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...

Control of mRNA stability is critical for expression of short-lived transcripts from cytokines and protooncogenes. Regulation involves an AU-rich element (ARE) in the 3 untranslated region (3UTR) and cognate trans-acting factors thought to promote either degradation or stabilization of the mRNA. In this study we present a novel approach using somatic cell genetics designed to identify regulators of interleukin-3 (IL-3) mRNA turnover. Mutant cell lines were generated from diploid HT1080 cells transfected with a reporter construct containing green fluorescent protein (GFP) linked to the IL-3 3UTR. GFP was expressed at low levels due to rapid decay of the mRNA. Following chemical mutagenesis and selection of GFP-overexpressing cells, we could isolate three mutant clones (slowA, slowB, and slowC) with a specific, trans-acting defect in IL-3 mRNA degradation, while the stability of IL-2 and tumor necrosis factor alpha reporter transcripts was not affected. Somatic cell fusion experiments revealed that the mutants are genetically recessive and form two complementation groups. Expression of the tristetraprolin gene in both groups led to reversion of the mutant phenotype, thereby linking this gene to the IL-3 mRNA degradation pathway. The genetic approach described here should allow identification of the defective functions by gene transfer and is also applicable to the study of other mRNA turnover pathways.

BackgroundVascular smooth muscle cell (VSMC) senescence and apoptosis are involved in atherosclerotic plaque vulnerability. Arginase‐II (Arg‐II) has been shown to promote vascular dysfunction and plaque vulnerability phenotypes in mice through uncoupling of endothelial nitric oxide synthase and activation of macrophage inflammation. The function of Arg‐II in VSMCs with respect to plaque vulnerability is unknown. This study investigated the functions of Arg‐II in VSMCs linking to plaque vulnerability.Methods and ResultsIn vitro studies were performed on VSMCs isolated from human umbilical veins, whereas in vivo studies were performed on atherosclerosis‐prone apolipoprotein E‐deficient (ApoE−/−) mice. In nonsenescent VSMCs, overexpressing wild‐type Arg‐II or an l‐arginine ureahydrolase inactive Arg‐II mutant (H160F) caused similar effects on mitochondrial dysfunction, cell apoptosis, and senescence, which were abrogated by silencing p66Shc or p53. The activation of p66Shc but not p53 by Arg‐II was dependent on extracellular signal‐regulated kinases (ERKs) and sequential activation of 40S ribosomal protein S6 kinase 1 (S6K1)—c‐Jun N‐terminal kinases (JNKs). In senescent VSMCs, Arg‐II and S6K1, ERK‐p66Shc, and p53 signaling levels were increased. Silencing Arg‐II reduced all these signalings and cell senescence/apoptosis. Conversely, silencing p66Shc reduced ERK and S6K1 signaling and Arg‐II levels and cell senescence/apoptosis. Furthermore, genetic ablation of Arg‐II in ApoE−/− mice reduced the aforementioned signaling and apoptotic VSMCs in the plaque of aortic roots.ConclusionsArg‐II, independently of its l‐arginine ureahydrolase activity, promotes mitochondrial dysfunction leading to VSMC senescence/apoptosis through complex positive crosstalk among S6K1‐JNK, ERK, p66Shc, and p53, contributing to atherosclerotic vulnerability phenotypes in mice.

The occurrence of pathologically stable mRNAs of protooncogenes, growth factors and cyclins has been proposed to contribute to experimental and human oncogenesis. In normal resting cells, mRNAs containing an AU-rich element (ARE) in their 3 0 untranslated region are subjected to rapid degradation. Tristetraprolin (TTP) is an RNA-binding zinc-finger protein that promotes decay of ARE-containing mRNAs. Here we report that TTP acts as a potent tumor suppressor in a v-H-ras-dependent mast cell tumor model, where tumors express abnormally stable interleukin-3 (IL-3) mRNA as part of an oncogenic autocrine loop. Premalignant v-H-ras cells were transfected with TTP and injected into syngeneic mice. TTP expression delayed tumor progression by 4 weeks, and late appearing tumors escaped suppression by loss of TTP. When transfected into a fully established tumor line, TTP reduced cloning efficiency in vitro and growth of the inoculated cells in vivo. Transgenic TTP interfered with the autocrine loop by enhancing the degradation of IL-3 mRNA with concomitant reduction of IL-3 secretion. Our data establish the ARE as an antioncogenic target in a model situation, underline the importance of mRNA stabilization in oncogenesis and show for the first time that tumor suppression can be achieved by interfering with mRNA turnover.