Reactive Oxygen Species-generating Mitochondrial DNA Mutation Up-regulates Hypoxia-inducible Factor-1α Gene Transcription via Phosphatidylinositol 3-Kinase-Akt/Protein Kinase C/Histone Deacetylase Pathway

Nobuko Koshikawa,Jun-Ichi Hayashi,Akira Nakagawara,Keizo Takenaga

doi:10.1074/jbc.m109.054221

Nobuko Koshikawa, Jun-Ichi Hayashi + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.054221

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Abstract

Lewis lung carcinoma-derived high metastatic A11 cells constitutively overexpress hypoxia-inducible factor (HIF)-1alpha mRNA compared with low metastatic P29 cells. Because A11 cells exclusively possess a G13997A mutation in the mitochondrial NADH dehydrogenase subunit 6 (ND6) gene, we addressed here a causal relationship between the ND6 mutation and the activation of HIF-1alpha transcription, and we investigated the potential mechanism. Using trans-mitochondrial cybrids between A11 and P29 cells, we found that the ND6 mutation was directly involved in HIF-1alpha mRNA overexpression. Stimulation of HIF-1alpha transcription by the ND6 mutation was mediated by overproduction of reactive oxygen species (ROS) and subsequent activation of phosphatidylinositol 3-kinase (PI3K)-Akt and protein kinase C (PKC) signaling pathways. The up-regulation of HIF-1alpha transcription was abolished by mithramycin A, an Sp1 inhibitor, but luciferase reporter and chromatin immunoprecipitation assays indicated that Sp1 was necessary but not sufficient for HIF-1alpha mRNA overexpression in A11 cells. On the other hand, trichostatin A, a histone deacetylase (HDAC) inhibitor, markedly suppressed HIF-1alpha transcription in A11 cells. In accordance with this, HDAC activity was high in A11 cells but low in P29 cells and in A11 cells treated with the ROS scavenger ebselene, the PI3K inhibitor LY294002, and the PKC inhibitor Ro31-8220. These results suggest that the ROS-generating ND6 mutation increases HIF-1alpha transcription via the PI3K-Akt/PKC/HDAC pathway, leading to HIF-1alpha protein accumulation in hypoxic tumor cells.

Highlights

Hypoxia is a common characteristic of locally advanced solid tumors
The results showed that D6 and A11 cells expressed a larger amount of hypoxia-inducible factor (HIF)-1␣ mRNA than P29 and P34 cells (Fig. 1A)
The results showed that the half-life of HIF-1␣ mRNA in A11 cells was nearly equal to that in P29 cells (ϳ8 h) (Fig. 1C)

Summary

Introduction

Hypoxia is a common characteristic of locally advanced solid tumors. Hypoxic tumor cells activate many genes, including those related to cell survival, glycolysis, and angiogenesis, and invasion and metastasis to adapt to and escape from the microenvironment [5, 6]. We report here that certain ROS-generating mtDNA mutations can stimulate HIF-1␣ transcription via the phosphatidylinositol 3-kinase (PI3K)/protein kinase C (PKC)/ histone deacetylase (HDAC) pathway. To examine a causal relationship between the ND6 mutation and HIF-1␣ transcription, we examined HIF-1␣ mRNA levels in trans-mitochondrial cybrids, P29mtA11 and A11mP29 cells, which carry mtDNA from A11 and P29 cells and nuclear DNA from P29 and A11 cells, respectively.

Results

Conclusion