Temporal induction of intestinal epithelial hypoxia-inducible factor-2α is sufficient to drive colitis

Abstract

Hypoxia is a notable feature of inflammatory bowel disease and chronic induction of hypoxia-inducible factor (HIF)-1α and HIF-2α (endothelial PAS domain protein 1, EPAS1) play important, but opposing, roles in its pathogenesis. While activation of HIF-1α decreases intestinal inflammation and is beneficial in colitis, activation of HIF-2α exacerbates colitis and increases colon carcinogenesis in animal models, primarily due to the role of epithelial HIF-2α in mounting a potent inflammatory response. Previous work from our laboratory showed that mice overexpressing intestinal epithelial HIF-2α led to massive intestinal inflammation and decreased survival. As oxygen homeostasis and HIFs are critical in embryonic development, it is not clear whether the observed intestinal inflammatory response was secondary to developmental defects. To address this question, the present study used a mouse model to temporally modulate expression of intestinal epithelial HIF-2α to assess its role in mediating inflammatory response. Remarkably, activation of HIF-2α in intestinal epithelial cells in adult mice increased expression of proinflammatory mediators; however, no decrease in survival was observed. Furthermore, in an acute model of colitis, activation of HIF-2α was sufficient to exacerbate colitis. These data confirm our previous finding that epithelial HIF-2α mediates inflammatory response and demonstrates that activation of HIF-2α is sufficient to exacerbate colitis.NEW & NOTEWORTHY Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disease of the intestinal tract. Hypoxia and activation of its downstream transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α are notable features of IBD. HIF-1α has well-characterized protective roles in IBD; however, the role of HIF-2α has been less studied. Using novel HIF-2α mouse models, we show that activation of HIF-2α in intestinal epithelial cells is sufficient to exacerbate colitis.

Keywords: colitis; hypoxia; hypoxia-inducible factor-2α.

Graphical abstract

Fig. 1.

Temporal model of intestinal hypoxia-inducible factor-2α (HIF-2α) activation. A: schematic of a temporal HIF-2α-overexpressing mouse model (Vil-ER^T2Hif2α^LSL/LSL). B: Western blot analysis of HIF-2α from scraped intestinal mucosal cells of Vil-ER^T2Hif2α^LSL/LSL or littermate control Hif-2α^+/+ mice. Quantitative PCR analysis for the expression of divalent metal transporter 1 (Dmt1), duodenal cytochrome b (DcytB), ferroportin (Fpn) in scraped mucosal cells from the small intestine (C) and colon (D) of Vil-ER^T2Hif2α^LSL/LSL or littermate control, Hif-2α^+/+ mice. *P < 0.05, **P < 0.01, ***P < 0.001 for the difference between Vil-ER^T2Hif2α^LSL/LSL compared with littermate Hif-2α^+/+ mice. Four mice were assessed from each group.

Fig. 2.

Temporal activation of intestinal hypoxia-inducible factor-2α (HIF-2α) does not lead to any basal histological changes. Representative hematoxylin-and-eosin staining of small intestine (duodenum) (A, E) and colon (B, F) from littermate control Hif2α^+/+ and Vil-ER^T2Hif2α^LSL/LSL mice at 1 wk and 4 wk, respectively. Representative Ki67 staining and quantitation of small intestine (C, G) and colon (D, H) from Hif2α^+/+ and Vil-ER^T2Hif2α^LSL/LSL mice at 1 wk and 4 wk, respectively. Four mice were assessed from each group.

Fig. 3.

Temporal activation of intestinal hypoxia-inducible factor-2α (HIF-2α) activates proinflammatory targets in the intestine. Quantitative PCR analysis of Steap4, Tnf-α, and Cxcl1 in the small intestine (A) and colon (B) from Vil-ER^T2Hif2α^LSL/LSL or Hif2α^+/+ mice. *P < 0.05, **P < 0.01, ***P < 0.001 for the difference between Vil-ER^T2Hif2α^LSL/LSL and littermate control Hif2α^+/+ mice. Four mice were assessed from each group.

Fig. 4.

Temporal activation of intestinal hypoxia-inducible factor-2α (HIF-2α) exacerbates colitis. A: schematic of DSS-induced colitis model. Colon length (n = 6; B), representative hematoxylin-and-eosin staining of colons and inflammation score from Hif2α^+/+ and Vil-ER^T2Hif2α^LSL/LSL mice (n = 6; C), Ki67 staining and quantitation of colon from Hif2α^+/+ and Vil-ER^T2Hif2α^LSL/LSL mice (n = 4; D), and qPCR analysis for the expression of Steap4 and Tnf-α in scraped colonic mucosal cells from Vil-ER^T2Hif2α^LSL/LSL or Hif2α^+/+ mice (n = 5; E). *P < 0.05, **P < 0.01 for the differences between Vil-ER^T2Hif2α^LSL/LSL compared with littermate control Hif2α^+/+ mice.

Fig. 5.

RNA-Seq analysis in the colons from Hif2α^Flx/Flx and Hif2α^ΔIE mice. A: expression of HIF-2α in Hif2α^Flx/Flx and Hif2α^ΔIE mice. Quantitative PCR analysis for the expression of Steap4 (B), Cxcl1 (C), and Tnf-α (D) in scraped colonic mucosal cells from Hif2α^Flx/Flx and Hif2α^ΔIE mice. E: heat map generated from RNA-seq data on scrapped colonic mucosal cells isolated from Hif2α^Flx/Flx and Hif2α^ΔIE mice. F: list of top 15 KEGG pathways altered in Hif2α^Flx/Flx and Hif2α^ΔIE mice. G: qPCR array analysis from scraped colonic mucosal cells from Vil-ER^T2Hif2α^LSL/LSL and Hif2α^+/+. H: list of top 10 KEGG pathways with respective altered genes in Hif2α^+/+ and Vil-ER^T2Hif2α^LSL/LSL mice. Three mice were assessed for each group. *P < 0.05, **P < 0.01, ***P < 0.001 for the differences between Hif2α^ΔIE or Vil-ER^T2Hif2α^LSL/LSL mice compared with littermate control mice.