The mammalian intestinal epithelium is a continuously developing system. Each intestinal crypt contains a single, multipotent stem cell which undergoes rapid proliferation. Descendants of this stem cell become the four principal cell types that constitute the gut epithelium: absorptive, goblet, enteroendocrine, and Paneth cells. This coupled cell proliferation and differentiation is subject to stringent regulation. Expression of many intestinal genes follow a gradient of distribution along the duodenal-colon (horizontal) axis, and the crypt-villus and crypt-surface epithelium (vertical axes of the small and large intestines, respectively. The unique region- specific, cell-specific, and spatial-specific nature of gene regulation renders the intestinal epithelium attractive for studying the mechanism controlling cell proliferation and differentiation. We previously isolated a gene, named A4, that is transcriptionally activated upon differentiation of the human colonic epithelial cell line HT29-18. Expression of A4 is enriched in the intestinal epithelium and follows an increasing, horizontal gradient from the small to the large intestine. In addition, expression of A4 is abundant in the crypt and follows a diminishing, vertical gradient along the crypt-villus and crypt-surface epithelium axIs of the small and large intestine, respectively. These salient features suggest that A4 may serve as an excellent marker of crypt cell differentiation, and that understanding the mechanism regulating A4 expression in vitro may shed some light on the mechanism controlling gut epithelial differentiation. Within the project period of the current Program Project, we have made significant progress in understanding the regulation of the A4 promoter during HT29 differentiation. Specifically, we identified a positive cis-element in the proximal A4 promoter that interacts with a DNA-binding protein (putative transcription factor) with a differentiation- dependent activity. In addition, we showed that A4 is a member of the proteolipid family of proteins and is a membrane protein of the endoplasmic reticulum with putative ion channel activity.
Two Specific Aims are proposed in the competitive renewal application for Project 5: (1) to isolate and characterize the differentiation- dependent DNA-binding factor (named GATG-binding protein) that interacts with the A4 promoter and to study its effect on epithelial differentiation, and (2) to further characterize the function of A4 as an ion channel and as a potential mediator of differentiation. These studies should help understand the mechanism controlling intestinal epithelial differentiation.

Project Start
Project End
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
6
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Aihara, Eitaro; Montrose, Marshall H (2014) Importance of Ca(2+) in gastric epithelial restitution-new views revealed by real-time in vivo measurements. Curr Opin Pharmacol 19:76-83
Singh, Varsha; Yang, Jianbo; Chen, Tiane-e et al. (2014) Translating molecular physiology of intestinal transport into pharmacologic treatment of diarrhea: stimulation of Na+ absorption. Clin Gastroenterol Hepatol 12:27-31
Lin, Rong; Murtazina, Rakhilya; Cha, Boyoung et al. (2011) D-glucose acts via sodium/glucose cotransporter 1 to increase NHE3 in mouse jejunal brush border by a Na+/H+ exchange regulatory factor 2-dependent process. Gastroenterology 140:560-71
Hartley, Jane Louise; Zachos, Nicholas C; Dawood, Ban et al. (2010) Mutations in TTC37 cause trichohepatoenteric syndrome (phenotypic diarrhea of infancy). Gastroenterology 138:2388-98, 2398.e1-2
Zachos, Nicholas C; Hodson, Caleb; Kovbasnjuk, Olga et al. (2008) Elevated intracellular calcium stimulates NHE3 activity by an IKEPP (NHERF4) dependent mechanism. Cell Physiol Biochem 22:693-704
Li, Xuhang; Donowitz, Mark (2008) Fractionation of subcellular membrane vesicles of epithelial and nonepithelial cells by OptiPrep density gradient ultracentrifugation. Methods Mol Biol 440:97-110
Lee, Aven; Rayfield, Andrew; Hryciw, Deanne H et al. (2007) Na+-H+ exchanger regulatory factor 1 is a PDZ scaffold for the astroglial glutamate transporter GLAST. Glia 55:119-29
Donowitz, Mark; Singh, Siddharth; Salahuddin, Farah F et al. (2007) Proteome of murine jejunal brush border membrane vesicles. J Proteome Res 6:4068-79
Murtazina, Rakhilya; Kovbasnjuk, Olga; Zachos, Nicholas C et al. (2007) Tissue-specific regulation of sodium/proton exchanger isoform 3 activity in Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) null mice. cAMP inhibition is differentially dependent on NHERF1 and exchange protein directly activated by cAMP in ileum versus J Biol Chem 282:25141-51
Donowitz, Mark; Li, Xuhang (2007) Regulatory binding partners and complexes of NHE3. Physiol Rev 87:825-72

Showing the most recent 10 out of 110 publications