Relaxin augments BMP-2-induced osteoblast differentiation and bone formation

J Bone Miner Res. 2014 Jul;29(7):1586-96. doi: 10.1002/jbmr.2197.

Abstract

Relaxin (Rln), a polypeptide hormone of the insulin superfamily, is an ovarian peptide hormone that is involved in a diverse range of physiological and pathological reactions. In this study, we investigated the effect of Rln on bone morphogenetic protein 2 (BMP-2)-induced osteoblast differentiation and bone formation. Expression of Rln receptors was examined in the primary mouse bone marrow stem cells (BMSCs) and mouse embryonic fibroblast cell line C3H/10T1/2 cells by RT-PCR and Western blot during BMP-2-induced osteoblast differentiation. The effect of Rln on osteoblast differentiation and mineralization was evaluated by measuring the alkaline phosphatase activity, osteocalcin production, and Alizarin red S staining. For the in vivo evaluation, BMP-2 and/or Rln were administered with type I collagen into the back of mice, and after 3 weeks, bone formation was analyzed by micro-computed tomography (µCT). Western blot was performed to determine the effect of Rln on osteoblast differentiation-related signaling pathway. Expression of Rxfp 1 in BMSCs and C3H/10T1/2 cells was significantly increased by BMP-2. In vitro, Rln augmented BMP-2-induced alkaline phosphatase expression, osteocalcin production, and matrix mineralization in BMSCs and C3H/10T1/2 cells. In addition, in vivo administration of Rln enhanced BMP-2-induced bone formation in a dose-dependent manner. Interestingly, Rln synergistically increased and sustained BMP-2-induced Smad, p38, and transforming growth factor-β activated kinase (TAK) 1 phosphorylation. BMP-2-induced Runx 2 expression and activity were also significantly augmented by Rln. These results show that Rln enhanced synergistically BMP-2-induced osteoblast differentiation and bone formation through its receptor, Rxfp 1, by augmenting and sustaining BMP-2-induced Smad and p38 phosphorylation, which upregulate Runx 2 expression and activity. These results suggest that Rln might be useful for therapeutic application in destructive bone diseases.

Keywords: BMP; BONE; RELAXIN; RXFP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bone Morphogenetic Protein 2 / pharmacology*
  • Calcification, Physiologic / drug effects
  • Cell Differentiation / drug effects*
  • Cell Line
  • Core Binding Factor Alpha 1 Subunit / metabolism
  • Humans
  • MAP Kinase Kinase Kinases / metabolism
  • Mice, Inbred C57BL
  • Osteoblasts / cytology*
  • Osteoblasts / drug effects
  • Osteoblasts / enzymology
  • Osteogenesis / drug effects*
  • Phosphorylation / drug effects
  • Protein Binding / drug effects
  • Receptors, G-Protein-Coupled / metabolism
  • Relaxin / pharmacology*
  • Smad Proteins / metabolism
  • p38 Mitogen-Activated Protein Kinases / metabolism

Substances

  • Bone Morphogenetic Protein 2
  • Core Binding Factor Alpha 1 Subunit
  • RXFP1 protein, mouse
  • Receptors, G-Protein-Coupled
  • Runx2 protein, mouse
  • Smad Proteins
  • Relaxin
  • p38 Mitogen-Activated Protein Kinases
  • MAP Kinase Kinase Kinases
  • MAP kinase kinase kinase 7