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.
Highlights
Functions of short, cytosolic splice Bcl2 protein isoforms are unknown
SNix Is Disproportionately Up-regulated in Cardiac Hypertrophy— Transcriptional regulation of Nix in murine and human cardiac hypertrophy has been observed by microarray profiling, Northern blot analysis, quantitative RT-PCR, and activity of a cardiac transgenic Nix promoter/luciferase promoter [5, 22, 23]
The C-terminal truncated form of Nix was described a decade ago as a product of alternate mRNA splicing in the heart [5] (Fig. 1, A and B), but its regulation in heart disease has not been reported because microarrays and conventional Northern blot analyses do not distinguish between Nix and splice isoform of Nix (sNix) transcripts
Summary
Results: TNF␣ provokes complexing of the splice isoform of Nix (sNix) to NFB p65/RelA, their nuclear translocation, gene promoter binding, and suppressed expression. Several Bcl family proteins are expressed both as mitochondrial-targeted full-length and as cytosolic truncated alternately spliced isoforms. Expressed shorter Bcl family isoforms can heterotypically bind to and prevent mitochondrial localization of their full-length analogs, suppressing their activity by sequestration. ChIP sequencing of TNF␣-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 TNF␣-stimulated cytoplasmic-nuclear shuttling of the alternately spliced non-mitochondrial Nix isoform and uncover a role for sNix as a modulator of TNF␣/NFB-stimulated cardiac gene expression.
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