Bin Tang1, Jing Ma2, Xiaoqin Ha3, Yuanqiang Zhang4, Yuan Xing5. 1. Department of International Medical, China-Japan Friendship Hospital, Beijing 100029, China. 2. Department of Traditional Chinese Medicine, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. 3. Department of Clinical Laboratory Medicine, Lanzhou General Hospital of Lanzhou Military Region, People's Liberation Army, Key Laboratory of Stem Cell and Gene Drug in Gansu Province, Lanzhou 730000, China. 4. Department of Histology and Embryology, Fourth Military Medical University, Xi'an 710032, China. Electronic address: zhangyq@fmmu.edu.cn. 5. Department of Clinical Laboratory Medicine, Lanzhou General Hospital of Lanzhou Military Region, People's Liberation Army, Key Laboratory of Stem Cell and Gene Drug in Gansu Province, Lanzhou 730000, China. Electronic address: lzxingyuan1987@126.com.
Abstract
AIMS: The pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) specifically regulates phospho-Ser473 of protein kinase B (PKB, Akt) opposing cell survival during myocardial ischemia/reperfusion (I/R). Previous studies demonstrated PHLPP1 expression level was controlled by several mechanisms. However, the regulation mechanism of cardiac PHLPP1 expression following myocardial I/R remains unknown. MAIN METHODS: The current study utilized the mouse model of myocardial I/R injury in vivo and the neonatal rat ventricular myocytes (NRVMs) of hypoxia/reoxygenation (H/R) injury in vitro. Expression of PHLPP1, nuclear factor-kappa B (NF-κB) and pNF-κB were determined by western blot. The expression of PHLPP1 and translocation of NF-κB was assessed by immunofluorescence. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NF-κB to the promoter region of phlpp1 gene. KEY FINDINGS: Myocardial I/R had no effect on cardiac PHLPP1 expression following I/R (30 min/2 h) but decreased after 4 h reperfusion. In vitro, H/R (4 h/1 h) and tumor necrosis factor-alpha (TNF-α)-stimulation resulted in upregulation of PHLPP1 in NRVMs, which was blocked with etanercept. Yet, H2O2-induced oxidative stress had no obvious effect on PHLPP1 expression of NRVMs at early stage but N-acetylcysteine (NAC) pretreatment increased PHLPP1 levels after 4 h H2O2 stimulation. TNF-α and H/R led to both expression and transcriptional activity of NF-κB, accompany with higher expression of PHLPP1. Pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor, prevented the response not only in TNF-α-treated cardiomyocytes but also in H/R-treated group. SIGNIFICANCE: These results implicated that TNF-α involved in cardiac PHLPP1 upregulation during reoxygenation, which was mediated by NF-κB transcriptional activity.
AIMS: The pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) specifically regulates phospho-Ser473 of protein kinase B (PKB, Akt) opposing cell survival during myocardial ischemia/reperfusion (I/R). Previous studies demonstrated PHLPP1 expression level was controlled by several mechanisms. However, the regulation mechanism of cardiac PHLPP1 expression following myocardial I/R remains unknown. MAIN METHODS: The current study utilized the mouse model of myocardial I/R injury in vivo and the neonatal rat ventricular myocytes (NRVMs) of hypoxia/reoxygenation (H/R) injury in vitro. Expression of PHLPP1, nuclear factor-kappa B (NF-κB) and pNF-κB were determined by western blot. The expression of PHLPP1 and translocation of NF-κB was assessed by immunofluorescence. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NF-κB to the promoter region of phlpp1 gene. KEY FINDINGS: Myocardial I/R had no effect on cardiac PHLPP1 expression following I/R (30 min/2 h) but decreased after 4 h reperfusion. In vitro, H/R (4 h/1 h) and tumor necrosis factor-alpha (TNF-α)-stimulation resulted in upregulation of PHLPP1 in NRVMs, which was blocked with etanercept. Yet, H2O2-induced oxidative stress had no obvious effect on PHLPP1 expression of NRVMs at early stage but N-acetylcysteine (NAC) pretreatment increased PHLPP1 levels after 4 h H2O2 stimulation. TNF-α and H/R led to both expression and transcriptional activity of NF-κB, accompany with higher expression of PHLPP1. Pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor, prevented the response not only in TNF-α-treated cardiomyocytes but also in H/R-treated group. SIGNIFICANCE: These results implicated that TNF-α involved in cardiac PHLPP1 upregulation during reoxygenation, which was mediated by NF-κB transcriptional activity.
Authors: Changli Zhang; Madeline P Smith; George K Zhou; Alon Lai; Robert C Hoy; Victoria Mroz; Olivia M Torre; Damien M Laudier; Elizabeth W Bradley; Jennifer J Westendorf; James C Iatridis; Svenja Illien-Jünger Journal: Cell Death Dis Date: 2019-10-03 Impact factor: 8.469