Effects Of Estrogen On Adiponectin Regulation Of Human Osteoblast Differentiation And OPG/RANKL Expression

Part one Relationships Between Serum Adiponectin, Leptin Levels and Bone Mineral Density, and Bone Biochemical Barkers in Chinese WomenObjectivesAdiponectin and leptin, as the main circulating peptides secreted by adipose tissue, are potential contributors to bone metabolism. However, their associations with bone mineral density (BMD) are unclear. The present study was to investigate whether these serum adipocytokines' levels are associated with BMD and bone turnover markers.Materials and MethodsIn this study,336 postmenopausal and 265 premenopausal Chinese women were included. Serum concentrations of adiponectin, leptin, bone-specific alkaline phosphatase (BAP) and bone cross-linked N-telopeptides of type I collagen (NTX) were measured with ELISA. Serum levels of 25-hydroxyvitamin D, parathyroid hormone (PTH), insulin, estradiol (E2), total testerone were detected by radioimmunoassay (RIA). BMD values were measured using DXA at the total body, lumbar spine, total hip, and total forearm. Body compositions including lean tissue mass and body fat mass were also measured by DXA. The correlations between adiponectin, leptin and BMD, were made using Pearson's correlation analysis and Partial correlation analysis. Multivariate linear stepwise analysis was performed to determine how much of the variance in BMD at various skeletal regions could be explained by age, BMI, lean mass, smoking habits, calcium intake, physical activity, adiponectin, etc.ResultsIn postmenopausal Chinese women, the multiple linear stepwise regression analysis showed that year since menopause, lean mass, E2 and adiponectin, but not fat mass and leptin, were independent predictors of BMD (P＜0.05 or P＜0.01). However, in premenopausal Chinese women, adiponectin was not the predictor of BMD. The significant positive correlations between adiponectin and BAP, NTX and NTX/BAP were found only in postmenopausal women even adjusted by both age and fat mass (all P<0.05).ConclusionsAdiponectin was an independent predictor of BMD, and positively correlated with bone turnover biochemical markers in postmenopausal women, but not in premenopausal women. It suggested that adiponectin might exert a negative effect on bone mass by promoting excessive bone resorption associated with bone loss. However, these effects may be mediated by menopausal status. Chapter two Effects of E2 on Adiponectin-Regulated Human Osteoblasts DifferentiationObjectivesOur study showed that adiponectin could exert a negative effect on bone mass in postmenopausal women, but not in premenopausal women. This suggested the action of adiponectin on BMD might be influenced by the levels of circulating estrogen in women. The present study was undertaken to investigate the effect of estrogen (Estradiol, E2) on adiponectin-regulated human osteoblast (HOB) differentiation.MethodsHOBs were treated with a-minimum essential medium (a-MEM) or a-MEM containing 10μg/mL adiponectin or 10μg/mL adiponectin together with 10-10～10-8M E2 for 48 hours. HOBs were also exposed toα-MEM containing 10μg/mL adiponectin alone or together with 10-8 M E2 for 12～48 hours. In addition, HOBs were treated with a-MEM containing 10μg/mL adiponectin and10-8M E2, adding p38 agonist, estrogen receptor antagonist or not respectively, for 48 hours. HOBs differentiation were assessed by measuring ALP activity and osteocalcin (OC) secretion. ALP activity was determined by using p-nitrophenyl phosphate as substrate, OC secretion was measured by using a specific radioimmunoassay (RIA) kit, p38 and p-p38 were analysed by Western Blot.ResultsAdiponectin stimulated ALP activity, OC secretion and phosphorylation of p38 kinase, whereas E2 inhibited ALP activity, OC secretion (all P<0.01) and phosphorylation of p38 kinase significantly. In cells treated with adiponectin together with E2, E2 significantly decreased adiponectin-induced ALP activity, OC secretion (P<0.05 or P<0.01) and p38 activation in a dose-or time-dependent manner. However, the ALP activity and OC secretion depressed by E2 were increased significantly by addition of p38 agonist (P<0.05) or estrogen receptor antagonist (P<0.01).ConclusionThese data indicated that E2 supressed adiponectin-induced HOB differentiation via inhibiting the phosphorylation of MAPK p38 kinase. Chapter three The Effects of E2 on Adiponectin-Modulated the Expression of OPG and RANKL in Human OsteoblastsObjectivesAs it was showed, that E2 supressed adiponectin to promote HOB differentiation by inhibiting the phosphorylation of MAPK p38 kinase. This study was further undertaken to investigate the effects of E2 on adiponectin-regulated osteoprotegerin (OPG) and receptor activator of nuclear factor-KB ligand (RANKL) expression in HOB.Materials and MethodsHOBs were treated with a-MEM or a-MEM containing 10μg/mL adiponectin alone or together with 10-10～10-8 M E2 for 48 hours. Those were also exposed to a-MEM containing 10μg/mL adiponectin alone or together with 10-8M E2 for 12～48 hours. In addition, HOBs were treated with a-MEM containing 10μg/mL adiponectin and 10-8 M E2, adding p38 agonist, estrogen receptor antagonist or not respectively, for 48 hours. Real-time quantitative PCR (RT-PCR) was used to detect OPG and RANKL mRNA expression, and ELISA was used to detect their protein expression in cultured HOB respectively.ResultsAdiponectin inhibited OPG mRNA expression and protein secretion but stimulated RANKL mRNA expression and protein secretion (all P<0.01). Whereas, E2 stimulated OPG mRNA expression and protein secretion but inhibited RANKL mRNA expression and protein secretion (all P<0.01). In cells treated with adiponectin and E2 together, E2 enhanced adiponectin-regulated OPG mRNA and protein expression while attenuated RANKL mRNA and protein expression in a dose-and time-dependent manner (P<0.05 or P＜0.01),but these effects of E2 were reversed by addition of p38 agonist or estrogen receptor antagonist (all P＜0.01).ConclusionsE2 supressed the action of adiponectin on modulation of OPG and RANKL expression through MAPK/p38 pathway. This might be a mechanism that BMD was negatively correlated with adiponectin levels in postmenopausal women but not in premenopausal women.