Received for publication December 14, 2005.
Revised February 23, 2006.
Accepted for publication February 27, 2006.

EVALUATION OF UROTHELIAL STRETCH-INDUCED COX-2 EXPRESSION IN NOVEL HUMAN CELL CULTURE AND PORCINE IN VIVO URETERAL OBSTRUCTION MODELS

Abstract

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 hours. We evaluated COX-2 expression with RT-PCR, and immunoblotting. We cultured primary human urothelial cells on stretch plates, applied stretch for up to 48 hours, 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 hours, maintaining induction for 24 hours. In cell culture, stretch induced COX-2 steady-state mRNA and protein within the first 3 hours of stretch, maintaining this induction for over 6 hours. 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.

Key words: cell culture, cell stretch, cyclooxygenase-2, in vivo model, mRNA stability, mechanotransduction

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