Phosphatidylinositol 3-Kinase Signaling Enhances Intestinal Crypt Epithelial Cell Recovery after Radiation

Abstract

Intestinal stem cell (ISC) signaling maintains the balance of self-renewal and differentiation. Herein, the role of phosphatidylinositol 3-kinase (PI3K) signaling in ISC responses to radiation was interrogated using Villin-Cre pik3r1^lox/lox (p85^ΔIEC) mice and p85α-deficient human enteroids (shp85α). Lethal whole-body irradiation in mice was performed to monitor PI3K-mediated survival responses. Rectal biopsies from patients with radiation proctitis were examined by immunohistochemistry for the PI3K/Akt- and Wnt-target survivin. The intestinal epithelial cells (IECs) from p85^ΔIEC mice showed increased protein levels of phosphorylated phosphatase and tensin homolog, phosphorylated Akt^Ser473, survivin, cyclin D1, and ρ-β-catenin^Ser552, as well as increased mRNA for ISC/progenitor cell. In situ hybridization showed that enhanced PI3K signaling reduced leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5⁺) cells but enhanced AXIS inhibition protein 2 (Axin2⁺) cells. The shp85α enteroids showed increased mRNA expression of Wnt targets and transcription factor ASCL2, needed for dedifferentiation-mediated restoration of ablated ISCs. The p85α-deficient enteroids showed reduced HES1 mRNA and increases in secretory (ATOH1/MATH1) signaling determinants GFI1 and SPDEF, indicative of reduced NOTCH signaling. Seahorse analyses and phosphorylated p38 staining in IEC^Δp85 mice indicated that enhanced PI3K signaling led to increased IEC mitochondrial respiration and reactive oxygen species generation. Expression of survivin correlated with the radiation injury in patients. The current data indicate that PI3K signaling increased mitochondrial reactive oxygen species generation and ISC activation that improved IEC recovery from radiation-induced injury. The results suggest that increasing PI3K signaling and induced mitochondrial respiration may improve mucosal healing in patients with radiation injury.