Probes for INS

Aim: Reactive oxygen species (ROS) produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by NADPH oxidases. In the intestine, due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut, and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut.

Results: NoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS induced colon cancer, NoxO1 has a protective role and may influence the population of natural killer cells.

Conclusion: NoxO1 affects colon epithelium homeostasis and prevents inflammation.

Lgr5+ stem cells are crucial to gut epithelium homeostasis; however, how these cells are maintained is not fully understood. Zinc finger HIT-type containing 1 (Znhit1) is an evolutionarily conserved subunit of the SRCAP chromosome remodeling complex. Currently, the function of Znhit1 in vivo and its working mechanism in the SRCAP complex are unknown. Here we show that deletion of Znhit1 in intestinal epithelium depletes Lgr5+ stem cells thus disrupts intestinal homeostasis postnatal establishment and maintenance. Mechanistically, Znhit1 incorporates histone variant H2A.Z into TSS region of genes involved in Lgr5+ stem cell fate determination, including Lgr5, Tgfb1 and Tgfbr2, for subsequent transcriptional regulation. Importantly, Znhit1 promotes the interaction between H2A.Z and YL1 (H2A.Z chaperone) by controlling YL1 phosphorylation. These results demonstrate that Znhit1/H2A.Z is essential for Lgr5+ stem cell maintenance and intestinal homeostasis. Our findings identified a dominant role of Znhit1/H2A.Z in controlling mammalian organ development and tissue homeostasis in vivo.

Abstract

BACKGROUND & AIMS:

Inflammation affects regeneration of the intestinal epithelia; long noncoding RNAs (lncRNAs) regulate cell functions, such as proliferation, differentiation, and migration. We investigated the mechanisms by which the lncRNA H19, imprinted maternally expressed transcript (H19) regulates regeneration of intestinal epithelium using cell cultures and mouse models of inflammation.

METHODS:

We performed RNA-sequencing transcriptome analyses of intestinal tissues from mice with lipopolysaccharide (LPS)-induced sepsis to identify lncRNAs associated with inflammation; findings were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization analyses of intestinal tissues from mice with sepsis or dextran sulfate sodium (DSS)-induced mucosal wound healing and patients with ulcerative colitis compared to healthy individuals (controls). We screened cytokines for their ability to induce expression of H19 in HT-29 cells and intestinal epithelial cells (IECs), and confirmed findings in crypt epithelial organoids derived from mouse small intestine. IECs were incubated with different signal transduction inhibitors and effects on H19 lncRNA levels were measured. We assessed intestinal epithelial proliferation or regeneration in H19ΔEx1/+ mice given LPS or DSS vs wild-type littermates (control mice). H19 was overexpressed in IECs using lentiviral vectors and cell proliferation was measured. We performed RNA antisense purification, RNA immunoprecipitation, and luciferase reporter assays to study functions of H19 in IECs.

RESULTS:

In RNA-sequencing transcriptome analysis of lncRNA expression in intestinal tissues from mice, we found that levels of H19 lncRNA changed significantly with LPS exposure. Levels of H19 lncRNA increased in intestinal tissues of patients with ulcerative colitis, micewith LPS-induced and polymicrobial sepsis, or mice with DSS-induced colitis, compared with controls. Increased H19 lncRNA localized to epithelial cells in the intestine, regardless of Lgr5 messenger RNA expression. Exposure of IECs to interleukin 22 (IL22) increased levels of H19 lncRNA with time and dose, which required STAT3 and protein kinase A activity. IL22 induced expression of H19 in mouse intestinal epithelial organoids within 6 hours. Exposure to IL22 increased growth of intestinal epithelial organoids derived from control mice, but not H19ΔEx1/+ mice. Overexpression of H19 in HT-29 cells increased their proliferation. Intestinal mucosa healed more slowly after withdrawal of DSS from H19ΔEx1/+ mice vs control mice. Crypt epithelial cells from H19ΔEx1/+ mice proliferated more slowly than those from control miceafter exposure to LPS. H19 lncRNA bound to p53 and microRNAs that inhibit cell proliferation, including microRNA 34a and let-7; H19 lncRNA binding blocked their function, leading to increased expression of genes that promote regeneration of the epithelium.

CONCLUSIONS:

The level of lncRNA H19 is increased in inflamed intestinal tissues from mice and patients. The inflammatory cytokine IL22 induces expression of H19 in IECs, which is required for intestinal epithelial proliferation and mucosal healing. H19 lncRNA appears to inhibit p53 protein and microRNA 34a and let-7 to promote proliferation of IECs and epithelial regeneration.