Molecular mechanism of fibronectin-induced chemotactic migration

我们发现，21K的Fib2片段在极低的浓度下具有趋化活性，而完整的Fn几乎没有这种活性。完整的FN抑制ST-13前脂肪细胞的脂肪细胞分化，而源于FN分子氨基端纤维蛋白结合区的24K片段显著促进了ST-13前脂肪细胞的分化。完整FN的蛋白降解方式可能是FN功能激活的重要决定因素。首先，我们研究了炎性蛋白水解酶(纤溶酶、中性粒细胞组织蛋白酶G和弹性蛋白酶)和基质金属蛋白酶(基质分解酶、基质溶解酶、明胶酶A和间质胶原酶)对活性FN片段释放的影响。明胶酶A在产生趋化纤维蛋白2片段方面是最活跃的。中性粒细胞弹性蛋白酶能有效释放分化刺激纤维蛋白1片段。除了这种…之外蛋白酶对趋化迁移的间接作用更多，一些细胞内依赖的半胱氨酸蛋白酶直接参与了对FN的趋化迁移。其次，尝试用21K Fib 2片段固定凝胶亲和层析柱分离与21K Fib 2片段相关的细胞表面受体蛋白。在应用^&lt；125&gt；i-Labed NIH-L13细胞提取物后，用GRGDSP多肽、BSA溶液和Fib 2片段去除的Fn片段混合物洗脱柱，使得能够专门去除与亲和凝胶非特异性结合的蛋白质。结果表明，用21K的Fib2片段可洗脱出一个尖锐的放射性峰，并在其中检测到三条蛋白质条带，其Mr分别为30 kDa、60 kDa和200 kDa。将21K的Fib-2片段和细胞表面受体与双反应剂DSS进行交联，也表明Fib-2片段与具有相同MRS的蛋白质结合，这些蛋白质有望成为介导Fib-2片段效应的受体。较少

We have shown that proteolytic degradation of FN yields biological activities not present in intact FN.We found that 21K Fib 2 fragment has a chemotactic activity at extremely low concentrations that intact FN hardly show the activity. Adipocyte differentiation of ST-13 preadipocytes is inhibited by intact FN, but the 24K fragment originated fromtheamino-terminal fibrin-binding domain of FN molecule dramatically stimulated the differentiation. The mode of proteolytic degradation of intact FN may be an important determinant for activation of FN function. First, we examined effects of inflammatory proteases (plasmin, neutrophil cathepsin G and elastase) and matrix metalloproteinases (stromelysin, matrilysin, gelatinase A, and interstitial collagenase) on the release of active FN fragments. Gelatinase A was found to be the most active in generating chemotactic Fib 2 fragments. Differentiation stimuratory Fib 1 fragments were effectively released by neutrophil elastase. In addition to such … More indirect action of proteases on the chemotactic migration, some intracellular Ca^<2+> dependentcysteine proteases were shown to involve directly in the chemotactic migration in response to FN.Second, a cell surface receptor protein for the 21K Fib 2 fragment was tried to isolate by an affinity column using 21K Fib 2 fragment-fixed gel. Washing the column with GRGDSP peptide, BSA solution, and then the Fib 2 fragment-depleted FN fragments mixture, after applying the ^<125>I-labed NIH-L13 cell extract, enabled to remove exclusively the proteins binding nonspecifically to the affinity gel. As a result, one sharp radioactive peak could be eluted with the 21K Fib 2 fragment, in which three protein bands with Mr.of 30kDa, 60kDa, and >200kDa were detected by aotoradiography. Cross-linking of the 21K Fib 2 fragment and cell surface receptor with bireactive agent DSS also showed that the Fib 2 fragment binds to proteins with the same Mrs. These proteins would be expected to be receptors mediating the Fib 2 fragment effects. Less