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Many transcription factors contain proline- or glutamine-rich activation domains. Here it is shown that simple homopolymeric stretches of these amino acids can activate transcription when fused to the DNA binding domain of GAL4 factor. In vitro, activity increased with polymer length, whereas in cell transfection assays maximal activity was achieved by 10 to 30 glutamines or about 10 prolines. Similar results were obtained when glutamine stretches were placed within a [GAL4]-VP16 chimeric protein. Because these stretches are encoded by rapidly evolving triplet repeats (microsatellites), they may be the main cause for modulation of transcription factor activity and thus result in subtle or overt genomic effects.

Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.Endothelium-derived nitric oxide (NO), synthesized from L-arginine by endothelial nitric oxide synthase (eNOS), plays a critical role in the regulation of vascular tone and the maintenance of vascular integrity by modulating various processes. It promotes vasodilatation, inhibits platelet activation and prevents vascular smooth muscle cell proliferation and/or migration (4). Abnormalities in eNOS gene expression or activation, which result in decreased NO production, are thought to contribute to the pathogenesis of various cardiovascular disorders such as atherosclerosis and hypertension (49).eNOS-mediated NO generation is a highly regulated cellular event. The mechanisms controlling eNOS activity involve multiple regulatory steps including gene expression, co-and posttranslational modification, intracellular localization, cofactors and phosphorylation (20). Although eNOS was originally described as a constitutive enzyme with enzyme activation achieved via calmodulin (CaM) binding in response to increased Ca 2ϩ , recent studies indicate that a variety of stimuli can modulate its expression at the transcriptional (15) and/or posttranscriptional level (6). Moreover, it is evidenced that phosphorylation of eNOS also modulates eNOS activity independent of Ca 2ϩ /CaM. Studies showed that Ser-1177 of human eNOS (Ser-1179 of bovine sequence) is phosphorylated directly by protein kinase B (PKB)/Akt (10, 18, 37), which results in an increase in electron flux through the reductase domain and increase in NO production (36). Shear stress and insulin have been shown to activate eNOS through the activation of PKB (14,41). eNOS phosphorylation at Ser-1177 by PKB therefore represents another important regulatory mechanism of eNOS activation in addition to Ca 2ϩ /CaM-depe...

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Background-Arginase competes with endothelial nitric oxide synthase (eNOS) for the substrate L-arginine and decreases NO production. This study investigated regulatory mechanisms of arginase activity in endothelial cells and its role in atherosclerosis. Methods and Results-In human endothelial cells isolated from umbilical veins, thrombin concentration-and timedependently stimulated arginase enzymatic activity, reaching a 1.9-fold increase (PϽ0.001) at 1 U/mL for 24 hours. The effect of thrombin was prevented by C3 exoenzyme or the HMG-CoA reductase inhibitor fluvastatin, which inhibit RhoA, or by the ROCK inhibitors Y-27632 and HA-1077. Adenoviral expression of constitutively active RhoA or ROCK mutants enhanced arginase activity (Ϸ3-fold, PϽ0.001), and the effect of active RhoA mutant was inhibited by the ROCK inhibitors. Neither thrombin nor the active RhoA/ROCK mutants affected arginase II protein level, the only isozyme detectable in the cells. Moreover, a significantly higher arginase II activity (1.5-fold, not the protein level) and RhoA protein level (4-fold) were observed in atherosclerotic aortas of apoE Ϫ/Ϫ compared with wild-type mice. Interestingly, L-arginine (1 mmol/L), despite a significantly higher eNOS expression in aortas of apoE Ϫ/Ϫ mice, evoked a more pronounced contraction, which was reverted to a greater vasodilation by the arginase inhibitor L-norvaline (20 mmol/L) compared with the wild-type animals (nϭ5, PϽ0.001). Conclusions-Thrombin