LIM Mineralization Protein-1 Enhances Bone Morphogenetic Protein-2-Mediated Osteogenesis Through Activation of ERK1/2 MAPK Pathway and Upregulation of Runx2 Transactivity

J Bone Miner Res. 2015 Aug;30(8):1523-35. doi: 10.1002/jbmr.2481. Epub 2015 May 26.

Abstract

LIM mineralization protein-1 (LMP-1) is an intracellular regulator of bone formation. Upregulation of bone morphogenetic proteins (BMPs) and stabilization of BMP/Smad signaling have been proven to be the key mechanisms through which LMP-1 enhances osteogenesis. However, how LMP-1 regulates BMPs expression and related bone formation remains unclear. In this study, a LMP-1-induced osteogenesis cell model was used to study the molecular action of LMP-1 on BMP-2 expression and bone formation. The results show that overexpression of LMP-1 significantly increases, whereas downregulation of endogenous LMP-1 decreases BMP-2 expression and bone formation. Antagonism of BMP-2 with noggin or short hairpin BMP-2 significantly attenuates the osteoinductive effect of LMP-1, suggesting that the osteoinductive effect of LMP-1 is mediated by BMP-2. LMP-1 regulation of BMP-2 is found to occur at the transcription level using a luciferase reporter assay with a reporter construct containing a BMP-2 promoter. A promoter deletion assay reveals that -1000/-500 bp is the key regulated region by LMP-1. A Runx2-binding site is then located at -934/-920 bp and confirmed by luciferase assay using a reporter construct containing repeats of this Runx2-binding site and the site-directed mutagenesis analysis. Overexpression of LMP-1 significantly increases Runx2 expression. Downregulation of Runx2 expression significantly decreases BMP-2 promoter activity and BMP-2 expression. A ChIP assay demonstrates that LMP-1 increases the interaction between Runx2 and BMP-2 promoter. A luciferase reporter assay using the OSE2 promoter containing a Runx2-binding site confirms that Runx2 transactivity can be upregulated by LMP-1. Moreover, inhibiting the activation of different pathways with specific pathway inhibitors reveals that ERK1/2 MAPK activation is essential for LMP-1-induced upregulation of Runx2 transactivity and subsequent BMP-2 expression. In conclusion, our novel findings describe a positive regulatory effect of LMP-1 on BMP-2 expression and BMP-2-mediated osteogenesis. This effect occurs through activation of ERK1/2 pathway and subsequent upregulation of Runx2 transactivity.

Keywords: BMP-2; ERK1/2; LMP-1; OSTEOGENESIS; RUNX2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Animals
  • Bone Morphogenetic Protein 2 / biosynthesis*
  • Bone Morphogenetic Protein 2 / genetics
  • Cell Line
  • Core Binding Factor Alpha 1 Subunit / genetics
  • Core Binding Factor Alpha 1 Subunit / metabolism
  • Cytoskeletal Proteins / genetics
  • Cytoskeletal Proteins / metabolism*
  • Gene Expression Regulation / physiology
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • LIM Domain Proteins / genetics
  • LIM Domain Proteins / metabolism*
  • MAP Kinase Signaling System / physiology*
  • Mice
  • Mitogen-Activated Protein Kinase 3 / genetics
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Osteoblasts / cytology
  • Osteoblasts / metabolism*
  • Osteogenesis / physiology*
  • Rats
  • Rats, Sprague-Dawley

Substances

  • Adaptor Proteins, Signal Transducing
  • Bmp2 protein, mouse
  • Bmp2 protein, rat
  • Bone Morphogenetic Protein 2
  • Core Binding Factor Alpha 1 Subunit
  • Cytoskeletal Proteins
  • Intracellular Signaling Peptides and Proteins
  • LIM Domain Proteins
  • Pdlim7 protein, mouse
  • Pdlim7 protein, rat
  • Runx2 protein, mouse
  • Runx2 protein, rat
  • Mitogen-Activated Protein Kinase 3