Elucidating the Role of Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII ) in Skeletal Muscle: Implications for Metabolism

Lisa Crowther (2010). Elucidating the Role of Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII ) in Skeletal Muscle: Implications for Metabolism PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Lisa Crowther
Thesis Title Elucidating the Role of Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII ) in Skeletal Muscle: Implications for Metabolism
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2010-12
Thesis type PhD Thesis
Supervisor George Muscat
Total pages 246
Total colour pages 16
Total black and white pages 230
Subjects 06 Biological Sciences
Abstract/Summary Nuclear hormone receptors (NRs) are ligand activated DNA binding proteins that regulate genes involved in growth and differentiation, reproduction, morphogenesis and metabolism. Orphan nuclear receptors are gene products that were identified as belonging to the NR superfamily on the basis of sequence identity, but the endogenous molecules regulating their activity are unknown. These proteins have been denoted orphan NRs, and represent promising pharmacological targets, due to the possibility of identifying small molecules which could act as ligands of orphan nuclear receptors, and control functions involved in disease. The chicken ovalbumin upstream promoter transcription factors I and II (COUP-TFI and II) are orphan members of the nuclear receptor superfamily that control biological processes such as organogenesis and neurogenesis and are implicated in homeostasis. They also modulate the activity of genes with fundamental roles in metabolic pathways, such as bile acid synthesis (1), ketogenesis (2), cholesterol transport (3,4), fatty acid β-oxidation (5) and glucose homeostasis and insulin sensitivity (6). Skeletal muscle is a major mass peripheral tissue that accounts for ~40% of the total body mass and of energy expenditure, and is a major site of fatty acid and glucose oxidation. Accordingly, skeletal muscle plays a considerable part in the progression of dyslipidemia, diabetes and obesity, major risk factors for cardiovascular disease. While the role of COUP-TFII in regulation of metabolic genes in liver and adipose tissue has been studied extensively in vitro, its role in skeletal muscle metabolism has been less well characterised. A previous study undertaken in this laboratory indicated a pivotal role for COUP-TFs in the regulation of lipid and energy homeostasis in skeletal muscle cells, however both COUP-TFI and COUP-TFII were attenuated and it was of interest to elucidate the involvement of COUP-TFI and/or COUP-TFII in these effects and determine gene targets specific to COUP-TFI and/or COUP-TFII. In the current study, skeletal muscle specific and ubiquitously expressed COUP-TFI and COUP-TFII gain and loss of function expression vectors were constructed, and expression and activity of the constructs in cell culture were tested. Constructs were generated to express VP16-COUP-TFII, full length native COUP-TFII, and truncated COUP-TFII lacking the helix 12 region (COUP-TFIIΔH12) driven by both the SV40 promoter for ubiquitous expression, as well as vectors driven by the human skeletal muscle alpha actin (HSA) promoter, for skeletal muscle specific expression. COUP-TFII was demonstrated to activate the an MCAD heterologous reporter vector, and COUP-TFIIΔH12 repressed this activation. Stable C2C12 cell lines expressing COUP-TFII and COUP-TFIIΔH12 were generated to examine the roles of COUP-TFII in skeletal muscle cells in vitro. COUP-TFII mRNA and protein was observed to be downregulated following differentiation of myoblasts to myotubes, however stable expression of ectopic COUP-TFII and COUP-TFIIΔH12 in C2C12 cells led to modulation of metabolic, myogenic and nuclear receptor gene expression including genes involved in glucose homeostasis, and several genes previously shown to be modulated in skeletal muscle following exercise were also regulated in these cells. ChIP analysis and promoter assays demonstrated binding and activation of the promoter of Glut4, one of the target genes identified, through an SP1 site. In light of previous research demonstrating involvement of COUP-TFII in the LXR mediated induction of SREBP-1c in skeletal muscle, cross-talk between COUP-TFII and LXR was analysed in vitro. Expression of LXRα, LXRβ, COUP-TFII and COUP-TFI mRNA in C2C12 myotubes remained unchanged following LXR agonist treatment, while SREBP-1c expression increased. Reporter assays demonstrated that COUP-TFII activated a nuclear response element of the SREBP-1c promoter, encompassing a putative COUP binding site. ChIP analysis demonstrated COUP-TFII recruitment to this putative COUP binding site after T0901317 treatment, and LXR was also recruited to this region as well as the previously characterised downstream LXRE region. Furthermore, co-immunoprecipitation demonstrated interaction between COUP-TFII and LXRs in C2C12 cells with and without T0901317 treatment. These findings further characterised cross-talk between LXR and COUP-TFII in skeletal muscle. In order to investigate the role of COUP-TFII in skeletal muscle in vivo, we aimed to produce mice with skeletal muscle specific expression of the truncated dominant negative mutant COUP-TFIIΔH12. Two founder transgenic mice were identified with the pHSA-COUP-TFIIΔH12 transgene integrated into the genome. However, analysis of transgenic offspring of these mice demonstrated that mRNA expression of the ectopic COUP-TFIIΔH12 was very low in skeletal muscle compared to the endogenous COUP-TFII mRNA expression, and these mice were not suitable for further analysis. The pHSA-COUP-TFII transgene was then used for pronuclear injection and one founder was identified at the end of my candidature. Transgenic offspring from this founder analysed by another member of the laboratory also had no increased mRNA or protein expression of COUP-TFII, despite integration of the transgene. These observations, as well as observation in vitro that expression of ectopic COUP-TFII in myoblasts was downregulated following differentiation, indicated that total levels of COUP-TFII are tightly regulated in differentiated skeletal muscle cells in vitro and in vivo.
Keyword Coup-tf
Nuclear Receptors
Skeletal Muscle
Additional Notes 35-37, 39-41, 55, 106, 134, 157, 178, 203-206, 224.

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Created: Mon, 25 Jul 2011, 10:06:12 EST by Miss Lisa Crowther on behalf of Library - Information Access Service