651 VERAPAMIL AND PENTOXIFYLLINE INHIBIT TGF-BETA-STIMULATED TIMP-3 PRODUCTION IN PEYRONIE'S DISEASE FIBROBLASTS: EVIDENCE OF CAMP- AND CALCIUM-DEPENDENT SIGNALING CROSSTALK

Abstract

You have accessJournal of UrologyPenis/Testis/Urethra: Benign & Malignant Disease II1 Apr 2010651 VERAPAMIL AND PENTOXIFYLLINE INHIBIT TGF-BETA-STIMULATED TIMP-3 PRODUCTION IN PEYRONIE'S DISEASE FIBROBLASTS: EVIDENCE OF CAMP- AND CALCIUM-DEPENDENT SIGNALING CROSSTALK Marcello Del Carlo, Ada Cole, and Laurence Levine Marcello Del CarloMarcello Del Carlo More articles by this author , Ada ColeAda Cole More articles by this author , and Laurence LevineLaurence Levine More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1025AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The calcium channel blocker verapamil and the phosphodiesterase type 4 (PDE4) inhibitor pentoxifylline have been reported to be useful as a treatment alternative for patients with Peyronie's Disease (PD). The fibrotic effects of TGF-beta overexpression in PD are well documented but the role of cAMP and calcium as secondary intermediates of TGF-beta signaling is poorly understood. The purpose of this study was to determine the effects of these anti-fibrotic agents on TGF-beta mediated tissue inhibitor of matrix metalloproteinase (TIMP) production as a potential mechanism of action. METHODS Fibroblasts were isolated from fibrotic plaque tissue removed from patients undergoing surgical correction for PD. The effects of verapamil (100 uM) and pentoxifylline (500 uM) on TGF-beta (10 ng/ml) mediated TIMP-3 production was analyzed by immunoblotting conditioned media from monolayer cultures 48 hours after stimulation, and reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify TIMP-3 mRNA in cells 8 hours after stimulation. Intracellular [calcium] fluxes were analyzed by confocal microscopy (lambda-ex = 340/380 nm, lambda-em = 510 nm) using Fura-2/AM (2 uM) loaded cells in the presence and absence of either the PKA activator forskolin (10 uM) or the PKA inhibitor H89 (10 uM) to modulate intracellular [cAMP]. Luciferase reporter constructs were transfected in cells to quantify CREB binding activity. RESULTS Pre-treatment with either verapamil or pentoxifylline reduced TGF-beta mediated TIMP-3 mRNA expression and protein production, and also prevented increased cytosolic calcium fluxes observed in cells stimulated with TGF-beta. Similar results were obtained with the calcium channel blocker nifedipine and the PKA activator forskolin. Pretreatment with either the cell-permeable calcium-chelator BAPTA/AM (5 uM) or the PKA inhibitor H89 had no effect. Treatment with either verapamil or pentoxifylline alone significantly induced CREB activity, which was marginally attenuated in the presence of TGF-beta. CONCLUSIONS Decreased TGF-beta mediated TIMP-3 expression represents an attractive mechanism for the anti-fibrotic effects displayed by verapamil and pentoxifylline therapy in patients with PD. The increased activity of cAMP-sensitive calcium channels caused by TGF-beta overexpression may represent an important biochemical mechanism for the localized fibrosis observed in PD. Chicago, IL© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e255 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Marcello Del Carlo More articles by this author Ada Cole More articles by this author Laurence Levine More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...