The role of insulin-like growth factor binding protein-3 (IGFBP-3) in the proliferation and differentiation of L6 myogenic cells and porcine embryonic myogenic cells (PEMC)

There are six high affinity insulin-like growth factor binding proteins (IGFBP 1--6) that regulate IGF biological activity. Among them, IGFBP-3 has been shown to suppress or stimulate proliferation of cultured cells depending upon the cell types and the assay conditions. IGFBP-3 has been shown to suppress both IGF-I and Long-R3-IGF-I-stimulated proliferation of embryonic porcine myogenic cells (PEMC). In this study, exogenous addition of recombinant porcine IGFBP-3 (rplGFBP-3) suppresses the proliferation of L6 myogenic cells via both IGF-dependent and -independent mechanisms. Our data also show that rpIGFBP-3 causes IGF-independent anti-proliferatioe actions without increasing the level of phosphosmad-2 in L6 cultures. In addition, rpIGFBP-3 has been shown to inhibit L6 cell differentiation only via an IGF-dependent pathway. Forced expression of pIGFBP-3 in L6 myogenic cells indicates that endogenous pIGFBP-3 plays a similar role to exogenous rpIGFBP-3 in regulating the proliferation and differentiation of L6 myogenic cells. Using a DNA-based RNA interference (RNAi) technique, endogenous pIGFBP-3 is suppressed in cultured PEMC, which provides an excellent model in which to conduct pIGFBP-3 loss-of-function studies. Addition of rpIGFBP-3 to the cultured PEMC inhibits cell differentiation in serum-containing medium via an IGF-I-dependent pathway. IGFBP-3 mRNA level has been found to be relatively high in PEMC at 48 h post plating, but drops by 72 h post-plating, thereafter, increasing with differentiation. The pattern of IGFBP-3 protein expression mirrors that of IGFBP-3 mRNA expression over time. Immunofluorescent staining data reveal that IGFBP-3 protein localizes in both the cytoplasm and nucleus of proliferating PEMC but is located primarily in the cytoplasm of differentiated PEMC. Furthermore, IGFBP-3 is enriched in nucleus when PEMC are treated with TGFbeta1, which may contribute to the antiproliferative actions of TGFbeta1. Real-time RT-PCR data reveal that the members of IGFs system, TGFbeta1 and myostatin mRNA expressions are differentially regulated during the proliferation and differentiation of PEMC. The studies in this area provide new information about the role of IGFBP-3 and potential interactions of these important growth factors during porcine muscle growth and development, which will allow us to understand more about how to produce meat efficiently.