**Profiling Inflammatory Genes and Signaling Pathways in Rheumatoid Synoviocytes for RA Light Therapy**

Yasuko Shibata and Yoshimitsu Abiko

*Department of Biochemistry and Molecular Biology Nihon University School of Dentistry at Matsudo Japan* 

### **1. Introduction**

152 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

Sun, Y., Berger, E. J., Zhao, C., Jay, G. D., An, K. N. & Amadio, P. C. (2006). Expression and

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Rheumatoid arthritis (RA) is an autoimmune joint disease characterized by inflammation and destruction of the articular surfaces and bone. During joint movement, synovial tissues contribute to mechanical load bearing by changing their shape. These elastic synovial membranes are an early target of rheumatic inflammation, and together with chondrocytes, become a primary source of inflammatory factors (e.g., cytokines) that enter the synovial fluid (Dayer, 2004, Firestein, 2007). The pathophysiological steps leading to RA include inflammation, proliferation of synovial cells, and attachment to and invasion of adjacent cartilage and bone by fibroblast-like cells derived from rheumatoid synoviocytes (RA-FLSs) (Firestein, 1996, Pap et al., 2000, Tolboom et al., 2005, Bartok & Firestein, 2010).

In the healthy synovium, one to three layers of synoviocytes, the macrophage-like type A and the more abundant fibroblast-like type B (also referred to as synovial fibroblasts), form the synovial lining layer separating the synovial sublining layer of loose connective tissue from the joint cavity (Iwanaga et al., 2000). The joint damage observed in RA is mainly mediated by macrophage/macrophage-like cell-derived cytokines, such as interleukin (IL)- 1, IL-6 and tumor necrosis factor (TNF)-, which induce neutral protease production by FLSs and articular chondrocytes (Houssiau et al., 1988, Feldmann & Maini, 1999, Dayer, 2002). Although IL-1 and TNF- share many biological activities that are relevant in RA, early studies of experimental arthritis models have demonstrated that IL-1 plays a predominant role in cartilage destruction via inflammatory processes that include the activation of matrix metalloproteinases (MMPs) (Borghaei et al., 1998) and the inhibition of the synthesis of extracellular matrix (ECM) molecules (Mauviel et al., 1988). In particular, IL-1 is readily detected, long after the onset of RA, at high levels in the synovial fluid of RA patients. IL-1 can significantly alter the expression of a variety of genes, including inflammatory mediators such as cytokines and MMPs (Sun & Yokota, 2002, Suzuki et al., 2010). In addition, IL-1 stimulates bone resorption by activating osteoclasts (Goldring, 2003). That said, there is little comprehensive information available on the effects of IL-1 on fibroblast-like synoviocytes (FLSs).

RA-FLSs not only mediate tissue destruction, but also are considered to play a major role in initiating and driving RA in concert with inflammatory cells (Huber, 2006, Bartok &

Profiling Inflammatory Genes

was signaling-related (Fig. 1).

significant.

**2.1 IL-1-induced inflammatory signaling pathways** 

and Signaling Pathways in Rheumatoid Synoviocytes for RA Light Therapy 155

Our aims were to identify potential mediators of RA inflammation and joint destruction induced by the inflammatory cytokine IL-1 and to investigate the mechanisms responsible for their stimulation. Of the 8,746 genes on the Focus array HG-8500 GeneChip, 4,909 from untreated cells and 5,073 from IL-1-treated cells were classified as marginally expressed or present. After the cut-offs for induction (≥2.0-fold) and suppression (≤0.5-fold) by IL-1 were applied, 120 genes (74 upregulated and 46 downregulated genes) were identified as being affected by IL-1. When we then classified the IL-1-inducible gene expression in MH7A cells with respect to six functionalities based on BiologicalProcess words, we found that IL-1 conspicuously increased the expression of inflammation and apoptosis/proliferation-related genes, although the largest group of genes affected by IL-1

The genes that were upregulated (n=74) or down-regulated (n=46) by IL-1 were classified into 6 groups based on the GO-BiologicalProcess terms listed in the GeneSpring 6.2 software program. Fig. 1. Classification of genes whose expression in MH7A cells was affected by IL-1.

We used IPA to gain a better understanding of how differentially expressed genes were integrated into specific regulatory and signaling networks. A data set containing gene identifiers adhering to the 2-fold (120 genes) change used in microarray experiments was uploaded into the IPA software application. Each gene identifier was mapped to its corresponding gene object and then overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. The networks of these genes were then algorithmically generated based on their connectivity, after which a functional analysis of the networks identified the biological functions that were the most

We attempted to visualize the IL-1-induced genes within the canonical IPA pathway, which involves IL-1, IL-6, and TNF- signaling (Fig. 2A, canonical pathway name: IL-6 signaling). Four IL-1-inducible genes (>2.0-fold), IL-1, IL-6, IL-8, and IB, were situated within this atlas. As shown in Fig. 2A, activation of NF-B signaling is also important for the secretion of IL-8, IL-1 and IL-6. We previously used an ELISA to show that TNF- was not expressed in MH7A cells, regardless of whether they were exposed to IL-1 (0.1 unit/ml) (Shibata et al., 2005). Likewise, the results we obtained using the GeneChip microarray indicated no expression of TNF- in MH7A cells under our experimental conditions. IL-1 did induce its own expression, which may be important for its accumulation during the inflammation in RA.

Furthermore, high levels of NF-IL6 (CEBPB) expression were consistently observed.

Firestein, 2010). However, the original roles of single RA-FLS, and their relationship with macrophage-like type B cells in the rheumatoid synovium are poorly understood, although many case reports of RA patients, and many experimental reports using the synovium tisuues/cells from RA patients have been published. The utilization of cells such as the MH7A human RA-FLSs (Cell Bank, Riken Bioresource Center, Ibaraki, Japan), which are established cells isolated from the knee joint of an RA patient that retain the morphological and functional characteristics of primary synovial cells should provide guidance to clarify the mechanisms of onset of RA and to the development of useful treatments for RA. The MH7A cells are an immortalized RA-FLS line that stains positively for IL-1R, intercellular adhesion molecule-1 (ICAM-1), CD16, CD40, CD80, and CD95 and has been used extensively to investigate the molecular mechanisms underlying RA (Miyazawa et al., 1998). It has been shown, for example, that IL-1 enhances the production of IL-6, IL-8, and MMPs in MH7A cells (Shibata et al., 2005, Han et al., 2006) in a manner similar to that seen in the parental FLSs, although the immortalized cells grow more rapidly than the parental cells. These results clearly indicate the usefulness of MH7A cells for investigating the regulation of rheumatoid FLSs and the IL-1 signal transduction pathway to develop a future RA therapy.

In what is becoming known as the "post-genomic era", many new technologies and methodologies are being developed to take advantage of the recent progress in genomic research. Among these, gene expression profiling has become an invaluable tool in functional genomics. DNA microarrays, cDNA subtraction, and the serial analysis of gene expression have all emerged as leading transcript profiling technologies for the global analysis of biological systems. One of the high throughput technologies, high-density oligonucleotide genechip microarrays, makes it possible to simultaneously measure the relative abundance of numerous mRNAs within a cell. To better understand the direct IL-1-induced changes in gene expression that might promote inflammatory responses in MH7A cells, in the present study we used an Affymetrix HG-8500 Focus array GeneChip to analyze the patterns of gene expression, and then used the Ingenuity Pathway Analysis (IPA) software program to investigate the signaling pathways leading to the IL-1-induced gene expression. In addition, we used a biotin label-based antibody array to measure the levels of 507 human target proteins, including cytokines, chemokines, growth factors, angiogenic factors, proteases, and soluble receptors, among others, in the cell culture supernatants. We analyzed the expression profiles of both the genes and proteins to identify the key molecules involved in articular rheumatism, which could be related to the induction of inflammation and pannus formation in the synovial tissues of RA patients. In addition, we propose the possibility of a new therapy using linear polarized infrared light, that induces the suppression of inflammation and pain for RA patients with no apparent sideeffects.

#### **2. Microarray analysis**

MH7A human RA-FLSs (Riken Bioresource Center, Ibaraki) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and penicillin-streptomycin at 37°C under a 5% CO2 atmosphere. Total RNA from the MH7A cells was treated according to the manufacturer's protocol, and then hybridized to a Human Genome Focus Array HG-8500 GeneChip (Affymetrix Inc.).

Firestein, 2010). However, the original roles of single RA-FLS, and their relationship with macrophage-like type B cells in the rheumatoid synovium are poorly understood, although many case reports of RA patients, and many experimental reports using the synovium tisuues/cells from RA patients have been published. The utilization of cells such as the MH7A human RA-FLSs (Cell Bank, Riken Bioresource Center, Ibaraki, Japan), which are established cells isolated from the knee joint of an RA patient that retain the morphological and functional characteristics of primary synovial cells should provide guidance to clarify the mechanisms of onset of RA and to the development of useful treatments for RA. The MH7A cells are an immortalized RA-FLS line that stains positively for IL-1R, intercellular adhesion molecule-1 (ICAM-1), CD16, CD40, CD80, and CD95 and has been used extensively to investigate the molecular mechanisms underlying RA (Miyazawa et al., 1998). It has been shown, for example, that IL-1 enhances the production of IL-6, IL-8, and MMPs in MH7A cells (Shibata et al., 2005, Han et al., 2006) in a manner similar to that seen in the parental FLSs, although the immortalized cells grow more rapidly than the parental cells. These results clearly indicate the usefulness of MH7A cells for investigating the regulation of rheumatoid FLSs and the IL-1 signal transduction pathway to develop a future RA

In what is becoming known as the "post-genomic era", many new technologies and methodologies are being developed to take advantage of the recent progress in genomic research. Among these, gene expression profiling has become an invaluable tool in functional genomics. DNA microarrays, cDNA subtraction, and the serial analysis of gene expression have all emerged as leading transcript profiling technologies for the global analysis of biological systems. One of the high throughput technologies, high-density oligonucleotide genechip microarrays, makes it possible to simultaneously measure the relative abundance of numerous mRNAs within a cell. To better understand the direct IL-1-induced changes in gene expression that might promote inflammatory responses in MH7A cells, in the present study we used an Affymetrix HG-8500 Focus array GeneChip to analyze the patterns of gene expression, and then used the Ingenuity Pathway Analysis (IPA) software program to investigate the signaling pathways leading to the IL-1-induced gene expression. In addition, we used a biotin label-based antibody array to measure the levels of 507 human target proteins, including cytokines, chemokines, growth factors, angiogenic factors, proteases, and soluble receptors, among others, in the cell culture supernatants. We analyzed the expression profiles of both the genes and proteins to identify the key molecules involved in articular rheumatism, which could be related to the induction of inflammation and pannus formation in the synovial tissues of RA patients. In addition, we propose the possibility of a new therapy using linear polarized infrared light, that induces the suppression of inflammation and pain for RA patients with no apparent side-

MH7A human RA-FLSs (Riken Bioresource Center, Ibaraki) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and penicillin-streptomycin at 37°C under a 5% CO2 atmosphere. Total RNA from the MH7A cells was treated according to the manufacturer's protocol, and then hybridized to a Human Genome Focus Array HG-8500

therapy.

effects.

**2. Microarray analysis**

GeneChip (Affymetrix Inc.).

Our aims were to identify potential mediators of RA inflammation and joint destruction induced by the inflammatory cytokine IL-1 and to investigate the mechanisms responsible for their stimulation. Of the 8,746 genes on the Focus array HG-8500 GeneChip, 4,909 from untreated cells and 5,073 from IL-1-treated cells were classified as marginally expressed or present. After the cut-offs for induction (≥2.0-fold) and suppression (≤0.5-fold) by IL-1 were applied, 120 genes (74 upregulated and 46 downregulated genes) were identified as being affected by IL-1. When we then classified the IL-1-inducible gene expression in MH7A cells with respect to six functionalities based on BiologicalProcess words, we found that IL-1 conspicuously increased the expression of inflammation and apoptosis/proliferation-related genes, although the largest group of genes affected by IL-1 was signaling-related (Fig. 1).

The genes that were upregulated (n=74) or down-regulated (n=46) by IL-1 were classified into 6 groups based on the GO-BiologicalProcess terms listed in the GeneSpring 6.2 software program.

Fig. 1. Classification of genes whose expression in MH7A cells was affected by IL-1.

We used IPA to gain a better understanding of how differentially expressed genes were integrated into specific regulatory and signaling networks. A data set containing gene identifiers adhering to the 2-fold (120 genes) change used in microarray experiments was uploaded into the IPA software application. Each gene identifier was mapped to its corresponding gene object and then overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. The networks of these genes were then algorithmically generated based on their connectivity, after which a functional analysis of the networks identified the biological functions that were the most significant.

#### **2.1 IL-1-induced inflammatory signaling pathways**

We attempted to visualize the IL-1-induced genes within the canonical IPA pathway, which involves IL-1, IL-6, and TNF- signaling (Fig. 2A, canonical pathway name: IL-6 signaling). Four IL-1-inducible genes (>2.0-fold), IL-1, IL-6, IL-8, and IB, were situated within this atlas. As shown in Fig. 2A, activation of NF-B signaling is also important for the secretion of IL-8, IL-1 and IL-6. We previously used an ELISA to show that TNF- was not expressed in MH7A cells, regardless of whether they were exposed to IL-1 (0.1 unit/ml) (Shibata et al., 2005). Likewise, the results we obtained using the GeneChip microarray indicated no expression of TNF- in MH7A cells under our experimental conditions. IL-1 did induce its own expression, which may be important for its accumulation during the inflammation in RA. Furthermore, high levels of NF-IL6 (CEBPB) expression were consistently observed.

Profiling Inflammatory Genes

modified from (Shibata et al., 2009a)]

Fig. 3. The effect of Bay11-7085 on the production of IL-8

**2.2 Arithmetic IL-8 production through the PGE2 receptor synthesis** 

cells.

and Signaling Pathways in Rheumatoid Synoviocytes for RA Light Therapy 157

induced IL-8 expression (Matsusaka et al., 1993). We would therefore anticipate that these transcription factors would play an important role in the increased expression of IL-8 mRNA elicited by IL-1, but no increases in the expression for NFKB1 (NF-B), RELA (RelA), or CEBPB (NF-IL-6) were seen in the microarray experiment. On the other hand, in resting cells, NF-B is trapped in the cytoplasm through its interaction with its inhibitor, IkB, and a crucial step in the activation of NF-B is the stimulus-induced phosphorylation of IB by IB kinases (IKKs). To study the extent to which IKKs modulate IL-1 induced IL-8 production, MH7A cells were pretreated with Bay11-7085, an inhibitor of IB phosphorylation, for 1 hr before stimulation with IL-1(Shibata et al., 2009a). As shown in Fig. 3, Bay11-7085 completely blocked the release of IL-8 from IL-1-stimulated MH7A

**A.** The IL-1-induced signal transduction pathways leading to the production of IL-8. Gray symbols indicate genes that were up-regulated by IL-1. White symbols indicate genes whose expression was increased less than 2.0-fold by IL-1. **B.** The effects of Bay11-7085 on the production of IL-8. MH7A cells were first incubated with the IKK/ inhibitor Bay11-7085 for 1 hr, and then with IL-1 for 2 hr. The levels of IL-8 in medium conditioned by the cells were measured using an ELISA. [This figure is

PGE2, a product of the cyclooxygenation of arachidonic acid, is a potent mediator of the immune response and inflammation, and contributes to the pathogenesis of RA (Robinson et al., 1975, Trebino et al., 2003). PGE2 also displays a complex regulatory function affecting IL-8 gene expression, which is dependent on the concentration of PGE2 and on the specific cell type involved. At physiological and pathological concentrations of up to 100 μM, PGE2 is capable of upregulating endogenous IL-8 expression in human colonic epithelial cells (Yu & Chadee, 1998). However, there have been many reports that PGE2 has no effect on neutrophil-derived IL-8 induced by LPS (Wertheim et al., 1993); that it down-regulates IL-8 in response to LPS in human alveolar macrophages and blood monocytes (Standiford et al., 1992); and suppresses the production of chemokines, including IL-8, in human macrophages

**A.** The IL-1 and IL-6 signaling pathways as determined by IPA analysis. Grayed symbols indicate genes that were upregulated by IL-1; white symbols indicate genes whose expression was not increased by IL-1. **B.** A scatter plot of 8,793 genes. Genes whose expression ratio was changed by >2 fold by IL-1 are positioned outside the lines. **C.** Dose-dependent effects of IL-1 on the gene expression. MH7A cells were examined after treatment with fresh medium containing the indicated concentration of IL-1, and the cells were incubated for an additional 2 hr.

Fig. 2. The IL-1 and IL-6 signaling pathways and the results of the RT-PCR analysis

Based on the structural analysis, the chemokine superfamily can be divided into two groups: the C-X-C and the C-C subfamilies (Baggiolini et al., 1994). IL-8 belongs to the first group and acts mainly on neutrophils (Hebert & Baker, 1993). Our microarray experiments showed that IL-8 transcription was the most dramatically upregulated (20.8 fold) in MH7A cells exposed to IL-1. It is also noteworthy that the expression of other chemokines, including CXCL-1 (4.1-fold), -2 (4.0-fold) and -3 (2.7-fold), three potent stimulators of neutrophil activation and tissue infiltration was also enhanced. Leukocyte recruitment from the microvasculature to sites of inflammation is a sequential process that includes rolling, activation, firm adhesion, and finally, transmigration through the vessel wall (King & Hammer, 2001). Although macrophages, neutrophils, and endothelial cells are all thought to be primary sources of IL-8, which is known to be elevated at inflammatory sites such as the synovial fluid of RA patients, fibroblast-like and macrophage-like cells of the synovial membrane, together with the infiltrating leucocytes, are the major source of these mediators in the rheumatoid synovial pannus (Rathanaswami et al., 1993). In the GeneChip microarray experiment, an IL-1 concentration close to the levels seen clinically was used to stimulate the MH7A cells. The gene expression of IL-8, Gro- (CXCL1), Gro- (CXCL2), IL-1, and IL-6 in scatter plots is shown in Fig. 2B. According to a RT-PCR analysis, the levels of IL-8, CXCL1, CXCL2, IL-1, and IL-6 mRNA in MH7A cells were all found to be dosedependently increased by IL-1 (Fig. 2C). Therefore, IL-8 appears to play a major role in the initiation and maintenance of inflammatory responses.

Within the 5'-flanking region of the human IL-8 promoter are a number of motifs with the potential to bind various transcription factors in a cell and stimulus-dependent manner. In particular, the transcription factors NF-B and NF-IL-6 are both involved in stimulus-

**A.** The IL-1 and IL-6 signaling pathways as determined by IPA analysis. Grayed symbols indicate genes that were upregulated by IL-1; white symbols indicate genes whose expression was not increased by IL-1. **B.** A scatter plot of 8,793 genes. Genes whose expression ratio was changed by >2 fold by IL-1 are positioned outside the lines. **C.** Dose-dependent effects of IL-1 on the gene expression. MH7A cells were examined after treatment with fresh medium containing the indicated

Fig. 2. The IL-1 and IL-6 signaling pathways and the results of the RT-PCR analysis

Based on the structural analysis, the chemokine superfamily can be divided into two groups: the C-X-C and the C-C subfamilies (Baggiolini et al., 1994). IL-8 belongs to the first group and acts mainly on neutrophils (Hebert & Baker, 1993). Our microarray experiments showed that IL-8 transcription was the most dramatically upregulated (20.8 fold) in MH7A cells exposed to IL-1. It is also noteworthy that the expression of other chemokines, including CXCL-1 (4.1-fold), -2 (4.0-fold) and -3 (2.7-fold), three potent stimulators of neutrophil activation and tissue infiltration was also enhanced. Leukocyte recruitment from the microvasculature to sites of inflammation is a sequential process that includes rolling, activation, firm adhesion, and finally, transmigration through the vessel wall (King & Hammer, 2001). Although macrophages, neutrophils, and endothelial cells are all thought to be primary sources of IL-8, which is known to be elevated at inflammatory sites such as the synovial fluid of RA patients, fibroblast-like and macrophage-like cells of the synovial membrane, together with the infiltrating leucocytes, are the major source of these mediators in the rheumatoid synovial pannus (Rathanaswami et al., 1993). In the GeneChip microarray experiment, an IL-1 concentration close to the levels seen clinically was used to stimulate the MH7A cells. The gene expression of IL-8, Gro- (CXCL1), Gro- (CXCL2), IL-1, and IL-6 in scatter plots is shown in Fig. 2B. According to a RT-PCR analysis, the levels of IL-8, CXCL1, CXCL2, IL-1, and IL-6 mRNA in MH7A cells were all found to be dosedependently increased by IL-1 (Fig. 2C). Therefore, IL-8 appears to play a major role in the

Within the 5'-flanking region of the human IL-8 promoter are a number of motifs with the potential to bind various transcription factors in a cell and stimulus-dependent manner. In particular, the transcription factors NF-B and NF-IL-6 are both involved in stimulus-

concentration of IL-1, and the cells were incubated for an additional 2 hr.

initiation and maintenance of inflammatory responses.

induced IL-8 expression (Matsusaka et al., 1993). We would therefore anticipate that these transcription factors would play an important role in the increased expression of IL-8 mRNA elicited by IL-1, but no increases in the expression for NFKB1 (NF-B), RELA (RelA), or CEBPB (NF-IL-6) were seen in the microarray experiment. On the other hand, in resting cells, NF-B is trapped in the cytoplasm through its interaction with its inhibitor, IkB, and a crucial step in the activation of NF-B is the stimulus-induced phosphorylation of IB by IB kinases (IKKs). To study the extent to which IKKs modulate IL-1 induced IL-8 production, MH7A cells were pretreated with Bay11-7085, an inhibitor of IB phosphorylation, for 1 hr before stimulation with IL-1(Shibata et al., 2009a). As shown in Fig. 3, Bay11-7085 completely blocked the release of IL-8 from IL-1-stimulated MH7A cells.

**A.** The IL-1-induced signal transduction pathways leading to the production of IL-8. Gray symbols indicate genes that were up-regulated by IL-1. White symbols indicate genes whose expression was increased less than 2.0-fold by IL-1. **B.** The effects of Bay11-7085 on the production of IL-8. MH7A cells were first incubated with the IKK/ inhibitor Bay11-7085 for 1 hr, and then with IL-1 for 2 hr. The levels of IL-8 in medium conditioned by the cells were measured using an ELISA. [This figure is modified from (Shibata et al., 2009a)]

Fig. 3. The effect of Bay11-7085 on the production of IL-8

### **2.2 Arithmetic IL-8 production through the PGE2 receptor synthesis**

PGE2, a product of the cyclooxygenation of arachidonic acid, is a potent mediator of the immune response and inflammation, and contributes to the pathogenesis of RA (Robinson et al., 1975, Trebino et al., 2003). PGE2 also displays a complex regulatory function affecting IL-8 gene expression, which is dependent on the concentration of PGE2 and on the specific cell type involved. At physiological and pathological concentrations of up to 100 μM, PGE2 is capable of upregulating endogenous IL-8 expression in human colonic epithelial cells (Yu & Chadee, 1998). However, there have been many reports that PGE2 has no effect on neutrophil-derived IL-8 induced by LPS (Wertheim et al., 1993); that it down-regulates IL-8 in response to LPS in human alveolar macrophages and blood monocytes (Standiford et al., 1992); and suppresses the production of chemokines, including IL-8, in human macrophages

Profiling Inflammatory Genes

and Signaling Pathways in Rheumatoid Synoviocytes for RA Light Therapy 159

**A.** The gray symbols indicate the IL-1-inducible genes (>2.0-fold) in the apoptosis signaling pathway. **B.** The time-dependent effects of IL-1 on the gene expression of cIAP (BIRC3), which negatively

It has been suggested that RA is driven by a network of closely connected interdependent pathogenic mechanisms involving innate and adaptive immunity that ultimately lead to synovial inflammation and aggressive synovial overproliferation during the terminal destructive phase (Firestein, 2003). Furthermore, in a chronically inflamed microenvironment, these destructive changes are driven by invasive synovial hyperplasia and neovascularization (pannus formation) at the interface between the synovium and intraarticular space. During the process of synovial pannus formation, FLSs actively contribute to the inflamed and destructive local microenvironment by secreting a variety of inflammatory mediators, which lead to tissue degradation. In response to IL-1 and TNF-, FLSs also secrete angiogenic growth factors, such as vascular endothelial growth factor (VEGF) (Nagashima et al., 1999) and basic fibroblast growth factor (bFGF), which are essential for the pathophysiological neovascularization in RA joints. In addition, we have identified that BIRC3 is upregulated, making it a candidate mediator of FLS overproliferation in RA. BIRC3 encodes cIAP, which is a key negative regulator of apoptotic signaling in FLSs. The resulting inhibition of FLS apoptosis in the inflamed area would be expected to contribute to the

A RayBio biotin label-based human antibody array I (RayBiotech, Inc.) was used to measure the expression levels of 507 proteins in culture medium conditioned by MH7A cells. Table 1 presents the results of the biotin label-based antibody array analysis of secreted proteins that categorized interleukins, and molecules involved in ECM metabolism. IL-12 p70 and IL-8 were

regulates apoptotic signaling, were examined using MH7A cells.

Fig. 4. An atlas of an apoptosis signaling pathway

formation of the pannus in the RA joint.

**3. Protein chip analysis** 

**3.1 ECM degradation** 

(Takayama et al., 2002). Furthermore, PGE2 alone had little detectable effect on IL-8, although a small enhancement of the mRNA and protein levels of IL-6 has been observed in human synovial fibroblasts (Agro et al., 1996). In RA research, while PGE2 alone has limited effects on synovial cell production of IL-8, its effects are significant in the context of IL-1 stimulation; endogenous PGE2 may alter the cytokines secreted by mesenchymally-derived cells.

A variety of transcription factors, including NF-κB, NF-IL6, activator protein-1 and octamer-1, have been shown to regulate IL-8 gene transcription (Matsusaka et al., 1993). Caristi *et al*  (Caristi et al., 2005) have shown that, in human T cells, PGE2 induces IL-8 synthesis through an NF-B-independent pathway via its EP1- and EP4-type receptors (PGE2EP1 and PGE2EP4, respectively). We have demonstrated that PGE2 at physiological, as well as pathological, concentrations induced IL-8 production thorough the increased expression of PGE2EP4 receptors in synovial fibroblasts of RA *in vitro* (Shibata et al., 2009b). The present results were of particular interest, since the effect of PGE2 on IL-8 production was found in the IL-1-pretreated MH7A cells. We demonstrated that the newly expressed-PGE2 receptors are involved in this process.

These findings show the complexity with which PGE2 regulates IL-8 synthesis by inhibiting or enhancing its production depending on the cell types and environmental conditions. Therefore, we proposed that, in IL-1-stimulated synovial fibroblasts in RA patients, PGE2 induces IL-8 mRNA transcription by the activation of different signal transduction pathways from the conventional IL-1-stimulated pathways, including PGE2 receptor EP4-triggered pathways. IL-1 enhanced the gene expression of IL8 and PTGER4 (Shibata et al., 2009b), subsequently IL-8 production was enhanced by IL-1 and PGE2 from environmental neutrophils/macrophages in the synovial tissues. These results may therefore highlight a new important role for PGE2 in regulating IL-8 production by the synovial fibroblasts of RA patients, confirming the pro-inflammatory activity of this prostaglandin.

#### **2.3 Apoptotic signaling**

Inflammation of the synovial membrane results in the development of aggressive granulation tissue, called "pannus". Pannus tissue is composed mainly of inflammatory cells such as macrophages and FLSs. By a microarray analysis, the apoptosis/proliferation-related molecules included those involved in processes associated with negative/positive regulation of cell proliferation, apoptosis/anti-apoptosis, cell death, cell proliferation and regulation of the cell cycle, and a large number of these genes were influenced by IL-1 (Fig. 1). Interestingly, whereas the expression of the gene encoding the apoptosis inhibitor cIAP (BIRC3) was enhanced (4.8-fold), the expression of those encoding the cell death inducing molecule FOSL2 (FOS-like antigen 2, 0.4-fold)) and the cell proliferation-related molecule EDNRA (endothelin receptor type A, 0.2-fold) were both suppressed. We attempted to apply the IL-1-inducible genes to a typical pathway atlas involving apoptotic signaling (Fig. 4A). The expression of BIRC3, which encodes the anti-apoptotic protein cIAP, implies that IL-1 signaling leads to inhibition of apoptotic signaling, as expression of cIAP should exert an inhibitory effect on caspase 3 activity (Rothe et al., 1995, Budihardjo et al., 1999). To confirm the increased cIAP production in MH7A cells, we found that levels of the BIRC3 transcript were substantially increased in the IL-1-induced MH7A cells (Fig. 4B).

(Takayama et al., 2002). Furthermore, PGE2 alone had little detectable effect on IL-8, although a small enhancement of the mRNA and protein levels of IL-6 has been observed in human synovial fibroblasts (Agro et al., 1996). In RA research, while PGE2 alone has limited effects on synovial cell production of IL-8, its effects are significant in the context of IL-1 stimulation; endogenous PGE2 may alter the cytokines secreted by mesenchymally-derived

A variety of transcription factors, including NF-κB, NF-IL6, activator protein-1 and octamer-1, have been shown to regulate IL-8 gene transcription (Matsusaka et al., 1993). Caristi *et al*  (Caristi et al., 2005) have shown that, in human T cells, PGE2 induces IL-8 synthesis through an NF-B-independent pathway via its EP1- and EP4-type receptors (PGE2EP1 and PGE2EP4, respectively). We have demonstrated that PGE2 at physiological, as well as pathological, concentrations induced IL-8 production thorough the increased expression of PGE2EP4 receptors in synovial fibroblasts of RA *in vitro* (Shibata et al., 2009b). The present results were of particular interest, since the effect of PGE2 on IL-8 production was found in the IL-1-pretreated MH7A cells. We demonstrated that the newly expressed-PGE2

These findings show the complexity with which PGE2 regulates IL-8 synthesis by inhibiting or enhancing its production depending on the cell types and environmental conditions. Therefore, we proposed that, in IL-1-stimulated synovial fibroblasts in RA patients, PGE2 induces IL-8 mRNA transcription by the activation of different signal transduction pathways from the conventional IL-1-stimulated pathways, including PGE2 receptor EP4-triggered pathways. IL-1 enhanced the gene expression of IL8 and PTGER4 (Shibata et al., 2009b), subsequently IL-8 production was enhanced by IL-1 and PGE2 from environmental neutrophils/macrophages in the synovial tissues. These results may therefore highlight a new important role for PGE2 in regulating IL-8 production by the synovial fibroblasts of RA patients, confirming the pro-inflammatory activity of this

Inflammation of the synovial membrane results in the development of aggressive granulation tissue, called "pannus". Pannus tissue is composed mainly of inflammatory cells such as macrophages and FLSs. By a microarray analysis, the apoptosis/proliferation-related molecules included those involved in processes associated with negative/positive regulation of cell proliferation, apoptosis/anti-apoptosis, cell death, cell proliferation and regulation of the cell cycle, and a large number of these genes were influenced by IL-1 (Fig. 1). Interestingly, whereas the expression of the gene encoding the apoptosis inhibitor cIAP (BIRC3) was enhanced (4.8-fold), the expression of those encoding the cell death inducing molecule FOSL2 (FOS-like antigen 2, 0.4-fold)) and the cell proliferation-related molecule EDNRA (endothelin receptor type A, 0.2-fold) were both suppressed. We attempted to apply the IL-1-inducible genes to a typical pathway atlas involving apoptotic signaling (Fig. 4A). The expression of BIRC3, which encodes the anti-apoptotic protein cIAP, implies that IL-1 signaling leads to inhibition of apoptotic signaling, as expression of cIAP should exert an inhibitory effect on caspase 3 activity (Rothe et al., 1995, Budihardjo et al., 1999). To confirm the increased cIAP production in MH7A cells, we found that levels of the BIRC3 transcript were substantially increased in

cells.

prostaglandin.

**2.3 Apoptotic signaling** 

the IL-1-induced MH7A cells (Fig. 4B).

receptors are involved in this process.

**A.** The gray symbols indicate the IL-1-inducible genes (>2.0-fold) in the apoptosis signaling pathway. **B.** The time-dependent effects of IL-1 on the gene expression of cIAP (BIRC3), which negatively regulates apoptotic signaling, were examined using MH7A cells.

Fig. 4. An atlas of an apoptosis signaling pathway

It has been suggested that RA is driven by a network of closely connected interdependent pathogenic mechanisms involving innate and adaptive immunity that ultimately lead to synovial inflammation and aggressive synovial overproliferation during the terminal destructive phase (Firestein, 2003). Furthermore, in a chronically inflamed microenvironment, these destructive changes are driven by invasive synovial hyperplasia and neovascularization (pannus formation) at the interface between the synovium and intraarticular space. During the process of synovial pannus formation, FLSs actively contribute to the inflamed and destructive local microenvironment by secreting a variety of inflammatory mediators, which lead to tissue degradation. In response to IL-1 and TNF-, FLSs also secrete angiogenic growth factors, such as vascular endothelial growth factor (VEGF) (Nagashima et al., 1999) and basic fibroblast growth factor (bFGF), which are essential for the pathophysiological neovascularization in RA joints. In addition, we have identified that BIRC3 is upregulated, making it a candidate mediator of FLS overproliferation in RA. BIRC3 encodes cIAP, which is a key negative regulator of apoptotic signaling in FLSs. The resulting inhibition of FLS apoptosis in the inflamed area would be expected to contribute to the formation of the pannus in the RA joint.
