Distinct Pathways Regulate Proapoptotic Nix and BNip3 in Cardiac Stress

Anita S. Gálvez,Kelly M. Regula,Gerald W. Dorn,Yehia Marreez,Eric W. Brunskill,Lorrie A. Kirschenbaum,Bonnie J. Benner

doi:10.1074/jbc.m509056200

Anita S. Gálvez, Kelly M. Regula + Show 5 more

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https://doi.org/10.1074/jbc.m509056200

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Abstract

Up-regulation of myocardial Nix and BNip3 is associated with apoptosis in cardiac hypertrophy and ischemia, respectively. To identify mechanisms of gene regulation for these critical cardiac apoptosis effectors, the determinants of Nix and BNip3 promoter activation were elucidated by luciferase reporter gene expression in neonatal rat cardiac myocytes. BNip3 transcription was increased by hypoxia but not by phenylephrine (10 microM), angiotensin II (100 nM), or isoproterenol (10 microM). In contrast, Nix transcription was increased by phenylephrine but not by isoproterenol, angiotensin II, or hypoxia. Since phenylephrine stimulates cardiomyocyte hypertrophy via protein kinase C (PKC), the effects of phorbol myristate acetate (PMA, 10 nM for 24 h) and adenoviral PKC expression were assessed. PMA and PKC alpha, but not PKC epsilon or dominant negative PKC alpha, increased Nix transcription. Multiple Nix promoter GC boxes bound transcription factor Sp-1, and basal and PMA- or PKC alpha-stimulated Nix promoter activity was suppressed by mithramycin inhibition of Sp1-DNA interactions. In vivo determinants of Nix expression were evaluated in Nix promoter-luciferase (NixP) transgenic mice that underwent ischemia-reperfusion (1 h/24 h), transverse aortic coarctation (TAC), or cross-breeding with the G(q) overexpression model of hypertrophy. Luciferase activity increased in G alpha(q)-NixP hearts 3.2 +/- 0.4-fold and in TAC hearts 2.8 +/- 0.4-fold but did not increase with infarction-reperfusion. NixP activity was proportional to the extent of TAC hypertrophy and was inhibited by mithramycin. These studies revealed distinct mechanisms of transcriptional regulation for cardiac Nix and BNip3. BNip3 is hypoxia-inducible, whereas Nix expression was induced by G alpha(q)-mediated hypertrophic stimuli. PKC alpha, a G(q) effector, transduced Nix transcriptional induction via Sp1.

Highlights

The presence of several hypoxia-response elements (HRE) in the human Nix promoter has been noted previously [20] and forms a basis for the assumption that, like BNip3, Nix is regulated in experimental cardiac hypoxia [21]
Nix is the apoptotic effector for myocardial hypertrophy, whereas BNip3 is the apoptotic effector for cardiac hypoxia
These observations impacted our understanding of disease-specific pathways for programmed cardiomyocyte suicide and defined separate targets for therapeutics directed at preventing apoptosis in ischemic myocardial injury versus cardiac hypertrophy decompensation

Summary

Introduction

W. B.), and a grant from the Canadian Institute for Health Research (CIHR) Importance to two members of the BH3-only subgroup of Bcl2-like proteins, BNip and Nix [7,8,9,10] These two factors are each expressed in the heart, localized to mitochondria, and sufficient to induce apoptosis via the intrinsic, or mitochondrial, pathway [8, 10, 11]. Previous reports have defined in detail the physiological mechanisms and molecular determinants of BNip gene regulation; BNip expression varies between tissue types [16], and in the heart, is strikingly increased in response to in vitro cardiomyocyte hypoxia/acidosis or in vivo transient myocardial ischemia [8, 9, 14]. When compared with BNip, the basal pattern of Nix expression differs between

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