Rho expression and activation in vascular smooth muscle cells.
Phenotypic modulation of smooth muscle cells (SMC) involves dramatic changes in expression and organization of contractile and cytoskeletal proteins, but little is known of how this process is regulated. The present study used a cell culture model to investigate the possible involvement of RhoA, a known regulator of the actin cytoskeleton. In rabbit aortic SMC seeded into primary culture at moderate density, Rho activation was high at two functionally distinct time-points, first as cells modulated to the "synthetic" phenotype, and again upon confluence and return to the "contractile" phenotype. Rho expression increased with time, such that maximal expression occurred upon return to the contractile state. Transient transfection of synthetic state cells with constitutively active RhoA (Val14RhoA) caused a reduction in cell size and reorganization of cytoskeletal proteins to resemble that of the contractile phenotype. Actin and myosin filaments were tightly packed and highly organised while vimentin localised to the perinuclear region; focal adhesions were enlarged and concentrated at the cell periphery. Conversely, inhibition of endogenous Rho by C3 exoenzyme resulted in complete loss of contractile filaments without affecting vimentin distribution; focal adhesions were reduced in size and number. Treatment of synthetic state SMC with known regulators of SMC phenotype, heparin and thrombin, caused a modest increase in Rho activation. Long-term confluence and serum deprivation induced cells to return to a more contractile phenotype and this was augmented by heparin and thrombin. The results implicate RhoA for a role in regulating SMC phenotype and further show that activation of Rho by heparin and thrombin correlates with the ability of these factors to promote the contractile phenotype.
Centre for Research in Vascular Biology, Department of Anatomical Sciences, University of Queensland, Queensland 4072, Australia.
Nathalie F
NF
Worth
Gordon R
GR
Campbell
Julie H
JH
Campbell
Barbara E
BE
Rolfe
D000818:Animals; D002448:Cell Adhesion; D002454:Cell Differentiation / Q000502:physiology; D048430:Cell Shape; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004789:Enzyme Activation; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D010641:Phenotype; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D003598:Cytoskeletal Proteins; D011993:Recombinant Fusion Proteins; D020741:rho GTP-Binding Proteins