Abstract
Prostanoid synthesis via cyclooxygenase (COX)-2 induction during urothelial stretch is central to nociception, inflammation, contractility, and proliferation caused by urinary tract obstruction. We used our primary human urothelial cell stretch model published previously to evaluate the signaling mechanisms responsible for stretch-induced COX-2 expression in urothelial cells. To determine intracytosolic calcium concentrations ([Ca2+]i), primary human urothelial cells were grown on flexible membranes and loaded with Fura-2 acetoxymethyl ester (AM). We determined [Ca2+]i using a fluorescent scope during stretch. Additional cells were treated with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-AM, stretched, and COX-2 mRNA and protein were evaluated by real-time polymerase chain reaction and immunoblotting. To evaluate protein kinase C (PKC) in this system, cells were stretched and fractionated into membrane, cytosol, and nucleus. Fractions were immunoblotted for PKCα, β1, and ζ, the predominant isoforms in urothelial cells. We treated additional cells with increasing concentrations of either bisindolylmaleimide-I or a peptide PKC pseudosubstrate inhibitor, and COX-2 mRNA and protein were evaluated after stretching. Furthermore, we transfected urothelial cells with siRNA against each of the inducible PKC isoforms in these cells and evaluated the stretch-induced COX-2 response. Stretch of urothelial cells activated calcium flux and PKC translocation to membrane and nucleus. Pharmacological inhibition indicated that stretch-induced COX-2 expression is dependent on calcium and PKC, and biochemical knockdown experiments indicated that PKCζ is the predominant isoform mediating stretch-induced COX-2 expression. Elucidating the signaling mechanism of stretch-induced COX-2 expression may identify therapeutic targets.
Footnotes
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ABBREVIATIONS: COX, cyclooxygenase; PKC, protein kinase C; AM, acetoxymethyl ester; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DTT, dithiothreitol; PP, psuedosubstrate peptide; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; TEDK, Tris-HCl/KCl/EDTA/dithiothreitol/protease inhibitor cocktail; siRNA, small interfering RNA; KT-5720, (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i] [1,6]benzodiazocine-10-carboxylic acid hexyl ester.
- Received April 6, 2007.
- Accepted October 9, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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