Nrf2-ARE-Dependent Alterations in Zinc Transporter mRNA Expression in HepG2 Cells

<div><p>Zinc transporters are solute carrier family members. To date, 10 zinc transporters (ZnTs) and 14 Zrt-, Irt-like proteins (ZIPs) have been identified. ZnTs control intracellular zinc levels by effluxing zinc from the cytoplasm into the extracellular fluid, intracellular vesicles, and organelles; ZIPs also contribute to control intracellular zinc levels with influxing zinc into the cytoplasm. Recently, changes in zinc transporter expression have been observed in some stress-induced diseases, such as Alzheimer’s disease and diabetes mellitus. However, little is known regarding the mechanisms that regulate zinc transporter expression. To address this, we have investigated the effect of a well-established stress response pathway, the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway, on zinc transporter mRNA levels. Exposure to 10⁻⁴ M <i>tert</i>-butylhydroquinone (<i>t</i>-BHQ), which activates Nrf2-ARE signaling, for 6 h significantly increases ZnT-1, ZnT-3, and ZnT-6 mRNAs levels, and significantly decreases ZnT-10 and ZIP-3 mRNA levels. These changes are not observed with 10⁻⁶ M <i>t</i>-BHQ, which does not activate Nrf2-ARE signaling. Furthermore, <i>t</i>-BHQ exposure does not affect metal responsive element transcription, a <i>cis</i> element that is activated in response to intracellular free zinc accumulation. From these results, we believe that the transcription of ZnT-1, ZnT-3, ZnT-6, ZnT-10, and ZIP-3 is influenced by the Nrf2-ARE signal transduction pathway.</p></div

Antioxidant responsive element (ARE) luciferase reporter activity following <i>t</i>-BHQ treatment.

<p>HepG2 cells were cotransfected with pGL4.37, a firefly luciferase vector encoding the ARE, and the <i>Renilla</i> luciferase vector pGL4.74 prior to treatment with the denoted concentration of <i>t</i>-BHQ for 6 h. Firefly and <i>Renilla</i> luciferase activities were subsequently determined and firefly luciferase activity was normalized to the <i>Renilla</i> luciferase activity to control for transfection efficiency. Data are expressed as relative activity values compared with vehicle control (0.1% DMSO = 100%). Bars indicate the mean ± SD of 3 (10⁻⁶ M <i>t</i>-BHQ) or 4 (Control and 10⁻⁴ M <i>t</i>-BHQ) samples. ***p < 0.001 compared with control.</p

The effect of <i>t</i>-BHQ treatment on metal transcriptional factor-1 (MTF-1) mRNA expression.

<p>HepG2 cells were treated with the denoted concentration of <i>t</i>-BHQ or medium including 0.1% (v/v) DMSO as a control for 6 h. The mRNA level was determined using qRT-PCR with SYBR^® Green. Data are normalized to ß-actin expression. Bars represent the mean ± SD of 4 samples.</p

<i>t</i>-BHQ dose-dependent changes in HepG2 cell cytotoxicity.

<p>Cells were treated with the denoted concentration of <i>t</i>-BHQ or vehicle (0.1% (v/v) DMSO) for 6 h and cell viability was assessed using the WST-8 assay. Values represent the mean ± SD of 5 samples. ***p < 0.001 compared with control (100 ± 4.8%).</p

Metal responsive element (MRE)-luciferase reporter activity following <i>t</i>-BHQ treatment.

<p>HepG2 cells were cotransfected with the firefly luciferase vector encoding the MRE, pGL4.40, and the pGL4.74 <i>Renilla</i> luciferase vector. Transfected cells were treated with control (0.1% (v/v) DMSO), <i>t</i>-BHQ, or 0.1 mM ZnSO₄ (as a positive control) for 6 h and luciferase activities were determined. Firefly luciferase activity was normalized to <i>Renilla</i> luciferase activity to control for transfection efficiency. Data are expressed as relative activity values compared with control (0.1% DMSO = 100%). Bars indicate the mean ± SD of 4 samples. ***p < 0.001 compared with control.</p

The effect of <i>tert</i>-butylhydroquinone (<i>t</i>-BHQ) treatment on zinc transporter mRNA levels.

<p>HepG2 cells were treated with the denoted concentration of <i>t</i>-BHQ for 6 h. The expression of each mRNA was determined by qRT-PCR with SYBR^® Green. Data are normalized to ß-actin mRNA expression. Values represent the mean ± standard deviation (SD) of 4 samples. *p < 0.05, ***p < 0.001 compared with vehicle treated control (0.1% dimethyl sulfoxide; DMSO) cells.</p

Quantitative real-time polymerase chain reaction (qRT-PCR) primer sequences and product sizes.

<p>Quantitative real-time polymerase chain reaction (qRT-PCR) primer sequences and product sizes.</p

Dose-dependent alteration in heme oxygenase-1 mRNA expression following <i>t</i>-BHQ treatment.

<p>HepG2 cells were treated with the denoted concentration of <i>t</i>-BHQ or vehicle (0.1% (v/v) DMSO) for 6 h prior to quantification of mRNA expression by qRT-PCR with SYBR^® Green. Data are normalized to ß-actin expression. Bars indicate the mean ± SD of 4 samples. ***p < 0.001 compared with control.</p