Human BCL-G regulates secretion of inflammatory chemokines but is dispensable for induction of apoptosis by IFN-γ and TNF-α in intestinal epithelial cells

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-G S (short) and BCL-G L (long)were highly expressed in healthy gut tissue, and that their mRNA levels decreased in active inflammatory bowel diseases (for BCL-G S ) and colorectal cancer (for BCL-G S/L ). In vitro studies revealed that IFN-γ and TNF-α synergised to upregulate BCL-G S/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-G S/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.

Bioinformatics Analysis of Differentially Expressed Genes and Protein–Protein Interaction Networks Associated with Functional Pathways in Ulcerative Colitis

“…The combination of CCL20 and CCR6 may be responsible for the immunosuppressive effect of marrow-derived suppressor cells [ 34 ]. Similarly, Woznicki et al [ 35 ] found that the CCL20 and CCL5 chemokines are involved in the immune response in the human intestine. CCL20 may be an important regulatory molecule in the pathogenesis of IBD.…”

Lung Epithelial Regulation of BCL2 Related Protein A1 (BCL2A1) by Coronaviruses (SARS-CoV) and Type I Interferon Signaling

“…TNF-α and IFN-γ synergistically upregulated BCL2L14 and induced apoptosis in colon epithelial cell lines. However, Th1 cytokine-mediated colon epithelial apoptosis was independent of BCL2L14 expression and STAT1dependent (58). Furthermore, studies in knockout mice and cells suggested that contrary to the earlier claims, BCL-G/BCL2L14 was not involved in pro-apoptotic function (59).…”

Regulation of CEACAM Family Members by IBD-Associated Triggers in Intestinal Epithelial Cells, Their Correlation to Inflammation and Relevance to IBD Pathogenesis

“…β-actin was used as housekeeping gene. The 2 −ΔΔ Ct method was used to calculate the relative changes in each of the genes relative to the expression of β-actin determined from real-time qRT-PCR experiments ( 28 , 29 ).…”