Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

Active intestinal stem cells in the intestinal epithelium are prone to injury by ionizing radiation. We previously showed that upon radiation-induced injury, normally quiescent reserve ISCs (rISCs) marked by B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) are activated by MUSASHI1 (MSI1) and exit from the quiescent state to regenerate the intestinal epithelium. This study aims to further establish the mechanism that regulates activation of Bmi1-CreER;Rosa26eYFP (Bmi1-CreER) rISCs following γ radiation-induced injury. Bmi1-CreER mice were treated with tamoxifen to initiate lineage tracing of BMI1 (eYFP+) cells and exposed to 12 Gy of total body γ irradiation (TBI) or sham. Intestinal tissues were collected and analyzed by immunofluorescence, Western blot, qRT-PCR, ELISA, and ChIP-PCR. After irradiation, increased expression of Msi1 in eYFP+ cells was accompanied by increased expression of Axin2, a WNT marker. Promoter studies of the Msi1 gene indicated that Msi1 is a WNT target gene. Co-culture of stromal cells isolated from irradiated mice stimulated Bmi1-CreER-derived organoids regeneration more effectively than those from sham mice. Expression of WNT ligands, including Wnt2b, Wnt4, Wnt5a, and Rspo3, was increased in irradiated stromal cells compared to sham-treated stromal cells. Moreover, expression of the Sonic Hedgehog (SHH) effector, Gli1, was increased in stromal cells from irradiated mice. This was correlated with an increased expression of SHH in epithelial cells post-irradiation, indicating epithelial-stromal interaction. Finally, pre-injury treatment with SHH inhibitor cyclopamine significantly reduced intestinal epithelial regeneration and Msi1 expression post-irradiation. Upon ionizing radiation-induced injury, intestinal epithelial cells increase SHH secretion, stimulating stromal cells to secrete WNT ligands. WNT activators induce Msi1 expression in the Bmi1-CreER cells. This stromal-epithelial interaction leads to Bmi1-CreER rISCs induction and epithelial regeneration.