Abstract
Proteins of the BCL-2 family are evolutionarily conserved modulators of apoptosis that function as sensors of cellular integrity. Over the past three decades multiple BCL-2 family members have been identified, many of which are now fully incorporated into regulatory networks governing the mitochondrial apoptotic pathway. For some, however, an exact role in cell death signalling remains unclear. One such ‘orphan’ BCL-2 family member is BCL-G (or BCL2L14). In this study we analysed gastrointestinal expression of human BCL-G in health and disease states, and investigated its contribution to inflammation-induced tissue damage by exposing intestinal epithelial cells (IEC) to IFN-γ and TNF-α, two pro-inflammatory mediators associated with gut immunopathology. We found that both BCL-G splice variants — BCL-GS (short) and BCL-GL (long) — were highly expressed in healthy gut tissue, and that their mRNA levels decreased in active inflammatory bowel diseases (for BCL-GS) and colorectal cancer (for BCL-GS/L). In vitro studies revealed that IFN-γ and TNF-α synergised to upregulate BCL-GS/L and to trigger apoptosis in colonic epithelial cell lines and primary human colonic organoids. Using RNAi, we showed that synergistic induction of IEC death was STAT1-dependent while optimal expression of BCL-GS/L required STAT1, NF-κB/p65 and SWI/SNF-associated chromatin remodellers BRM and BRG1. To test the direct contribution of BCL-G to the effects of IFN-γ and TNF-α on epithelial cells, we used RNAi- and CRISPR/Cas9-based perturbations in parallel with isoform-specific overexpression of BCL-G, and found that BCL-G was dispensable for Th1 cytokine-induced apoptosis of human IEC. Instead, we discovered that depletion of BCL-G differentially affected secretion of inflammatory chemokines CCL5 and CCL20, thus uncovering a non-apoptotic immunoregulatory function of this BCL-2 family member. Taken together, our data indicate that BCL-G may be involved in shaping immune responses in the human gut in health and disease states through regulation of chemokine secretion rather than intestinal apoptosis.
Highlights
Introduction Proteins of the BCL2 family are central regulators of the intrinsic pathway of apoptosis, whichOfficial journal of the Cell Death Differentiation AssociationWoznicki et al Cell Death and Disease (2020)11:68 and the multi-domain pro-apoptotic effector proteins[1]
BCL-GS expression was restricted to male reproductive organs, while BCL-GL was detected in a wide range of tissues[4]
High mRNA levels of BCL-GL were detected in the spleen and lymph nodes, while less immunologically active sites like heart and brain showed no detectable expression of either BCLG variant
Summary
Woznicki et al Cell Death and Disease (2020)11:68 and the multi-domain pro-apoptotic effector proteins (such as BAK)[1]. Together, they regulate the integrity of the mitochondrial outer membrane, whose permeabilisation leads to the release of various apoptogenic factors such as cytochrome c, SMAC or oxidoreductase AIF. They regulate the integrity of the mitochondrial outer membrane, whose permeabilisation leads to the release of various apoptogenic factors such as cytochrome c, SMAC or oxidoreductase AIF These in turn amplify apoptotic signalling through activation of caspase-9, neutralisation of pro-survival IAPs or caspase-independent DNA fragmentation[2]. The promoter region of BCL-G harbours p53-, IRF-1- and STAT1-binding sites, and BCL-G induction was observed during p53-mediated apoptosis[9] and following stimulation with type I and type II interferons[10]
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