Evaluation of changes produced by cadmium on the expression of genes regulated by NF-Kb in macrophages and their dependence of the ZIP8 gene expression

Abstract

Atherosclerosis is the main risk factor for cardiovascular diseases and cigarette smoking is a major risk factor to develop cardiovascular diseases and atherosclerosis. A transcriptome study of circulating monocytes from 936 individuals from Germany identified a gene coexpression network linking, at least in part, smoking with atherosclerosis. It showed that the shortest paths connecting smoking with increased number of atherosclerotic plaques dependend on the over-expression of the GAS6 and ZIP8 genes. Previously, it has been shown that GAS6 has pleiotropic effects in the development and stabilization of atherosclerotic plaques, in part by stimulating production of pro-inflammatory cytokines. ZIP8 imports zinc into the cell, which inhibits pro-inflammatory response in leukocytes through blocking of activation of NF-κB and its nuclear translocation. However, ZIP8 can also import cadmium present in cigarette smoke, leading to cytotoxicity. The identified network suggested a new mechanism by which cadmium that is present in the cigarette smoke may promote pro-atherogenic effects by deregulation of an inflammatory signaling pathway. We tested this hypothesis by evaluating the effect of cadmium on the expression of genes regulated by NF-κB in macrophages and their dependence of the ZIP8 gene expression by Real-Time quantitative reverse transcription PCR, gene expression microarrays and ELISA. All experiments were performed in primary cultures of monocytes from healthy donors that were differentiated into three distinct subpopulations of macrophages: M0, M1 and M2. The results showed that the treatment with cadmium decreases ZIP8 gene expression in the three macrophage phenotypes and increased GAS6 gene expression in M1 macrophages, while decreased in M2 macrophages. Also, GAS6 gene expression is increased when the ZIP8 gene expression is diminished by an interfering RNA, showing a relationship between both genes. Moreover the treatment with cadmium has a stronger effects on the transcription activity of anti-inflammatory M2 macrophages compared with pro-inflammatory M1 macrophages, finding 185 suggestive differentially expressed genes (0.05 < FDR ≤ 0.1) in M1 macrophages and 4,472 genes differentially expressed (FDR ≤ 0.05) and 1,227 suggestive differentially expressed genes in M2 macrophages. According to our hypothesis we found “TNF-α signaling via NF-κB” enriched in M2 macrophages. In addition, 116 differentially expressed genes in M2, are regulated by NF-κB, showing that cadmium activates NF-κB, probably by reactive oxygen species production, promoting pro-inflammatory effects that could favor proatherogenic effects in macrophages M2.