Date of Award

12-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular Therapeutics and Cell Signaling

Research Advisor

Edwards A. Park Ph.D.

Committee

George A. Cook, Ph.D. Marshall B. Elam, M.D., Ph.D., FAHA Rao N. Gadiparthi, Ph.D. Roderick T. Hori, Ph.D.

Keywords

Gene regulation, Inverse recruitment, Nuclear corepressors, Nuclear receptor, PLA2, Thyroid hormone

Abstract

Rationale: Low grade inflammation has been correlated with elevated risk of hepatic steatosis and atherosclerosis. Secretory phospholipase A2 group IIA (PLA2g2a) enhances the progression of several chronic inflammatory diseases including arthritis and atherosclerosis. The potential linkage of hypothyroidism with inflammation led us to examine the modulation of sPLA2 expression by thyroid hormone (T3) in liver.

Objective: Most of the studies of phospholipase A2 group IIA (PLA2g2a) have been conducted with macrophages and vascular smooth muscle cells with regard to atherosclerosis. The liver is one of the major contributors to the total pool of extracellular PLA2g2a. The aim of the present study was to characterize the regulation of PLA2g2a and other sPLA2 genes by T3 in liver. The second aim was to identify the mechanism by which T3 inhibits the expression of PLA2g2a gene.

Methods and Results: In the first part of my project, I assessed the effect of T3 on sPLA2 gene expression in liver. I found that T3 inhibited the expression of the PLA2g2a in human and rat hepatocytes. Thyroid hormone status regulated the expression of endogenous PLA2g2a in both rats and mice. Other sPLA2 isoforms including PLA2g1b, PLA2g3 and PLA2g5 were also suppressed by T3 in liver. PLA2g2a was induced by cytokines and high fat diet. Both the cytokine and fatty acid mediated induction of PLA2g2a gene was blocked by T3. In an effort to dissect the mechanism of repression by T3, I cloned the PLA2g2a gene and identified a negative T3 response element in the promoter. This thyroid receptor (TRβ) binding site differs considerably from consensus T3 stimulatory elements. Using in vitro and in vivo binding assays, I demonstrated that TRβ bound directly to the PLA2g2a promoter. In present studies, I found that disruption of the corepressor binding site in TRβ decreased the T3 inhibition of PLA2g2a. Conversely a mutation in coactivator binding site had no effect on the T3 mediated inhibition of PLA2g2a. Knockdown of nuclear corepressor (NCoR1) or silencing mediator for retinoid and thyroid receptors (SMRT) by siRNA blocked the T3 inhibition of PLA2g2a. Using chromatin immunoprecipitation assays, I showed that NCoR1 and SMRT were associated with the PLA2g2a gene in the presence of T3.

Conclusion: The thyroid hormone status modulates the expression of PLA2g2a and other sPLA2 isoforms. Both hyperthyroid rats and mice had significantly lower levels of PLA2g2a as compared to hypothyroid rats and mice. My data suggest that the thyroid status modulates aspects of the inflammatory response. In contrast with the established role of T3 to recruit coactivators to TRβ, my experiments demonstrate a novel inverse recruitment mechanism in which unliganded TRβ is stimulatory while liganded TRβ recruits corepressors to inhibit PLA2g2a expression. In conclusion, present study provides a cellular mechanism by which T3 inhibits PLA2g2a expression.

DOI

10.21007/etd.cghs.2013.0286

Comments

One year embargo expired December 2014

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