Two Pathways for Cyclooxygenase-2 Protein Degradation in Vivo

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 (⌬18 COX-2) knock-in mouse lacking this 18-amino acid segment. ⌬18 COX-2 knock-in mice do not exhibit the renal or reproductive abnormalities of COX-2 null mice. ⌬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 ⌬18 COX-2 mice overexpress ⌬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 ⌬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 ⌬18 COX-2 protein levels are similar in kidneys and spleens from WT and ⌬18 COX-2 mice. There must be compensatory mechanisms to maintain constant COX-2 levels in these tissues.

Enzymes of the Cyclooxygenase Pathways of Prostanoid Biosynthesis

“…PGHS-1 and PGHS-2 have three common N-glycosylation sites at Asn-68, Asn-144 and Asn-410 25-30 that appear to be glycosylated co-translationally 30,31 (Figures 2 and 3). NMR and mass spectral analyses of the carbohydrate chains of PGHSs indicate that the structures of the N-glycosyl moieties are of the form (Man) n (GlcNAc) 2 (n = 6-9) 32-34 .…”

Polarization of the Innate Immune Response by Prostaglandin E2: A Puzzle of Receptors and Signals

“…N-Glycosylation of Asn594 leads to entry of COX-2 into the endoplasmic reticulum-associated degradation pathway. The second COX-2 degradation process is substrate turnover dependent and is independent of N-glycosylation at Asn594 (Wada et al, 2009). Notably, COX-2 protein stability has been found to be enhanced by atorvastatin, which may influence dendritic cell (DC) function and counteract some of the untoward effects associated with sustained inhibition of COX-2 (Alvarez et al, 2009b).…”

A Cyclooxygenase-2-dependent Prostaglandin E2 Biosynthetic System in the Golgi Apparatus

“…Finally, with respect to degradation, we note that in addition to ERAD COX-2 is subject to substrate turnover-induced degradation (29,30). This latter process does not require N-glycosylation of Asn-594.…”

“…We have reported that properly folded COX-2 can be degraded via two distinct pathways including AA turnover-dependent degradation and constitutive degradation involving the ER-associated degradation (ERAD) pathway (29,30). Others have shown that COX-2 can be ubiquitinated and degraded through the 26 S proteasome (32,35).…”

“…1A). N-Glycosylation of Asn-594 within the degron occurs post-translationally and is central to its ERAD (29,30,35). An N594A mutation in COX-2 blocks enzyme degradation.…”