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Autophagy is an evolutionarily conserved intracellular process and is considered one of the main catabolism pathways. In the process of autophagy, cells are digested nonselectively or selectively to recover nutrients and energy, so it is regarded as an antiaging process. In addition to the essential role of autophagy in cellular homeostasis, autophagy is a stress response mechanism for cell survival. Here, we review recent literature describing the pathway of autophagy and its role in different bone cell types, including osteoblasts, osteoclasts, and osteocytes. Also discussed is the mechanism of autophagy in bone diseases associated with bone homeostasis, including osteoporosis and Paget's disease. Finally, we discuss the application of autophagy regulators in bone diseases. This review aims to introduce autophagy, summarize the understanding of its relevance in bone physiology, and discuss its role and therapeutic potential in the pathogenesis of bone diseases such as osteoporosis.

Obesity is characterized by the expansion of adipose tissue which is partially modulated by adipogenesis. In the present study, we identified five differentially expressed genes by incorporating two adipogenesis-related datasets from the GEO database and their correlation with adipogenic markers. However, the role of scavenger receptor class A member 3 (SCARA3) in obesity-related disorders has been rarely reported. We found that Scara3 expression in old adipose tissue-derived mesenchymal stem cells (Ad-MSCs) was lower than it in young Ad-MSCs. Obese mice caused by deletion of the leptin receptor gene (db/db) or by a high-fat diet both showed reduced Scara3 expression in inguinal white adipose tissue. Moreover, hypermethylation of SCARA3 was observed in patients with type 2 diabetes and atherosclerosis. Data from the CTD database indicated that SCARA3 is a potential target for metabolic diseases. Mechanistically, JUN was predicted as a transcriptional factor of SCARA3 in different databases which is consistent with our further bioinformatics analysis. Collectively, our study suggested that SCARA3 is potentially associated with age-related metabolic dysfunction, which provided new insights into the pathogenesis and treatment of obesity as well as other obesity-associated metabolic complications.

Skeletal aging is characterized by low bone turnover and increased marrow fat accumulation. However, the underlying mechanism remains poorly defined. Here we show that pro-inflammatory and senescent subtypes of immune cells, including macrophages and neutrophils, accumulated in bone marrow and secreted abundant grancalcin (GCA) during aging. Elevation of bone marrow GCA level in young mice leads to premature skeletal aging. Deletion of GCA in neutrophils and macrophages rejuvenates skeletal aging. Mechanistically, we identify that GCA binds to Plexin-b2 receptor and inactivates its downstream pathway. Depletion of plexin-b2 in bone marrow mesenchymal stem cells abrogates the rejuvenated bone phenotype of GCA-knockout mice. Moreover, GCA neutralizing antibody is generated and shows a certain rejuvenating effect on bone. Given that immune cells are involved into diverse pathologic processes, targeting this fundamental mechanism to prevent skeletal aging offers novel treatment strategy not only for osteoporosis, but also for multiple age-related diseases.

Bone mesenchymal stem cells (BMSCs) are progenitor cells isolated from bone marrow, which keep potential to differentiate into several kinds of cells including osteoblasts and adipocytes. A dynamic mutual regulation exists between osteogenesis and adipogenesis processes. Long non-coding RNA (lncRNA) performs diverse functions in biological activities including regulation of BMSCs commitment. Evidence has shown that lncRNA regulates key signaling pathways including TGFβ/BMP, Wnt and Notch pathways, and several transcription factors in BMSCs differention. Dysregulation of lncRNA in BMSCs leads to disruption of osteo-adipogenesis difffrentiation and results in impairment of bone homeostasis. In this review, we focus on the role of lncRNA in several critical signaling pathways that involved in regulation of osteo-adipogenesis of BMSC and prospects the potential clinical application of lncRNA.

Numerous studies of single stranded endogenous ~ 22 nt microRNAs (miRNAs) have demonstrated their diverse biological functions including control of cell differentiation, cell cycle and pathological conditions. Recent studies suggest the application of miRNAs as ideal biomarkers and therapeutic targets. miRNAs play a vital role as post-transcriptional regulators of gene expression which controls osteoblasts mediated bone formation and osteoclasts related bone remodeling. Also, miRNAs regulate the differentiation of osteoblasts and osteogenesis. The differentiation of osteoblasts is crucial to the regulation of bone homeostasis and it is involved in triggering several signaling pathways like TGF-β, BMP and Wnt, which are regulated by miRNAs. This chapter summarizes the role of miRNAs in osteoblasts differentiation and emphasizes their potential therapeutic role in metabolic bone disorders.

With the increasing aging population, aging‐associated diseases are becoming epidemic worldwide, including aging‐associated metabolic dysfunction. However, the underlying mechanisms are poorly understood. In the present study, we aimed to investigate the role of microRNA miR‐188 in the aging‐associated metabolic phenotype. The results showed that the expression of miR‐188 increased gradually in brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) of mice during aging. MiR‐188 knockout mice were resistant to the aging‐associated metabolic phenotype and had higher energy expenditure. Meanwhile, adipose tissue‐specific miR‐188 transgenic mice displayed the opposite phenotype. Mechanistically, we identified the thermogenic‐related gene Prdm16 (encoding PR domain containing 16) as the direct target of miR‐188. Notably, inhibition of miR‐188 expression in BAT and iWAT of aged mice by tail vein injection of antagomiR‐188 ameliorated aging‐associated metabolic dysfunction significantly. Taken together, our findings suggested that miR‐188 plays an important role in the regulation of the aging‐associated metabolic phenotype, and targeting miR‐188 could be an effective strategy to prevent aging‐associated metabolic dysfunction., 1) MiR‐188 expression increased gradually in the BAT and iWAT of mice during aging. 2) MiR‐188 knockout mice were resistant to aging‐associated metabolic phenotype and had higher energy expenditure, whereas adipose tissue‐specific miR‐188 transgenic mice had the opposite effects. 3) Inhibiting miR‐188 expression in BAT and iWAT of aged mice by tail vein injection of antagomiR‐188 significantly ameliorated the aging‐associated metabolic phenotype, and targeting miR‐188 might be an effective way to prevent aging‐associated metabolic dysfunction.

Summary Skeletal aging is characterized by low bone turnover and marrow fat accumulation. However, the underlying mechanism for this imbalance is unclear. Here, we show that during aging in rats and mice proinflammatory and senescent subtypes of immune cells, including macrophages and neutrophils, accumulate in the bone marrow and secrete abundant grancalcin. The injection of recombinant grancalcin into young mice was sufficient to induce premature skeletal aging. In contrast, genetic deletion of Gca in neutrophils and macrophages delayed skeletal aging. Mechanistically, we found that grancalcin binds to the plexin-b2 receptor and partially inactivates its downstream signaling pathways, thus repressing osteogenesis and promoting adipogenesis of bone marrow mesenchymal stromal cells. Heterozygous genetic deletion of Plexnb2 in skeletal stem cells abrogated the improved bone phenotype of Gca-knockout mice. Finally, we developed a grancalcin-neutralizing antibody and showed that its treatment of older mice improved bone health. Together, our data suggest that grancalcin could be a potential target for the treatment of age-related osteoporosis.

Osteoarthritis (OA) causes the destruction of joints. Its pathogenesis is still under investigation, and there is no effective disease-modifying therapy. Here, we report that elevated cyclooxygenase-2 (COX-2) expression in the osteocytes of subchondral bone causes both spontaneous OA and rheumatoid arthritis (RA). The knockout of COX-2 in osteocytes or treatment with a COX-2 inhibitor effectively rescues the structure of subchondral bone and attenuates cartilage degeneration in spontaneous OA (STR/Ort) mice and tumor necrosis factor-α transgenic RA mice. Thus, elevated COX-2 expression in subchondral bone induces both OA-associated and RA-associated joint cartilage degeneration. The inhibition of COX-2 expression can potentially modify joint destruction in patients with arthritis.

Summary Age-dependent loss of hypothalamic neural stem cells (htNSCs) is important for the pathological consequences of aging; however, it is unclear what drives the senescence of htNSCs. Here, we report that a long non-coding RNA, Hnscr, is abundantly expressed in the htNSCs of young mice but decreases markedly in middle-aged mice. We show that depletion of Hnscr is sufficient to drive the senescence of htNSCs and aging-like phenotypes in mice. Mechanistically, Hnscr binds to Y-box protein 1 (YB-1) to prevent its degradation and thus the attenuation of transcription of the senescence marker gene p16INK4A. Through molecular docking, we discovered that a naturally occurring small compound, theaflavin 3-gallate, can mimic the activity of Hnscr. Treatment of middle-aged mice with theaflavin 3-gallate reduced the senescence of htNSCs while improving aging-associated pathology. These results point to a mediator of the aging process and one that can be pharmacologically targeted to improve aging-related outcomes.

Insulin resistance is the major pathological characteristic of type 2 diabetes, and the elderly often develop insulin resistance. However, the deep-seated mechanisms for aging-related insulin resistance remain unclear. Here, we showed that nanosized exosomes released by bone marrow mesenchymal stem cells (BM-MSCs) of aged mice could be taken up by adipocytes, myocytes, and hepatocytes, resulting in insulin resistance both in vivo and in vitro. Using microRNA (miRNA) array assays, we found that the amount of miR-29b-3p was dramatically increased in exosomes released by BM-MSCs of aged mice. Mechanistically, SIRT1 (sirtuin 1) was identified to function as the downstream target of exosomal miR-29b-3p in regulating insulin resistance. Notably, utilizing an aptamer-mediated nanocomplex delivery system that down-regulated the level of miR-29b-3p in BM-MSCs-derived exosomes significantly ameliorated the insulin resistance of aged mice. Meanwhile, BM-MSCs-specific overexpression of miR-29b-3p induced insulin resistance in young mice. Taken together, these findings suggested that BM-MSCs-derived exosomal miR-29b-3p could modulate aging-related insulin resistance, which may serve as a potential therapeutic target for aging-associated insulin resistance.

Mesenchymal stem/progenitor cells (MSPCs) undergo rapid self-renewal and differentiation, contributing to fast skeletal growth during childhood and puberty. It remains unclear whether these cells change their properties during late puberty to young adulthood, when bone growth and accrual decelerate. Here we show that MSPCs in primary spongiosa of long bone in mice at late puberty undergo normal programmed senescence, characterized by loss of nestin expression. MSPC senescence is epigenetically controlled by the polycomb histone methyltransferase enhancer of zeste homolog 2 (Ezh2) and its trimethylation of histone H3 on Lysine 27 (H3K27me3) mark. Ezh2 maintains the repression of key cell senescence inducer genes through H3K27me3, and deletion of Ezh2 in early pubertal mice results in premature cellular senescence, depleted MSPCs pool, and impaired osteogenesis as well as osteoporosis in later life. Our data reveals a programmed cell fate change in postnatal skeleton and unravels a regulatory mechanism underlying this phenomenon., Mesenchymal stem cells are essential for bone development, but it is unclear if their activity is maintained after late puberty, when bone growth decelerates. The authors show that during late puberty in mice, these cells undergo senescence under the epigenetic control of Ezh2.

We previously reported that Runx2/miR-3960/miR-2861 regulatory feedback loop stimulates osteoblast differentiation. However, the effect of this feedback loop on the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) remains unclear. Our recent study showed that miR-2861 and miR-3960 expression increases significantly duringβ-glycerophosphate-induced osteogenic transdifferentiation of VSMCs. Overexpression of miR-2861 or miR-3960 in VSMCs enhancesβ-glycerophosphate-induced osteoblastogenesis, whereas inhibition of miR-2861 or miR-3960 expression attenuates it. MiR-2861 or miR-3960 promotes osteogenic transdifferentiation of VSMCs by targeting histone deacetylase 5 or Homeobox A2, respectively, resulting in increased runt-related transcription factor 2 (Runx2) protein production. Furthermore, overexpression of Runx2 induces miR-2861 and miR-3960 transcription, and knockdown of Runx2 attenuatesβ-glycerophosphate-induced miR-2861 and miR-3960 transcription in VSMCs. Thus, our data show that Runx2/miR-3960/miR-2861 positive feedback loop plays an important role in osteogenic transdifferentiation of VSMCs and contributes to vascular calcification.

A specific bone vessel subtype, strongly positive for CD31 and endomucin (CD31hiEmcnhi), is identified as coupling angiogenesis and osteogenesis. The abundance of type CD31hiEmcnhi vessels decrease during ageing. Here we show that expression of the miR-497∼195 cluster is high in CD31hiEmcnhi endothelium but gradually decreases during ageing. Mice with depletion of miR-497∼195 in endothelial cells show fewer CD31hiEmcnhi vessels and lower bone mass. Conversely, transgenic overexpression of miR-497∼195 in murine endothelium alleviates age-related reduction of type CD31hiEmcnhi vessels and bone loss. miR-497∼195 cluster maintains the endothelial Notch activity and HIF-1α stability via targeting F-box and WD-40 domain protein (Fbxw7) and Prolyl 4-hydroxylase possessing a transmembrane domain (P4HTM) respectively. Notably, endothelialium-specific activation of miR-195 by intravenous injection of aptamer-agomiR-195 stimulates CD31hiEmcnhi vessel and bone formation in aged mice. Together, our study indicates that miR-497∼195 regulates angiogenesis coupled with osteogenesis and may represent a potential therapeutic target for age-related osteoporosis., H-type endothelium, defined by the high expression of CD31 and endomucin, is found in the bone where it promotes angiogenesis and osteogensis. Here Yang et al. show that the miR-497∼195 cluster regulates the generation and maintenance of the H-type endothelium by controlling the levels of Notch regulator Fbxw7 and the HIF regulator P4HTM.

Osteogenesis during bone modeling and remodeling is coupled with angiogenesis. A recent study showed that a specific vessel subtype, strongly positive for CD31 and endomucin (CD31(hi)Emcn(hi)), couples angiogenesis and osteogenesis. Here, we found that platelet-derived growth factor-BB (PDGF-BB) secreted by preosteoclasts induces CD31(hi)Emcn(hi) vessel formation during bone modeling and remodeling. Mice with depletion of PDGF-BB in the tartrate-resistant acid phosphatase-positive cell lineage show significantly lower trabecular and cortical bone mass, serum and bone marrow PDGF-BB concentrations, and fewer CD31(hi)Emcn(hi) vessels compared to wild-type mice. In the ovariectomy (OVX)-induced osteoporotic mouse model, serum and bone marrow levels of PDGF-BB and numbers of CD31(hi)Emcn(hi) vessels are significantly lower compared to sham-operated controls. Treatment with exogenous PDGF-BB or inhibition of cathepsin K to increase the number of preosteoclasts, and thus the endogenous levels of PDGF-BB, increases CD31(hi)Emcn(hi) vessel number and stimulates bone formation in OVX mice. Thus, pharmacotherapies that increase PDGF-BB secretion from preosteoclasts offer a new therapeutic target for treating osteoporosis by promoting angiogenesis and thus bone formation.

MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. However, no study has investigated the role of miRNA in postmenopausal osteoporosis. Here, we report that miR-503 was markedly reduced in circulating progenitors of osteoclasts-CD14(+) peripheral blood mononuclear cells (PBMCs) from postmenopausal osteoporosis patients compared with those from postmenopausal healthy women. Overexpression of miR-503 in CD14(+) PBMCs inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. Conversely, silencing of miR-503 in CD14(+) PBMCs promoted osteoclastogenesis. RANK, which is activated by the binding of RANKL and inducing osteoclast differentiation, was confirmed to be a target of miR-503. In vivo, silencing of miR-503 using a specific antagomir in ovariectomy (OVX) mice increased RANK protein expression, promoted bone resorption, and decreased bone mass, whereas overexpression of miR-503 with agomir inhibited bone resorption and prevented bone loss in OVX mice. Thus, our study revealed that miR-503 plays an important role in the pathogenesis of postmenopausal osteoporosis and contributes to a new therapeutic way for osteoporosis.

Adipocytes differentiate from mesenchymal stem cells (MSCs) in a process known as adipogenesis. The programme of adipogenesis is regulated by the sequential activation of transcription factors and several signaling pathways. There is growing evidence indicating that a class of small non-coding single-stranded RNAs known as "microRNAs (miRNAs)" also are involved in this process. In this review, we summarize the biology and functional mechanisms of miRNAs in adipocyte differentiation. In addition, we further discuss the miRNAs profiling, the miRNAs function and miRNAs target prediction in the adipogenesis.

Mesenchymal stem cells (MSCs) participate in the repair/remodelling of many tissues, where MSCs commit to different lineages dependent on the cues in the local microenvironment. Here we show that TGFβ-activated RhoA/ROCK signalling functions as a molecular switch regarding the fate of MSCs in arterial repair/remodelling after injury. MSCs differentiate into myofibroblasts when RhoA/ROCK is turned on, endothelial cells when turned off. The former is pathophysiologic resulting in intimal hyperplasia, whereas the latter is physiological leading to endothelial repair. Further analysis revealed that MSC RhoA activation promotes formation of an extracellular matrix (ECM) complex consisting of connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF). Inactivation of RhoA/ROCK in MSCs induces matrix metalloproteinase-3-mediated CTGF cleavage, resulting in VEGF release and MSC endothelial differentiation. Our findings uncover a novel mechanism by which cell–ECM interactions determine stem cell lineage specificity and offer additional molecular targets to manipulate MSC-involved tissue repair/regeneration., It is unclear what regulates the fate of mesenchymal stem cells (MSCs) in arterial repair following injury. Here, the authors show that MSC differentiation following injury is triggered by RhoA which in turn stimulates the release of connective tissue growth factor and vascular endothelial growth factor.

microRNAs (miRNAs) play pivotal roles in osteoblast differentiation. However, the mechanisms of miRNAs regulating osteoblast mineralization still need further investigation. Here, we performed miRNA profiling and identified that miR-93 was the most significantly downregulated miRNA during osteoblast mineralization. Overexpression of miR-93 in cultured primary mouse osteoblasts attenuated osteoblast mineralization. Expression of the Sp7 transcription factor 7 (Sp7, Osterix), a zinc finger transcription factor and critical regulator of osteoblast mineralization, was found to be inversely correlated with miR-93. Then Sp7 was confirmed to be a target of miR-93. Overexpression of miR-93 in cultured osteoblasts reduced Sp7 protein expression without affecting its mRNA level. Luciferase reporter assay showed that miR-93 directly targeted Sp7 by specifically binding to the target coding sequence region (CDS) of Sp7. Experiments such as electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and promoter luciferase reporter assay confirmed that Sp7 bound to the promoter of miR-93. Furthermore, overexpression of Sp7 reduced miR-93 transcription, whereas blocking the expression of Sp7 promoted miR-93 transcription. Our study showed that miR-93 was an important regulator in osteoblast mineralization and miR-93 carried out its function through a novel miR-93/Sp7 regulatory feedback loop. Our findings provide new insights into the roles of miRNAs in osteoblast mineralization. © 2012 American Society for Bone and Mineral Research.

Recently, a membrane-based estrogen receptor (ER), ER-α36, was identified and cloned that transduces membrane-initiated estrogen signaling such as activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway. Here we show that the postmenopausal level of estradiol (E2) induces mitogenic, antiapoptotic, and antiosteogenic effects and proapoptotic effects in postmenopausal osteoblasts and osteoclasts with high levels of ER-α36 expression, respectively. We also found that ER-α36 mediated the effects of postmenopausal-level E2 on proliferation, apoptosis, and differentiation of osteoblasts through transient activation of the MAPK/ERK pathway, whereas ER-α36-mediated postmenopausal-level E2 induces apoptosis of osteoclasts through prolonged activation of the MAPK/ERK pathway with the involvement of reactive oxygen species. We also show that the levels of ER-α36 expression in bone are positively associated with bone mineral density but negatively associated with bone biochemical markers in postmenopausal women. Thus the higher levels of ER-α36 expression are required for preserving bone mass in postmenopausal and menopausal women who become osteoporotic if ER-α36-mediated activities are dysregulated. © 2011 American Society for Bone and Mineral Research.

Preptin, a newly isolated 34-amino-acid peptide hormone that is cosecreted with insulin and amylin from pancreatic beta-cells, has emerged as a regulatory element in bone metabolism, but its mechanism remains unclear. We assessed the effects of preptin on proliferation and differentiation of human osteoblasts and investigated the mechanism involved. Our results demonstrated that preptin promoted human osteoblasts proliferation and alkaline phosphatase activity. Suppression of connective tissue growth factor (CTGF), which was upregulated by preptin in a dose- and time-dependent manner, with small interfering RNA (siRNA) abolished the preptin-induced human osteoblasts proliferation and differentiation. Preptin induced activation of ERK mitogen-activated protein kinase (MAPK), but not p38 or JNK in human osteoblasts. Furthermore, pretreatment of human osteoblasts with the ERK inhibitor PD98059 abolished the preptin-induced CTGF secretion and blocked the promoting effect of preptin on osteoblasts proliferation and differentiation. These data demonstrated that preptin is involved in bone anabolism mediated by ERK/CTGF in human osteoblasts and may contribute to the preservation of bone mass observed in hyperinsulinemic states, such as obesity.

Progesterone (P) has been suggested as a bone-trophic hormone. Previous studies have shown that P promoted bone formation by stimulating the proliferation and differentiation of osteoblasts. But, the effect of P on apoptosis of osteoblast in vitro has not been reported. We propose that P may promote bone formation by suppressing the apoptosis of osteoblast. The present study was performed to investigate the effect of P on apoptosis of murine MC3T3-E1 osteoblastic cells. Cell apoptosis was measured by acidine orange/ethidium bromide (AO/EB) staining and sandwich-enzyme-immunoassay. Progesterone receptor (PR), cytochrome c, caspase-9 and caspase-3 protein levels were determined by Western blot analysis. The enzyme substrate was also used to assess the activation of caspase-3 and caspase-9. Progesterone suppressed MC3T3-E1 cells apoptosis induced by serum deprivation, and this effect was blocked by a PR antagonist RU486. Furthermore, the suppressive effects of P on cytochrome c release and caspase-9 and caspase-3 activation in serum-deprived MC3T3-E1 cells were also reversed by RU486. Our study demonstrated that P protects osteoblast against apoptosis through PR and the downstream mitochondrial pathway. Thus, the data suggest that the effects of P on osteoblast apoptosis may contribute to the mechanisms by which P exerts its action on bone formation.

Tissue inhibitor of metalloproteinases (TIMPs) plays an essential role in the regulation of bone metabolism. Here we report that recombinant tissue metalloproteinase inhibitor-3 (TIMP-3) protein induces the apoptosis of MC3T3-E1 osteoblasts. Cell apoptosis was detected by sandwich-enzyme-immunoassay. Fas and Fasl protein levels were determined by Western blot analysis. The enzyme substrate was used to assess the activation of caspase-3 and caspase-8. The phosphorylation of JNK, p38 and ERK1/2 was examined by Western blot analysis. The ELISA suggested that TIMP-3 promoted MC3T3-E1 cell apoptosis. TIMP-3 treatment induced the expression of Fas and Fasl proteins, and the activation of caspase-8 and caspase-3. TIMP-3 treatment induced p38 and ERK phosphorylation. SB203580 and PD98059, the inhibitor of p38 and ERK, respectively, abolished the TIMP-3 effect on osteoblast apoptosis. In conclusion, the signal pathway through which TIMP-3 induces MC3T3-E1 cell apoptosis, mediated by Fas and involves the p38 and ERK signal transduction pathways.

WISP3 is essential for maintaining cartilage integrity mainly by regulating the expression of collagen II, and mutations of WISP3 linked to spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDT-PA) can compromise this function and lead to cartilage loss. The aim of this study was to evaluate the effect of WISP3 on insulin-like growth factor (IGF) signaling in human chondrocytes, investigate whether WISP3 up-regulates collagen II through the IGF signaling pathway, and compare IGF signaling between wild-type and mutant WISP3. Experimental results suggest that WISP3 up-regulates collagen II expression and inhibits the activation of IGF-IR, IRS-1, and ERK kinase in human chondrocytes, and mutation of WISP3 augments IGF signaling in human chondrocytes. In addition to the IGF signaling pathway, WISP3 might up-regulate collagen II expression through an IGF-independent signaling cascade.

L-carnitine (LC), an amino acid with a major role in cellular energy metabolism, has positive effects on bone metabolism. However, the effect of LC on apoptosis of osteoblast in vitro has not been reported. The aim of this study was to investigate the action of LC on apoptosis of mouse osteoblastic cell line MC3T3-E1. Cell apoptosis was measured by sandwich-enzyme-immunoassay. Release of cytochrome c from mitochondria into cytosol and Bcl-2, Bax protein levels were determined by Western blot analysis. The enzyme substrate was used to assess the activation of caspase-3 and caspase-9. LC inhibited MC3T3-E1 cell apoptosis induced by serum deprivation. Our study also shows that LC decreased cytochrome c release and caspase-3 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Furthermore, LC protected against MC3T3-E1 cell apoptosis induced by the glucocorticoid (GC) dexamethasone (Dex).

Background Osteoblast-derived matrix metalloproteinse-1 (MMP-1), MMP-2 and tissue inhibitor of metalloproteinase-1 (TIMP-1) play a role in bone metabolism by degrading bone matrix. Methods We measured MMP-1, MMMP-2, TIMP-1 and associated results with age and bone metabolism in 591 Chinese women aged 20–80 y. Results Serum MMP-1, MMP-2, and TIMP-1 concentrations exhibited positive correlation with age. Serum concentrations of MMP-1 were higher in 40–69 y old women. The concentrations of MMP-2 were significantly increased in the 50–69 y olds. Serum TIMP-1 concentrations were significantly lower in women aged 30–59 y, and then these were followed by an increase at > 60 y olds. We found a significant negative weaker correlation between MMP-2 and BMD. But multiple linear stepwise regression analysis showed that MMP-2 was not a determinant factor for BMD. There were significant positive correlations between MMP-2 and bone alkaline phosphatase (BAP), osteocalcin (OC), and cross-linked N-telopeptides of type I collagen (NTX). Conclusions The serum concentrations of MMP-1, MMP-2, and TIMP-1 exhibit age-related changes, and circulating MMP-2 and bone turnover are related.

Taurine is found in bone tissue, but its function in skeletal tissue is not fully understood. The present study was undertaken to investigate regulation of gene expression of connective tissue growth factor (CTGF), and the roles of mitogen-activated protein kinases (MAPKs) in murine osteoblast MC3T3-E1 cells treated with taurine. Western blot analysis showed taurine stimulated CTGF protein secretion in a dose- and time-dependent manner. Taurine induced activation of extracellular signal-regulated kinase (ERK), but not p38 and c-jun N-terminal Kinase (JNK), in osteoblasts. Furthermore, pretreatment of osteoblasts with the ERK inhibitor PD98059 abolished the taurine-induced CTGF production. These data indicate that taurine induces CTGF secretion in MC3T3-E1 cells mediated by the ERK pathway, and suggest that osteoblasts are direct targets of taurine.

Our study indicates that recombinant adiponectin induced RANKL and inhibited OPG expression in human osteoblasts through the AdipoR1/p38 MAPK pathway, and these responses contributed to the adiponectin-induced osteoclasts formation in the co-culture of osteoblast and peripheral blood monocytes systems. These findings showed that adiponectin increased osteoclast formation indirectly through stimulating RANKL and inhibiting OPG production in osteoblasts. It also suggests the pharmacological nature of recombinant adiponectin that indirectly induces osteoclasts formation. Introduction: Recently, adiponectin has emerged as an element in the regulation of bone metabolism, but the mechanism remains. This study was undertaken to investigate the action of adiponectin on osteoclastogenesis through revealing RANKL and osteoprotegerin (OPG) expression in osteoblasts and osteoclast formation. Materials and Methods: Real-time quantitative PCR and ELISA were used to detect RANKL and OPG mRNA and protein expression in cultured human osteoblasts. The involved signal pathway was studied using mitogen-activated protein kinase (MAPK) inhibitor and adiponectin receptor 1 (AdipoR1) siRNA. The effects of recombinant adiponectin on osteoclasts formation also were examined in the co-culture systems of osteoblast and peripheral blood monocytes (PBMCs) systems or purified CD14 + PBMCs cultures. Results: Our study showed that recombinant adiponectin induced RANKL and inhibited OPG mRNA expression in human osteoblasts in a dose- and time-dependent manner. Adiponectin also increased soluble RANKL and decreased OPG secretion in osteoblasts conditioned media. Suppression of AdipoR1 with siRNA abolished the adiponectin-regulated RANKL and OPG mRNA expression in osteoblasts. Furthermore, pretreatment of osteoblasts with the MAPK inhibitor SB203580 abolished adiponectin-regulated RANKL and OPG mRNA expression. Adiponectin induced osteoclast formation in the co-culture systems of osteoblast and PBMCs systems, and OPG entirely blocked this response. However, adiponectin had no direct effect on the differentiation of osteoclast precursor purified CD14 + PBMCs. Conclusions: These data indicate that recombinant adiponectin induced RANKL and inhibited OPG expression in human osteoblasts through the AdipoR1/p38 MAPK pathway, and these responses contributed to the adiponectin-induced osteoclast formation in the co-culture of osteoblast and PBMCs systems. These findings showed that adiponectin increased osteoclast formation indirectly through stimulating RANKL and inhibiting OPG production in osteoblasts. It suggests the pharmacological nature of recombinant adiponectin that indirectly induces osteoclasts formation.

Taurine influences bone metabolism and is taken up by cells via a specific transport system, the taurine transporter (TAUT). We report a link between taurine and bone homeostasis by demonstrating transcription and translation of TAUT in bone-forming cells. TAUT was expressed in human primary osteoblasts, the human osteosarcoma osteoblast-like cell line MG63, and the mouse osteoblastic cell line MC3T3-E1. Immunostaining with polyclonal antibodies also demonstrated the presence of TAUT in both human and murine osteoblasts. TAUT mRNA expression and [(3)H]taurine uptake increased during differentiation of MG63 cells in culture. Supplementation of culture medium with taurine enhanced alkaline phosphatase activity and osteocalcin secretion. The regulation and detailed function of taurine and TAUT in bone remain unclear, but our findings suggest a functional role for them in bone homeostasis.

We investigated the action of tissue inhibitor of metalloproteinase-1 (TIMP-1) on apoptosis and differentiation of mouse bone marrow stromal cell line MBA-1. TIMP-1 did not affect alkaline phosphatase (ALP) activity, suggesting that it is not involved in osteoblastic differentiation in MBA-1 cells. However, TIMP-1 inhibited MBA-1 apoptosis induced by serum deprivation in a dose-dependent manner. Our study also showed increased Bcl-2 protein expression and decreased Bax protein expression with TIMP-1 treatment. TIMP-1 decreased cytochrome c release and caspase-3 activation in MBA-1 cells. TIMP-1 activated phosphatidylinositol 3-kinase (PI3-kinase) and c-Jun N-terminal kinase (JNK), and the PI3-kinase inhibitor LY294002 or the JNK inhibitor SP600125 abolished its antiapoptotic activity. To investigate whether antiapoptotic action of TIMP-1 was mediated through its inhibition on MMP activities, we constructed mutant TIMP-1 by side-directed mutagenesis, which abolished the inhibitory activity of MMPs by deletion of Cys1 to Ala4. Wild-type TIMP-1 and mutant TIMP-1 expression plasmids were transfected in MBA-1 cells, and results showed that mutant TIMP-1 still protected the induced MBA-1 cell against apoptosis. These data suggest that TIMP-1 antiapoptotic actions are mediated via the PI3-kinase and JNK signaling pathways and independent of TIMP-1 inhibition of MMP activities.

Adipocytes can highly and specifically express adiponectin, and the adiponectin receptor (AdipoR) has been detected in bone-forming cells. The present study was undertaken to investigate the action of adiponectin on osteoblast proliferation and differentiation. AdipoR1 protein was detected in human osteoblasts. Adiponectin promoted osteoblast proliferation and resulted in a dose- and time-dependent increase in alkaline phosphatase (ALP) activity, osteocalcin and type I collagen production, and an increase in mineralized matrix. Suppression of AdipoR1 with small-interfering RNA (siRNA) abolished the adiponectin-induced cell proliferation and ALP expression. Adiponectin induces activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal Kinase (JNK), but not ERK1/2 in osteoblasts, and these effects were blocked by suppression of AdipoR1 with siRNA. Furthermore, pretreatment of osteoblasts with the JNK inhibitor SP600125 abolished the adiponectin-induced cell proliferation. p38 inhibitor SB203580 blocked the adiponectin-induced ALP activity. These data indicate that adiponectin induces human osteoblast proliferation and differentiation, and the proliferation response is mediated by the AdipoR/JNK pathway, while the differentiation response is mediated via the AdipoR/p38 pathway. These findings suggest that osteoblasts are the direct targets of adiponectin.

Parathyroid hormone (PTH) can stimulate bone resorption by increasing the activity and the number of osteoclasts in bone tissue by several mechanisms. Recently, osteoblast-derived membrane-type matrix metalloproteinase-1 (MT1-MMP) has been implied to play an important role in the process of bone resorption by degrading bone matrix. In the present study, we observed the effects of PTH (1-34) on MT1-MMP production, and the role of the protein kinase A (PKA) and protein kinase C (PKC) pathways in the regulation of MT1-MMP in cultures of human osteoblast-like MG-63 cells. By Northern blot and Western immunoblot analysis, we found, unexpectedly, that PTH (1-34) inhibited MT1-MMP mRNA and protein expression in a dose- and time-dependent manner in MG-63 cells. The PKA antagonist H-89 blunted PTH (1-34)-mediated decreases in MT1-MMP protein synthesis. Forskolin, a PKA agonist, decreased MT1-MMP expression, which was similar to the action of PTH on MT1-MMP expression, in MG-63 cells. Staurosporine, a PKC inhibitor, also blocked the inhibition by PTH. We suggest that both the PKA and PKC pathways are involved in MT1-MMP downregulation by PTH. Furthermore, we found that PTH (1-34) induced the expression of receptor activator of nuclear factor (NF)-KappaB ligand (RANKL) mRNA in a dose- and time-dependent manner in MG-63 cells, and this effect of PTH on RANKL mRNA expression was nearly parallel to the effects of MT1-MMP downregulation, implying a correlation between MT1-MMP and RANKL expression. Our findings suggest that the decreased MT1-MMP expression induced by PTH may be involved in RANKL signaling in osteoblasts, and may play a role in the activation of bone resorption.

Estriol has been showed to prevent bone loss in osteoporotic rats and postmenopausal women, but the mechanisms remain unclear. In the present study, we evaluated the effect of estriol on osteoblastic MG-63 cells in vitro, and compared its action with 17beta-estradiol (E2). Cell proliferation was determined by measuring total cell numbers and [3H]thymidine incorporation. Cell function was studied by measuring alkaline phosphatase (ALP) activity and secreted osteocalcin. Our data showed that estriol stimulated MG-63 cells proliferation in a dose-dependent manner, but had no influence on ALP activity in MG-63 cells and osteocalcin production. Compared with estriol treatment, E2 showed a stronger proliferation. Estrogen receptor (ER) alpha and beta expression in MG-63 cells can be detected by Western immunoblot analysis, and the proliferative response to E2 and estriol can be all abrogated by ER antagonist ICI 182,780. In conclusion, estriol stimulates osteoblastic MG-63 cells proliferation, but has no effects on differentiation. The proliferative response to estriol is mediated by the ER. These results suggest that estriol has an effect on osteoblastic proliferation, and this may contribute to its actions on prevention of bone loss.

To study the effects of progesterone (P) and 18-methyl levonorgestrel (LNG) on the expression of P receptors (PRA and PRB), c-fos, c-jun, and osteocalcin in the human osteosarcoma cell line MG-63 and human normal osteoblasts in order to understand the mechanism of progestin action on the proliferation and differentiation of osteoblasts.Cell proliferation was tested by MTT assay. The expression of PR, c-fos, c-jun, and osteocalcin was measured by semi-quantitative RT-PCR, Western immunoblot or immunocytochemistry.Progesterone and LNG (10(-10)-10(-6)M) stimulated cell proliferation in a dose-dependent manner. Progesterone and LNG did not affect the expression of PRA and PRB mRNA and protein, but c-fos and c-jun mRNA and protein were upregulated in a dose-dependent manner. The expression of osteocalcin mRNA was also increased in human osteoblasts in a dose and time-dependent manner with greater effects of LNG than P, while the expression of osteocalcin mRNA in MG-63 cells was not changed by P or LNG.Progesterone and LNG promote osteocalcin gene transcription by stimulating the expression of c-fos and c-jun, and result in osteoblast proliferation and differentiation.

Bone marrow mesenchymal stem cells (BMSCs) exhibit an age-dependent reduction in osteogenesis that is accompanied by an increased propensity toward adipocyte differentiation. This switch increases adipocyte numbers and decreases the number of osteoblasts, contributing to age-related bone loss. Here, we found that the level of microRNA-188 (miR-188) is markedly higher in BMSCs from aged compared with young mice and humans. Compared with control mice, animals lacking miR-188 showed a substantial reduction of age-associated bone loss and fat accumulation in bone marrow. Conversely, mice with transgenic overexpression of miR-188 in osterix+ osteoprogenitors had greater age-associated bone loss and fat accumulation in bone marrow relative to WT mice. Moreover, using an aptamer delivery system, we found that BMSC-specific overexpression of miR-188 in mice reduced bone formation and increased bone marrow fat accumulation. We identified histone deacetylase 9 (HDAC9) and RPTOR-independent companion of MTOR complex 2 (RICTOR) as the direct targets of miR-188. Notably, BMSC-specific inhibition of miR-188 by intra-bone marrow injection of aptamer-antagomiR-188 increased bone formation and decreased bone marrow fat accumulation in aged mice. Together, our results indicate that miR-188 is a key regulator of the age-related switch between osteogenesis and adipogenesis of BMSCs and may represent a potential therapeutic target for age-related bone loss.

The purpose of this study was to investigate the association of two novel β3-adrenergic receptor (ADRB3) gene polymorphisms (Ser165Pro and Ser257Pro) with type 2 diabetes (T2DM) in the Chinese population.A total of 650 patients with T2DM and 1337 health volunteers were enrolled to conduct the association study. Two candidate polymorphisms were recreated by site-directed mutagenesis and tested for their effect on ADRB3 expression and function in stable transfected human embryonic kidney 293 and Chinese hamster ovary-K1 cells. Real-time PCR, Western blot, confocal microscopy, and cAMP assay were used to determine mRNA, protein expression, trafficking, and ADRB3 function, respectively.We found that both polymorphisms were significantly associated with T2DM (odds ratio = 2.060 and 95% confidence interval = 1.303-3.258 for Ser165Pro and odds ratio = 7.588, 95% confidence interval = 1.639-35.138 for Ser257Pro). Patients with T2DM with the Ser165Pro C allele had higher hemoglobin A1c, fasting plasma glucose and postprandial plasma glucose values than those in TT genotypes. We also found that patients with T2DM with the Ser257Pro C allele had lower fasting serum insulin, postprandial serum insulin, and homeostasis model assessment for insulin resistance levels than TT genotype carriers. Further in vitro study indicated that cell lines stably expressing Ser165Pro and Ser257Pro mutants of the ADRB3 gene showed impaired cAMP accumulation activity. However, both polymorphisms had no effect on ADRB3 expression and trafficking.Ser165Pro and Ser257Pro polymorphisms affected ADRB3 function and were significantly associated with susceptibility to and development of T2DM.

Wisp3gene mutation was shown to cause spondyloepiphyseal dysplasia tarda with progressive arthropathy (SRDT-PA), but the underlying mechanism is not clear. To clarify this mechanism, we constructed the wild and mutatedWisp3expression vectors and transfected into human chondrocytes lines C-20/A4;Wisp3proteins subcellular localization, cell proliferation, cell apoptosis, andWisp3-mediated gene expression were determined, and dynamic secretion of collagen in transfected chondrocytes was analyzed by14C-proline incorporation experiment. MutatedWisp3protein increased proliferation activity, decreased apoptosis of C-20/A4 cells, and aggregated abnormally in cytoplasm. Expression of collagen II was also downregulated in C-20/A4 cells transfected with mutatedWisp3. Wild typeWisp3transfection increased intracellular collagen content and extracellular collagen secretion, but the mutatedWisp3lost this function, and the peak phase of collagen secretion was delayed in mutatedWisp3transfected cells. Thus abnormal protein distribution, cell proliferation, collagen synthesis, and secretion inWisp3mutated chondrocytes might contribute to the pathogenesis of SEDT-PA.

MicroRNAs (miRNAs) play crucial roles in bone metabolism. In the present study, we found that miR-148a is dramatically upregulated during osteoclastic differentiation of circulating CD14+ peripheral blood mononuclear cells (PBMCs) induced by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Overexpression of miR-148a in CD14+ PBMCs promoted osteoclastogenesis, whereas inhibition of miR-148a attenuated osteoclastogenesis. V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) is a transcription factor negatively regulating RANKL-induced osteoclastogenesis. miR-148a directly targeted MAFB mRNA by binding to the 3′ untranslated region (3′UTR) and repressed MAFB protein expression. In vivo, our study showed that silencing of miR-148a using a specific antagomir-inhibited bone resorption and increased bone mass in mice receiving ovariectomy (OVX) and in sham-operated control mice. Furthermore, our results showed that miR-148a levels significantly increased in CD14+ PBMCs from lupus patients and resulted in enhanced osteoclastogenesis, which contributed to the lower bone mineral density (BMD) in lupus patients compared with normal controls. Thus, our study provides a new insight into the roles of miRNAs in osteoclastogenesis, and contributes to a new therapeutic pathway for osteoporosis. © 2013 American Society for Bone and Mineral Research.

Adiponectin may exert a negative effect on bone metabolism by regulating osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) expression. However, the action of adiponectin on bone may be influenced by estrogen in women. The present study was undertaken to investigate the effects of 17β-estradiol (E2) on adiponectin-regulated OPG and RANKL expression in human osteoblast. Human osteoblasts were treated with α-MEM containing 10 μg/ml adiponectin alone or together with 10 − 10 to 10 − 8 M E2 for 12–48 h. Cells were also treated with α-MEM containing 10 μg/ml adiponectin together with 10 − 8 M E2 plus p38 agonist-anisomycin or estrogen receptor (ER) antagonist ICI182780 for 48 h. The effects of E2 were also investigated by knockdown of ERs or overexpression of p38 MAPK in osteoblasts. Further, we examined the effects of E2 on adiponectin-dependent osteoclastogenesis by the co-culture systems of osteoblast and CD14 + peripheral blood monocytes (PBMCs). Real-time quantitative PCR (RT-PCR) and ELISA were used to detect OPG/RANKL mRNA and their corresponding protein expression, Western Blot was used to analyze the phosphorylated p38 (p-p38) levels. The results showed that E2 blocked adiponectin-induced p38 phosphorylation, decreased adiponectin-regulated OPG/RANKL mRNA and protein expression in a dose- and time-dependent manner. ICI182780 or knockdown of ERs abolished the effects of E2 on adiponectin-dependent p38 phosphorylation and OPG/RANKL expression. Furthermore, anisomycin or overexpression of p38 also reserved the effects of E2 on adiponectin-dependent p38 phosphorylation and OPG/RANKL expression. E2 inhibited adiponectin-dependent osteoclastogenesis in the co-culture systems of osteoblast and CD14 + PBMCs, whereas anisomycin, ICI182780, knockdown of ERs and overexpression of p38 significantly reversed this response. In conclusions, our findings demonstrated, through blocking the activation of adiponectin-induced p38 MAPK, E2 suppressed the adiponectin-regulated OPG/RANKL expression and then inhibited osteoclastogenesis, which suggested that estrogen would suppress the effect of adiponectin on bone metabolism.

Osteoclasts, which are derived from monocyte-macrophage precursors, are exclusive bone resorption cells. Limited evidence indicates that a class of small non-coding single-stranded RNAs known as "microRNAs" (miRNAs) is also involved in bone resorption. Although various miRNAs regulate cell proliferation and differentiation, few miRNAs have been reported to play a key role in the regulation of osteoclast differentiation. In this short review, the biology and functional mechanisms of miRNAs in osteoclastogenesis are summarized. The profiling, function, and target prediction of miRNAs are discussed as well.

Aims Omentin-1 (also known as intelectin-1) is a recently identified visceral adipose tissue-derived cytokine that is inversely related to obesity. Our previous study showed that omentin-1 inhibits osteoblastic differentiation of calcifying vascular smooth muscle cells (CVSMCs) in vitro . This study was undertaken to investigate the effects of omentin-1 on arterial calcification and bone metabolism in vivo . Methods and results In vitro , omentin-1 stimulated production of osteoprotegerin (OPG) and inhibited production of receptor activator for nuclear factor κB ligand (RANKL) in both CVSMCs and osteoblasts. In vivo , adenovirus-mediated over-expression of omentin-1 attenuated arterial calcification and bone loss in OPG −/− mice. All these in vitro and in vivo actions were abrogated by blockade of the PI3K–Akt signalling pathway. Furthermore, omentin-1 reduced serum levels of RANKL, tartarate-resistant acid phosphatase-5b and osteocalcin, all of which are increased dramatically in OPG −/− mice. Conclusion These data suggest that omentin-1 ameliorates arterial calcification and bone loss in vivo through the regulation of the RANK signalling pathway.

Omentin-1 inhibited osteoblast differentiation in vitro. In co-culture systems of osteoblasts and osteoclast precursors, omentin-1 reduced osteoclast formation by stimulating osteoprotegerin (OPG) and inhibiting receptor activator for nuclear factor κB ligand (RANKL) production in osteoblasts. In vivo, adenovirus-mediated overexpression of omentin-1 suppressed bone turnover and restored bone mineral density (BMD) and bone strength in ovariectomized mice. Omentin-1 (also intelectin-1) is a recently identified visceral adipose tissue-derived cytokine that is highly abundant in plasma. This study was undertaken to investigate the effects of omentin-1 on bone metabolism. Osteoblast differentiation was assessed by measuring alkaline phosphatase activity, osteocalcin production and matrix mineralization. OPG and RANKL protein expression and secretion in osteoblasts were detected by Western blot and ELISA, respectively. The effect of recombinant omentin-1 on osteoclast formation was examined in co-culture systems of osteoblasts and osteoclast precursors. The effects of intravenous administration of adenoviral-delivered omentin-1 on bone mass, bone strength, and bone turnover were also examined in ovariectomized mice. In vitro, omentin-1 inhibited osteoblast differentiation, while it had no direct effect on osteoclast differentiation; it also reduced osteoclast formation in the co-culture systems through stimulating OPG and inhibiting RANKL production in osteoblasts. In vivo, adenovirus-mediated overexpression of omentin-1 partially restored BMD and bone strength in ovariectomized mice, accompanied by decreased levels of plasma osteocalcin and tartrate-resistant acid phosphatase-5b and lower serum RANKL/OPG ratios. The present study suggests that omentin-1 ameliorates bone loss induced by estrogen deficiency via downregulating the RANKL/OPG ratio.

Our recent study showed that miR-2861 promotes osteoblast differentiation by targeting histone deacetylase 5, resulting in increased runt-related transcription factor 2 (Runx2) protein production. Here we identified another new microRNA (miRNA) (miR-3960) that played a regulatory role in osteoblast differentiation through a regulatory feedback loop with miR-2861. miR-3960 and miR-2861 were found clustered at the same loci. miR-3960 was transcribed during bone morphogenic protein 2 (BMP2)-induced osteogenesis of ST2 stromal cells. Overexpression of miR-3960 promoted BMP2-induced osteoblastogenesis. However, the inhibition of miR-3960 expression attenuated the osteoblastogenesis. Homeobox A2 (Hoxa2), a repressor of Runx2 expression, was confirmed to be a target of miR-3960. Electrophoretic mobility shift assay and chromatin immunoprecipitation experiments confirmed that Runx2 bound to the promoter of the miR-3960/miR-2861 cluster. Furthermore, overexpression of Runx2 induced miR-3960/miR-2861 transcription, and block of Runx2 expression attenuated BMP2-induced miR-3960/miR-2861 transcription. Here we report that miR-3960 and miR-2861, transcribed together from the same miRNA polycistron, both function in osteoblast differentiation through a novel Runx2/miR-3960/miR-2861 regulatory feedback loop. Our findings provide new insights into the roles of miRNAs in osteoblast differentiation.

Bone tissue arises from mesenchymal stromal cells (MSCs) differentiated into the osteoblast lineage by genetic and epigenetic mechanisms. Emerging evidence indicates that the class of small non-coding single-stranded RNAs known as "microRNAs (miRNAs)" also plays a critical role in this process.In this short review, we summarize the biology and functional mechanisms of miRNAs. Importantly, we discuss miRNA expression, miRNA function, miRNA target prediction, miRNA overexpression and inhibition methods applied in osteoblastogenesis.We discuss the potential therapeutic opportunities and challenges for improving the treatment of bone-related diseases by using miRNAs as a therapeutic target.Although various microRNAs regulate cell proliferation and differentiation, only a few miRNAs has been reported so far to play a key role in the regulation of osteoblast differentiation and bone formation.

Insulin receptor substrate 1 (IRS1) is an essential molecule for the intracellular signaling of IGF1 and insulin, which are potent anabolic regulators of bone metabolism. Osteoblastic IRS1 is essential for maintaining bone turnover; however, the mechanism underlying this regulation remains unclear. To clarify the role of IRS1 in bone metabolism, we employed RNA interference to inhibit IRS1 gene expression and observed the effects of silencing this gene on the proliferation and differentiation of and the expression of matrix metallopeptidase (MMP) and tumor necrosis factor receptor superfamily, member 11b (TNFRSF11B) in MC3T3-E1 cells. Our results showed that IRS1 short hairpin RNAs can effectively suppress the expression of IRS1, and inhibit the phosphorylation of AKT in IRS1 pathway; reduce the expression of MMP2, MMP3, MMP13, and MMP14, decrease the expression of TNFRSF11B and RANKL (also known as tumor necrosis factor (ligand) superfamily, member 11), however increase the RANKL/TNFRSF11B ratio; decrease cell survival, proliferation, and mineralization, and impair the differentiation of MC3T3-E1 cells. The downregulation of IRS1 had no effect on the expression of MMP1. Our findings suggest that IRS1 not only promotes bone formation and mineralization but also might play roles in bone resorption partly via the regulation of MMPs and RANKL/TNFRSF11B ratio, thus regulates the bone turnover.

Background Assessment of renal function in patients undergoing coronary artery bypass grafting (CABG) is important. Cystatin C has been proposed as an improved indicator of renal function. The aim of this study was to assess cystatin C as an early marker of changes in glomerular filtration rate (GFR) after CABG. Methods Blood samples were collected from 61 CABG patients at different time points. Using 51Cr-ethylenediaminetetraacetic acid (51Cr-EDTA) clearance as a ‘gold standard’, we compared the correlations and non-parametric receiver operator characteristic curves of serum cystatin C, serum creatinine and 24 h creatinine clearance (Ccr). Results The inverse of cystatin C correlated better with 51Cr-EDTA than those of serum creatinine and Ccr ( r = 0.8578, 0.6771 and 0.6929, respectively). Cystatin C exhibited significantly superior diagnostic accuracy for detecting GFR 2 compared with serum creatinine ( P = 0.013) and Ccr ( P = 0.025); for detecting GFR 2, cystatin C had similar diagnostic accuracy to Ccr ( P = 0.812) but was superior to creatinine ( P = 0.033). At the best cut-off value, cystatin C had sensitivity 89% and specificity 93% for detecting GFR 2, sensitivity 86% and specificity 96% for detecting GFR 2. Conclusions Cystatin C is a better marker for detecting small temporary changes of GFR in CABG patients. This may allow better identification of patients with renal impairment.

Background The relationship between the levels of gonadotropic hormones and bone metabolism-related cytokines in Chinese women is unclear. We investigated the relationship between FSH and LH and OPG, leptin, TGF-β1, and TGF-β2 in Chinese women. Methods A cross-sectional study of 694 Chinese women, aged 20 to 82 y was conducted. Levels of serum FSH, LH, OPG, leptin, TGF-β1, and TGF-β2 were determined. Results In premenopausal females, serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels seemly showed no correlation with the cytokine levels. In perimenopausal females, serum FSH and LH levels showed significant positive correlation with osteoprotegerin (OPG) and transforming growth factor-β2 (TGF-β2) levels (r = 0.286 to 0.405, all P = 0.000), whereas they showed negative correlation with TGF-β1 levels (r = − 0.413 and − 0.354, all P = 0.000). In postmenopausal females, FSH and LH levels showed positive correlation with OPG levels (r = 0.247 and 0.241, all P = 0.000), negative correlation with leptin and TGF-β1 levels (r = − 0.234 to − 0.319, all P = 0.000), and no correlation with TGF-β2 levels. Multiple linear regression stepwise analysis revealed the following results. In premenopausal females, 2.0% and 1.5% of the changes in LH could be explained by OPG and leptin, respectively, while 1.9% of the changes in OPG could be explained by LH. In perimenopausal females, the determinants of OPG and TGF-β1 on FSH were 10.9% and 17.0%, respectively, and the determinants of OPG, TGF-β1 and TGF-β2 on LH were 4.5%, 4.9% and 16.4%, respectively. The determinants of FSH and LH on OPG were 14.5% and 2.5%, respectively. The determinant of FSH on TGF-β1 was 4.5%, while the determinant of LH on TGF-β2 was 16.4%. In postmenopausal females, the determinants of leptin and OPG on FSH were 10.2% and 2.8%, respectively, and the determinants of OPG and TGF-β1 on LH were 5.8% and 2.3%, respectively. The determinant of FSH on OPG, leptin and TGF-β1 were 6.1%, 3.4% and 9.2%. Conclusions These results indicate that age-related gonadotropic hormone levels are associated with changes in OPG, TGF-β1, TGF-β2 and leptin, and change with menopausal status.

Arterial calcification is common, but the mechanisms remain unclear. This study was undertaken to investigate the arterial calcification in adiponectin-deficient mice in vivo and the effects of adiponectin on cultured vascular smooth muscle cells in vitro. Alizarin red S staining was used to detect arterial calcification of adiponectin(-/-) mice. Alkaline phosphatase activity, osteocalcin secretion, and Runx2 protein expression were examined in cultured calcifying vascular smooth muscle cells (CVSMCs). The involved signal pathway was studied using a mitogen-activated protein kinase (MAPK) inhibitor and adiponectin receptor 1 (AdipoR1) siRNA. Adiponectin(-/-) mice developed slight arterial calcification after being fed with normal chow diet for 30 wk. Adenovirus-mediated supplement of adiponectin attenuated arterial calcification in these mice. On cultured CVSMCs, adiponectin inhibited ALP activity, osteocalcin secretion, Runx2 protein expression, and the formation of mineralized nodules. Adiponectin receptor 1 (AdipoR1) protein was detected in CVSMCs, and adiponectin activated p38 mitogen-activated protein kinase. Furthermore, inhibition of AdipoR1 expression or p38 activation reversed the effects of adiponectin on ALP activity. These results showed that adiponectin inhibited osteoblastic differentiation of CVSMCs through the AdipoR1/p38 signaling pathway. Our findings showed that adiponectin(-/-) mice developed arterial calcification, and this could be attributed to the loss of inhibitory action of adiponectin on osteoblastic differentiation of CVSMCs. It suggested that adiponectin plays a protective role against arterial calcification.

Background Transforming growth factor-β 1 (TGF-β1), TGF-β2, osteoprotegerin (OPG), and leptin are important cytokines in the regulation of bone remodeling. We investigated the relationship of TGF-β1 and TGF-β2 concentrations with those of OPG and leptin in Chinese females. Methods The serum concentrations of TGF-β1, TGF-β2, OPG, and leptin were measured by ELISA in 459 healthy Chinese females aged 25–80 y. Results The mean values (± SD) of the serum concentrations of TGF-β1, TGF-β2, OPG, and leptin in Chinese females were 29.7 ± 1.69 μg/l, 13.7 ± 3.86 μg/l, 3.81 ± 1.96 pmol/l, and 10.5 ± 2.01 μg/l, respectively. Further, the serum TGF-β1 concentrations of postmenopausal women were significantly lower than those of perimenopausal and premenopausal women (24.3 ± 1.59 vs 33.4 ± 1.69 and 37.6 ± 1.64, respectively), while the TGF-β2 concentrations of postmenopausal women were significantly higher than those of perimenopausal and premenopausal women (14.6 ± 3.91 vs 13.5 ± 3.93 and 11.7 ± 2.68, respectively). The serum TGF-β1 concentration was found to be significantly negatively correlated with age (r = − 0.335, P = 0.000) and the TGF-β2 concentration, to be significantly positively correlated with age (r = 0.230, P = 0.000). The TGF-β1 concentration was found to be significantly negatively correlated with both TGF-β2 (r = − 0.261, P = 0.000) and OPG (r = − 0.313, P = 0.000) concentrations; a significantly positive correlation was found between the TGF-β1 and leptin concentrations (r = 0.164, P = 0.000) and between TGF-β2 and OPG concentrations (r = 0.432, P = 0.000). Conclusion These results provide age-related reference values of TGF-β1 and TGF-β2 in Chinese adult women, and reveal the relationships between these cytokines.

Background Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) may play an important role in bone mass regulation in postmenopausal women. Methods A cross-sectional study of 699 healthy Chinese women, aged 20 to 82 y, was conducted. Serum FSH and LH and BMD were measured at the posteroanterior (PA) spine, lateral spine, total hip, and distal forearm. Results The geometric mean values (± SD) of serum FSH and LH in premenopausal women were 3.94 ± 2.08 and 7.51 ± 2.58 IU/l, respectively, and in postmenopausal women were 28.8 ± 1.88 and 25.6 ± 1.95 IU/l, respectively. The correlation of FSH to BMD at different skeletal regions (r = − 0.597 – − 0.492, P = 0.000) was higher than that of LH to BMD (r = − 0.452 – − 0.332, P = 0.000). The prevalences of osteoporosis for the quartiles of FSH at various skeletal sites were 0.57%, 0.43%, 27.1%, and 30.9%, respectively; and of LH were 2.14%, 4.43%, 19.5%, and 26.0%, respectively. The prevalence of osteoporosis in 3rd and 4th quartile was more significantly increased than the 1st and 2nd quartile. Conclusions These data suggest that FSH and LH levels in circulation are associated with BMD changes and osteoporosis occurrence in Chinese women.

Visfatin (also known as pre-B cell colony-enhancing factor or PBEF) is a novel adipocytokine that is highly expressed in visceral fat and upregulated in obesity and type 2 diabetes mellitus. Visfatin binds to and activates the insulin receptor (IR), thereby exerting insulin-mimetic effects in various cell lines. IR has been detected in osteoblasts, which is consistent with the role of insulin as an important osteotropic hormone. This study investigated the actions of visfatin on human primary osteoblasts. The expression and tyrosine phosphorylation of IR, IR substrate-1 (IRS-1), and IRS-2 were determined by immunoprecipitation and immunoblotting. Cell proliferation was determined by measuring [(3)H]thymidine incorporation and cell number. Glucose uptake was determined by measuring 2-[(3)H]deoxyglucose incorporation. Real-time quantitative reverse-transcription polymerase chain reaction (PCR) was used for determining alkaline phosphatase (ALP), osteocalcin, and type I collagen mRNA expression. Enzyme-linked immunosorbent assay and radioimmunoassay were used for measuring ALP activity, osteocalcin secretion, and type I collagen production. We found that visfatin induced tyrosine phosphorylation of IR, IRS-1, and IRS-2. Moreover, the effects of visfatin - glucose uptake, proliferation, and type I collagen enhancement of cultured human osteoblast-like cells - bore a close resemblance to those of insulin and were inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester, a specific inhibitor of IR tyrosine kinase activity. We also unexpectedly found that visfatin downregulated osteocalcin secretion from human osteoblast-like cells. These data indicate that the regulation of glucose uptake, proliferation, and type I collagen production by visfatin in human osteoblasts involves IR phosphorylation, the same signal-transduction pathway used by insulin.