Ethanol-induced downregulation of the angiotensin AT2 receptor in murine fibroblasts is mediated by PARP-1.
Molecular mechanisms accompanying ethanol-induced cytotoxicity remain to be defined. The renin-angiotensin system with its respective receptors, the angiotensin AT1 and AT2 receptor (AT1R and AT2R), has been implicated in these processes. The AT2R seems to counteract the pro-inflammatory, pro-hypertrophic, and pro-fibrotic actions of the AT1R and is involved in cellular differentiation and tissue repair. Recently, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel negative transcriptional regulator of the AT2R. However, the complex interactions between ethanol, PARP-1, and the AT2R are largely unknown. In this in vitro study, we aimed to clarify whether acute ethanol treatment modifies AT2R promoter activity or AT2R mRNA and protein levels and whether PARP-1 is involved in ethanol-mediated regulation of the AT2R. Murine fibroblasts of the R3T3 and MEF line (murine embryonic fibroblasts) were exposed to ethanol for 24h. AT2R promoter activity, mRNA and protein levels were analyzed with and without PARP-1 inhibition and in PARP-1 knockout MEF cells. Expression of PARP-1 was analyzed over course of time, and cell viability and DNA fragmentation were measured on single-cell level by flow cytometry. Ethanol exposition induced substantial downregulation of the AT2R on promoter, mRNA and protein levels in a dose-dependent manner. Pharmacological inhibition or ablation of PARP-1 completely abolished this effect. Ethanol treatment did not have any effect on AT1R mRNA and protein levels in MEF cells. Further, acute ethanol treatment promoted DNA fragmentation and caused transcriptional induction of PARP-1. Our findings reveal that PARP-1 is an upstream transcriptional regulator of the AT2 receptor in the context of ethanol exposure and represses the AT2R gene in fibroblasts in vitro. Variations in expression of the potentially tissue-protective AT2R might contribute to ethanol-mediated pathology.
Menk
Mario
M
Department of Anesthesiology and Intensive Care Medicine, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum/Campus Charité Mitte, 13353 Berlin, Germany. mario.menk@charite.de
von Haefen
Clarissa
C
Funke-Kaiser
Heiko
H
Sifringer
Marco
M
Schefe
Jan H
JH
Kirsch
Sebastian
S
Seidel
Kerstin
K
Reinemund
Jana
J
Steckelings
Ulrike M
UM
Unger
Thomas
T
Spies
Claudia D
CD
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival; D053938:DNA Fragmentation; D015536:Down-Regulation / Q000235:genetics*; D000431:Ethanol / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009447:Neuroblastoma; D016716:PC12 Cells; D000071137:Poly (ADP-Ribose) Polymerase-1; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D044139:Receptor, Angiotensin, Type 2 / Q000032:analysis / Q000235:genetics*
D006360:Heat-Shock Proteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D012333:RNA, Messenger; D044139:Receptor, Angiotensin, Type 2; D000431:Ethanol; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases