Graduation Date

Spring 5-7-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pathology & Microbiology

First Advisor

Dr. Hamid Band

Abstract

All adult organs are endowed with a small pool of resident stem cells that must be maintained throughout life to provide for cell turnover during homeostasis and tissue repair following any injury. The unique ability to self-renew as well as to differentiate into functional cells of organs in which they reside makes stem cells essential for the maintenance of organ systems. It is now becoming increasingly evident that aberrant activity of stem cells contributes to diseases such as cancer or tissue/organ atrophy. Thus, it is critical to better understand the mechanisms that regulate adult stem cells. Ligand-activated receptor tyrosine kinases (RTKs) represent an important means to regulate adult stem cells. We and others have established the CBL-family ubiquitin ligases as key negative regulators of RTKs. However, physiological roles of CBL proteins in intestinal epithelial homeostasis are unknown. Recently, we have shown that CBL and CBL-B function as redundant but essential regulators of hematopoietic stem cell (HSC) quiescence and their combined deletion in HSCs of mice leads to a rapidly-fatal myeloproliferative disorder, mimicking a similar leukemic disease associated with mutations of CBL in humans. CBL and CBL-B have also been shown to help maintain asymmetric neural stem cell division. Any role of CBL proteins in the regulation of epithelial stem cells has not been reported. To address such a role, we generated a novel mouse model (Cbl-flox/flox; Cbl-b-null; Lgr5-EGFP-IRES-CreERT2; Rosa26-LacZ) to confer concurrent tamoxifen-inducible loss of CBL and CBL-B in the Lgr5-expressing intestinal epithelial stem cells (IESCs). Tamoxifen injection in this inducible CBL/CBL-B double knockout (iDKO) mouse model resulted in a rapid and significant reduction in the Lgr5-High IESC pool with a concomitant increase in the Lgr5-Lo transit amplifying cells. Lineage tracing using LacZ-staining revealed an increase in the number of blue progeny in the iDKOs, suggesting an increased IESC commitment to differentiation. Of the progeny, iDKO animals showed a propensity towards enterocyte and goblet cell fate at the expense of Paneth cells. Loss of IESCs in iDKO mice led to slower recovery from intestinal epithelial injury due to X-ray radiation of the abdomen. In vitro deletion of CBL proteins in the crypt culture recapitulated the loss of self renewal phenotype and was associated with hyperactivation of MAPK pathway and downregulation of Wnt pathway effector TCF4. These results demonstrate a novel requirement of CBL/CBL-B in the maintenance of a well-studied epithelial stem cell compartment, the IESC, and suggest that CBL proteins by regulating MAPK pathway protect IESCs from exhaustion.

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