INCREASED PI3K/AKT-INDUCED SURVIVIN DRIVES INTESTINAL CRYPT HEALING IN RESPONSE TO RADIATION INJURY VIA ENHANCED MITOCHONDRIAL-DERIVED ROS SIGNALING

Intestinal stem cell (ISC) homeostasis underlies signaling events that maintain the delicate balance of active self-renewal and passive differentiation. However, under certain contexts, ISC function is irreversibly compromised—requiring committed intestinal epithelial cell (IEC) lineages to dedifferentiate and regain “stemness”. Here, we investigated the underlying signaling mechanisms involved in intestinal mucosal healing in response to radiation injury.

The role of PI3K signaling in ISC reconstitution following radiation injury was interrogated using Villin-Cre pik3r1^lox/lox (p85^ΔIEC) mice and human enteroids (shp85α), deficient in epithelial expression of the regulatory subunit, p85α. Isolated IECs/tissues from p85^ΔIEC mice and human enteroids were examined for proteins/ genes associated with ISC/progenitor cells (PC), paneth cell, mitochondrial complexes, glycolysis, Wnt and notch signaling, using western blot, RT-qPCR and in situ hybridization. The proliferation zone of IECs was assessed by BrdU incorporation in vivo. Mitochondrial function of IECs was evaluated by oxygen consumption rate (OCR) and ROS production using seahorse and DCFH-DA assays, respectively. Lethal whole-body irradiation was performed to monitor PI3K-mediated time-course survival responses. Surgical resections from radiation proctitis were examined for survival and apoptotic markers using IHC, to assess IEC responses to radiation injury.

The IECs from p85^ΔIEC mice showed increased protein levels of p-PTEN, p-Akt^Ser473, survivin, cyclinD1 and ρ-β-catenin^Ser552 as well as increased mRNA for ISC/PC. In situ hybridization showed that enhanced PI3K signaling reduced Lgr5+ cells but expansion of Axin2+ cells. The shp85α enteroids showed increased transcripts of Wnt-targets and transcription factor Ascl2, needed for dedifferentiation-mediated restoration of ablated ISCs. The p85a-deficient enteroids also showed reduced Hes1 mRNA and increase in secretory (Atoh1/Math1) signaling determinants Gfi1 and Spdef, indicative of reduced NOTCH signaling. Seahorse analyses and p-p38 staining in p85^ΔIEC mice suggesting that enhanced PI3K signaling led to increased IEC mitochondrial respiration and ROS generation. Surprisingly, we found that expression of the Wnt target, survivin correlated with radiation injury in patients.

The current data establish that PI3K signaling increases mitochondrial ROS generation and ISC activation that improves IEC recovery from radiation-induced injury. The results suggest that increasing mitochondrial respiration and/or PI3K signaling may be attractive avenues for improving mucosal healing from radiation injury in patients.