Up-regulation of cofilin-1 in cell senescence associates with morphological change and p27kip1 -mediated growth delay.

Morphological change is an explicit characteristic of cell senescence, but the underlying mechanisms remains to be addressed. Here, we demonstrated, after a survey of various actin-binding proteins, that the post-translational up-regulation of cofilin-1 was essential for the reduced rate of actin depolymerization morphological enlargement in senescent cells. Additionally, up-regulated cofilin-1 mainly existed in the serine-3 phosphorylated form, according to the 2D gel immunoblotting assay. The up-regulation of cofilin-1 was also detected in aged mammalian tissues. The over-expression of wild-type cofilin-1 and constitutively phosphorylated cofilin-1 promoted cell senescence with an increased cell size. Additionally, senescent phenotypes were also reduced by knockdown of total cofilin-1, which led to a decrease in phosphorylated cofilin-1. The senescence induced by the over-expression of cofilin-1 was dependent on p27Kip1 , but not on the p53 and p16INK4 expressions. The knockdown of p27Kip1 alleviated cell senescence induced by oxidative stress or replicative stress. We also found that the over-expression of cofilin-1 induced the expression of p27Kip1 through transcriptional suppression of the transcriptional enhancer factors domain 1 (TEAD1) transcription factor. The TEAD1 transcription factor played a transrepressive role in the p27Kip1  gene promoter, as determined by the promoter deletion reporter gene assay. Interestingly, the down-regulation of TEAD1 was accompanied by the up-regulation of cofilin-1 in senescence. The knockdown and restoration of TEAD1 in young cells and old cells could induce and inhibit p27Kip1 and senescent phenotypes, respectively. Taken together, the current data suggest that cofilin-1/TEAD1/p27Kip1 signaling is involved in senescence-related morphological change and growth arrest.

Elevated Lifetime Lead Exposure Impedes Osteoclast Activity and Produces an Increase in Bone Mass in Adolescent Mice.

The heavy metal lead (Pb) has a deleterious effect on skeletal health. Because bone mass is maintained through a balance of bone formation and resorption, it is important to understand the effect of Pb levels on osteoblastic and osteoclastic activity. Pb exposure is associated with low bone mass in animal models and human populations; however, the correlation between Pb dosing and corresponding bone mass has been poorly explored. Thus, mice were exposed to increasing Pb and at higher levels (500 ppm), there was unexpectedly an increase in femur-tibial bone mass by 3 months of age. This is contrary to several studies alluded to earlier. Increased bone volume (BV) was accompanied by a significant increase in cortical thickness of the femur and trabecular bone that extended beyond the epiphyseal area into the marrow cavity. Subsequent evaluations revealed an increase in osteoclast numbers with high Pb exposure, but a deficiency in osteoclastic activity. These findings were substantiated by observed increases in levels of the resorption-altering hormones calcitonin and estrogen. In addition we found that pro-osteoclastic nuclear factor-kappa beta (NF-κB) pathway activity was dose dependently elevated with Pb, both in vivo and in vitro. However, the ability of osteoclasts to resorb bone was depressed in the presence of Pb in media and within test bone wafers. These findings indicate that exposure to high Pb levels disrupts early life bone accrual that may involve a disruption of osteoclast activity. This study accentuates the dose dependent variation in Pb exposure and consequent effects on skeletal health.

Heavy Metal Ion Regulation of Gene Expression: MECHANISMS BY WHICH LEAD INHIBITS OSTEOBLASTIC BONE-FORMING ACTIVITY THROUGH MODULATION OF THE Wnt/ß-CATENIN SIGNALING PATHWAY.

Exposure to lead (Pb) from environmental sources remains an overlooked and serious public health risk. Starting in childhood, Pb in the skeleton can disrupt epiphyseal plate function, constrain the growth of long bones, and prevent attainment of a high peak bone mass, all of which will increase susceptibility to osteoporosis later in life. We hypothesize that the effects of Pb on bone mass, in part, come from depression of Wnt/ß-catenin signaling, a critical anabolic pathway for osteoblastic bone formation. In this study, we show that depression of Wnt signaling by Pb is due to increased sclerostin levels in vitro and in vivo. Downstream activation of the ß-catenin pathway using a pharmacological inhibitor of GSK-3ß ameliorates the Pb inhibition of Wnt signaling activity in the TOPGAL reporter mouse. The effect of Pb was determined to be dependent on sclerostin expression through use of the SOST gene knock-out mice, which are resistant to Pb-induced trabecular bone loss and maintain their mechanical bone strength. Moreover, isolated bone marrow cells from the sclerostin null mice show improved bone formation potential even after exposure to Pb. Also, our data suggest that the TGFß canonical signaling pathway is the mechanism by which Pb controls sclerostin production. Taken together these results support our hypothesis that the osteoporotic-like phenotype observed after Pb exposure is, in part, regulated through modulation of the Wnt/ß-catenin pathway.

Biphasic calcium phosphate nano-composite scaffolds reinforced with bioglass provide a synthetic alternative to autografts in a canine tibiofibula defect model.

BACKGROUND: Bone grafting is commonly used to repair bone defects. As the porosity of the graft scaffold increases, bone formation increases, but the strength decreases. Early attempts to engineer materials were not able to resolve this problem. In recent years, nanomaterials have demonstrated the unique ability to improve the material strength and toughness while stimulating new bone formation. In our previous studies, we synthesized a nano-scale material by reinforcing a porous ß-tricalcium phosphate (ß-TCP) ceramic scaffold with Na2O-MgO-P2O5-CaO bioglass (ß-TCP/BG). However, the in vivo effects of the ß-TCP/BG scaffold on bone repair remain unknown. METHODS: We investigated the efficacy of ß-TCP/BG scaffolds compared to autografts in a canine tibiofibula defect model. The tibiofibula defects were created in the right legs of 12 dogs, which were randomly assigned to either the scaffold group or the autograft group (six dogs per group). Radiographic evaluation was performed at 0, 4, 8, and 12 weeks post-surgery. The involved tibias were extracted at 12 weeks and were tested to failure via a three-point bending. After the biomechanical analysis, specimens were subsequently processed for scanning electron microscopy analysis and histological evaluations. RESULTS: Radiographic evaluation at 12 weeks post-operation revealed many newly formed osseous calluses and bony unions in both groups. Both the maximum force and break force in the scaffold group (n = 6) were comparable to those in the autograft group (n = 6, P > 0.05), suggesting that the tissue-engineered bone repair achieved similar biomechanical properties to autograft bone repair. At 12 weeks post-operation, obvious new bone and blood vessel formations were observed in the artificial bone of the experimental group. CONCLUSIONS: The results demonstrated that new bone formation and high bone strength were achieved in the ß-TCP/BG scaffold group, and suggested that the ß-TCP/BG scaffold could be used as a synthetic alternative to autografts for the repair of bone defects.

Icariin Augments Bone Formation and Reverses the Phenotypes of Osteoprotegerin-Deficient Mice through the Activation of Wnt/ ß -Catenin-BMP Signaling.

Icariin has been mostly reported to enhance bone fracture healing and treat postmenopausal osteoporosis in ovariectomized animal model. As another novel animal model of osteoporosis, there is few publication about the effect of Icariin on osteoprotegerin-deficient mice. Therefore, the goal of this study is to find the effect on bone formation and underlying mechanisms of Icariin in osteoprotegerin (OPG) knockout (KO) mice. We found that Icariin significantly stimulated new bone formation after local injection over the surface of calvaria at the dose of 5 mg/kg per day. With this dose, Icariin was also capable of significantly reversing OPG-deficient-induced bone loss and bone strength reduction. Real-time PCR analysis showed that Icariin significantly upregulated the expression of BMP2, BMP4, RUNX2, OC, Wnt1, and Wnt3a in OPG KO mice. Icariin also significantly increased the expression of AXIN2, DKK1, TCF1, and LEF1, which are the direct target genes of ß -catenin signaling. The in vitro studies showed that Icariin induced osteoblast differentiation through the activation of Wnt/ ß -catenin-BMP signaling by in vitro deletion of the ß -catenin gene using ß -catenin(fx/fx) mice. Together, our findings demonstrate that Icariin significantly reverses the phenotypes of OPG-deficient mice through the activation of Wnt/ ß -catenin-BMP signaling.

Runx1 is critical for PTH-induced onset of mesenchymal progenitor cell chondrogenic differentiation.

Parathyroid hormone (PTH) plays a critical role in the regulation of chondrogenesis. In this study, we have found for the first time that Runt-related transcription factor 1 (Runx1) contributes to PTH-induced chondrogenesis. Upon PTH treatment, limb bud mesenchymal progenitor cells in micromass culture showed an enhanced chondrogenesis, which was associated with a significant increase of chondrogenic marker gene expression, such as type II collagen and type X collagen. Runx1 was also exclusively expressed in cells treated with PTH at the onset stage of chondrogenesis. Knockdown of Runx1 completely blunted PTH-mediated chondrogenesis. Furthermore, PTH induced Runx1 expression and chondrogenesis were markedly reduced by inhibition of protein kinase A (PKA) signaling. Taken together, our present study indicates that chondrogenesis induced by PTH in mesenchymal progenitor cells is mediated by Runx1, which involves the activation of PKA. These data provide a novel insight into understanding the molecular mechanisms behind PTH-enhanced cartilage regeneration.

TIMP2 deficient mice develop accelerated osteoarthritis via promotion of angiogenesis upon destabilization of the medial meniscus.

Vascular invasion into the normally avascular articular surface is a hallmark of advanced osteoarthritis (OA). In this study, we demonstrated that the expression of tissue inhibitor of metalloproteinases-2 (TIMP2), an anti-angiogenic factor, was present at high levels in normal articular chondrocytes, and was drastically decreased shortly after destabilization of the medial meniscus (DMM). We also investigated the anti-angiogenic properties of TIMP2 via knockout. We hypothesized that the loss of TIMP2 could accelerate osteoarthritis development via promotion of angiogenesis. Loss of TIMP2 led to increased periarticular vascular formation 1 month post DMM, compared to wild-type mice, and did so without altering the expression pattern of matrix metalloproteinases and vascular endothelial growth factors. The increased vascularization eventually resulted in a severe degeneration of the articular surface by 4 months post DMM. Our findings suggest that reduction of TIMP2 levels and increased angiogenesis are possible primary events in OA progression. Inhibiting or delaying angiogenesis by TIMP2 expression or other anti-angiogenic therapies could improve OA prevention and treatment.

Lead induces an osteoarthritis-like phenotype in articular chondrocytes through disruption of TGF-ß signaling.

Lead remains a significant environmental toxin, and we believe we may have identified a novel target of lead toxicity in articular chondrocytes. These cells are responsible for the maintenance of joint matrix, and do so under the regulation of TGF-ß signaling. As lead is concentrated in articular cartilage, we hypothesize that it can disrupt normal chondrocyte phenotype through suppression of TGF-ß signaling. These experiments examine the effects of lead exposure in vivo and in vitro at biologically relevant levels, from 1 nM to 10 µM on viability, collagen levels, matrix degrading enzyme activity, TGF-ß signaling, and articular surface morphology. Our results indicate that viability was unchanged at levels ≤100 µM Pb, but low and high level lead in vivo exposure resulted in fibrillation and degeneration of the articular surface. Lead treatment also decreased levels of type II collagen and increased type X collagen, in vivo and in vitro. Additionally, MMP13 activity increased in a dose-dependent manner. Active caspase 3 and 8 were dose-dependently elevated, and treatment with 10 µM Pb resulted in increases of 30% and 500%, respectively. Increasing lead treatment resulted in a corresponding reduction in TGF-ß reporter activity, with a 95% reduction at 10µM. Levels of phosphoSmad2 and 3 were suppressed in vitro and in vivo and lead dose-dependently increased Smurf2. These changes closely parallel those seen in osteoarthritis. Over time this phenotypic shift could compromise maintenance of the joint matrix.

Gene expression profile of steroid-induced necrosis of femoral head of rats.

The key to treating steroid-induced necrosis of femoral heads (SINFH) is early diagnosis. Dramatic improvements in diagnosis could be made if the pathogenesis of SINFH was more fully understood; however, the underlying mechanism of this disease is currently unknown. To explore the potential mechanism of SINFH, we performed gene array analysis on a rat model of the disease and compare the expression profile with that of normal rats. A quantitative RT-PCR and immunohistochemistry (IHC) assays were used to confirm the microarray results. Compared to the control group, 190 genes in the experimental group were differentially expressed, with 52 up-regulated and 138 down-regulated. Of these genes, 102 are known (deposited in GenBank), while 88 of them are unknown. The known genes can be divided into several families according to their biological functions, such as oxidative stress, apoptosis, signal transduction, angiogenesis, extracellular matrix, lipid metabolism, and transcription related genes. The results of quantitative RT-PCR and IHC were consistent with gene chip results. Our findings indicate that many genes involved in diverse signaling pathways were differentially expressed between SINFH rats and normal rats. Furthermore, our findings suggest that the development of SINFH is a complicated and dynamic process affected by multiple factors and signaling pathways and regulated by various genes.

Classic yin and yang tonic formula for osteopenia: study protocol for a randomized controlled trial.

BACKGROUND: Osteoporosis is a growing worldwide problem, with the greatest burden resulting from fractures. Nevertheless, the majority of fractures in adults occur in those with "osteopenia" (bone mineral density (BMD) only moderately lower than young normal individuals). Since long-term drug therapy is an expensive option with uncertain consequences and side effects, natural herbal therapy offers an attractive alternative. The purpose of this study is to evaluate the effect on BMD and safety of the Classic Yin and Yang Tonic Formula for treatment of osteopenia and to investigate the mechanism by which this efficacy is achieved. METHODS/DESIGN: We propose a multicenter double-blind randomized placebo-controlled trial to evaluate the efficacy and safety of the Classic Yin and Yang Tonic Formula for the treatment of osteopenia. Participants aged 55 to 75 with low bone mineral density (T-score between -1 and -2.5) and kidney deficiency in TCM will be included and randomly allocated into two groups: treatment group and control group. Participants in the treatment group will be treated with Classic Yin and Yang Tonic Granule, while the controlled group will receive placebo. Primary outcome measure will be BMD of the lumbar spine and proximal femur using dual-energy X-ray absorptiometry. Secondary outcomes will include pain intensity measured with visual analogue scales, quality of life, serum markers of bone metabolism, indices of Neuro-endocrino-immune network and safety. DISCUSSION: If the Classic Yin and Yang Tonic Formula can increase bone mass without adverse effects, it may be a novel strategy for the treatment of osteoporosis. Furthermore, the mechanism of the Chinese medical formula for osteoporosis will be partially elucidated. TRIAL REGISTRATION: This study is registered at ClinicalTrials.gov, NCT01271647.

Osthole stimulates osteoblast differentiation and bone formation by activation of beta-catenin-BMP signaling.

Osteoporosis is defined as reduced bone mineral density with a high risk of fragile fracture. Current available treatment regimens include antiresorptive drugs such as estrogen receptor analogues and bisphosphates and anabolic agents such as parathyroid hormone (PTH). However, neither option is completely satisfactory because of adverse effects. It is thus highly desirable to identify novel anabolic agents to improve future osteoporosis treatment. Osthole, a coumarin-like derivative extracted from Chinese herbs, has been shown to stimulate osteoblast proliferation and differentiation, but its effect on bone formation in vivo and underlying mechanism remain unknown. In this study, we found that local injection of Osthole significantly increased new bone formation on the surface of mouse calvaria. Ovariectomy caused evident bone loss in rats, whereas Osthole largely prevented such loss, as shown by improved bone microarchitecture, histomorphometric parameters, and biomechanical properties. In vitro studies demonstrated that Osthole activated Wnt/beta-catenin signaling, increased Bmp2 expression, and stimulated osteoblast differentiation. Targeted deletion of the beta-catenin and Bmp2 genes abolished the stimulatory effect of Osthole on osteoblast differentiation. Since deletion of the Bmp2 gene did not affect Osthole-induced beta-catenin expression and the deletion of the beta-catenin gene inhibited Osthole-regulated Bmp2 expression in osteoblasts, we propose that Osthole acts through beta-catenin-BMP signaling to promote osteoblast differentiation. Our findings demonstrate that Osthole could be a potential anabolic agent to stimulate bone formation and prevent estrogen deficiency-induced bone loss.

Changes of cervical dorsal root ganglia induced by compression injury and decompression procedure: a novel rat model of cervical radiculoneuropathy.

Our study aimed to establish a model of compression injury of cervical dorsal root ganglia (DRG) in the rat and to investigate the pathological changes following compression injury and decompression procedures. Thirty rats were divided into three groups: control group receiving sham surgery, compression group undergoing surgery to place a micro-silica gel on C6 DRG, and decompression group with subsequent decompression procedure. The samples harvested from the different groups were examined with light microscopy, ultrastructural analysis, and horseradish peroxidase (HRP) retrograde tracing techniques. Apoptosis of DRG neurons was demonstrated with TUNEL staining. Changes in PGE2 and PLA2 in DRG neurons were detected with enzyme-linked immunosorbent assay (ELISA). Local expression of vascular endothelial growth factor (VEGF) was monitored with immunohistochemistry. DRG neurons in the compression group became swollen with vacuolar changes in cytoplasm. Decompression procedure partially ameliorated the resultant compression pathology. Ultrastructural examination showed a large number of swollen vacuoles, demyelinated nerve root fibers, absence of Schwann cells, and proliferation in the surrounding connective tissues in the compression group. Compared to the control group, the compression group showed a significant decrease in the number of the HRP-labeled cells and a significant increase in levels of PGE2 and PLA2, in the expression of VEGF protein, and in the number of apoptotic DRG neurons. These findings demonstrate that compression results in local inflammation, followed by increased apoptosis and upregulation of VEGF. We conclude that such a model provides a tool to study the pathogenesis and treatment of cervical radiculoneuropathy.

Environmental agents affect skeletal growth and development.

In this treatise we will examine complexities in the development and function of cells of the musculoskeletal system. Specifically, the role of chondrocytes and their ontogeny and osteoblasts and their ontogeny will be discussed as they regulate cartilage and bone formation. This background information will provide the foundation for evaluating the effects of environmental toxicants on skeletal development. A number of agents such as heavy metals (i.e. lead) and polycyclic aromatic hydrocarbons (i.e. pesticides and cigarette smoke) interact with cells of the skeletal system and adversely affect development. These agents have not been of major research interest, nevertheless, given changes in the environmental profile of the United States and other developed countries, it is important that we understand their effects in bone and cartilage. Research in this area will identify strategies that may be used to help prevent musculoskeletal diseases due to toxicant exposure.

Environmental toxicants may modulate osteoblast differentiation by a mechanism involving the aryl hydrocarbon receptor.

UNLABELLED: The AHR mediates many of the toxicological effects of aromatic hydrocarbons. We show that AHR expression in osteoblasts parallels the induction of early bone-specific genes involved in maturation. The AHR may not only mediate the effects of toxicants, but with an as yet unidentified ligand, be involved in the differentiation pathways of osteoblasts. INTRODUCTION: Metabolic bone diseases arise as a result of an imbalance in bone cell activities. Recent evidence suggests that environmental toxicants may be contributing factors altering these activities. One candidate molecule implicated in mediating the toxic effects of exogenous compounds is the aryl hydrocarbon receptor (AHR). MATERIALS AND METHODS: Osteoblasts isolated from neonatal rat calvaria were analyzed for AHR expression by quantitative PCR, Western blot, and immunohistochemistry. In addition, AHR activation was evaluated by electromobility gel shift assay and fluorescence microscopy. RESULTS: Our findings showed AHR expression in mature osteoblasts in vivo. The pattern of AHR expression peaks after alkaline phosphatase and before induction of osteocalcin. We first show that AHR functions as a transactivating receptor in osteoblasts, as evidenced by its ligand-dependent migration to the nucleus and its association with known dioxin response elements. AHR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) mediated the induction of cytochrome p450 1A1 and cycloxygenase-2 protein levels. This effect could be inhibited by the potent AHR antagonist, 3'4 methoxynitroflavone. Furthermore, lead treatment of osteoblasts upregulates the expression of AHR mRNA and protein levels, supporting a novel mechanism whereby lead in the skeleton may increase the sensitivity of bone cells to toxicant exposure. CONCLUSIONS: These data imply that the AHR mediates the effects of aromatic toxicants on bone and that AHR expression is regulated during osteoblast differentiation.