Respiratory Uncoupling Induces δ-Aminolevulinate Synthase Expression through a Nuclear Respiratory Factor-1-dependent Mechanism in HeLa Cells

John O Holloszy,Clay F Semenkovich,Bing Li

doi:10.1074/jbc.274.25.17534

John O Holloszy, Clay F Semenkovich + Show 1 more

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https://doi.org/10.1074/jbc.274.25.17534

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Abstract

Nuclear respiratory factor (NRF)-1 appears to be important for the expression of several respiratory genes, but there is no direct evidence that NRF-1 transduces a physiological signal into the production of an enzyme critical for mitochondrial biogenesis. We generated HeLa cells containing plasmids allowing doxycycline-inducible expression of uncoupling protein (UCP)-1. In the absence of doxycycline, UCP-1 mRNA and protein were undetectable. In the presence of doxycycline, UCP-1 was expressed and oxygen consumption doubled. This rise in oxygen consumption was associated with an increase in NRF-1 mRNA. It was also associated with an increase in NRF-1 protein binding activity as determined by electrophoretic mobility shift assay using a functional NRF-1 binding site from the delta-aminolevulinate (ALA) synthase promoter. Respiratory uncoupling also caused a time-dependent increase in protein levels of ALA synthase, an early marker for mitochondrial biogenesis. ALA synthase induction by respiratory uncoupling was prevented by transfecting cells with an oligonucleotide antisense to the region of the NRF-1 initiation codon; a scrambled oligonucleotide with the same base composition had no effect. Respiratory uncoupling increases oxygen consumption and lowers energy reserves. In HeLa cells, uncoupling also increases ALA synthase, an enzyme critical for mitochondrial respiration, but only if translatable mRNA for NRF-1 is available. These data suggest that the transcription factor NRF-1 plays a key role in cellular adaptation to energy demands by translating physiological signals into an increased capacity for generating energy.

Highlights

Tissues adapt to an increased need for energy by increasing mitochondria, resulting in an enhanced capacity to generate ATP by oxidative phosphorylation
HeLa cells were isolated that contained both pTet-On (Fig. 1A), directing constitutive expression of a transcriptional activator that binds to a tetracycline-responsive element (TRE) in the presence of doxycycline, and pUCP-1 (Fig. 1B), which contains a TRE
In the absence of doxycycline, control human ␤-actin message was detected in pTet-On/pUCP-1 HeLa cells, but there was no expression of uncoupling protein (UCP)-1 mRNA in multiplex RT-PCR assays of total RNA (Fig. 2A, ϪDox)

Summary

Introduction

Tissues adapt to an increased need for energy by increasing mitochondria, resulting in an enhanced capacity to generate ATP by oxidative phosphorylation. Functional binding sites for NRF-1 were subsequently described in several nuclear genes critical for mitochondrial biogenesis, including ALA synthase [9] and human mitochondrial transcription factor A (TFAM) [10]. HeLa cells, the source for the initial purification of the NRF-1 protein [12], were engineered to inducibly express uncoupling protein (UCP)-1, and ALA synthase protein was assayed as a marker for stimulation of the assembly of mitochondrial respiratory complexes.

Results

Conclusion