Site-specific proteolysis of cyclooxygenase-2: a putative step in inflammatory prostaglandin E(2) biosynthesis.
Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in inflammatory prostanoid biosynthesis. Transcriptional, post-transcriptional, and post-translational covalent modifications have been defined as important levels of regulation for COX-2 gene expression. Here, we describe a novel regulatory mechanism in primary human cells involving regulated, sequence-specific proteolysis of COX-2 that correlates with its catalytic activity and ultimately, the biosynthesis of prostaglandin E(2) (PGE(2)). Proinflammatory cytokines induced COX-2 expression and its proteolysis into stable immunoreactive fragments of 66, 42-44, 34-36, and 28 kDa. Increased COX-2 activity (PGE(2) release) was observed coincident with the timing and degree of COX-2 proteolysis with correlation analysis confirming a linear relationship (R(2) = 0.941). Inhibition of induced COX-2 activity with non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 selective inhibitors also abrogated cleavage. To determine if NSAID inhibition of proteolysis was related to drug-binding-induced conformational changes in COX-2, we assayed COX-inactive NSAID derivatives that fail to bind COX-2. Interestingly, these compounds suppressed COX-2 activity and cleavage in a correlated manner, thus suggesting that the observed NSAID-induced inhibition of COX-2 cleavage occurred through COX-independent mechanisms, presumably through the inhibition of proteases involved in COX-2 processing. Corroborating this observation, COX-2 cleavage and activity were mutually suppressed by calpain/cathepsin protease inhibitors. Our data suggest that the nascent intracellular form of COX-2 may undergo limited proteolysis to attain full catalytic capacity.
Mancini
Arturo
A
Department of Anatomy and Cell Biology, McGill University, Montreal, (QC), Canada.
Jovanovic
Dragan V
DV
He
Qing W
QW
Di Battista
John A
JA
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism; D001171:Arthritis, Juvenile / Q000276:immunology; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D015232:Dinoprostone / Q000096:biosynthesis* / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006801:Humans; D007249:Inflammation / Q000378:metabolism*; D053583:Interleukin-1beta / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013583:Synovial Membrane / Q000166:cytology
D000894:Anti-Inflammatory Agents, Non-Steroidal; D015853:Cysteine Proteinase Inhibitors; D053583:Interleukin-1beta; D010446:Peptide Fragments; D051546:Cyclooxygenase 2; D015232:Dinoprostone