Abstract
BackgroundUcp3 is an integral protein of the inner mitochondrial membrane with a role in lipid metabolism preventing deleterious effects of fatty acids in states of high lipid oxidation. Ucp3 is expressed in brown adipose tissue and skeletal muscle and controlled by a transcription factor complex including PPARalpha, MyoD and the histone acetyltransferase p300. Several studies have demonstrated interaction of these factors with chicken ovalbumin upstream promoter transcription factor II (Coup-TFII). This nuclear receptor is involved in organogenesis and other developmental processes including skeletal muscle development, but also co-regulates a number of metabolic genes. In this study we in silico analyzed the upstream region of Ucp3 of the Djungarian hamster Phodopus sungorus and identified several putative response elements for Coup-TFII. We therefore investigated whether Coup-TFII is a further player in the transcriptional control of the Ucp3 gene in rodents.ResultsBy quantitative PCR we demonstrated a positive correlation of Coup-TFII and Ucp3 mRNA expression in skeletal muscle and brown adipose tissue in response to food deprivation and cold exposure, respectively. In reporter gene assays Coup-TFII enhanced transactivation of the Ucp3 promoter conveyed by MyoD, PPARalpha, RXRalpha and/or p300. Using deletions and mutated constructs, we identified a Coup-TFII enhancer element 816–840 bp upstream of the transcriptional start site. Binding of Coup-TFII to this upstream enhancer was confirmed in electrophoretic mobility shift and supershift assays.ConclusionTranscriptional regulation of the Coup-TFII gene in response to starvation and cold exposure seems to be the regulatory mechanism of Ucp3 mRNA expression in brown adipose and skeletal muscle tissue determining the final appropriate rate of transcript synthesis. These findings add a crucial component to the complex transcriptional machinery controlling expression of Ucp3. Given the substantial evidence for a function of Ucp3 in lipid metabolism, Coup-TFII may not only be a negative regulator of glucose responsive genes but also transactivate genes involved in lipid metabolism.
Highlights
Uncoupling protein 3 (Ucp3) is an integral protein of the inner mitochondrial membrane with a role in lipid metabolism preventing deleterious effects of fatty acids in states of high lipid oxidation
Ucp3 introns were amplified with exonic primers based on the known hamster cDNA [GenBank: AF271265] and on the exon 1 sequence obtained as described above
Coup-TFII is a strong activator of Ucp3 gene transcription by binding to an upstream element in the Ucp3 promoter as shown by luciferase assays and electrophoretic mobility shift assays
Summary
Ucp is an integral protein of the inner mitochondrial membrane with a role in lipid metabolism preventing deleterious effects of fatty acids in states of high lipid oxidation. Several studies have demonstrated interaction of these factors with chicken ovalbumin upstream promoter transcription factor II (Coup-TFII) This nuclear receptor is involved in organogenesis and other developmental processes including skeletal muscle development, and co-regulates a number of metabolic genes. Increased levels of circulating free fatty acids (FFA) are common to all these physiological states; infusion experiments imply that these are the primary cause for Ucp upregulation [9]. It has been suggested, though not proven experimentally, that Ucp is a fatty acid anion carrier [10]
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