INTEGRIN-INDUCED PROTEIN-KINASE-C-ALPHA AND C-EPSILON TRANSLOCATION TO FOCAL ADHESIONS MEDIATES VASCULAR SMOOTH-MUSCLE CELL SPREADING

The extracellular matrix influences the cellular spreading of vascular smooth muscle cells (VSMCs) via integrin receptors. However, the intr acellular signaling mechanisms are still incompletely understood. We i nvestigated the hypothesis that VSMCs binding to fibronectin activates the protein kinase C (PKC) pathway, causes differential intracellular PKC isoform translocation, and mediates cell spreading. VSMCs binding to poly-L-lysine or preincubated with Arg-Gly-Asp (RGD) peptides were used as controls. Diacylglycerol (DAG) and phospholipase D (PLD) acti vity were measured by thin-layer chromatography. Intracellular distrib ution of PKC isoforms was assessed by confocal microscopy. VSMCs bindi ng to fibronectin induced focal adhesions and cell spreading within 30 minutes. Fibronectin induced a rapid increase in DAG content, peaking at 10 minutes with a sustained response for <1 hour. In contrast, PLD activity was not influenced by specific binding to fibronectin. PKC i soforms alpha, delta, epsilon, and zeta were assessed by confocal micr oscopy. Fibronectin induced a PKC isoform translocation to the cell nu cleus and to focal adhesions within minutes. The nuclear PKC alpha imm unoreactivity was transiently increased. PKC isoforms alpha and epsilo n were both translocated to focal adhesions. The intracellular distrib utions of other PKC isoforms were not influenced by fibronectin. The e ffects of fibronectin on DAG generation, the translocation of PKC alph a and PKC epsilon, and cell spreading were all abolished by the incuba tion with RGD peptides. Downregulation of PKC isoforms alpha and epsil on with specific antisense oligodinucleotides resulted in a significan t inhibition of cell spreading. Our results show that integrins induce intracellular signaling in VSMCs via DAG and PKC. PKC isoform alpha i s translocated to the nucleus, whereas PKC isoforms alpha and epsilon are translocated to focal adhesions. Both isoforms seem to play a role in inside-out integrin signaling and cell spreading.