Estrogen and/or Estrogen Receptor α Inhibits BNIP3-Induced Apoptosis and Autophagy in H9c2 Cardiomyoblast Cells

Bih-Cheng Chen,Chia-Yao Shen,Yueh-Min Lin,Chih-Yang Huang,Yi-Jiun Weng,Yueh-Sheng Chen,Marthandam Shibu,Vijaya Viswanadha,Hsin-Yueh Liang,Chien-Kuo Han

doi:10.3390/ijms19051298

Abstract

The process of autophagy in heart cells maintains homeostasis during cellular stress such as hypoxia by removing aggregated proteins and damaged organelles and thereby protects the heart during the times of starvation and ischemia. However, autophagy can lead to substantial cell death under certain circumstances. BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), a hypoxia-induced marker, has been shown to induce both autophagy and apoptosis. A BNIP3-docked organelle, e.g., mitochondria, also determines whether autophagy or apoptosis will take place. Estrogen (E2) and estrogen receptor (ER) alpha (ERα) have been shown to protect the heart against mitochondria-dependent apoptosis. The aim of the present study is to investigate the mechanisms by which ERα regulates BNIP3-induced apoptosis and autophagy, which is associated with hypoxic injury, in cardiomyoblast cells. An in vitro model to mimic hypoxic injury in the heart by engineering H9c2 cardiomyoblast cells to overexpress BNIP3 was established. Further, the effects of E2 and ERα in BNIP3-induced apoptosis and autophagy were determined in BNIP3 expressing H9c2 cells. Results from TUNEL assay and Immunoflourecense assay for LC3 puncta formation, respectively, revealed that ERα/E2 suppresses BNIP3-induced apoptosis and autophagy. The Western blot analysis showed ERα/E2 decreases the protein levels of caspase 3 (apoptotic marker), Atg5, and LC3-II (autophagic markers). Co-immunoprecipitation of BNIP3 and immunoblotting of Bcl-2 and Rheb showed that ERα reduced the interaction between BNIP3 and Bcl-2 or Rheb. The results confirm that ERα binds to BNIP3 causing a reduction in the levels of functional BNIP3 and thereby inhibits cellular apoptosis and autophagy. In addition, ERα attenuated the activity of the BNIP3 promoter by binding to SP-1 or NFκB sites.

Highlights

Autophagy is a process of cellular self-degradation that occurs at low basal levels in the heart and facilitates the removal of damaged organelles, cytosolic proteins and pathogens [1]
Binding of the C-terminal transmembrane domain of BNIP3 to the mitochondrial membrane initiates an apoptotic cascade that results in the depolarization and opening of mitochondrial permeability transition pores (MPTP), a process that leads to mitochondrial dysfunction and subsequent cell death via apoptosis necrosis or autophagy [5,6,7,8,9,10]
Our results showed that Estrogen Receptor α (ERα) and E2 attenuated the effectors of autophagy in cells that overexpressed BNIP3 (Figure 3B)

Summary

Introduction

Autophagy is a process of cellular self-degradation that occurs at low basal levels in the heart and facilitates the removal of damaged organelles, cytosolic proteins and pathogens [1]. WWee aallssoo eevvaalluuaatteedd tthhee eexxpprreessssiioonn ooff BBNNIIPP33 pprrootteeiinn iinn cceellllss tthhaatt hhaadd bbeeeenn eexxppoosseedd ttoo tthhee EERRαα iinnhhiibbiittoorr mmeellaattoonniinn ((1100––1133~~1100––88MM)). TThhee lleevveell ooff BBNNIIPP33 pprrootteeiinn iinnccrreeaasseedd iinn aa ccoonncceennttrraattiioonn--ddeeppeennddeenntt mmaannnneerr;; ((BB)) TTeett--oonn EERRαα HH99cc cceellllss wweerree ttrraannssffeecctteedd wwiitthh BBNNIIPP33 ((66 μμgg)),, iinnccuubbaatteedd ffoorr 66hh,,aannddwweerreetthheenneexxppoosseeddttooddooxxyyccyycclliinnee((00,,11,,22μμgg//mmLL)) iinn sseerruumm--ffrreeee mmeeddiiuumm ffoorr 1188 hh. The present study shows that BNIP3 induces apoptosis in a caspase-dependent manner which is in accordance with our previous studies on hypoxia-associated effects on H9c2 cells as well as in neonatal rat ventricular myocytes (NRVMs) [60,61,62,63,64]. A previous study showed that under conditions such as hypoxia, the survival signaling pathway in heart cells is down-regulated and FOXO3a-induced BNIP3 expression contributes to increased autophagy and apoptosis [60]. The expression of BNIP3 was attenuated by the transcriptional inactivation level in cells that were exposed to ERα

Cell Culture and Transfection

Western Blot Analysis

DNA Fragmentation

RT-PCR

Findings

Luciferase Assay