sexes of many species (Simpson, 2003).

**1. Introduction** 

within the cells.

with c-kit ligand, granulocyte-macrophage colony-stimulating factor, and TNFalpha. *J. Immunol.* 154: 5851-61.


## **Estrogens Involvement in the Physiopathology of Articular Cartilage**

Safa Moslemi, Magali Demoor, Karim Boumediene, Philippe Galera and Laure Maneix *Laboratory of Extracellular Matrix and Pathology, University of Caen/Lower-Normandy, Faculty of Medicine, Caen France* 

## **1. Introduction**

40 Challenges in Rheumatology

Tak PP. (2000). Examination of the synovium and synovial fluid, *in* Firestein GS, Panayi GS

Thomas R, Davis LS, Lipsky PE. (1994). Rheumatoid synovium is enriched in mature

Vandooren B, Cantaert T, ter Borg M, et al. (2008). Tumor necrosis factor drives cadherin 11 expression in rheumatoid inflammation. *Arthritis Rheum.* 58: 3051-62. Zvaifler NJ, Steinman RM, Kaplan G, Lau LL, Rivelis M. (1985). Identification of

alpha. *J. Immunol.* 154: 5851-61.

Oxford University Press, New York, pp.55-68.

rheumatoid arthritis. *J. Clin. Invest.* 76: 789-800.

antigen-presenting dendritic cells. *J. Immunol.* 152: 2613-23.

with c-kit ligand, granulocyte-macrophage colony-stimulating factor, and TNF-

& Wollheim RA. (eds.), *Rheumatoid arthritis: Frontiers on pathogenesis and treatment*,

immunostimulatory dendritic cells in the synovial effusions of patients with

Life expectancy increases in developed countries and results in a high prevalence of agerelated diseases namely in women. Elderly subjects generate a huge demand on the social and health services linked to their disability and dependence. Besides the own effects of aging, the estrogenic deficiency, especially during menopause, is the major cause of degenerative diseases such as osteoporosis, skin aging, Alzheimer's disease and osteoarthritis (OA) (Antonicelli et al., 2008; Candore et al., 2006; Elders, 2000; Felson & Nevitt, 1998; Pietschmann et al., 2008; Stovall & Pinkerton, 2008). OA is a worldwide public health problem which may affect different sites of the skeleton. In the Western world, at least 10% of the population has OA symptoms and 80% of the population will be potentially affected after the age of 70 years. Knee OA represents one of the major causes of morbidity and disability in relation to the worsening of quality of life (Elders, 2000). Pathogenesis of knee OA involves multiple factors including gender, weight, and genetics. Although agedependent degenerative pathologies, especially OA, involve complex mechanisms in which the imbalance of the cytokines/growth factors plays a crucial role, it is more and more obvious that estrogens participate, with their receptors and modulators, to all of these diseases affecting connective tissues (Calleja-Agius & Brincat, 2009). Estrogens are steroidal hormones irreversibly synthesized from androgens *via* a crucial enzyme of steroidogenesis called aromatase, a member of cytochrome P450 superfamily, encoded by the *CYP19* gene and being expressed in both sexes of many species (Simpson, 2003).

Although non genomic action of estrogens is now recognized but these sexual hormones act often as signaling molecules that exert their effects by binding to estrogen receptors (ER) within the cells.

The estrogen-receptor complex interacts then with DNA to change the expression of estrogen-responsive genes. The two known estrogen receptors, ER and ER, are present in numerous tissues other than those associated with reproduction, including bone, liver, heart, brain and cartilage and being selectively expressed in some targets; for instance ER is selectively expressed in lung and intestine. The presence of ER and ER was detected in articular chondrocytes of different species, especially in human, rat, pig, cow and rabbit

Estrogens Involvement in the Physiopathology of Articular Cartilage 43

intra- and/or paracrine actions. Due to their lipophilic feature, estrogen can diffuse into the cell through plasma membrane and induce estrogen-dependent intracellular signaling pathways and/or bind the intracellular receptors especially ER stimulating an estrogen-

In mammals, estrogens produced locally in different tissues, may exert various biological effects but are also responsible of the development of some pathological process such as hormone-dependent cancers. Estrogens exert their physiological and pathological effects through specific receptors considered as nuclear factors by binding target genes at a specific

The first study on estrogen receptor, published by Toft & Groski in 1966, demonstrated that a specific protein of rat uterus was able to bind specifically estrogens. Later, the gene of this protein has been cloned (Walter et al., 1985) and sequenced (Green et al., 1986) from mammal cancer epithelium (MCF-7) of a patient suffering from breast cancer. This protein is now recognized as ER. A second gene (ER) has been also cloned from a cDNA library of the rat prostatic cells (Kuiper et al., 1996), being expressed selectively in some tissues and

It is now established that the majority of the normal and pathological action of estrogens is generally mediated through these two estrogen receptors ERand ER which are members of nuclear receptor super family including progesterone, glucocorticoids, androgens and vitamin D receptors (Nuclear Receptor Nomenclature Committee, 1999; Germain et al., 2006; Mangelsdorf et al., 1995). In human, ERis encoded by *ESR1* gene located on chromosome 6 (6q25.1) whereas ER is encoded by *ESR2* gene located on chromosome 14 (14q23.2). Both *ESR1* and *ESR2* genes encoding the estrogen receptor may undergo alternative splicing of mRNA. Most of these transcripts differ only in their 5'UTR (Untranslated Region) and will be mainly translated into a long form of ER, recognized as ER66 (Flouriot et al., 1998). However, a second form of ER protein, derived from alternative splicing of exon 1A mRNA or a site of alternative translation initiation (AUG codon 174) was discovered and named ER46 (Barrailler et al. 1999; Flouriot et al., 2000). After translation, the 46 kDa isoform is truncated of the 173 first amino acids of the long form of 66 kDa. ER46 protein is thus composed of 422 amino acids, whereas ER66 has 595 amino acids. Both ER isoforms are capable of inducing a physiological response after ligand binding. The 46 kDa isoform can heterodimerize with ER66 and competitively inhibit the functions of ligand-independent *trans* activation of the long form of the receptor

ERand ER530 amino acids) are structurally organized in six distinct functional domains (A to F) (Fig. 1). There are structural and functional similarities more or less strong between ER and ER whose homology percentages vary significantly depending on the area considered. DNA binding domain (DBD) is conserved at about 97%, which means that both receptors can bind the same *cis* nucleotide sequences and thus activate transcription of identical target genes. However, there is only 55% homology at the level of ligand binding domain (LBD), indicating that ER and ER have different ligand binding specificity. Finally, ER has only a truncated form of ligand-independent *trans* activation domain (AF-1), thus limiting its *trans* activation ability (Hall & McDonnell, 1999; Pearce &

dependent response in target cells.

*cis* sequence called Estrogen Responsive Element (ERE).

presenting more affinity for phytoestrogens.

**2.2 ER structure** 

(Flouriot et al., 2000).

Jordan, 2004).

(Classen et al., 2001; Dayani et al., 1988; Oshima et al., 2007; Ushiyama et al., 1999). The action of 17-estradiol (17-E2) on the cartilage appears to be intimately linked to the sex of individuals since it was shown that the binding capacity of 17-E2 to its receptor was significantly higher in chondrocytes derived from male rather than female individuals (Nasatzky et al., 1994). In addition, 17-E2 can stimulate the nuclear expression of its own receptors in chondrocytes and create therefore a loop that reinforces the activation effects of the hormone in this cell type (Richmond et al., 2000). Moreover, a strong correlation was found between the polymorphism of *ESR1* gene encoding ER and increased risk of OA in several populations (Bergink et al. 2003; Valdes et al., 2006). All these observations tend to prove that the cartilage is an estrogen-sensitive tissue, in which 17-E2 would be likely to play a preponderant role in regulating chondrocyte homeostasis in joint diseases such as OA.

Even thought hormone replacement therapy (HRT) is the most efficient treatment against, at least, some of the degenerative pathologies mentioned above but studies confirm that risks linked to HRT can exceed the discounted benefits. As a consequence, it becomes important to address the question of the age-associated disorders and to find new targets to treat them. Selective Estrogen Receptor Modulators (SERM) like tamoxifen, raloxifen, fulvestrant and genistein have shown to be useful and attracted the attention of researchers (Cotter & Cashman, 2003; Khalil, 2010; Riggs & Hartmann 2003). A characteristic that distinguishes these substances from pure receptor agonists and antagonists is that their action is different in various tissues, thereby giving the possibility to selectively inhibit or stimulate estrogenlike action in various tissues. For instance tamoxifen (a first generation SERM) and raloxifen have been clinically used as antagonists of ER against breast cancer while they could be potentially agonists in bone and growth plate (Chagin et al, 2007; Nilsson et al, 2003). It has been proposed that these SERM could be used to replace natural estrogens to induce growth plate fusion reducing thereby the final height in girls expected to achieve extreme tall stature. Moreover, phytoestrogens (flavones, isoflavones, lignans) have aroused much interest as natural SERM and potential substitutes in the hormonal treatment of postmenopausal women, but they require much further investigation regarding their mechanism(s) of action and their safety (Dodin et al, 2003).

## **2. Estrogen synthesis and action**

## **2.1 Estrogen synthesis** *via* **aromatase**

Estrogens are steroidal hormones composed from 18 atoms of carbon and produced essentially in gonads, ovary and testis, but also in non reproductive tissues such as bone and adipose tissue. These lipophilic compounds irreversibly synthesized from androgens *via* a crucial enzyme of steroidogenesis called aromatase, a member of cytochrome P450 superfamily, encoded by the *CYP19* gene and being expressed in both sexes of many species. In pre-menopoausal women, estrogens such as 17-E2 (the more estrogenic) and estrone are synthesized in ovary during follicular phase whereas estriol being produced essentially by placenta during pregnancy. After menopause, when ovary activity disappears, production of 17-E2 from testosterone *via* aromatase is assumed by peripheral tissues like liver, adipose tissue, bone, vascular endothelium, chondrocyte and synovial cells (Simpson, 2003; Takeuchi et al., 2007; Tanko et al., 2008). Consequently, circulating 17-E2 concentrations decrease drastically to 0.04-0.21 nM to reach those found in man (Chambliss & Shaul, 2002). Thus, estrogen action being localized and switch from endocrine to auto-, intra- and/or paracrine actions. Due to their lipophilic feature, estrogen can diffuse into the cell through plasma membrane and induce estrogen-dependent intracellular signaling pathways and/or bind the intracellular receptors especially ER stimulating an estrogendependent response in target cells.

## **2.2 ER structure**

42 Challenges in Rheumatology

(Classen et al., 2001; Dayani et al., 1988; Oshima et al., 2007; Ushiyama et al., 1999). The action of 17-estradiol (17-E2) on the cartilage appears to be intimately linked to the sex of individuals since it was shown that the binding capacity of 17-E2 to its receptor was significantly higher in chondrocytes derived from male rather than female individuals (Nasatzky et al., 1994). In addition, 17-E2 can stimulate the nuclear expression of its own receptors in chondrocytes and create therefore a loop that reinforces the activation effects of the hormone in this cell type (Richmond et al., 2000). Moreover, a strong correlation was found between the polymorphism of *ESR1* gene encoding ER and increased risk of OA in several populations (Bergink et al. 2003; Valdes et al., 2006). All these observations tend to prove that the cartilage is an estrogen-sensitive tissue, in which 17-E2 would be likely to play a preponderant role in regulating chondrocyte homeostasis in joint diseases such as

Even thought hormone replacement therapy (HRT) is the most efficient treatment against, at least, some of the degenerative pathologies mentioned above but studies confirm that risks linked to HRT can exceed the discounted benefits. As a consequence, it becomes important to address the question of the age-associated disorders and to find new targets to treat them. Selective Estrogen Receptor Modulators (SERM) like tamoxifen, raloxifen, fulvestrant and genistein have shown to be useful and attracted the attention of researchers (Cotter & Cashman, 2003; Khalil, 2010; Riggs & Hartmann 2003). A characteristic that distinguishes these substances from pure receptor agonists and antagonists is that their action is different in various tissues, thereby giving the possibility to selectively inhibit or stimulate estrogenlike action in various tissues. For instance tamoxifen (a first generation SERM) and raloxifen have been clinically used as antagonists of ER against breast cancer while they could be potentially agonists in bone and growth plate (Chagin et al, 2007; Nilsson et al, 2003). It has been proposed that these SERM could be used to replace natural estrogens to induce growth plate fusion reducing thereby the final height in girls expected to achieve extreme tall stature. Moreover, phytoestrogens (flavones, isoflavones, lignans) have aroused much interest as natural SERM and potential substitutes in the hormonal treatment of postmenopausal women, but they require much further investigation regarding their

Estrogens are steroidal hormones composed from 18 atoms of carbon and produced essentially in gonads, ovary and testis, but also in non reproductive tissues such as bone and adipose tissue. These lipophilic compounds irreversibly synthesized from androgens *via* a crucial enzyme of steroidogenesis called aromatase, a member of cytochrome P450 superfamily, encoded by the *CYP19* gene and being expressed in both sexes of many species. In pre-menopoausal women, estrogens such as 17-E2 (the more estrogenic) and estrone are synthesized in ovary during follicular phase whereas estriol being produced essentially by placenta during pregnancy. After menopause, when ovary activity disappears, production of 17-E2 from testosterone *via* aromatase is assumed by peripheral tissues like liver, adipose tissue, bone, vascular endothelium, chondrocyte and synovial cells (Simpson, 2003; Takeuchi et al., 2007; Tanko et al., 2008). Consequently, circulating 17-E2 concentrations decrease drastically to 0.04-0.21 nM to reach those found in man (Chambliss & Shaul, 2002). Thus, estrogen action being localized and switch from endocrine to auto-,

mechanism(s) of action and their safety (Dodin et al, 2003).

**2. Estrogen synthesis and action 2.1 Estrogen synthesis** *via* **aromatase** 

OA.

In mammals, estrogens produced locally in different tissues, may exert various biological effects but are also responsible of the development of some pathological process such as hormone-dependent cancers. Estrogens exert their physiological and pathological effects through specific receptors considered as nuclear factors by binding target genes at a specific *cis* sequence called Estrogen Responsive Element (ERE).

The first study on estrogen receptor, published by Toft & Groski in 1966, demonstrated that a specific protein of rat uterus was able to bind specifically estrogens. Later, the gene of this protein has been cloned (Walter et al., 1985) and sequenced (Green et al., 1986) from mammal cancer epithelium (MCF-7) of a patient suffering from breast cancer. This protein is now recognized as ER. A second gene (ER) has been also cloned from a cDNA library of the rat prostatic cells (Kuiper et al., 1996), being expressed selectively in some tissues and presenting more affinity for phytoestrogens.

It is now established that the majority of the normal and pathological action of estrogens is generally mediated through these two estrogen receptors ERand ER which are members of nuclear receptor super family including progesterone, glucocorticoids, androgens and vitamin D receptors (Nuclear Receptor Nomenclature Committee, 1999; Germain et al., 2006; Mangelsdorf et al., 1995). In human, ERis encoded by *ESR1* gene located on chromosome 6 (6q25.1) whereas ER is encoded by *ESR2* gene located on chromosome 14 (14q23.2). Both *ESR1* and *ESR2* genes encoding the estrogen receptor may undergo alternative splicing of mRNA. Most of these transcripts differ only in their 5'UTR (Untranslated Region) and will be mainly translated into a long form of ER, recognized as ER66 (Flouriot et al., 1998). However, a second form of ER protein, derived from alternative splicing of exon 1A mRNA or a site of alternative translation initiation (AUG codon 174) was discovered and named ER46 (Barrailler et al. 1999; Flouriot et al., 2000). After translation, the 46 kDa isoform is truncated of the 173 first amino acids of the long form of 66 kDa. ER46 protein is thus composed of 422 amino acids, whereas ER66 has 595 amino acids. Both ER isoforms are capable of inducing a physiological response after ligand binding. The 46 kDa isoform can heterodimerize with ER66 and competitively inhibit the functions of ligand-independent *trans* activation of the long form of the receptor (Flouriot et al., 2000).

ERand ER530 amino acids) are structurally organized in six distinct functional domains (A to F) (Fig. 1). There are structural and functional similarities more or less strong between ER and ER whose homology percentages vary significantly depending on the area considered. DNA binding domain (DBD) is conserved at about 97%, which means that both receptors can bind the same *cis* nucleotide sequences and thus activate transcription of identical target genes. However, there is only 55% homology at the level of ligand binding domain (LBD), indicating that ER and ER have different ligand binding specificity. Finally, ER has only a truncated form of ligand-independent *trans* activation domain (AF-1), thus limiting its *trans* activation ability (Hall & McDonnell, 1999; Pearce & Jordan, 2004).

Estrogens Involvement in the Physiopathology of Articular Cartilage 45

In this pathway, ERs will follow the classical mechanism described above except that they interact at the nuclear level with various transcriptional activator or repressor factors to regulate transcription of many estrogen-dependent genes that lack an ERE (Kushner et al.,

Indeed, most estrogen-dependent genes have not necessarily in their regulatory regions a consensus ERE sequence. Therefore, estrogen will be involved in signaling pathways called ERE-independent which imply an interaction of ER with promoter of target gene through other transcription factors. In this case, the region of the receptor DBD does not bind to DNA, but participates in protein-protein interactions or recruitment of co-regulatory proteins to regulate expression of many genes. This mechanism of action is frequently used by nuclear receptors, which significantly complicates the decrypting of the effects induced by the ER (Gottlicher et al., 1998). Many of ER/protein interactions, which occur in cells to regulate transcription of many target genes, are composed for example of ER/AP-1 complex (c-Fos/c-Jun) (Ascenzi et al., 2006; Duan et al., 2008; Kushner et al. 2000; Matthews et al. 2006 ; Paech et al., 1997, Uht et al 1997; Webb et al., 1999,), ER/Sp complex (Ascenzi et al., 2006; He et al., 2005; Kim et al., 2003; Saville et al., 2000; Stoner et al., 2004), and ER/NF-B complex (Galien & Garcia, 1997; Stein & Yang, 1995). This latter is the only known

For instance, after being activated by 17-E2 binding, ER interact with the transcription factor NF-B to control the transcriptional repression of certain genes such as IL-6 gene, which is involved in cartilage catabolism. Furthermore, in articular chondrocytes, it has been shown that 17-E2 can counteract the effects of IL-1 by inhibiting nuclear translocation of the p65 subunit of NF-B and therefore, binding of p65 to inducible nitric oxide synthase (iNOS) gene promoter (Richette et al., 2007). In addition, it has been suggested that ER and NF-B compete for binding to the same transcriptional co-activators (p300/CBP and PCAF), and that in the case of activation of transcription by 17-E2 through ER, the pool of these coactivators is mobilized predominantly by ER at the expense of NF-B (Ansari & Gandy,

Therefore, estrogen/ER complex allows developing multiple physiological responses following hormonal stimulation by different signaling pathways and inter-connections with

The non-genomic effects are defined as any action that does not involve transcriptional activity resulting from direct or indirect interaction of a nuclear receptor with the regulatory sequences of hormone-regulated genes. These effects are very rapid (in the order of several seconds to several minutes), incompatible with gene activation or protein synthesis. The effects of non-genomic estrogen receptors are often linked to signaling pathways that involve G protein-coupled receptors, ion channels or receptors linked to enzymes. 17-E2 could modulate intracellular calcium or cAMP production and activate MAPK/ERK, PLC, PKA, or that of PI3K signaling pathways (Marino et al., 2002) (Fig 2). All these effects are not modified by inhibitors of transcription (actinomycin D) or translation (cycloheximide) (Losel & Wehling, 2003), confirming that they do not depend on a genomic action. In 1998, Beyer and Raab, by coupling 17-E2 to BSA (Bovine Serum Albumin), thereby preventing it from crossing the plasma membrane, observed that 17-E2 modulates intracellular calcium. Thus,

**2.3.2 ERE-independent genomic pathway**

2000; Paech et al., 1997; Sabbah et al., 1999; Safe, 2001).

mechanism of transcriptional repression induced by ER (Fig. 2).

different *trans* factors and *cis* elements at the DNA level of target cells.

**2.3.3 Non genomic signaling pathway** 

2007).

Fig. 1. Structures and functional domains of estrogen receptors (ER)

Structures, functional domains and sequence homology percentage (*italic*) of and isoforms. AF-1, ligand-independent *trans* activation domain; AF-2, ligand-dependent *trans* activation domain; DBD, DNA Binding Domain; hsp90, heat-shock protein 90; LBD, Ligand Binding Domain; NLS, Nuclear Localization Signal. These 3 isoforms, ERα46, ERα66 and ERβ are expressed in articular chondrocytes.

## **2.3 Genomic and non genomic action of estrogen**

It is well established that estrogens act at target genes level to modulate their transcription *via* ER binding, so playing a transcriptional factor role recognized as ligand-dependent mechanism (Jensen & DeSombre, 1973). However, estrogen action becomes more complicated following the discovery of ER, or different ER and ER isoforms but also with the identification of new plasma-associated membrane receptors. ER and ER regulate thus estrogen-dependent genes expression through two distinct mechanisms: EREdependent genomic pathway and ERE-independent genomic pathway.

## **2.3.1 ERE-dependent genomic pathway**

In this mechanism ER and ER, following ligand fixation (estrogen, phytoestrogen, SERM), are subjected to homo- or hetero-dimerization, then ligand/ER complex move to the nucleus to bind directly ERE *cis* element (AGGTCAxxxTGACCT) (Hall et al., 2001) present in the promoter of target genes (Fig. 2). The determination of consensus sequences ERE and the fixation of ER will help to recruit transcriptional factors such as FOXA1 (Forkhead Box A1) or GATA4 (GATA binding protein 4), which will first ensure the chromatin remodeling necessary for ER binding. Ligand binding also allows the recruitment of transcription cofactors such as SRC-1 (Steroid Receptor Coactivator-1), GRIP-1 (Glucocorticoid Receptor Interacting Protein-1), CBP (CREB Binding Protein)/p300, or TRAP220 (Thyroid Hormone Receptor Activating Protein 220) (McKenna et al., 1999). The classic genomic pathway requires the activity of the two *trans* activation domains AF-1 and AF-2 that will allow the sequential and cyclic recruitment of different co-factors of transcription (Metivier et al., 2001).

## **2.3.2 ERE-independent genomic pathway**

44 Challenges in Rheumatology

Fig. 1. Structures and functional domains of estrogen receptors (ER)

dependent genomic pathway and ERE-independent genomic pathway.

cyclic recruitment of different co-factors of transcription (Metivier et al., 2001).

**2.3 Genomic and non genomic action of estrogen** 

**2.3.1 ERE-dependent genomic pathway** 

Structures, functional domains and sequence homology percentage (*italic*) of and isoforms. AF-1, ligand-independent *trans* activation domain; AF-2, ligand-dependent *trans* activation domain;

DBD, DNA Binding Domain; hsp90, heat-shock protein 90; LBD, Ligand Binding Domain; NLS, Nuclear Localization Signal. These 3 isoforms, ERα46, ERα66 and ERβ are expressed in articular chondrocytes.

It is well established that estrogens act at target genes level to modulate their transcription *via* ER binding, so playing a transcriptional factor role recognized as ligand-dependent mechanism (Jensen & DeSombre, 1973). However, estrogen action becomes more complicated following the discovery of ER, or different ER and ER isoforms but also with the identification of new plasma-associated membrane receptors. ER and ER regulate thus estrogen-dependent genes expression through two distinct mechanisms: ERE-

In this mechanism ER and ER, following ligand fixation (estrogen, phytoestrogen, SERM), are subjected to homo- or hetero-dimerization, then ligand/ER complex move to the nucleus to bind directly ERE *cis* element (AGGTCAxxxTGACCT) (Hall et al., 2001) present in the promoter of target genes (Fig. 2). The determination of consensus sequences ERE and the fixation of ER will help to recruit transcriptional factors such as FOXA1 (Forkhead Box A1) or GATA4 (GATA binding protein 4), which will first ensure the chromatin remodeling necessary for ER binding. Ligand binding also allows the recruitment of transcription cofactors such as SRC-1 (Steroid Receptor Coactivator-1), GRIP-1 (Glucocorticoid Receptor Interacting Protein-1), CBP (CREB Binding Protein)/p300, or TRAP220 (Thyroid Hormone Receptor Activating Protein 220) (McKenna et al., 1999). The classic genomic pathway requires the activity of the two *trans* activation domains AF-1 and AF-2 that will allow the sequential and In this pathway, ERs will follow the classical mechanism described above except that they interact at the nuclear level with various transcriptional activator or repressor factors to regulate transcription of many estrogen-dependent genes that lack an ERE (Kushner et al., 2000; Paech et al., 1997; Sabbah et al., 1999; Safe, 2001).

Indeed, most estrogen-dependent genes have not necessarily in their regulatory regions a consensus ERE sequence. Therefore, estrogen will be involved in signaling pathways called ERE-independent which imply an interaction of ER with promoter of target gene through other transcription factors. In this case, the region of the receptor DBD does not bind to DNA, but participates in protein-protein interactions or recruitment of co-regulatory proteins to regulate expression of many genes. This mechanism of action is frequently used by nuclear receptors, which significantly complicates the decrypting of the effects induced by the ER (Gottlicher et al., 1998). Many of ER/protein interactions, which occur in cells to regulate transcription of many target genes, are composed for example of ER/AP-1 complex (c-Fos/c-Jun) (Ascenzi et al., 2006; Duan et al., 2008; Kushner et al. 2000; Matthews et al. 2006 ; Paech et al., 1997, Uht et al 1997; Webb et al., 1999,), ER/Sp complex (Ascenzi et al., 2006; He et al., 2005; Kim et al., 2003; Saville et al., 2000; Stoner et al., 2004), and ER/NF-B complex (Galien & Garcia, 1997; Stein & Yang, 1995). This latter is the only known mechanism of transcriptional repression induced by ER (Fig. 2).

For instance, after being activated by 17-E2 binding, ER interact with the transcription factor NF-B to control the transcriptional repression of certain genes such as IL-6 gene, which is involved in cartilage catabolism. Furthermore, in articular chondrocytes, it has been shown that 17-E2 can counteract the effects of IL-1 by inhibiting nuclear translocation of the p65 subunit of NF-B and therefore, binding of p65 to inducible nitric oxide synthase (iNOS) gene promoter (Richette et al., 2007). In addition, it has been suggested that ER and NF-B compete for binding to the same transcriptional co-activators (p300/CBP and PCAF), and that in the case of activation of transcription by 17-E2 through ER, the pool of these coactivators is mobilized predominantly by ER at the expense of NF-B (Ansari & Gandy, 2007).

Therefore, estrogen/ER complex allows developing multiple physiological responses following hormonal stimulation by different signaling pathways and inter-connections with different *trans* factors and *cis* elements at the DNA level of target cells.

## **2.3.3 Non genomic signaling pathway**

The non-genomic effects are defined as any action that does not involve transcriptional activity resulting from direct or indirect interaction of a nuclear receptor with the regulatory sequences of hormone-regulated genes. These effects are very rapid (in the order of several seconds to several minutes), incompatible with gene activation or protein synthesis. The effects of non-genomic estrogen receptors are often linked to signaling pathways that involve G protein-coupled receptors, ion channels or receptors linked to enzymes. 17-E2 could modulate intracellular calcium or cAMP production and activate MAPK/ERK, PLC, PKA, or that of PI3K signaling pathways (Marino et al., 2002) (Fig 2). All these effects are not modified by inhibitors of transcription (actinomycin D) or translation (cycloheximide) (Losel & Wehling, 2003), confirming that they do not depend on a genomic action. In 1998, Beyer and Raab, by coupling 17-E2 to BSA (Bovine Serum Albumin), thereby preventing it from crossing the plasma membrane, observed that 17-E2 modulates intracellular calcium. Thus,

Estrogens Involvement in the Physiopathology of Articular Cartilage 47

it appears that palmitoylation of the classical form of the receptor is necessary for its membrane addressing (Acconcia et al., 2005; Ellmann et al., 2009). In addition, it has been demonstrated that ER could be anchored to the plasma membrane through interactions with many membrane proteins such as caveolin 1 and 2, striatine or with adapter proteins like Shc

In absence of 17-E2, ER can be activated by phosphorylation *via* protein kinases A or C by extracellular signals like growth factors or cytokines, neurotransmitters, or by cell cycle regulators (Le Goff et al., 1994). Epidermal growth factor (EGF) mimics the effects of 17-E2 in the mouse uterus. Similarly, insulin, insulin-like growth factor-I (IGF-I), dopamine or transforming growth factor- (TGF-) may activate ER. The main targets of these growth factors are the many serine residues present in the AF-1 domain of ER particularly Ser118

In summary, estrogens are involved in many signaling pathways, allowing fine control of

OA is a worldwide public health problem which may affect different sites of the skeleton. In the Western world, at least 10% of the population present OA symptoms and 80% of the population will be potentially affected after the age of 70 years. Knee OA represents one of the major causes of morbidity and disability in relation to the worsening of quality of life. Pathogenesis of knee OA involves multiple factors including gender, weight, and genetics. Association between OA and estrogen deficiency during menopause has been firstly evoked

The hypothesis that estrogen deficiency may promote the development of OA has then been relied on the results of observational epidemiological studies. It is known that the frequency of knee OA is higher in women than in men; it is worsened after menopause and is lower in women receiving hormone replacement therapy (HRT) (Oliveria et al., 1995; Wilson et al., 1990). Although contradictions exist, some studies have shown that hysterectomy may also be associated with OA suggesting the potential role of estrogens to prevent these age-related diseases (Inoue et al., 1995; Spector et al., 1988; Spector et al., 1991). In addition, women with metastatic breast cancer when treated with aromatase inhibitors develop joint pains and cessation of aromatase inhibitors therapy resolved this joint pain (Burstein & Winer, 2007; Crew et al., 2007); aromatase inhibitors are widely used as adjuvant therapy in postmenopausal women with ER positive breast cancer (for review see Moslemi & Seralini, 2005). Moreover, an association of estrogen receptor (ER and ER) polymorphisms has been found in patients with OA compared to unaffected subjects. All these data suggest a protective role of estrogen and HRT on OA through functional isoforms of ER. Such a protective effect of estrogen on the development of OA may suggest two potential mechanisms: a direct effect on cartilage and an indirect effect through modifications of sub-chondral bone remodelling. Nevertheless, some clinical studies based on symptomatic parameters failed to report any effect of HRT on cartilage metabolism, that's why further studies are required to clearly demonstrate beneficial effects of estrogens

and p130 Cas (Crk -associated substrate) (Cheskis et al., 2007).

**2.3.4 Ligand-independent signaling pathway** 

**3. Estrogen involvement in osteoarthritis** 

**3.1 Epidemiological observations and clinical data** 

by Cecil & Archer in 1925 following clinical observation.

on molecular regulations of articular cartilage homeostasis.

and Ser167 (Nilsson et al., 2001).

cell and tissue functions.

#### Fig. 2. Different pathways involved in estrogen receptors (ER) signaling.

These different pathways occur in estrogen sensitive cells including ERE-dependent genomic, EREindependent genomic (*via* Sp1, AP-1 and NF-B) and membrane associated ER/G protein mechanisms. NF-B pathway is the only inhibitory mechanism. In articular chondrocytes ER66 homodimer complex binds predominantly Sp1 proteins to activate GC-box mediated t*rans* activation of target genes such as Uridine diphospho-glucose deshydrogenase (UDPGD) and type II collagen (type II Col). Other mechanisms mediated by ER46 , ER, ER and ER homo- and/or heterodimer and finally membrane associated ER /protein G need to be elucidated in articular chondrocytes. Note that 17-E2 production from androgens like testosterone takes place *via* aromatase (*CYP19*) locally (intracrine effect) or in other cells (paracrine or endocrine effects). Adc, Adenylate cyclase; AP-1, Activating Protein-1; ERK, Extracellular signal Regulated Kinase; ERE, Estrogen Responsive Element; G, G protein; Hsp-90; Heat-shock protein 90; MAPK, Mitogen-Activated Protein Kinase; NF-B, Nuclear Factor-B; PI3K, Phosphatidyl Inositol 3-Kinase; PKA, Protein Kinase A; PLC, Phospholipase C; SERM, Selective Estrogen Receptor Modulator; Sp1, Specific Protein 1.

they confirm that this action is induced by a membrane ER. The existence of membrane estrogen receptor was discovered in the late 1970s in endometrial cells (Pietras & Szego, 1977). Various studies tempted to show that these membrane receptors are structurally similar to the classical cytoplasmic ER and that the and forms are represented (Chambliss & Shaul, 2002; Pappas et al., 1995; Razandi et al., 1999). More recently, a novel isoform of ER was highlighted: ER36. This 36 kDa protein lacks the *trans* activation domains AF-1 and AF-2 and has a DBD and a partial LBD, suggesting a membrane localization for this isoform (Wang et al., 2006). Also, estrogen binding sites localized at the membrane and the cytoplasm were detected in MCF-7 (Harrington et al., 2006). Since ER does not have a transmembrane domain, it appears that palmitoylation of the classical form of the receptor is necessary for its membrane addressing (Acconcia et al., 2005; Ellmann et al., 2009). In addition, it has been demonstrated that ER could be anchored to the plasma membrane through interactions with many membrane proteins such as caveolin 1 and 2, striatine or with adapter proteins like Shc and p130 Cas (Crk -associated substrate) (Cheskis et al., 2007).

## **2.3.4 Ligand-independent signaling pathway**

46 Challenges in Rheumatology

Fig. 2. Different pathways involved in estrogen receptors (ER) signaling.

Estrogen Receptor Modulator; Sp1, Specific Protein 1.

These different pathways occur in estrogen sensitive cells including ERE-dependent genomic, EREindependent genomic (*via* Sp1, AP-1 and NF-B) and membrane associated ER/G protein mechanisms. NF-B pathway is the only inhibitory mechanism. In articular chondrocytes ER66 homodimer complex binds predominantly Sp1 proteins to activate GC-box mediated t*rans* activation of target genes such as Uridine diphospho-glucose deshydrogenase (UDPGD) and type II collagen (type II Col). Other

mechanisms mediated by ER46 , ER, ER and ER homo- and/or heterodimer and finally

membrane associated ER /protein G need to be elucidated in articular chondrocytes. Note that 17-E2 production from androgens like testosterone takes place *via* aromatase (*CYP19*) locally (intracrine effect) or in other cells (paracrine or endocrine effects). Adc, Adenylate cyclase; AP-1, Activating Protein-1; ERK, Extracellular signal Regulated Kinase; ERE, Estrogen Responsive Element; G, G protein; Hsp-90; Heat-shock protein 90; MAPK, Mitogen-Activated Protein Kinase; NF-B, Nuclear Factor-B; PI3K, Phosphatidyl Inositol 3-Kinase; PKA, Protein Kinase A; PLC, Phospholipase C; SERM, Selective

they confirm that this action is induced by a membrane ER. The existence of membrane estrogen receptor was discovered in the late 1970s in endometrial cells (Pietras & Szego, 1977). Various studies tempted to show that these membrane receptors are structurally similar to the classical cytoplasmic ER and that the and forms are represented (Chambliss & Shaul, 2002; Pappas et al., 1995; Razandi et al., 1999). More recently, a novel isoform of ER was highlighted: ER36. This 36 kDa protein lacks the *trans* activation domains AF-1 and AF-2 and has a DBD and a partial LBD, suggesting a membrane localization for this isoform (Wang et al., 2006). Also, estrogen binding sites localized at the membrane and the cytoplasm were detected in MCF-7 (Harrington et al., 2006). Since ER does not have a transmembrane domain, In absence of 17-E2, ER can be activated by phosphorylation *via* protein kinases A or C by extracellular signals like growth factors or cytokines, neurotransmitters, or by cell cycle regulators (Le Goff et al., 1994). Epidermal growth factor (EGF) mimics the effects of 17-E2 in the mouse uterus. Similarly, insulin, insulin-like growth factor-I (IGF-I), dopamine or transforming growth factor- (TGF-) may activate ER. The main targets of these growth factors are the many serine residues present in the AF-1 domain of ER particularly Ser118 and Ser167 (Nilsson et al., 2001).

In summary, estrogens are involved in many signaling pathways, allowing fine control of cell and tissue functions.

## **3. Estrogen involvement in osteoarthritis**

## **3.1 Epidemiological observations and clinical data**

OA is a worldwide public health problem which may affect different sites of the skeleton. In the Western world, at least 10% of the population present OA symptoms and 80% of the population will be potentially affected after the age of 70 years. Knee OA represents one of the major causes of morbidity and disability in relation to the worsening of quality of life. Pathogenesis of knee OA involves multiple factors including gender, weight, and genetics. Association between OA and estrogen deficiency during menopause has been firstly evoked by Cecil & Archer in 1925 following clinical observation.

The hypothesis that estrogen deficiency may promote the development of OA has then been relied on the results of observational epidemiological studies. It is known that the frequency of knee OA is higher in women than in men; it is worsened after menopause and is lower in women receiving hormone replacement therapy (HRT) (Oliveria et al., 1995; Wilson et al., 1990). Although contradictions exist, some studies have shown that hysterectomy may also be associated with OA suggesting the potential role of estrogens to prevent these age-related diseases (Inoue et al., 1995; Spector et al., 1988; Spector et al., 1991). In addition, women with metastatic breast cancer when treated with aromatase inhibitors develop joint pains and cessation of aromatase inhibitors therapy resolved this joint pain (Burstein & Winer, 2007; Crew et al., 2007); aromatase inhibitors are widely used as adjuvant therapy in postmenopausal women with ER positive breast cancer (for review see Moslemi & Seralini, 2005). Moreover, an association of estrogen receptor (ER and ER) polymorphisms has been found in patients with OA compared to unaffected subjects. All these data suggest a protective role of estrogen and HRT on OA through functional isoforms of ER. Such a protective effect of estrogen on the development of OA may suggest two potential mechanisms: a direct effect on cartilage and an indirect effect through modifications of sub-chondral bone remodelling. Nevertheless, some clinical studies based on symptomatic parameters failed to report any effect of HRT on cartilage metabolism, that's why further studies are required to clearly demonstrate beneficial effects of estrogens on molecular regulations of articular cartilage homeostasis.

Estrogens Involvement in the Physiopathology of Articular Cartilage 49

17-E2 increase TGF- expression whereas supra-physiologic doses decrease strongly its expression (Saggese et al., 1993). The concept of dose-dependence is confirmed by the majority of the studies on 17-E in the articular cartilage where physiological doses of the hormone show to be protective when considering the structural integrity of tissue while supraphysiologic concentrations are often deleterious (Richette et al., 2003) (Fig. 3). Experiments on chondrocytes from ovariectomized monkeys treated with 17-E showed that this hormone increases concomitantly the expression of the binding protein of IGF-I (IGFBP-2) and synthesis of PG (Richmond et al., 2000). In addition, the synovial fluid of these animals contains IGF-I twice more than untreated animals. Thereafter, it was shown that estrogen deficiency increases the sensitivity of cell response to certain pro-inflammatory cytokines through an increase in the number of receptors or cytokine cofactors amplifying consequently the effects of catabolic cytokines in these cells. Estrogen treatment in ovariectomized animals significantly reduced the production and secretion of pro-inflammatory cytokines in articular chondrocytes. This anti-inflammatory effect of 17-E2 was highlighted by Le Bail et al. (2001) who showed that the localized production of 17-E2 by synovial cells has inhibited the IL-6 secretion by articular chondrocytes. In addition, 17-E2 can also reduce production of pro-inflammatory prostaglandins through a decrease in the mRNA steady-state levels of cyclooxygenase-2

The majority of the beneficial effects of 17-E2 in articular chondrocytes is focused on the inhibition of catabolic pathways. Thus, Lee et al. (2003) found that 17-E2 decreases the secretion of the metalloproteinase MMP-1 in human osteoarthritic articular chondrocytes. In addition, 17-E2 can antagonize the degradation of PGs and the expression and activity of MMP-1, MMP-3 and MMP-13 enzymes induced by IL-1 in rabbit articular chondrocytes (Richette et al., 2004). Especially, the effects of 17-E2 on the expression of MMP-13 seem to be transmitted through the indirect binding of ER66 at an AP-1 site in the promoter of MMP-13 gene (Lu et al., 2006). Like the mode of action of 17-E2 on the expression levels of TGF-, the effects of the hormone on the expression of MMPs and their inhibitors TIMPs (tissue inhibitors of MMPs) appear to be dose-dependent. Low doses of 17-E2 decreased MMP-1/TIMP-1 and MMP-3/TIMP-1 ratios while higher concentrations have the opposite effect (Song et al., 2003).

The generation of reactive oxygen species (ROS) contributes actively to the cartilage ECM degradation in OA, in particular by inducing a decrease in the synthesis of PGs. However, due to the structure of their phenol nuclei, 17-E2 and its metabolites have anti-oxidant features (Liehr & Roy, 1998) that protect the cartilage degradation induced by ROS (Claassen et al., 2005). It is now established that 17-E2 is a modulator of the redox status of

During the arthritic process, overproduction of nitric oxide (NO) is a consequence of the action of proinflammatory cytokines such as IL-1 or TNF- that promote the activation of inducible nitric oxide synthase (iNOS) in OA chondrocytes. The iNOS gene is under the control of an estrogen-dependent promoter. Indeed, estrogen deficiency activates transcription of this promoter while a replacement therapy by 17-E2 inhibits it in ovariectomized mice (Cuzzocrea et al., 2003). 17-E2 can counteract the deleterious effects induced by IL-1 in rabbit articular chondrocytes by reducing the binding capacity of NF-B

(COX-2) in bovine articular chondrocytes (Morisset et al., 1998).

**4.2 Metalloproteinases (MMPs) expression and activity** 

**4.3 Anti-oxidant effects** 

chondrocytes.

## **3.2 Biological** *in vivo* **and** *in vitro* **studies**

*In vivo* animal studies indicate that estrogens may have a protective effect against OA, reversing or reducing the cartilage degradation in ovariectomized mice, rats, sheeps and monkeys (Cuzzocrea et al., 2003; Ham et al., 2002; Høegh-Andersen et al., 2004; Oestergaard et al., 2006). However, the way by which estrogens act to prevent the pathogenesis of OA in these models remains unclear. It is supposed that estrogens prevent cartilage degradation by increasing production of growth factors such as insulin growth factor-I (IGF-I) and suppressing pro-inflammatory cytokines expression such as interleukin-6 (IL-6). A few data exist about the role of estrogen in regulating the synthesis of matrix compounds. *In vivo*, 17-E2 prevents the degradation of collagen type II in the women treated with HRT (Mouritzen et al., 2003) but *in vitro*, it does not seem capable of modulating neosynthesis nor secretion of type II collagen in articular chondrocytes in primary culture (Ab-Rahim et al., 2008; Claassen et al., 2006). The measurement of serum C-terminal telopeptide of type II collagen (CTX-II) in bovine articular cartilage explants has determined that 17-E2 significantly protected the cartilage degradation induced by tumor necrosis factor- (TNF-) and oncostatin M (Oestergaard et al., 2006). In similar *ex vivo* experiments, 17-E2 can increase glycosaminoglycan (GAG) content of articular explants (Englert et al., 2006). Finally, the double invalidation of ER and ER gene receptors (double knock-out ER -/-, ER -/-) in mice aged of 6 months increases the number and size of osteophytes as well as a thinning of the sub-chondral plate, without changing the cartilage degradation (Sniekers et al., 2009). We demonstrated recently that 17-E2 (the most potent among all estrogens) at physiologic doses, and ER66 (wild type receptor) but not ER46 (AF-1 deleted receptor) could up-regulate at both mRNA and protein levels UDP-glucose dehydrogenase (UDPGD) in primary cultured articular chondrocytes *via* specific protein 1 (Sp1) binding sites (Maneix et al., 2008). This enzyme is responsible of UDP-glucuronate synthesis which is the main component of GAG chains polymerization in cartilage: it plays an essential role in the elongation of GAG chains and their attachment to the axial protein of proteoglycans (PGs). Its decarboxylation provides UDP xylose, which serves to anchor chains of chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulphate (HS). Moreover, we also established that 17-E2 increases the gene expression of type II collagen (*COL2A1*), the main collagen of hyaline cartilage, *via trans* activation domains AF-1 of ER66 in coordination with the transcription factors Sp1, Sp3, p300 and Soxs (Maneix et al, 2010). It would thus be a genomic mechanism ERE-independent. Finally, our preliminary results also showed that phytoestrogens such as apigenin and genistein could up-regulate the expression of UDPGD (unpublished data).

Overall, these data indicate that estrogen/phytoestrogens and their receptors can be considered as potent regulators of chondrocyte homeostasis and are pro-anabolic for extracellular matrix synthesis. This may have potential applications in the tissue engineering of articular cartilage and offer new perspectives to prevent and/or to treat OA.

## **4. Mechanisms of estrogen action in OA**

## **4.1 Effects on growth factors and cytokines**

17-E2 can firstly interact with pathways affecting the synthesis and secretion of the key growth factors involved in the regulation of cartilage metabolism. So, TGF- expression in the iliac crest chondrocytes is influenced by 17-E2 in a biphasic manner: low concentrations of

*In vivo* animal studies indicate that estrogens may have a protective effect against OA, reversing or reducing the cartilage degradation in ovariectomized mice, rats, sheeps and monkeys (Cuzzocrea et al., 2003; Ham et al., 2002; Høegh-Andersen et al., 2004; Oestergaard et al., 2006). However, the way by which estrogens act to prevent the pathogenesis of OA in these models remains unclear. It is supposed that estrogens prevent cartilage degradation by increasing production of growth factors such as insulin growth factor-I (IGF-I) and suppressing pro-inflammatory cytokines expression such as interleukin-6 (IL-6). A few data exist about the role of estrogen in regulating the synthesis of matrix compounds. *In vivo*, 17-E2 prevents the degradation of collagen type II in the women treated with HRT (Mouritzen et al., 2003) but *in vitro*, it does not seem capable of modulating neosynthesis nor secretion of type II collagen in articular chondrocytes in primary culture (Ab-Rahim et al., 2008; Claassen et al., 2006). The measurement of serum C-terminal telopeptide of type II collagen (CTX-II) in bovine articular cartilage explants has determined that 17-E2 significantly protected the cartilage degradation induced by tumor necrosis factor- (TNF-) and oncostatin M (Oestergaard et al., 2006). In similar *ex vivo* experiments, 17-E2 can increase glycosaminoglycan (GAG) content of articular explants (Englert et al., 2006). Finally, the double invalidation of ER and ER gene receptors (double knock-out ER -/-, ER -/-) in mice aged of 6 months increases the number and size of osteophytes as well as a thinning of the sub-chondral plate, without changing the cartilage degradation (Sniekers et al., 2009). We demonstrated recently that 17-E2 (the most potent among all estrogens) at physiologic doses, and ER66 (wild type receptor) but not ER46 (AF-1 deleted receptor) could up-regulate at both mRNA and protein levels UDP-glucose dehydrogenase (UDPGD) in primary cultured articular chondrocytes *via* specific protein 1 (Sp1) binding sites (Maneix et al., 2008). This enzyme is responsible of UDP-glucuronate synthesis which is the main component of GAG chains polymerization in cartilage: it plays an essential role in the elongation of GAG chains and their attachment to the axial protein of proteoglycans (PGs). Its decarboxylation provides UDP xylose, which serves to anchor chains of chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulphate (HS). Moreover, we also established that 17-E2 increases the gene expression of type II collagen (*COL2A1*), the main collagen of hyaline cartilage, *via trans* activation domains AF-1 of ER66 in coordination with the transcription factors Sp1, Sp3, p300 and Soxs (Maneix et al, 2010). It would thus be a genomic mechanism ERE-independent. Finally, our preliminary results also showed that phytoestrogens such as apigenin and genistein could up-regulate the expression of UDPGD

Overall, these data indicate that estrogen/phytoestrogens and their receptors can be considered as potent regulators of chondrocyte homeostasis and are pro-anabolic for extracellular matrix synthesis. This may have potential applications in the tissue engineering

17-E2 can firstly interact with pathways affecting the synthesis and secretion of the key growth factors involved in the regulation of cartilage metabolism. So, TGF- expression in the iliac crest chondrocytes is influenced by 17-E2 in a biphasic manner: low concentrations of

of articular cartilage and offer new perspectives to prevent and/or to treat OA.

**4. Mechanisms of estrogen action in OA 4.1 Effects on growth factors and cytokines** 

**3.2 Biological** *in vivo* **and** *in vitro* **studies** 

(unpublished data).

17-E2 increase TGF- expression whereas supra-physiologic doses decrease strongly its expression (Saggese et al., 1993). The concept of dose-dependence is confirmed by the majority of the studies on 17-E in the articular cartilage where physiological doses of the hormone show to be protective when considering the structural integrity of tissue while supraphysiologic concentrations are often deleterious (Richette et al., 2003) (Fig. 3). Experiments on chondrocytes from ovariectomized monkeys treated with 17-E showed that this hormone increases concomitantly the expression of the binding protein of IGF-I (IGFBP-2) and synthesis of PG (Richmond et al., 2000). In addition, the synovial fluid of these animals contains IGF-I twice more than untreated animals. Thereafter, it was shown that estrogen deficiency increases the sensitivity of cell response to certain pro-inflammatory cytokines through an increase in the number of receptors or cytokine cofactors amplifying consequently the effects of catabolic cytokines in these cells. Estrogen treatment in ovariectomized animals significantly reduced the production and secretion of pro-inflammatory cytokines in articular chondrocytes. This anti-inflammatory effect of 17-E2 was highlighted by Le Bail et al. (2001) who showed that the localized production of 17-E2 by synovial cells has inhibited the IL-6 secretion by articular chondrocytes. In addition, 17-E2 can also reduce production of pro-inflammatory prostaglandins through a decrease in the mRNA steady-state levels of cyclooxygenase-2 (COX-2) in bovine articular chondrocytes (Morisset et al., 1998).

#### **4.2 Metalloproteinases (MMPs) expression and activity**

The majority of the beneficial effects of 17-E2 in articular chondrocytes is focused on the inhibition of catabolic pathways. Thus, Lee et al. (2003) found that 17-E2 decreases the secretion of the metalloproteinase MMP-1 in human osteoarthritic articular chondrocytes. In addition, 17-E2 can antagonize the degradation of PGs and the expression and activity of MMP-1, MMP-3 and MMP-13 enzymes induced by IL-1 in rabbit articular chondrocytes (Richette et al., 2004). Especially, the effects of 17-E2 on the expression of MMP-13 seem to be transmitted through the indirect binding of ER66 at an AP-1 site in the promoter of MMP-13 gene (Lu et al., 2006). Like the mode of action of 17-E2 on the expression levels of TGF-, the effects of the hormone on the expression of MMPs and their inhibitors TIMPs (tissue inhibitors of MMPs) appear to be dose-dependent. Low doses of 17-E2 decreased MMP-1/TIMP-1 and MMP-3/TIMP-1 ratios while higher concentrations have the opposite effect (Song et al., 2003).

## **4.3 Anti-oxidant effects**

The generation of reactive oxygen species (ROS) contributes actively to the cartilage ECM degradation in OA, in particular by inducing a decrease in the synthesis of PGs. However, due to the structure of their phenol nuclei, 17-E2 and its metabolites have anti-oxidant features (Liehr & Roy, 1998) that protect the cartilage degradation induced by ROS (Claassen et al., 2005). It is now established that 17-E2 is a modulator of the redox status of chondrocytes.

During the arthritic process, overproduction of nitric oxide (NO) is a consequence of the action of proinflammatory cytokines such as IL-1 or TNF- that promote the activation of inducible nitric oxide synthase (iNOS) in OA chondrocytes. The iNOS gene is under the control of an estrogen-dependent promoter. Indeed, estrogen deficiency activates transcription of this promoter while a replacement therapy by 17-E2 inhibits it in ovariectomized mice (Cuzzocrea et al., 2003). 17-E2 can counteract the deleterious effects induced by IL-1 in rabbit articular chondrocytes by reducing the binding capacity of NF-B

Estrogens Involvement in the Physiopathology of Articular Cartilage 51

remodeling: osteoblasts that synthesize bone matrix and osteoclasts that are responsible for the resorption (bone loss) of existing bone. Estrogens inhibit osteoclasts activation and are therefore considered as inhibitors of bone resorption. The predominant mechanism that appears to be involved is the inhibition of IL-6 synthesis, the main cytokine involved in the activation of resorption. This inhibition of IL-6 synthesis by estrogens is mediated through the modification of NF-B binding on IL-6 gene promoter; there is no direct binding of ER to DNA (Ray et al., 1994). Consequently, an increase of bone resorption, generally favoring the occurrence of osteoporosis, is frequently seen in women after menopause which is related to the decrease of circulating estrogen levels during this period (Reginster et al., 2003). In this

Paradoxically, the frequency of OA and osteoporosis in postmenopausal women are most often inversely correlated, as reflected in the levels of osteocalcin, a marker of bone turnover, which are generally lower among women with OA than women without OA (for review: Dequeker et al., 2003). Patients suffering from OA of the knee are generally less subjected to bone loss. The increase in bone mass is a risk factor for incidence and development of knee and hip osteoarthritis. It was suggested that an increase in bone density in the area of the sub-chondral bone may induce bone stiffness and accelerate cartilage destruction. Thus, high bone density is associated with an increase of OA prevalence of the hip, hand and knee in postmenopausal women. However, high bone density increases risk of knee OA but protects against the progression of the disease once it is established. Therefore, bone loss in people with OA which is already established accelerates the progression of the disease. Indeed, Jacobsen et al. (2007) showed that the reduction of intra-articular space in the hip was correlated with the decrease in mineral density of sub-chondral bone in postmenopausal women. It was suggested that the alteration of bone structure can cause changes in the load distribution within the joint and promote the development of OA (Sniekers et al. 2008). In this case, the favorable effect of estrogen on cartilage may be due in part to the anti-resorption effect of 17-E2 on bone.

**5. Phytoestrogen and Selective Estrogen Receptor Modulators (SERM)** 

During the second half of the 20th century, HRT has been repeatedly promoted as the only pharmacological approach allowing a global prevention of all disorders related to or potentiated by estrogen deprivation. Recently, the risk/benefit profile of HRT has been severely challenged because of apparent increased risks of invasive breast cancer, coronary heart disease events, stroke and pulmonary embolism among treated women. These new findings imply a careful reassessment of the current evidence justifying the prescription of HRT for the prevention of the management of chronic disorders. The many contradictions recorded concerning benefit/risk effects of HRT are often related to differences in methodology and criteria used in measuring the effects of estrogen therapy in clinical studies. Indeed, analysis based strictly on symptomatic parameters showed that HRT had no effect or even adverse effects on the progression of OA (Von Muhlen et al., 2002). Conversely, more advanced techniques of magnetic resonance imaging showed that recipients of long term HRT (5 years or more) had a volume of articular cartilage largest in the knee than women having never taken any treatments (Wluka et al., 2001). As to the evolution of medical imaging techniques and advances in the diagnosis of OA, it appears that estrogens have a strong potential for preventing disease and preserving the structural integrity of the articulation among postmenopausal women. Thus, recent clinical trials of

context, HRT could effectively prevent postmenopausal osteoporosis.

Fig. 3. Role of 17-E2 and estrogen receptor ER /ER on joint cartilage homeostasis.

Col II, type II collagen; IGF-I, Insulin-like Growth Factor-I; IL, Interleukin; iNOS, Inducible Nitrogen Oxide Synthase; MMPs, Metalloproteinases; PGs, Proteoglycans; ROS, Reactive Oxygen Species; SERM, Selective Estrogen Receptor Modulators; TIMPs, Tissue Inhibitors MMPs; TGF-, Transforming Growth Factor-; TNF-, Tumor Necrosis Factor-; UDPGD, Uridine Diphospho-Glucose Dehydrogenase. The roles of SERM and phytoestrogens need to be clarified.

in the promoter of iNOS gene resulting in inhibition of nuclear translocation of this factor (Richette et al., 2007). This results in an inhibition of iNOS gene transcription and a subsequent reduction in NO production by chondrocytes.

## **4.4 Matrix components turn-over**

To better understand the physiopathology of articular cartilage in osteoarthritis, Høegh-Andersen et al. (2004) have validated an experimental model of ovariectomized rats with modification in cartilage structure representative of *in vivo* pathological changes observed in early human osteoarthritis. In animals aged from 5 to 7 months, estrogen deficiency increases the erosion of the articular surface and accelerates the renewal of matrix molecules. This animal model also showed that the effectiveness of HRT in the prevention of cartilage loss is increased when estrogen is administered to the animal from its operation and not after a period of 3 weeks (Oestergaard et al. 2006). These works need to be compared with the many epidemiological data which showed that the benefits of HRT are strengthened when patients are treated at the first signs of menopause and continued their treatment over a period exceeding 5 years. The importance of prevention in the treatment of damaged cartilage appears essential.

## **4.5 Sub-chondral bone regulation**

Within the joint, cartilage is not the only target tissue for estrogens. 17-E2 also controls the renewal of the sub-chondral bone by maintaining a balance between the two players in bone

Fig. 3. Role of 17-E2 and estrogen receptor ER /ER on joint cartilage homeostasis.

roles of SERM and phytoestrogens need to be clarified.

**4.4 Matrix components turn-over** 

damaged cartilage appears essential.

**4.5 Sub-chondral bone regulation** 

subsequent reduction in NO production by chondrocytes.

Col II, type II collagen; IGF-I, Insulin-like Growth Factor-I; IL, Interleukin; iNOS, Inducible Nitrogen Oxide Synthase; MMPs, Metalloproteinases; PGs, Proteoglycans; ROS, Reactive Oxygen Species; SERM, Selective Estrogen Receptor Modulators; TIMPs, Tissue Inhibitors MMPs; TGF-, Transforming Growth Factor-; TNF-, Tumor Necrosis Factor-; UDPGD, Uridine Diphospho-Glucose Dehydrogenase. The

in the promoter of iNOS gene resulting in inhibition of nuclear translocation of this factor (Richette et al., 2007). This results in an inhibition of iNOS gene transcription and a

To better understand the physiopathology of articular cartilage in osteoarthritis, Høegh-Andersen et al. (2004) have validated an experimental model of ovariectomized rats with modification in cartilage structure representative of *in vivo* pathological changes observed in early human osteoarthritis. In animals aged from 5 to 7 months, estrogen deficiency increases the erosion of the articular surface and accelerates the renewal of matrix molecules. This animal model also showed that the effectiveness of HRT in the prevention of cartilage loss is increased when estrogen is administered to the animal from its operation and not after a period of 3 weeks (Oestergaard et al. 2006). These works need to be compared with the many epidemiological data which showed that the benefits of HRT are strengthened when patients are treated at the first signs of menopause and continued their treatment over a period exceeding 5 years. The importance of prevention in the treatment of

Within the joint, cartilage is not the only target tissue for estrogens. 17-E2 also controls the renewal of the sub-chondral bone by maintaining a balance between the two players in bone remodeling: osteoblasts that synthesize bone matrix and osteoclasts that are responsible for the resorption (bone loss) of existing bone. Estrogens inhibit osteoclasts activation and are therefore considered as inhibitors of bone resorption. The predominant mechanism that appears to be involved is the inhibition of IL-6 synthesis, the main cytokine involved in the activation of resorption. This inhibition of IL-6 synthesis by estrogens is mediated through the modification of NF-B binding on IL-6 gene promoter; there is no direct binding of ER to DNA (Ray et al., 1994). Consequently, an increase of bone resorption, generally favoring the occurrence of osteoporosis, is frequently seen in women after menopause which is related to the decrease of circulating estrogen levels during this period (Reginster et al., 2003). In this context, HRT could effectively prevent postmenopausal osteoporosis.

Paradoxically, the frequency of OA and osteoporosis in postmenopausal women are most often inversely correlated, as reflected in the levels of osteocalcin, a marker of bone turnover, which are generally lower among women with OA than women without OA (for review: Dequeker et al., 2003). Patients suffering from OA of the knee are generally less subjected to bone loss. The increase in bone mass is a risk factor for incidence and development of knee and hip osteoarthritis. It was suggested that an increase in bone density in the area of the sub-chondral bone may induce bone stiffness and accelerate cartilage destruction. Thus, high bone density is associated with an increase of OA prevalence of the hip, hand and knee in postmenopausal women. However, high bone density increases risk of knee OA but protects against the progression of the disease once it is established. Therefore, bone loss in people with OA which is already established accelerates the progression of the disease. Indeed, Jacobsen et al. (2007) showed that the reduction of intra-articular space in the hip was correlated with the decrease in mineral density of sub-chondral bone in postmenopausal women. It was suggested that the alteration of bone structure can cause changes in the load distribution within the joint and promote the development of OA (Sniekers et al. 2008). In this case, the favorable effect of estrogen on cartilage may be due in part to the anti-resorption effect of 17-E2 on bone.

## **5. Phytoestrogen and Selective Estrogen Receptor Modulators (SERM)**

During the second half of the 20th century, HRT has been repeatedly promoted as the only pharmacological approach allowing a global prevention of all disorders related to or potentiated by estrogen deprivation. Recently, the risk/benefit profile of HRT has been severely challenged because of apparent increased risks of invasive breast cancer, coronary heart disease events, stroke and pulmonary embolism among treated women. These new findings imply a careful reassessment of the current evidence justifying the prescription of HRT for the prevention of the management of chronic disorders. The many contradictions recorded concerning benefit/risk effects of HRT are often related to differences in methodology and criteria used in measuring the effects of estrogen therapy in clinical studies. Indeed, analysis based strictly on symptomatic parameters showed that HRT had no effect or even adverse effects on the progression of OA (Von Muhlen et al., 2002). Conversely, more advanced techniques of magnetic resonance imaging showed that recipients of long term HRT (5 years or more) had a volume of articular cartilage largest in the knee than women having never taken any treatments (Wluka et al., 2001). As to the evolution of medical imaging techniques and advances in the diagnosis of OA, it appears that estrogens have a strong potential for preventing disease and preserving the structural integrity of the articulation among postmenopausal women. Thus, recent clinical trials of

Estrogens Involvement in the Physiopathology of Articular Cartilage 53

hyluronan overproduction is considered as an early reaction in OA followed by PG loss and collagen degradation (Stracke et al., 2001). However, additional research is critical to determine how phytoestrogens act on cartilage cells to obtain a more complete

From epidemiological, clinical, *in vivo* and *in vitro* studies, a huge amount of data consistent with the fact that estrogens and their receptors can now be considered among the main players involved in the chondrocyte homeostasis and participate in cartilage protection from degradation and erosion occurring during menopause. Indeed, once ligand/ER complex is formed, estrogens such as 17-E2 could act with ER to induce or to inhibit *trans* activation of target genes of chondrocytes, the predominant cells of articular cartilage allowing expression of the specific chondrogenic markers such as UDPGD, PGs, type II collagen or inhibition of catabolic markers such as metalloproteinases and interleukins. There are different pathways by which estrogen and ER may interact with target genes in chondrocytes but it seems that Sp1 mediated *trans* activation being preferentially used in this cell type. In this mechanism ER needs AF-1 sequence (ligand-independent *trans* activation domain) to exert its action on GC boxes *via* Sp1 in target genes. In addition, some molecules sharing structural and functional features with estrogens like SERM and phytoestrogens can mimic estrogenic action and are therefore useful to repair cartilage erosion or might contribute at least to protect premenopausal joint cartilage and to maintain its homeostasis in a prevention strategy but these molecules need further investigation and deserves more attention from the scientific community to prove their safety and efficacy.

Osteoarthritis, cartilage, chondrocytes, estrogens, phytoestrogens, selective estrogen

Ab-Rahim, S., Selvaratnam, L. & Kamarul, T. (2008). The effect of TGF-beta1 and beta-

chondrocyte cultures. *Cell Biol. Int.,* Vol. 32, No. 7, (Jul 2008), pp. (841-847) Acconcia, F., Ascenzi, P., Bocedi, A., Spisni, E., Tomasi, V., Trentalance, A., Visca, P. &

Ansari, R.A. & Gandy, J. (2007). Determining the *trans*repression activity of xenoestrogen on

Antonicelli R, Olivieri F, Morichi V, Urbani E, Mais V. (2008). Prevention of cardiovascular

Arjmandi, B.H., Khalil, D.A., Lucas, E.A., Smith, B.J., Sinichi, N., Hodges, S.B., Juma, S.,

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estradiol on glycosaminoglycan and type II collagen distribution in articular

Marino, M. (2005). Palmitoylation-dependent estrogen receptor alpha membrane localization: regulation by 17beta-estradiol. *Mol. Biol. Cell,* Vol. 16, No. 1, (Jan 2005),

nuclear factor-kappa B in Cos-1 cells by estrogen receptor-alpha. *Int. J. Toxicol.,* Vol.

events in early menopause: a possible role for hormone replacement therapy. *Int J* 

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understanding of the effects.

**6. Conclusion** 

**7. Key points** 

**8. References** 

pp. (231-237)

consortium "Women's Health Initiative" showed that women treated with equine estrogens over a period of 7 years had 17% lower risk to undergo a hip replacement compared to control group (Cirillo et al., 2006). Finally, an *in vivo* study performed on 180 ovariectomized monkeys for 3 years found that HRT reduced the severity of arthritic lesions, through attenuation of the PGs loss and reducing the number of osteophytes in these animals (Ham et al., 2002). Given its chondroprotective potential, it would seem that HRT is capable of modulating chondrocyte metabolism. Studies have also shown that SERM and phytoestrogens may have beneficial effect on cartilage metabolism and to alleviate OA symptoms (Arjmandi et al, 2004; Bassleer et al., 1996; Guiducci et al., 2005; Tsai et al., 1992). It has been shown that when administered at a clinically relevant dose in young male rats, tamoxifen causes persistent retardation of longitudinal and cortical radial bone growth through systemic suppression of IGF-I production and local effects on the growth plate cartilage; it increases in chondrocytes proliferation/apoptosis and decreases the number of hypertophic chondrocytes (Karimian et al., 2008). Similarly, raloxifen could act as estrogen agonist on the growth plate of ovariectomized immature rabbits, accelerating growth plate senescence and thus hastening epiphyseal fusion (Nilsson et al, 2003).

Besides estrogens, natural molecules such as phytoestrogens (estrogen-like compounds in plants) sharing structural and functional homologies with endogenous estrogens, could also act (even thought at micromolar concentrations) as agonists and/or antagonists in hormonesensitive target cells. Scientists are now interested in the tissue-selective activities of phytoestrogens considering anti-estrogenic effects in reproductive tissue that could help to reduce the risk of hormone-associated cancers (breast, uterine, ovarian, and prostate), while estrogenic effects on bone and cardiovascular system for instance could favor the maintenance of bone density and protect against atherosclerosis respectively. Moreover, it has been suggested that the consumption of dietary phytoestrogens (soja isoflavones, lignans, etc.) may have beneficial effects on bone health at all stages of life. That's why phytoestrogens have aroused much interest as potential substitutes in HRT of postmenopausal women, but they require much further investigation regarding their mechanism of action and their safety. Like estrogens, phytoestrogens may act *via* genomic and non genomic pathways. The most relevant molecular actions of phytoestrogens are those mediated by ERs. They may act on protein tyrosine kinase, MAP kinase, topoisomerase II and at all stages of cell cycle and apoptosis. They can also change the response to growth factors and cytokines. Genistein up to 100 M reduces the production of lipopolysaccharide (LPS)-stimulated pro-inflammatory molecules (COX-2, NO) but not that of COX-1 responsible of releasing prostaglandins in normal human chondrocytes (Hooshmand et al., 2007). When tested on human chondrocytes and chondrocytic cell line CHON-002, bavachin, a flavonid phytoestrogen isolated from *Psoralea corylifolia*, potentially protected cartilage from inflammation-mediated damage in joints of OA through decreasing IL-1beta-induced activation of IKK-IB alpha-NF-B signaling pathway (Cheng et al., 2010). Formononetin, a phytoestrogen isolated from *Astragalus membranaceus* showed to have biphasic positive effects on human normal osteoblasts and OA sub-chondral osteoblasts by modifying their biological synthetic capacities (Huh et al., 2010). Using female bovine articular chondrocytes, it has been demonstrated that the stimulating effect of insulin on GAGs sulfate incorporation was enhanced significantly after preincubation of cells with 10-11 -10-5 M daidzein or 10-9 -10-5 M genistein but not by 17-E2 (Claassen et al., 2008). More recently, xanthohumol, a prenylflavonoid extracted from hop, showed to prevent hyaluronan overproduction as well as PG and collagen loss in bovine chondrocytes; hyluronan overproduction is considered as an early reaction in OA followed by PG loss and collagen degradation (Stracke et al., 2001). However, additional research is critical to determine how phytoestrogens act on cartilage cells to obtain a more complete understanding of the effects.

## **6. Conclusion**

52 Challenges in Rheumatology

consortium "Women's Health Initiative" showed that women treated with equine estrogens over a period of 7 years had 17% lower risk to undergo a hip replacement compared to control group (Cirillo et al., 2006). Finally, an *in vivo* study performed on 180 ovariectomized monkeys for 3 years found that HRT reduced the severity of arthritic lesions, through attenuation of the PGs loss and reducing the number of osteophytes in these animals (Ham et al., 2002). Given its chondroprotective potential, it would seem that HRT is capable of modulating chondrocyte metabolism. Studies have also shown that SERM and phytoestrogens may have beneficial effect on cartilage metabolism and to alleviate OA symptoms (Arjmandi et al, 2004; Bassleer et al., 1996; Guiducci et al., 2005; Tsai et al., 1992). It has been shown that when administered at a clinically relevant dose in young male rats, tamoxifen causes persistent retardation of longitudinal and cortical radial bone growth through systemic suppression of IGF-I production and local effects on the growth plate cartilage; it increases in chondrocytes proliferation/apoptosis and decreases the number of hypertophic chondrocytes (Karimian et al., 2008). Similarly, raloxifen could act as estrogen agonist on the growth plate of ovariectomized immature rabbits, accelerating growth plate

Besides estrogens, natural molecules such as phytoestrogens (estrogen-like compounds in plants) sharing structural and functional homologies with endogenous estrogens, could also act (even thought at micromolar concentrations) as agonists and/or antagonists in hormonesensitive target cells. Scientists are now interested in the tissue-selective activities of phytoestrogens considering anti-estrogenic effects in reproductive tissue that could help to reduce the risk of hormone-associated cancers (breast, uterine, ovarian, and prostate), while estrogenic effects on bone and cardiovascular system for instance could favor the maintenance of bone density and protect against atherosclerosis respectively. Moreover, it has been suggested that the consumption of dietary phytoestrogens (soja isoflavones, lignans, etc.) may have beneficial effects on bone health at all stages of life. That's why phytoestrogens have aroused much interest as potential substitutes in HRT of postmenopausal women, but they require much further investigation regarding their mechanism of action and their safety. Like estrogens, phytoestrogens may act *via* genomic and non genomic pathways. The most relevant molecular actions of phytoestrogens are those mediated by ERs. They may act on protein tyrosine kinase, MAP kinase, topoisomerase II and at all stages of cell cycle and apoptosis. They can also change the response to growth factors and cytokines. Genistein up to 100 M reduces the production of lipopolysaccharide (LPS)-stimulated pro-inflammatory molecules (COX-2, NO) but not that of COX-1 responsible of releasing prostaglandins in normal human chondrocytes (Hooshmand et al., 2007). When tested on human chondrocytes and chondrocytic cell line CHON-002, bavachin, a flavonid phytoestrogen isolated from *Psoralea corylifolia*, potentially protected cartilage from inflammation-mediated damage in joints of OA through decreasing IL-1beta-induced activation of IKK-IB alpha-NF-B signaling pathway (Cheng et al., 2010). Formononetin, a phytoestrogen isolated from *Astragalus membranaceus* showed to have biphasic positive effects on human normal osteoblasts and OA sub-chondral osteoblasts by modifying their biological synthetic capacities (Huh et al., 2010). Using female bovine articular chondrocytes, it has been demonstrated that the stimulating effect of insulin on GAGs sulfate incorporation was enhanced significantly after preincubation of cells with 10-11 -10-5 M daidzein or 10-9 -10-5 M genistein but not by 17-E2 (Claassen et al., 2008). More recently, xanthohumol, a prenylflavonoid extracted from hop, showed to prevent hyaluronan overproduction as well as PG and collagen loss in bovine chondrocytes;

senescence and thus hastening epiphyseal fusion (Nilsson et al, 2003).

From epidemiological, clinical, *in vivo* and *in vitro* studies, a huge amount of data consistent with the fact that estrogens and their receptors can now be considered among the main players involved in the chondrocyte homeostasis and participate in cartilage protection from degradation and erosion occurring during menopause. Indeed, once ligand/ER complex is formed, estrogens such as 17-E2 could act with ER to induce or to inhibit *trans* activation of target genes of chondrocytes, the predominant cells of articular cartilage allowing expression of the specific chondrogenic markers such as UDPGD, PGs, type II collagen or inhibition of catabolic markers such as metalloproteinases and interleukins. There are different pathways by which estrogen and ER may interact with target genes in chondrocytes but it seems that Sp1 mediated *trans* activation being preferentially used in this cell type. In this mechanism ER needs AF-1 sequence (ligand-independent *trans* activation domain) to exert its action on GC boxes *via* Sp1 in target genes. In addition, some molecules sharing structural and functional features with estrogens like SERM and phytoestrogens can mimic estrogenic action and are therefore useful to repair cartilage erosion or might contribute at least to protect premenopausal joint cartilage and to maintain its homeostasis in a prevention strategy but these molecules need further investigation and deserves more attention from the scientific community to prove their safety and efficacy.

## **7. Key points**

Osteoarthritis, cartilage, chondrocytes, estrogens, phytoestrogens, selective estrogen receptor modulators, hormone replacement therapy, menopause.

## **8. References**


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**Part 3** 

**Clinical Manifestations and** 

**Diagnosis of Rheumatic Diseases** 

