Li-Juan Guo

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.

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-125a TNF receptor-associated factor 6 to inhibit osteoclastogenesis

MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. In the present study, we found that miR-125a was dramatically down-regulated during macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclastogenesis of circulating CD14+ peripheral blood mononuclear cells (PBMCs). Overexpression of miR-125a in CD14+ PBMCs inhibited osteoclastogenesis, while inhibition of miR-125a promoted osteoclastogenesis. TNF receptor-associated factor 6 (TRAF6), a transduction factor for RANKL/RANK/NFATc1 signal, was confirmed to be a target of miR-125a. EMSA and ChIP assays confirmed that NFATc1 bound to the promoter of the miR-125a. Overexpression of NFATc1 inhibited miR-125a transcription, and block of NFATc1 expression attenuated RANKL-regulated miR-125a transcription. Here, we reported that miR-125a played a biological function in osteoclastogenesis through a novel TRAF6/ NFATc1/miR-125a regulatory feedback loop. It suggests that regulation of miR-125a expression may be a potential strategy for ameliorating metabolic disease.

Relationship between serum omentin-1 level and bone mineral density in girls with anorexia nervosa

Background: Adolescents with anorexia nervosa (AN) have low bone mineral density (BMD). Omentin-1, the main circulating adipocytokine, plays an important role in bone metabolism in healthy individuals. However, their association with bone metabolism in AN is unknown. Methods: Serum omentin-1, bone turnover biochemical markers, and BMD were determined in 26 girls with AN and 24 healthy girls (15–18 years old). Results: Omentin-1 levels increased in AN subjects, and the differences became greater after controlling for fat mass. Omentin-1 was negatively correlated with BMD. In the multiple linear stepwise regression analysis, omentin-1, body mass index and lean mass, but not fat mass, were independent predictors of BMD for the combined group. Significant negative correlations were found between omentin-1 and bone-specific alkaline phosphatase, bone cross-linked N-telopeptides of type I collagen. Omentin-1 was also independently associated with BMD and bone turnover markers in the AN and control groups considered separately. Conclusions: Omentin-1 was an independent predictor of BMD in adolescents with AN, and negatively correlated with bone turnover markers. This suggested that omentin-1 may exert a negative effect on bone mass by inhibiting bone formation in girls with AN.

Relationship of circulating MMP–2, MMP–1, and TIMP–1 levels with bone biochemical markers and bone mineral density in postmenopausal Chinese women

IntroductionOsteoblast-derived matrix metalloproteinase (MMP)–2, MMP–1 and tissue inhibitor of metalloproteinase (TIMP)–1 have been shown to play a role in bone metabolism by degrading the bone matrix.MethodsThe present study was performed to investigate the relationships between serum MMP–2, MMP–1, or TIMP–1 levels and bone mineral density (BMD), as well as bone biochemical markers, in 297 Chinese postmenopausal women aged 42–80 years.ResultsWe found a significant negative weak correlation between MMP–2 and BMD at various skeletal regions. After adjustment for age and BMI, the correlation with BMD at the femoral neck and total hip disappeared. Multiple linear stepwise regression analysis showed that MMP–2 was not a determinant factor for BMD. The significant positive correlations between MMP–2 and bone cross-linked N–telopeptides of type I collagen (NTX), alkaline phosphatase (BAP), and osteocalcin (OC) and were found, and remained significant after adjustment for age and BMI. Moreover, serum MMP–2 concentrations were significantly higher in postmenopausal women with osteoporosis than in age-matched normal controls. There were no significant correlations between MMP–1, TIMP–1 and BMD. There were no significant relationships between MMP–1 and BAP, OC, and NTX. The associations between TIMP–1 and BAP and OC were not specific and constant.ConclusionsIn conclusion, our results suggest that circulating MMP–2 and markers of bone turnover are correlated, and serum MMP–2 levels may rise with increase in bone turnover.

Tissue inhibitor of matrix metalloproteinase-1 suppresses apoptosis of mouse bone marrow stromal cell line MBA-1

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.

A novel microRNA regulates osteoclast differentiation via targeting protein inhibitor of activated STAT3 (PIAS3)

MicroRNAs (miRNAs) involve in the regulation of a wide range of physiological processes. Recent studies suggested that miRNAs might play a role in osteoclast differentiation. Here, we identify a new miRNA (miR-9718) in primary mouse osteoclasts that promotes osteoclast differentiation by repressing protein inhibitor of activated STAT3 (PIAS3) at the post-transcriptional level. MiR-9718 was found to be transcribed during osteoclastogenesis, which was induced by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Overexpression of miR-9718 in RAW 264.7 cells promoted M-CSF and RANKL-induced osteoclastogenesis, whereas inhibition of miR-9718 attenuated it. PIAS3 was predicted to be a target of miR-9718. Luciferase reporter gene validated the prediction. Transfection of pre-miR-9718 in RAW 264.7 cells induced by both M-CSF and RANKL inhibited expression of PIAS3 protein, while the mRNA levels of PIAS3 were not attenuated. In vivo, our study showed that silencing of miR-9718 using a specific antagomir inhibited bone resorption and increased bone mass in mice receiving ovariectomy (OVX) and in sham-operated control mice. Thus, our study showed that miR-9718 played an important role in osteoclast differentiation via targeting PIAS3 both in vitro and in vivo.

miR-23a/b regulates the balance between osteoblast and adipocyte differentiation in bone marrow mesenchymal stem cells

Age-related osteoporosis is associated with the reduced capacity of bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteoblasts instead of adipocytes. However, the molecular mechanisms that decide the fate of BMSCs remain unclear. In our study, microRNA-23a, and microRNA-23b (miR-23a/b) were found to be markedly downregulated in BMSCs of aged mice and humans. The overexpression of miR-23a/b in BMSCs promoted osteogenic differentiation, whereas the inhibition of miR-23a/b increased adipogenic differentiation. Transmembrane protein 64 (Tmem64), which has expression levels inversely related to those of miR-23a/b in aged and young mice, was identified as a major target of miR-23a/b during BMSC differentiation. In conclusion, our study suggests that miR-23a/b has a critical role in the regulation of mesenchymal lineage differentiation through the suppression of Tmem64.

Stimulation of RANKL and inhibition of membrane-type matrix metalloproteinase-1 expression by parathyroid hormone in normal human osteoblasts.

Receptor activator of NF‐κB (RANK) ligand (RANKL), expressed by cells of the osteoblast lineage binds to RANK, induces signaling and a gene expression cascade that leads to osteoclast differentiation and activation. Recently, osteoblast‐derived membrane‐type matrix metalloproteinases‐1 (MT1‐MMP) have been implicated in the process of bone resorption by degrading bone matrix. In the present study, we investigated the effects of parathyroid hormone [PTH (1–34)] on RANKL and MT1‐MMP production in cultured normal human osteoblast‐like cells (hOB). In reverse transcription polymerase chain reaction studies, we observed that PTH (1–34) induced RANKL messenger ribonucleic acid (mRNA) expression. Activity assays demonstrated that PTH (1–34) simultaneously inhibited MT1‐MMP protein expression in a dose‐ and time‐dependent manner. The effect of PTH (1–34) on MT1‐MMP production was parallel to that on RANKL expression, suggesting a tight inverse relationship between MT1‐MMP and RANKL expression. Our findings indicated that the decreased MT1‐MMP expression by PTH may be involved in RANKL signaling in osteoblasts and activation of osteoclasts.