Hypoxia-inducible factor-1 (HIF-1) takes part in the transcriptional activation of hypoxia-responsive genes. HIF-1␣, a subunit of HIF-1, is rapidly degraded under normoxic conditions by the ubiquitin-proteosome system. Hypoxia up-regulates HIF-1␣ by inhibiting its degradation, thereby allowing it to accumulate to high levels with 3-6 h of hypoxia treatment and decreasing thereafter. In vascular tissues, prostacyclin (prostaglandin I 2 (PGI 2 )) is a potent vasodilator and inhibitor of platelet aggregation and is known as a vasoprotective molecule. However, the role of PGI 2 in HIF-1 activation has not been studied. In the present study, we investigated the effect of PGI 2 on HIF-1 regulation in human umbilical vein endothelial cells under prolonged hypoxia (12 h). Augmentation of PGI 2 via adenovirus-mediated gene transfer of both cyclooxygenase-1 and PGI 2 synthase activated HIF-1 by stabilizing HIF-1␣ in cells under prolonged hypoxia or the hypoxia-normoxia transition but not under normoxia. Exogenous H 2 O 2 abolished PGI 2 -and catalase-induced HIF-1␣ up-regulation, which suggests that degradation of HIF-1␣ under prolonged hypoxia is through a reactive oxygen species-dependent pathway. Moreover, PGI 2 attenuated NADPH oxidase activity by suppressing Rac1 and p47 phox expression under hypoxia. These data demonstrate a novel function of PGI 2 in down-regulating reactive oxygen species production by attenuating NADPH oxidase activity, which stabilizes HIF-1␣ in human umbilical vein endothelial cells exposed to prolonged hypoxia.Hypoxia induces a number of cellular responses, such as angiogenesis, erythropoiesis, and glycolysis, through both gene regulation and post-translational modification of proteins. Hypoxia-inducible factor-1 (HIF-1) 2 takes part in the transcriptional activation of hypoxia-responsive genes through binding to the hypoxia-responsive element (HRE) in the promoter or enhancer regions and activating a number of genes (1-4). HIF-1 is a heterodimer composed of HIF-1␣ and HIF-1. HIF-1␣ is rapidly degraded under normoxic conditions by the ubiquitin-proteosome system, whereas HIF-1 is constitutively expressed (5). Under hypoxia, HIF-1␣ has been shown to be up-regulated to high levels at 3-6 h and decrease thereafter (6, 7). A number of studies have focused on the mechanism of HIF-1␣ stabilization under hypoxia for 4 -6 h. Recently, a natural antisense HIF-1␣ was suggested to down-regulate HIF-1␣ in A549 cells under prolonged hypoxia (6). However, regulation of HIF-1␣ under prolonged hypoxia (12 h) in endothelial cells remains largely unknown.Under normoxic conditions, HIF-1␣ is regulated through hydroxylation of proline residues by prolyl hydroxylase enzymes (8, 9). The von Hippel-Lindau tumor suppressor protein (pVHL) associates with hydroxylated HIF-1␣ and targets it for ubiquitination and rapid degradation (10). Under hypoxia, prolyl hydroxylase is inactivated through oxygen-sensing mechanisms, and the unmodified HIF-1␣ accumulates, permitting dimerization with HIF-1 (3, 11). Various redox-de...
Cyclooxygenase-2 (COX-2) plays major roles in diverse physiological and pathological processes such as inflammation and tumorigenesis. Transcriptional control of COX-2 has been extensively investigated and characterized, but its post-translational control is less clear. Here, we report a novel mechanism by which COX-2 is degraded. Protein levels of caveolin-1 (Cav-1) and COX-2 showed an inverse relation in colon cancer cell lines. COX-2 proteins in lung and colon tissues were higher in Cav-1 null mice than in wild-type mice. RNAi knockdown of Cav-1 increased COX-2 protein level and decreased ubiquitinated COX-2 accumulation. In addition, deletion of the carboxy (C)-terminus of COX-2, which contains a unique 19-amino acid segment compared with COX-1, resulted in reduced Cav-1 binding and attenuated COX-2 degradation. COX-1 and green fluorescence protein containing the C-terminus of COX-2 resulted in enhanced degradation. Our findings suggest that Cav-1 binds COX-2 in endoplasmic reticulum (ER) and carries it for degradation via ER associated degradation. The C-terminal region of COX-2 is required for Cav-1 binding and degradation. These results indicate a novel function of Cav-1 in controlling COX-2 expression, which may regulate physiological functions and have tumor suppression effects.
Background: Caveolin-1 assists in COX-2 degradation through the proteasome pathway. Results: Caveolin-1 enhances interactions among COX-2, Derlin-1, and the p97-Ufd1 complex and assists in COX-2 retrotranslocation and ubiquitination. Conclusion: Caveolin-1 is a cofactor facilitating COX-2 degradation via a Derlin-1-p97 pathway. Significance: Results represent a novel model for Derlin-1-mediated N-glycosylated protein degradation facilitated by caveolin-1.
Acute hepatic injury caused by inflammatory liver disease is associated with high mortality. This study examined the role of caveolin‐1 (Cav‐1) in lipopolysaccharide (LPS) and D‐galactosamine (GalN)‐induced fulminant hepatic injury in wild type and Cav‐1‐null (Cav‐1−/−) mice. Hepatic Cav‐1 expression was induced post‐LPS/GalN treatment in wild‐type mice. LPS/GalN‐treated Cav‐1−/− mice showed reduced lethality and markedly attenuated liver damage, neutrophil infiltration and hepatocyte apoptosis as compared to wild‐type mice. Cav‐1 deletion significantly reduced LPS/GalN‐induced caspase‐3, caspase‐8 and caspase‐9 activation and pro‐inflammatory cytokine and chemokine expression. Additionally, Cav‐1−/− mice showed suppressed expression of Toll‐like receptor 4 (TLR4) and CD14 in Kupffer cells and reduced expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 in liver cells. Cav‐1 deletion impeded LPS/GalN‐induced inducible nitric oxide synthase expression and nitric oxide production and hindered nuclear factor‐κB (NF‐κB) activation. Taken together, Cav‐1 regulated the expression of mediators that govern LPS‐induced inflammatory signalling in mouse liver. Thus, deletion of Cav‐1 suppressed the inflammatory response mediated by the LPS‐CD14‐TLR4‐NF‐κb pathway and alleviated acute liver injury in mice.
Prostacyclin (PGI2), a potent vasodilator and platelet antiaggregatory eicosanoid, is cytoprotective in cerebral circulation. It is synthesized from arachidonic acid (AA) by the sequential action of cyclooxygenase- (COX-) 1 or 2 and prostacyclin synthase (PGIS). Because prostacyclin is unstable in vivo, PGI2 analogs have been developed and demonstrated to protect against brain ischemia. This work attempts to selectively augment PGI2 synthesis in mixed glial culture or in a model of Parkinson's disease (PD) by direct adenoviral gene transfer of prostacyclin biosynthetic enzymes and examines whether it confers protection in cultures or in vivo. Confluent mixed glial cultures actively metabolized exogenous AA into PGE2 and PGD2. These PGs were largely NS398 sensitive and considered as COX-2 products. Gene transfer of AdPGIS to the cultures effectively shunted the AA catabolism to prostacyclin synthesis and concurrently reduced cell proliferation. Furthermore, PGIS overexpression significantly reduced LPS stimulation in cultures. In vivo, adenoviral gene transfer of bicistronic COX-1/PGIS to substantia nigra protected 6-OHDA- induced dopamine depletion and ameliorated behavioral deficits. Taken together, this study shows that enhanced prostacyclin synthesis reduced glial activation and ameliorated motor dysfunction in hemiparkinsonian rats. Prostacyclin may have a neuroprotective role in modulating the inflammatory response in degenerating nigra-striatal pathway.
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