Ramy Naguib Attia
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Document Type: |
Doctoral Dissertation |
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Name: |
Ramy Naguib Attia |
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Email Address: |
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Title: |
Regulation of Pyruvate Dehydrogenase Kinase 4 by Thyroid Hormone: Role of Peroxisome Proliferator Activated Receptor Gamma Coactivator-1 Alpha and CCAAT Enhancer Binding Protein. |
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Degree: |
Doctor of Philosophy |
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Program: |
Integrated Program in Biomedical Sciences |
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Track, if in
Integrated Program in Biomedical Sciences |
Cell Biology and Biochemistry |
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Research Advisor: |
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Advisor's Email: |
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Committee Members: |
George A. Cook, Ph.D. |
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John V. Cox, Ph.D. |
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Marshall B. Elam, M.D., Ph.D., FAHA |
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Dale Parker Suttle, Jr., Ph.D. |
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Keywords: |
C/EBP beta, PGC-1 alpha, Pyruvate dehydrogenase kinase 4, Thyroid hormone |
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Availability: |
World-Wide Web Access |
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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/EBP§) 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/EBP§ was identified as an important accessory
factor in the T3 induction of the PDK4 gene. Knock-down of C/EBP§ 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.
Attached File(s)
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Revised 31 March 2009