Abstract
COX-2, formally known as prostaglandin endoperoxide H synthase-2 (PGHS-2), catalyzes the committed step in prostaglandin biosynthesis. COX-2 is induced during inflammation and is overexpressed in colon cancer. In vitro, an 18-amino acid segment, residues 595-612, immediately upstream of the C-terminal endoplasmic reticulum targeting sequence is required for N-glycosylation of Asn(594), which permits COX-2 protein to enter the endoplasmic reticulum-associated protein degradation system. To determine the importance of this COX-2 degradation pathway in vivo, we engineered a del595-612 PGHS-2 (Delta 18 COX-2) knock-in mouse lacking this 18-amino acid segment. Delta 18 COX-2 knock-in mice do not exhibit the renal or reproductive abnormalities of COX-2 null mice. Delta 18 COX-2 mice do have elevated urinary prostaglandin E(2) metabolite levels and display a more pronounced and prolonged bacterial endotoxin-induced febrile response than wild type (WT) mice. Normal brain tissue, cultured resident peritoneal macrophages, and cultured skin fibroblasts from Delta 18 COX-2 mice overexpress Delta 18 COX-2 relative to WT COX-2 expression in control mice. These results indicate that COX-2 can be degraded via the endoplasmic reticulum-associated protein degradation pathway in vivo. Treatment of cultured cells from WT or Delta 18 COX-2 mice with flurbiprofen, which blocks substrate-dependent degradation, attenuates COX-2 degradation, and treatment of normal mice with ibuprofen increases the levels of COX-2 in brain tissue. Thus, substrate turnover-dependent COX-2 degradation appears to contribute to COX-2 degradation in vivo. Curiously, WT and Delta 18 COX-2 protein levels are similar in kidneys and spleens from WT and Delta 18 COX-2 mice. There must be compensatory mechanisms to maintain constant COX-2 levels in these tissues.
Highlights
There are two prostaglandin endoperoxide H synthases (PGHSs)2 called PGHS-1 and prostaglandin endoperoxide H synthase-2 (PGHS-2)
The ⌬18 COX-2 protein is catalytically indistinguishable from native COX-2, but unlike wild type (WT) COX-2, it is unable to undergo N-glycosylation of Asn594 and degradation via the endoplasmic reticulum-associated degradation” (ERAD) pathway (17)
In combination with other in vitro studies of the mechanism of COX-2 protein degradation (17, 18), our results indicate that the ERAD pathway participates in COX-2 protein degradation in vivo
Summary
Materials—129X1/SvJ genomic DNA (stock number 000691) was obtained from The Jackson Laboratory. 2.0 ϫ 105 cells were plated in 96-well plates and incubated for 30 – 60 min in a CO2 incubator at which time the media were removed and fresh RPMI 1640 medium supplemented with 10% HFBS, penicillin, and streptomycin was added. After 24 h, the media were changed to 100 l of RPMI 1640 medium containing penicillin and streptomycin with or without inhibitors, and the cells were incubated for 30 min. An additional 100 l of RPMI 1640 medium containing penicillin, streptomycin, and 200 ng/ml LPS was added to each well, and the cells were incubated for different times, and the supernatants were harvested and kept at Ϫ80 °C until analyzed.
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