Mechanisms Of The Effects Of Apelin On The Proliferation And Apoptosis Of Human Osteoblasts

Mechanisms of the Effects of Apelin on the Proliferation and Apoptosis of Human OsteoblastsObjective: Apelin is a recently discovered peptide that is produced in a variety of tissues and has a lot of biological functions. Adipocytes can express and secrete apelin. APJ mediates the biological effects of apelin. The aim of this study was to characterize apelin and APJ expression in osteoblasts and to investigate the effects of apelin on human osteoblasts proliferation and differentiation.Methods: Alizarin Red S staining was used for assessment of human osteoblasts matrix mineralization. The expression of apelin and APJ mRNA in primary-cultured human osteoblasts, human osteoblast-like MG-63 cells and mouse MC3T3-E1 osteoblasts were determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR); Western blot detected apelin and APJ protein expression in primary-cultured human osteoblasts. Human osteoblasts proliferation was assessed by measuring [~3H]thymidine (2μCi/ml) incorporation into trichloroacetic acid (TCA)-insoluble material and by cell count. Cell apoptosis was analyzed by ELISA. Alkaline phosphatase (ALP) activity was measured using ALP ELISA kit, osteocalcin (OC) was measured by RIA, Procollagen I C-terminal propeptide (PICP) in the cell-conditioned medium was measured using an ELISA Kit. The expression of Bcl-2, Bax, caspase-3, cytochrome C, p-Akt, Akt, p-JNK, JNK, p-p38, p38, p-ERK1/2 and ERK1/2 were measured using Western blot, and the expression of PI3-kinase p85αwas detected by immuno-precipitation. RNA interference was used to down-regulate the expression of APJ in human osteoblasts. PI3-K inhibitor LY294002 and Akt inhibitor HIMO were also used for the signaling pathway study.Results: (1) Apelin and APJ mRNA were expressed in primary-cultured human osteoblasts, osteoblast-like MG63 cells and MC3T3-E1 osteoblasts. Western blot detected apelin and APJ protein expression in primary-cultured human osteoblasts. (2) ALP activity, OC and typeⅠcollagen secretion were not affected by apelin. (3) Apelin stimulated human osteoblasts proliferation and inhibited human osteoblasts apoptosis, suppression of APJ with siRNA abolished apelin's effects on human osteoblasts proliferation and apoptosis. (4) Western blot analysis showed that apelin increased Bcl-2 protein and decreased Bax protein expression, and also blocked the release of cytochrome c and activation of caspase-3.(5) Apelin activated the phosphorylation of phosphatidylinositol 3-kinase(PI3-K)and Akt; these effects were blocked by suppression of APJ with siRNA; pretreatment of osteoblasts with PI3-kinase inhibitor LY294002 or Akt inhibitor HIMO abolished apelin-induced activation of Akt; furthermore, LY294002 or HIMO abolished apelin's mitogenic and anti-apoptotic activity in human osteoblasts.Conclusion: Human osteoblasts express apelin and APJ and apelin enhances human osteoblast proliferation and inhibits its apoptosis through APJ/PI3-K/Akt pathway, but has no effect on osteoblast differentiation.