Abstract
Syndecan-4 is a ubiquitously expressed transmembrane heparan sulphate proteoglycan acting in concert with integrins in the formation of focal adhesions and stress fibres. Signalling events studied thus far suggest the formation of a ternary complex between syndecan-4, phosphatidylinositol 4,5-bisphosphate and protein kinase C alpha (PKCalpha). Syndecan-4 clustering at the cell surface has also been associated with RhoA-dependent signalling, but the relationship between PKCalpha and RhoA has not been resolved. Here we present evidence that syndecan-4, PKCalpha and RhoA are in a linear pathway necessary for the formation and maintenance of stress fibres in primary rat embryo fibroblasts. Inhibition of PKCalpha activity through the use of specific pharmacological inhibitors, a dominant-negative construct, or siRNA downregulation of protein levels, attenuated focal adhesion formation and the maintenance of stress fibres. However, these effects could be bypassed through independent activation of RhoA with lysophosphatidic acid, but not by clustering of syndecan-4 with ligand. Furthermore, inhibition of PKCalpha could block the increase in the GTP levels of RhoA induced by clustering of syndecan-4 at the cell surface. All these data point to a mechanism whereby syndecan-4 signals to RhoA in a PKCalpha-dependent manner and PKCalpha directly influences RhoA activity.
Highlights
Cell adhesion is a complex, multi-stage process during which cells adhering to extracellular matrix (ECM)-coated planar substrates assemble focal contacts with the underlying substratum
rat embryo fibroblasts (REFs) cells express PKC␣, ␦, ⑀, and PKC constitutes a family of related serine and threonine kinases and includes three subfamilies: the ‘conventional’ PKCs (␣,  and ␥), which require diacylglycerol (DAG) and Ca2+ for their activation; the ‘novel’ PKCs (␦, ⑀, and ) which are DAG-dependent but Ca2+-independent; and the ‘atypical’ PKCs ( and ) for which the mechanisms of activation are still incompletely understood (Mellor and Parker, 1998)
As a first stage to ascertain the role of PKC in stress fibre formation in rat embryo fibroblasts (REFs), PKC expression in these cells was determined (Fig. 1A)
Summary
Cell adhesion is a complex, multi-stage process during which cells adhering to extracellular matrix (ECM)-coated planar substrates assemble focal contacts with the underlying substratum. Following attachment on the ECM protein fibronectin (FN), Rac and Cdc appear to be activated and contribute to cell spreading through actin polymerization and the formation of focal complexes (Clark et al, 1998; Price et al, 1998; del Pozo et al, 2000). At this stage RhoA activity is suppressed (Ren et al, 1999; Arthur and Burridge, 2001). The assembly of stress fibres and subsequent maturation of focal complexes into focal adhesions correlate with, and are dependent on, activation of RhoA signalling (Ren et al, 1999; Rottner et al, 1999)
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