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

Cell Death Dis. 2020 Jan 27;11(1):68. doi: 10.1038/s41419-020-2263-0.

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.

MeSH terms

  • Apoptosis / drug effects*
  • Apoptosis / genetics
  • CRISPR-Cas Systems
  • Cell Line, Tumor
  • Chemokine CCL20 / metabolism
  • Chemokine CCL5 / metabolism
  • Chemokines / metabolism*
  • Colorectal Neoplasms / genetics
  • Colorectal Neoplasms / metabolism
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism*
  • Gene Knockout Techniques
  • Humans
  • Inflammatory Bowel Diseases / genetics
  • Inflammatory Bowel Diseases / metabolism
  • Interferon-gamma / pharmacology*
  • Intestinal Mucosa / metabolism*
  • NF-kappa B / metabolism
  • Organoids / drug effects
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-bcl-2 / metabolism*
  • STAT1 Transcription Factor / genetics
  • STAT1 Transcription Factor / metabolism
  • Transcription Factors / metabolism
  • Tumor Necrosis Factor-alpha / pharmacology*
  • Up-Regulation

Substances

  • BCL2L14 protein, human
  • CCL20 protein, human
  • CCL5 protein, human
  • Chemokine CCL20
  • Chemokine CCL5
  • Chemokines
  • NF-kappa B
  • Proto-Oncogene Proteins c-bcl-2
  • SMARCA2 protein, human
  • STAT1 Transcription Factor
  • STAT1 protein, human
  • Transcription Factors
  • Tumor Necrosis Factor-alpha
  • Interferon-gamma