Alternative splicing of endothelial cell fibronectin mRNA in the IIICS region. Functional significance.

Transforming growth factor-beta 1 (TGF-beta 1) is thought to play a role in modulating vascular cell function in vivo. In vitro, it decreases endothelial cell proliferation and migration. We postulated that these biologic activities could be mediated through TGF-beta 1 modulation of specific gene expression. Therefore we differentially screened a human umbilical vein endothelial cell cDNA library with cDNAs prepared from both untreated and TGF-beta 1-treated bovine aortic endothelial cells. Using this technique, we isolated many TGF-beta 1-induced cDNA clones. Sequence analysis of these cDNAs showed that many of them corresponded to alternatively spliced fibronectin mRNAs. These fibronectin clones all contained the extradomain I (ED I) but three different forms of the type III connecting segment (IIICS). These different fibronectin cDNAs were expressed in bacteria and the recombinant proteins used to study the effects of IIICS alternative splicing on cell attachment, spreading, and migration in bovine aortic endothelial and smooth muscle cells and B16F10 melanoma cells. The results of these experiments show that attachment and spreading of bovine aortic endothelial and smooth muscle cells depend primarily on the presence of the Arg-Gly-Asp-Ser (RGDS) sequence in the recombinant fibronectin proteins. However attachment and spreading of bovine aortic endothelial cells are modulated by alternative splicing in the IIICS region. Specifically splicing of the IIICS region decreases spreading and increases migration rates of the endothelial cells. On the contrary, using a cell line (B16F10 melanoma cells) that is known not to require the RGDS sequence for adhesion confirmed previous findings that B16F10 melanoma cells do not require the presence of the RGDS sequence for attachment and spreading. Indeed B16F10 cells were able to attach and spread on two recombinant proteins that did not contain the RGDS sequence. However attachment and spreading of B16F10 were dramatically inhibited when a 75-base pair DNA fragment was removed from the 5' end of the IIICS region. These results suggest that various regions of the fibronectin molecule may be able to interact with different cell populations to promote cell attachment and spreading, and that alternative splicing may modulate this process.