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Life Sci. 2014 Feb 6;96(1-2):18-25. doi: 10.1016/j.lfs.2013.12.017. Epub 2013 Dec 22.

Rho-kinase limits BMP-4-stimulated osteocalcin synthesis in osteoblasts: regulation of the p38 MAP kinase pathway.

Author information

1
Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan; Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.
2
Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan; Department of Clinical Laboratory, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511, Japan. Electronic address: tokuda@ncgg.go.jp.
3
Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.
4
Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan.

Abstract

AIM:

We previously reported that bone morphogenetic protein-4 (BMP-4) stimulates the synthesis of osteocalcin via p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells, whereas p44/p42 MAP kinase plays as a negative regulator in the synthesis. In the present study, we investigated whether Rho-kinase is involved in BMP-4-stimulated osteocalcin synthesis in MC3T3-E1 cells.

MAIN METHODS:

The levels of osteocalcin were measured by ELISA. The phosphorylation of each protein kinase was analyzed by Western blotting. The mRNA levels of osteocalcin were determined by real-time RT-PCR.

KEY FINDINGS:

BMP-4 induced the phosphorylation of myosin phosphatase targeting subunit-1 (MYPT-1), a substrate of Rho-kinase. Y27632 or fasudil, specific inhibitors of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly amplified the BMP-4-stimulated osteocalcin synthesis in a dose-dependent manner. The osteocalcin mRNA expression levels induced by BMP-4 were enhanced by Y27632 or fasudil. BMP-4-stimulated osteocalcin release was significantly up-regulated in Rho-knocked down cells with Rho A-siRNA. Y27632 or fasudil failed to affect the BMP-4-induced phosphorylation of SMAD1 or p44/p42 MAP kinase. On the other hand, Y27632 or fasudil markedly strengthened the phosphorylation levels of p38 MAP kinase induced by BMP-4.

SIGNIFICANCE:

These results strongly suggest that Rho-kinase negatively regulates BMP-4-stimulated osteocalcin synthesis via the p38 MAP kinase pathway in osteoblasts.

KEYWORDS:

BMP-4; MAP kinase; Osteoblast; Osteocalcin; Rho-kinase

PMID:
24368139
DOI:
10.1016/j.lfs.2013.12.017
[Indexed for MEDLINE]
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