BACKGROUND/AIMS: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element(s) (ARE) in target gene promoters, enabling oxidatively stressed cells to respond in order to restore redox homeostasis. Post-translational modifications (PTMs) that mediate activation of Nrf2, in the cytosol and its release from Keap1, have been extensively studied but PTMs that impact its biology after activation are beginning to emerge. In this regard, PTMs like acetylation, phosphorylation, ubiquitination and sumoylation contribute towards the Nrf2 subcellular localization, and its transactivation function. We previously demonstrated that Nrf2 traffics to the promyelocytic leukemia-nuclear bodies (PML-NB), where it is a target for modification by small ubiquitin-like modifier (SUMO) proteins (sumoylation), but the site(s) for SUMO conjugation have not been determined. In this study, we aim to identify SUMO-2 conjugation site(s) and explore the impact, sumoylation of the site(s) have on Nrf2 stability, nuclear localization and transcriptional activation of its target gene expression upon oxidative stress. METHODS: The putative SUMO-binding sites in Nrf2 for human isoform1 (NP_006155.2) and mouse homolog (NP_035032.1) were identified using a computer-based SUMO-predictive software (SUMOplot™). Site-directed mutagenesis, immunoblot analysis, and ARE-mediated reporter gene assays were used to assess the impact of sumoylation on these site(s) in vitro. Effect of mutation of these sumoylation sites of Nrf2 on expression of Heme Oxygenase1 (HO-1) was determined in HEK293T cell. RESULTS: Eight putative sumoylation sites were identified by SUMOplot™ analysis. Out of the eight predicted sites only one 532LKDE535 of human (h) and its homologous 524LKDE527 of mouse (m) Nrf2, exactly matches the SUMO-binding consensus motif. The other high probability SUMO-acceptor site identified was residue K110, in the motifs 109PKSD112 and 109PKQD112 of human and mouse Nrf2, respectively. Mutational analysis of putative sumoylation sites (human (h)/mouse (m) K110, hK533 andmK525)showed that these residues are needed for SUMO-2 conjugation, nuclear localization and ARE driven transcription of reporter genes and the endogenous HO-1 expression by Nrf2. These residues also stabilized Nrf2, as evident from shorter half-lives of the mutant protein compared to wild-type Nrf2. CONCLUSION: Our findings indicate that SUMO-2 mediated sumoylation of K110 and K533 in human Nrf2 regulates in part its transcriptional activity by enhancing its stabilization and nuclear localization.
Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632, suggesting protease-activated receptor coupling to Gq and G12/13, respectively. These data suggest a vascular bed specific differential coupling of protease-activated receptors to the signaling pathways that regulate endothelial nitric oxide synthase and nitric oxide production that may be responsible for endothelial dysfunction associated with cardiovascular disease.
Protease‐activated receptor‐3 (PAR‐3) is widely recognized as a non‐signaling elusive member of a G protein‐coupled receptor family known to regulate the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway via phosphorylation of its regulatory sites Thr495 and Ser1177. The eNOS/NO pathway is responsible for vascular smooth muscle cell relaxation and disruption of this pathway leads to endothelial dysfunction, laminar shear stress and vascular inflammation, a precursor to cellular induced hypertension. Using human coronary artery endothelial cells (HCAECs) as a model, we demonstrated that PAR‐3 is highly co‐expressed with PAR‐1 and ‐2 and can be activated using a synthetic peptide ligand, TFRGAP. We hypothesize that PAR‐3 can signal independently of PAR‐1 and ‐2 to regulate eNOS via site‐ specific phosphorylation and induce pro‐inflammation via the NFkB pathway. Analysis using eNOS specific antibodies showed that 500uM of TFRGAP lead to the phosphorylation of the positive site of eNOS‐Ser1177 and the negative site ‐Thr495, 30 seconds and 15 minutes respectively. Pretreatment of HCAECs with 10uM SCH79797, a selective PAR‐1 inhibitor, did not block the increase in phosphorylation at both eNOS regulatory sites stimulated by TFRGAP. Similar results where found in a stable PAR‐1 deficient cell line of HCAECs which confirmed the signaling potential of PAR‐3 using thrombin and the PAR‐3 activating peptide. Our data suggests that PAR‐3 is able to regulate eNOS/NO signaling via site‐specific phosphorylation that is independent of PAR‐1 activation. In additional studies cytokine production was measured using an ELISA after PAR‐3 activation. Data from these studies will lead to a better understanding of endothelial dysfunction and vascular inflammation while identifying potential drug targets for cardiovascular diseases. Grant Funding Source: The project [poster] described was supported by CTSA award No. UL1TR000445
Protease‐activated receptor‐3 (PAR‐3) is widely recognized as a non‐signaling elusive member of a G protein‐coupled receptor family known to regulate the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway via phosphorylation of its regulatory sites. Failure of the eNOS/NO pathway leads to endothelial dysfunction, a precursor to cellular induced hypertension. Using human coronary artery endothelial cells (HCAECs) as a model for human endothelium, we demonstrated that PAR‐3 is highly co‐expressed with PAR‐1 and ‐2 and can be activated using a synthetic peptide ligand, SFNGGP‐NH2. We hypothesize that PAR‐3 can signal independently of PAR‐1 and ‐2 to regulate eNOS via site specific phosphorylation and induce proi‐nflammation via the NFkB pathway. Analysis using eNOS specific antibodies showed that 50uM of SFNGGP lead to the phosphorylation of the positive site of eNOS at ‐Ser1177 and the negative site ‐Thr495 in a time‐dependent manner. Pretreatment of HCAECs with 10uM SCH79797, a selective PAR‐1 inhibitor, did not block the increase in phosphorylation at both eNOS regulatory sites stimulated by SFNGGP. We created a stable PAR‐1 deficient cell line of HCAECs using lentiviral particles to further establish the signaling potential of PAR‐3 in the presence of thrombin and the PAR‐3 activating peptide. Our data suggests that PAR‐3 is able to regulate eNOS/NO signaling via site specific phosphorylation that is independent of PAR‐1 activation and cause pro‐inflammation with the secretion of cytokines. Understanding the signaling pathways responsible for eNOS‐induced NO and cytokine production will lead to a better understanding of endothelial dysfunction and vascular inflammation in cardiovascular diseases.
Protease‐activated receptor‐3 (PAR‐3) is widely recognized as a non‐signaling elusive member of a G protein‐coupled receptor family known to regulate the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway via phosphorylation of its regulatory sites. Failure of the eNOS/NO pathway leads to endothelial dysfunction, a precursor to cellular induced hypertension. Using human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) as a model for human endothelium, we demonstrated that PAR‐3 is highly co‐expressed with PAR‐1 and ‐2 and can be activated using a synthetic peptide, SFNGGP‐NH2. We hypothesize that PAR‐3 can signal independently of PAR‐1 and ‐2 to regulate eNOS. Analysis using eNOS specific antibodies showed that 50uM of SFNGGP lead to the phosphorylation of the positive site of eNOS at ‐Ser1177 and the negative site ‐Thr495 in a time‐dependent manner in both cell lines. Human endothelial cells pretreated with 10uM of SCH79797, a selective PAR‐1 inhibitor, and then stimulated with SFNGGP; we observed an increase in phosphorylation at both eNOS regulatory sites. Using siRNA, transient knock‐down of PAR‐1 confirms PAR‐3 independent signaling potential. Our data suggests that PAR‐3 is able to regulate eNOS/NO signaling via site specific phosphorylation that is independent of PAR‐1 activation. Cardiovascular disease is the leading cause of death among Americans, where hypertension can be a silent aggressor. Studying the signaling pathways responsible for eNOS‐induced NO production will lead to a better understanding of endothelial dysfunction in cardiovascular diseases.NIH‐NIGMS‐SCORE and Porter Physiology Developmental Fellowship
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