Surf4 collaborates with derlin-2 and derlin-1 to mediate cyclooxygenase-2 translocation to the cytosol for degradation.

Derlin family members participate in the retrotranslocation of endoplasmic reticulum (ER) lumen proteins to the cytosol for ER-associated degradation (ERAD); however, the proteins facilitating this retrotranslocation remain to be explored. Using CRISPR library screening, we have found that derlin-2 and surfeit locus protein 4 (Surf4) are candidates to facilitate degradation of cyclooxygenase-2 (COX-2, also known as PTGS2). Our results show that derlin-2 acts upstream of derlin-1 and that Surf4 acts downstream of derlin-2 and derlin-1 to facilitate COX-2 degradation. Knockdown of derlin-2 or Surf4 impedes the ubiquitylation of COX-2 and the interaction of COX-2 with caveolin-1 (Cav-1) and p97 (also known as VCP) in the cytosol. Additionally, COX-2 degradation is N-glycosylation dependent. Although derlin-2 facilitates degradation of N-glycosylated COX-2, the interaction between derlin-2 and COX-2 is independent of COX-2 N-glycosylation. Derlin-1, Surf4 and p97 preferentially interact with non-glycosylated COX-2, whereas Cav-1 preferentially interacts with N-glycosylated COX-2, regardless of the N-glycosylation pattern. Collectively, our results reveal that Surf4 collaborates with derlin-2 and derlin-1 to mediate COX-2 translocation from the ER lumen to the cytosol. The derlin-2-derlin-1-Surf4-Cav-1 machinery might represent a unique pathway to accelerate COX-2 degradation in ERAD.

Deletion of caveolin-1 attenuates LPS/GalN-induced acute liver injury in mice.

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.

Caveolin-1 interacts with Derlin-1 and promotes ubiquitination and degradation of cyclooxygenase-2 via collaboration with p97 complex.

Caveolin-1 (Cav-1) interacts with and mediates protein trafficking and various cellular functions. Derlin-1 is a candidate for the retrotranslocation channel of endoplasmic reticulum proteins. However, little is known about how Derlin-1 mediates glycosylated protein degradation. Here, we identified Cav-1 as a key player in Derlin-1- and p97-mediated cyclooxygenase 2 (COX-2) ubiquitination and degradation. Derlin-1 augmented the interaction of Cav-1 and COX-2 and mediated the degradation of COX-2 in a COX-2 C terminus-dependent manner. Suppression of Cav-1 decreased the ubiquitination of COX-2, and mutation of Asn-594 to Ala to disrupt N-glycosylation at the C terminus of COX-2 reduced the interaction of COX-2 with Cav-1 but not Derlin-1. Moreover, suppression of p97 increased the ubiquitination of COX-2 and up-regulated COX-2 but not COX-1. Cav-1 enhanced the interaction of p97 with Ufd1 and Derlin-1 and collaborated with p97 to interact with COX-2. Cav-1 may be a cofactor in the interaction of Derlin-1 and N-glycosylated COX-2 and may facilitate Derlin-1- and p97 complex-mediated COX-2 ubiquitination, retrotranslocation, and degradation.

Enhanced prostacyclin synthesis by adenoviral gene transfer reduced glial activation and ameliorated dopaminergic dysfunction in hemiparkinsonian rats.

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.

Caveolin-1 facilitates cyclooxygenase-2 protein degradation.

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.

Superoxide dismutase and catalase inhibit oxidized low-density lipoprotein-induced human aortic smooth muscle cell proliferation: role of cell-cycle regulation, mitogen-activated protein kinases, and transcription factors.

Several antioxidant enzymes, including copper, zinc-superoxide dismutase (Cu, Zn-SOD) and catalase, have been suggested to be protective against the proliferation of vascular smooth muscle cells exposed to oxidative stress. In the present study, we investigated effects of Cu, Zn-SOD and/or catalase on oxLDL-induced proliferation of, and intracellular signaling in, human aortic smooth muscle cells (HASMCs). HASMCs were transfected with adenovirus carrying the human Cu, Zn-SOD gene and/or the human catalase gene. This resulted in a high level of Cu, Zn-SOD and/or catalase overexpression and decreased oxLDL-induced proliferation. Cu, Zn-SOD and/or catalase also arrested cell cycle progression, which was associated with decreased expression of cyclin D1, cyclin E, CDK2, and CDK4 and upregulation of p21(Cip1) and p27(Kip1). Phosphorylation studies on ERK1/2, JNK, and p38, three major subgroups of mitogen activator protein kinases, demonstrated that Cu, Zn-SOD and/or catalase overexpression suppressed ERK1/2 and JNK phosphorylation. Gel-mobility shift analysis showed that oxLDL caused an increase in the DNA binding activity of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB), which was inhibited by Cu, Zn-SOD and/or catalase overexpression. These results provide the first evidence that overexpression of Cu, Zn-SOD and/or catalase in HASMCs attenuates the cell proliferation caused by oxLDL stimulation and that this inhibitory effect is mediated via downregulation of ERK1/2 and JNK phosphorylation and AP-1 and NF-kappaB inactivation. These observations support the feasibility of the increase of Cu, Zn-SOD and/or catalase expression in human smooth muscle cells as a means of protection against oxidant injury.

Induction of prostacyclin/PGI2 synthase expression after cerebral ischemia-reperfusion.

Prostacyclin (PGI2), a potent vasodilator and inhibitor of platelet aggregation and leukocyte activation, is crucial in vascular diseases such as stroke. Prostacyclin synthase (PGIS) is the key enzyme for PGI2 synthesis. Although expression of PGIS was noted in the brain, its role in ischemic insult remains unclear. Here we reported the temporal and spatial expression of PGIS mRNA and protein after 60-min transient ischemia. Northern blot and in situ hybridization revealed a delayed increase of PGIS mRNA in the ischemic cortex at 24- to 72-h after ischemia; PGIS was detected mainly in the ipsilateral penumbra area, pyriform cortex, hippocampus, and leptomeninges. Western blot and immunohistochemical analysis revealed that PGIS proteins were expressed temporally and spatially similar to PGIS mRNA. PGIS was heavily colocalized with PECAM-1 to endothelial cells at the leptomeninges, large and small vessels, and localized to neuronal cells, largely at the penumbra area. A substantial amount of PGIS was also detected in the macrophage and glial cells. To evaluate its role against ischemic infarct, we overexpressed PGIS by adenoviral gene transfer. When infused 72 h before ischemia (- 72 h), Adv-PGIS reduced infarct volume by approximately 50%. However, it had no effect on infarct volume when infused immediately after ischemia (0 h). Eicosanoid analysis revealed selective elevation of PGI2 at - 72 h while PGI2 and TXB2 were both elevated at 0 h, altering the PGI2/thromboxane A2 (TXA2) ratio from 10 to 4. These findings indicate that PGIS protects the brain by enhancing PGI2 synthesis and creating a favorable PGI2/TXA2 ratio.

Stabilization of hypoxia-inducible factor-1{alpha} by prostacyclin under prolonged hypoxia via reducing reactive oxygen species level in endothelial cells.

Hypoxia-inducible factor-1 (HIF-1) takes part in the transcriptional activation of hypoxia-responsive genes. HIF-1alpha, a subunit of HIF-1, is rapidly degraded under normoxic conditions by the ubiquitin-proteosome system. Hypoxia up-regulates HIF-1alpha 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-1alpha 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-1alpha up-regulation, which suggests that degradation of HIF-1alpha 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-1alpha in human umbilical vein endothelial cells exposed to prolonged hypoxia.

Superoxide dismutase inhibits the expression of vascular cell adhesion molecule-1 and intracellular cell adhesion molecule-1 induced by tumor necrosis factor-alpha in human endothelial cells through the JNK/p38 pathways.

OBJECTIVE: Expression of adhesion molecules on endothelial cells and subsequent leukocyte recruitment are critical early events in the development of atherosclerosis. We tried to study possible effects of Cu/Zn superoxide dismutase (SOD) on adhesion molecule expression and its underlying mechanism in the prevention and treatment of cardiovascular disorders. METHODS AND RESULTS: Human aortic endothelial cells (HAECs) were transfected with adenovirus carrying the human SOD gene (AdSOD) to investigate whether SOD expression in HAECs attenuated tumor necrosis factor (TNF)-alpha-induced reactive oxygen species production and adhesion molecule expression and to define the mechanisms involved. SOD expression significantly suppressed TNF-alpha-induced expression of vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1 and reduced the binding of the human neutrophils to TNF-alpha-stimulated HAECs. SOD expression suppressed c-JUN N-terminal kinase and p38 phosphorylation. It also attenuated intracellular superoxide anion production and NADPH oxidase activity in TNF-alpha-treated HAECs. CONCLUSIONS: These results provide evidence that SOD expression in endothelial cells attenuates TNF-alpha-induced superoxide anion production and adhesion molecule expression, and that this protective effect is mediated by decreased JNK and p38 phosphorylation and activator protein-1 and nuclear factor kappaB inactivation. These results suggest that SOD has antiinflammatory properties and may play important roles in the prevention of atherosclerosis and inflammatory response. Superoxide dismutase overexpression in endothelial cells attenuates tumor necrosis factor alpha-induced superoxide anion production and adhesion molecule expression, and this effect is mediated by decreased JNK and p38 phosphorylation and AP-1 and nuclear factor B inactivation. These results suggest that superoxide dismutase may play an important role in the prevention of atherosclerosis and inflammatory response.