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. PI3-K/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 noninsu-lin-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.

High serum levels of menatetrenone in male patients with ossification of the posterior longitudinal ligament.

STUDY DESIGN: This work was performed to investigate the role of vitamin K (VK) in the pathogenesis of ossification of posterior longitudinal ligament (OPLL), by analyzing the biochemical markers of the blood samples of OPLL patients and responses of ligament cells derived from OPLL lesion to VK2. OBJECTIVES: The pathogenesis of OPLL, classified as a form of diffuse idiopathic skeletal hyperostosis, is still unclear. In this study, we investigated the role of menaquinone (VK2) in patients with OPLL (OPLL patients) and the effects of VK2 on ligament cells isolated from OPLL lesion. METHODS: Serum levels of intact osteocalcin, glu-osteocalcin, MK-4, -7 (VK2 variants) and other minerals in spot blood samples were measured in 24 OPLL patients and in 24 age-matched control patients (non-OPLL patients). The cultured cells isolated from an OPLL patient were treated with MK-4. Alkaline phosphatase (Al-p) activity and osteocalcin release were measured after 2 weeks of culture. RESULTS: In the clinical study, the serum MK-4 in male OPLL patients was significantly higher than that in male non-OPLL patients. However, among female patients, the difference was not significant. Although the serum osteocalcin in females was significantly higher than that in males, there was no significant difference between the OPLL and non-OPLL groups. In in vitro study, MK-4 did not increase Al-p activity in the ligament cells isolated from nonossified region of OPLL patient. Osteoblastic activity of the cultured cells was not stimulated by MK-4. CONCLUSION: From these results and previous reports, we propose the possibility of the impediment in VK2 metabolism in OPLL patients. The results also implicate the gender tendency in OPLL, because the difference of serum level of MK-4 in OPLL patients was significant only in male.

Involvement of bone morphogenic protein-2 (BMP-2) in the pathological ossification process of the spinal ligament.

OBJECTIVE: To investigate the function of bone morphogenic protein-2 (BMP-2) in the ossification of the spinal ligament (OSL). METHODS: Total RNA was prepared from the cultured spinal ligament cells from patients with OSL and analysed by reverse transcription-polymerase chain reaction using specific primers for BMP-2. BMP-2 mRNA expression in ligament tissues was examined by in situ hybridization. Spinal ligament cells from patients without OSL were treated with BMP-2 and examined for alkaline phosphatase activity. RESULTS: Expression of the BMP-2 gene was detected in cultured spinal ligament cells. In ligament tissues, BMP-2 mRNA was present in the chondrocyte-like cells in the fibrocartilage zone. Exogenous BMP-2 increased alkaline phosphatase activity in spinal ligament cells from patients without OSL. CONCLUSION: The BMP-2 gene is expressed in the spinal ligaments of OSL patients, and exogenous BMP-2 stimulates osteogenic differentiation of spinal ligament cells. The expression of BMP-2 in the spinal ligaments could be a clue in elucidating how heterotrophic osteogenesis develops in ligament tissue.