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. 2013 May 31;288(22):15745-59.
doi: 10.1074/jbc.M112.439844. Epub 2013 Apr 15.

NADPH oxidase 4 regulates cardiomyocyte differentiation via redox activation of c-Jun protein and the cis-regulation of GATA-4 gene transcription

Thomas V A Murray  1 , Ioannis Smyrnias, Ajay M Shah, Alison C Brewer
Affiliations

NADPH oxidase 4 regulates cardiomyocyte differentiation via redox activation of c-Jun protein and the cis-regulation of GATA-4 gene transcription

Thomas V A Murray et al. J Biol Chem. .

Abstract

NADPH oxidase 4 (Nox4) generates reactive oxygen species (ROS) that can modulate cellular phenotype and function in part through the redox modulation of the activity of transcription factors. We demonstrate here the potential of Nox4 to drive cardiomyocyte differentiation in pluripotent embryonal carcinoma cells, and we show that this involves the redox activation of c-Jun. This in turn acts to up-regulate GATA-4 expression, one of the earliest markers of cardiotypic differentiation, through a defined and highly conserved cis-acting motif within the GATA-4 promoter. These data therefore suggest a mechanism whereby ROS act in pluripotential cells in vivo to regulate the initial transcription of critical tissue-restricted determinant(s) of the cardiomyocyte phenotype, including GATA-4. The ROS-dependent activation, mediated by Nox4, of widely expressed redox-regulated transcription factors, such as c-Jun, is fundamental to this process.

Keywords: AP-1 Transcription Factor; Cardiac Development; Cardiogenesis; Nox; Nox4; Reactive Oxygen Species (ROS); Redox Regulation.

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Figures

FIGURE 1.
FIGURE 1.
Efficient cardiotypic differentiation in P19 CL6 cells is dependent upon Nox4. A, Q-PCR analysis of GATA-4 mRNA in differentiating P19 CL6 cells. B, GATA-4 protein levels in differentiating P19 CL6 cells. β-Actin is included as a loading control. C, Q-PCR analysis of Nox4 mRNA in P19 CL6 cells over the indicated differentiation time course. D, Q-PCR analysis of Nox4 mRNA levels in undifferentiated P19 CL6 transduced for 72 h with either AdshRNANox4 (black bar) or control AdshGFP (open bar), at an m.o.i. of 100. E, Q-PCR analysis of GATA-4 mRNA levels in P19 CL6 cells during the indicated differentiation time course. Cells were transduced with either AdshRNANox4 (black bars) or control AdshGFP (gray bars) at an m.o.i. of 100 and allowed to differentiate for up to 7 days as indicated. F, GATA-4 proteins levels at day 4 of differentiation after transduction with either AdshGFP or AdshNox4. G, Q-PCR analysis of GATA-4 mRNA in undifferentiated P19 CL6 cells transduced with either AdβGal or AdNox4 for 24 h. H, Q-PCR analysis of MEF2C and Nkx 2.5 mRNA in P19 CL6 cells after 4 days of differentiation. Cells were transduced with for 72 h with either AdshRNANox4 (black bar) or control AdshGFP (open bar), at an m.o.i. of 100. I, semi-quantitative RT-PCR analysis of α-MHC mRNA levels in equivalent samples to those assessed in E, at day 7 of differentiation. All Q-PCR values are shown normalized to β-actin and are plotted in arbitrary units (A.U.) relative to appropriate control. **, p < 0.01; *, p < 0.05.
FIGURE 2.
FIGURE 2.
Activation of GATA-4 transcription is dependent upon H2O2 production and requires p22Phox. A, extracellular H2O2 production in cell culture media from undifferentiated (day 0) and differentiated (day 4) P19CL6 cells treated with either 10 μm DPI or 0.1% DMSO (vehicle control) for 6 h prior to assay. B, H2O2 production in cell culture media from undifferentiated P19 CL6 cells (NT (not transduced); open bar) or from cells at day 4 of differentiation transduced with either AdshGFP (black bar) or AdshRNANox4 (gray bar). C, Q-PCR analysis of GATA-4 mRNA levels in P19 CL6 cells after H2O2 treatment. Cells were treated with 10 nm H2O2 for 24 h and cultured for a further 3 days before analysis. D, Western blot analysis of p22Phox protein levels in undifferentiated P19 CL6 cells, 72 h after transfection with control scrambled siRNA (Scram) or siRNA directed against p22Phox (si p22phox). β-Actin is included as a loading control. E, Q-PCR analysis of GATA-4 mRNA levels in P19 CL6 cells transfected with control scrambled siRNA (Scram; black bar) or siRNA directed against p22Phox (si p22phox) for 24 h and subsequently induced to differentiate for 4 days as indicated.
FIGURE 3.
FIGURE 3.
Functional analysis of the 5′-upstream sequence of the GATA-4 gene. A, structural organization of the mouse GATA-4 gene showing alternative first exons E1a, E1b, and E1b′ (not to scale). B, semi-quantitative RT-PCR analysis of alternative GATA-4 first exon usage in mRNA isolated from undifferentiated P19 CL6 cells (see “Experimental Procedures”). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is included as a positive control. C, semi-quantitative RT-PCR analysis of alternative GATA-4 first exon expression in mRNA isolated from undifferentiated P19 CL6 cells (day 0) and cells at day 4 of differentiation. GAPDH is again included as a loading control. D, luciferase reporter activity resulting from undifferentiated P19CL6 cells (day 0; open bars) or cells at day 4 of differentiation (day 4; black bars), cultured in the presence of vehicle control, or 4 mm tiron or 10 μm DPI as indicated. Cells were transfected with a GATA-4 promoter construct (GATA-4 prom) or the promoterless control vector (empty control), as indicated, for 24 h prior to the measurement of luciferase activity. E, luciferase activity resulting from undifferentiated P19 CL6 cells transduced with either AdNox4 (black bars) or AdβGal (open bars) as control, subsequently transfected with a GATA-4 reporter plasmid or control promoterless vector as indicated, and incubated for 24 h under normal growth conditions or in the presence of tiron (4 mm) for 6 h prior to luciferase activity measurement as indicated. In all cases, luciferase activity, normalized to that of the co-transfected SV40-Renilla luciferase control vector, is expressed as RLU. All data are presented as mean ± S.E. *, p < 0.05; n.s., not significant.
FIGURE 4.
FIGURE 4.
c-Jun binds to a conserved sequence within the GATA-4 promoter region. A, bio-oligonucleotide pulldown assay of a 1.4-kb biotinylated mouse GATA-4 promoter fragment, incubated with nuclear extract from P19 CL6 cells at day 4 of differentiation. Affinity-purified DNA-transcription factor complexes were analyzed for transcription factors c-Jun, p53, and NF-κB by Western blot analyses as indicated (G4). Control (Con) represents equivalent experiment performed in the absence of biotinylated DNA. Input represents equivalent amount of nuclear extract, prior to affinity purification. B, sequence comparison of human, mouse, and rat GATA-4 promoter regions immediately proximal to and including exon E1a. Potential AP-1 (c-Jun)-binding sites are outlined in boxes. Asterisks indicate nucleotide homology between all three species. Nucleotides are numbered relative to the CAP site of each gene (arrowed, +1). C, 100-bp biotinylated DNA fragments comprising individual putative AP-1 sites within the mouse GATA-4 promoter (A–C, respectively) were incubated with nuclear extracts prepared from P19 CL6 cells at day 4 of differentiation. Affinity-purified complexes were analyzed for c-Jun binding by Western blot. Again, nuclear extract was run as a control (input). Loading control denotes (equivalent) levels of oligonucleotides bound to beads, released by incubation with formamide (see under “Experimental Procedures”), and separated by agarose gel electrophoresis. D, oligonucleotides comprising the wild type site A (Wt) or a mutated site A (Mut) AP-1-binding site, subjected to pulldown assay as described in C. E, ChIP analyses of chromatin isolated from P19 CL6 cells after 4 days of differentiation. Equivalent aliquots of chromatin were immunoprecipitated using antibodies to c-Jun, p53, or nonimmune (Rabbit) IgG as indicated. The input fraction corresponds to a 1:100 dilution of initial starting chromatin solution before immunoprecipitation.
FIGURE 5.
FIGURE 5.
Functional analyses of the AP-1 site in the GATA-4 promoter. A, luciferase activity resulting from undifferentiated (day 0; open bars) or differentiated (day 4; black bars) P19 CL6 cells, transfected with either a WT GATA-4 promoter-luciferase plasmid (Wt) or the same plasmid containing a mutated AP-1-binding site (Mut) as indicated. B, luciferase activity resulting from undifferentiated P19CL6 cells, again transfected with either the WT or mutated promoter-luciferase constructs and additionally transduced with AdNox4 or AdβGal. In all cases, luciferase activity was measured 24 h after transfection and transduction and was normalized to that of the co-transfected SV40-Renilla luciferase vector and expressed as RLU. C and D, histograms depicting the fold induction, relative to controls, of the WT and mutated GATA-4 promoter activities upon either differentiation or Nox4 transduction, respectively. All data are presented as mean ± S.E. *, p < 0.05. n.s., not significant.
FIGURE 6.
FIGURE 6.
Nox4 increases GATA-4 expression via activation of c-Jun. A, Western blot analyses of phosphorylated (Phospho-) and total c-Jun levels in nuclear extracts from undifferentiated P19 CL6 cells, transduced with either AdβGal or AdNox4 for 24 h prior to harvesting. B, luciferase activity resulting from undifferentiated P19 CL6 cells transfected with an AP-1-luciferase reporter construct (black bars) or minimal promoter-containing control plasmid (open bars), and additionally transduced with AdβGal or AdNox4 as indicated for 24 h prior to harvesting. C, fold induction of Nox4-induced increase in AP-1 luciferase reporter activity in the presence or absence of DPI. Cells were transduced with AdNox4 for 24 h and treated overnight with 1 μm DPI before assaying. D, Western blot analysis of c-Jun protein levels in undifferentiated P19 CL6 cells, 48 h after transfection with control siRNA (siCon) or siRNA directed against c-Jun (siJun). β-Actin is included as a loading control. E, Q-PCR analysis of GATA-4 mRNA levels in undifferentiated P19 CL6 cells (untreated control; open bar) or P19 CL6 cells transfected with control siRNA (siCon; black bar) or siRNA directed against c-Jun (siJun) for 24 h, and subsequently induced to differentiate for 4 days as indicated. F, luciferase activity resulting from undifferentiated P19 CL6 cells transfected with control siRNA (si Con; open bar) or siRNA directed against c-Jun (si c-Jun; black bar), and incubated for 24 h. All cells were subsequently transfected with a GATA-4 reporter plasmid and transduced with either AdβGal or AdNox4 as indicated and cultured for a further 24 h. G, fold induction of Nox4-induced increase in AP-1 luciferase reporter activity in the presence or absence of a JNK inhibitor. Cells were transduced with AdNox4 for 24 h and treated overnight with 20 μm JNK inhibitor II before assaying. H, fold induction of Nox4-induced increase in GATA-4 luciferase reporter activity in the presence or absence of a JNK inhibitor. Cells were transduced with AdNox4 for 24 h and treated overnight with 20 μm JNK inhibitor II before assaying. For all reporter assays luciferase activity was normalized to that of a co-transfected SV40-Renilla luciferase vector and is expressed as RLU or fold change as indicated. All data are presented as mean ± S.E. *, p < 0.05.
FIGURE 7.
FIGURE 7.
Nox4 activates c-Jun in NRCs but does not affect GATA-4 mRNA expression levels. A, representative Western blot analyses of Nox4 protein levels in whole cell lysates or phospho- and total c-Jun protein levels in nuclear extracts of NRCs transduced with either AdβGal or AdNox4 for 24 h. Actin is included as a loading control. B, densitometric analysis of the proportion of c-Jun phosphorylation, derived from three separate Western blot analyses. C, ELISA-based analyses of binding activity of c-Jun in nuclear extracts of NRCs transduced with AdβGal-transduced (open bar) or AdNox4-transduced (black bar) as in A. D, luciferase activity resulting from NRCs transduced for 24 h with either AdβGal or AdNox4 as indicated and subsequently transfected with an AP-1-luciferase reporter construct (black bars) or minimal promoter-containing control plasmid (open bars) and cultured for a further 24 h. Luciferase activity was normalized to that from the co-transfected SV40 Renilla luciferase control vector and is expressed as RLU. E, Q-PCR analysis of GATA-4 mRNA levels in NRCs transduced with AdβGal or AdNox4 for 24 h. F, representative Western blot analyses of GATA-4 protein levels in whole-cell lysates of NRCs transduced with either AdβGal or AdNox4 for 24 h. Actin is included as a loading control. All data are presented as mean ± S.E. *, p < 0.05; n.s., not significant.
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