Shirine E. Usmani

Rho/ROCK and MEK/ERK activation by transforming growth factor- α induces articular cartilage degradation

Identification and characterization of therapeutic targets for joint conditions, such as osteoarthritis (OA), is exceedingly important for addressing the increasing burden of disease. Transforming growth factor-α (TGFα) is upregulated by articular chondrocytes in experimentally induced and human OA. To test the potential involvement of TGFα, which is an activator of epidermal growth factor receptor (EGFR) signaling, in joint degeneration and to identify signaling mechanisms mediating articular chondrocyte responses to TGFα, rat chondrocytes and osteochondral explants were treated with TGFα and various inhibitors of intracellular signaling pathways. Stimulation of EGFR signaling in articular chondrocytes by TGFα resulted in the activation of RhoA/ROCK (Rho kinase), MEK (MAPK/ERK kinase)/ERK (extracellular-signal-regulated kinase), PI3K (phosphoinositide 3-kinase) and p38 MAPK (mitogen-activated protein kinase) pathways. Modification of the chondrocyte actin cytoskeleton was stimulated by TGFα, but inhibition of only Rho or ROCK activation prevented morphological changes. TGFα suppressed expression of anabolic genes including Sox9, type II collagen and aggrecan, which were rescued only by inhibiting MEK/ERK activation. Furthermore, catabolic factor upregulation by TGFα was prevented by ROCK and p38 MAPK inhibition, including matrix metalloproteinase-13 and tumor necrosis factor-α, which are well known to contribute to cartilage digestion in OA. To assess the ability of TGFα to stimulate degradation of mature articular cartilage, type II collagen and aggrecan cleavage fragments were analyzed in rat osteochondral explants exposed to exogenous TGFα. Normal articular cartilage contained low levels of both cleavage fragments, but high levels were observed in the cartilage treated with TGFα. Selective inhibition of MEK/ERK and Rho/ROCK activation greatly reduced or completely prevented excess type II collagen and aggrecan degradation in response to TGFα. These data suggest that TGFα is a strong stimulator of cartilage degradation and that Rho/ROCK and MEK/ERK signaling have critical roles in mediating these effects.

Transforming growth factor alpha controls the transition from hypertrophic cartilage to bone during endochondral bone growth

UNLABELLED We have recently identified transforming growth factor alpha (TGFα) as a novel growth factor involved in the joint disease osteoarthritis. The role of TGFα in normal cartilage and bone physiology however, has not been well defined. PURPOSE The objective of this study was to determine the role of TGFα in bone development through investigation of the Tgfa knockout mouse. METHODS The gross skeletons as well as the cartilage growth plates of Tgfa knockout mice and their control littermates were examined during several developmental stages ranging from newborn to ten weeks old. RESULTS Knockout mice experienced skeletal growth retardation and expansion of the hypertrophic zone of the growth plate. These phenotypes were transient and spontaneously resolved by ten weeks of age. Tgfa knockout growth plates also had fewer osteoclasts along the cartilage/bone interface. Furthermore, knockout mice expressed less RUNX2, RANKL, and MMP13 mRNA in their cartilage growth plates than controls did. CONCLUSIONS Tgfa knockout mice experience a delay in bone development, specifically the conversion of hypertrophic cartilage to true bone. The persistence of the hypertrophic zone of the growth plate appears to be mediated by a decrease in MMP13 and RANKL expression in hypertrophic chondrocytes and a resulting reduction in osteoclast recruitment. Overall, TGFα appears to be an important growth factor regulating the conversion of cartilage to bone during the process of endochondral ossification.

Reduction in disease progression by inhibition of transforming growth factor α-CCL2 signaling in experimental posttraumatic osteoarthritis.

Transforming growth factor α (TGFα) is increased in osteoarthritic (OA) cartilage in rats and humans and modifies chondrocyte phenotype. CCL2 is increased in OA cartilage and stimulates proteoglycan loss. This study was undertaken to test whether TGFα and CCL2 cooperate to promote cartilage degradation and whether inhibiting either reduces disease progression in a rat model of posttraumatic OA.

Transforming growth factor-alpha induces endothelin receptor A expression in osteoarthritis.

Previously, our lab identified transforming growth factor‐alpha (TGFα) as a novel factor involved in osteoarthritis (OA) in a surgical model of the disease. In the same study, we also observed increased transcript levels for endothelin receptor A (ET(A)R), a known contributor to cartilage pathology. To investigate the connection between TGFα and endothelin signaling in OA, primary articular chondrocytes and osteochondral explants were isolated from Sprague–Dawley rats and treated with vehicle or TGFα. Expression of ET(A)R protein and its encoding gene Ednra was assessed. Chondrocytes and cartilage explants were also treated with the endothelin receptor A/B antagonist Bosentan, in order to determine whether TGFα effects could be blocked. TGFα induced expression of ET(A)R protein and its encoding gene Ednra. In primary chondrocyte cultures, Bosentan did not block TGFα responses of the anabolic genes Sox9, Agc1, and Col2a1, but reduced the induction of Mmp13 and Ednra transcripts by TGFα. In osteochondral explants, the inhibitor partially blocked TGFα reduction of type II collagen, as well as induction of MMP‐13 and type II collagen neoepitopes. TGFα induces ET(A)R expression in articular chondrocytes and receptor antagonism appears to block some TGFα‐induced catabolic effects in a three‐dimensional organ culture system. Thus, TGFα may be a therapeutic target upstream of ET(A)R in OA.

Context-specific protection of TGFα null mice from osteoarthritis.

Transforming growth factor alpha (TGFα) is a growth factor involved in osteoarthritis (OA). TGFα induces an OA-like phenotype in articular chondrocytes, by inhibiting matrix synthesis and promoting catabolic factor expression. To better understand TGFα’s potential as a therapeutic target, we employed two in vivo OA models: (1) post-traumatic and (2) aging related OA. Ten-week old and six-month old male Tgfa null mice and their heterozygous (control) littermates underwent destabilization of the medial meniscus (DMM) surgery. Disease progression was assessed histologically using the Osteoarthritis Research Society International (OARSI) scoring system. As well, spontaneous disease progression was analyzed in eighteen-month-old Tgfa null and heterozygous mice. Ten-week old Tgfa null mice were protected from OA progression at both seven and fourteen weeks post-surgery. No protection was seen however in six-month old null mice after DMM surgery, and no differences were observed between genotypes in the aging model. Thus, young Tgfa null mice are protected from OA progression in the DMM model, while older mice are not. In addition, Tgfa null mice are equally susceptible to spontaneous OA development during aging. Thus, TGFα might be a valuable therapeutic target in some post-traumatic forms of OA, however its role in idiopathic disease is less clear.

CXC chemokine ligand 12a enhances chondrocyte proliferation and maturation during endochondral bone formation

OBJECTIVE We investigated the roles of CXC chemokine ligand 12a (CXCL12a), also known as stromal cell-derived factor-1α (SDF-1α), in endochondral bone growth, which can give us important clues to understand the role of CXCL12a in osteoarthritis (OA). METHODS Primary chondrocytes and tibial explants from embryonic 15.5 day-old mice were cultured with recombinant mouse CXCL12a. To assess the role of CXCL12a in chondrogenic differentiation, we conducted mesenchymal cell micromass culture. RESULTS In tibia organ cultures, CXCL12a increased total bone length in a dose-dependent manner through proportional effects on cartilage and bone. In accordance with increased length, CXCL12a increased the protein level of proliferation markers, such as cyclin D1 and proliferating cell nuclear antigen (PCNA), in primary chondrocytes as well as in tibia organ culture. In addition, CXCL12a increased the expression of Runx2, Col10 and MMP13 in primary chondrocytes and tibia organ culture system, implying a role of CXCL12a in chondrocyte maturation. Micromass cultures of limb-bud mesenchymal progenitor cells (MPCs) revealed that CXCL12a has a limited effect on early chondrogenesis, but significantly promoted maturation of chondrocytes. CXCL12a induced the phosphorylation of p38 and Erk1/2 MAP kinases and IκB. The increased expression of cyclin D1 by CXCL12a was significantly attenuated by inhibitors of MEK1 and NF-κB. On the other hand, p38 and Erk1/2 MAP kinase and NF-κB signaling were associated with CXCL12a-induced expression of Runx2 and MMP13, the marker of chondrocyte maturation. CONCLUSION CXCL12a promoted the proliferation and maturation of chondrocytes, which strongly suggest that CXCL12a may have a negative effect on articular cartilage and contribute to OA progression.

215 TRANSFORMING GROWTH FACTOR ALPHA AND ENDOTHELIN RECEPTOR A SIGNALING IN OSTEOARTHRITIS

gene expression in response to manipulating the actin cytoskeleton in chondrocytes. Methods: Chondrocytes isolated from embryonic mice growth plates were plated in high density monolayer cultures and treated for 24 hours with vehicle, 10mM Y27632 (inhibits Rho/ROCK signaling), 1mM cytochalasin D (inhibits actin polymerization) or 50 nM jasplakinolide (promotes actin polymerization). RNA was isolated, and then hybridized to Affymetrix MOE4.0 chips. Bioinformatic analysis (MAS5.0) was performed and probe sets demonstrating a significant, at least 1.5-fold change and reliable signal were considered for further analysis. These data were compared to our previous microarray analyses of chondrocyte differentiation in vitro and in vivo (growth plate zones of microdissected tibia, time course of micromass cultures differentiating from mesenchymal cells to hypertrophy) we have previously analyzed. Results: Inhibition of actin polymerization by cytochalasin D resulted in the most compelling data in comparison to other actin manipulations. We observed that treating growth plates with cytochalasin D resulted in very large bones consisting entirely of cells with hypertrophic morphology. Analysis of the gene set revealed that most genes that were upregulated were similarly regulated in other models of chondrocyte differentiation to hypertrophy, suggesting that cytochalasin D stimulates chondrocyte hypertrophy, a novel finding. We continued our studies by comparing gene sets with other models of chondrocyte hypertrophy and identified the nuclear receptor, Rora as a common gene upregulated in all 3 models studied. We found that Rora is most highly expressed in the hypertrophic portion of the growth plate and is expressed throughout growth plates of cytochalasin D treated bones. Additionally, multiple targets of Rora are also increased in chondrocytes treated with cytochalasin D, such as fatty acid binding protein 4, lipoprotein lipase and CD36 (a receptor previously implicated in osteoarthritis). We also show that stimulation of Rora by its ligand cholesterol results in a phenotype similar to chondrocyte hypertrophy. These data suggest that cholesterol signaling through Rora contributes to chondrocyte hypertrophy. Interestingly, because it has been shown in other cell types that Rora expression is regulated by Hif1a, we assessed and confirmed increased levels of Hif1a in the hypertrophic region of the growth plate and throughout the growth plates of bones treated with cytochalasin D. Similarly, Rora and Hif1a expression was upregulated in chondrocytes with cartilagespecific deletion of the Rac1 gene. Conclusions: Identification of regulators of chondrocyte hypertrophy will be essential in preventing pathological differentiation of articular chondrocytes in osteoarthritis. Our data implicate the nuclear receptor Rora and its activator cholesterol in stimulating chondrocyte hypertrophy. These data have far reaching implications for preventing and possibly minimalizing the severity of osteoarthritis.

MOLECULAR CONTROL OF ARTICULAR CARTILAGE DEGENERATION BY TRANSFORMING GROWTH FACTOR ALPHA

Background: Articular cartilage degeneration is a hallmark of osteoarthritis (OA). We previously identified increased expression of transforming growth factor alpha (TGF?) and chemokine (C-C motif) ligand 2 (CCL2) in articular cartilage from a rat modelof OA (1,2). We subsequently reported that TGF? signalling modified chondrocyte cytoskeletal organization, increased catabolic and decreased anabolic gene expression and suppressed Sox9. Due to other roles in chondrocytes, we hypothesized that the effects ofTGF? on chondrocytes are mediated by Rho/ROCK and MEK/ERK signaling pathways. Methods: Primary cultures of chondrocytes and articularosteochondral explants were treated with pharmacological inhibitors of MEK1/2(U0126), ROCK (Y27632), Rho (C3), p38 MAPK (SB202190) and PI3K (LY294002) to elucidate pathway involvement. Results: Using G-LISA we determined that stimulation of primary chondrocytes with TGF? activates RhoA. Reciprocally, inhibition of RhoA/ROCK but not other signalling pathways prevents modification of the actin cytoskeleton in responseto TGF?. Inhibition of MEK/ERKsignaling rescued suppression of anabolic gene expression by TGF? including SOX9 mRNA and protein levels. Inhibition of MEK/ERK, Rho/ROCK, p38 MAPK and PI3K signalling pathways differentially controlled the induction of MMP13 and TNF? gene expression. TGF? also induced expression of CCL2 specifically through MEK/ERK activation. In turn, CCL2 treatment induced the expression of MMP3 and TNF?. Finally, we assessed cartilage degradation by immunohistochemical detection of type II collagen cleavage fragments generated by MMPs. Blockade of RhoA/ROCK and MEK/ERK signalling pathways reduced the generation of type IIcollagen cleavage fragments in response to TGF? stimulation. Conclusions: Rho/ROCK signalling mediates TGF?-induced changes inchondrocyte morphology, while MEK/ERK signalling mediates the suppression ofSox9 and its target genes, and CCL2 expression. CCL2, in turn, induces the expression of MMP3 and TNF?, two potent catabolic factors known to be involved in OA. These pathways may represent strategic targets for interventional approaches to treating cartilage degeneration in osteoarthritis. References: 1. Appleton CTG et al. Arthritis Rheum 2007;56:1854-68. 2. Appleton CTG et al. Arthritis Rheum 2007; 56:3693-705.

Global gene expression analyses in early experimental osteoarthritis reveal novel players in articular cartilage degenerations

Introduction: Articular cartilage degeneration is a hallmark of osteoarthritis (OA). We sought to identify dysregulated genes in degenerating cartilage and hypothesized that altered growth factor expression causes cartilage degradation in OA. Methods: Genome-wide microarray analysis of RNA harvested directly from sham (control) and degenerating articular cartilage was performed using our previously characterized pre-clinical rat model of knee OA1. Known OA genes were validated using RNA samples (real-time PCR) and histological sections (immunofluorescence) from independent animals. Functional studies in chondrocytes and articular cartilage explants investigated the effects of one identified factor (transforming growth factor alpha (TGF-α)) on cartilage degeneration. The effects of TGF-α on primary chondrocyte morphology, proliferation, gene expression, and SOX9 transcription factor expression were assessed. Results: Dysregulated gene expression profiles in degenerating cartilage included known OA genes2. Microarray expression profiles were consistently validated at RNA and protein levels by alternative methods. Several genes previously unstudied in OA cartilage were upregulated, including growth factors (e.g. TGF-α and kit ligand), cell surface receptors (e.g. endothelin type A receptor), and proteases (e.g. cathepsin S). Functional studies demonstrated that TGF-α alters chondrocyte morphology through re-organization of the actin cytoskeleton. TGF-α also stimulated primary chondrocyte proliferation and chondrocyte cluster formation in cartilage explants. Chondrocyte expression of anabolic genes and total collagen protein levels were reduced by TGF-α, while expression of catabolic factors increased. Finally, TGF-α reduced both the expression of total SOX9 and levels of phosphorylated (active) SOX9. Conclusions: Our microarray study identified numerous factors previously unstudied in OA cartilage, including increased levels of TGF-α. Functional studies determined that TGF-α promotes cartilage degeneration through chondrocyte proliferation and catabolic factor expression. Further, TGF-α inhibits chondrocyte anabolism, likely through a mechanism involving suppression of SOX9. References: 1C.T.G. Appleton et al. (2007). Arthritis Res Ther 9(1):R13. 2C.T.G. Appleton et al. (2007), Arthritis Rheum In Press.