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By Jamshed Arslan Pharm.D.
Nickel (Ni) is a naturally abundant metallic element. It is a major component of stainless steel, coins, and many other items of daily use. Disturbingly, Ni exposure is associated with cancer and several diseases of the lung, kidney and cardiovascular system. Despite being carcinogenic, Ni's ability to cause DNA mutations and induce oxidative stress is low. To explain this paradox and investigate how the impact of carcinogen exposure may last for months or even years, researchers at the New York University and University of Virginia examined Ni-induced changes in transcription and cellular regulation in human epithelial cells from lung and urinary bladder. They found that Ni induces epithelial-mesenchymal transition (EMT) by epigenetically activating ZEB1. As a result of such irreversible activation of EMT master regulator ZEB1, the invasive mesenchymal phenotype in Ni-exposed cells persisted even after the termination of exposure.
Immunohistochemistry-Paraffin: [E-Cadherin Antibody (7H12) NBP2-19051] - Human kidney distal tubules stained with E-CAD antibody. Secondary antibody: Donkey anti-Mouse Alexa Fluor 555. Imaging with a 40x objective from Zeiss Observer microscope with Apotome2. Image from verified customer review.
Nickel induces epithelial-mesenchymal transition (EMT)
The researchers exposed non-malignant human bronchial epithelial cells to a non-cancerous dose of Ni. RNA-seq revealed that a subset of genes was persistently down- or up-regulated even two weeks after the removal of Ni from the culture medium. Functional enrichment analysis, used to determine overrepresented classes of genes, showed that the major pathway associated with these differentially expressed genes was epithelial-mesenchymal transition (EMT) regulation. The mesenchymal phenotype in Ni-exposed cells was confirmed when a downregulation of epithelial markers (CDH1 and CLDN1) was observed concomitantly with an upregulation of mesenchymal marker (FN1), at both mRNA and protein levels. Likewise, wound-healing and transwell invasion assays revealed heightened migratory and invasive abilities of these Ni-exposed cells.
Similar findings in a non-invasive human bladder cancer cell line led to the conclusion that chronic Ni exposure induces EMT. The next step was to find the mechanistic basis behind this phenomenon.
Nickel-induced EMT is ZEB1-dependent
Gene expression analysis of the EMT signaling pathway showed ZEB1, a negative regulator of CDH1, to be among the highly upregulated genes in Ni-exposed cells as compared to untreated cells. Analysis of mRNA and protein levels corroborated these findings. Knocking down ZEB1 through shRNA, recovered CDH1 protein and decreased the invasive and migratory abilities of Ni-exposed cells. These results showed the ZEB1-dependent induction of EMT by Ni.
To explore possible epigenetic basis of this observation, the team observed ZEB1 promoter by performing chromatin immunoprecipitation (ChIP)-qPCR analysis. They found that histone modifications related to gene activation (H3K4me3) at ZEB1 promoter in Ni-exposed human epithelial cells remained similar to untreated cells. However, the levels of histone modifications associated with gene repression (H3K27me3) decreased significantly. Likewise, a persistent downregulation of miRNAs that act as ZEB1 repressors (miR-200/205), which could be reversed by ZEB1 depletion, meant that ZEB1 suppresses these miRNAs in Ni-exposed cells.
In short, certain histone modifications and the suppression of ZEB1-repressing miRNAs lead to a sustained ZEB1 upregulation, which in turn promotes a highly migratory and invasive mesenchymal phenotype in Ni-exposed cells.
Significance
This study provides a plausible explanation to why environmental exposure to certain contaminants persists long after the termination of exposure. It helps understanding diseases associated with chronic exposure to a physiologically relevant dose of Ni.
Learn more about tools for epigenetic analysis
Jamshed Arslan, Pharm D.
University of Alabama at Birmingham, School of Medicine
Dr. Arslan studies cell signaling in mitochondrial defects in C. elegans
and transgenic mice.
References
Jose, Cynthia C., et al. "Nickel Exposure Induces Persistent Mesenchymal Phenotype in Human Lung Epithelial Cells through Epigenetic Activation of ZEB1." Molecular Carcinogenesis, vol. 57, no. 6, 2018, pp. 794 – 806. doi: 10.1002/mc.22802.
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