McCusker R H, Clemmons D R
Department of Animal Sciences, University of Illinois, Urbana 61801.
Endocrinology. 1994 May;134(5):2095-102. doi: 10.1210/endo.134.5.7512498.
The insulin-like growth factors (IGF-I and IGF-II) stimulate muscle cell proliferation and/or differentiation (depending upon the culture conditions). They also increase IGF-binding protein (IGFBP) levels in muscle cell conditioned media and in some instances there is a direct correlation 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), mouse myocytes (BC3H-I) 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 [125I]IGF-I binding capacity 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 BC3H-1 cells responded to transforming growth factor beta 1 (TGF-beta 1), in a dose-dependent manner, with suppressed conditioned media levels of IGFBP-4 and IGFBP-5 but TGF-beta 1 did not affect IGFBP-2 levels in pVSM cell media. TGF-beta 1 (5 ng/ml) suppressed IGFBP levels (CM [125I]IGF-I binding capacity) in L6 and BC3H-1 cell media by 48% and 61%, respectively. IGFBP-5 levels, in BC3H-1 cell media, were decreased by treatment with either basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF). Neither treatment affected IGFBP-2 levels. 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 stimulatory 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 were also suppressed 34% and 84% by 5 U/ml thrombin, respectively. The inhibitory activity of thrombin was specific, i.e. reversible by hirudin and was not due to direct IGFBP proteolysis. Since suramin and staurosporine increased media levels of the IGFBP, this suggests that constitutive secretion of TGF-beta 1, bFGF, or EGF might provide a tonic suppressive 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. Depressed IGFBP secretion may play a key role in determining muscle cell responsiveness to either the mitogenic or differentiation stimulating activity of the IGFs.
胰岛素样生长因子（IGF-I和IGF-II）刺激肌肉细胞增殖和/或分化（取决于培养条件）。它们还会提高肌肉细胞条件培养基中IGF结合蛋白（IGFBP）的水平，在某些情况下，IGFBP分泌的表观速率与肌肉细胞增殖之间存在直接关联。我们利用大鼠骨骼肌（L6）、小鼠心肌细胞（BC3H-I）和猪血管平滑肌（pVSM）细胞在体外研究了其他细胞因子对肌肉细胞IGFBP条件培养基水平的影响，以确定这种关系是否得以维持。通过[125I]IGF-I结合能力测定和蛋白质印迹分析来测量条件培养基（CM）中的IGFBP水平。免疫印迹表明，BC3H-1和L6细胞分泌IGFBP-5（分子量31 - 32,000），L6细胞分泌IGFBP-4，而pVSM细胞分泌IGFBP-2（分子量34,000）。L6和BC3H-1细胞均以剂量依赖方式对转化生长因子β1（TGF-β1）产生反应，其条件培养基中IGFBP-4和IGFBP-5的水平受到抑制，但TGF-β1不影响pVSM细胞培养基中IGFBP-2的水平。TGF-β1（5 ng/ml）使L6和BC3H-1细胞培养基中的IGFBP水平（CM [125I]IGF-I结合能力）分别降低了48%和61%。用碱性成纤维细胞生长因子（bFGF）或表皮生长因子（EGF）处理可降低BC3H-1细胞培养基中IGFBP-5的水平。两种处理均不影响IGFBP-2的水平。相反，在L6成肌细胞中，bFGF增加了培养基中IGFBP-4和IGFBP-5的水平；L6细胞对EGF无反应。胰岛素增加了L6和BC3H-1细胞培养基中IGFBP-4和IGFBP-5的水平。这种刺激作用被TGF-β1（L6和BC3H-1细胞）或bFGF（BC3H-1细胞）显著抑制。5 U/ml凝血酶也分别使L6和BC3H-1细胞CM中的IGFBP水平降低了34%和84%。凝血酶的抑制活性具有特异性，即水蛭素可使其逆转，且并非由于直接的IGFBP蛋白水解作用。由于苏拉明和星形孢菌素增加了培养基中IGFBP的水平，这表明TGF-β1、bFGF或EGF的组成性分泌可能提供了一种调节IGFBP分泌的抑制机制。因此，我们的结果支持以下结论：几种细胞因子可抑制肌肉细胞分泌IGFBP-4和IGFBP-5，但不影响IGFBP-2。IGFBP分泌降低可能在决定肌肉细胞对IGF的促有丝分裂或分化刺激活性的反应中起关键作用。
