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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

Stress kinase signaling, p53 levels, and cell viability 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. (A) 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. (B) Cells were examined for viability using a trypan blue exclusion assay. Gray bars indicate control cultures and black bars indicate rapamycin treated cultures. The concentration of ethidium bromide is indicated beneath each bar. Differences between cultures exposed to ethidium bromide and unexposed cultures, which are significant at P < 0.05 are indicated by an asterisk and differences between control and rapamycin treated cultures under identical conditions are indicated by a pound sign.
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Figure 8: Stress kinase signaling, p53 levels, and cell viability 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. (A) 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. (B) Cells were examined for viability using a trypan blue exclusion assay. Gray bars indicate control cultures and black bars indicate rapamycin treated cultures. The concentration of ethidium bromide is indicated beneath each bar. Differences between cultures exposed to ethidium bromide and unexposed cultures, which are significant at P < 0.05 are indicated by an asterisk and differences between control and rapamycin treated cultures under identical conditions are indicated by a pound sign.

Mentions: We examined downstream targets of the mechanistic target of rapamycin (mTOR) pathway, and found the serine phosphorylation of S6 ribosomal protein, the insulin receptor substrate 1 (IRS-1), and Akt were all reduced following exposure to ethidium bromide consistent with reduced mTOR activity (Figure 7). In addition, the phosphorylation of the p38 MAPK stress kinase was increased by exposure to ethidium bromide as was the phosphorylation of the p38MAPK downstream target, heat shock protein 27 (hsp27) (Figure 8A). Levels of p53 protein were increased in cells exposed to ethidium bromide (Figure 8A) while neither the phosphorylation of 38, hsp27, nor the increase in p53 occurred if cells were treated with rapamycin (Figure 8). Treatment of cells with rapamycin also significantly increased viability at 7 days of exposure to ethidium bromide at the highest concentrations used in our studies (Figure 8B).


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

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

Stress kinase signaling, p53 levels, and cell viability 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. (A) 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. (B) Cells were examined for viability using a trypan blue exclusion assay. Gray bars indicate control cultures and black bars indicate rapamycin treated cultures. The concentration of ethidium bromide is indicated beneath each bar. Differences between cultures exposed to ethidium bromide and unexposed cultures, which are significant at P < 0.05 are indicated by an asterisk and differences between control and rapamycin treated cultures under identical conditions are indicated by a pound sign.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 8: Stress kinase signaling, p53 levels, and cell viability 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. (A) 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. (B) Cells were examined for viability using a trypan blue exclusion assay. Gray bars indicate control cultures and black bars indicate rapamycin treated cultures. The concentration of ethidium bromide is indicated beneath each bar. Differences between cultures exposed to ethidium bromide and unexposed cultures, which are significant at P < 0.05 are indicated by an asterisk and differences between control and rapamycin treated cultures under identical conditions are indicated by a pound sign.
Mentions: We examined downstream targets of the mechanistic target of rapamycin (mTOR) pathway, and found the serine phosphorylation of S6 ribosomal protein, the insulin receptor substrate 1 (IRS-1), and Akt were all reduced following exposure to ethidium bromide consistent with reduced mTOR activity (Figure 7). In addition, the phosphorylation of the p38 MAPK stress kinase was increased by exposure to ethidium bromide as was the phosphorylation of the p38MAPK downstream target, heat shock protein 27 (hsp27) (Figure 8A). Levels of p53 protein were increased in cells exposed to ethidium bromide (Figure 8A) while neither the phosphorylation of 38, hsp27, nor the increase in p53 occurred if cells were treated with rapamycin (Figure 8). Treatment of cells with rapamycin also significantly increased viability at 7 days of exposure to ethidium bromide at the highest concentrations used in our studies (Figure 8B).

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