Urokinase-receptor-mediated phenotypic changes in vascular smooth muscle cells require the involvement of membrane rafts.
The cholesterol-enriched membrane microdomains lipid rafts play a key role in cell activation by recruiting and excluding specific signalling components of cell-surface receptors upon receptor engagement. Our previous studies have demonstrated that the GPI (glycosylphosphatidylinositol)-linked uPAR [uPA (urokinase-type plasminogen activator) receptor], which can be found in lipid rafts and in non-raft fractions, can mediate the differentiation of VSMCs (vascular smooth muscle cells) towards a pathophysiological de-differentiated phenotype. However, the mechanism by which uPAR and its ligand uPA regulate VSMC phenotypic changes is not known. In the present study, we provide evidence that the molecular machinery of uPAR-mediated VSMC differentiation employs lipid rafts. We show that the disruption of rafts in VSMCs by membrane cholesterol depletion using MCD (methyl-beta-cyclodextrin) or filipin leads to the up-regulation of uPAR and cell de-differentiation. uPAR silencing by means of interfering RNA resulted in an increased expression of contractile proteins. Consequently, disruption of lipid rafts impaired the expression of these proteins and transcriptional activity of related genes. We provide evidence that this effect was mediated by uPAR. Similar effects were observed in VSMCs isolated from Cav1Z(-/-) (caveolin-1-deficient) mice. Despite the level of uPAR being significantly higher after the disruption of the rafts, uPA/uPAR-dependent cell migration was impaired. However, caveolin-1 deficiency impaired only uPAR-dependent cell proliferation, whereas cell migration was strongly up-regulated in these cells. Our results provide evidence that rafts are required in the regulation of uPAR-mediated VSMC phenotypic modulations. These findings suggest further that, in the context of uPA/uPAR-dependent processes, caveolae-associated and non-associated rafts represent different signalling membrane domains.
Kiyan
Julia
J
Hannover Medical School, Carl-Neuberg Strasse 1, D-30625 Hannover, Germany. kiian.ioulia@mh-hannover.de
Smith
Graham
G
Haller
Hermann
H
Dumler
Inna
I
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism; D054337:Cell Dedifferentiation / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D005372:Filipin / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D020868:Gene Silencing; D006801:Humans; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D051242:Caveolin 1; C524898:PLAUR protein, human; C524899:Plaur protein, mouse; D055293:Receptors, Urokinase Plasminogen Activator; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D005372:Filipin; D014568:Urokinase-Type Plasminogen Activator