Inhibition of Rac1 promotes BMP-2-induced osteoblastic differentiation

M Onishi,T Yamashita,Y Fujita,H Yoshikawa

doi:10.1038/cddis.2013.226

Abstract

Small G proteins of the Rho family are pivotal regulators of several signaling networks. The Ras homolog family (Rho) and one of its targets, Rho-associated protein kinase (ROCK), participate in a wide variety of biological processes, including bone formation. A previous study has demonstrated that the ROCK inhibitor Y-27632 enhanced bone formation induced by recombinant human bone morphogenetic protein-2 (BMP-2) in vivo and in vitro. However, the effect of other Rho family members, such as Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle 42 (Cdc42), on bone formation remains unknown. In this study, we investigated whether Rac1 also participates in BMP-2-induced osteogenesis. Expression of a dominant-negative mutant of Rac1 enhanced BMP-2-induced osteoblastic differentiation in C2C12 cells, whereas a constitutively active mutant of Rac1 attenuated that effect. Knockdown of T-lymphoma invasion and metastasis 1 (Tiam1), a Rac-specific guanine nucleotide exchange factor, enhanced BMP-2-induced alkaline phosphatase activity. Further, we demonstrated that BMP-2 stimulated Rac1 activity. These results indicate that the activation of Rac1 attenuates osteoblastic differentiation in C2C12 cells.

Highlights

It has been shown that the function of Bone morphogenetic proteins (BMPs) is modulated by the small GTPase Ras homolog family A (RhoA) and Rhoassociated protein kinase (Rho kinase, ROCK).[4,5] We have shown that the expression of a dominant-negative ROCK mutant in mouse stromal ST2 cells induced osteoblastic differentiation, whereas the expression of a constitutively active ROCK mutant attenuated osteoblastic differentiation.[4]
C2C12 cells were transfected with a dominant-negative mutant of Rac[1] (RacDN), a constitutively active mutant of Rac[1] (RacCA), or with a control plasmid, with or without recombinant human bone morphogenetic protein-2 (rhBMP-2)
To examine the role of triple functional domain (Trio) or Tiam[1] in C2C12 osteoblastic differentiation induced by BMP2, we examined the BMP-2-induced alkaline phosphatase (ALP) activity in these cells

Summary

Introduction

It has been shown that the function of BMPs is modulated by the small GTPase Ras homolog family A (RhoA) and Rhoassociated protein kinase (Rho kinase, ROCK).[4,5] We have shown that the expression of a dominant-negative ROCK mutant in mouse stromal ST2 cells induced osteoblastic differentiation, whereas the expression of a constitutively active ROCK mutant attenuated osteoblastic differentiation.[4] Continuous supply of a specific ROCK inhibitor (Y-27632) enhanced ectopic bone formation induced by BMP-2.4 Propagating these effects, neogenin acts as a receptor for BMPs and activates RhoA.[5] Knockdown of neogenin in mouse C2C12 myoblasts promoted BMP-2-induced osteoblastic differentiation, whereas overexpression of neogenin suppressed this process.[5] These findings suggest that RhoA–ROCK signaling negatively regulates BMP-induced osteoblastic differentiation. We assessed the role of Rac[1] in BMP-2-induced osteogenesis

Methods

Results

Conclusion