Autophagy gene deletion in the intestinal epithelium delays YAP-mediated regeneration post-injury

Background and rationale: Homeostasis of the intestinal epithelium is maintained by actively dividing intestinal stem cells (aISC). When aISCs are depleted via tissue damaging agents such as chemotherapy, irradiation, and inflammation, other cells aid in epithelial regeneration. These other, non-aISCs must be injury resistant relative to aISCs and must also have the capacity for self-renewal and multipotency. We call non-aISC regenerative cells facultative stem cells. Previous work from our lab showed that intestinal epithelial cells with relatively high autophagy levels can act as facultative stem cells, but the mechanism by which high autophagy levels contribute to regeneration post-injury is unknown. One key pathway implicated in regeneration of the intestinal epithelium is the YAP/TAZ signaling pathway; however, the link between autophagy and YAP signaling in intestinal epithelial regeneration is unknown. The objective of this study is to define the extent to which autophagy-high cells utilize YAP/TAZ signaling to promote intestinal regeneration. Methods: We subjected mice (wildtype, inducible intestinal epithelial-specific Atg7 knockout (Atg7DIEC), and control (WTDIEC) to 12Gy whole-body irradiation (IR) followed by histological and molecular evaluation at 1, 2, and 3.5 days post-IR to evaluate YAP signaling during epithelial damage and regeneration. Autophagy status of single cells was determined via fluorescence-activated cell sorting (FACS) of cells stained with the autophagic vesicle dye CytoID, which we previously confirmed distinguishes cells with high versus low autophagy. Sorted, CytoID-stained, EpCAM+, live cells were evaluated for organoid formation efficiency with or without treatment with autophagy inhibitors (bafilomycin or Ulk1 inhibitor) or large tumor suppressor (LATS) kinase inhibitor to assess the role of YAP activation in organoid formation in cells with differential autophagy levels. Results and Conclusions: We found that autophagy (LC3-II western) and YAP (YAP target gene qPCR: Cyr61 and YAP-mediated fetal-like genes: Ly6a, Tacstd2, Clu, and Anxa1) were increased at 2 days post-IR in wildtype mice. Atg7DIEC mice at 3.5 days post-IR showed a significant increase in YAP activation compared to WTDIEC mice. LATS inhibitor enhanced organoid formation efficiency in cells regardless of autophagy levels, but the percent increase in organoid formation between control and LATS inhibitor-treated cells was significantly higher in cells with low versus high autophagy. When we treated sorted EpCAM+ cells with bafilomycin or Ulk1 inhibitor to block autophagic flux, we observed a decrease in organoid formation capacity that was rescued with addition of the LATS inhibitor. Taken together, our data show that autophagy loss leads to an increase in YAP signaling that is not associated with enhanced regeneration. These findings suggests that there is either a delay in the regenerative response in autophagy deficient cells or upregulation of YAP in cells that do not require autophagy. Lastly, our in vitro data suggest that one of the pathways— autophagy or YAP signaling— must be active to promote for organoid formation. Ongoing studies are using genetic models to define mechanistic links between autophagy and YAP/TAZ signaling in vivo and in vitro. This research was funded NIH grants F31DK141082 (KK), R01DK124369 (KEH), and R01DK142682 (KEH). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.