The Contributions of Integrin Affinity and Integrin-Cytoskeletal Engagement in Endothelial and Smooth Muscle Cell Adhesion to Vitronectin

Abstract

The serine proteinase inhibitor, plasminogen activator inhibitor type-1 (PAI-1), binds to the adhesion protein vitronectin with high affinity at a site that is located directly adjacent to the vitronectin RGD integrin binding sequence. The binding of PAI-1 to vitronectin sterically blocks integrin access to this site and completely inhibits the binding of purified integrins to vitronectin; however, its inhibition of endothelial and smooth muscle cell adhesion to vitronectin is at most 50 -75%. Because PAI-1 binds vitronectin with ϳ10 -100-fold higher affinity than purified integrins, we have analyzed the mechanism whereby these cells are able to overcome this obstacle. Our studies exclude proteolytic removal of PAI-1 from vitronectin as the mechanism, and show instead that cell adhesion in the presence of PAI-1 is dependent on integrin-cytoskeleton engagement. Disrupting endothelial or smooth muscle cell actin polymerization and/or focal adhesion assembly reduces cell adhesion to vitronectin in the presence of PAI-1 to levels similar to that observed for the binding of purified integrins to vitronectin. Furthermore, endothelial cell, but not smooth muscle cell adhesion to vitronectin in the presence of PAI-1 requires both polymerized microtubules and actin, further demonstrating the importance of the cytoskeleton for integrin-mediated adhesion. Finally, we show that cell adhesion in the presence of PAI-1 leads to colocalization of PAI-1 with the integrins ␣v␤3 and ␣v␤5 at the cell-matrix interface.Cell adhesion receptors play important roles in maintaining cell anchorage and polarity in addition to promoting migration and differentiation. Many of the known adhesion receptors belong to the integrin family of noncovalent heterodimeric transmembrane proteins that are expressed by most cell types. The integrin family consists of at least 18 ␣-and 8 ␤-subunits that combine to form as many as 24 different ␣␤ dimers (1). The extracellular domains of both integrin subunits are required for binding to adhesion proteins. In addition, integrins need physiological concentrations of divalent cations such as calcium or magnesium. Integrins have relatively short cytoplasmic tails that interact with an intracellular protein complex, termed focal contacts, which include adaptor proteins and kinases. The focal contacts control the linkage between the cytoskeleton and integrins and mediate intracellular signaling. Integrins direct forces generated by the cytoskeleton onto the extracellular matrix (ECM) 2 to produce the traction necessary for cell adhesion and migration (2). Understanding the mechanism and control of integrin-mediated cell adhesion and migration is of special interest because of their importance in processes such as wound healing and angiogenesis as well as pathologies associated with these processes.In general, integrins recognize short linear peptide sequences on adhesion proteins, the most prevalent being arginine-glycine-aspartic acid (RGD), which is found in many ECM proteins such as fibronectin, collagens, ...

PAI1 stimulates assembly of the fibronectin matrix in osteosarcoma cells through crosstalk between the αvβ5 and α5β1 integrins

“…A recent study has shown that cells adhering to vitronectin in the presence of PAI1 are only partially inhibited from attachment (Stefansson et al, 2007). Those cells that can overcome the inhibitory effects of PAI1 on adhesion are partially spread and do not assemble either focal contacts or stress fibers.…”

Role of Nonmuscle Myosin II in Migration of Wharton's Jelly-Derived Mesenchymal Stem Cells

“…These are components of ECM and their upregulation indicates that they could be playing a role in stronger adherence of blebbistatin-treated cells to the substratum [20,21]. Similarly, VTN, a serum-spreading protein responsible for spreading of cells and adhesion [22], was found to be upregulated in our blebbistatin-treated WJ-MSCs, which by morphology were flattened and more spread out than the control WJ-MSCs. MMPs play a role in the turnover and degradation of ECM components and basement membranes, which influences many biological processes such as proliferation, migration, and angiogenesis [23].…”

Plasminogen activator inhibitor-1 inhibits prostate tumor growth through endothelial apoptosis

“…It is possible that competition between PAI-1 and cell membrane integrin already bound to vitronectin favors the latter because of its higher affinity. Once bound to its ligand, the integrin-mediated endothelial cell adhesion is further strengthened by the formation of cell-substratum focal contacts (35). During active angiogenesis and in the newly established vasculature, however, angiogenic endothelial cells actively break focal contact and further readhesion to vitronectin is inhibited by a high local concentration of PAI-1 in the tumor microenvironment.…”