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Inhibition of mTOR Prevents ROS Production Initiated by Ethidium Bromide-Induced Mitochondrial DNA Depletion.

Nacarelli T, Azar A, Sell C - Front Endocrinol (Lausanne) (2014)

Bottom Line: Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA.These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate.Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Drexel University College of Medicine , Philadelphia, PA , USA.

ABSTRACT
The regulation of mitochondrial mass and DNA content involves a complex interaction between mitochondrial DNA replication machinery, functional components of the electron transport chain, selective clearance of mitochondria, and nuclear gene expression. In order to gain insight into cellular responses to mitochondrial stress, we treated human diploid fibroblasts with ethidium bromide at concentrations that induced loss of mitochondrial DNA over a period of 7 days. The decrease in mitochondrial DNA was accompanied by a reduction in steady state levels of the mitochondrial DNA binding protein, TFAM, a reduction in several electron transport chain protein levels, increased mitochondrial and total cellular ROS, and activation of p38 MAPK. However, there was an increase in mitochondrial mass and voltage dependent anion channel levels. In addition, mechanistic target of rapamycin (mTOR) activity, as judged by p70S6K targets, was decreased while steady state levels of p62/SQSTM1 and Parkin were increased. Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA. These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate. Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.

No MeSH data available.


Related in: MedlinePlus

Steady state levels of mitochondrial proteins following ethidium bromide exposure. Human fibroblast cell cultures grown in the presence or absence of 1 nM rapamycin were exposed to increasing doses of ethidium bromide for a period of 7 days. At this time, total cellular protein extracts were prepared and analyzed by immunoblot as described in Section “Materials and Methods” for a subset of mitochondrial resident proteins. The levels of beta-actin are presented as a control for equal protein loading. The immunoblot presented is a representative blot of a minimum of two measurements with similar results.
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Figure 5: Steady state levels of mitochondrial proteins following ethidium bromide exposure. Human fibroblast cell cultures grown in the presence or absence of 1 nM rapamycin were exposed to increasing doses of ethidium bromide for a period of 7 days. At this time, total cellular protein extracts were prepared and analyzed by immunoblot as described in Section “Materials and Methods” for a subset of mitochondrial resident proteins. The levels of beta-actin are presented as a control for equal protein loading. The immunoblot presented is a representative blot of a minimum of two measurements with similar results.

Mentions: In order to gain further insight into the changes in mitochondrial mass following ethidium bromide exposure, we examined the steady state levels of several resident mitochondrial proteins. Consistent with the reduction in mitochondrial DNA content, the steady state levels of the mitochondrial transcription factor (TFAM) decreased in both control and rapamycin treated HDFs upon exposure to ethidium bromide (Figure 5). In contrast, the steady state levels of the VDAC increased in control HDF cultures upon exposure to ethidium bromide (Figure 5). The baseline levels of VDAC were lower than controls in rapamycin treated cultures but these levels did not change upon exposure to ethidium bromide (Figure 5).


Inhibition of mTOR Prevents ROS Production Initiated by Ethidium Bromide-Induced Mitochondrial DNA Depletion.

Nacarelli T, Azar A, Sell C - Front Endocrinol (Lausanne) (2014)

Steady state levels of mitochondrial proteins following ethidium bromide exposure. Human fibroblast cell cultures grown in the presence or absence of 1 nM rapamycin were exposed to increasing doses of ethidium bromide for a period of 7 days. At this time, total cellular protein extracts were prepared and analyzed by immunoblot as described in Section “Materials and Methods” for a subset of mitochondrial resident proteins. The levels of beta-actin are presented as a control for equal protein loading. The immunoblot presented is a representative blot of a minimum of two measurements with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109433&req=5

Figure 5: Steady state levels of mitochondrial proteins following ethidium bromide exposure. Human fibroblast cell cultures grown in the presence or absence of 1 nM rapamycin were exposed to increasing doses of ethidium bromide for a period of 7 days. At this time, total cellular protein extracts were prepared and analyzed by immunoblot as described in Section “Materials and Methods” for a subset of mitochondrial resident proteins. The levels of beta-actin are presented as a control for equal protein loading. The immunoblot presented is a representative blot of a minimum of two measurements with similar results.
Mentions: In order to gain further insight into the changes in mitochondrial mass following ethidium bromide exposure, we examined the steady state levels of several resident mitochondrial proteins. Consistent with the reduction in mitochondrial DNA content, the steady state levels of the mitochondrial transcription factor (TFAM) decreased in both control and rapamycin treated HDFs upon exposure to ethidium bromide (Figure 5). In contrast, the steady state levels of the VDAC increased in control HDF cultures upon exposure to ethidium bromide (Figure 5). The baseline levels of VDAC were lower than controls in rapamycin treated cultures but these levels did not change upon exposure to ethidium bromide (Figure 5).

Bottom Line: Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA.These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate.Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Drexel University College of Medicine , Philadelphia, PA , USA.

ABSTRACT
The regulation of mitochondrial mass and DNA content involves a complex interaction between mitochondrial DNA replication machinery, functional components of the electron transport chain, selective clearance of mitochondria, and nuclear gene expression. In order to gain insight into cellular responses to mitochondrial stress, we treated human diploid fibroblasts with ethidium bromide at concentrations that induced loss of mitochondrial DNA over a period of 7 days. The decrease in mitochondrial DNA was accompanied by a reduction in steady state levels of the mitochondrial DNA binding protein, TFAM, a reduction in several electron transport chain protein levels, increased mitochondrial and total cellular ROS, and activation of p38 MAPK. However, there was an increase in mitochondrial mass and voltage dependent anion channel levels. In addition, mechanistic target of rapamycin (mTOR) activity, as judged by p70S6K targets, was decreased while steady state levels of p62/SQSTM1 and Parkin were increased. Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA. These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate. Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.

No MeSH data available.


Related in: MedlinePlus