title: Thrombin-induced rapid geranylgeranylation of RhoA as an essential process for RhoA activation in endothelial cells.
abstract: RhoA plays a critical signaling role in thrombin-induced endothelial dysfunction. The possible thrombin regulation of geranylgeranylation, a lipid modification, of unprocessed RhoA and the significance of the geranylgeranylation in RhoA activation in endothelial cells (ECs) are not well understood. The amounts of the unprocessed and geranylgeranylated forms of RhoA in non-stimulated cultured human aortic ECs were 31 +/- 8 and 69 +/- 8% total cellular RhoA, respectively (n = 6, p < 0.0001), as determined by the Triton X-114 partition method. Thrombin-induced rapid conversion of most of the unprocessed RhoA into the geranylgeranylated form within 1 min through stimulating geranylgeranyltransferase I (GGTase I) activity. Thrombin-induced rapid geranylgeranylation was inhibited by acute short term (3 min) pretreatment with atorvastatin as well as by an inhibitor of GGTase I (GGTI-286). Thrombin also rapidly stimulated GTP loading of RhoA, which was blocked by acute pretreatment with either atorvastatin or GGTI-286. These observations indicate the dependence of thrombin stimulation of RhoA on the rapid geranylgeranylation of unprocessed RhoA. Importantly, the addition of geranylgeranylpyrophosphate to ECs pretreated with atorvastatin quickly reversed the atorvastatin inhibition of thrombin stimulation of RhoA. These results suggest that geranylgeranylation of unprocessed RhoA may limit thrombin-induced full activation of RhoA in ECs. Cytoskeleton analysis demonstrated that atorvastatin and GGTI-286 inhibited thrombin-induced stress fiber formation. We provide the evidence that, in thrombin-stimulated ECs, the unprocessed form of RhoA is rapidly geranylgeranylated to become the mature form, which then is converted into GTP-bound active RhoA.
authors.affiliation: First Department of Internal Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ohkawara
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Ishibashi
authors.forename: Takayuki
authors.initials: T
authors.lastname: Sakamoto
authors.forename: Koichi
authors.initials: K
authors.lastname: Sugimoto
authors.forename: Kenji
authors.initials: K
authors.lastname: Nagata
authors.forename: Keiko
authors.initials: K
authors.lastname: Yokoyama
authors.forename: Nobuo
authors.initials: N
authors.lastname: Sakamoto
authors.forename: Masashi
authors.initials: M
authors.lastname: Kamioka
authors.forename: Isao
authors.initials: I
authors.lastname: Matsuoka
authors.forename: Shigetomo
authors.initials: S
authors.lastname: Fukuhara
authors.forename: Naotoshi
authors.initials: N
authors.lastname: Sugimoto
authors.forename: Yoh
authors.initials: Y
authors.lastname: Takuwa
authors.forename: Yukio
authors.initials: Y
authors.lastname: Maruyama
mesh_terms: D000199:Actins / Q000378:metabolism; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D000069059:Atorvastatin; D015153:Blotting, Western; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D003720:Densitometry; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006538:Heptanoic Acids / Q000494:pharmacology; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D050356:Lipid Metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D017368:Protein Prenylation; D011758:Pyrroles / Q000494:pharmacology; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D000199:Actins; D003902:Detergents; C437105:GGTI 286; D006538:Heptanoic Acids; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D011758:Pyrroles; D006153:Guanosine Diphosphate; D011092:Polyethylene Glycols; D006160:Guanosine Triphosphate; D017830:Octoxynol; C010615:Nonidet P-40; D000069059:Atorvastatin; D013917:Thrombin; D020742:rhoA GTP-Binding Protein; D007930:Leucine