Stretch induction of cyclooxygenase-2 expression in human urothelial cells is calcium- and protein kinase C zeta-dependent.

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 ([Ca(2+)](i)), primary human urothelial cells were grown on flexible membranes and loaded with Fura-2 acetoxymethyl ester (AM). We determined [Ca(2+)](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 PKCalpha, beta1, and zeta, 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 PKCzeta is the predominant isoform mediating stretch-induced COX-2 expression. Elucidating the signaling mechanism of stretch-induced COX-2 expression may identify therapeutic targets.

1,25-Dihydroxyvitamin D3 selectively translocates PKCalpha to nuclei in ROS 17/2.8 cells.

We have investigated protein kinase C (PKC) regulation by 1,25-(OH)2D3 in the rat osteosarcoma cell line ROS 17/2.8 since previous reports have implicated PKC in the 1,25-(OH)2D3-mediated regulation of osteocalcin gene expression (J. Biol. Chem. 267 (1992) 12562; Endocrinology 136 (1995) 5685). Here we report that 1,25-(OH)2D3 increased PKCalpha, but not PKCbetaI, epsilon or zeta, levels in the nuclear fraction in a time-dependent manner. Unlike PMA, 1,25-(OH)2D3 did not alter the association of any of the expressed PKC isoenzymes with the plasma membrane. Treatment with 20 nM 1,25-(OH)2D3 for 15 min, 30 min, 1 h and 24 h increased PKCalpha levels in the nuclear fraction by 2.3- to 2.6-fold. Nuclear PKCalpha expression was also increased with doses of 1,25-(OH)2D3 as low as a 0.05 nM. 1,25-(OH)2D3-mediated stabilization of osteocalcin mRNA (Arch. Biochem. Biophys. 332 (1996) 142) was inhibited with bisindolylmaleimide treatment, suggesting that PKCalpha may be involved in the 1,25-(OH)2D3-mediated regulation of osteocalcin gene expression.

PI3K mediates stretch-induced COX-2 expression during urinary tract obstruction.

BACKGROUND AND PURPOSE: Stretch-induced cyclooxygenase-2 (COX-2) expression occurs in urothelial cells during urinary tract obstruction (UTO). This increases COX-2-dependent prostanoid synthesis in stretched urothelial cells. These prostanoids then act on afferent neurons and smooth muscle cells in the ureter to amplify nociceptive and contractile responses, respectively. We previously used a unilateral ureteral obstruction (UUO) mouse model and a primary human urothelial cell (HUC) stretch model to describe ureteral COX-2 expression during UTO. The current study was performed to determine whether phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways are necessary for stretch-induced COX-2 expression in urothelial cells. MATERIALS AND METHODS: Adult male CD-1 mice were treated with 25% dimethyl sulfoxide/phosphate buffered saline or PI3K inhibitor LY294002 (3 mg/kg, 30 mg/kg) for 1 hour before performing UUO for up to 4 hours. Obstructed and contralateral mouse ureters were analyzed via immunohistochemistry or Western blotting to assess in vivo stretch-induced COX-2 expression. In addition, HUCs were cyclically stretched (5%-20% displacement, 12 cycles/min) on collagen I-coated stretch plates and assessed for COX-2 expression via Western blotting. RESULTS: Histologic analyses of obstructed ureters show that urothelial cells stretch in response to external obstruction, COX-2 expression increases in the stretched urothelial cells, and no infiltrating immune cells were present under the conditions of the study. PI3K inhibitor LY294002 (30 mg/kg) attenuated in vivo stretch-induced COX-2 expression. LY294002 or RNA-interference also attenuated (HUC) stretch-induced COX-2 expression in vitro. Furthermore, the results also show that LY294002 inhibits stretch-induced protein kinase C (PKCζ) activation previously identified upstream of stretch-induced COX-2 expression in HUCs. CONCLUSIONS: The results indicate that PI3K is a mediator of stretch-induced COX-2 expression in urothelial cells. Identifying molecules that couple urothelial cell stretch to COX-2 expression may provide targets of drug action for effective therapeutics for UTO.

Evaluation of urothelial stretch-induced cyclooxygenase-2 expression in a mouse ureteral obstruction model.

Prostanoids play a major role in the nociceptive response to ureteral obstruction. Cyclooxygenases (COXs) 1 and 2 catalyze the rate-limiting step in prostanoid synthesis; COX-2 is the more inducible isoform. Previous studies in human and animal models have shown that COX-2 is highly induced during ureteral obstruction. Our objective was to characterize acute COX-2 induction in a reproducible mouse model. Unilateral ureteral ligation was performed, and obstruction was maintained for 2, 4, 6, 8, or 12 hours. We evaluated COX-2 protein expression using Western immunoblotting, and found that ureteral obstruction induced COX-2 expression ninefold within 6 hours. This is the first report to characterize in vivo temporal stretch-induced COX-2 expression in a mouse model. This model will be critical for elucidation of COX-2 signaling pathways and may eventually help to identify novel therapeutic targets for treating ureteral obstruction.

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.

Suppression of 15-hydroxyprostaglandin dehydrogenase messenger RNA concentration, protein expression, and enzymatic activity during human ureteral obstruction.

Prostanoids produce significant effects in the ureter, particularly in response to obstruction. Ureteral obstruction is associated with increased prostanoid synthesis via cyclooxygenase induction; however, prostaglandin degradation mediated by 15-hydroxyprostaglandin dehydrogenase (PGDH) has not been evaluated in the ureter. The purpose of this study was to determine whether PGDH steady-state mRNA, protein, and enzyme activity are altered in the human ureter during obstruction. Human ureteral segments from patients undergoing donor nephrectomy (normal segments) or ureteral stricture repair (obstructed segments) were obtained with proper informed consent. We evaluated PGDH steady-state mRNA relative to ribosomal protein S26 reference gene by reverse transcription-polymerase chain reaction and Vistra Green fluoroimaging. We determined PGDH protein content relative to glyceraldehyde-3-phosphate dehydrogenase by immunoblotting and PGDH localization by immunohistochemistry. PGDH enzymatic activity was determined by measurement of conversion of 15-hydroxy- to 15-keto-prostaglandin using thin layer chromatography separation. We found that PGDH mRNA and protein were decreased 4- to 6-fold, and enzyme activity was decreased >3-fold in obstructed human ureter relative to normal controls. PGDH was localized to the urothelial cells, with little or no expression in smooth muscle. Our results indicate that PGDH mRNA, protein, and enzyme activity are suppressed in the human ureter during obstruction. Increased concentrations of prostanoids subsequent to ureteral obstruction seem to be due to decreased degradation as well as increased synthesis. Modulation of prostanoid degradation may have therapeutic relevance in obstructive disorders of the ureter.