Down-regulation of peroxisome proliferator activated receptor γ coactivator 1α induces oxidative stress and toxicity of 1-(4-Chlorophenyl)-benzo-2,5-quinone in HaCaT human keratinocytes

Abstract

Peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α) is a transcriptional coactivator that is known to regulate oxidative stress response by enhancing the expression of antioxidant genes. We have shown previously that 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone-metabolite of 4-monochlorobiphenyl (PCB3) induces oxidative stress and toxicity in human skin keratinocytes, and breast and prostate epithelial cells. In this study, we investigate whether PGC-1α regulates oxidative stress and toxicity in 4-ClBQ treated HaCaT human keratinocytes. Results showed significant down-regulation in the expression of PGC-1α and catalase in 4-ClBQ treated HaCaT cells. Down-regulation of PGC-1α expression was associated with 4-ClBQ induced increase in the steady-state levels of cellular reactive oxygen species (ROS) and toxicity. Overexpression of pgc-1α enhanced the expression of catalase and suppressed 4-ClBQ induced increase in cellular ROS levels and toxicity. These results suggest that pgc-1α mediates 4-ClBQ induced oxidative stress and toxicity in HaCaT cells presumably by regulating catalase expression.

Keywords: Catalase; Oxidative stress; PCB3; PCB3-quinone; PGC-1α; Polychlorinated biphenyls.

Fig. 1

pgc-1α expression is inhibited in 4-ClBQ treated HaCaT cells. Quantitative RT-PCR and immunoblotting assays were used to measure pgc-1α (A) mRNA and (B) protein levels in control and 4-ClBQ treated cells at 24 h after the addition of 4-ClBQ. Quantitation of results from the immunoblots are shown in (C). (D) pgc-1α mRNA expression in control and 24 h of 3.0 μM PCB3 treated cells. Fold change was calculated relative to untreated control cells. Asterisks represent statistical significance compared to cells that were not treated with 4-ClBQ; p < 0.05, n = 3; error bars represent standard deviation of three independent experiments.

Fig. 2

4-ClBQ treatment decreases catalase expression and activity in HaCaT cells. Quantitative RT-PCR, immunoblotting and biochemical assays were used to measure catalase (A) mRNA expression, protein levels (B and C) and activity (D) in 4-ClBQ treated cells at 24 h after the addition of 4-ClBQ. Fold change was calculated relative to untreated control cells. Asterisks represent statistical significance compared to cells that were not treated with 4-ClBQ; p < 0.05, n = 3; error bars represent standard deviation of three independent experiments.

Fig. 3

Overexpression of pgc-1α suppresses 4-ClBQ induced down-regulation of catalase expression in HaCaT cells. Quantitative RT-PCR and immunoblotting assays were used to measure PGC-1α (A) mRNA and (B) protein levels in vector-alone and PGC-1α plasmid DNA transfected cells. Catalase expression in control and 4-ClBQ treated vector-alone and PGC-1α plasmid DNA transfected cells was measured using quantitative RT-PCR (C), immunoblotting (D and E), and native gel-electrophoresis based activity assay (F). Asterisks represent statistical significance compared to vector-alone transfected cells in absence of 4-ClBQ treatment; # indicates statistical significance compared to vector-alone transfected cells in presence of 4-ClBQ treatment; p < 0.05, n = 3; error bars represent standard deviation of three independent experiments.

Fig. 4

Overexpression of pgc-1α suppresses 4-ClBQ induced oxidative stress and toxicity in HaCaT cells. Flow cytometry measurements of MitoSOX Red oxidation were used to determine cellular ROS levels in control and 4-ClBQ treated vector-alone and pgc-1α overexpressing cells at the end of 24 h of treatment: (A) Representative histograms and (B) fold-change in MitoSOX Red oxidation. (C) Flow cytometry measurements of PI-positive cells and (D) a clonogenic assay was used to measure toxicity in control and 4-ClBQ treated vector-alone and pgc-1α overexpressing cells. Fold change was calculated relative to vector-alone transfected cells in absence of 4-ClBQ treatment. Asterisks represent statistical significance compared to vector-alone transfected cells in absence of 4-ClBQ treatment; # indicates statistical significance compared to vector-alone transfected cells in presence of 4-ClBQ treatment; p < 0.05, n = 3; error bars represent standard deviation of three independent experiments.