Date of Award

5-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cell Biology and Biochemistry

Research Advisor

Edwards A. Park, Ph.D.

Committee

George A. Cook, Ph.D. John V. Cox, Ph.D. Marshall B. Elam, M.D., Ph.D., FAHA Dale Parker Suttle, Jr., Ph.D.

Abstract

Pyruvate dehydrogenase kinase 4 (PDK4) regulates pyruvate oxidation through the phosphorylation and inhibition of the pyruvate dehydrogenase complex (PDC). The PDC catalyzes the conversion of pyruvate to acetyl-CoA and it is an important control point in glucose and pyruvate metabolism. Previous studies had reported that PDK4 gene expression is induced by thyroid hormone (T3). These studies did not investigate the mechanisms by which T3 regulated PDK4 gene expression. I have examined the role of the thyroid hormone receptor (TR), transcriptional coactivators especially the peroxisome proliferator activated receptor gamma coactivator-1 (PGC-1α) and other transcription factors that act as accessory factors in T3 actions.

Thyroid hormone receptors (TRα or TRβ) are part of the nuclear receptor family and have been implicated in the induction of many genes by T3. The nuclear receptors constitute a broad class of transcription factors that have the ability to bind to DNA and regulate the expression of the genes in a ligand dependent manner. To identify a binding site for the TRβ in the promoter of the PDK4 gene, I transfected serial deletions of this promoter ligated to the luciferase reporter gene into the human hepatoma (HepG2) cells. The TRβ binding site was characterized by gel shift mobility assays, site directed mutagenesis and in vivo and in vitro luciferase constructs transfection techniques. In addition, I have used the chromatin immunoprecipitation (ChIP) technique to confirm the binding of the TRβ to the PDK4 gene promoter.

Transcription coregulators bind to nuclear receptors to facilitate or inhibit the transcription of the target genes. I have explored the role of several coregulators including the transcriptional coactivator peroxisome proliferator activated receptor gamma coactivator-1 (PGC-1α). Previous studies demonstrated that PGC-1α could induce the PDK4 gene. Here, I found that T3 increases PGC-1α abundance and association with the PDK4 gene. In addition, adenoviral shRNA-mediated knock-down of PGC-1α reduced the T3 induction of PDK4 and several other genes. These data suggest that PGC-1α participates in the T3 induction of multiple genes in the liver. In addition, I investigated the role of several transcription factors including estrogen related receptor (ERRα), CCAAT/enhancer binding protein beta (C/EBPß) and the forkhead transcription factor (FOXO1) in the activation of the PDK4 gene by T3. ERRα and FoxO1 recruit PGC-1α to the proximal PDK4 promoter and are involved in the synergistic stimulation of the PDK4 gene by T3 and PGC-1α. C/EBPß was identified as an important accessory factor in the T3 induction of the PDK4 gene. Knock-down of C/EBPß with adenoviral shRNA decreased both the basal expression and T3 stimulation of PDK4. Overall, my results showed that T3 induces PDK4 gene expression through a TRβ binding site in the promoter of the PDK4 gene and that the two transcription coregulators PGC-1α and C/EBPβ enhance this induction.

Finally, I have tested the ability of T3 to induce a group of transcriptional regulators and metabolic genes. After 24 hours, 27 gens were found to be induced by T3 including several coactivators. These results suggest that T3 may induce a network of transcriptional regulators to further amplify the actions of T3.

Thus, my studies have characterized the mechanism of the activation of an important metabolic gene, PDK4 gene, by T3 and identified the transcription factors that are involved in this mechanism. Also, my studies have provided a broad insight of the hepatic transcription factors network that work coordinately to facilitate the action of the T3 in the liver. This work may open a door to further investigate therapeutic targets in this network to modulate metabolic disease processes that are affected by the thyroid hormone.

DOI

10.21007/etd.cghs.2009.0019

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