Characterization of Fasting-Induced p21 Expression and Protection of Intestinal Stem Cells

<p>The cyclin dependent kinase inhibitor, p21, plays a key role in cell cycle. Additionally, the p21 gene, <italic>Cdkn1a</italic> (hereafter <italic>p21</italic>) is often used as a marker of cellular stress. To investigate <italic>p21</italic> promoter activity under basal conditions and in response to various forms of stress, we generated knock-in imaging reporter mice that express firefly luciferase (<italic>FLuc</italic>) under the control of the endogenous p21 promoter. I demonstrated that FLuc expression and bioluminescence detection mirrored endogenous p21 protein levels and promoter activity in vivo. Contrary to previously known roles for p53-mediate expression of <italic>p21</italic>, imaging of reporter cells demonstrated that p53 prevented the ERK/MAPK pathway from activating <italic>p21</italic> expression when quiescent cells were stimulated with serum to re-enter the cell cycle. In addition, low light bioluminescence imaging identified <italic>p21</italic> expression in specific regions of individual organs that were not previously observed including the paraventricular, arcuate and dorsomedial nuclei of the hypothalamus - regions that detect nutrient levels in the blood stream and regulate metabolism throughout the body. These results suggested a link between <italic>p21</italic> expression and metabolic regulation. I found that food deprivation (fasting) potently induced <italic>p21</italic> expression in tissues involved in metabolic regulation, including liver, pancreas, and hypothalamic nuclei. The ability of fasting to induce <italic>p21</italic> expression was found to be independent of p53, but dependent on the transcription factor FOXO1, which was bound to the <italic>p21</italic> promoter region only in fasted mice. Previous work has shown that short-term fasting protects mice from what would normally be lethal doses of etoposide. I hypothesized that p21 may be involved in this protection, as <italic>p21</italic> expression increased in response to both fasting and DNA damage. I demonstrated that fasting prior to a high dose of etoposide treatment enhanced survival by protecting small intestinal stem cells, but that p21 was not required for this protection. While high dose etoposide treatment caused complete destruction of the crypt-villus architecture and near complete loss of small intestinal stem cells in free-feeding mice, fasting prior to etoposide treatment enabled stem cell survival and subsequent reconstitution of the small intestinal crypts and villi. Using LacZ reporter mice and lineage tracing, I found that both crypt base columnar (CBC) and +4 stem cells contributed to crypt restoration in the fasted mice. Though etoposide in fed and fasted mice induced similar amounts of DNA double strand breaks (DSBs) immediately following treatment, resolution of DNA DSBs, as measured by loss of &gamma;H2AX staining, occurred more quickly in stem cells of fasted mice compared to those of fed mice.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biology and Biomedical Sciences: Molecular Cell Biology

Year dc:date.available

2014

Author dc:creator

• Tinkum, Kelsey Lynn

Contributors dc:contributor

• Helen Piwnica-Worms

Language dc:language

English (en)

DOI

https://doi.org/10.7936/K7BZ641C

OAI identifier oai:identifier

oai:openscholarship.wustl.edu:etd-2357