Mitochondrial Transcription Factor A Induction by Redox Activation of Nuclear Respiratory Factor 1

Claude A Piantadosi,Hagir B Suliman

doi:10.1074/jbc.m508805200

Abstract

The nuclear expression of mitochondrial transcription factor A (Tfam), which is required for mitochondrial DNA (mtDNA) transcription and replication, must be linked to cellular energy needs. Because respiration generates reactive oxygen species as a side-product, we tested the idea that reactive oxygen species regulate Tfam expression through phosphorylation of nuclear respiratory factor (NRF-1) and binding to the Tfam promoter. In mitochondria-rich rat hepatoma cells that overexpress NRF-1, basal and oxidant-induced increases were found in Tfam expression and mtDNA content. Specific binding of NRF-1 to Tfam promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. NRF-1-Tfam binding was augmented under pro-oxidant conditions. NRF-1 gene silencing produced 1:1 knockdown of Tfam expression and decreased mtDNA content. To evaluate oxidation-reduction (redox) regulation of NRF-1 in Tfam expression, blockade of upstream phosphatidylinositol 3-kinase was used to demonstrate loss of oxidant stimulation of NRF-1 phosphorylation and Tfam expression. The oxidant response was also abrogated by specific inhibition of Akt/protein kinase B. Examination of the NRF-1 amino acid sequence revealed an Akt phosphorylation consensus at which site-directed mutagenesis abolished NRF-1 phosphorylation by Akt. Finally, Akt phosphorylation and NRF-1 translocation predictably lacked oxidant regulation in a cancer line having no PTEN tumor suppressor (HCC1937 cells). This study discloses novel redox regulation of NRF-1 phosphorylation and nuclear translocation by phosphatidylinositol 3,4,5-triphosphate kinase/Akt signaling in controlling Tfam induction by an anti-oxidant pro-survival network.

Highlights

Tfam, like other high mobility group-domain proteins, binds, unwinds, and bends DNA without respect to sequence specificity [4]
Communication between mitochondria and the nucleus is essential to energy homeostasis, and close coordination is especially important because the electron transport chain produces reactive oxygen species (ROS) [19], which are damaging to mitochondrial DNA (mtDNA) and loss of oxidative phosphorylation proteins
Tfam transcript analysis indicated a 5-fold higher expression level in hepatoma relative to normal hepatocytes (Fig. 1A). On this basis it was anticipated that transcription factors involved in Tfam gene expression would be up-regulated in rat hepatoma cells

Summary

Introduction

Like other high mobility group-domain proteins, binds, unwinds, and bends DNA without respect to sequence specificity [4]. To investigate whether ROS participate in retrograde signaling from mitochondria to nucleus, we tested the hypothesis in rat cells that peroxide regulates Tfam expression through NRF-1 phosphorylation and Tfam promoter binding to service mtDNA maintenance. Nuclear protein was incubated with 32P-labeled NRF-1 recognition site from rat Tfam gene promoter (Ϫ205/Ϫ185) (lanes 1 and 2).

Results

Conclusion