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.

Effects of lovastatin on Rho isoform expression, activity, and association with guanine nucleotide dissociation inhibitors

“…Therefore, it is difficult to predict how inhibition of Rho prenylation in vivo will affect the activation state of Rho. The prevailing view is that statins inhibit Rho signaling by preventing Rho membrane association, and block agonist-induced Rho activation [12][13][14]30,35,36]. These studies generally examined short-term (< 6 h) effects of statins on acute, GEFmediated Rho activation.…”

RhoA and Rac1 Changes in the Atherosclerotic Lesions of WHHLMI Rabbits

“…The sources of most conventional reagents for protein and immunohistochemical analyses were described previously 8,9,15,16) .…”

“…The levels of GTP-bound forms of RhoA and Rac1 of the aortas were determined by pull-down assays as described previously 8,9,18) . The removed aortas were cut into small pieces and lysed in a buffer (50 mmol/L Tris, pH 7.2, 500 mmol/L NaCl, 10 mmol/L MgCl2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 20 g/mL antipain, 20 g/mL leupeptin, and 1 mmol/L PMSF).…”

“…Unprocessed and geranylgeranylated forms of RhoA and Rac1 were separated by the Triton X-114 partition method as described previously 9) . The aortas were cut into small pieces and solubilized with a buffer containing 50 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 5 mmol/L MgCl2, 200 mol/L GDP, 1 mmol/L dithiothreitol, 1 mmol/L pefabloc, 20 mol/L each of leupeptin, aprotinin and soybean trypsin inhibitor, and then sonicated.…”

“…Rac1 (Ras-related C3 botulinum toxin substrate1) is a small (~21 kDa) GTPase protein, a member of the Rac subfamily of the Rho family of GTPases. This molecule is a pleiotropic regulator of many cellular processes including the cell cycle, cell-cell adhesion of unprocessed RhoA in thrombin-stimulated endothelial cells 9) . It has been shown that RhoA and Rac1 are expressed in the remodeled vascular wall in vivo 3,[10][11][12] .…”