Metallothionein isoform 2A expression is inducible and protects against ROS-mediated cell death in rotenone-treated HeLa cells

Abstract

The role of MT (metallothionein) gene expression was investigated in rotenone-treated HeLa cells to induce a deficiency of NADH:ubiquinone oxidoreductase (complex I). Complex I deficiency leads to a diversity of cellular consequences, including production of ROS (reactive oxygen species) and apoptosis. HeLa cells were titrated with rotenone, resulting in dose-dependent decrease in complex I activity and elevated ROS production at activities lower than 33%. Expression of MT2A (MT isoform 2A), but not MT1A or MT1B RNA, was significantly inducible by rotenone (up to 7-fold), t-BHP (t-butyl hydroperoxide; 5-fold) and CdCl2 (50-fold), but not ZnCl2. Myxothiazol treatment did not elevate either ROS or MT2A levels, which supports a ROS-related mechanism for rotenone-induced MT2A expression. To evaluate the role of MT2A expression, MT2A and MT1B were overexpressed in HeLa cells and treated with rotenone. Compared with control and MT1B-overexpressing cells, ROS production was significantly lower and cell viability higher in MT2A-overexpressing HeLa cells when ROS production was enhanced by treatment with t-BHP. Mitochondrial membrane potential was noticeably less reduced in both MT-overexpressing cell lines. MT2A overexpression in rotenone-treated cells also significantly reduced or delayed apoptosis induction, as measured by caspase 3/7 activity and cytosolic nucleosome enrichment. We conclude that MT2A offers significant protection against the main death-causing consequences of rotenone-induced complex I deficiency in HeLa cells. Our results are in support of the protective role against oxidative stress ascribed to MTs and provide evidence that MT2A expression may be a beneficial downstream adaptive response in complex I-deficient cells.

Figure 1. Evaluation of RNA transcription level variation of housekeeping genes

Variation in real-time PCR-generated Ct values of selected housekeeping genes of HeLa cells treated with rotenone (0–10 μM), CdCl₂ (12.5 μM), ZnCl₂ (250 μM) and t-BHP (0–1 μM) are shown. Incubations were carried out for 24 h (rotenone, metals) and 3 h (t-BHP). The median values are indicated by small squares, 25–75% percentiles are indicated by the boxes and minimum and maximum values indicated by whiskers. Genes are GAPDH, β-actin, β₂-microglobulin (β-2-MG), RNA polymerase II (RP2) and 18 S rRNA.

Figure 2. MT2A RNA expression in rotenone-treated HeLa cells

MT2A RNA expression and total MT protein levels in cells were determined as described in the Experimental section. Normalized RNA expression levels are expressed as the mean ratio ±S.D. (n=3) relative to untreated cells (open bar). Asterisks indicate statistically significant values (P<0.05) compared with the untreated cells. Expression levels were compared in cells treated with rotenone, metal inducers CdCl₂ (12.5 μM) and ZnCl₂ (250 μM), t-BHP and myxothiazol as indicated. The value for Cd-induced expression is indicated above the bar.

Figure 3. ROS production in rotenone-treated HeLa cells

ROS production was measured fluorimetrically in cells treated with rotenone, metal inducers CdCl₂ (12.5 μM) and ZnCl₂ (250 μM), t-BHP and myxothiazol using the ROS-sensitive probe, DCFDA, and normalized to protein content. Values are means±S.D. (n=8) and expressed as percentage relative to untreated cells.

Figure 4. ATP levels in rotenone-treated MT-overexpressing HeLa cells

Total cellular ATP content was measured in rotenone-treated MT1B- (pIRESneo2-MT1B-transfected; light grey bars), MT2A-overexpressing (pIRESneo2-MT2A-transfected; dark grey bars) and control (pIRESneo2-transfected; open bars) HeLa cells as described in the Experimental section. Values were normalized to protein content and expressed as a mean percentage of untreated cells (±S.D., n=4). *P<0.05 when compared with untreated cells of the same cell line; †P<0.05 when compared with control cell line at the same treatment; ‡P<0.05 when comparing MT2A- with MT1B-overexpressing cell line at the same treatment.

Figure 5. ROS production in rotenone- and t-BHP-treated MT-overexpressing HeLa cells

ROS production was measured in control (open bars), MT1B- (light grey bars) and MT2A- (dark grey bars) overexpressing cell lines treated with rotenone or t-BHP. Values, which were normalized to protein content, are means±S.D. (n=8) and expressed as percentage relative to untreated cells. *P<0.05 when compared with untreated cells of the same cell line; †P<0.05 when compared with control cell line at the same treatment; ‡P<0.05 when comparing MT2A- with MT1B-overexpressing cell line at the same treatment.

Figure 6. Cell viability in rotenone- and t-BHP-treated MT-overexpressing HeLa cells

Cell viability was measured using the MTT test in MT-overexpressing (MT1B, light grey bars; MT2A, dark grey bars) and control cell lines (open bars) treated with rotenone or t-BHP as described in the Experimental section. Values, which were normalized to protein content, are means±S.D. (n=3) and expressed as percentage viability relative to untreated cells. Acetic acid (6%) was used as positive control (PC). *P<0.05 when compared with untreated cells of the same cell line; †P<0.05 when compared with control cell line at the same treatment; ‡P<0.05 when comparing MT2A- with MT1B-overexpressing cell line at the same treatment.

Figure 7. Assessment of mitochondrial membrane potential in rotenone-treated MT-overexpressing cells

Membrane potential of control (A), MT1B- (B) and MT2A-overexpressing (C) HeLa cells treated with 0, 100 and 1000 nM rotenone was visualized by confocal microscopy after TMRM staining.

Figure 8. Caspase 3/7 activation in rotenone-treated MT-overexpressing HeLa cells

Caspase 3/7 activity was measured in MT-overexpressing (MT1B, light grey bars; MT2A, dark grey bars) and control cell lines (open bars) treated with rotenone as described in the Experimental section. Reaction velocities are indicated as change in relative fluorescence units (RFU) per μg of total protein. Staurosporine treatment (1 μg/ml for 2 h) was the positive control (PC) for caspase activation. Values are means±S.D. (n=3). *P<0.05 when compared with untreated cells of the same cell line; †P<0.05 when compared with control cell line at the same treatment; ‡P<0.05 when comparing MT2A- with MT1B-overexpressing cell line at the same treatment.

Figure 9. Cytosolic nucleosome enrichment in rotenone-treated MT-overexpressing HeLa cells

Cytosolic nucleosome enrichment was determined in MT1B- (light grey bars), MT2A- (dark grey bars) overexpressing and control (open bars) cells treated with rotenone for 24 h or staurosporine (1 μg/ml for 2 h) as positive control (PC). Values, normalized relative to protein content, are indicated as a mean fold change (±S.D., n=3) relative to untreated cells. *P<0.05 when compared with untreated cells of the same cell line; †P<0.05 when compared with control cell line at the same treatment; ‡P<0.05 when comparing MT2A with MT1B-overexpressing cell line at the same treatment.