Distinct nuclear gene expression profiles in cells with mtDNA depletion and homoplasmic A3243G mutation

https://doi.org/10.1016/j.mrfmmm.2005.02.002Get rights and content

Abstract

The pathobiochemical pathways determining the wide variability in phenotypic expression of mitochondrial DNA (mtDNA) mutations are not well understood. Most pathogenic mtDNA mutations induce a general defect in mitochondrial respiration and thereby ATP synthesis. Yet phenotypic expression of the different mtDNA mutations shows large variations that are difficult to reconcile with ATP depletion as sole pathogenic factor, implying that additional mechanisms contribute to the phenotype. Here, we use DNA microarrays to identify changes in nuclear gene expression resulting from the presence of the A3243G diabetogenic mutation and from a depletion of mtDNA (ρ0 cells). We find that cells respond mildly to these mitochondrial states with both general and specific changes in nuclear gene expression. This observation indicates that cells can sense the status of mtDNA. A number of genes show divergence in expression in ρ0 cells compared to cells with the A3243G mutation, such as genes involved in oxidative phosphorylation. As a common response in A3243G and ρ0 cells, mRNA levels for extracellular matrix genes are up-regulated, while the mRNA levels of genes involved in ubiquitin-mediated protein degradation and in ribosomal protein synthesis is down-regulated. This reduced expression is reflected at the level of cytosolic protein synthesis in both A3243G and ρ0 cells.

Our finding that mitochondrial dysfunction caused by different mutations affects nuclear gene expression in partially distinct ways suggests that multiple pathways link mitochondrial function to nuclear gene expression and contribute to the development of the different phenotypes in mitochondrial disease.

Introduction

Mitochondrial dysfunction caused by mutations in the mitochondrial genome is related to a variety of diseases such as type two diabetes mellitus (DM2), cancer and neuro-muscular diseases [1]. Mitochondria are involved in multiple cellular processes of which ATP production by oxidative phosphorylation (OXPHOS) is the most prominent one. However, mitochondria also accommodate other processes, such as the tricarboxylic acid cycle and fatty acid oxidation. In addition, mitochondrial function is linked to calcium, iron and ROS signalling and apoptotic pathways. The variation in clinical phenotype of mitochondrial diseases is difficult to explain by merely a reduced respiration rate [2]. Rather the consequences of additional mitochondrial dysfunction on retrograde signalling pathways may determine the distinct nature of the clinical manifestation [3]. A genome-wide differential gene transcription profile of normal cells and cells with dysfunctioning mitochondria is expected to give insight in the pathobiochemical pathways affected in mitochondrial disease [4], [5].

In order to investigate how mitochondrial mutations affect the nuclear gene expression profile we created 143B cybrid cells with mitochondrial DNA being the only variable [6]. The first state of respiratory dysfunction is induced by an A–G conversion at location 3243 in the tRNAleu gene of the mitochondrial DNA. This mutation causes maternally inherited diabetes and deafness (MIDD) [7] in most carriers and associates also with the neuromuscular MELAS syndrome [8]. Another state of mitochondrial dysfunction is induced by a depletion of mtDNA (ρ0 cells). Using cybrid cells with 100% wild-type mitochondrial DNA, cybrid cells with 100% A3243G mutant mitochondrial DNA of the same haplotype and ρ0 cells, we found both common mitochondrial-defect and MIDD-specific responses in nuclear gene expression.

Section snippets

Cell culture, cell characteristics and GeneChip hybridisation

The cybrid cells used in this report have been previously described [2], [9]. In short, 143B osteosarcoma cells were treated with ethidium bromide to create ρ0 cells devoid of mitochondrial DNA. Next, ρ0 cells were fused with enucleated cells from a MIDD patient, generating clones with different but stable heteroplasmy levels for the 3243 mutation. Two apparently homoplasmic mutants (VM48 and VM50) and two apparently homoplasmic wild-type (VW6 and VW7) cybrid clones were selected and used as

Cell characteristics and quality controls

The cybrid cell lines selected for gene expression profiling were comparable to each other with respect to cell morphology, doubling time (range 13–18 h) and cell viability (∼4% trypan blue stained cells). The average oxygen consumption of the wild-type cybrids was 1.3 ± 0.3 fmol/(min cell) (n = 10), while this value for the 100% 3243 mutant cybrids was 0.1 ± 0.1 fmol/(min cell) (n = 5). ρ0 cells did not respire at all. Mitochondrial copy numbers of the wild type and the 3243 mutant cybrids were comparable

Discussion

With the aim to elucidate pathways involved in mitochondrial-nuclear genome cross-talk, we have undertaken a genome-wide analysis of the alterations in nuclear gene expression in response to two different types of mtDNA mutations that both provoke mitochondrial dysfunction such as loss of mtDNA encoded proteins and respiration [8], [19]. The transmitochondrial fusion technique developed by King and Attardi [6] permitted the generation of cybrid cell lines with the mtDNA genome being the only

Acknowledgements

We thank Marchien van de Sande for her excellent overall technical assistance. The microarray hybridisations were performed by Eveline Mank at the Leiden Genome Technology Center.

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