Tumor necrosis factor-alpha upregulated PHLPP1 through activating nuclear factor-kappa B during myocardial ischemia/reperfusion

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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 (30min/2h) but decreased after 4h reperfusion. In vitro, H/R (4h/1h) 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 4h 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.

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