A Nucleus-targeted Alternately Spliced Nix/Bnip3L Protein Isoform Modifies Nuclear Factor κB (NFκB)-mediated Cardiac Transcription

• Published in: The Journal of biological chemistry Vol. 288; no. 22; pp. 15455 - 15465
• Main Authors: Chen, Yun, Decker, Keith F., Zheng, Dali, Matkovich, Scot J., Jia, Li, Dorn, Gerald W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 31-05-2013; American Society for Biochemistry and Molecular Biology
• Subjects: Active Transport, Cell Nucleus - physiology; Alternative Splicing - physiology; Animals; Bcl-2 Family Proteins; Cardiovascular Disease; Cell Biology; Cell Line; Cell Nucleus - genetics; Cell Nucleus - metabolism; Gene Expression Regulation - physiology; Gene Regulation; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mice; Mice, Mutant Strains; Mitochondrial Proteins - genetics; Mitochondrial Proteins - metabolism; Muscle Proteins - genetics; Muscle Proteins - metabolism; Myoblasts, Cardiac - cytology; Myoblasts, Cardiac - metabolism; NF-kappa B (NF-KB); Protein Isoforms - genetics; Protein Isoforms - metabolism; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; sNix; Transcription Factor RelA - genetics; Transcription Factor RelA - metabolism; Transcription, Genetic - physiology; Tumor Necrosis Factor (TNF); Tumor Necrosis Factor-alpha - genetics; Tumor Necrosis Factor-alpha - metabolism; Cardiovascular Disease; Gene Regulation; NF-kappa B (NF-KB); Tumor Necrosis Factor (TNF); Bcl-2 Family Proteins; sNix
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.452342

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.
Background: Functions of short, cytosolic splice Bcl2 protein isoforms are unknown.
Results: TNFα provokes complexing of the splice isoform of Nix (sNix) to NFκB p65/RelA, their nuclear translocation, gene promoter binding, and suppressed expression.
Conclusion: sNix is a modifier of TNFα-mediated cardiomyocyte gene transcription.
Significance: sNix-TNFα interactions comprise a previously undescribed mechanism for cross-talk between extrinsic and intrinsic apoptosis pathways.