Xian-Ping Wu

Adiponectin Stimulates RANKL and Inhibits OPG Expression in Human Osteoblasts Through the MAPK Signaling Pathway

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.

A RUNX2/MIR-3960/MIR-2861 regulatory feedback loop during mouse osteoblast differentiation

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.

Application of micro-ct assessment of 3-d bone microstructure in preclinical and clinical studies

As the mechanical competence of trabecular bone is a function of its apparent density and 3-D distribution, assessment of 3-D trabecular structural characteristics may improve our ability to understand the pathophysiology of osteoporosis, to test the efficacy of pharmaceutical intervention, and to estimate bone biomechanical properties. We have studied ovariectomy-induced osteopenia in rats and its treatment with agents such as estrogen and sodium fluoride. We have demonstrated that 3-D micro-computed tomography (µCT) can directly quantify mouse trabecular and cortical bone structure with an isotropic resolution of 6 µm3. µCT is also useful for studying osteoporosis in mice and phenotypes of mice with gene manipulation, such as SHIP-knockout mice, which are severely osteoporotic due to increased numbers of hyperresorptive osteoclasts, PTHrP heterozygous-null mice, and mice with Zmpste24 deficiency. µCT can quantify osteogenesis in mouse Ilizarov leg-lengthening procedures, osteoconduction in a rat cranial defect model, and structural changes in arthritic rabbits, rats, and mice. In clinical studies, we evaluated longitudinal changes in the iliac crests. Paired bone biopsies from the same premenopausal and postmenopausal women showed the changes in 3-D trabecular structure, such as decreased trabecular thickness, shifting of trabecular model from platelike structure to rodlike structure, and decreased degree of anisotropy were remarkable. Treatment with PTH in postmenopausal women with osteoporosis significantly improved trabecular morphology with a shift toward a more platelike structure, increased trabecular connectivity density, and increased cortical thickness. Paired bone biopsy specimens from the iliac crest in postmenopausal women with osteoporosis before and an average of 2 years after beginning of estrogen replacement therapy demonstrated that posttreatment biopsies showed a significant change in the ratio of plates to rods and statistically insignificant changes in other 3-D trabecular parameters. Thus, µCT can characterize 3-D structure of various animal models, and the longitudinal changes in 3-D bone microarchitectural integrity that deteriorates in the transmenopausal period, is preserved with HRT, and is improved with PTH treatment in postmenopausal women.

miR-148a regulates osteoclastogenesis by targeting V-maf musculoaponeurotic fibrosarcoma oncogene homolog B.

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.

Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 via JNK and PI3-K/Akt signaling pathways.

The aim of this study was to investigate the effects of apelin on proliferation and apoptosis of mouse osteoblastic MC3T3-E1 cells. APJ was expressed in MC3T3-E1 cells. Apelin did not affect Runx2 expression, alkaline phosphatase (ALP) activity, osteocalcin and type I collagen secretion, suggesting that it has no effect on osteoblastic differentiation of MC3T3-E1 cells. However, apelin stimulated MC3T3-E1 cell proliferation and inhibited cell apoptosis induced by serum deprivation. Our study also shows that apelin decreased cytochrome c release and caspase-3, capase-8 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Apelin activated c-Jun N-terminal kinase (JNK) and Akt (phosphatidylinositol 3-kinase downstream effector), and the JNK inhibitor SP600125, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO) inhibited its effects on proliferation and serum deprivation-induced apoptosis. Furthermore, apelin protected against apoptosis induced by the glucocorticoid dexamethasone or TNF-alpha. Apelin stimulates proliferation and suppresses serum deprivation-induced apoptosis of MC3T3-E1 cells and these actions are mediated via JNK and PI3-K/Akt signaling pathways.

Apelin suppresses apoptosis of human osteoblasts.

Objectives: Apelin is a recently discovered peptide that is the endogenous ligand for the orphan G-protein-coupled receptor APJ. Adipocytes can express and secrete apelin. Osteoblast can express apelin and APJ. The aim of this study was to investigate the action of apelin on apoptosis of human osteoblasts. Results: Apelin inhibited human osteoblasts apoptosis induced by serum deprivation. Suppression of APJ with small-interfering RNA (siRNA) abolished the anti-apoptotic activity of apelin. Our study also showed an increased Bcl-2 protein expression and decreased Bax protein expression under the treatment of apelin. Apelin decreased cytochrome c release and caspase-3 activation in human osteoblasts. Apelin activated phosphatidylinositol-3 kinase (PI-3 kinase) and Akt. The apelin-induced activation of Akt was blocked by suppression of APJ with siRNA. LY294002 (a PI-3 kinase inhibitor) or 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO; an Akt inhibitor) abolished apelin induced activation of Akt, and, LY294002 or HIMO abolished the anti-apoptotic activity of apelin. Furthermore, apelin protects against apoptosis induced by the glucocorticoid dexamethasone. Conclusions: Apelin suppresses serum deprivation-induced apoptosis of human osteoblasts and the anti-apoptotic action is mediated via the APJ/PI-3 kinase/Akt signaling pathway.

MiR‐503 Regulates Osteoclastogenesis via Targeting RANK

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.

miR-93/Sp7 function loop mediates osteoblast mineralization

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.

Apelin and its receptor are expressed in human osteoblasts.

OBJECTIVES Apelin is a recently discovered peptide that is the endogenous ligand for the orphan G-protein-coupled receptor APJ. Adipocytes can express and secrete apelin. The aim of this study was to characterize apelin and APJ expression in human osteoblasts and to investigate the effects of apelin on osteoblasts. RESULTS Apelin and APJ were expressed in human osteoblasts. Apelin stimulated proliferation of human osteoblasts, but had no effect on alkaline phosphatase (ALP) activity, osteocalcin and type I collagen production in human osteoblasts. Suppression of APJ with small-interfering RNA (siRNA) abolished the apelin-induced cell proliferation. Apelin induced activation of Akt (Phosphatidylinositol-3 kinase downstream effector), but not MAPKs, such as c-jun N-terminal Kinase (JNK), p38 and ERK1/2 in human osteoblasts. This effect was blocked by suppression of APJ with siRNA. Furthermore, LY294002 (PI3 kinase inhibitor) blocked the activation of Akt by apelin and abolished the apelin-induced cell proliferation. CONCLUSIONS Human osteoblasts express apelin and APJ and apelin enhances human osteoblast proliferation, but has no effect on osteoblast differentiation, and APJ/PI3 kinase/Akt pathway is involved in the proliferation response. These findings suggest that apelin may function as a mitogenic agent for osteoblasts.

Apelin suppresses apoptosis of human vascular smooth muscle cells via APJ/PI3-K/Akt signaling pathways.

Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in regulating vascular remodeling during cardiovascular diseases. Apelin is the endogenous ligand for the G-protein-coupled receptor APJ and plays an important role in the cardiovascular system. However, the mechanisms of apelin on apoptosis of VSMCs have not been elucidated. Using a culture of human VSMCs as a model for the study of apoptosis, the relationship between apelin and apoptosis of human VSMCs and the signal pathway involved were investigated. Using western blotting, we confirmed that VSMCs could express APJ. To evaluate the possible role of apelin in VSMC apoptosis, we assessed its effect on apoptosis of human VSMCs. The results showed that apelin inhibited human VSMCs apoptosis induced by serum deprivation. Suppression of APJ with small-interfering RNA (siRNA) abolished the anti-apoptotic activity of apelin. Apelin increased Bcl-2 protein expression, but decreased Bax protein expression. An increase in activation of extracellular signal-regulated protein kinase (ERK) and Akt (a downstream effector of phosphatidylinositol 3-kinase) was shown after apelin stimulation. Suppression of APJ with siRNA abolished the apelin-induced activation of ERK and Akt. LY294002 (a PI3-K inhibitor) blocked apelin-induced activation of Akt and abolished the apelin-induced antiapoptotic activity. Our study suggests that apelin suppresses serum deprivation-induced apoptosis of human VSMCs, and that the anti-apoptotic action is mediated through the APJ/PI3-K/Akt signaling pathways.