A blood pressure-associated variant of the SLC39A8 gene influences cellular cadmium accumulation and toxicity

Abstract

Genome-wide association studies have revealed a relationship between inter-individual variation in blood pressure and the single nucleotide polymorphism rs13107325 in the SLC39A8 gene. This gene encodes the ZIP8 protein which co-transports divalent metal cations, including heavy metal cadmium, the accumulation of which has been associated with increased blood pressure. The polymorphism results in two variants of ZIP8 with either an alanine (Ala) or a threonine (Thr) at residue 391. We investigated the functional impact of this variant on protein conformation, cadmium transport, activation of signalling pathways and cell viability in relation to blood pressure regulation. Following incubation with cadmium, higher intracellular cadmium was detected in cultured human embryonic kidney cells (HEK293) expressing heterologous ZIP8-Ala391, compared with HEK293 cells expressing heterologous ZIP8-Thr391. This Ala391-associated cadmium accumulation also increased the phosphorylation of the signal transduction molecule ERK2, activation of the transcription factor NFκB, and reduced cell viability. Similarly, vascular endothelial cells with the Ala/Ala genotype had higher intracellular cadmium concentration and lower cell viability than their Ala/Thr counterpart following cadmium exposure. These results indicate that the ZIP8 Ala391-to-Thr391 substitution has an effect on intracellular cadmium accumulation and cell toxicity, providing a potential mechanistic explanation for the association of this genetic variant with blood pressure.

Figure 1.

Results of computational analyses of ZIP8 Ala391-to-Thr391. (A) A comparison of DNA sequences around the rs13107325 site in 23 mammals. (B) Output of ConSurf Server analysis based on 142 reference sequences, the arrow points the amino acid position of Ala391. (C) Graphical interpretation of the transmembrane helical domains predicted by TMHMM. The number at the top denotes the amino acid position, the Ala391-to-Thr391 substitution is circled in pink, which locates outside the cell membrane. (D) Structural perturbation induced by the Ala391-to-Thr391 substitution as shown by Robetta secondary protein structure prediction. The image shows an overlapped simulation of the two proteins. The Thr391 residue (represented by the blue circle) is at a distance of 6 Å´ from the Ala391 residue (represented by the yellow circle).

Figure 2.

Effect of ZIP8 Ala391-to-Thr391 on intracellular Cd²⁺accumulation. (A) Immunoblotting analysis of HEK293 cells transfected with the pcDNA-ZIP8-Ala391, pcDNA-ZIP8-Thr391, or pcDNA3.1(+) plasmid, with an anti-ZIP8 antibody and an antibody for the loading control protein β-actin. Upper panel: a representative immunoblot image; Lower panel: mean (± SEM) values of ZIP8 band intensities standardized against β-actin band intensities. (B) Intracellular Cd²⁺ concentrations in HEK293 cells transfected with the pcDNA-ZIP8-Ala391 or pcDNA-ZIP8-Thr391 plasmid, standardized against transfection efficiency and subtracted from intracellular Cd²⁺ concentrations of HEK293 cells transfected with the empty pcDNA3.1(+) plasmid. Data shown are mean (± SEM) values from three experiments, with duplicate for each plasmid in each experiment. (C) Immunoblotting analysis of HUVECs of the Ala/Ala or Ala/Thr genotype with an anti-ZIP8 antibody and an antibody for the loading control protein Hsc-70. Upper panel: a representative immunoblot image; lower panel: mean (± SEM) values of ZIP8 band intensities standardized against Hsc-70 band intensities. (D) Intracellular Cd²⁺ concentrations in HUVECs of the Ala/Ala or Ala/Thr genotype, standardized against ZIP8 levels (represented by ZIP8 band intensities with normalization against β-actin band intensities). Data shown are mean (± SEM) values from 3 experiments, with duplicate for each plasmid in each experiment. * Indicates P < 0.05 by paired Student’s t-test.

Figure 3.

Effect of ZIP8 Ala391-to-Thr391 on Cd²⁺-induced cell death. (A) Digital light microscopic images of HEK293 cells transfected with the pcDNA-ZIP8-Ala391, pcDNA-ZIP8-Thr391, or pcDNA3.1(+) plasmid, followed by a 24 h incubation with Cd²⁺ (1 µmol/l). (B) Percentages of viable HEK239 cells transfected with the pcDNA-ZIP8-Ala391, pcDNA-ZIP8-Thr391, or pcDNA3.1(+) plasmid, followed by a 2 h incubation with Cd²⁺ (1 µmol/l), determined by MTS assay. (C) Relative amounts of LDH in media conditioned by HEK239 cells transfected with the pcDNA-ZIP8-Ala391, pcDNA-ZIP8-Thr391, or pcDNA3.1(+) plasmid, followed by a 24 h incubation with Cd²⁺ (1 µmol/l). (D) Relative amounts of LDH in media conditioned by HUVECs of the Ala/Ala or Ala/Thr genotype, followed by a 24 h incubation with Cd²⁺ (1 µmol/l). Data shown in (B), (C) and (D) are mean (± SEM) values from 3 experiments, with triplicate for each plasmid in each experiment. *Indicates P < 0.05 by Wilcoxon test.

Figure 4.

Effect of ZIP8 Ala391-to-Thr391 on ERK phosphorylation in cells incubated with Cd²⁺. HEK239 cells transfected with the pcDNA-ZIP8-Ala391, pcDNA-ZIP8-Thr391, or pcDNA3.1(+) plasmid, were incubated with Cd²⁺ (1 µmolL) for 24 h, followed by immunoblotting analyses of ZIP8, total ERK1, phosphorylated ERK1, total ERK2, and phosphorylated ERK2. (A) Representative immunoblot images. (B) Mean (± SEM) values of relative intensities of the ZIP8 band. (C) Mean (± SEM) values of the ratio of phosphorylated ERK1 band intensity versus total ERK1 band intensity, standardized against ZIP8 band intensity. (D) Mean (± SEM values of the ratio of phosphorylated ERK2 band intensity versus total ERK2 band intensity, standardized against ZIP8 band intensity. Data shown in (B) to (D) are from three experiments, with triplicate for each plasmid in each experiment. * Indicates P < 0.05 by paired Student’s t-test.

Figure 5.

Effect of ZIP8 Ala391-to-Thr391 on NFκB activity in cells incubated with Cd²⁺. (A) HEK293 cells were transfected with either the pcDNA-ZIP8-Ala391, pcDNA-ZIP8-Thr391, or pcDNA3.1(+) plasmid, as well as a firefly luciferase reporter plasmid containing copies of the NFκB responsive element upstream of the firefly luciferase reporter gene, and a renilla luciferase plasmid to serve a reference for transfection efficiency. The transfected cells were incubated with Cd²⁺ (1 μmol/l) for 2 h, and NFκB activity measured by dual-luciferase assays. Data shown are mean (± SEM) luciferase activity from three experiments, with triplicate for each plasmid in each experiment. (B) HUVECs of the Ala/Ala or Ala/Thr genotype were subjected to immunoblotting analyses of the phospho-IKKα/β. Shown on the left is a representative immunoblot image, and on the right are the relative phospho-IKKα/β band intensities standardized against loading control protein (Hsc-70) band intensities in different genotypes. Data shown are mean (±SEM) values from 3 experiments of 4 samples for each genotype, conducted in duplicate. * Indicates P < 0.05 by paired Student’s t-test.