EFFECTS OF CYTOKINES ON INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN SECRETION BY MUSCLE-CELLS IN-VITRO

The insulin-like growth factors (IGF-I and IGF-II) stimulate muscle ce ll proliferation and/or differentiation (depending upon the culture co nditions). They also increase IGF-binding protein (IGFBP) levels in mu scle cell conditioned media and in some instances there is a direct co rrelation between the apparent rate of IGFBP secretion and muscle cell proliferation. We have investigated the effect of other cytokines on muscle cell IGFBP conditioned media levels using rat skeletal (L6), mo use myocytes (BC3H-1) and porcine Vascular smooth (pVSM) muscle cells in vitro to determine if this relationship is maintained. IGFBP levels in conditioned media (CM) were measured by an I-125IGF-I binding ca pacity assay and by western blot analysis. Immunoblots indicated that BC3H-1 and L6 cells secrete IGFBP-5 (31-32,000 M(r)), L6 cells secrete IGFBP-4, and pVSM cells secrete IGFBP-2 (34,000 M(r)). Both L6 and BC 3H-1 cells responded to transforming growth factor beta(1) (TGF-beta(1 )), in a dose-dependent manner, with suppressed conditioned media leve ls of IGFBP-4 and IGFBP-5 but TGF-beta(1) did not affect IGFBP-2 level s in pVSM cell media. TGF-beta(1) (5 ng/ml) suppressed IGFBP levels (C M I-125IGF-I binding capacity) in L6 and BC3H-1 cell media by 48% an d 61%, respectively. IGFBP-5 levels, in BC3H-1 cell media, were decrea sed by treatment with either basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF). Neither treatment affected IGFBP-2 leve ls. In contrast in L6 myoblasts, bFGF increased media levels of IGFBP- 4 and IGFBP-5; L6 cells were not responsive to EGF. Insulin increased IGFBP-4 and IGFBP-5 levels in L6 and BC3H-1 cell media. This stimulato ry effect was markedly suppressed by either TGF-beta(1) (L6 and BC3H-1 cells) or bFGF (BC3H-1 cells). L6 and BC3H-1 cell CM IGFBP levels wer e also suppressed 34% and 84% by 5 U/ml thrombin, respectively. The in hibitory activity of thrombin was specific, i.e. reversible by hirudin and was not due to direct IGFBP proteolysis. Since suramin and stauro sporine increased media levels of the IGFBP, this suggests that consti tutive secretion of TGF-beta(1), bFGF, or EGF might provide a tonic su ppressive mechanism for controlling IGFBP secretion. Thus, our results support the conclusion that the secretion of IGFBP-4 and IGFBP-5, but not IGFBP-2, by muscle cells was suppressed by several cytokines. Dep ressed IGFBP secretion may play a key role in determining muscle cell responsiveness to either the mitogenic or differentiation stimulating activity of the IGFs.