A nucleus-targeted alternately spliced Nix/Bnip3L protein isoform modifies nuclear factor κB (NFκB)-mediated cardiac transcription

Abstract

Several Bcl2 family proteins are expressed both as mitochondrial-targeted full-length and as cytosolic truncated alternately spliced isoforms. Recombinantly expressed shorter Bcl2 family isoforms can heterotypically bind to and prevent mitochondrial localization of their full-length analogs, thus suppressing their activity by sequestration. This "sponge" role requires 1:1 expression stoichiometry; absent this an alternate role is suggested. Here, RNA sequencing revealed coordinate regulation of BH3-only protein Nix/Bnip3L (Nix) and its alternately spliced soluble form (sNix) in hearts, but relative sNix/Nix expression of ∼1:10. Accordingly, we examined other putative functions of sNix. Although Nix expressed in H9c2 rat myoblasts localized to mitochondria, sNix showed variable cytoplasmic and nuclear distribution. Tumor necrosis factor α (TNFα) induced rapid and complete sNix nucleoplasmic translocation concomitant with nuclear translocation of the p65/RelA subunit of NFκB. sNix co-localized and co-precipitated with p65/RelA after TNFα stimulation; TNFα-induced sNix nuclear translocation did not occur in p65/RelA null murine embryonic fibroblasts. ChIP sequencing of TNFα-stimulated H9c2 cells revealed sNix suppression of p65/RelA binding to a subset of weaker DNA binding sites, accounting for its ability to alter gene expression in cultured cells and in vivo mouse hearts. These findings reveal TNFα-stimulated cytoplasmic-nuclear shuttling of the alternately spliced non-mitochondrial Nix isoform and uncover a role for sNix as a modulator of TNFα/NFκB-stimulated cardiac gene expression. Transcriptional co-regulation of sNix and Nix, combined with sNix posttranslational regulation by TNFα, comprises a previously unknown mechanism for molecular cross-talk between extrinsic death receptor and intrinsic mitochondrial apoptosis pathways.

Keywords: Bcl-2 Family Proteins; Cardiovascular Disease; Gene Regulation; NF-kappa B (NF-KB); Tumor Necrosis Factor (TNF); sNix.

FIGURE 1.

Alternate mRNA splice isoform, sNix, is regulated in cardiac hypertrophy. A, schematic diagram depicting sNix and Nix cDNA structures. The white box inside the sNix open reading frame is an alternate exon encoding an early TAA termination codon (not to exact scale). B, top, aligned cDNA nucleotide sequences of sNix and Nix C-terminal regions showing alternately spliced insert (red) and position of termination codons (green). Bottom, corresponding aligned amino acid sequence. C, ethidium-stained gel showing sNix and Nix PCR products in nontransgenic (ntg) and Gα_q transgenic (Gq) hearts; positions of PCR primers spanning the alternate exon are shown by arrows in B. D, quantitative sNix (left) and Nix (right) mRNA expression assayed by RNA sequencing of nontransgenic (white) and Gα_q (black) hearts (n = 4 each, * = p < 0.05). E, immunoblot analyses showing mitochondrial Nix and cytosolic sNix protein levels in two nontransgenic and two Gα_q hearts. Protein quantification was conducted using ImageJ and normalized by COX IV and GAPDH, respectively. F, quantitative analysis of splice isoform expression of other members of the Bcl2 family assayed as in D.

FIGURE 2.

sNix nuclear translocation protects against TNFα-induced H9c2 cell death. A and B, confocal micrographs of H9c2 cells infected with control adeno-β-gal (top row), adeno-FLAG-Nix (middle row), or adeno-FLAG-sNix (bottom row). A, H9c2 cells under typical tissue culture conditions. Red = MitoTracker red; green = FLAG; blue = DAPI nuclear stain. B, H9c2 cells after 24-h serum deprivation without (veh, vehicle) or 60 min after treatment with TNFα (20 ng/ml) or PDGF (20 ng/ml). Red = lamin a/c labeling nuclear membrane; green = FLAG; blue = DAPI nuclear stain. C, fluorescent micrographs (left) showing live (green) and dead (red) H9c2 cells infected with control adeno-β-gal, adeno-FLAG-Nix, or adeno-FLAG-sNix after vehicle or TNFα (50 ng/ml) treatment for 24 h. The percentage of dead cells was calculated from three independent wells under each condition (right).

FIGURE 3.

Association and co-translocation of sNix and NFκB p65/RelA to cell nuclei after TNFα stimulation. A, confocal micrographs of H9c2 cells expressing pDsRed modified red fluorescent protein-p65/RelA (red) β-gal control (top) or FLAG-Nix (middle) or FLAG-sNix (bottom). FLAG epitope stains green. Columns show nuclear translocation of red p65/RelA and sNix, but not Nix, 60 and 120 min after the addition of (20 ng/ml) TNFα. Co-localization of sNix and p65/RelA in cytosol before TNFα addition is indicated by yellow fluorescence at time 0. B, co-immunoprecipitation of sNix and p65/RelA from cytosol in unstimulated H9c2 cells and nucleoplasm in TNFα-stimulated H9c2 cells. fx = fraction; IP = immunoprecipitating antibody; IB = immunoblotting antibody. Tubulin and lamin B are loading controls for cytosol and nuclear fractions, respectively. C, the absence of TNFα-stimulated sNix nuclear translocation in p65/RelA null MEFs. Red = lamin a/c labeling nuclear membrane; green = FLAG; blue = DAPI nuclear stain. D. TNFα-stimulated sNix nuclear translocation in wild-type MEFs.

FIGURE 4.

TNFα-stimulated NFκB signaling is suppressed after sNix expression in H9c2 cardiac myoblasts. A, heat map depiction of genome-wide ChIP-seq signal intensity of H9c2 cells as a function of proximity to bioinformatically predicted p65/RelA binding sites. Results are shown with and without TNFα and sNix. chr, chromosome. B, five genetic examples showing individual p65/RelA ChIP-seq intensities for the different experimental conditions and corresponding heat map plots. C, top two transcription factor motifs (RELA and REL) discovered at repeat-masked p65/RelA binding sites using the MEME Suite. Top 100 p65/RelA peaks (by ChIP-seq -fold change) were used for de novo motif search. D, summary of gene ontology analysis using DAVID for 71 TNFα-regulated genes. The gene ontology categories are ranked by the log₁₀ p values for disproportionate representation. E, TNFα-induced gene regulation (log₂ of -fold induction for the 71 TNFα-regulated genes) in the absence and presence of sNix. A paired two-tailed t test was used to calculate the p value. Insets to the right show individual regulation of sNix suppressed (top) and nonsuppressed mRNAs.

FIGURE 5.

Validating studies showing that sNix suppresses TNFα-induced p65/RelA occupancies and inhibits TNFα-stimulated NFκB target gene expression. A, representative ChIP-qPCR determinations of TNFα and sNix effects on p65/RelA binding intensities within three sNix-suppressible H9c2 cell-expressed NFκB target genes. B, group mean data and intergroup comparisons for ChIP-qPCR and RT-qPCR for the same genes as in A. Data are mean ± S.D. of three PCR reactions. Rel enrich, relative enrichment.

FIGURE 6.

Cardiomyocyte-expressed sNix is localized in nuclei and cytoplasm and alters cardiac gene expression without inducing apoptosis or cardiomyopathy. A, immunoblot analysis of FLAG-Nix and -sNix myocardial fractions in Nix and sNix transgenic mouse hearts. S = supernatant cytosolic fraction labeled by α-tubulin (α-tubul); 10p = 10,000 × g pellet enriched in mitochondrial proteins marked by COX IV; Nuc = nuclear fraction labeled by lamin. B, representative M-mode echocardiograms (top) and fluorescent TUNEL staining (bottom) from control, Nix, and sNix hearts. Quantitative group echocardiographic measures are under “Results.” C, quantitative group data for TUNEL labeling (n = 5 each group). cont, control. D, unsupervised hierarchical clustering of sNix-regulated cardiac gene expression. Left, raw reads; right, normalized reads. Each column is one individual mouse heart. E, results of gene ontology analysis of sNix-regulated cardiac mRNAs.