Liprin-α4 Is Required for Nickel Induced Receptor Protein Tyrosine Phosphatase-Leukocyte Antigen Related Receptor F (RPTP-LAR) Activity

Liprin-α4 was strongly induced following nickel (II) chloride exposure in a variety of cell types including BEAS-2B, A549, BEP2D and BL41 cells. Liprin-α4, a member of the Liprin alpha family, has seven isoforms but only three of these variants were detected in BEAS-2B cells (004, 201 and 202). The level of Liprin-α4 variants 201 and 004 were highly increased in BEAS-2B cells in response to nickel. We showed that Liprin-α4 bound directly to the cytoplasmic region of RPTP-LAR (receptor protein tyrosine phosphatase-leukocyte antigen-related receptor F). The cytoplasmic region of RPTP-LAR contains two phosphatase domains but only the first domain shows activity. The second domain interacts with other proteins. The phosphatase activity was increased both following nickel treatment and also in the presence of nickel ions in cell extracts. Liprin-α4 knock-down lines with decreased expression of Liprin-α4 variants 004 and 201 exhibited greater nickel toxicity compared to controls. The RPTP-LAR phosphatase activity was only slightly increased in a Liprin-α4 knock-down line. Liprin-α4 appeared necessary for the nickel induced tyrosine phosphatase activity. The presence of Liprin-α4 and nickel increased tyrosine phosphatase activity that reduced the global levels of tyrosine phosphorylation in the cell

Effects of nickel treatment on H3K4 trimethylation and gene expression.

Occupational exposure to nickel compounds has been associated with lung and nasal cancers. We have previously shown that exposure of the human lung adenocarcinoma A549 cells to NiCl(2) for 24 hr significantly increased global levels of trimethylated H3K4 (H3K4me3), a transcriptional activating mark that maps to the promoters of transcribed genes. To further understand the potential epigenetic mechanism(s) underlying nickel carcinogenesis, we performed genome-wide mapping of H3K4me3 by chromatin immunoprecipitation and direct genome sequencing (ChIP-seq) and correlated with transcriptome genome-wide mapping of RNA transcripts by massive parallel sequencing of cDNA (RNA-seq). The effect of NiCl(2) treatment on H3K4me3 peaks within 5,000 bp of transcription start sites (TSSs) on a set of genes highly induced by nickel in both A549 cells and human peripheral blood mononuclear cells were analyzed. Nickel exposure increased the level of H3K4 trimethylation in both the promoters and coding regions of several genes including CA9 and NDRG1 that were increased in expression in A549 cells. We have also compared the extent of the H3K4 trimethylation in the absence and presence of formaldehyde crosslinking and observed that crosslinking of chromatin was required to observe H3K4 trimethylation in the coding regions immediately downstream of TSSs of some nickel-induced genes including ADM and IGFBP3. This is the first genome-wide mapping of trimethylated H3K4 in the promoter and coding regions of genes induced after exposure to NiCl(2). This study may provide insights into the epigenetic mechanism(s) underlying the carcinogenicity of nickel compounds

A list of the top 50 highest nickel-induced genes with qualified H3K4me3 peaks within 5,000 bp flanking the TSSs from crosslinked and non-crosslinked chromatins isolated from nickel-treated and control A549 cells and the fold induction in gene expression determined by RNA-Seq between nickel-treated and control A549 cells was listed.

<p>*RIMKLA was not one of the top50 genes with qualified H3K4me3 peaks in non-crosslinked chromatin because no peaks were identified within 5000 bp flanking the TSS.</p><p>**ZNF292 was the top 51 gene with qualified H3K4me3 peaks in crosslinked chromatin and the top 50 gene in non-crosslinked chromatin.</p

Information of ChIP-Seq Data Sets from crosslinked and non-crosslinked chromatins isolated from control or nickel-treated A549 cells.

<p>Information of ChIP-Seq Data Sets from crosslinked and non-crosslinked chromatins isolated from control or nickel-treated A549 cells.</p

Global profiling of all nickel-induced genes and the top 50 highest nickel-induced genes with qualified H3K4me3 peaks within 5,000 bp upstream or downstream of TSS (center line at 0) isolated from (A) crosslinked chromatin and (B) non-crosslinked chromatin from untreated control (red) and nickel-treated (blue) A549 cells.

<p>Global profiling of all nickel-induced genes and the top 50 highest nickel-induced genes with qualified H3K4me3 peaks within 5,000 bp upstream or downstream of TSS (center line at 0) isolated from (A) crosslinked chromatin and (B) non-crosslinked chromatin from untreated control (red) and nickel-treated (blue) A549 cells.</p

RNA-Seq analysis of the expression of (A) CA9, (B) NDRG1, (C) ADM, and (D) IGFBP3 genes induced by nickel treatment (lower panel) compared to untreated control (upper panel) in A549 cells and aligned to the exons and introns of their respective genes.

<p>RNA-Seq analysis of the expression of (A) CA9, (B) NDRG1, (C) ADM, and (D) IGFBP3 genes induced by nickel treatment (lower panel) compared to untreated control (upper panel) in A549 cells and aligned to the exons and introns of their respective genes.</p

Real Time-PCR analysis of the relative level of induction (fold induction depicted as %) of expression of VEGFA, ZNF654, HIG2, IFI44L and PPFIA4 genes in nickel-treated PBMCs compared to untreated control cells.

<p>Real Time-PCR analysis of the relative level of induction (fold induction depicted as %) of expression of VEGFA, ZNF654, HIG2, IFI44L and PPFIA4 genes in nickel-treated PBMCs compared to untreated control cells.</p

Global profiling of all nickel-induced genes and the top 50 highest nickel-induced genes with qualified H3K4me3 peaks within 5,000 bp upstream or downstream of TSS (center line at 0) isolated from crosslinked chromatin from untreated (red) and nickel-treated (blue) peripheral blood mononuclear cells (PBMCs).

<p>Global profiling of all nickel-induced genes and the top 50 highest nickel-induced genes with qualified H3K4me3 peaks within 5,000 bp upstream or downstream of TSS (center line at 0) isolated from crosslinked chromatin from untreated (red) and nickel-treated (blue) peripheral blood mononuclear cells (PBMCs).</p

Real Time-PCR (RT-PCR) analysis of the relative level of induction (fold induction depicted as %) of expression of CA9, NDRG1, ADM, and IGFBP3 genes in nickel-treated A549 cells compared to untreated control cells.

<p>Real Time-PCR (RT-PCR) analysis of the relative level of induction (fold induction depicted as %) of expression of CA9, NDRG1, ADM, and IGFBP3 genes in nickel-treated A549 cells compared to untreated control cells.</p