Single‐Cell Sequencing Reveals Distinct Expression Patterns of the Integrated Stress Response (ISR) Pathway Regulating Cell Survival in Secretory Cells in Colon of Hnrnp I Knockout Mice

Abstract

ObjectivesHeterogeneous nuclear ribonucleoprotein I (Hnrnp I) is an RNA‐binding protein functions during pre‐mRNA splicing and regulation of alternative splicing events. Previously we found that ablation of Hnrnp I activates NF‐κB signaling in intestinal epithelial cells (IECs), resulting in increased nuclear translocation of P65 (NF‐κB subunit) and upregulation of proinflammatory cytokines and chemokines. Integrated stress response (ISR) signaling was linked to activation of canonical NF‐κB pathway. Therefore, in this study, we aimed to investigate ISR in the colon of Hnrnp I knockout mice. We hypothesize that cellular stress from Hnrnp I knockout triggers ISR, consequently affects the cellular dynamics among secretory cell types on colon epithelium (enteroendocrine, Paneth and goblet cells).MethodsTotal RNA was isolated from total colon cell preparations from WT and KO mice. Single‐cell RNAseq libraries (biological replicates, n=3) were prepared using Chromium Single‐Cell Gene Expression NextGem (v3.1, 10X Genomics). Analyses from raw counts were performed using the Seurat (v3.2.0) R package using default parameters, a computational cell calling algorithm was used to identify cell type using two published annotated mouse cell datasets. Pathway analysis was performed for secretary cell clusters including enteroendocrine cells (EECs), Paneth cells, and goblet cellsResultsComparing to WT, number of EECs in colon was significantly increased in KO mice. Total mRNA level of Atf4, the principal regulator of ISR, was significantly increased in colon secretory cells in KO mice. We also examined mRNA expression of ATF4 target genes in secretory cells. Specifically, gene encoding the regulator of apoptosis and autophagy, members of Bcl2 family, Bnip3, was significantly increased in secretory cells in KO mice, while Mlx and Gabarapl2 (Atg8), both are involved in apoptosis and autophagy signaling pathways, have a trend of increase in secretory cells in KO mice, all comparing to WT.ConclusionsCellular stress from Hnrnp I knockout activated ISR in IECs, specially in those secretory cell types – EECs, Paneth, and goblet cells. The activation of ISR led to both autophagy and apoptosis signaling in these cells through the upregulation of Bnip3, Mlxand Gabarapl2. Taken together, our results suggest that in response to the cellular stresses from the depletion of Hnrnp I in IECs, ISR activation in colonic secretory cells, stimulating apoptosis and autophagy pathway signaling, particularly in EECs, resulting in the proliferation of EECs in KO. The overall manipulation by ISR signaling in the colon secretary cells may present a critical early response to modulations in colon, potentially leading to perturbations to homeostasis among host colon epithelia, colon residential immunity, and gut microbiota.