Date Available

5-18-2022

Year of Publication

2020

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Medicine

Department/School/Program

Microbiology, Immunology, and Molecular Genetics

Advisor

Dr. Terrence Barrett

Abstract

Signaling events governing intestinal stem cell (ISC) homeostasis maintain the delicate balance of active self-renewal and passive differentiation to replenish intestinal epithelial cells (IEC) every 3-5 days. However, under certain contexts, ISC function is irreversibly compromised—requiring committed IEC lineages to dedifferentiate and regain “stemness”. In the current studies, we examine the signaling events driving epithelial cell responses to injury to expose pathologic failures in the healing response. Our specific goal is to tease out the cellular contexts that promote dedifferentiation to design effective therapeutics for disease conditions compromising ISC function.

First, we generated a novel transgenic animal using the epithelial-specific Villin promoter to constitutively ablate the critical negative regulator in the Phosphotidyl-inositol 3 Kinase (PI3K) signaling pathway, p85α, in IECs. At baseline, removal of p85α led to increased activation of PI3K/Akt, as seen through elevations in phosphorylated PTEN, Akt, and GSK3β. The consequence of the persistent activation was a shifting of cells out of the stem cell niche, into more differentiated the proliferative progenitor pool, as seen by a decrease in Lgr5+ cells per crypt and an increase in Axin2+ transcripts by in situ hybridization. Further investigations revealed PI3K activation led to a bolstered secretory lineage, increased total number and size of Paneth cells within the crypt through reduced Notch signaling. Subsequent studies mechanistically clarify mitochondrial bioenergentics in p85-deficient epithelial cells demonstrate enhanced State III respiration to drive production of ROS. ROS activates p38-MAPK to mount observed differentiation pressure. Using whole-body irradiation to target ISCs, we revealed that p85-deficient crypts underwent restitution at a faster pace than their control counterparts. The consequence of the enhanced regeneration was an increased organism survival after lethal irradiation. Transcript flux revealed that p85-deficient crypts increased Wnt and PI3K signaling targets, specifically Lgr5, Axin2 and Survivin earlier in the regenerative process, suggesting the increased secretory progenitor pool could enact dedifferentiation mechanisms to replenish ISCs. Using human samples of radiation-induced intestinal injury, we demonstrate the requirement of Wnt-target Survivin protein expression in IEC survival, suggesting a conserved mechanism and possible avenue for future therapeutic intervention.

Next, using isolated crypt epithelial cells from patients with inflammatory bowel disease (IBD), we establish that steroid-treated (prednisone) IBD patients harbor aberrant Wnt/β-catenin and NFκB signaling in IECs despite clinical improvement. At the molecular level, human IECs had significantly blunted cytosolic accumulation of Axin2 protein and subsequently decreased nuclear localization of downstream transcriptional activator, p-β-cateninSer552. Using a validated murine model of IBD, Dextran Sodium Sulfate-induced colitis (DSS colitis), we appreciated conserved blunting of inflammation-induced Wnt activation following dexamethasone administration. Studies in isolated murine ISC cultures revealed that the blunting of Wnt activation occurred in the absence of inflammatory stimulus—suggesting steroids directly affect ISC activation by interfering with β-catenin transcriptional activity. By tracking β-catenin-directed TCF/LEF transcriptional activity with a stably-transfected luciferase construct, we identified the signaling disruption occurred downstream of the β-catenin destruction complex, and resulted in limited ISC activation and mobilization in response to injury. Appropriately, primary ISC cultures from mice demonstrated that at high doses, steroid therapy inappropriately limits ISC activation sufficient Wnt ligands in culture. This leads to a depleted ISC pool and preventing mucosal restitution after injury.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2020.196

Funding Information

Work presented in the current thesis was supported by the University of Kentucky NIH CTSA TL1 Training Grant; 5TL1TR000115-05 (2015-2017)

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