Cultured porcine myogenic cells produce insulin-like growth factor binding protein-3 (IGFBP-3) and transforming growth factor beta-1 stimulates IGFBP-3 production.

Abstract

Insulin-like growth factor binding proteins (IGFBP) may act locally as autocrine or paracrine regulators of insulin-like growth factor activity in specific tissues such as muscle. Although secretion of IGFBP by cultured myogenic cell lines has been examined, little is known about secretion of IGFBP by primary myogenic cell cultures. This may be because primary myogenic cultures contain non-muscle cells (fibroblasts) that complicate interpretation of IGFBP determinations. We have circumvented this problem by subculturing nonfusing cells from extensively fused porcine myogenic cultures and comparing the IGFBP production of these nonfusing, porcine muscle-derived cells with that of primary porcine myogenic cell cultures. Immunoprecipitation with specific antibodies and 125I-IGF-I ligand blot analysis showed that myogenic cultures secreted IGFBP-3 (doublet band, 43 kDa and 39 kDa), IGFBP-2 (34 kDa), IGFBP-4 (30 and 24 kDa), and IGFBP-5 (30 and 28 kDa). Muscle-derived fibroblasts secreted no detectable IGFBP-3 but approximately 10 times more IGFBP-2 than did myogenic cell cultures. Treatment of myogenic cultures for 24 h with transforming growth factor (TGF) beta-1 caused a concentration-dependent increase in IGFBP-3 secretion with a maximum 1.5-fold increase occurring at .5 ng of TGF beta-1/mL. In contrast, TGF beta-1 treatment did not stimulate detectable IGFBP-3 secretion by muscle-derived fibroblast cultures. Northern analysis of total RNA using a porcine IGFBP-3 probe revealed that TGF beta-1 treatment resulted in a fourfold increase in the steady-state level of IGFBP-3 mRNA in myogenic cultures. Insulin-like growth factor binding protein-3 mRNA was not detectable in fibroblast cultures either before or after TGF beta-1 treatment. This is the first report of IGFBP-3 secretion by cultured myogenic cells.

Effects of endotoxin lipopolysaccharide administration on the somatotropic axis

“…Although it has been reported that IGFBP-3 is GH and IGF-I dependent (Spagnoli & Rosenfeld 1997), acute LPS administration, in a higher dose than in the present study (1 mg/kg), increases hepatic IGFBP-3 levels together with a significant decrease in serum GH and IGF-I levels (Fan et al 1994). These results suggest that other factors may modulate the serum concentration of IGFBP-3, and are in accordance with other data previously reported showing that IL-1, TNF and transforming growth factor (TGF-) induce IGFBP-3 synthesis (Olney et al 1995, Hembree et al 1996, Han et al 1997. Since LPS administration has been shown to increase not only IL-1 and TNF but also hepatic TGF mRNA levels (Masuhara 1995), these can be possible mechanisms by which LPS increases circulating IGFBP-3.…”

Effect of recombinant porcine IGF-binding protein-3 on proliferation of embryonic porcine myogenic cell cultures in the presence and absence of IGF-I

“…Cultured PEMCs produce IGFBP-3, and levels of IGFBP-3 mRNA and protein decrease significantly during differentiation and then increase after differentiation is complete (Hembree et al 1996, Yang et al 1999. These changes in IGFBP-3 mRNA concentration during differentiation suggest that IGFBP-3 may play a role in myogenesis and/or alterations in proliferation of cultured PEMCs during differentiation.…”

“…Binding proteins were detected with 125 I-IGF-I as described previously (Hembree et al 1996, Yang et al 1999.…”

“…IGFBP-3 is produced by a number of different cell types including porcine embryonic myogenic cells (PEMCs) and porcine muscle satellite cells (Hembree et al 1996, Dahlfors & Arnqvist 2000, Granata et al 2000, Jennische & Hall 2000, Wabitsch et al 2000, Drivdahl et al 2001, Kveiborg et al 2001, Yi et al 2001. Although IGFBP-3 has generally been shown to suppress proliferation of cultured cells, it may also stimulate proliferation depending upon the cell type and the assay conditions (Baxter 2000.…”

Zinc partitions IGFs from soluble IGF binding proteins (IGFBP)-5, but not soluble IGFBP-4, to myoblast IGF type 1 receptors

“…Cultured skeletal myoblasts do not appear to have abundant cell-associated IGFBPs, but secrete either a single IGFBP or a wide variety of combinations of soluble IGFBPs including IGFBP-2, -3, -4, -5, -6 and -7 (rp1) (McCusker et al 1989b, Ernst et al 1992, James et al 1993, McFarland et al 1993, Kou et al 1994, McCusker & Clemmons 1994, 1998, Bach et al 1995, Ernst & White 1996, Hembree et al 1996, Damon et al 1997, Ewton et al 1998, Musaro & Rosenthal 1999, Yang et al 1999, Bayol et al 2000, Crown et al 2000, Haugk et al 2000, Meadows et al 2000, Foulstone et al 2001, Rousse et al 2001, Yi et al 2001. With such an array of IGFBP profiles, it is difficult to interpret the specific role of a given IGFBP to myoblast function although clearly IGFBPs can act as autocrine and paracrine growth and differentiation modulators (McCusker & Clemmons 1988, Hodgkinson et al 1989, Tollefsen et al 1989, Bach et al 1994, Ewton & Florini 1995, Rotwein et al 1995, Silverman et al 1995, Ernst et al 1996, Ernst & White 1996, James et al 1996, Stewart et al 1996, Damon et al 1998a,b, Fligger et al 1998, Rousse et al 1998, Meadows et al 2000, Foulstone et al 2001…”