Rho-kinase negatively regulates thyroid hormone-stimulated osteocalcin synthesis in osteoblasts

Evidence is accumulating that Rho-associated kinase (Rho-kinase) plays important roles not only in vascular smooth muscle cell contraction, but also in a variety of cellular functions, including bone metabolism. In the present study, we investigated the involvement of Rho-kinase in the osteocalcin synthesis induced by triiodothyronine (T₃) in osteoblast-like MC3T3-E1 cells. T₃ time-dependently induced phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632, a specific inhibitor of Rho-kinase, attenuated the MYPT-1 phosphorylation induced by T₃. T₃-stimulated osteocalcin release was significantly enhanced by Y27632. Fasudil, another Rho-kinase inhibitor, amplified the osteocalcin release induced by T₃. T₃-stimulated osteocalcin release was significantly augmented in Rho-knockdown cells with Rho A-siRNA. Y27632 and fasudil also increased the mRNA expression level of osteocalcin induced by T₃. These results strongly suggest that T₃ stimulates the activation of Rho-kinase in osteoblasts, which functions as a negative regulator of T₃-stimulated osteocalcin synthesis.