Sottile J, Hocking D C, Langenbach K J
Department of Medicine, Center for Cardiovascular Research and Department of Pharmacology and Physiology, University of Rochester Medical Center, Box 679, Rochester, NY 14642, USA. jane_sottile@urmc. rochester.edu.
J Cell Sci. 2000 Dec;113 Pt 23:4287-99. doi: 10.1242/jcs.113.23.4287.
Many aspects of cell behavior are regulated by cell-extracellular matrix interactions, including cell migration and cell growth. We previously showed that the addition of soluble fibronectin to collagen-adherent fibronectin-null cells enhances cell growth. This growth-promoting effect of fibronectin depended upon the deposition of fibronectin into the extracellular matrix; occupancy and clustering of fibronectin-binding integrins was not sufficient to trigger enhanced cell growth. To determine whether the binding of integrins to fibronectin's RGD site is required for fibronectin-enhanced cell growth, the ability of fibronectin lacking the integrin-binding RGD site (FN(Delta)RGD) to promote cell growth was tested. FN(Delta)RGD promoted cell growth when used as an adhesive substrate or when added in solution to collagen-adherent fibronectin-null cells. Addition of FN(Delta)RGD to collagen-adherent fibronectin-null cells resulted in a 1.6-1.8x increase in cell growth in comparison with cells grown in the absence of fibronectin. The growth-promoting effects of FN(Delta)RGD and wild-type fibronectin were blocked by inhibitors of fibronectin polymerization, including the anti-fibronectin antibody, L8. In addition, FN(Delta)RGD-induced cell growth was completely inhibited by the addition of heparin, and was partially blocked by either heparitinase-treatment or by addition of recombinant fibronectin heparin-binding domain. Heparin and heparitinase-treatment also partially blocked the growth-promoting effects of wild-type fibronectin, as well as the deposition of wild-type fibronectin into the extracellular matrix. These data suggest that cell surface heparan-sulfate proteoglycans contribute to the growth-promoting effects of FN(Delta)RGD and wild-type fibronectin. Addition of heparin, treatment with heparitinase, or incubation with monoclonal antibody L8 all inhibited the formation of short linear FN(Delta)RGD fibrils on the cell surface. Inhibitory (beta)1 integrin antibodies had no effect on FN(Delta)RGD fibril formation, FN(Delta)RGD-induced cell growth, or cell adhesion on FN(Delta)RGD-coated substrates. These data suggest that fibronectin fibril formation can promote cell growth by a novel mechanism that is independent of RGD-integrin binding, and that involves cell surface proteoglycans.
细胞行为的许多方面都受到细胞与细胞外基质相互作用的调节，包括细胞迁移和细胞生长。我们之前表明，向黏附于胶原蛋白的纤连蛋白缺失细胞中添加可溶性纤连蛋白可促进细胞生长。纤连蛋白的这种促生长作用取决于纤连蛋白在细胞外基质中的沉积；纤连蛋白结合整合素的占据和聚集不足以触发细胞生长增强。为了确定整合素与纤连蛋白的RGD位点结合对于纤连蛋白增强的细胞生长是否必要，测试了缺乏整合素结合RGD位点的纤连蛋白（FN(Δ)RGD）促进细胞生长的能力。当用作黏附底物或添加到溶液中与黏附于胶原蛋白的纤连蛋白缺失细胞一起培养时，FN(Δ)RGD可促进细胞生长。与在无纤连蛋白条件下生长的细胞相比，向黏附于胶原蛋白的纤连蛋白缺失细胞中添加FN(Δ)RGD可使细胞生长增加1.6至1.8倍。FN(Δ)RGD和野生型纤连蛋白的促生长作用被纤连蛋白聚合抑制剂所阻断，包括抗纤连蛋白抗体L8。此外，添加肝素可完全抑制FN(Δ)RGD诱导的细胞生长，而用硫酸乙酰肝素酶处理或添加重组纤连蛋白肝素结合域则可部分阻断该生长。肝素和硫酸乙酰肝素酶处理也部分阻断了野生型纤连蛋白的促生长作用以及野生型纤连蛋白在细胞外基质中的沉积。这些数据表明，细胞表面硫酸乙酰肝素蛋白聚糖有助于FN(Δ)RGD和野生型纤连蛋白的促生长作用。添加肝素、用硫酸乙酰肝素酶处理或与单克隆抗体L8孵育均抑制了细胞表面短线性FN(Δ)RGD纤维的形成。抑制性β1整合素抗体对FN(Δ)RGD纤维形成、FN(Δ)RGD诱导的细胞生长或细胞在FN(Δ)RGD包被底物上的黏附均无影响。这些数据表明，纤连蛋白纤维形成可通过一种独立于RGD - 整合素结合且涉及细胞表面蛋白聚糖的新机制促进细胞生长。
