Establishment of γ-secretase-deficient goblet-like cells: A novel in vitro platform to dissect regulatory mechanisms of mucus production in the intestinal epithelium

Intestinal goblet cells play a crucial role in producing mucus that forms the protective mucosal barrier. However, the complex in vivo interactions among microbial, dietary, and immune factors make it challenging to dissect the intrinsic regulatory mechanisms underlying goblet cell mucus production. In this study, we established a novel HT-29-derived cell line lacking Presenilin-1 and Nicastrin, two core components of γ-secretase, a membrane-associated protease complex that cleaves multiple transmembrane proteins, including Notch receptors. Similar to γ-secretase-inhibited HT-29 cells, our newly established cell line suppressed Notch signaling, upregulated ATOH1, and increased the expression of MUC2, a major colonic Mucin and a goblet cell marker, thereby maintaining a stable mature goblet-like phenotype with continuous activation of the transcriptional program driving mucus secretion. Therefore, we designated it as induced Mature Intestinal Goblet-like cells (iMIGs). In contrast, RBPJ-deficient HT-29 cells exhibited an unexpected decrease in MUC2 expression, suggesting the involvement of additional γ-secretase-dependent pathways beyond canonical Notch signaling. Furthermore, inhibition of the MEK/ERK pathway, which also regulates goblet cell differentiation in vivo, reduced both ATOH1 and MUC2 expression, indicating that iMIGs retain in vivo-like regulatory mechanisms for mucus production. Under dextran sulfate sodium-induced colitis-mimicking conditions, iMIGs enhanced MUC2 expression and exhibited altered endoplasmic reticulum stress responses, independent of intestinal microbiota or immune cell influence. These findings demonstrate that iMIGs provide a versatile in vitro platform for investigating the molecular regulation of mucus production in goblet cells under both physiological and pathological conditions, including inflammatory bowel disease.

Goblet cell; Inflammatory bowel disease; MUC2; Mucus production; Notch signaling; γ-secretase.