Dickkopf-1 directs periosteal bone formation in two murine models of inflammatory arthritis.

OBJECTIVE: The Wnt signalling antagonist Dickkopf-1 (DKK1) inhibits osteoblast differentiation and function and has been described to play a central role in promoting bone loss, while blockade of DKK1 increases bone formation. We investigated the effects of DKK1 on periosteal new bone formation in two murine models of inflammatory arthritis, the antigen-induced arthritis (AIA) and K/BxN serum transfer arthritis (STA) models. METHOD: The flare variant of AIA was induced in wild-type mice and a blocking antibody to DKK1, control rat immunoglobulin G (IgG), or phosphate-buffered saline (PBS) was administered starting on day 14, a time at which inflammation and erosions are known to be established. Knees were assessed for histological inflammation and periosteal new bone formation was quantitated. In addition, STA was generated in transgenic (Tg) mice with osteoblast-specific overexpression of Dkk1 and littermate controls. New bone formation around the wrists of these mice was quantified by micro-computed tomography. RESULTS: Blockade of DKK1 in arthritic mice resulted in significantly more periosteal new bone formation compared to mice treated with control rat IgG or PBS. Conversely, in the setting of increased Dkk1 expression, arthritic Dkk1 Tg mice developed significantly less periosteal new bone than arthritic controls. CONCLUSION: DKK1 is a regulator of periosteal bone formation in inflammatory arthritis. Thus, regulation of DKK1 may be considered as a therapeutic approach in inflammatory diseases in which patients suffer from excessive periosteal bone formation, such as spondyloarthritis.

Healing of erosions in rheumatoid arthritis remains elusive: results with 24 months of the anabolic agent teriparatide.

Objective: Erosion healing in rheumatoid arthritis (RA) is difficult to demonstrate. This extension study aimed to determine whether 2 years of teriparatide (TPTD) produces erosion healing. Method: Subjects in a previous 12 month randomized controlled trial of TPTD in RA were invited to receive 12 additional months of open-label TPTD. Eleven of the 24 original subjects were enrolled in the extension study, six of whom received TPTD in the final 12 months only. Subjects receiving 24 months of TPTD were assessed for reduction in erosion volume from baseline using computed tomography. We also compared erosion volumes between 12 and 24 months of TPTD. Large erosions in subjects receiving TPTD for 24 months were examined for volume change. Results: In the six patients who received 24 months of TPTD, there was no significant change in erosion volume at the metacarpophalangeal (MCP) and proximal interphalangeal (PIP) joints compared with baseline. The six subjects who received 24 months of TPTD had similar changes in erosion volume to the five who received 12 months of TPTD, in MCP (p = 0.17) and PIP (p = 0.63) joints. Assessment of large erosions in those receiving TPTD for 24 months showed no evidence of erosion healing. Conclusion: While this extension study was too small to be conclusive, we observed no evidence of reduction in erosion volume with the addition of TPTD for 24 months in subjects with RA in whom disease activity was controlled on a tumour necrosis factor inhibitor. This is consistent with our negative findings at 12 months.

Effects of Teriparatide on Joint Erosions in Rheumatoid Arthritis: A Randomized Controlled Trial.

OBJECTIVE: Articular erosions correlate with disability in rheumatoid arthritis (RA). Biologic agents reduce erosion progression in RA, but erosion healing occurs infrequently. This study was undertaken to assess the effects of the anabolic agent teriparatide on joint erosion volume in RA patients treated with a tumor necrosis factor inhibitor (TNFi). METHODS: We conducted a randomized controlled trial in 24 patients with erosive RA, osteopenia, and disease activity controlled by TNFi treatment for at least 3 months. Half were randomized to receive teriparatide for 1 year and the others constituted a wait-list control group. Subjects and primary rheumatologists were not blinded with regard to treatment assignment, but all outcomes were assessed in a blinded manner. The primary outcome measure was change in erosion volume determined by computed tomography at 6 anatomic sites. Significance within each hand and anatomic site was based on a 2-tailed test, with P values less than 0.05 considered significant. RESULTS: Baseline characteristics of the treatment groups were well balanced. After 52 weeks, the median change in erosion volume in the teriparatide group was -0.4 mm3 (interquartile range [IQR] -34.5, 29.6) and did not differ significantly from that in controls (median change +9.1 mm3 [IQR -29.6, 26.4]) (P = 0.28). No significant difference in change in erosion volume was noted at the radius, ulna, or metacarpophalangeal joints. Bone mineral density improved at the femoral neck and lumbar spine in the teriparatide group. CONCLUSION: Our findings indicate that teriparatide treatment for 1 year does not significantly reduce erosion volume in the hands or wrists of patients with established RA with disease activity controlled by TNFi treatment.

Inflammation in arthritis induces expression of BMP3, an inhibitor of bone formation.

OBJECTIVES: Inflammation in diseases such as rheumatoid arthritis (RA) stimulates osteoclast-mediated articular bone erosion and inhibits osteoblast-mediated bone formation, leading to a net loss of bone. Pro-inflammatory cytokines and antagonists of the Wnt signalling pathway have been implicated in the inhibition of osteoblast differentiation and activity in RA, contributing to the erosive process and impairing erosion healing. Importantly, osteoblast differentiation and function are also regulated by the osteogenic bone morphogenetic protein (BMP) signalling pathway, which is antagonized by BMP3. We therefore examined the potential role of BMP3 in inflammatory arthritis. METHOD: Two murine models of RA, K/BxN serum transfer arthritis (STA) and antigen-induced arthritis (AIA), were used to establish the temporal expression of BMP3 and the cellular sources of BMP3 mRNA and protein in inflammatory arthritis. To determine the effects of inflammation on the expression of BMP3 in osteoblasts, murine calvarial osteoblasts were treated with pro-inflammatory cytokines and BMP3 expression was assessed. RESULTS: In both murine models of RA, BMP3 mRNA and protein are highly expressed by osteoblasts lining inflammation-bone interfaces late in the course of arthritis. Synovial tissues are not a significant source of BMP3. BMP3 expression is induced in osteocalcin-expressing osteoblasts in vitro following stimulation by tumour necrosis factor (TNF). CONCLUSIONS: These data implicate BMP3 as a novel factor that may act locally to contribute to the erosive process and inhibit the repair of articular bone in RA through inhibition of osteoblast differentiation and function.

Role of vascular channels as a novel mechanism for subchondral bone damage at cruciate ligament entheses in osteoarthritis and inflammatory arthritis.

OBJECTIVES: The purpose of this work was to test whether normal peri-entheseal vascular anatomy at anterior and posterior cruciate ligaments (ACL and PCL) was associated with distribution of peri-entheseal bone erosion/bone marrow lesions (BMLs) in inflammatory arthritis (IA) and osteoarthritis (OA). METHODS: Normal microanatomy was defined histologically in mice and by 3 T MRI and histology in 21 cadaveric knees. MRI of 89 patients from the Osteoarthritis Initiative and 27 patients with IA was evaluated for BMLs at ACL and PCL entheses. Antigen-induced arthritis (AIA) in mice was evaluated to ascertain whether putative peri-entheseal vascular regions influenced osteitis and bone erosion. RESULTS: Vascular channels penetrating cortical bone were identified in knees of non-arthritic mice adjacent to the cruciate ligaments. On MRI of normal cadavers, vascular channels adjacent to the ACL (64% of cases) and PCL (71%) entheses were observed. Histology of 10 macroscopically normal cadaveric specimens confirmed the location of vascular channels and associated subclinical changes including subchondral bone damage (80% of cases) and micro-cyst formation (50%). In the AIA model, vascular channels clearly provided a site for inflammatory tissue entry and osteoclast activation. MRI showed BMLs in the same topographic locations in both patients with early OA (41% ACL, 59% PCL) and IA (44%, 33%). CONCLUSION: The findings show that normal ACL and PCL entheses have immediately adjacent vascular channels which are common sites of subtle bone marrow pathology in non-arthritic joints. These channels appear to be key determinants in bone damage in inflammatory and degenerative arthritis.

Activated human T cells express alternative mRNA transcripts encoding a secreted form of RANKL.

Receptor activator of nuclear factor-kappaB-ligand (RANKL), encoded by the gene TNFSF11, is required for osteoclastogenesis, and its expression is upregulated in pathologic bone loss. Transcript variants of TNFSF11 messenger RNA (mRNA) have been described that encode a membrane-bound and a putative secreted form of RANKL. We identify a TNFSF11 transcript variant that extends the originally identified transcript encoding secreted RANKL. We demonstrate that this TNFSF11 transcript variant is expressed by the human osteosarcoma cell line, Saos-2, and by both primary human T cells and Jurkat T cells. Of relevance to the production of RANKL in pathologic bone loss, expression of this secreted TNFSF11 transcript is upregulated in Jurkat T cells and primary human T cells upon activation. Furthermore, this transcript can be translated and secreted in Jurkat T cells in vitro and is able to support osteoclast differentiation. Our data highlight the complexity of the TNFSF11 genomic locus, and demonstrate the potential for the expression of alternate mRNA transcripts encoding membrane-bound and secreted forms of RANKL. Implications of alternate mRNA transcripts encoding different RANKL protein isoforms should be carefully considered and specifically examined in future studies, particularly those implicating RANKL in pathologic bone loss.

RANKL protein is expressed at the pannus-bone interface at sites of articular bone erosion in rheumatoid arthritis.

OBJECTIVES: Receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) have been demonstrated to be critical regulators of osteoclast generation and activity. In addition, RANKL has been implicated as an important mediator of bone erosion in rheumatoid arthritis (RA). However, the expression of RANKL and OPG at sites of pannus invasion into bone has not been examined. The present study was undertaken to further elucidate the contribution of this cytokine system to osteoclastogenesis and subsequent bone erosion in RA by examining the pattern of protein expression for RANKL, OPG and the receptor activator of NF-kappaB (RANK) in RA at sites of articular bone erosion. METHODS: Tissues from 20 surgical procedures from 17 patients with RA were collected as discarded materials. Six samples contained only synovium or tenosynovium remote from bone, four samples contained pannus-bone interface with adjacent synovium and 10 samples contained both synovium remote from bone and pannus-bone interface with adjacent synovium. Immunohistochemistry was used to characterize the cellular pattern of RANKL, RANK and OPG protein expression immediately adjacent to and remote from sites of bone erosion. RESULTS: Cellular expression of RANKL protein was relatively restricted in the bone microenvironment; staining was focal and confined largely to sites of osteoclast-mediated erosion at the pannus-bone interface and at sites of subchondral bone erosion. RANK-expressing osteoclast precursor cells were also present in these sites. OPG protein expression was observed in numerous cells in synovium remote from bone but was more limited at sites of bone erosion, especially in regions associated with RANKL expression. CONCLUSIONS: The pattern of RANKL and OPG expression and the presence of RANK-expressing osteoclast precursor cells at sites of bone erosion in RA contributes to the generation of a local microenvironment that favours osteoclast differentiation and activity. These data provide further evidence implicating RANKL in the pathogenesis of arthritis-induced joint destruction.

Angiopoietin-1 is expressed in the synovium of patients with rheumatoid arthritis and is induced by tumour necrosis factor alpha.

OBJECTIVES: To examine the potential role of the angiogenic growth factor angiopoietin-1 (Ang-1) in inflammatory arthritis. METHODS: Eighteen synovial tissue samples were obtained from 17 patients with a clinical diagnosis of rheumatoid arthritis (RA) and compared with six synovial tissue samples from six patients with osteoarthritis (OA). Ang-1 expression in synovial tissues was determined by immunohistochemistry and in situ hybridisation. Ang-1 mRNA and protein expression were also examined by northern blot analysis and enzyme linked immunosorbent assay (ELISA) in cultured synovial fibroblasts and human umbilical vein endothelial cells (HUVECs) before and after treatment with tumour necrosis factor (TNF)alpha. RESULTS: Ang-1 protein expression was detected by immunohistochemistry in 16/18 RA synovial tissue samples. Ang-1 protein was frequently observed in the synovial lining layer and in cells within the sublining synovial tissue, in both perivascular areas and in areas remote from vessels. In contrast, Ang-1 was only weakly detected in these sites in OA samples. Ang-1 mRNA and protein were also expressed in cultured synovial fibroblasts derived from patients with RA. In addition, induction of Ang-1 mRNA and protein was observed by northern blot analysis and ELISA after stimulation of RA synovial fibroblasts, but not HUVECs, with the proinflammatory cytokine TNF alpha. CONCLUSIONS: Ang-1 mRNA and protein are expressed in the synovium of patients with RA, and are up regulated in synovial fibroblasts by TNF alpha. Ang-1 may therefore be an important regulator of angiogenesis in inflammatory arthritis.

Bone destruction in arthritis.

Rheumatoid arthritis (RA) is characterised by the presence of an inflammatory synovitis accompanied by destruction of joint cartilage and bone. Destruction of cartilage matrix results predominantly from the action of connective tissue proteinases released by RA synovial tissues, chondrocytes, and pannus tissue. Several lines of evidence in RA and in animal models of arthritis support a role for osteoclasts in the pathogenesis of bone erosions. RA synovial tissues produce a variety of cytokines and growth factors that may increase osteoclast formation, activity, and/or survival. These include interleukin 1alpha (IL1alpha) and beta, tumour necrosis factor alpha (TNFalpha), IL11, IL17, and macrophage colony stimulating factor (M-CSF). Receptor activator of NFkappaB ligand (RANKL) is an essential factor for osteoclast differentiation and also functions to augment T cell-dendritic cell cooperative interactions. CD4+ T cells and synovial fibroblasts derived from RA synovium are sources of RANKL. Furthermore, in collagen induced arthritis (CIA), blockade with osteoprotegerin (OPG), a decoy receptor for RANKL, results in protection from bone destruction. To further evaluate the role of osteoclasts in focal bone erosion in arthritis, arthritis was generated in the RANKL knockout mouse using a serum transfer model. Despite ongoing inflammation, the degree of bone erosion in arthritic RANKL knockout mice, as assessed by microcomputed tomography and correlated histopathological analysis, was dramatically reduced compared with that seen in arthritic control mice. Cartilage damage was present in both the arthritic RANKL knockout mice and in arthritic control littermates, with a trend toward milder cartilage damage in the RANKL knockout mice. This study supports the hypothesis that osteoclasts play an important part in the pathogenesis of focal bone erosion in arthritis, and reveals distinct mechanisms of cartilage destruction and bone erosion in this animal model of arthritis. Future directions for research in this area include the further investigation of a possible direct role for the RANKL/RANK/OPG system in cartilage metabolism, and the possible role of other cell types and cytokines in bone erosion in arthritis.

TRANCE/RANKL knockout mice are protected from bone erosion in a serum transfer model of arthritis.

There is considerable evidence that osteoclasts are involved in the pathogenesis of focal bone erosion in rheumatoid arthritis. Tumor necrosis factor-related activation-induced cytokine, also known as receptor activator of nuclear factor-kappaB ligand (TRANCE/RANKL) is an essential factor for osteoclast differentiation. In addition to its role in osteoclast differentiation and activation, TRANCE/RANKL also functions to augment T-cell dendritic cell cooperative interactions. To further evaluate the role of osteoclasts in focal bone erosion in arthritis, we generated inflammatory arthritis in the TRANCE/RANKL knockout mouse using a serum transfer model that bypasses the requirement for T-cell activation. These animals exhibit an osteopetrotic phenotype characterized by the absence of osteoclasts. Inflammation, measured by clinical signs of arthritis and histopathological scoring, was comparable in wild-type and TRANCE/RANKL knockout mice. Microcomputed tomography and histopathological analysis demonstrated that the degree of bone erosion in TRANCE/RANKL knockout mice was dramatically reduced compared to that seen in control littermate mice. In contrast, cartilage erosion was present in both control littermate and TRANCE/RANKL knockout mice. These results confirm the central role of osteoclasts in the pathogenesis of bone erosion in arthritis and demonstrate distinct mechanisms of cartilage destruction and bone erosion in this animal model of arthritis.

Plasma cell differentiation requires the transcription factor XBP-1.

Considerable progress has been made in identifying the transcription factors involved in the early specification of the B-lymphocyte lineage. However, little is known about factors that control the transition of mature activated B cells to antibody-secreting plasma cells. Here we report that the transcription factor XBP-1 is required for the generation of plasma cells. XBP-1 transcripts were rapidly upregulated in vitro by stimuli that induce plasma-cell differentiation, and were found at high levels in plasma cells from rheumatoid synovium. When introduced into B-lineage cells, XBP-1 initiated plasma-cell differentiation. Mouse lymphoid chimaeras deficient in XBP-1 possessed normal numbers of activated B lymphocytes that proliferated, secreted cytokines and formed normal germinal centres. However, they secreted very little immunoglobulin of any isotype and failed to control infection with the B-cell-dependent polyoma virus, because plasma cells were markedly absent. XBP-1 is the only transcription factor known to be selectively and specifically required for the terminal differentiation of B lymphocytes to plasma cells.

The role of TNF-receptor family members and other TRAF-dependent receptors in bone resorption.

The contribution of osteoclasts to the process of bone loss in inflammatory arthritis has recently been demonstrated. Studies in osteoclast biology have led to the identification of factors responsible for the differentiation and activation of osteoclasts, the most important of which is the receptor activator of NF-kappa B ligand/osteoclast differentiation factor (RANKL/ODF), a tumor necrosis factor (TNF)-like protein. The RANKL/ODF receptor, receptor activator of NF-kappa B (RANK), is a TNF-receptor family member present on both osteoclast precursors and mature osteoclasts. Like other TNF-family receptors and the IL-1 receptor, RANK mediates its signal transduction via TNF receptor-associated factor (TRAF) proteins, suggesting that the signaling pathways activated by RANK and other inflammatory cytokines involved in osteoclast differentiation and activation are interconnected.

Mechanisms of bone loss in inflammatory arthritis: diagnosis and therapeutic implications.

Rheumatoid arthritis represents an excellent model in which to gain insights into the local and systemic effects of joint inflammation on skeletal tissues. Three forms of bone disease have been described in rheumatoid arthritis. These include: focal bone loss affecting the immediate subchondral bone and bone at the joint margins; periarticular osteopenia adjacent to inflamed joints; and generalized osteoporosis involving the axial and appendicular skeleton. Although these three forms of bone loss have several features in common, careful histomorphometric and histopathological analysis of bone tissues from different skeletal sites, as well as the use of urinary and serum biochemical markers of bone remodeling, provide compelling evidence that different mechanisms are involved in their pathogenesis. An understanding of these distinct pathological forms of bone loss has relevance not only with respect to gaining insights into the different pathological mechanisms, but also for developing specific and effective strategies for preventing the different forms of bone loss in rheumatoid arthritis.

Tumor biology: use of tiled images in conjunction with measurements of cellular proliferation and death in response to drug treatments.

Tumor growth is dependent on the balance between cell proliferation and cell death, and these events occur heterogenously within an individual tumor. We present a methodology that provides integrative information about cell kinetics, cell death, and cell growth within individual tumors in animals treated with cytotoxic chemotherapeutic agents. Using HCT-116 and NCI-H460 cells, human colonic adenocarcinoma and non-small cell lung cells, respectively, traditional xenograft studies were performed. The tumor-bearing animals were treated with cyclophosphamide (Cytoxan), gemcitabine (Gemzar), or mitomycin C, and extensive analysis of the tumors was studied. Cell kinetics were evaluated by measuring the apoptotic and proliferation indices. The ability to image an entire tumor section using "tiling" by creating a large montage from many high-resolution images makes it possible to identify regional differences within areas of tumor and to demonstrate differences in these tumor regions after treatment with selected chemotherapeutic agents. Two specific areas within tumors have been identified: (a) areas of viable cells within the cell cycle, determined by bromodeoxyuridine and/or morphological characteristics determined by hematoxylin staining; and (b) areas of necrosis determined by the absence of bromodeoxyuridine and proliferating cell nuclear antigen-labeled cells coupled with morphological changes. By standardizing the tumor size to 100 mm2, different patterns of tumor responses to chemotherapeutic agents were determined. By creating such tiled images and by quantitating cell cycle kinetics, it is possible to gain a more complete understanding of tumor growth and response to treatment, leading to the development of more reliable methods for assessing the clinical behavior of anticancer drugs.

Pathogenesis of bone erosions in rheumatoid arthritis.

Patients with rheumatoid arthritis are at risk for the development of a generalized form of bone loss affecting the axial and appendicular skeleton. In addition, juxta-articular osteopenia and focal erosion of marginal and subchondral bone are commonly seen. The pathogenesis of focal bone erosions is an area of active investigation. Studies of tissue sections from sites of bone erosion in rheumatoid arthritis and in animal models of inflammatory arthritis have identified multinucleated cells with the phenotype of osteoclasts in bone resorption lacunae in these sites, suggesting that osteoclasts mediate a component of this pathologic bone loss. Numerous soluble and cell-membrane factors produced by rheumatoid synovial tissues are likely to play a role in the initiation and progression of bone erosions. In addition, recent studies suggest a role for T lymphocytes and their products in osteoclast-mediated bone loss. This paper reviews the cellular mechanisms and factors implicated in bone erosions in rheumatoid arthritis, and discusses the possible therapeutic strategies suggested by these findings.

Amyloid myopathy masquerading as polymyositis.

OBJECTIVE: It is not well appreciated that the clinical presentation of amyloid myopathy can mimic that of polymyositis. By retrospective clinicopathologic analysis we determined distinctive features of amyloid myopathy that differentiate the 2 diseases. METHODS: Two patients with clinical and histologic evidence of an inflammatory myopathy had fatal outcomes despite appropriate treatment for polymyositis. Their clinical course and original pathologic specimens were reviewed. In addition, original tissue samples were obtained and analyzed using Congo red staining and immunoperoxidase. RESULTS: The initial diagnosis of polymyositis was supported in both cases by muscle biopsies showing inflammatory infiltrates and elevations of creatine phosphokinase and by classic electromyography. Retrospective evaluation of the initial muscle biopsies disclosed subtle but incontrovertible evidence of vascular amyloid. Further analysis of the original specimens confirmed the presence of immunoglobin light chain (AL) amyloid. CONCLUSION: Amyloid myopathy can mimic polymyositis. Both can have similar clinical symptoms, as well as inflammatory infiltrates on muscle biopsy. Failure to recognize amyloid myopathy deprives patients of potentially life prolonging treatment. Congo red staining and immunohistochemical analysis of tissue could prevent misdiagnosis.

Synovial tissue in rheumatoid arthritis is a source of osteoclast differentiation factor.

OBJECTIVE: Osteoclast differentiation factor (ODF; also known as osteoprotegerin ligand, receptor activator of nuclear factor kappaB ligand, and tumor necrosis factor-related activation-induced cytokine) is a recently described cytokine known to be critical in inducing the differentiation of cells of the monocyte/macrophage lineage into osteoclasts. The role of osteoclasts in bone erosion in rheumatoid arthritis (RA) has been demonstrated, but the exact mechanisms involved in the formation and activation of osteoclasts in RA are not known. These studies address the potential role of ODF and the bone and marrow microenvironment in the pathogenesis of osteoclast-mediated bone erosion in RA. METHODS: Tissue sections from the bone-pannus interface at sites of bone erosion were examined for the presence of osteoclast precursors by the colocalization of messenger RNA (mRNA) for tartrate-resistant acid phosphatase (TRAP) and cathepsin K in mononuclear cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to identify mRNA for ODF in synovial tissues, adherent synovial fibroblasts, and activated T lymphocytes derived from patients with RA. RESULTS: Multinucleated cells expressing both TRAP and cathepsin K mRNA were identified in bone resorption lacunae in areas of pannus invasion into bone in RA patients. In addition, mononuclear cells expressing both TRAP and cathepsin K mRNA (preosteoclasts) were identified in bone marrow in and adjacent to areas of pannus invasion in RA erosions. ODF mRNA was detected by RT-PCR in whole synovial tissues from patients with RA but not in normal synovial tissues. In addition, ODF mRNA was detected in cultured adherent synovial fibroblasts and in activated T lymphocytes derived from RA synovial tissue, which were expanded by exposure to anti-CD3. CONCLUSION: TRAP-positive, cathepsin K-positive osteoclast precursor cells are identified in areas of pannus invasion into bone in RA. ODF is expressed by both synovial fibroblasts and by activated T lymphocytes derived from synovial tissues from patients with RA. These synovial cells may contribute directly to the expansion of osteoclast precursors and to the formation and activation of osteoclasts at sites of bone erosion in RA.

The nuclear factor of activated T cells (NFAT) transcription factor NFATp (NFATc2) is a repressor of chondrogenesis.

Nuclear factor of activated T cells (NFAT) transcription factors regulate gene expression in lymphocytes and control cardiac valve formation. Here, we report that NFATp regulates chondrogenesis in the adult animal. In mice lacking NFATp, resident cells in the extraarticular connective tissues spontaneously differentiate to cartilage. These cartilage cells progressively differentiate and the tissue undergoes endochondral ossification, recapitulating the development of endochondral bone. Proliferation of already existing articular cartilage cells also occurs in some older animals. At both sites, neoplastic changes in the cartilage cells occur. Consistent with these data, NFATp expression is regulated in mesenchymal stem cells induced to differentiate along a chondrogenic pathway. Lack of NFATp in articular cartilage cells results in increased expression of cartilage markers, whereas overexpression of NFATp in cartilage cell lines extinguishes the cartilage phenotype. Thus, NFATp is a repressor of cartilage cell growth and differentiation and also has the properties of a tumor suppressor.