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Published on: March 14, 2011
Induction of antioxidant enzymes by FAK in a human leukemic cell line, HL-60
Yoshiko Sonoda1, Naomi Aiba, Ryouko Utsubo
1Department of Biochemistry, Kyoritsu College of Pharmacy, Shibakoen 1-5-30, Minato, Tokyo 105-8512, Japan. sonoda-ys@kyoritsu-ph.ac.jp
Abstract:
We have established several focal adhesion kinase (FAK) cDNA-transfected HL-60 (HL-60/FAK) cells which were highly resistant to oxidative stress-induced apoptosis. To identify target genes that are involved in HL-60/FAK cells, we performed cDNA microarray screening using apoptosis-chip. There, we identified the decrease of glutathione peroxidase (GPx). This result prompted us to investigate the changes of antioxidant enzymes. Here, we demonstrate that lipid peroxidation was suppressed after treatment with hydrogen peroxide in HL-60/FAK cells but not vector-transfected HL-60 (HL-60/Vect) cells. Furthermore, we demonstrate that HL-60/FAK cells have higher basal reactive oxygen species (ROS) levels than the parental HL-60 or HL-60/Vect cells, while ROS accumulation by hydrogen peroxide treatment was almost the same in these cells. Basal activity and mRNA expression of antioxidant enzymes, particularly of GSH reductase (GRe), phospholipid hydroperoxide glutathione peroxidase (PHGPx) were markedly elevated in HL-60/FAK cells. In contrast, GPx and catalase levels were decreased in HL-60/FAK cells. Further, a Src family kinases inhibitor, PP2, suppressed GRe and PHGPx mRNA by inactivation of FAK and c-Src in HL-60/FAK cells. These results suggest that FAK upregulates antioxidant enzymes and suppresses lipid peroxidation, resulting in the anti-apoptotic state for oxidative stress.
Insights
Focal adhesion kinase (FAK) enhances cellular resistance to oxidative stress by upregulating antioxidant enzymes, suppressing lipid peroxidation, and preventing apoptosis. This involves increased GSH reductase and PHGPx, despite decreased GPx and catalase.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Oxidative stress induces apoptosis, a programmed cell death pathway.
- Focal adhesion kinase (FAK) plays a role in cellular signaling and survival.
- Understanding FAK's role in oxidative stress resistance is crucial for developing therapeutic strategies.
Purpose of the Study:
- To investigate the molecular mechanisms by which FAK confers resistance to oxidative stress-induced apoptosis.
- To identify target genes regulated by FAK in response to oxidative stress.
Main Methods:
- Establishment of FAK-transfected HL-60 cells (HL-60/FAK) resistant to oxidative stress.
- cDNA microarray screening using an apoptosis-chip to identify differentially expressed genes.
- Measurement of lipid peroxidation and reactive oxygen species (ROS) levels.
- Assessment of antioxidant enzyme activity and mRNA expression (GPx, GRe, PHGPx, catalase).
- Inhibition of FAK and c-Src using PP2 to study their effect on antioxidant enzyme expression.
Main Results:
- HL-60/FAK cells exhibited suppressed lipid peroxidation and similar ROS accumulation compared to control cells under hydrogen peroxide treatment.
- Basal levels of reactive oxygen species (ROS) were higher in HL-60/FAK cells.
- mRNA expression and activity of glutathione reductase (GRe) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) were significantly elevated in HL-60/FAK cells.
- Expression of glutathione peroxidase (GPx) and catalase was decreased in HL-60/FAK cells.
- Inhibition of FAK and c-Src with PP2 reduced GRe and PHGPx mRNA levels.
Conclusions:
- FAK upregulates key antioxidant enzymes (GRe, PHGPx) and suppresses lipid peroxidation, contributing to resistance against oxidative stress-induced apoptosis.
- FAK-mediated signaling, involving c-Src, plays a critical role in maintaining the antioxidant defense system.
- These findings highlight FAK as a potential therapeutic target for conditions involving oxidative stress and apoptosis.
