Osteoarthritis (OA) is a degenerative joint disease, and the mechanism of its pathogenesis is poorly understood. Recent human genetic association studies showed that mutations in the Frzb gene predispose patients to OA, suggesting that the Wnt/␤-catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for ␤-catenin in this disease has not been reported. Because tissue-specific activation of the ␤-catenin gene (targeted by Col2a1-Cre) is embryonic lethal, we specifically activated the ␤-catenin gene in articular chondrocytes in adult mice by generating ␤-catenin conditional activation (cAct) mice through breeding of ␤-catenin fx(Ex3)/fx(Ex3) mice with Col2a1-CreER T2 transgenic mice. Deletion of exon 3 of the ␤-catenin gene results in the production of a stabilized fusion ␤-catenin protein that is resistant to phosphorylation by GSK-3␤. In this study, tamoxifen was administered to the 3-and 6-mo-old Col2a1-CreER T2 ;␤-catenin fx(Ex3)/wt mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of ␤-catenin protein was detected by immunostaining in articular cartilage tissues of ␤-catenin cAct mice. In 5-mo-old ␤-catenin cAct mice, reduction of Safranin O and Alcian blue staining in articular cartilage tissue and reduced articular cartilage area were observed. In 8-mo-old ␤-catenin cAct mice, cell cloning, surface fibrillation, vertical clefting, and chondrophyte/osteophyte formation were observed. Complete loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area were also found in ␤-catenin cAct mice. Expression of chondrocyte marker genes, such as aggrecan, Mmp-9, Mmp-13, Alp, Oc, and colX, was significantly increased (3-to 6-fold) in articular chondrocytes derived from ␤-catenin cAct mice. Bmp2 but not Bmp4 expression was also significantly upregulated (6-fold increase) in these cells. In addition, we also observed overexpression of ␤-catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of ␤-catenin signaling in articular chondrocytes in adult mice leads to the premature chondrocyte differentiation and the development of an OA-like phenotype. This study provides direct and definitive evidence about the role of ␤-catenin in the development of OA.

The Notch pathway has recently been implicated in mesenchymal progenitor cell (MPC) differentiation from bone marrow-derived progenitors. However, whether Notch regulates MPC differentiation in an RBPjκ-dependent manner, specifies a particular MPC cell fate, regulates MPC proliferation and differentiation during early skeletal development or controls specific Notch target genes to regulate these processes remains unclear. To determine the exact role and mode of action for the Notch pathway in MPCs during skeletal development, we analyzed tissue-specific loss-of-function (Prx1Cre; Rbpjkf/f), gain-of-function (Prx1Cre; Rosa-NICDf/+) and RBPjκ-independent Notch gain-of-function (Prx1Cre; Rosa-NICDf/+; Rbpjkf/f) mice for defects in MPC proliferation and differentiation. These data demonstrate for the first time that the RBPjκ-dependent Notch signaling pathway is a crucial regulator of MPC proliferation and differentiation during skeletal development. Our study also implicates the Notch pathway as a general suppressor of MPC differentiation that does not bias lineage allocation. Finally, Hes1 was identified as an RBPjκ-dependent Notch target gene important for MPC maintenance and the suppression of in vitro chondrogenesis.

Objective. Osteoarthritis is a degenerative joint disease whose molecular mechanism is currently unknown. Wnt/␤-catenin signaling has been demonstrated to play a critical role in the development and function of articular chondrocytes. To determine the role of ␤-catenin signaling in articular chondrocyte function, we generated Col2a1-ICAT-transgenic mice to inhibit ␤-catenin signaling in chondrocytes.Methods. The expression of the ICAT transgene was determined by immunostaining and Western blot analysis. Histologic analyses were performed to determine changes in articular cartilage structure and morphology. Cell apoptosis was determined by TUNEL staining and the immunostaining of cleaved caspase 3 and poly(ADP-ribose) polymerase (PARP) proteins. Expression of Bcl-2, Bcl-x L , and Bax proteins and caspase 9 and caspase 3/7 activities were examined in primary sternal chondrocytes isolated from 3-day-old neonatal Col2a1-ICAT-transgenic mice and their wild-type littermates and in primary chicken and porcine articular chondrocytes.Results. Expression of the ICAT transgene was detected in articular chondrocytes of the transgenic mice. Associated with this, age-dependent articular cartilage destruction was observed in Col2a1-ICATtransgenic mice. A significant increase in cell apoptosis in articular chondrocytes was identified by TUNEL staining and the immunostaining of cleaved caspase 3 and PARP proteins in these transgenic mice. Consistent with this, Bcl-2 and Bcl-x L expression were decreased and caspase 9 and caspase 3/7 activity were increased, suggesting that increased cell apoptosis may contribute significantly to the articular cartilage destruction observed in Col2a1-ICAT-transgenic mice.Conclusion. Inhibition of ␤-catenin signaling in articular chondrocytes causes increased cell apoptosis and articular cartilage destruction in Col2a1-ICATtransgenic mice.

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Chondrogenesis and endochondral ossification are the cartilage differentiation processes that lead to skeletal formation and growth in the developing vertebrate as well as skeletal repair in the adult. The exquisite regulation of these processes, both in normal development and in pathologic situations, is impacted by a number of different types of stress. These include normal stressors such as mechanical loading and hypoxia as well pathologic stressors such as injury and/or inflammation and environmental toxins. This article provides an overview of the processes of chondrogenesis and endochondral ossification and their control at the molecular level. A summary of the influence of the most well-understood normal and pathologic stressors on the differentiation program is also presented. Introduction to cartilageCartilage is a connective tissue that is comprised primarily of matrix (mainly collagens and proteoglycans) containing relatively sparse populations of chondrocytes, which perform matrix-generation and maintenance functions. During the development and growth of vertebrates, chondrogenesis is the dynamic cellular process that leads to the establishment of various types of cartilage, including hyaline, fibrous, and elastic cartilage. Hyaline cartilage is found in craniofacial structures, the trachea and bronchial tubes, the articular surfaces of diarthrodial joints, and the growth plate (GP) of long bones. GPs are responsible for driving the process of limb lengthening and bone growth during development pre- and postnatally. This type of bone growth involves the process of endochondral ossification (otherwise known as bone formation). The type of cartilage that is most prominent and most susceptible to both normal and pathologic forms of stress is the hyaline cartilage of the limb and trunk skeleton, which originates from the differentiation of condensed mesenchymal cells into clusters of cartilage cells known as chondrocytes. These cartilage anlagen preform the skeleton and provide a framework for endochondral bone development, a process that involves chondrocyte maturation and matrix mineralization in the GPs. The cells of each skeletal element proceed through a multi-step differentiation process generating both the mature GP cartilage, which controls skeletal growth during early and adolescent development, and the permanent articular cartilage (AC) found at the joint surface of all long bones.The processes of chondrogenesis and endochondral bone formation are not restricted to the developing skeletal system. In fact, following stress-related injuries, such as fractures of endochondral bone, the developmental programs of chondrogenesis and chondrocyte proliferation, maturation, hypertrophy, and terminal differentiation are reinitiated at the site of injury. Additionally, stress-related cartilage diseases such as osteoarthritis (OA) also have marked effects on the differentiation and maintenance of AC during adult life. This is why much attention has been paid to studying the cellular and molecular mechanism...

Obesity is a risk factor for osteoarthritis (OA), the greatest cause of disability in the US. The impact of obesity on OA is driven by systemic inflammation, and increased systemic inflammation is now understood to be caused by gut microbiome dysbiosis. Oligofructose, a nondigestible prebiotic fiber, can restore a lean gut microbial community profile in the context of obesity, suggesting a potentially novel approach to treat the OA of obesity. Here, we report that - compared with the lean murine gut - obesity is associated with loss of beneficial Bifidobacteria, while key proinflammatory species gain in abundance. A downstream systemic inflammatory signature culminates with macrophage migration to the synovium and accelerated knee OA. Oligofructose supplementation restores the lean gut microbiome in obese mice, in part, by supporting key commensal microflora, particularly Bifidobacterium pseudolongum. This is associated with reduced inflammation in the colon, circulation, and knee and protection from OA. This observation of a gut microbiome-OA connection sets the stage for discovery of potentially new OA therapeutics involving strategic manipulation of specific microbial species inhabiting the intestinal space.

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).

Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-␤ signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3 −/− chondrocytes. BMP signaling is increased in TGF-␤ deficiency and is required for accelerated chondrocyte maturation.Introduction: Disruption of TGF-␤ signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures. Materials and Methods: Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3 −/− mice. Expressions of maturational markers, as well as genes involved in TGF-␤ and BMP signaling were examined. Chondrocytes were treated with TGF-␤ and BMP-2, and effects on maturation-related genes and BMP/TGF-␤ responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures. Results: Expression of colX and other maturational markers was markedly increased in Smad3−/− chondrocytes. Smad3 −/− chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF-␤1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-␤ toward BMP signaling. In Smad3 −/− chondrocytes, alternative TGF-␤ signaling pathways remained responsive, as shown by luciferase assays. These non-Smad3-dependent TGF-␤ pathways reduced colX expression and alkaline phosphatase activity in TGF-␤-treated Smad3 −/− cultures, but only partially. In contrast, Smad3 −/− chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor. Conclusions: These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-␤ signaling with important implications for osteoarthritis and cartilage diseases.