Integrin-induced protein kinase Cα and Cε translocation to focal adhesions mediates vascular smooth muscle cell spreading

Abstract

The extracellular matrix influences the cellular spreading of vascular smooth muscle cells (VSMCs) via integrin receptors. However, the intracellular signaling mechanisms are still incompletely understood. We investigated 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) activity were measured by thin-layer chromatography. Intracellular distribution of PKC isoforms was assessed by confocal microscopy. VSMCs binding 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 isoforms α, δ, ε, and ζ were assessed by confocal microscopy. Fibronectin induced a PKC isoform translocation to the cell nucleus and to focal adhesions within minutes. The nuclear PKCα immunoreactivity was transiently increased. PKC isoforms α and ε were both translocated to focal adhesions. The intracellular distributions of other PKC isoforms were not influenced by fibronectin. The effects of fibronectin on DAG generation, the translocation of PKCα and PKCε, and cell spreading were all abolished by the incubation with RGD peptides. Downregulation of PKC isoforms α and ε with specific antisense oligodinucleotides resulted in a significant inhibition of cell spreading. Our results show that integrins induce intracellular signaling in VSMCs via DAG and PKC. PKC isoform α is translocated to the nucleus, whereas PKC isoforms α and ε are translocated to focal adhesions. Both isoforms seem to play a role in inside-out integrin signaling and cell spreading.