LIM Mineralization Protein‐1 Enhances Bone Morphogenetic Protein‐2–Mediated Osteogenesis Through Activation of ERK1/2 MAPK Pathway and Upregulation of Runx2 Transactivity. (26th May 2015) - Pan, Hehai
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. © 2015 American Society for Bone and Mineral Research.