**5.3. CCNs in chondrogenesis and osteogenesis**

The CCN proteins appear to play a critical role in the establishment of the skeletal system and have been shown to have positive and negative regulatory roles in skeletal development, as demonstrated through cell culture experiments and animal models. Collectively, these studies demonstrate that CCN proteins promote differentiation and proliferation of chondrocytes, osteoblasts, and vascular endothelial cells, which are important in both endochondral and intramembranous ossification.

Cartilage is first seen in the developing embryo in the areas that serve as templates for endochondral ossification, as well as in Meckel's cartilage in the head area [1]. Both CCN1 and CCN2 promote chondrogenic and osteoblastic differentiation [232,233]. CCNs can interact with members of the TGF‐β and BMP family via the chordin‐like homology found in the VWC domain, and modulate their binding affinity for their respective receptors [234]. CCN2 is important during embryogenesis and bone formation, including during proliferation of mesenchymal cells, their differentiation into chondrocytes, and condensation of these cells into the cartilage model that will form bone [1,235]. CCN2 expression is highest in the vascular tissue and the maturing chondrocytes of the embryo. TGF‐β, which induces CCN2, directs the condensation of these cells and regulates secretion of other matricellular proteins such as fibronectin [235]. Cells from CCN2 knockout mice do not undergo this condensation process in vitro and have less synthesis of cartilage proteoglycans [236]. Neutralizing antibodies, siRNA, and antisense oligonucleotides have all been used to demonstrate that reduced or absence of CCN2 prevents condensation from occurring [1,235]. Overexpression of CCN2 causes an increase in the length and density of bones [237]. In vertebral bodies, downregulation of CCN2 is necessary for chondrocyte differentiation [235]. These studies demonstrate that the spatial and temporal regulation of CCNs is an important component of development.

Development of cartilage involves several different transcription factors, such as Sox 9, that control this process, as well as growth factors that interact with cellular receptors to facilitate various cellular functions [2]. Interaction with the extracellular matrix has been implicated in development as well. Various epigenetic mechanisms and several different microRNAs also are important in the development of cartilage [3,4].

BMPs are important during skeletogenesis [238] and are known to be regulated by CCN2 [234]. Expression of CCN2 is observed in both the perichondrium and the chondrocytes during development [239]. BMPs play a role in the repair of cartilage as well as during development [240]. BMP2 causes proliferation of chondrocytes during development, while BMP4 does not [241]. CCN2 and CCN3 can bind BMP2 and abrogate its ability to promote chondrogenic and osteogenic differentiation [242,243], respectively, whereas CCN4 binding to BMP2 enhances its function in osteogenesis [244]. CCN2 also binds BMP4 and blocks its signaling capabilities [234]. This binding thus modulates the activity of BMP‐9. CCN2 also modulates the Wnt pathway in *Xenopus* embryos by binding to LRP6 co‐receptor [245]. CCN2 expression in chondrocytes is controlled by both Rac 1 and actin pathways that are mediated by TGF‐β/Smad signaling. CCN4 is expressed in developing mesenchymal, pre‐osteoblastic, and cartilage cells and is believed to regulate skeletal growth and repair [246]. A truncated form of CCN4, WISP1v, has been shown to regulate the differentiation of chondrocytes toward endochondral ossification [247].

Chondrocytes are usually considered quiescent in the adult, although this can change during pathological processes such as osteoarthritis. Cartilage has very little capacity to regenerate, even though progenitor cells are present in the adult [6]. This lack of repair ability causes a number of disease issues as humans age, including the most prevalent form of degenerative joint disease, osteoarthritis [7]. In adult skeletal systems, CCN2 is highly expressed in the osteoblasts lining metaphyseal trabeculae and in osteogenic surfaces lining fracture calluses, suggesting that its upregulation in these areas may contribute to bone growth and fracture repair [248].

#### **5.4. CCNs in cartilage and bone pathology**

Several types of pathologies are observed in cartilage and bone. In cartilage, the most common is osteoarthritis, which significantly increases as the population ages. Disorders of the inter‐ vertebral disc are observed as well, and also increase with aging. Wounding and subsequent repair of cartilage occurs and can mimic the progression of osteoarthritis. Various types of chondritis, or inflammation of cartilage, are observed in various regions of the body. Congen‐ ital defects in cartilage include conditions such as chondrodysplasia, an example of which would be achondroplasia, causing dwarfism. Finally, chondromas or chondrosarcomas, tumors that can either become or are cancer, can occur within this tissue.

### *5.4.1. Intervertebral disc*

CCN2 is induced by signaling from retinoids in cartilage in the growth plate [230]. CCN2 causes differentiation of the chondrocytes and is important for matrix deposition and signal‐ ing from cellular receptors [226,231]. Retinoids are important signaling molecules involved

The CCN proteins appear to play a critical role in the establishment of the skeletal system and have been shown to have positive and negative regulatory roles in skeletal development, as demonstrated through cell culture experiments and animal models. Collectively, these studies demonstrate that CCN proteins promote differentiation and proliferation of chondrocytes, osteoblasts, and vascular endothelial cells, which are important in both endochondral and

Cartilage is first seen in the developing embryo in the areas that serve as templates for endochondral ossification, as well as in Meckel's cartilage in the head area [1]. Both CCN1 and CCN2 promote chondrogenic and osteoblastic differentiation [232,233]. CCNs can interact with members of the TGF‐β and BMP family via the chordin‐like homology found in the VWC domain, and modulate their binding affinity for their respective receptors [234]. CCN2 is important during embryogenesis and bone formation, including during proliferation of mesenchymal cells, their differentiation into chondrocytes, and condensation of these cells into the cartilage model that will form bone [1,235]. CCN2 expression is highest in the vascular tissue and the maturing chondrocytes of the embryo. TGF‐β, which induces CCN2, directs the condensation of these cells and regulates secretion of other matricellular proteins such as fibronectin [235]. Cells from CCN2 knockout mice do not undergo this condensation process in vitro and have less synthesis of cartilage proteoglycans [236]. Neutralizing antibodies, siRNA, and antisense oligonucleotides have all been used to demonstrate that reduced or absence of CCN2 prevents condensation from occurring [1,235]. Overexpression of CCN2 causes an increase in the length and density of bones [237]. In vertebral bodies, downregulation of CCN2 is necessary for chondrocyte differentiation [235]. These studies demonstrate that the

spatial and temporal regulation of CCNs is an important component of development.

are important in the development of cartilage [3,4].

Development of cartilage involves several different transcription factors, such as Sox 9, that control this process, as well as growth factors that interact with cellular receptors to facilitate various cellular functions [2]. Interaction with the extracellular matrix has been implicated in development as well. Various epigenetic mechanisms and several different microRNAs also

BMPs are important during skeletogenesis [238] and are known to be regulated by CCN2 [234]. Expression of CCN2 is observed in both the perichondrium and the chondrocytes during development [239]. BMPs play a role in the repair of cartilage as well as during development [240]. BMP2 causes proliferation of chondrocytes during development, while BMP4 does not [241]. CCN2 and CCN3 can bind BMP2 and abrogate its ability to promote chondrogenic and

in the cartilage becoming hypertrophic during endochondral ossification.

**5.3. CCNs in chondrogenesis and osteogenesis**

178 Composition and Function of the Extracellular Matrix in the Human Body

intramembranous ossification.

CCN2 is highly expressed in the cartilage present in the intervertebral disc, just as it is in other types of cartilage. The intervertebral disc contains the nucleus pulposus, and this is surrounded by the annulus fibrosus composed of fibrocartilage. The annulus fibrosus contains mainly the fibrillar collagen type I, although type II collagen is present along the inner surface as in reference [249]. Proper function of the annulus fibrosus is important in disc function, and lack of intact matrix can lead to disc instability [249]. Herniation of the nucleus pulposus is sometimes implicated as a cause of back pain [11] and increased expression of CCN2 can be correlated with discs that are painful [250].

#### *5.4.2. Osteoarthritis and cartilage wound healing*

Osteoarthritis is the most common form of degenerative joint disease [7]. During the disease process, chondrocytes undergo cell death due to lack of ability to renew, particularly with aging [251]. The initial degenerative changes that occur are termed chondropenia, although this is not often identified as osteoarthritis [252]. Upregulation of the catabolic pathways occurs, causing cartilage degradation, and alteration of the phenotype of the chondrocytes is ultimately responsible for loss of articular cartilage [253]. Much of this change in cartilage structure and function occurs via interactions with matrix and signaling from both matrix and growth factors [254,255]. Initial disease presents with an upregulation of collagen type II synthesis, and a change in collagen type from type II to types I and III [40,256]. A decrease in aggrecan is observed during the pathogenesis of osteoarthritis, while other proteoglycans such as decorin and fibromodulin are increased [257]. Aging causes decreased matrix deposition subsequent to increases in degradation by MMPs as well, causing additional issues with tissue integrity [255]. MMPs and aggrecanase are upregulated during osteoarthritis disease progres‐ sion [258] causing additional cartilage loss. These processes are very similar to what occurs following an injury to cartilage and its subsequent repair [259].

CCN2 is increased in chondrocytes isolated from human osteoarthritis cartilage [260–262]. Increases in this matricellular protein can lead to fibrosis, and it is possible that mechanisms used to deliver CCN2 to degenerating discs may cause some repair to occur [249] similar to what is seen in an experimental model of osteoarthritis [263]. CCN3 has been shown to be upregulated in an osteoarthritis mouse model [264] and in human osteoarthritis as well [265]. Mutation of CCN3 in mice causes a loss of normal joint function and disease that appears similar to osteoarthritis in humans [266].

#### *5.4.3. Chondromas/chondrosarcomas*

A chondroma is a benign tumor of cartilage that may be present within bone and can cause fractures due to its growth pattern. Normally, little is done with these tumors unless there is a danger of fracture. Chondrosarcomas are cancerous tumors that can occur at any age, unlike other sarcomas. These are refractory to treatment and can be highly metastatic. The expression of CCNs can be correlated with chondrosarcoma grades and thus may be useful in clinical identification of these tumors [267]. The same may be true for chondromas as well.

#### *5.4.4. Osteosarcomas*

Osteosarcoma is the most common type of cancer that develops in bone, occurring most frequently in children and young adults. Most primary tumors develop in the areas of bone that are growing rapidly such as near the ends of long bones surrounding the knee—the distal femur or the proximal tibia. The proximal humerous is the next most common site, although osteosarcoma can develop in any bone. Evidence suggests that osteosarcoma might originate from mesenchymal cells with osteoblastic features [268,269]. CCN1 expression correlates with poor prognosis of osteosarcoma and overexpression of CCN1 increases cell proliferation and metastatic potential of tumor cell lines [270] and CCN1 knockdown reverses this phenomenon [271]. In humans, CCN3 is associated with increased lung metastasis in osteosarcoma patients [272] and CCN4 expression was shown to be higher in bone from osteosarcoma patients compared to normal tissue [273].

#### *5.4.5. Ewing sarcoma*

aging [251]. The initial degenerative changes that occur are termed chondropenia, although this is not often identified as osteoarthritis [252]. Upregulation of the catabolic pathways occurs, causing cartilage degradation, and alteration of the phenotype of the chondrocytes is ultimately responsible for loss of articular cartilage [253]. Much of this change in cartilage structure and function occurs via interactions with matrix and signaling from both matrix and growth factors [254,255]. Initial disease presents with an upregulation of collagen type II synthesis, and a change in collagen type from type II to types I and III [40,256]. A decrease in aggrecan is observed during the pathogenesis of osteoarthritis, while other proteoglycans such as decorin and fibromodulin are increased [257]. Aging causes decreased matrix deposition subsequent to increases in degradation by MMPs as well, causing additional issues with tissue integrity [255]. MMPs and aggrecanase are upregulated during osteoarthritis disease progres‐ sion [258] causing additional cartilage loss. These processes are very similar to what occurs

CCN2 is increased in chondrocytes isolated from human osteoarthritis cartilage [260–262]. Increases in this matricellular protein can lead to fibrosis, and it is possible that mechanisms used to deliver CCN2 to degenerating discs may cause some repair to occur [249] similar to what is seen in an experimental model of osteoarthritis [263]. CCN3 has been shown to be upregulated in an osteoarthritis mouse model [264] and in human osteoarthritis as well [265]. Mutation of CCN3 in mice causes a loss of normal joint function and disease that appears

A chondroma is a benign tumor of cartilage that may be present within bone and can cause fractures due to its growth pattern. Normally, little is done with these tumors unless there is a danger of fracture. Chondrosarcomas are cancerous tumors that can occur at any age, unlike other sarcomas. These are refractory to treatment and can be highly metastatic. The expression of CCNs can be correlated with chondrosarcoma grades and thus may be useful in clinical

Osteosarcoma is the most common type of cancer that develops in bone, occurring most frequently in children and young adults. Most primary tumors develop in the areas of bone that are growing rapidly such as near the ends of long bones surrounding the knee—the distal femur or the proximal tibia. The proximal humerous is the next most common site, although osteosarcoma can develop in any bone. Evidence suggests that osteosarcoma might originate from mesenchymal cells with osteoblastic features [268,269]. CCN1 expression correlates with poor prognosis of osteosarcoma and overexpression of CCN1 increases cell proliferation and metastatic potential of tumor cell lines [270] and CCN1 knockdown reverses this phenomenon [271]. In humans, CCN3 is associated with increased lung metastasis in osteosarcoma patients [272] and CCN4 expression was shown to be higher in bone from osteosarcoma patients

identification of these tumors [267]. The same may be true for chondromas as well.

following an injury to cartilage and its subsequent repair [259].

180 Composition and Function of the Extracellular Matrix in the Human Body

similar to osteoarthritis in humans [266].

*5.4.3. Chondromas/chondrosarcomas*

compared to normal tissue [273].

*5.4.4. Osteosarcomas*

Ewing sarcoma is the second most common type of bone cancer that predominantly affects children. CCN3 is expressed in approximately 30% of Ewing Sarcoma cases and its expression correlates with lower survival [272]. Elevated CCN3 expression also correlated with recur‐ rences and metastases compared to primary tumors in a study that examined 170 human Ewing sarcoma specimens by immunohistochemistry. In this same study, a low level of CCN3 expression was associated with improved patient prognosis [274].

#### *5.4.6. Chondrodysplasias*

Mutations in the major collagen present in hyaline and elastic cartilage, type II, cause several chondrodysplasias [81]. Mutation of type XI collagen also causes skeletal issues due to issues with the structure of the growth plate, in mice with this defect [275]. Mice lacking collagen IX appear normal but have alterations in the cartilage forming the growth plate and ultimately these mice develop osteoarthritis when older [276]. Mutations cause skeletal issues in humans as well, as do mutations in type X collagen. Mice lacking collagen X have several phenotypes, including no obvious changes, metaphyseal dysplasia, and death soon after birth [81,83,277].

#### *5.4.7. CCNs in cartilage and bone pathophysiology*

The role of CCNs in cartilage function involves several family members of this class of matricellular proteins. CCN1‐6 have all been identified in cartilage matrix, and all CCN genes have an increase in their expression in osteoarthritis or rheumatoid arthritis [265]. CCN3 is integral to the proliferation of chondrocytes, while CCN1, CCN2, and CCN6 are involved in later states of maturation, proliferation, and the calcification of cartilage matrix during endochondral ossification. CCN4 and CCN5 also participate in the differentiation and calcification of cartilage [231]. CCN2 causes proper bone strength, shape, and length, while the counteraction by CCN3 regulates these structural processes.

CCN1 knockout mice die during development, making analysis of their skeleton difficult; however, this protein causes proliferation of chondrocytes and secretion of matrix in vitro [278]. CCN1 is present in chondrocytes in the proliferative and pre‐hypertrophic zones during endochondral bone formation [278]. CCN1 signals through the WNT signaling pathway to cause maturation of chondrocytes isolated from sternal cartilage, with overexpression of CCN1 causing damage to chondrocytes [279]. Induction of CCN1 by β‐catenin causes maturation of chondrocytes, while overexpression using a cartilage‐specific promoter causes chondrodys‐ plasia [279]. CCN1 is decreased in expression during the differentiation of mesenchymal stem cells into chondrocytes and osteoblasts, indicating that it may be important in the maintenance of stem cells [280].

CCN2 promotes differentiation and proliferation of chondrocytes as well as osteoblasts (see reference [47]). The presence of CCN2 is an important modulator of the deposition of cartilage matrix. CCN2 also is a major factor in the induction of fibrosis [281]. This growth factor causes secretion of collagen type II and can induce cells to proliferate and subsequently differentiate [282]. Lack of CCN2 causes issues with bone growth due to lack of hypertrophic zone cartilage growth and loss of angiogenesis [283,284] and causes death immediately after birth due to respiratory failure [239]. CCN2 binds several growth factors, including bone morphogenetic proteins and TGF‐β, and can affect cartilage function in this manner [226].

Addition of CCN2 to damaged cartilage in rats can cause enhanced healing [263]. Although exogenous administration of matrix metalloproteases can cause damage to cartilage similar to osteoarthritis [285,286], the use of these inhibitors does not aid in the treatment of the disease [287]. Overexpression of CCN2 using a col2a1 promoter caused reversal of some aging‐related changes present in the articular cartilage of aged mice by enhancing matrix deposition and proliferation of chondrocytes. In addition, changes characteristic of cartilage degeneration were reversed, such as the expression of collagens I or X and the presence of MMPs 9 and 13 [288]. These mice also showed greater levels of matrix and faster ossification during endo‐ chondral bone growth [237].

CCN2 binds to receptor activator of NF‐kappa B (RANK) as seen by plasmon resonance analysis, and enhances RANK signaling. This indicates its importance in the formation of osteoclasts [289].

Fibroblast growth factor causes an increase in BMPs, and this binding causes repair of articular cartilage defects. Different BMPs have differing effects on this repair, but overall BMP3 is an important component during repair of articular cartilage [279].

CCN3 decreases proliferation in several different cell types [290]. CCN3 appears to be downregulated by PTHrP [231], which is involved in the growth of bone. CCN3 may regulate apoptosis under conditions of serum deprivation. The presence of this CCN decreased levels of both proteoglycan and collagen, which may mimic the cartilage environment in which no vascularization occurs and thus conditions are somewhat hypoxic [291]. Bone regeneration in mice that lack CCN3 is enhanced [292]. Loss of CCN2 causes an increase in the expression of CCN3 with a concomitant decrease in proliferation due to the presence of this matricellular protein. This deletion also caused reduced differentiation of chondrocytes due to the upregu‐ lation of CCN3 [231]. Loss of CCN2 causes reductions in aggrecan and types II and X collagen during development, a process which mimics the matrix loss that occurs with aging during osteoarthritis development [226]. Overexpression of CCN2 has effects that counter these reductions [288]. Overexpression of CCN4 caused an effect on cartilage differentiation by changing the function of another growth factor, TGF‐β3. Mice that completely lacked CCN4 did repair surgical defects well, while mice that expressed CCN4 demonstrated some recovery from this injury [293]. Lack of another member of the CCN family, CCN6, causes a disease in humans that is a form of childhood arthritis, progressive pseudorheumatoid dysplasia [294]. The function of CCN6 in normal cartilage is not well understood, although its expression is high in osteoarthritis [295].

CCN1 and CCN2 expression is elevated during fracture repair in the long bones throughout the reparative phase of the callus, notably in proliferating chondrocytes and osteoblasts [296,297]. Abrogation of CCN1 by antibodies inhibits bone fracture healing in mice [298]. Further, recombinant CCN2 protein promotes the repair of articular cartilage in a rat osteoar‐ thritis model [263]. These studies suggest that CCN proteins may play important roles in the homeostasis of bone and cartilage tissues.
