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

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 ([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.

EP2 Receptor Activates Dual G Protein Signaling Pathways that Mediate Contrasting Proinflammatory and Relaxatory Responses in Term Pregnant Human Myometrium

“…An association between PGHS-2 regulation and calcium (37)(38)(39), as well as PGHS-2 and cAMP (40 -42), has been reported before in other systems. We have previously shown that nonreceptor-mediated elevation of cAMP can stimulate expression of PGHS-2 in pregnant human myometrium (24).…”

Increased cyclooxygenase-2 expression and prostaglandin E2production in pressurized renal medullary interstitial cells

“…Several in vitro studies have demonstrated that mechanical stretch activates COX-2 expression in bladder smooth muscle and vascular endothelial cells as well as renal podocytes, urothelial cells, and osteoblasts (11,15,17,20). Both in urothelial cells (17) and podocytes (20) increased COX-2 protein level was observed after stretching for 6 h in agreement with our data showing significant induction of COX-2 protein abundance in RMICs pressurized for 6 h. Many cellular signal transduction cascades of COX-2 induction have been identified, including those involving mitogen-activated protein kinases, PKA, PKC, NF-B, extracellular matrix proteins, and glycogen synthase kinase-3␤. However, most of these studies involved hormone, mitogen-induced, or hypertonic conditions, and data investigating mechanosensing pathways of COX-2 induction remain sparse.…”

“…Several in vitro studies have demonstrated increased COX-2 expression in numerous cell culture models, including renal podocytes, urothelial cells, vascular endothelial cells, and osteoblasts, which have been subjected to mechanical stretch (11,15,17,20). These data led us to hypothesize that elevated pressure increases COX-2 expression and activity in RMICs isolated from rats.…”

LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

“…A reduction of calcium efflux can also contribute to an increase in [Ca 2+ ] i (Elliott and Sapolsky, 1993). Nevertheless calcium dependent increase in COX-2 gene transcription has been reported (Puga et al, 1997;Pham et al, 2006;Jerde et al, 2008), suggesting that CT may increase COX-2 expression by elevating [Ca 2+ ] i in cardiomyocytes.…”