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Originally published In Press as doi:10.1074/jbc.M509056200 on November 16, 2005

J. Biol. Chem., Vol. 281, Issue 3, 1442-1448, January 20, 2006
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Distinct Pathways Regulate Proapoptotic Nix and BNip3 in Cardiac Stress*

Anita S. Gálvez{ddagger}, Eric W. Brunskill{ddagger}, Yehia Marreez{ddagger}, Bonnie J. Benner{ddagger}, Kelly M. Regula§1, Lorrie A. Kirschenbaum§, and Gerald W. Dorn, II{ddagger}2

From the {ddagger}Department of Internal Medicine, University of Cincinnati, Cincinnati Ohio 45267-0542 and §The Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R2H 2A6, Canada

Received for publication, August 16, 2005 , and in revised form, November 4, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 µM), angiotensin II (100 nM), or isoproterenol (10 µM). 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 Gq 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 Gq effector, transduced Nix transcriptional induction via Sp1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cardiomyocyte apoptosis contributes to functional deterioration in ischemic, hypertrophic, and dilated cardiomyopathies (1–5). A critical but poorly understood feature of the cardiomyocyte cell death program is stress-mediated induction of gene expression for several pro-apoptotic factors belonging to the Bcl2 family of apoptosis-regulating proteins (4, 6). Recent studies of apoptosis gene induction in cardiac hypoxia and hypertrophy decompensation have assigned particular importance to two members of the BH3-only subgroup of Bcl2-like proteins, BNip3 and Nix (7–10). 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). The potential for BNip3 or Nix, alone or in association with other Bcl2 family proteins (12, 13), to disrupt mitochondrial integrity by communicating with the mitochondrial permeability transition pore has suggested to some that a major function of the BH3-only proteins is to determine the on/off state of the mitochondrial permeability transition pore (14). Indeed, mitochondrial disruption may have especially profound consequences for the heart as myocardium is enriched in mitochondria and has a high rate of energy utilization (15).

We examined the hypothesis that cardiac regulation of these two closely related mitochondrial death proteins would, because of their functional similarities, be distinct, i.e. that each was the apoptotic effector of a different cell stress pathway. Previous reports have defined in detail the physiological mechanisms and molecular determinants of BNip3 gene regulation; BNip3 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). In cultured carcinoma cells, hypoxic induction of BNip3 was reported to be hypoxia-inducible factor-1{alpha}-(HIF-1{alpha})3 dependent, consistent with the presence of multiple hypoxia-response elements (HRE) that are canonical HIF-1{alpha}-binding sites in the 5' promoter region of the BNip3 gene (17). Nix, on the other hand, is constitutively expressed in many tissues, including the heart (18, 19), and has not been reported to increase with myocardial hypoxia/ischemia (14). Instead, Nix expression is strikingly increased in Gq-dependent cardiac hypertrophies (10).

Notwithstanding the absence of positive data for Nix transcriptional regulation in ischemic cardiac tissue, the presence of three putative HREs in the human Nix promoter (20) and a report of hypoxic induction of Nix in cultured tumor cell lines (17) have led to general acceptance of the notion that cardiac Nix and BNip3 are both hypoxia-inducible (21) and are regulated in a similar manner, by similar pathways (11). The biological advantage of such a high degree of functional and regulatory overlap is not immediately obvious. In contrast, distinct regulatory mechanisms for Nix and BNip3 would constrain apoptotic responses to uniquely defined conditions, which might be optimal for cell death programming in a terminally differentiated organ, such as the heart.

Herein, we have described the cloning, characterization, and in vitro and in vivo functional analyses of the mouse Nix promoter. When compared with BNip3, the basal pattern of Nix expression differs between tissue types, and in the heart, Nix is induced by entirely different physiological stimuli. Hypertrophy-inducible cardiac Nix expression was mediated by protein kinase C (PKC) {alpha} activation and dependent upon binding of the transcription factor Sp1. These results contradicted accepted dogma regarding hypoxic induction of cardiac Nix and established that Nix and BNip3 represent the terminal effectors of distinct stress-mediated intrinsic cardiomyocyte apoptosis pathways for hypertrophy and hypoxia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of the Mouse Nix and BNip3 Gene Promoters and Creation of Promoter-Reporter Constructs—We employed a PCR-based strategy using high fidelity DNA polymerase to amplify the intergenic region between Nix and protein phosphatase 2a (PP2a), comprising the upstream Nix promoter. A 7.4-kb DNA fragment was PCR-amplified from C57/BL6 genomic DNA utilizing the primers 5'-CTAGCTAGCTCTTGCACCATCTTGCCTGGT-3' and 5'-CTGGAGCTCCCGTGCTCACTGTTGAGGCCAGCG-3'. To facilitate cloning, the oligonucleotides were engineered with NheI and SacI sites (underlined), respectively. The PCR products were digested with NheI and SacI and subcloned into the XbaI and SacI sites of pBS (Stratagene, La Jolla, CA) The Nix promoter region was verified by DNA sequence analysis. The 7.4-kb Nix promoter was subsequently digested with EcoRV, EcoRI, SacI, and PstI to generate a series of 5' deletions that were cloned into pGL3-Basic (Promega, Madison, WI) to create Nix promoter-luciferase reporter constructs. A similar strategy was used to create a pGL3-Basic luciferase reporter construct from the 2.3-kb human BNip3 promoter.

Cardiomyocyte Culture and Transfection—Ventricular myocytes were isolated from 1–2-day-old Sprague-Dawley rats and cultured as described (8). For promoter-reporter studies, 1.0 µg of plasmid DNA was transfected using FuGENE 6 transfection reagent (Roche Applied Science). After 24 h, transfected cardiomyocyte cultures were infected with PKC adenoviri (multiplicity of infection of 10), cultivated under hypoxic conditions (95% N2, 5% CO2, with less than 1% O2 by oxygen sensor), or treated with chemical agents (10 nM phorbol myristate acetate in dimethyl sulfoxide, 10 µM phenylephrine, 10 µM isoproterenol, or 100 nM or 1 µM angiotensin II).

Generation of Nix Promoter-Luciferase Transgenic Mice—The– 5.362-kb Nix promoter-luciferase construct, linearized and separated from vector DNA, was injected into the male pronucleus of FVBN single cell mouse embryos, implanted into pseudopregnant females. Three founders were identified by genomic Southern analysis of tail clip DNA, and colonies were established. There was no apparent phenotype in any of the promoter-reporter transgenic mouse lines.

Acute pressure overloading by microsurgical transverse aortic coarctation (TAC) for 4 days and infarction-reperfusion (1-h occlusion of left anterior descending artery followed by 24 h reperfusion) were modeled as described (10, 22). Animals were treated in accordance with approved University of Cincinnati Institutional Animal Care and Use Committee protocols.

mRNA Analysis—Northern blots used 2 µg of poly(A)+ mRNA, hybridized with 32P-labeled cDNAs for Nix or BNip3. For Nix, blots were also hybridized to a 700-bp fragment of the extreme 3'-untranslated region, which recognizes only the higher molecular weight Nix transcript (10). A mouse multiple tissue Northern blot was purchased from Clontech Laboratories, Inc.

Luciferase Assays—Myocytes were harvested, lysed in cell culture lysis reagent (Promega), and clarified by centrifugation at 1,000 x g, and the supernatant was immediately assayed for luciferase activity. Hearts and other organs were weighed after removal and immediately homogenized in 1 ml of cell culture lysis reagent 1x (Promega) and centrifuged at 10,000 x g for 20 min, and fresh supernatants were assayed for luciferase activity in a Berthold luminometer (Sirius) using the Luciferase assay system from Promega. Relative luciferase activity was normalized to protein content (Bradford assay). Data are reported as induction relative to empty vector (pGL3-Basic) for myocytes or relative luminescence units per microgram of protein (relative luminescence units/µg protein) for tissue samples.

Electrophoretic Mobility Shift Assays—Gel mobility shift assays were performed essentially as described previously (23). Briefly, 20,000 cpm of 32P-double-stranded DNA (5 pmol/ul) was incubated with 300 ng of human recombinant Sp1 protein in the presence or absence of a 50-fold molar excess of unlabeled competing oligonucleotide for 20 min. DNA-protein complexes were resolved by electrophoresis through 5% polyacrylamide gels and visualized by autoradiography. Probe sequences were as follows (the position of the GC box is bold and underlined): Sp1A, 5'-GCAGACGCAGAAAGGGGGCGGGGGAACTCGACTTGTTG-3'; Sp1Amut, 5'-GCAGACGCAGAAAAGGGTTCGGGGGAACTCGACTTGTTG-3', Sp1B, 5'-CAGCTCGCGAATGCCCCGCCCAGCCCGGCCTGGTC-3'; Sp1C, 5'-TGGATACTGCAGGCGTGGGAGGGGTTCCATTCAGGCCCC-3'.

Confocal Immunohistochemistry Studies—Deparafinized sections underwent antigen retrieval by heating in 10 mM citric buffer/2 mM EDTA, pH 6.2, and were immunolabeled with anti-Sp1 (PEP-2) from Santa Cruz Biotechnology or anti-luciferase clone mAB21 from Upstate%20Biotechnology">Upstate Biotechnology. Detection used anti-mouse conjugated with Alexa Fluor 488 or anti-rabbit conjugated with Alexa Fluor 568 IgGs (Molecular Probes). Sections were examined on a Nikon PCM2000 laser confocal microscope.

Statistical Analysis—Results are reported as means ± S.E. All transfections were performed in duplicate. Experimental groups were compared using Student's t test or one-way analysis of variance. A Bonferroni test was used for post-hoc comparisons, with p < 0.05 indicating significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vivo Nix and BNip3 Gene Expression—Mouse Nix (GenBankTM accession number AF067395 [GenBank] ) is located on chromosome 14D1, immediately 3' of PP2a. We used PCR to amplify this intergenic region. Sequencing of the Nix promoter revealed the absence of consensus TATA or CCAAT transcription initiator elements but the presence of several GC boxes 5' to the transcription start site, which is typical of TATA-less promoters (Fig. 1A).

The presence of several HREs 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). Indeed, the mouse Nix promoter and human BNip3 promoter are similar in that both possess multiple putative HRE and GC box motifs (Fig. 1A). To determine whether Nix and BNip3 are co-regulated, we performed comparative Northern blot analysis for these two transcripts across different mouse tissues. Nix expression appeared constitutive, with modest transcript levels in almost all tissues assayed, whereas BNip3 transcript levels varied greatly between different organs, being highest in heart, liver, and kidney (Fig. 1B). These distinct patterns of gene expression across tissues suggested that, in addition to having distinct basal expression patterns, the Nix and BNip3 genes might be differentially induced.

In Vitro Analysis of Nix and BNip3 Promoters—To identify conditions for differential regulation of Nix and BNip3 in the heart, 5'-flanking sequences of each gene were linked to a luciferase reporter and transiently expressed in cultured neonatal rat cardiac myocytes (NRCM). Culture of NRCM in a hypoxic environment (<1% 02) increased BNip3, but not Nix, promoter-reporter activity (Fig. 1C). The transcriptional response to cardiac-acting neurohormones also differed. Nix promoter activity was induced by phenylephrine, a potent Gq-coupled hypertrophic agonist in this system (24, 25), but not isoproterenol or angiotensin II. In contrast, BNip3 promoter activity was not induced by any of these agents, and transcription in response to phenylephrine and isoproterenol was significantly repressed. Thus, the in vitro NRCM promoter-reporter system demonstrated distinct regulation of Nix and BNip3 to environmental and hormonal stressors; BNip3 is exclusively induced by hypoxia, whereas Nix is induced by the hypertrophic agonist phenylephrine. Since the mechanisms for hypoxic BNip3 induction have previously been studied in detail (9), and those for hypertrophic induction of Nix are entirely unknown, subsequent studies focused on Nix.


Figure 1
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  		FIGURE 1.
Nix and BNip3 promoter structure and functional analysis. A, top, mouse Nix promoter sequence. Consensus sites are shown for GC box elements (black ovals) and the HIF-1 hypoxia-responsive element (white boxes). Restriction sites used for deletion analysis are depicted. Bottom, BNip3 gene promoter fragment and consensus sites. B, differential expression of Nix and BNip3 in mouse tissues. 2 µg of poly(A)+ RNA/lane was hybridized with Nix cDNA (top) or 3' fragment (middle, IMAGE: 656945, (700-bp fragment of the extreme 3'-untranslated region)) and BNip3 cDNA (bottom). SK, skeletal. C, Nix and BNip3 promoter-luciferase reporter activities in NRCM. Results are shown as luciferase activity as a percent of control (vehicle-treated) cells, which was ~130-fold that of cells transfected with empty pGL3-Basic vector. NRCM were stimulated with phenylephrine (PE,10 µM), isoproterenol (ISO,10 µM), or angiotensin II (ANGII, 100 nM or 1 µM) for 24 h or were exposed to hypoxia for 6, 24, and 48 h. Normoxic cells were used as control. n = 3 each, performed with duplicate determinations. **, p < 0.01 versus vehicle or normoxic cells.

 
The functional significance of individual cis-elements in the Nix promoter was assessed in assays of basal and stimulated activity of 5' deletion Nix promoter-luciferase reporter constructs transfected into NRCM and by electrophoretic mobility shift assay. As depicted in Fig. 2A, basal Nix promoter-reporter activity diminished in proportion to the length of the constructs, from –5.36 kb to –0.19 kb (Fig. 2A). A Nix promoter-reporter construct containing the complete ~7-kb PP2a-Nix intergenic region had a very low level of basal transcriptional activity, likely attributable to regulatory elements from the PP2a gene (data not shown), and was not studied further. Thus, loss of GC boxes corresponded with loss of basal promoter activity. As previous reports have linked responsiveness of GC box-rich promoters in several genes with regulated binding of the transcription factor, Sp1, to GC elements (23, 26–29), we used electrophoretic mobility shift assays to determine whether the same mechanism could be operative for Nix (Fig. 2, B and C). When compared with a synthetic oligonucleotide encoding an authentic Sp1 binding site (cont), an identical band was seen with a nucleotide spanning the –8 to –1 region (probe A), the –141 to –134 region (probe B), and the –322 to –316 region (probe C) of the Nix promoter but not probe A with a mutated GC box. Competition with excess unlabeled authentic Sp1 oligonucleotide eliminated the band, and the addition of anti-Sp1 antibody resulted in a supershift. These results demonstrated that Sp1 binds specifically to GC-rich motifs in the Nix promoter.

The mechanism for Sp1-mediated regulation of signaling genes has been reported as induction of this factor by PKC (23, 26–29). Our studies showed that phenylephrine, which is known to induce hypertrophy in NRCM by activating PKC (30, 31), specifically increased NRCM Nix promoter activity (Fig. 1C). Accordingly, we assessed the consequences on Nix transcription of directly activating PKC with phorbol myristate acetate (PMA, 10 nM for 24 h). As shown in Fig. 3A, PMA treatment increased Nix transcriptional activity in all constructs. Based on the observed parallel loss of Nix promoter activity and GC-rich elements in the series of 5'-truncated Nix promoter-reporter constructs and the observation that at least some of these elements bound the PMA-responsive transcription factor Sp1 (23), we considered that inhibition of promoter-Sp1 binding should depress Nix transcription. Indeed, disrupting Sp1 binding to GC boxes with mithramycin A (26) decreased basal activity and eliminated PMA responsiveness for each of the Nix promoter-reporter constructs (Fig. 3A).


Figure 2
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  		FIGURE 2.
Sp1 binding to the Nix promoter. A, deletion analysis of the 5' upstream region of Nix gene. The 5362-bp full-length promoter construct and four 5' deletion constructs (3853, 988, 320, and 186 bp) were analyzed for luciferase activity in NRCM. GC boxes and HIF-1 sites are shown as in Fig. 1. A, B, and C designate regions used to generate probes for gel shift analyses. n = 7; *, p < 0.05, **, p < 0.01 versus the longest construct. B, three 36-bp oligonucleotide probes derived from the mouse Nix promoter sequence with a Sp1 (A, B, or C) motif were end-labeled with [{gamma}-32P]ATP and incubated with or without recombinant human Sp1 protein (rhSp1). The arrow indicates the mobility-shifted probe and its binding nuclear protein complex. Sp1 consensus oligonucleotide (Sp1 c.o.) was added to the binding reaction at a 50-fold molar excess to demonstrate Sp1-binding specificity. As a positive control (cont), Sp1 consensus oligonucleotide was also used as probe. C, polyclonal anti-Sp1 (PEP2) antibody (Ab) added to the binding reaction supershifted the Sp1A probe. Sp1A mutated (Amut) probe did not bind Sp1.

 
In the above studies, phorbol ester stimulation of Nix promoter activity was employed as a pharmacological surrogate for endogenous diacylglycerol-mediated activation of PKC. To determine whether PKC per se was capable of inducing Nix transcription and to elucidate any preferential activity of relevant PKC isoforms in this function, NRCM transfected with –5.36-kb NixP were infected with adenovirus encoding either PKC{alpha} or PKC{epsilon}, the two most highly cardiac-expressed conventional and novel PKC isoforms, respectively (32). (Transfection of NRCM with adeno-PKC{delta} induced spontaneous cell death by apoptosis (not shown) and therefore could not be included in the comparison). As shown in Fig. 3B, PKC{alpha}, but not PKC{epsilon}, increased Nix promoter activity in non-treated cardiac myocytes to levels comparable with stimulation with PMA, consistent with a major role for this isoform. Dominant negative PKC{alpha} was without any effects on Nix transcription (Fig. 3B). Collectively, these studies delineate a Sp1-dependent mechanism for Nix transcriptional activation by PKC{alpha} in NRCM.

In Vivo Analysis of the Mouse Nix Promoter—The in vivo determinants of Nix transcriptional regulation were evaluated in three independent transgenic mouse lines expressing the –5.362-kb NixP-luciferase (NixP-luc) construct. Consistent with constitutive low level expression on the multiple-tissue Northern blot, basal luciferase activity was low in all organs sampled, although it was higher in hearts than in other organs tested (Fig. 4A, inset). When compared with baseline, cardiac NixP-luc activity was increased in acutely pressure overloaded hearts (108 ± 9 mm mercury for 4 days) and in NixP-luc mice crossed onto the G{alpha}q transgenic background (33) but not in hearts 24 h after 1 h of reversible ischemia of the left anterior descending coronary artery (Fig. 4A), a period of time previously determined to be sufficient for induction of BNip3 (8). Nix transcriptional activity, assayed with the luciferase reporter, was directly proportional to the magnitude of pressure overload-induced cardiac hypertrophy assessed as heart weight corrected for body weight (p < 0.05, r = 0.71; Fig. 4B).

To determine whether the mechanism for Nix induction in pressure overload and G{alpha}q-dependent cardiac hypertrophy was Sp1-mediated as suggested by the NRCM studies, NixP-luc activity was assayed in separate cohorts of G{alpha}q transgenic/NixP-luc mice treated for 14 days with mithramycin (400 µg/kg/day, intraperitoneal, Fig. 4C). As in NRCM (Fig. 3), in vivo suppression of hypertrophy-mediated Nix induction by mithramycin shows that Nix transcriptional regulation is dependent upon Sp1. Consistent with these findings, Sp1 expression was increased in parallel with Nix transcriptional activity (measured as luciferase protein expression) in NixP-luc mice crossed with G{alpha}q overexpressors (Fig. 4D).


Figure 3
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  		FIGURE 3.
PKC and SP1 regulation of the Nix promoter. A, the effect of PMA and mithramycin A (Mith) on Nix promoter activity. Myocytes transfected with Nix promoter constructs were exposed to 10 nM PMA for 24 h, with or without 500 nM mithramycin A or vehicle. n = 5; *, p < 0.05, **, p < 0.01 versus vehicle; ##, p < 0.01 versus PMA. DMSO, dimethyl sulfoxide. B, the effect of PKC adenoviral infection on Nix promoter activation. NRCM transfected with the full-length Nix promoter-luciferase construct were infected with adeno-PKC{alpha} (ad-PKC{alpha}) wild type (WT) or dominant negative (DN) or adeno-PKC{epsilon} (Ad-PKC{epsilon} wild type) for 24 h. Activity is reported as that relative to control cells infected with adeno-beta-galactosidase (beta-gal). n = 3, **, p < 0.01 versus beta-galactosidase; ##, p < 0.01 versus adenoviral infection.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The current results contradict the widely held notion that Nix and BNip3, the two major cardiac BH3-only proteins, are regulated by similar mechanisms (11, 21). Instead, each of these two powerful mitochondrial death proteins appeared to be induced by a different set of physiological and neurohormonal stimuli. Thus, Nix is the apoptotic effector for myocardial hypertrophy, whereas BNip3 is the apoptotic effector for cardiac hypoxia. These observations not only impacted our understanding of disease-specific pathways for programmed cardiomyocyte suicide but also defined separate targets for therapeutics directed at preventing apoptosis in ischemic myocardial injury versus cardiac hypertrophy decompensation.

Cardiac apoptosis is thought to have pathophysiological relevance in chronic heart failure and after myocardial infarction, which are the clinical syndromes corresponding to the circumstances under which Nix and BNip3 are reportedly up-regulated. In ischemic and dilated human cardiomyopathies, the prevalence of apoptotic cardiomyocytes is markedly increased (1, 2). Although it has not been possible to determine whether apoptosis is a contributory factor in human heart failure decompensation (or is simply a consequence of functional deterioration), experimental models have unambiguously demonstrated the potential for either chronic indolent (34) or acute severe (5) myocardial apoptosis to cause heart failure. Indeed, human pressure overload cardiac hypertrophy is associated with Nix induction (10), and inhibition of Nix with a dominant negative mutant defective in mitochondrial targeting is protective against apoptotic peripartum heart failure in the G{alpha}q transgenic model (10). These data suggest at least the strong possibility that programmed cell death in general, and Nix-mediated apoptosis in particular, contributes to hypertrophy decompensation and the progression of heart failure.

In contrast, BNip3 is not regulated in cardiac hypertrophy or by hypertrophic agonists but is dramatically increased in expression by hypoxia or ischemia (8, 9). The distinct regulatory pathways for Nix and BNip3 are of particular interest because the two proteins have virtually identical functions. Both are proapoptotic BH3-only members of the Bcl-2 superfamily of apoptotic regulatory proteins, both are targeted to mitochondria by a C-terminal transmembrane spanning sequence, and both must interact with other Bcl-2 proteins, such as Bax or Bad, to initiate apoptosis by cytochrome c release and activation of the intrinsic pathway (11, 14, 21). Indeed, there has been some confusion as to whether Nix and BNip3 were products of the same or different genes, contributed to by the original nomenclature for Nix as "BNip3L" (18, 19). The current studies showed that these related apoptotic factors are the products of different genes that are regulated by distinct physiological conditions in the heart. Although it is possible that Nix and BNip3 can also be co-regulated in other conditions, such as by hypoxia in human tumor cells (17), the striking differences in cardiac regulation imply unique pathophysiologies, despite apparent functional redundancy at the protein level. Gene targeting experiments will likely be needed to precisely define the contributions of Nix and BNip3 genes in the in vivo context.


Figure 4
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  		FIGURE 4.
In vivo Nix promoter-luciferase activity in transgenic mice. A, hearts from 8-week-old Nix promoter-luciferase mice were assayed for basal Nix promoter activity and for the effects of ischemia-reperfusion (I/R, 1 h/24 h), pressure overload induced by TAC, or G{alpha}q overexpression (G{alpha}q). n = 5; *, p < 0.05, **, p < 0.01 versus no treatment (–). B, Nix promoter-luciferase activity as a function of TAC hypertrophy, measured as heart weight corrected for body weight (HW/BW) in transgenic lines D and L; r = 0.71, p < 0.05. RLU, relative luminescence units. C, the inhibitory effect of 14 day treatment with mithramycin A (Mith) (400 µg/kg, daily) on G{alpha}q-mediated enhancement of Nix promoter activity. n = 6; **, p < 0.01 versus phosphate-buffered saline (PBS)-treated controls. D, luciferase (upper panel, green) and Sp1 (lower panel, red) immunohistochemistry of hearts from 8-week-old nontransgenic (NTG), NixP-luc and G{alpha}q-NixP-luc mice. Luciferase staining was negative in non-transgenic mice with a cytoplasmatic distribution in NixP-luc mice, and that increased in G{alpha}q-NixP-luc mice. Sp1 was constitutively nuclear in control samples but was increased in nuclei and cytoplasm of G{alpha}q-NixP-luc mice.

 
The molecular mechanisms of cardiac BNip3 induction have previously been elucidated in detail (8, 9). Hypoxia/acidosis is a powerful stimulus for BNip3 transcription, mediated by the proximal HRE in the promoter region. In contrast, Nix transcription was not increased by either in vitro hypoxia or angiotensin II application. Instead, it was increased by those stimuli that are associated with development of myocardial or cardiac myocyte hypertrophy, i.e. phenylephrine application, G{alpha}q overexpression, and pressure overload (10, 24, 25, 33). A known common mediator of each of these events, PKC{alpha} (30, 31, 33, 35), was sufficient to induce Nix promoter activity in cultured NRCM and may be the essential mediator of in vivo Nix induction as well, although the current studies cannot be definitive in this regard. However, it is worth noting that PKC{alpha} is transcriptionally increased and activated in Gq-dependent hypertrophy (33, 35) and that it has been shown to act synergistically with Gq to cause systolic and diastolic heart failure in experimental mouse models (32). Accordingly, transcriptional up-regulation of Nix is only one of many potentially deleterious effects of PKC{alpha} activation in myocardial hypertrophy.

In the case of the Nix gene, transcriptional induction was accomplished via Sp1 binding to GC-rich elements within the promoter. This pattern of transcriptional regulation has been described with phorbol ester enhancement of Sp1 binding to other signaling genes, including those for the thromboxane A2 receptor (23), manganese superoxide dismutase (27), interferon-{gamma} receptor (28), 8S-lipoxygenase (26), and deoxyribonuclease II (29). Of particular relevance to the current studies, Simpson and co-workers (36) also described a similar transcriptional signaling axis for norepinephrine, cardiomyocyte PKC{alpha}, and Sp1 that induces the skeletal actin promoter during in vitro NRCM hypertrophy. Suppression of in vivo G{alpha}q-stimulated Nix induction by mithramycin treatment established the relevance of this pathway to the intact organism.

These studies corrected the misperception that, like BNip3, cardiac Nix induction is regulated by hypoxia/ischemia. Rather, the signaling events leading to increased Nix expression involved activation of PKC{alpha} by hypertrophic agents, induction of Sp1, and its binding to GC box motifs in the promoter. In vivo,G{alpha}q overexpression and pressure overload activate this pathway. The distinct stimuli for, and mechanisms of, Nix and BNip3 transactivation in the heart reveal a tightly regulated and previously unrecognized system for stress-dependent manipulation of cell death in the heart. In the broader pathophysiological context, the delineation of opposing regulatory pathways for functionally identical apoptosis proteins identifies a multiplier effect whereby differentially modulated gene expression increases both the variety and the specificity of biological response.


   FOOTNOTES
 
* This work was supported in part by Grants HL59888 and HL58010 from the NHLBI, National Institutes of Health (to G. W. D.), National Institutes of Health Grant T32 HL07382 (to E. W. B.), and a grant from the Canadian Institute for Health Research (CIHR) (to L. A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of a CIHR studentship and North Award. Back

2 To whom correspondence should be addressed: Dept. of Internal Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0542. E-mail: dorngw{at}ucmail.uc.edu.

3 The abbreviations used are: HIF-1, hypoxia-inducible factor-1; HRE, hypoxia-response elements; PKC, protein kinase C; PMA, phorbol myristate acetate; NixP, Nix promoterluciferase; TAC, transverse aortic coarctation; PP2a, protein phosphatase 2a; NRCM, neonatal rat cardiac myocytes; luc, luciferase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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