Potential involvement of the interaction between insulin-like growth factor binding protein (IGFBP)-6 and LIM mineralization protein (LMP)-1 in regulating osteoblast differentiation.
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.
Strohbach
Cassandra
C
Jerry L. Pettis Memorial VA Center, Musculoskeletal Disease Center, Loma Linda, CA 92357, USA.
Kleinman
Sabrina
S
Linkhart
Thomas
T
Amaar
Yousef
Y
Chen
Shin-Tai
ST
Mohan
Subburaman
S
Strong
Donna
D
D048868:Adaptor Proteins, Signal Transducing; D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002113:Calcification, Physiologic; D002454:Cell Differentiation; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics; D003598:Cytoskeletal Proteins; D005786:Gene Expression Regulation; D006801:Humans; D047468:Immunoprecipitation; D018976:Insulin-Like Growth Factor Binding Protein 6 / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism; D060588:LIM Domain Proteins; D051379:Mice; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport; D016415:Sequence Alignment; D013347:Subcellular Fractions / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D015415:Biomarkers; D024042:Collagen Type I; D003598:Cytoskeletal Proteins; D018976:Insulin-Like Growth Factor Binding Protein 6; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; C484380:PDLIM7 protein, human; D000469:Alkaline Phosphatase