Date of Award

5-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cell Biology and Biochemistry

Research Advisor

Radhakrishna Rao, Ph.D.

Committee

Anjaparavanda Naren, Ph.D. Susan Senogles, Ph.D. Gabor Tigyi, M.D., Ph.D. Christopher Waters, Ph.D.

Keywords

acetaldehyde, occludin, PP2A

Abstract

Chronic alcohol consumption causes health problems including cancers, liver damage, and cirrhosis.These problems are exacerbated by endotoxins from the bacterial population of the colon, which can enter the bloodstream when gastrointestinal tight junctions, protein complexes that prevent paracellular passage of molecules, are compromised. The mechanisms of this barrier disruption are the subject of current study.

Acetaldehyde, produced as a result of ethanol breakdown by intestinal microflora as well as intestinal epithelia, is a known carcinogen. It compromises the barrier function of intestinal epithelial cells by causing irregularities in the phosphorylation of tight junction proteins. It also reacts with proteins, lipids, and DNA to cause cellular malfunction.

This project explored the roles of PP2A (a protein phosphatase) in acetaldehyde-mediated tight junction disruption, wound healing, and morphological differentiation. Intestinal epithelial cell culture in monolayers on permeable membranes were used to test the effects of acetaldehyde in the presence or absence of specific inhibitors on barrier function via transepithelial resistance and the paracellular passage of fluorescent molecules. Immunocytochemistry and confocal microscopy were used to track the localization of occludin in the cell lines tested, in cells cultured in monolayers or in a three-dimensional model in Matrigel. Results for acute administration of acetaldehyde were confirmed in mouse ileum.

Acute administration of acetaldehyde was demonstrated to have deleterious effects on the barrier function and wound healing of Caco-2 cells and mouse ileum, with varying degrees of attenuation of the effects of acetaldehyde via PP2A inhibition. Transepithelial resistance declined and inulin flux increased in the presence of acetaldehyde, as occludin was dephosphorylated and internalized by the cell in the presence of increased PP2A association and activity. PP2A inhibition by fostriecin, siRNA, or a specific peptide inhibitor not only rescued the cells from the disruption indicated by inulin flux, but also decreased the association of PP2A with occludin, resulting in preservation of threonine phosphorylation and localization of occludin at the tight junction. These results were confirmed by experiments with fostriecin in mouse ileum ex vivo. At all concentrations tested, acute exposure to acetaldehyde inhibited wound healing in an established in vitro model. Migration was not restored by PP2A inhibition, indicating that acetaldehyde's effects on wound healing were PP2A independent.

Chronic exposure to acetaldehyde disrupted differentiation in cells grown in Matrigel, as shown by changes in morphology, in a manner attenuated by fostriecin pretreatment. These results indicated that the known effects of acetaldehyde on cell differentiation may be PP2Adependent, and that intact tight junctions with appropriately-phosphorylated proteins may be important for maintaining the polarity of differentiated Caco-2 cells.

In conclusion, acetaldehyde disrupted tight junction barrier via occludin dephosphorylation by PP2A to cause leaky epithelia and deficient differentiation of cells. It slowed wound healing in a PP2A-independent manner. All of these findings contribute to an understanding of the mechanisms by which acetaldehyde causes health problems in alcoholics

DOI

10.21007/etd.cghs.2012.0075

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