Focal Adhesion Integrity Is Downregulated by the Alternatively Spliced Domain ofHumanTenascin

Tenascin, together with thrombospondin and SPARC, form a family of matrix proteins that, when added to bovine aortic endothelial cells, caused a dose-dependent reduction in the number of focal adhesion-positive cells to -50% of albumin-treated controls . For tenascin, a maximum response was obtained with 20-60 jug/ml of protein . The reduction in focal adhesions in tenascin-treated spread cells was observed 10 min after addition of the adhesion modulator, reached the maximum by 45 min, and persisted for at least 4 h in the continued presence of tenascin . This effect was fully reversible, was independent of de novo protein synthesis, and was neutralized by a polyclonal antibody to tenascin . Monoclonal antibodies to specific domains of tenascin (mAbs 81C6 and 127) were used to localize the active site to the alternatively CELL substrate adhesion typically involves cellular recognition of an adhesive matrix protein (e.g ., fibronectin, laminin, vitronectin, or fibrinogen) by an integrin . Integrins are a family of heterodimeric cell surface receptors that recognize specific domains in adhesive extracellular matrix proteins (reviewed in reference 21) . Subsequent to attachment of cells to a substrate via integrins, additional cell surface, cytoskeletal, and extracellular components become engaged in the adhesion process, resulting in cell spreading and the formation of stable adhesion plaques . The dynamics of a living organism require that cell substrate adhesion be regulated . During wound healing, embryogenesis, and metastasis, processes in which cells proliferate and migrate, cell-substrate adhesions disassemble and reform (2, 5, 44) . Previous studies have shown that exposure of cells to tumor-promoting phorbol esters (17, 36) or to certain growth factors (18, 19) can cause loss of focal adhesions. More recently, studies demonstrate that certain extracellular matrix molecules may participate in destabilizing or modulating cell-substrate contacts . Murphy-Ullrich © The Rockefeller University Press, 0021-9525/91/11/1127/10 $2 . 00 TheJournal of Cell Biology, Volume 115, Number4, November 1991 1127-1136 1127 spliced segment of tenascin . Furthermore, a recombinant protein corresponding to the alternatively spliced segment (fibronectin type III domains 6-12) was expressed in Escherichia coli and was active in causing loss of focal adhesions, whereas a recombinant form of a domain (domain 3) containing the RGD sequence had no activity. Chondroitin-6-sulfate effectively neutralized tenascin activity, whereas dermatan sulfate and chondroitin-4-sulfate were less active and heparan sulfate and heparin were essentially inactive . Studies suggest that galactosaminoglycans neutralize tenascin activity through interactions with cell surface molecules . Overall, our results demonstrate that tenascin, thrombospondin, and SPARC, acting as soluble ligands, are able to provoke the loss of focal adhesions in wellspread endothelial cells . and Hook showed that thrombospondin (TSP)' caused a loss of focal adhesion plaques from spread bovine aortic endothelial (BAE) cells (30) . Sage et al . reported that SPARC (osteonectin) causes rounding of BAE cells (26, 35) . Tenascin (also referred to as hexabrachion or cytotactin) is a large oligomeric glycoprotein that has been implicated as an adhesion modulator ; however, its physiological role remains unclear. Tenascin is localized primarily in developing tissues and in tumors (reviewed in 8, 12, 13) and, like many matrix glycoproteins, it is composed of multiple domains, which may have discrete functions (12, 16, 22, 23, 37) . Alternative splicing ofmRNA results in multiple forms of the molecule with varying numbers of a 91 amino acid motif resembling fibronectin type III (FN-III) domains (16, 23, 43) . Tenascin is known to interact with other molecules of the extracellular matrix . There is a strong binding oftenascin to 1 . Abbreviations used in this paper: RAE, bovine aortic endothelial ; C-4-S, chondroitin-4-sulfate ; C-6-S, chondroitin-6-sulfate ; FN-III, fibronectin type III; IRM, interference reflection microscopy ; TSP, thrombospondin . on April 30, 2019 jcb.rupress. org Downloaded from http://doi.org/10.1083/jcb.115. 4.1127 Published Online: 15 November, 1991 | Supp Info: