PARP1对NFATs的调控在病理性心肌肥大中的作用
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摘要
目的探究多聚腺苷二磷酸核糖聚合酶1(PARP1)与活化T细胞核转录因子3(NFATc3)、NFATc4在病理性心肌肥大中的调控关系及其机制。方法采用异丙肾上腺素(ISO)刺激原代心肌细胞24 h,在细胞水平建立心肌肥大模型; SD大鼠皮下注射ISO,在动物水平建立心肌肥大模型。使用核蛋白抽提试剂盒分离胞质和胞核蛋白,免疫印迹法检测相应蛋白的出入核及蛋白表达水平变化;利用免疫荧光,检测NFATc3、NFATc4的亚细胞定位;采用腺病毒感染的方法进行PARP1过表达,通过转染小干扰RNA进行PAPR1的敲低。结果在细胞及动物水平上,ISO成功建立心肌肥大模型,均观察到ISO引起NFATc3和NFATc4在细胞核的聚集增加;同时,ISO可致PARP1的PAR化酶活性上升;单独过表达PARP1可诱导NFATc3和NFATc4入核增加,敲低PARP1抑制了ISO引起的入核增加,而给予PARP1抑制剂3AB则可一定程度逆转NFATc3和NFATc4的入核转位。结论ISO可能通过诱导PARP1酶活性的上调,进而促进NFATc3和NFATc4的转位入核,从而诱导心肌肥大的发生。
Aim To investigate the interaction and mechanism of PARP1 and NFATc3,NFATc4 in ISOinduced pathological cardiac hypertrophy. Methods To establish the model of cardiac hypertrophy in vitroand in vivo,primary neonatal rat cardiomyocytes were treated with ISO( 10 μmol·L~(-1)) for 24 h; SD rats were subcutaneously injected with 1. 2 mg·kg~(-1)·d~(-1) ISO for 7 d. The nuclear and cytoplasmic proteins wereseparated by Cellytic Nuclear Extraction Kit. The subcellular localization of NFATc3 and NAFTc4 were detected by Western blot and immunofluorescence. The recombinant adenovirus( Ad-PARP1) infection was used to overexpress PARP1 and knockdown PARP1 by transfecting with siRNA of PARP1 in cardiomyocytes.Results The models of cardiac hypertrophy were successfully built both in vivo and vitro by ISO. It was determined that NFATc3 and NFATc4 were transfered into the nuclear from the cytoplasm in primary neonatal rat cardiomyocytes( NRCMs) after being treated with ISO. And the enzymatic activity of PARP1 was boosted in ISO-treated group. Overexpression of PARP1 promo-ted the nuclear translocation of NFATc3 and NFATc4 in cardiomyocytes,while knockdown of PARP1 could reverse the nuclear translocation induced by ISO.PARP1 inhibitor 3 AB retarded ISO-induced nuclear transportation of NFATc3 and NFATc4 to some extent.Conclusions ISO leads to the up-regulation of enzymatic activity of PARP1 and promotes nuclear translocation of NFATc3 and NFATc4,thus aggravating cardiac hypertrophy.
Aim To investigate the interaction and mechanism of PARP1 and NFATc3,NFATc4 in ISOinduced pathological cardiac hypertrophy. Methods To establish the model of cardiac hypertrophy in vitroand in vivo,primary neonatal rat cardiomyocytes were treated with ISO( 10 μmol·L~(-1)) for 24 h; SD rats were subcutaneously injected with 1. 2 mg·kg~(-1)·d~(-1) ISO for 7 d. The nuclear and cytoplasmic proteins wereseparated by Cellytic Nuclear Extraction Kit. The subcellular localization of NFATc3 and NAFTc4 were detected by Western blot and immunofluorescence. The recombinant adenovirus( Ad-PARP1) infection was used to overexpress PARP1 and knockdown PARP1 by transfecting with siRNA of PARP1 in cardiomyocytes.Results The models of cardiac hypertrophy were successfully built both in vivo and vitro by ISO. It was determined that NFATc3 and NFATc4 were transfered into the nuclear from the cytoplasm in primary neonatal rat cardiomyocytes( NRCMs) after being treated with ISO. And the enzymatic activity of PARP1 was boosted in ISO-treated group. Overexpression of PARP1 promo-ted the nuclear translocation of NFATc3 and NFATc4 in cardiomyocytes,while knockdown of PARP1 could reverse the nuclear translocation induced by ISO.PARP1 inhibitor 3 AB retarded ISO-induced nuclear transportation of NFATc3 and NFATc4 to some extent.Conclusions ISO leads to the up-regulation of enzymatic activity of PARP1 and promotes nuclear translocation of NFATc3 and NFATc4,thus aggravating cardiac hypertrophy.
引文
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[6]郑宇静,左彤彤,封宇飞.靶向DNA损伤反应途径:PARP抑制剂抗肿瘤治疗研究进展[J].中国药理学通报,2018,34(2):157-61.[6]Zheng Y J,Zuo T T,Feng Y F.Targeting DNA damage response pathway:recent progress of PARP inhibitors in cancer therapy[J].Chin Pharmacol Bull,2018,34(2):157-61.
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[8]Li Q,Li Z M,Sun S Y,et al.PARP1 interacts with HMGB1 and promotes its nuclear export in pathological myocardial hypertrophy[J].Acta Pharmacol Sin,2019,40(5):589-98.
[9]Lu J,Zhang R W,Hong H Q et al.The poly(ADP-ribosyl)ation of Fox O3 mediated by PARP1 participates in isoproterenol-induced cardiac hypertrophy[J].Biochim Biophy Acta,2016,1863(12):3027-39.
[10]Osadchii,O E.Cardiac hypertrophy induced by sustained beta-adrenoreceptor activation:pathophysiological aspects[J].Heart Fail Rev,2007,12(1):66-86.
[11]王桂军,姚玉胜,王洪新.Ca2+/CaMKⅡ信号通路在肿瘤坏死因子-α诱导心肌肥大中的作用[J].中国药理学通报,2010,26(3):387-91.[11]Wang G J,Yao Y S,Wang H X.Ca2+/CaMKⅡare involved in tumor necrosis factorα-induced cardiomyocyte hypertrophy in rats[J].Chin Pharmacol Bull,2010,26(3):387-91.
[12]Mognol G P,Carneiro F R,Robbs B K,et al.Cell cycle and apoptosis regulation by NFAT transcription factors:new roles for an old player[J].Cell Death Dis,2016,7:e2199.
[13]Olabisi O A,Soto-Nieves N,Nieves E,et al.Regulation of transcription factor NFAT by ADP-ribosylation[J].Mol Cell Biol,2008,28(9):2860-71.
[14]Shin S Y,Yang J M,Choo S M,et al.System-level investigation into the regulatory mechanism of the calcineurin/NFAT signaling pathway[J].Cell Signal,2008,20(6):1117-24.
[2]Crabtree G R,Olson E N.NFAT signaling:choreographing the social lives of cells[J].Cell,2002,109(Suppl):S67-79.
[3]Li H,Gao S,Ye J T,et al.COX-2 is involved in ET-1-induced hypertrophy of neonatal rat cardiomyocytes:role of NFATc3[J].Mole Cell Endocrinol,2014,382(2):998-1006.
[4]Le K,Li R F,Xu S W,et al.PPARalpha activation inhibits endothelin-1-induced cardiomyocyte hypertrophy by prevention of NFATc4 binding to GATA-4[J].Arch Biochem Biophys,2012,518(1):71-8.
[5]Fiedler B,Lohmann S M,Smolenski A,et al.Inhibition of calcineurin-NFAT hypertrophy signaling by c GMP-dependent protein kinase type I in cardiac myocytes[J].Proc Natl Acad Sci USA,2002,99(17):11363-8.
[6]郑宇静,左彤彤,封宇飞.靶向DNA损伤反应途径:PARP抑制剂抗肿瘤治疗研究进展[J].中国药理学通报,2018,34(2):157-61.[6]Zheng Y J,Zuo T T,Feng Y F.Targeting DNA damage response pathway:recent progress of PARP inhibitors in cancer therapy[J].Chin Pharmacol Bull,2018,34(2):157-61.
[7]Wang L P,Li Z M,Tan Y Z,et al.PARP1 interacts with STAT3and retains active phosphorylated-STAT3 in nucleus during pathological myocardial hypertrophy[J].Mol Cell Endocrinol,2018,474:137-50.
[8]Li Q,Li Z M,Sun S Y,et al.PARP1 interacts with HMGB1 and promotes its nuclear export in pathological myocardial hypertrophy[J].Acta Pharmacol Sin,2019,40(5):589-98.
[9]Lu J,Zhang R W,Hong H Q et al.The poly(ADP-ribosyl)ation of Fox O3 mediated by PARP1 participates in isoproterenol-induced cardiac hypertrophy[J].Biochim Biophy Acta,2016,1863(12):3027-39.
[10]Osadchii,O E.Cardiac hypertrophy induced by sustained beta-adrenoreceptor activation:pathophysiological aspects[J].Heart Fail Rev,2007,12(1):66-86.
[11]王桂军,姚玉胜,王洪新.Ca2+/CaMKⅡ信号通路在肿瘤坏死因子-α诱导心肌肥大中的作用[J].中国药理学通报,2010,26(3):387-91.[11]Wang G J,Yao Y S,Wang H X.Ca2+/CaMKⅡare involved in tumor necrosis factorα-induced cardiomyocyte hypertrophy in rats[J].Chin Pharmacol Bull,2010,26(3):387-91.
[12]Mognol G P,Carneiro F R,Robbs B K,et al.Cell cycle and apoptosis regulation by NFAT transcription factors:new roles for an old player[J].Cell Death Dis,2016,7:e2199.
[13]Olabisi O A,Soto-Nieves N,Nieves E,et al.Regulation of transcription factor NFAT by ADP-ribosylation[J].Mol Cell Biol,2008,28(9):2860-71.
[14]Shin S Y,Yang J M,Choo S M,et al.System-level investigation into the regulatory mechanism of the calcineurin/NFAT signaling pathway[J].Cell Signal,2008,20(6):1117-24.