Regulation Of Proliferation And Differentiation Of MC3T3-E1 By CHIP

PEF-neu-CHIP vector was transfected to MC3T3-E1 cells. Then cells were cultured with α-mimal essential medium supplemented with 1.0mg/ml Geneticin(G418). The surviving (Geneticin-resistant ) cells were cloned. Finally overexpression of CHIP protein was detected by Western blot in MC3T3-E1-HA-CHIP stable cell line. MC3T3-E1 wild type cell line and CHIP-overexpressing cell lines were induced towards osteoblast with condition medium (10mM/mlglycerophosphatesodium and 50ug/ml ascorbic acid). Differentiation and mineralization of two cell lines were verified by assay of Alkaline phosphatase (AKP) activity , AKP staining, Von Kossa staining and Alizarin Red staining. The results showed that AKP activity of CHIP-overexpressing cell lines were lower than that of MC3T3-E1 wild type cell line. The same results were testified by AKP staining. Mineralization of CHIP-overexpressing cell lines were also lower than that of MC3T3-E1 wild type cell line . This result was proved by Von Kossa staining and Alizarin Red staining. All above results demonstrated that overexpression of CHIP inhibited differentiation and mineralization of MC3T3-E1 cell line. In order to discuss molecular mechanism of the underlying function of CHIP protein on differentiation and mineralization of MC3T3-E1 cell line ,luciferase assay and western blot were carried out. The results showed that transcription of Smad3 was down regulated by overexpression of CHIP with luciferase assay, and Smad3 protein was down regulated in CHIP-overexpressing cell lines by western blot. It implied that CHIP possibly degraded Smad3 protein and ngatively regulated Smad3-induced Alkaline phosphatase activity and mineralization in MC3T3-E1 cell line. Finally the cell proliferation assay was peformed, and the results showed that CHIP inhibited proliferation in CHIP-overexpressing cell lines.