Insulin Potentiates the Proliferation and Bone Morphogenetic Protein-2-Induced Osteogenic Differentiation of Rat Spinal Ligament Cells via Extracellular Signal-Regulated Kinase and Phosphatidylinositol 3-Kinase

Study Design. This study was designed to confirm the correlation of hyperinsulinemia with ossification of posterior longitudinal ligament ( OPLL) of the cervical spine in vitro. Objective. To investigate the effects of insulin on the proliferation, collagen synthesis, and osteogenic differentiation of isolated rat spinal ligament cell in the presence or absence of bone morphogenetic protein-2 ( BMP-2). Summary of Background Data. Noninsulin-dependent diabetes mellitus is an independent risk factor for the onset of OPLL, but the mechanism is still unknown. We have hypothesized that insulin may exert direct effects on the proliferation and osteogenic differentiation of spinal ligament cells. Methods. Cells isolated from rat spinal ligaments were stimulated by different concentrations of insulin in the presence or absence of BMP-2. The proliferation of the cell was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid ( MTT) and direct cell counting and the procollagen type I amino-terminal peptide was measured by enzyme-linked immunosorbent assay. The gene expressions of insulin receptor, alkaline phosphatase, osteocalcin and Runx2 were examined by quantitative reverse transcriptase-polymerase chain reaction. PI3K/Akt and extracellular signal-regulated kinase ( ERK) were analyzed by western blotting. Results. Insulin positively regulated the expression of its receptor in the cells and stimulated the proliferation of the cells in a time- and dose-dependent manner. Insulin alone did not affect the synthesis of procollagen type I amino-terminal peptide synthesis or osteogenesis differentiation in the cells. However, high concentration of insulin ( 1000 nmol/L) significantly promoted BMP-2-induced alkaline phosphatase expression and activation, which was associated with the up-regulation of PI3-K/Akt and down-regulation of ERK in the cells. Conclusion. Hyperinsulinemia may contribute to the onset and progression of OPLL in subjects with noninsulin-dependent diabetes mellitus by stimulating the proliferation and BMP-2-induced osteogenic differentiation of ligament cells via the activation of insulin receptor and PI3-K/Akt pathway and the suppression of ERK.