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VEGF stimulation of mitochondrial biogenesis: requirement of AKT3 kinase - PubMed

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VEGF stimulation of mitochondrial biogenesis: requirement of AKT3 kinase

Gary L Wright et al. FASEB J. 2008 Sep.

Abstract

The growth factor, vascular endothelial growth factor (VEGF), induces angiogenesis and promotes endothelial cell (EC) proliferation. Affymetrix gene array analyses show that VEGF stimulates the expression of a cluster of nuclear-encoded mitochondrial genes, suggesting a role for VEGF in the regulation of mitochondrial biogenesis. We show that the serine threonine kinase Akt3 specifically links VEGF to mitochondrial biogenesis. A direct comparison of Akt1 vs. Akt3 gene silencing was performed in ECs and has uncovered a discrete role for Akt3 in the control of mitochondrial biogenesis. Silencing of Akt3, but not Akt1, results in a decrease in mitochondrial gene expression and mtDNA content. Nuclear-encoded mitochondrial gene transcripts are also found to decrease when Akt3 expression is silenced. Concurrent with these changes in mitochondrial gene expression, lower O(2) consumption was observed. VEGF stimulation of the major mitochondrial import protein TOM70 is also blocked by Akt3 inhibition. In support of a role for Akt3 in the regulation of mitochondrial biogenesis, Akt3 silencing results in the cytoplasmic accumulation of the master regulator of mitochondrial biogenesis, PGC-1alpha, and a reduction in known PGC-1alpha target genes. Finally, a subtle but significant, abnormal mitochondrial phenotype is observed in the brain tissue of AKT3 knockout mice. These results suggest that Akt3 is important in coordinating mitochondrial biogenesis with growth factor-induced increases in cellular energy demands.

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Figures

Figure 1.
Figure 1.

VEGF stimulates a cellular program for mitochondrial biogenesis. Real-time RT-PCR analysis of gene expression from total RNA isolated from ECs either serum starved or treated with VEGF (20 ng/ml) for 6 h using primers listed in Table 1. Fold changes in gene expression of six nuclear encoded mitochondrial genes as compared to 18S rRNA expression as an internal control are shown. All real-time PCR was performed in triplicate.

Figure 2.
Figure 2.

Silencing of Akt3 inhibits mitochondrial gene expression. A) Western blot analysis of Akt1 and Akt3 expression, resulting from transfection of two different amounts of isoform-specific RNAi. B) Western blot analysis of total Akt3 protein levels in cells transfected with scrambled control (C) or Akt1 RNAi. The p85 PI3 kinase subunit is shown as a loading control. C) RT-PCR of total RNA isolated from ECs transfected with RNAi against scrambled control (SCR), Akt1 (1), or Akt3 (3), using primers directed against the mitochondrial encoded genes, COXI, COXII, and NADH dehydrogenase. The ribosomal subunit RNA S26 is shown as an internal control. All analyses were performed at least 3 independent times.

Figure 3.
Figure 3.

Akt3 is required for maintenance of mtDNA copy number. A) Southern blot analysis of total genomic DNA isolated from ECs transfected with either Akt1 or Akt3 RNAi and probed using COXII, a gene encoded by the mitochondria. Two independent transfections are shown. B) Real-time PCR of total genomic DNA isolated from ECs transfected using either control scrambled RNAi or RNAi directed against Akt1 (Akt1-) or Akt3 (Akt3-) using primers directed against COXII and the ribosomal subunit RNA S26 as an internal control. The graph represents the change in DNA copy number as a percentage of control. All real-time analyses were performed in triplicate.

Figure 4.
Figure 4.

Silencing of Akt3 results in a decreased QO2 and nuclear mitochondrial gene expression. A) Respiration of mitochondria in living cells was measured using a Clark-type O2 electrode in an oxygen consumption chamber. The graph represents nanomoles of O2 consumed per minute relative to total cell number, as assessed by quantitation of total protein. *P > 0.05. B) Left panels: fluorescent images of ECs cotransfected with control RNAi or Akt3 RNAi plus a GFP chimera containing a mitochondrial import sequence to specifically label mitochondria. Right panels: images of cells transfected with control or Akt3 RNAi followed by staining with rhodamine phallodin and Topro to visualize actin filaments and nuclei, respectively. C) RT-PCR analysis of Akt3 and TOM70 expression in cells transfected with scrambled control (SCR) or Akt3 RNAi and either serum starved or treated with VEGF (-V) (20 ng/ml) for 6 h.

Figure 5.
Figure 5.

Blockade of Akt3 expression results in exclusion of PGC-1α from the nucleus. A) Western blot analysis of total protein from cells serum starved (SS) then stimulated with VEGF for the times indicated and probed for PGC-1α expression. PI3K p85 subunit is shown as a loading control. B) Western blot analysis of PGC-1α expression in cells transfected with scrambled (SCR) or Akt3 RNAi. E2F is shown as a loading control. C) Confocal images of cells cotransfected with a GFP-PGC-1α expression construct and SCR or Akt3 RNAi. D) Confocal immunofluorescent images of cells cotransfected with a myc-PGC-1α expression construct and SCR or Akt3 RNAi. E) RT-PCR of total RNA isolated from ECs transfected with RNAi against either SCR or Akt3, using primers directed against the nuclear encoded mitochondrial genes NRF-1, MCAD, and PDβ. The ribosomal subunit RNA S26 is shown as an internal control.

Figure 6.
Figure 6.

Akt3-KO animals display fewer and larger mitochondria. Representative electron micrographs of brain sections in wild-type and Akt3-KO animals showing enlarged and fewer mitochondria.

Figure 7.
Figure 7.

Model of VEGF stimulation of mitochondrial biogenesis. VEGF stimulation results in the activation of Akt3, which controls the nuclear localization of the master regulator of mitochondrial biogenesis, PGC-1α, resulting in the increased expression of nuclear encoded mitochondrial gene expression.

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