Two pathways for cyclooxygenase-2 protein degradation in vivo.
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.
Wada
Masayuki
M
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Saunders
Thomas L
TL
Morrow
Jason
J
Milne
Ginger L
GL
Walker
Kimberly P
KP
Dey
Sudhansu K
SK
Brock
Thomas G
TG
Opp
Mark R
MR
Aronoff
David M
DM
Smith
William L
WL
D000818:Animals; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D055879:Gene Knock-In Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic
D051546:Cyclooxygenase 2