title: TRPV4 regulates insulin mRNA expression and INS-1E cell death via ERK1/2 and NO-dependent mechanisms.
abstract: TRPV4 is a Ca2+-permeable, nonselective cation channel. Recently, TRPV4 was implicated in controlling peripheral insulin sensitivity, insulin secretion and apoptosis of pancreatic beta cells. Here, we characterize the role and potential mechanisms of TRPV4 in regulating insulin mRNA expression and cell death in insulin producing INS-1E cells and rat pancreatic islets. TRPV4 protein production was downregulated by siRNA. Intracellular calcium level was measured using Fluo-3 AM. Gene expression was studied by real-time PCR. Phosphorylation of extracellular signal-regulated kinase (ERK1 and ERK2) was detected by Western blot. Nitric oxide (NO) production was assessed by chemiluminescent reaction. Reactive oxygen species (ROS) level was analysed using a fluorogenic dye (DCFDA). Cell death was evaluated by determination of cytoplasmic histone-associated DNA fragments. Downregulation of TRPV4 neither affected insulin mRNA expression nor INS-1E cell growth. By contrast, pharmacological TRPV4 activation by 100nmol/l GSK1016790A increased Ca2+levels in INS-1E cells and enhanced insulin mRNA expression after 1 and 3h, whereas a suppression of insulin mRNA expression was detected after 24h incubation. GSK1016790A increased ERK1/2 phosphorylation and NO production but not ROS production. Pharmacological blockade of ERK1/2 attenuated GSK1016790A-induced insulin mRNA expression. Inhibition of NO synthesis by l-NAME failed to affect insulin mRNA expression in GSK1016790A treated INS-1E cells. Furthermore, inhibition of NO production attenuated GSK1016790A-induced INS-1E cell death. In pancreatic islets, 100nmol/l GSK1016790A increased insulin mRNA levels after 3h without inducing cytotoxicity after 24h. In conclusion, TRPV4 differently regulates insulin mRNA expression in INS-1E cells via ERK1/2 and NO-dependent mechanisms.
authors.affiliation: Department of Animal Physiology and Biochemistry, Poznań University of Life Sciences, 60-637 Poznań, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Billert
authors.affiliation: Department of Animal Physiology and Biochemistry, Poznań University of Life Sciences, 60-637 Poznań, Poland. Electronic address: mskrzyps@up.poznan.pl.
authors.forename: M
authors.initials: M
authors.lastname: Skrzypski
authors.affiliation: Department of Animal Physiology and Biochemistry, Poznań University of Life Sciences, 60-637 Poznań, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Sassek
authors.affiliation: Department of Animal Physiology and Biochemistry, Poznań University of Life Sciences, 60-637 Poznań, Poland.
authors.forename: D
authors.initials: D
authors.lastname: Szczepankiewicz
authors.affiliation: Department of Animal Physiology and Biochemistry, Poznań University of Life Sciences, 60-637 Poznań, Poland.
authors.forename: T
authors.initials: T
authors.lastname: Wojciechowicz
authors.affiliation: Department of Ophthalmology, Charité University Medicine Berlin, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Mergler
authors.affiliation: Department of Hepatology and Gastroenterology, Interdisciplinary Centre of Metabolism, Endocrinology, Diabetes and Metabolism, Charité-University Medicine Berlin, 13353 Berlin, Germany; Department of Internal Medicine-Gastroenterology, Park-Klinik Weissensee, 13086 Berlin, Germany.
authors.forename: M Z
authors.initials: MZ
authors.lastname: Strowski
authors.affiliation: Department of Animal Physiology and Biochemistry, Poznań University of Life Sciences, 60-637 Poznań, Poland.
authors.forename: K W
authors.initials: KW
authors.lastname: Nowak
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D049109:Cell Proliferation / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007328:Insulin / Q000235:genetics* / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000378:metabolism / Q000473:pathology; D007930:Leucine / Q000008:administration & dosage / Q000031:analogs & derivatives; D020935:MAP Kinase Signaling System / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013449:Sulfonamides / Q000008:administration & dosage; D050916:TRPV Cation Channels / Q000235:genetics* / Q000378:metabolism
chemical_list: D007328:Insulin; C530602:N-(1-((4-(2-(((2,4-dichlorophenyl)sulfonyl)amino)-3-hydroxypropanoyl)-1-piperazinyl)carbonyl)-3-methylbutyl)-1-benzothiophene-2-carboxamide; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013449:Sulfonamides; D050916:TRPV Cation Channels; C486577:Trpv4 protein, rat; D009569:Nitric Oxide; D007930:Leucine; D002118:Calcium
keywords: Beta-cell; Calcium; Insulin; Islets; TRPV4