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Stretch Induction of Cyclooxygenase-2 Expression in Human Urothelial Cells Is Calcium- and Protein Kinase C -Dependent

The Uropharmacology and Endourology Research Laboratory; Departments of Pharmaceutical Sciences (T.J.J., W.S.M.), School of Veterinary Medicine (C.M.C., D.E.B.), College of Agricultural and Life Sciences (J.J.P.), and Surgery-Division of Urology (T.J.J., D.E.B., S.Y.N.), University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin

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 PKC, β1, and , 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 PKC is the predominant isoform mediating stretch-induced COX-2 expression. Elucidating the signaling mechanism of stretch-induced COX-2 expression may identify therapeutic targets.

Address correspondence to: Dr. Travis J. Jerde, University of Wisconsin Medical School, Department of Surgery, Division of Urology, K6-561 Clinical Science Center; 600 Highland Avenue, Madison, WI 53792. E-mail: jerde{at}surgery.wisc.edu