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

Immature mice are more susceptible to the detrimental effects of high fat diet on cancellous bone in the distal femur

AbstractWith the increasing prevalence of obesity among children and adolescents, it is imperative to understand the implications of early diet-induced obesity on bone health. We hypothesized that cancellous bone of skeletally immature mice is more susceptible to the detrimental effects of a high fat diet (HFD) than mature mice, and that removing excess dietary fat will reverse these adverse effects. Skeletally immature (5weeks old) and mature (20weeks old) male C57BL/6J mice were fed either a HFD (60% kcal fat) or low fat diet (LFD; 10% kcal fat) for 12weeks, at which point, the trabecular bone structure in the distal femoral metaphysis and third lumbar vertebrae were evaluated by micro-computed tomography. The compressive strength of the vertebrae was also measured. In general, the HFD led to deteriorations in cancellous bone structure and compressive biomechanical properties in both age groups. The HFD-fed immature mice had a greater decrease in trabecular bone volume fraction (BVF) in the femoral metaphysis, compared to mature mice (p=0.017 by 2-way ANOVA). In the vertebrae, however, the HFD led to similar reductions in BVF and compressive strength in the two age groups. When mice on the HFD were switched to a LFD (HFD:LFD) for an additional 12weeks, the femoral metaphyseal BVF in immature mice showed no improvements, whereas the mature mice recovered their femoral metaphyseal BVF to that of age-matched lean controls. The vertebral BVF and compressive strength of HFD:LFD mouse bones, following diet correction, were equivalent to those of LFD:LFD mice in both age groups. These data suggest that femoral cancellous metaphyseal bone is more susceptible to the detrimental effects of HFD before skeletal maturity and is less able to recover after correcting the diet. Negative effects of HFD on vertebrae are less severe and can renormalize with LFD:LFD mice after diet correction, in both skeletally immature and mature animals

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

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

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

The Pathobiology of Osteoarthritis in Obesity: The Role of Synovial Inflammation, Joint Insulin Resistance, and Dysbiosis of the Gut Microbiome

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology and Laboratory Medicine, 2018.Osteoarthritis (OA) is the most disabling diseases globally, and obesity is a major risk factor for its development, with 66% of individuals with OA being either overweight or obese. Despite this comorbid association, the mechanisms linking obesity to the development of OA have not been fully elucidated. Dogma purports that increased biomechanical loading in obesity is the main driver of the increased prevalence and acceleration of OA. However, recent studies, from our lab and others, have discovered that synovial inflammation and altered insulin signaling is associated the accelerated progression of OA in obese mice and humans. These findings led us to hypothesize that while increased joint loading may be a factor, obesity-induced pathobiological change in the synovium is the seminal driver of accelerated OA degeneration of joints. To test this hypothesis, we performed a transcriptomic analysis of synovial tissue from lean and obese mice at a time point prior to development of fulminant joint degeneration. Significant pro-inflammatory changes were observed in the obese synovium pre-degeneration, suggesting these changes may be critical in disease pathogenesis. Pathway analysis of RNAseq results revealed significant induction of numerous inflammatory pathways including activation of NF-kB, stimulation of macrophages and B Cells, and altered type 2 diabetes signaling associated with reduced insulin signaling. The macrophage and insulin findings align with detection of increased macrophage infiltration into mouse and human synovium, and the ability of insulin to protect human synoviocytes from catabolic changes induced by TNF and other inflammatory cytokines. Since it is established that insulin signaling is anti-inflammatory in various tissues, we further hypothesized that insulin plays an anti-inflammatory role in the synovial joint, and loss of signaling will lead to the spontaneous onset of OA. To test this hypothesis in vivo, we developed a genetic mouse model of synovial insulin resistance using the Cre-Lox system to knockout the insulin receptor (IR) in synoviocytes and superficial zone chondrocytes. IR knockout mice did not spontaneously develop OA, nor did they exhibit exacerbated disease following injury compared to control mice, indicating synovial insulin resistance alone is not sufficient to induce OA, and likely requires the contributing effects of obesity induced systemic inflammation as well. Finally, it is now established that the influence of obesity on type 2 diabetes and systemic inflammation is directly downstream of an obesity-related dysbiosis of the gut microbiome that is correctable via dietary supplementation with various indigestible prebiotic fibers. Based on this we finally hypothesized that i) obesity-induced dysbiosis of the gut microbiome is associated with increased inflammation systemically and in the synovial joint, and ii) correction of this dysbiosis with a prebiotic fiber will mitigate acceleration of OA joint degeneration in obesity. Analysis of the gut microbiome of obese mice revealed an almost complete loss of the anti-inflammatory microbe, Bifidobacterium pseudolongum. Remarkably, dietary supplementation with the indigestible fiber oligofructose corrected this dysbiosis, leading to a more than a 1,000-fold increase in Bifidobacterium pseudolongum in the obese gut microbiome. Associated with this shift were anti-inflammatory effects in the colon, the systemic circulation, and the synovium and protection from the obesity induced acceleration of cartilage degeneration. Taken together, findings presented here establish that pathobiological pro-inflammatory changes in the synovium are key precursors driving the accelerated disease progression in the OA of Obesity. Additionally, we implicate a dysbiotic gut microbiome as pathogenic in this context, and demonstrate that prebiotic correction of this dysbiosis is a novel therapeutic strategy for treating the OA of obesity, a disease for which there are no clinically accepted treatment approaches

Investigating the Molecular Mechanisms of Cigarette Smoke Inhibition of Fracture Healing

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Environmental Medicine, 2011.There is strong clinical evidence of a link between smoking and impaired fracture healing. Despite much effort, the molecular mechanisms underlying these effects remain poorly understood. Work described here investigated the novel hypothesis that polycyclic aromatic hydrocarbons in smoke impair fracture healing by a mechanism mediated by the Aryl Hydrocarbon Receptor. This work established that the AHR is expressed and is transcriptionally active during chondrogenesis and the prototypical PAH, benzo(a)pyrene, inhibits in vitro chondrogenesis while accelerating chondrocyte hypertrophy. These effects were AHR-dependent and were associated with formation of BaP-DNA adducts. In vivo studies demonstrated that doses of BaP below the Lowest Observed Adverse Effect Level impaired tibial fracture healing, characterized by less a smaller fracture callus at all doses and time points examined. Efforts to establish a mouse model of delayed fracture healing in response to mainstream smoke exposure were unsuccessful. Smoke-exposed mice had enhanced fracture healing characterized by increased VEGFa expression, vascularity, and biomechanical strength. Given the emerging importance of HIF1α/HIF2α as mediators of skeletal development, crosstalk between the AHR and Hypoxia Inducible Factor pathways was investigated as a mechanism for AHR-mediated skeletal toxicity. Work described here describes a novel mechanism of HIF/AHR crosstalk. While AHR signaling does not inhibit HIF signaling, hypoxia enhanced AHR signaling. These effects were due to a novel oxygen-dependent, stem cell-specific posttranscriptional regulation of AHR protein levels by HIF1/2α. It was determined this crosstalk is not crucial for bone formation, as investigation of embryonic bone growth in AHR-/- limbs revealed no defect, and fracture repair in AHR-/- mice is uncompromised. The work in this thesis provides the first characterization of AHR expression and function in chondrocytes which could mediate toxic effects of dioxins during bone growth. Detrimental effects of low doses of BaP on fracture healing were demonstrated. However, smoking was surprisingly beneficial to mouse fracture healing, which illustrated the difficulties of modeling cigarette smoke exposure. Furthermore, a novel mechanism of HIF1/2α-dependent AHR protein regulation that is unique to MSCs was described. Despite the apparent specificity of these effects to MSCs, the AHR does not play an endogenous role during bone formation