Integrin / 1-and / 3-mediated Endothelial Cell Migration Is Triggered Through Distinct Signaling Mechanisms

spectively, and these events take place in the absence of cytokines, growth factors, or chemoattractants. Cell attachment and spreading on these ligands occur in the absence of extracellular calcium, as does migration on collagen. In contrast, vitronectin-mediated migration is absolutely dependent on the presence of extracellular calcium. Cell contact with immobilized vitronectin or anti-Otv/~3 mAbs promotes a measurable rise in [Ca2+]~ which requires an extracellular calcium source, whereas collagen, or anti-ot2~t mAbs fail to promote this signaling event. In fact, vitronectin-mediated migration and the rise in intracellular calcium showed the same dose dependence on extracellular calcium. While vitronectin and collagen differ in their ability to induce a calcium influx both ligands or antibodies to their respective integrins promote an equivalent increase in intracellular pH consistent with activation of the Na/H antiporter an event independent of extracellular calcium. These results support two salient conclusions. Firstly, collagen and vitronectin, through their respective integrins, promote distinct intracellular signaling events. Secondly, the o~v~3 specific influx of calcium is not required for cell spreading yet appears to facilitate cellular migration on vitronectin. C ELLULAR migration plays a major role in development, wound healing, immune defense, and metastasis. To understand the molecular basis of cellular migration it is important to characterize the molecular event(s) underlying this biological process. It is clear that cell motility depends on the ability of cells to identify, attach and change shape in response to specific extracellular matrix components (Leavesley et al., 1992; Duband et al., 1991, 1990, 1986; Stoolman, 1989; Strauss et al., 1989; Tucker et al., 1988). Recent experimental evidence indicates that the integrin family of cell surface receptors not only mediate cellular adhesion and spreading but are directly involved in migration (Leavesley et al., 1992; Marks et al., 1991; Akiyama et al., 1989; Strauss et al., 1989; Tucker et al., 1988; McCarthy and Furcht, 1984). Integrins are known to promote highly stabilized adhesion to extracellular matrix (ECM) 1 proteins (Orlando and Cheresh, 1991), leading to reorganization of the actin-cytoskeleton, depending in part on the cytoplasmic tail of the integrin/~ subunit (Otey et al., 1990; Tapley et al., 1989; Horowitz, 1986). Thus, integrins provide a functional linkage between the extracellular environment and the intraceUular compartment. (Tamkun et al., 1986; Buck and Horowitz, 1987). 1. Abbreviations used in this paper: Ac LDL, acetylated low-density lip~ protein; ECM, extracellular matrix; HUVEC, human umbilical vein endothelial cells; pHi, intracellular pH. The nature of the integrin-induced transmembrane signal(s) leading to the locomotory response have yet to be defined, although some early integrin-stimulated second messengers have been described. For example, alterations in cytosolic free calcium ([Ca2+]0, and intracellular pH (pHi) are observed during interactions with ECM components (Jaconi et al., 1991; Ng-Sikorski et al., 1991; Ingber et al., 1990; Schwartz et al., 1991; Altieri et al., 1990). Recent studies have shown that endothelial cell adhesion to matrix proteins triggers a detectable influx of extraceUular calcium via voltage-independent calcium channels (Schwartz, 1993). Alterations in [Ca2+]t directly affect the function of several actin-binding proteins, altering the organization of the actin network (Stossel, 1989). Various Ca2+-sensitive kinases and phosphatases are associated with, and/or act upon the cytoskeleton (Gaudry et al., 1992; Hendey and Maxfield, 1992; Wilson et al., 1991). Thus [Ca2+]~ is a candidate integrin second messenger with potential to directly or indirectly regulate cytoskeletal structure and consequently cell spreading and motility. In the current study we have examined the potential role of calcium on endothelial cell migration. These studies were performed in the absence of any growth factors, cytokines, chemoattractants, or serum, allowing us to examine cellular migration due to individual ECM components. We demonstrate that endothelial cell attachment and spreading on vitronectin or collagen leads to an elevation of pHi which is independent of extracellular calcium. Cell contact with © The Rockefeller University Press, 0021-9525/93/04/163/8 $2.00 The Journal of Cell Biology, Volume 121, Number 1, April 1993 163-170 163 on M ay 5, 2017 D ow nladed fom Published April 1, 1993