Functional characterization of solute carrier family 39 member 8 variant in relation to hypertension and coronary artery disease.

Abstract

Hypertension and coronary artery disease are complex diseases with both environmental and genetic contributions. Genome-wide association studies (GWASs) have revealed a number of common genetic variants with modest effects on the disease susceptibility. One of the novel genetic variants (SNP rs13107325, C ! T, Ala391Thr) identified in GWASs has been associated with a number of CVD-related traits, including SBP, DBP, HDL-C, body mass index, and schizophrenia. This SNP is located at exon 8 of the solute carrier family 39 member 8 gene (SLC39A8), and results in an amino acid substitution from Alanine to Threonine at amino acid position 391. SLC39A8 was first found to be highly induced by Mycobacterium bovis BCG cell wall and TNF-α. The encoded protein ZIP8 is a divalent cation/bicarbonate cotransporter with different affinities for a number of metal ions. Recent studies revealed the major role of ZIP8 in the uptake of cadmium and manganese. The objective of this project is to investigate whether the Ala391Thr polymorphism has an impact on the structure or function of ZIP8, and the underlying mechanisms responsible for the deleterious cell events associated with cadmium exposure, that over time, may contribute to the development of hypertension. We showed that ZIP8 containing the Ala391 variant resulted in higher intracellular accumulation of Cd2+ at low dose and Zn2+ at physiological concentration. Lower cell proliferation and higher cell death were observed in transfected HEKs and HuVECs exposed to Cd2+. This was accompanied by stronger ERK1/2 and NF-ĸB activation in HEK293 possibly induced by Cd2+ uptake. We also showed some evidence of ZIP8 participation in HDL3-mediated cholesterol efflux, however this needs to be further validated in an improved experimental model. 6" " Bioinformatics analysis suggested a possible structural impact of Ala391Thr on ZIP8 function, which was supported by conservation analysis of the alanine amino acid. Membrane protein structure analysis predicted a shift of an α-helical domain possibly caused by the change of the amino acid hydrophillicity. The secondary structure predicted by the Robetta algorithm based on a similar homologue supported that this structural change may be caused by the Ala391Thr variation. Our data indicate that the amino acid substitution (Ala391Thr) caused by SNP rs13107325 alters the transport activity of ZIP8 on metal ions, leading to differential activation of ERK1/2 and NF-κB pathways, which subsequently results in different levels of cell death on human kidney and endothelial cells. This study sought to provide the preliminary mechanistic explanations for the association of rs1310725 with blood pressure and HDL-C following the GWAS. These observations have demonstrated a promising start for further investigation of SLC39A8 in the context of hypertensive mice or human populations. This may enable further pharmacological studies for the development of a potential therapeutic candidate for hypertension treatment.