Evaluation of urothelial stretch-induced cyclooxygenase-2 expression in novel human cell culture and porcine in vivo ureteral obstruction models.
Obstruction and stretch induce cyclooxygenase (COX)-2 expression and prostanoid synthesis in urinary tissues, causing pain, inflammation, hypercontractility, and cell proliferation. Our objective was to characterize acute COX-2 induction during in vivo ureteral obstruction, establish a cell culture model of urothelial stretch-induced COX-2 expression, and evaluate whether mechanotransduction could alter transcriptional and post-transcriptional regulation of COX-2. We performed laparoscopic unilateral ureteral ligation in pigs and allowed progression for 1, 2, 6, 24, or 48 h. We evaluated COX-2 expression with reverse transcriptase (RT)-polymerase chain reaction (PCR) and immunoblotting. We cultured primary human urothelial cells on stretch plates, applied stretch for up to 48 h, and measured COX-2 expression by RT-PCR and immunoblotting, transcription with run-on assays, and mRNA stability with actinomycin mRNA decay assays. In vivo ureteral obstruction induced COX-2 expression 4-fold within 6 h, maintaining induction for 24 h. In cell culture, stretch induced COX-2 steady-state mRNA and protein within the first 3 h of stretch, maintaining this induction for over 6 h. Three hours of stretch doubled COX-2 transcription relative to unstretched controls and increased COX-2 mRNA half-life 3-fold. This is the first report to characterize in vivo temporal stretch-induced COX-2 expression in the urothelium and establish a primary urothelial cell culture model for the study of stretch-induced COX-2 mechanisms. This is also the first report to identify alterations in steady-state COX-2 mRNA having components of both transcriptional and post-transcriptional regulation of stretch-regulated COX-2. Future elucidation of COX-2 signaling may identify novel therapeutic targets for treating stretch and distension of urinary tissues.
Jerde
Travis J
TJ
The Uropharmacology and Endourology Research Laboratory, University of Wisconsin Medical School, Department of Pharmaceutical Science, Division of Urology, K6-561 Clinical Science Center, 600 Highland Avenue, Madison, WI 53792, USA. jerde@surgery.wisc.edu
Mellon
William S
WS
Bjorling
Dale E
DE
Nakada
Stephen Y
SY
D000328:Adult; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000096:biosynthesis* / Q000235:genetics; D004195:Disease Models, Animal; D004790:Enzyme Induction; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013314:Stress, Mechanical; D013552:Swine; D014158:Transcription, Genetic; D014513:Ureter / Q000166:cytology / Q000201:enzymology*; D014517:Ureteral Obstruction / Q000201:enzymology*; D019459:Urothelium / Q000166:cytology / Q000201:enzymology*
D012333:RNA, Messenger; D051546:Cyclooxygenase 2