Functional And Mechanism Research Of Insulin Receptor Substrate 1 On The Cell Differentiation In Mouse Pre-osteoblasts And Pre-adipocytes

Objective:To observe the effects of insulin receptor substrate 1 (IRS-1) gene silence and core bingding factor alpha 1(cbfa-1) over-expression on the proliferation,differentiation and alkaline phosphatase(ALP),osteocalcin,bone morphogenetic proteins(BMPs), osteoprotegerin,osteopontin and osteonectin expression in MC3T3-E1 pre-osteoblasts and 3T3-L1 pre-adipocytes,and discover the mechanisms of IRS-1 involved in the differentiation of pre-adipocyte and pre-osteoblasts.Methods:Three short hairpin RNA(shRNA) fragments of the IRS-1 gene were constructed into pGenesil-1 vector,then transfected into pre-adipocyte and pre-osteoblast,over-expression of Cbfa-1/P56 or Cbfa-1/P57 was used as positive controls for bone biochemistry marker assay.Cell lysate and supernatant were collected for further use.The IRS-1 knock-down and the adipsin expression were validated by western blot,the alkaline phosphatase,osteocalcin,bone morphogenetic proteins,osteoprotegerin,osteopontin and osteonectin expression was analyzed by ELISA.The fat droplets formation of 3T3-L1 cells with or without IRS-1 knock down was identified by oil-red O staining,the mineralization of 3T3-L1 cells with IRS-1 knock down was detected by Von kossa's silver staining after induction by Vitamin c and 13-glycerol-phosphoric acid sodium.Results:The expression of IRS-1 was successfully silenced down by shRNA,osteopontin,ALP,osteonectin,osteoprotegerin, and BMP expressions were down-regulated significantly following the IRS-1 silence in MC3T3-E1 pre-osteoblast cells(P＜0.05),no difference was found in osteocalcin expression.However,in 3T3-L1 pre-adipocytes,IRS-1 silence led to the up-regulation of osteocalcin, ALP,osteoprotegerin and BMP,but the osteonectin expression was down-regulated in 3T3-L1 cell.Von kossa's silver staining showed that 3T3-L1 adipocytes with IRS-I knock down can be induced to form the mineralization nodules with the VitaminC andβ-glycerol-phosphoric acid sodium treatment.The cellular proliferation was decreased by IRS-1 knock down in MC3T3-E1 cells and 3T3-L1.Conclusion:IRS-1 promotes the cellular differentiation, proliferation and extracellular matrix formation both in MC3T3-E1 and 3T3-L1 cells.The first time we found that IRS-1 knock-down increased the ability of 3T3-L1 pre-adipocytes trans-differentiate into osteoblasts,therefore IRS-1 plays important roles in dual-directional differentiation of pre-osteoblasts and pre-adipocytes.