Akimov S S, Belkin A M
Department of Biochemistry, The Holland Laboratory, American Red Cross, Rockville, MD 20855, USA.
J Cell Sci. 2001 Aug;114(Pt 16):2989-3000. doi: 10.1242/jcs.114.16.2989.
Assembly of fibronectin into a fibrillar matrix is critical for regulation of cell growth and migration, embryogenesis and wound healing. We have previously shown that cell-surface tissue transglutaminase serves as an integrin-binding adhesion coreceptor for fibronectin. Here we report that transglutaminase strongly promotes fibronectin assembly mediated by alpha5beta1 integrin. This effect is independent from transglutaminase-mediated enzymatic crosslinking of fibronectin and separate from the ability of transglutaminase to stimulate cell spreading. Surface transglutaminase increases the binding of fibronectin to cells via interaction with its gelatin-binding domain that contains modules I6II1,2I7-9 and lacks integrin-binding motifs. The gelatin-binding fragment of fibronectin binds to surface transglutaminase on cells in suspension but does not interact with cell monolayers where surface transglutaminase is occupied by fibronectin. Surface transglutaminase colocalizes with growing fibronectin fibrils at early timepoints of matrix formation and remains codistributed with fibronectin matrices thereafter. The observed stimulation of matrix assembly by transglutaminase is blocked by the gelatin-binding fragment of fibronectin, but is not strongly perturbed by its N-terminal fragment consisting of modules I1-5. These results implicate an interaction between transglutaminase and the gelatin-binding domain of fibronectin in matrix assembly and suggest its role in initiation of fibrillogenesis. However, blocking antibodies against alpha5beta1 integrin or the cell-binding fragment of fibronectin that contains modules III2-11 most strongly suppress matrix formation and abolish the effects of transglutaminase. Hence, transglutaminase cooperates with but can not substitute for alpha5beta1 integrin in fibronectin assembly. Treatment of fibroblasts with transforming growth factor beta (TGFbeta) significantly increases surface expression of transglutaminase and its association with beta1 integrins, but not with alphaVbeta3 integrin. TGFbeta enhances the binding of fibronectin to the cell surface and elevates matrix formation, whereas antibody against transglutaminase or the gelatin-binding fragment of fibronectin suppresses these effects, indicating an involvement of transglutaminase in TGFbeta-dependent fibronectin assembly. Therefore, TGFbeta-induced fibronectin matrix deposition during normal wound healing or fibrotic disorders may depend on upregulation of integrin-associated surface transglutaminase.
纤连蛋白组装成纤维状基质对于细胞生长与迁移、胚胎发育及伤口愈合的调控至关重要。我们之前已表明，细胞表面组织转谷氨酰胺酶作为纤连蛋白的整合素结合黏附共受体。在此我们报告，转谷氨酰胺酶强烈促进由α5β1整合素介导的纤连蛋白组装。此效应独立于转谷氨酰胺酶介导的纤连蛋白酶促交联，且与转谷氨酰胺酶刺激细胞铺展的能力无关。表面转谷氨酰胺酶通过与其含I6II1、2I7 - 9模块且缺乏整合素结合基序的明胶结合域相互作用，增加纤连蛋白与细胞的结合。纤连蛋白的明胶结合片段与悬浮细胞上的表面转谷氨酰胺酶结合，但不与表面转谷氨酰胺酶被纤连蛋白占据的细胞单层相互作用。在基质形成的早期时间点，表面转谷氨酰胺酶与生长中的纤连蛋白纤维共定位，此后仍与纤连蛋白基质共分布。观察到的转谷氨酰胺酶对基质组装的刺激被纤连蛋白的明胶结合片段阻断，但未被其由I1 - 5模块组成的N端片段强烈干扰。这些结果表明转谷氨酰胺酶与纤连蛋白的明胶结合域在基质组装中有相互作用，并提示其在纤维形成起始中的作用。然而，针对α5β1整合素的阻断抗体或纤连蛋白含III2 - 11模块的细胞结合片段最强烈地抑制基质形成并消除转谷氨酰胺酶的作用。因此，在纤连蛋白组装中，转谷氨酰胺酶与α5β1整合素协同作用但不能替代它。用转化生长因子β（TGFβ）处理成纤维细胞显著增加转谷氨酰胺酶的表面表达及其与β1整合素的结合，但不与αVβ3整合素结合。TGFβ增强纤连蛋白与细胞表面的结合并提高基质形成，而针对转谷氨酰胺酶的抗体或纤连蛋白的明胶结合片段抑制这些效应，表明转谷氨酰胺酶参与TGFβ依赖的纤连蛋白组装。因此，在正常伤口愈合或纤维化疾病期间，TGFβ诱导的纤连蛋白基质沉积可能依赖于整合素相关表面转谷氨酰胺酶的上调。
