Preptin Induces Human Osteoblasts Proliferation And Differentiation Through The ERK/CTGF Signaling Pathway

ObjectiveTo investigate the proliferation and differentiation effects of preptin on human osteoblasts and to approach the mechanism.MethodsPrimary cultures of normal human osteoblasts were prepared from trabecular bone obtained during surgery following traffic accidents None of the bone donors had clinical symptoms or histories of bone metabolic disorders. The phenotypes of cells were characterized based on the expression levels of ALP, collagen type 1 and osteocalcin and the formation of mineralization nodules. To observe the dose and time effects of preptin on human osteoblasts proliferation and differentiation, human osteoblasts were subsequently treated with vehicle or 10-10,10-9,10-8 or 10-7 M preptin for 24 h in serum-free a-MEM. Human osteoblasts proliferation was assessed by measuring [3H] thy mi dine incorporation into trichloroacetic acid-insoluble material. Human osteoblasts differentiation was evaluated by ALP level released into the lysate solution. ALP activity was assayed by spectrophotometric measurement of p-nitrophenol. By determine the level of CTGF secreting into the culture medium to observe whether preptin indicing the expression of CTGF in osteoblasts and its dose and time effects. CTGF was assayed by Western blot. With small interfering RNA for CTGF (CTGF-siRNA) to inhibit human osteoblasts expressing CTGF, or with cell signal pathway inhibitors, PD98059, SP600125 or SB203580 for 2 h prior to treatment with 10-9 M preptin to block mitogen-activated protein kinase (MAPK) signal pathway of human osteoblasts, so that analyzed whether and how the MAPK/CTGF signaling pathway was involved in the preptin inducing human osteoblasts proliferation and differentiation. Western blot was used to assay p38MAPK, ERK1/2, JNK and p-p38, p-ERK1/2, p-JNK. ResultsOur results demonstrated that preptin promoted human osteoblasts proliferation and alkaline phosphatase activity. Suppression of connective tissue growth factor (CTGF), whi ch was upregul ated by prepti n i n 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.ConclusionsPreptin is involved in bone anabolism mediated by ERK/CTGF in human osteoblasts and may contribute to the increase of bone mass in obesity. CTGF is a downstream mediator for prepti n-induced proliferation and differentiation in human osteoblasts.