Notch signaling controls chondrocyte hypertrophy via indirect regulation of Sox9

RBPjk-dependent Notch signaling regulates both the onset of chondrocyte hypertrophy and the progression to terminal chondrocyte maturation during endochondral ossification. It has been suggested that Notch signaling can regulate Sox9 transcription, although how this occurs at the molecular level in chondrocytes and whether this transcriptional regulation mediates Notch control of chondrocyte hypertrophy and cartilage development is unknown or controversial. Here we have provided conclusive genetic evidence linking RBPjk-dependent Notch signaling to the regulation of Sox9 expression and chondrocyte hypertrophy by examining tissue-specific Rbpjk mutant (Prx1Cre;Rbpjk(f/f)), Rbpjk mutant/Sox9 haploinsufficient (Prx1Cre;Rbpjk(f/f);Sox9(f/+)), and control embryos for alterations in SOX9 expression and chondrocyte hypertrophy during cartilage development. These studies demonstrate that Notch signaling regulates the onset of chondrocyte maturation in a SOX9-dependent manner, while Notch-mediated regulation of terminal chondrocyte maturation likely functions independently of SOX9. Furthermore, our in vitro molecular analyses of the Sox9 promoter and Notch-mediated regulation of Sox9 gene expression in chondrogenic cells identified the ability of Notch to induce Sox9 expression directly in the acute setting, but suppresses Sox9 transcription with prolonged Notch signaling that requires protein synthesis of secondary effectors

DNA methyltransferase 3b regulates articular cartilage homeostasis by altering metabolism

Osteoarthritis (OA) is the most common form of arthritis worldwide. It is a complex disease affecting the whole joint but is generally characterized by progressive degradation of articular cartilage. Recent genome-wide association screens have implicated distinct DNA methylation signatures in OA patients. We show that the de novo DNA methyltransferase (Dnmt) 3b, but not Dnmt3a, is present in healthy murine and human articular chondrocytes and its expression decreases in OA mouse models and in chondrocytes from human OA patients. Targeted deletion of Dnmt3b in murine articular chondrocytes results in an early-onset and progressive postnatal OA-like pathology. RNA-Seq and methylC-Seq analyses of Dnmt3b loss-of-function chondrocytes show that cellular metabolic processes are affected. Specifically, TCA metabolites and mitochondrial respiration are elevated. Importantly, a chondroprotective effect was found following Dnmt3b gain of function in murine articular chondrocytes in vitro and in vivo. This study shows that Dnmt3b plays a significant role in regulating postnatal articular cartilage homeostasis. Cellular pathways regulated by Dnmt3b in chondrocytes may provide novel targets for therapeutic approaches to treat OA

Lead Exposure Inhibits Fracture Healing and Is Associated with Increased Chondrogenesis, Delay in Cartilage Mineralization, and a Decrease in Osteoprogenitor Frequency

Lead exposure continues to be a significant public health problem. In addition to acute toxicity, Pb has an extremely long half-life in bone. Individuals with past exposure develop increased blood Pb levels during periods of high bone turnover or resorption. Pb is known to affect osteoblasts, osteoclasts, and chondrocytes and has been associated with osteoporosis. However, its effects on skeletal repair have not been studied. We exposed C57/B6 mice to various concentrations of Pb acetate in their drinking water to achieve environmentally relevant blood Pb levels, measured by atomic absorption. After exposure for 6 weeks, each mouse underwent closed tibia fracture. Radiographs were followed and histologic analysis was performed at 7, 14, and 21 days. In mice exposed to low Pb concentrations, fracture healing was characterized by a delay in bridging cartilage formation, decreased collagen type II and type X expression at 7 days, a 5-fold increase in cartilage formation at day 14 associated with delayed maturation and calcification, and a persistence of cartilage at day 21. Fibrous nonunions at 21 days were prevalent in mice receiving very high Pb exposures. Pb significantly inhibited ex vivo bone nodule formation but had no effect on osteoclasts isolated from Pb-exposed animals. No significant effects on osteoclast number or activity were observed. We conclude that Pb delays fracture healing at environmentally relevant doses and induces fibrous nonunions at higher doses by inhibiting the progression of endochondral ossification

Expression and function of G-protein-coupled receptorsin the male reproductive tract

This review focuses on the expression and function of muscarinic acetylcholine receptors (mAChRs), α1-adrenoceptors and relaxin receptors in the male reproductive tract. The localization and differential expression of mAChR and α1-adrenoceptor subtypes in specific compartments of the efferent ductules, epididymis, vas deferens, seminal vesicle and prostate of various species indicate a role for these receptors in the modulation of luminal fluid composition and smooth muscle contraction, including effects on male fertility. Furthermore, the activation of mAChRs induces transactivation of the epidermal growth factor receptor (EGFR) and the Sertoli cell proliferation. The relaxin receptors are present in the testis, RXFP1 in elongated spermatids and Sertoli cells from rat, and RXFP2 in Leydig and germ cells from rat and human, suggesting a role for these receptors in the spermatogenic process. The localization of both receptors in the apical portion of epithelial cells and smooth muscle layers of the vas deferens suggests an involvement of these receptors in the contraction and regulation of secretion.Esta revisão enfatiza a expressão e a função dos receptores muscarínicos, adrenoceptores α1 e receptores para relaxina no sistema reprodutor masculino. A expressão dos receptores muscarínicos e adrenoceptores α1 em compartimentos específicos de dúctulos eferentes, epidídimo, ductos deferentes, vesícula seminal e próstata de várias espécies indica o envolvimento destes receptores na modulação da composição do fluido luminal e na contração do músculo liso, incluindo efeitos na fertilidade masculina. Além disso, a ativação dos receptores muscarínicos leva à transativação do receptor para o fator crescimento epidermal e proliferação das células de Sertoli. Os receptores para relaxina estão presentes no testículo, RXFP1 nas espermátides alongadas e células de Sertoli de rato e RXFP2 nas células de Leydig e germinativas de ratos e humano, sugerindo o envolvimento destes receptores no processo espermatogênico. A localização de ambos os receptores na porção apical das células epiteliais e no músculo liso dos ductos deferentes de rato sugere um papel na contração e na regulação da secreção.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de FarmacologiaUNIFESP, EPM, Depto. de FarmacologiaSciEL

Reduced COX-2 Expression in Aged Mice Is Associated With Impaired Fracture Healing

The cellular and molecular events responsible for reduced fracture healing with aging are unknown. Cyclooxygenase 2 (COX-2), the inducible regulator of prostaglandin E2 (PGE2) synthesis, is critical for normal bone repair. A femoral fracture repair model was used in mice at either 7–9 or 52–56 wk of age, and healing was evaluated by imaging, histology, and gene expression studies. Aging was associated with a decreased rate of chondrogenesis, decreased bone formation, reduced callus vascularization, delayed remodeling, and altered expression of genes involved in repair and remodeling. COX-2 expression in young mice peaked at 5 days, coinciding with the transition of mesenchymal progenitors to cartilage and the onset of expression of early cartilage markers. In situ hybridization and immunohistochemistry showed that COX-2 is expressed primarily in early cartilage precursors that co-express col-2. COX-2 expression was reduced by 75% and 65% in fractures from aged mice compared with young mice on days 5 and 7, respectively. Local administration of an EP4 agonist to the fracture repair site in aged mice enhanced the rate of chondrogenesis and bone formation to levels observed in young mice, suggesting that the expression of COX-2 during the early inflammatory phase of repair regulates critical subsequent events including chondrogenesis, bone formation, and remodeling. The findings suggest that COX-2/EP4 agonists may compensate for deficient molecular signals that result in the reduced fracture healing associated with aging

The Role of DNA Methylation in Common Skeletal Disorders

Bone is a complex connective tissue characterized by a calcified extracellular matrix. This mineralized matrix is constantly being formed and resorbed throughout life, allowing the bone to adapt to daily mechanical loads and maintain skeletal properties and composition. The imbalance between bone formation and bone resorption leads to changes in bone mass. This is the case of osteoporosis and osteoarthritis, two common skeletal disorders. While osteoporosis is characterized by a decreased bone mass and, consequently, higher susceptibly to fractures, bone mass tends to be higher in patients with osteoarthritis, especially in the subchondral bone region. It is known that these diseases are influenced by heritable factors. However, the DNA polymorphisms identified so far in GWAS explain less than 10% of the genetic risk, suggesting that other factors, and specifically epigenetic mechanisms, are involved in the pathogenesis of these disorders. This review summarizes current knowledge about the influence of epigenetic marks on bone homeostasis, paying special attention to the role of DNA methylation in the onset and progression of osteoporosis and osteoarthritis

Delayed fracture healing and increased callus adiposity in a C57BL/6J Murine model of obesity-associated type 2 diabetes mellitus

Introduction: Impaired healing and non-union of skeletal fractures is a major public health problem, with morbidity exacerbated in patients with diabetes mellitus (DM). DM is prevalent worldwide and affects approximately 25.8 million US adults, with >90% having obesity-related type 2 DM (T2DM). While fracture healing in type 1 DM (T1DM) has been studied using animal models, an investigation into delayed healing in an animal model of T2DM has not yet been performed. Methods: Male C57BL/6J mice at 5 weeks of age were placed on either a control lean diet or an experimental high-fat diet (HFD) for 12 weeks. A mid-diaphyseal open tibia fracture was induced at 17 weeks of age and a spinal needle was used for intra-medullary fixation. Mice were sacrificed at days 7, 10, 14, 21, 28, and 35 for micro-computed tomography (μCT), histology-based histomorphometry and molecular analyses, and biomechanical testing. Results: HFD-fed mice displayed increased body weight and impaired glucose tolerance, both characteristic of T2DM. Compared to control mice, HFD-fed mice with tibia fractures showed significantly (p<0.001) decreased woven bone at day 28 by histomorphometry and significantly (p<0.01) decreased callus bone volume at day 21 by μCT. Interestingly, fracture calluses contained markedly increased adiposity in HFD-fed mice at days 21, 28, and 35. HFD-fed mice also showed increased PPARγ immunohistochemical staining at day 14. Finally, calluses from HFD-fed mice at day 35 showed significantly (p<0.01) reduced torsional rigidity compared to controls. Discussion: Our murine model of T2DM demonstrated delayed fracture healing and weakened biomechanical properties, and was distinctly characterized by increased callus adiposity. This suggests altered mesenchymal stem cell fate determination with a shift to the adipocyte lineage at the expense of the osteoblast lineage. The up-regulation of PPARγ in fracture calluses of HFD-fed mice is likely involved in the proposed fate switching

EFFECTS OF INSULIN ON EARLY OA CHANGES

Objective. Obesity is a state of chronic inflammation that is associated with insulin resistance and type 2 diabetes mellitus (DM), as well as an increased risk of osteoarthritis (OA). This study was undertaken to define the links between obesity-associated inflammation, insulin resistance, and OA, by testing the hypotheses that 1) tumor necrosis factor (TNF) is critical in mediating these pathologic changes in OA, and 2) insulin has direct effects on the synovial joint that are compromised by insulin resistance. Methods. The effects of TNF and insulin on catabolic gene expression were determined in fibroblast-like synoviocytes (FLS) isolated from human OA synovium. Synovial TNF expression and OA progression were examined in 2 mouse models, high-fat (HF) diet–fed obese mice with type 2 DM and TNF-knockout mice. Insulin resistance was investigated in synovium from patients with type 2 DM. Results. Insulin receptors (IRs) were abundant in both mouse and human synovial membranes. Human OA FLS were insulin responsive, as indicated by the dose-dependent phosphorylation of IRs and Akt. In cultures of human OA FLS with exogenous TNF, the expression and release of MMP1, MMP13, and ADAMTS4 by FLS were markedly increased, whereas after treatment with insulin, these effects were selectively inhibited by >50%. The expression of TNF and its abundance in the synovium were elevated in samples from obese mice with type 2 DM. In TNF-knockout mice, increases in osteophyte formation and synovial hyperplasia associated with the HF diet were blunted. The synovium from OA patients with type 2 DM contained markedly more macrophages and showed elevated TNF levels as compared to the synovium from OA patients without diabetes. Moreover, insulin-dependent phosphorylation of IRs and Akt was blunted in cultures of OA FLS from patients with type 2 DM. Conclusion. TNF appears to be involved in mediating the advanced progression of OA seen in type 2 DM. While insulin plays a protective, antiinflammatory role in the synovium, insulin resistance in patients with type 2 DM may impair this protective effect and promote the progression of OA