Nuclear respiratory factor 1: a novel inhibitor of the human gene promoter of the cardiac isoform of acetyl-coenzyme a carboxylase

Introduction: Nuclear respiratory factor-1 (NRF-1) is a pivotal transcriptional modulator controlling the expression of nuclear genes encoding mitochondrial proteins. Activation of 5'-AMP-activated protein kinase (AMPK) has been associated with enhanced NRF-1 gene promoter binding activity. Furthermore, NRF-1 has been linked with increased gene expression of carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting mitochondrial fatty acid (FA) transfer enzyme. Upstream, the cardiac-enriched isoform of acetyl-CoA carboxylase (ACCl3) synthesises malonyl-CoA, a potent inhibitor of CPT-1 and FA 13-oxidation. Since ACCl3 induction elevates malonyl-CoA levels, we hypothesised that AMPK activates NRF-1 thereby inhibiting ACCl3 expression in the heart to ultimately increase FA !3-oxidation. Methods: A 1,317 bp human ACCl3 gene promoter-luciferase construct (pPll13-1317/+65-Luc) was transiently transfected into rat neonatal cardiomyocytes ± the following expression constructs: NRF-1 (pSG5-NRF-1) and dominant negative NRF-1 (pCMV-HA-dnNRF-1). To elucidate whether AMPK is involved in the transcriptional regulation of ACCl3 gene, the transfected cardiomyocytes were treated with AMPK activators, i.e. AICAR or metformin for 24 hours. Transfections were also performed with upstream stimulatory factor 1 (USF1), a known transactivator of the human ACCl3 gene promoter. In addition, the effect of NRF-1 on endogenous USF1 transcriptional activity was evaluated with a luciferase construct containing multiple copies of USF1-specific enhancer elements (pUSF1-Luc). Results: NRF-1 overexpression reduced ACCl3 gene promoter activity by 56 ± 8.8% (p<0.001 vs. pPlll3-1317/+65-Luc). Cotransfection with the dnNRF-1 construct abrogated this effect. Addition of AICAR dose-dependently decreased ACCl3 gene promoter activity (p<0.05 vs. untreated control). In contrast, NRF-1 attenuation of ACCl3 gene promoter activity was not altered by AICAR treatment. Exposure to metformin did not affect the ACCl3 gene promoter activity at baseline, and in response to NRF-1. NRF-1 inhibited the USF1-dependent upregulation of ACCl3 gene promoter activity by 58 ± 7.5% (p<0.001 vs. pPlll3-1317/+65-Luc + USF1), an effect reversed with the dnNRF-1 construct. In addition, NRF-1 reduced endogenous USF1 transcriptional activity by 55 ± 6.2% (p<0.001 vs. pUSF1-Luc). This effect was abolished with the dnNRF-1 construct. The NRF-1-mediated decrease in endogenous USF1 transcriptional activity was independent of AMPK activation with AICAR. The ACC~ mRNA expression level was unaffected at baseline, and by NRF-1 overexpression. However, AICAR (2 mM) treatment markedly reduced the ACC~ mRNA expression level (p<0.05 vs. vehicle) at baseline, but had no effect in response to NRF-1 overexpression. Conclusion: Our data reveal a unique, inhibitory role for NRF-1 in the transcriptional regulation of human ACC~ gene in the mammalian heart, independent of AMPK activation.

Grantor dc:publisher.institution

Department of Medicine

Year dc:date.issued

2008

Author dc:creator

• Adam, Tasneem

Advisor dc:contributor.advisor

• Hatter Cardiovascular, Essop, M F

• cardiac isoform

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/11427/41985

OAI identifier oai:identifier

oai:open.uct.ac.za:11427/41985