Wang Ning, Huo Rong, Cai Benzhi, Lu Yan, Ye Bo, Li Xiang, Li Faqian, Xu Haodong
Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, UCLA, 10833 Le Conte Ave., CHS 13-145E, Los Angeles, CA 90095-1732, USA.
Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA.
Free Radic Biol Med. 2016 Jul;96:34-44. doi: 10.1016/j.freeradbiomed.2016.04.003. Epub 2016 Apr 9.
Oxidants and canonical Wnt/β-catenin signaling have been shown to decrease cardiac Na(+) channel activity by suppressing NaV1.5 expression. Our aims are to determine if hydrogen peroxide (H2O2), one oxidant of reactive oxygen species (ROS), activates Wnt/β-catenin signaling and promotes β-catenin nuclear activity, leading to suppression of NaV1.5 expression and if this suppression requires the interaction of β-catenin with its nuclear partner, TCF4 (also called TCF7L2) to decrease SCN5a promoter activity. The results demonstrated that H2O2 increased β-catenin, but not TCF4 nuclear localization determined by immunofluorescence without affecting total β-catenin protein level. Furthermore, H2O2 exerted a dose- and time-dependent suppressive effect on NaV1.5 expression. RT-PCR and/or Western blot analyses revealed that overexpressing active form of β-catenin or stabilizing β-catenin by GSK-3β inhibitors, LiCl and Bio, suppressed NaV1.5 expression in HL-1 cells. In contrast, destabilization of β-catenin by a constitutively active GSK-3β mutant (S9A) upregulated NaV1.5 expression. Whole-cell recording showed that LiCl significantly inhibited Na(+) channel activity in these cells. Using immunoprecipitation (IP), we showed that β-catenin interacted with TCF4 indicating that β-catenin as a co-transfactor, regulates NaV1.5 expression through TCF4. Analyses of the SCN5a promoter sequences among different species by using VISTA tools indicated that SCN5a promoter harbors TCF4 binding sites. Chromatin IP assays demonstrated that both β-catenin and TCF4 were recruited in the SCN5a promoter, and regulated its activity. Luciferase promoter assays exhibited that β-catenin inhibited the SCN5a promoter activity at a dose-dependent manner and this inhibition required TCF4. Small interfering (Si) RNA targeting β-catenin significantly increased SCN5a promoter activity, leading to enhanced NaV1.5 expression. As expected, β-catenin SiRNA prevents H2O2 suppressive effects on both SCN5a promoter activity and NaV1.5 expression. Our findings indicate that H2O2 inhibits NaV1.5 expression by activating the Wnt/β-catenin signaling and β-catenin interacts with TCF4 to transcriptionally suppress cardiac NaV1.5 expression.
已有研究表明，氧化剂和经典Wnt/β-连环蛋白信号通路可通过抑制NaV1.5的表达来降低心脏钠通道活性。我们的目的是确定活性氧（ROS）的一种氧化剂过氧化氢（H2O2）是否会激活Wnt/β-连环蛋白信号通路并促进β-连环蛋白的核活性，从而导致NaV1.5表达受到抑制，以及这种抑制是否需要β-连环蛋白与其核伴侣TCF4（也称为TCF7L2）相互作用以降低SCN5a启动子活性。结果表明，通过免疫荧光检测发现H2O2增加了β-连环蛋白的核定位，但不影响TCF4的核定位，且不影响总β-连环蛋白蛋白水平。此外，H2O2对NaV1.5的表达具有剂量和时间依赖性的抑制作用。RT-PCR和/或蛋白质印迹分析显示，过表达β-连环蛋白的活性形式或用GSK-3β抑制剂LiCl和Bio稳定β-连环蛋白可抑制HL-1细胞中NaV1.5的表达。相反，组成型活性GSK-3β突变体（S9A）使β-连环蛋白不稳定，从而上调了NaV1.5的表达。全细胞记录显示，LiCl显著抑制了这些细胞中的钠通道活性。通过免疫沉淀（IP），我们发现β-连环蛋白与TCF4相互作用，这表明β-连环蛋白作为一种辅助转录因子，通过TCF4调节NaV1.5的表达。使用VISTA工具对不同物种的SCN5a启动子序列进行分析表明，SCN5a启动子含有TCF4结合位点。染色质免疫沉淀分析表明，β-连环蛋白和TCF4均被募集到SCN5a启动子中，并调节其活性。荧光素酶启动子分析显示，β-连环蛋白以剂量依赖性方式抑制SCN5a启动子活性，且这种抑制需要TCF4。靶向β-连环蛋白的小干扰（Si）RNA显著增加了SCN5a启动子活性，导致NaV1.5表达增强。正如预期的那样，β-连环蛋白SiRNA可防止H2O2对SCN5a启动子活性和NaV1.5表达的抑制作用。我们的研究结果表明，H2O2通过激活Wnt/β-连环蛋白信号通路抑制NaV1.5表达，且β-连环蛋白与TCF4相互作用以转录抑制心脏NaV1.5的表达。
