IntroductionOsteoarthritis (OA) is a degenerative joint disease affecting a large population of people. The mechanism of this highly prevalent disease is not fully understood. Currently there is no effective disease-modifying treatment for OA. The purpose of this study was two-fold: 1) to investigate the role of MMP13 in the development of OA; and 2) to evaluate the efficacy of the MMP13 inhibitor CL82198 as a pharmacologic treatment for preventing OA progression.MethodsTo investigate the role of the endogenous Mmp13 gene in OA development, tamoxifen was administered to two-week-old Col2CreER;Mmp13fx/fx (Mmp13Col2ER) and Cre-negative control mice for five days. OA was induced by meniscal-ligamentous injury (MLI) when the mice were 10 weeks old and MLI or sham-operated joints were harvested 4, 8, 12, or 16 weeks after surgery. To evaluate the efficacy of CL82198, MLI surgery was performed on 10-week-old wild type mice. CL82198 or saline was administered to the mice daily beginning immediately after the surgery for up to 16 weeks. The joint tissues collected from both experiments were evaluated by cartilage grading, histology/histomorphometry, immunohistochemistry (IHC), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The ability of CL82198 to inhibit MMP13 activity in vitro was confirmed by ELISA.ResultsThe OA progression was decelerated in Mmp13Col2ER mice 8, 12, and 16 weeks post-surgery. Cartilage grading by blinded observers confirmed decreased articular cartilage degeneration in Mmp13Col2ER mice at 8, 12 and 16 weeks compared to Cre-negative mice. Histomorphometric analysis demonstrated that Mmp13Col2ER mice had a higher articular cartilage area and thickness at 12 and 16 weeks post-surgery compared to the control mice. Results of IHC revealed greater type II collagen and proteoglycan expression in Mmp13Col2ER mice. Chondrocyte apoptosis, as determined by TUNEL staining, was higher in control mice compared to Mmp13Col2ER mice. CL82198 inhibited MMP13 activity in conditioned media from vehicle (> 85%) or bone morphogenetic protein 2 (BMP2)-treated (> 90%) primary murine sternal chondrocytes. Intraperitoneal injection of CL82198 decelerated MLI-induced OA progression, increased type II collagen and proteoglycan levels, and inhibited chondrocyte apoptosis compared to saline treatment as determined by OA grading, histology, histomorphometry, IHC, and TUNEL staining, respectively.ConclusionsMmp13 is critical for OA progression and pharmacologic inhibition of MMP13 is an effective strategy to decelerate articular cartilage loss in a murine model of injury-induced knee OA.

Understanding the molecular underpinnings of cancer is of critical importance to developing targeted intervention strategies. Identification of such targets, however, is notoriously difficult and unpredictable. Malignant cell transformation requires the cooperation of a few oncogenic mutations that cause substantial reorganization of many cell features 1 and induce complex changes in gene expression patterns 2-6 . Genes critical to this multi-faceted cellular phenotype thus only have been identified following signaling pathway analysis 7-10 or on an ad hoc basis 4, 11-14 . Our observations that cell transformation by cooperating oncogenic lesions depends on synergistic modulation of downstream signaling circuitry 15-17 suggest that malignant transformation is a highly cooperative process, involving synergy at multiple levels of regulation, including gene expression. Here we show that a large proportion of genes controlled synergistically by loss-of-function p53 and Ras activation are critical to the malignant state. Remarkably, 14 among 24 such 'cooperation response genes' (CRGs) were found to contribute to tumor formation in gene perturbation experiments. In contrast, only one in 14 perturbations of genes responding in a non-synergistic manner had a similar effect. Synergistic control of gene expression by oncogenic mutations thus emerges as an underlying key to malignancy and provides an attractive rationale for identifying intervention targets in gene networks downstream of oncogenic gain and loss-of-function mutations.To identify genes regulated synergistically by cooperating oncogenic mutations at genomic scale, we compared mRNA expression profiles of young adult murine colon (YAMC) cells

Objective. To determine whether Smurf2, an E3 ubiquitin ligase known to inhibit transforming growth factor ␤ (TGF␤) signaling, is expressed in human osteoarthritic (OA) cartilage and can initiate OA in mice.Methods. Human OA cartilage was obtained from patients undergoing knee arthroplasty. Samples were graded histologically using the Mankin scale and were examined immunohistochemically for Smurf2 expression. A transgene driven by the collagen 2␣1 promoter was used to overexpress Smurf2 in mice. Smurf2 overexpression in mouse sternal chondrocytes was confirmed by reverse transcription-polymerase chain reaction and Western blotting. Changes in articular cartilage area, chondrocyte number, and chondrocyte diameter were assessed histomorphometrically using OsteoMeasure software. Alterations in type X collagen and matrix metalloproteinase 13 (MMP-13) in articular chondrocytes were examined by in situ hybridization and immunohistochemistry, respectively. Joint bone phenotypes were evaluated by microfocal computed tomography. The effects of Smurf2 overexpression on TGF␤ signaling were examined using a luciferase-based reporter and immunoprecipitation/Western blotting.Results. Human OA cartilage strongly expressed Smurf2 as compared with nonarthritic human cartilage. By 8 months of age, Smurf2-transgenic mice exhibited decreased articular cartilage area, fibrillation, clefting, eburnation, subchondral sclerosis, and osteophytes. Increased expression of type X collagen and MMP-13 were also detected in articular cartilage from transgenic mice. Transgenic sternal chondrocytes showed reduced TGF␤ signaling as well as decreased expression and increased ubiquitination of pSmad3.Conclusion. Smurf2 is up-regulated during OA in humans, and Smurf2-transgenic mice spontaneously develop an OA-like phenotype that correlates with decreased TGF␤ signaling and increased pSmad3 degradation. Overall, these results suggest a role of Smurf2 in the pathogenesis of OA.Arthritis is the number one cause of disability in the US (1). It has been projected that by 2020, 59.4 million Americans will be affected (18.2%) (2). Osteoarthritis (OA), the most common form of arthritis, is a noninflammatory degenerative joint disease characterized by articular chondrocyte dysfunction, articular cartilage degradation, osteophyte formation, and subchondral sclerosis (3). There is limited understanding of the seminal molecular and/or cellular events in articular cartilage degeneration, and there are few therapeutic options for OA patients. Thus, understanding these events would have a tremendous impact on the development of more-effective therapeutic paradigms.Biochemical, genetic, and mechanical factors contribute to OA progression (4).

Objective The incidence of low back pain is extremely high and is often linked to intervertebral disc (IVD) degeneration. The mechanism of this disease is currently unknown. In this study, we have investigated the role of β-catenin signaling in IVD tissue function. Methods β-catenin protein levels were measured in disc samples derived from patients with disc degeneration and normal subjects by immunohistochemistry (IHC). To generate β-catenin conditional activation (cAct) mice, Col2a1-CreERT2 transgenic mice were bred with β-cateninfx(Ex3)/fx(Ex3) mice. Changes in disc tissue morphology and function were analyzed by micro-CT, histology and real-time PCR assays. Results We found that β-catenin protein was up-regulated in disc tissues from patients with disc degeneration. To assess the effects of increased β-catenin on disc tissue we generated β-catenin cAct mice. Overexpression of β-catenin in disc cells led to extensive osteophyte formation in 3- and 6-month-old β-catenin cAct mice which were associated with significant changes in the cells and extracellular matrix of disc tissues and growth plate. Gene expression analysis demonstrated that activation of β-catenin could enhance Runx2-dependent Mmp13 and Adamts5 expression. Moreover, genetic ablation of the Mmp13 or Adamts5 under β-catenin cAct background, or treatment of β-catenin cAct mice with a specific MMP13 inhibitor, ameliorated the mutant phenotype. Conclusions β-catenin signaling pathway plays a critical role in disc tissue function.