H. Haller et al., INTEGRIN-INDUCED PROTEIN-KINASE-C-ALPHA AND C-EPSILON TRANSLOCATION TO FOCAL ADHESIONS MEDIATES VASCULAR SMOOTH-MUSCLE CELL SPREADING, Circulation research, 82(2), 1998, pp. 157-165
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.