Abstract
Insulin‐like growth factor binding protein (IGFBP)‐6 has been reported to inhibit differentiation of myoblasts and osteoblasts. In the current study, we explored the mechanisms underlying IGFBP‐6 effects on osteoblast differentiation. During MC3T3‐E1 osteoblast differentiation, we found that IGFBP‐6 protein was down‐regulated. Overexpression of IGFBP‐6 in MC3T3‐E1 and human bone cells inhibited nodule formation, osteocalcin mRNA expression and ALP activity. Furthermore, accumulation of IGFBP‐6 in the culture media was not required for any of these effects suggesting that IGFBP‐6 suppressed osteoblast differentiation by an intracellular mechanism. A yeast two‐hybrid screen of an osteosarcoma library was conducted to identify intracellular binding partners to account for IGFBP‐6 inhibitory effects on osteoblast differentiation. LIM mineralizing protein (LMP‐1) was identified as a high affinity IGFBP‐6 binding partner. Physical interaction between IGFBP‐6 and LMP‐1 was confirmed by co‐immunoprecipitation. Fluorescent protein fusion constructs for LMP‐1 and IGFBP‐6 were transiently transfected into osteoblasts to provide evidence of subcellular locations for each protein. Coexpression of LMP‐1‐GFP and IGFBP‐6‐RFP resulted in overlapping subcellular localization of LMP‐1 and IGFBP‐6. To determine if there was a functional association of IGFBP‐6 and LMP‐1 as well as a physical association, we studied the effect of IGFBP‐6, LMP‐1 and their combination on type I procollagen promoter activity. LMP‐1 increased promoter activity while IGFBP‐6 reduced promoter activity, and coexpression of LMP‐1 with IGFBP‐6 abrogated IGFBP‐6 suppression. These studies provide evidence that overexpression of IGFBP‐6 suppresses human and murine osteoblast differentiation, that IGFBP‐6 and LMP‐1 physically interact, and supports the conclusion that this interaction may be functionally relevant. J. Cell. Biochem. 104: 1890–1905, 2008. © 2008 Wiley‐Liss, Inc.