Background: Sonic Hedgehog (SHH) signaling is crucial for embryonic development. Mutation in this pathway hinders cell migration, differentiation, proliferation, and, at times, tumorigenesis. We recently showed that SHH is induced in intestinal epithelial cells shortly after γ irradiation, and its inhibition impairs epithelial regeneration by abrogating epithelial-stromal crosstalk. However, the mechanisms regulating the activation of SHH upon injury of intestinal epithelial cells are unknown. Aim: To explore the mechanism that governs the induction of SHH signaling in intestinal epithelial cells upon irradiation. Methods: We performed computational analysis to identify putative binding sites of transcription factors (TFs) that bind to mouse and human SHH promoters using JASPAR (database of transcription factor binding profiles). Enriched intestinal epithelial cells (CD45^- CD326⁺) were isolated from Bmi1-Cre^ER mice exposed to 12 Gy total body irradiation or sham-treated for 24 hr and used to assess the expression levels of selected TFs. We determined the effect of two selected TFs, HIF1α and KLF4, on the Shh promoter activity using Gaussia Luciferase Assay. In addition, we performed Chromatin Immunoprecipitation (ChIP)-PCR using HIF1α and KLF4 antibodies to identify specific binding sites for these TFs within the Shh promoter. To assess whether HIF1α and KLF4 interact, we performed co-immunoprecipitation (co-IP) assays. In vivo experiments were performed with N=3, and statistical analysis was performed using a t-test with a p-value <0.5 as significant. Results: Using JASPAR, we identified 22 tentative TF-binding sites in mouse and human SHH promoters. Using RT-PCR, we showed that 24hr post-irradiation, Nfya, Klf4, Stat3, Foxj2, Hif1α, and Gata4 were significantly upregulated in enriched intestinal epithelial cells (CD45^- CD326⁺) of mice exposed to 12 Gy radiation compared to sham-irradiated mice. We identified one putative binding site for KLF4 and eight for HIF1α within the 1,600 bp of murine Shh promoter. Both TFs share a site around 210 bp of the Shh promoter. Luciferase assay performed at 72hr after co-transfection of Hif1α and Klf4 showed a 3-fold increase in the activity of the Shh promoter. ChIP-PCR assays showed that KLF4 binds to the 210-bp site, and HIF1α binds to six identified sites, including the 210-bp site. Using co-IP, we demonstrated that KLF4 and HIF1α physically interact with each other. Conclusions: HIF1α and KLF4 expression levels are induced upon radiation injury in the intestinal epithelium, and in vitro, they activate the Shh promoter by binding to a shared region of the promoter that involves physical interaction. These results demonstrate that HIF1α and KLF4 mechanistically regulate SHH expression in the intestinal epithelial cells following radiation injury.

Zhi Jian Wu

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