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. 2023 Mar 1;324(3):G159-G176.
doi: 10.1152/ajpgi.00244.2022. Epub 2022 Dec 20.

Enterocyte-specific deletion of metal transporter Zip14 (Slc39a14) alters intestinal homeostasis through epigenetic mechanisms

Felix R Jimenez-Rondan  1 , Courtney H Ruggiero  1 , Kelley Lobean McKinley  2 , Jin Koh  3 , John F Roberts  4 , Eric W Triplett  2 , Robert J Cousins  1
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"V体育安卓版" Enterocyte-specific deletion of metal transporter Zip14 (Slc39a14) alters intestinal homeostasis through epigenetic mechanisms

Felix R Jimenez-Rondan et al. Am J Physiol Gastrointest Liver Physiol. .

Abstract

Zinc has anti-inflammatory properties using mechanisms that are unclear. Zip14 (Slc39a14) is a zinc transporter induced by proinflammatory stimuli and is highly expressed at the basolateral membrane of intestinal epithelial cells (IECs). Enterocyte-specific Zip14 ablation (Zip14ΔIEC) in mice was developed to study the functions of this transporter in enterocytes. This gene deletion led to increased intestinal permeability, increased IL-6 and IFNγ expression, mild endotoxemia, and intestinal dysbiosis. RNA sequencing was used for transcriptome profiling. These analyses revealed differential expression of specific intestinal proinflammatory and tight junction (TJ) genes. Binding of transcription factors, including NF-κβ, STAT3, and CDX2, to appropriate promoter sites of these genes supports the differential expression shown with chromatin immunoprecipitation assays. Total histone deacetylase (HDAC), and specifically HDAC3, activities were markedly reduced with Zip14 ablation. Intestinal organoids derived from ΔIEC mice display TJ and cytokine gene dysregulation compared with control mice VSports手机版. Differential expression of specific genes was reversed with zinc supplementation of the organoids. We conclude that zinc-dependent HDAC enzymes acquire zinc ions via Zip14-mediated transport and that intestinal integrity is controlled in part through epigenetic modifications. NEW & NOTEWORTHY We show that enterocyte-specific ablation of zinc transporter Zip14 (Slc39a14) results in selective dysbiosis and differential expression of tight junction proteins, claudin 1 and 2, and specific cytokines associated with intestinal inflammation. HDAC activity and zinc uptake are reduced with Zip14 ablation. Using intestinal organoids, the expression defects of claudin 1 and 2 are resolved through zinc supplementation. These novel results suggest that zinc, an essential micronutrient, influences gene expression through epigenetic mechanisms. .

Keywords: Zip14; cytokines; inflammation; intestinal; zinc. V体育安卓版.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

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Graphical abstract
Figure 1.
Figure 1.
Enterocyte-specific Zip14 deletion causes systemic endotoxemia, mucosal inflammation, and increased intestinal permeability. A: comparative Zip14 mRNA expression in proximal small intestine (total int), intestinal epithelial cells (IEC), intestinal organoids, liver, lung, and skeletal muscle of Zip14F/F (control) or Zip14ΔIEC mice (n = 3/group). B: Western analyses of Zip14 protein in proximal small intestine (total int), IECs, and lung from F/F and ΔIEC mice (n = 3/group). C: chromatin immunoprecipitation (ChIP) assays of NF-κβ and STAT3 binding to Zip14 promoter using IEC chromatin from F/F and ΔIEC mice (n = 3/group). D: representative micrographs showing hematoxylin and eosin (H&E)-stained sections of jejunum from F/F and ΔIEC mice. Bar = 300 µm. E: IL-1β, IL-6, and IFNγ relative mRNA expression in IECs from F/F and ΔIEC mice (n = 3 or 4/group). F: indirect intestinal permeability measured as serum FITC, 2 h after administration of FITC-Dextran 4 kDa by gavage to F/F and ΔIEC mice (n = 3/group). G: serum endotoxin in F/F and ΔIEC mice (n = 5/group). H: alkaline phosphatase (Alpi) activity in IECs from F/F and ΔIEC mice (n = 5/group). Values are means ± SE; n, number of biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; Student’s t test.
Figure 2.
Figure 2.
Enterocyte-specific Zip14 ablation causes microbial dysbiosis. A: bar plot of microbial phyla relative abundance in fecal microbiota from Zip14ΔIEC and Zip14F/F control mice. (experiment 1) Individually housed mice in box cages (n = 5/group); (experiment 2) individually housed mice in suspended, wire cages and given drinking water with or without 15 µM Zn (n = 3/group). B: box plots of the genus Akkermansia mucinphilia of the Verrucomicrobiota phylum, showing abundance using the centered log ratio transformation of sequence counts of fecal microbiota from Zip14ΔIEC and Zip14F/F mice from (A). Boxes = interquartile range. Horizontal line = median. C: Shannon plots showing α diversity measures of fecal Verrucomicrobiota from Zip14ΔIEC and Zip14F/F control mice from (A). Boxes = interquartile range. Horizontal line = median. IEC, intestinal epithelial cell. n, number of biological replicates.
Figure 3.
Figure 3.
Zip14 knockout causes differential expression of genes required for intestinal homeostasis in intestinal epithelial cells and intestinal organoids. A: volcano plot showing transcript abundance in total RNA from proximal small intestine of WB-KO vs. WT mice as analyzed by RNA-sequencing. Significantly altered genes with a false discovery rate (FDR) adjusted P ≤ 0.05 and Log2FC >2 are highlighted in red. Fold change of abundance is expressed as a ratio of WB-KO to WT values. To validate our RNA-sequencing results, RNA was obtained from IECs derived from Zip14ΔIEC and Zip14 ΔF/F mice (n = 3–5/group) for qPCR assays to confirm upregulation (B) or downregulation (C). Bar graphs represent biological replicates (means ± SD). Statistical significance determined by t test and FDR-adjusted P value. Values are means ± SE; n, number of biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test. D: Western analysis of relevant intestinal proteins, some of which were dysregulated with Zip14 ablation, using protein lysates of IECs from ΔIEC and F/F mice (n = 3 or 4/group). Claudin-1, E-cadherin, FoxM1, and ZO-1 were from individual blots. The β-actin below each blot serves as the loading control for the corresponding blot above. Occludin and HuR were excised from the same blot. The β-actin below those two blots serves as the loading control. IEC, intestinal epithelial cell.
Figure 4.
Figure 4.
Differential expression of specific genes is retained in cultured intestinal organoids. A: phase contrast micrographs of 3-D intestinal organoids derived from F/F (left) and ΔIEC (right) mice after 10 days of culture. Bar = 400 µm. B: retention of differential gene expression pattern in the intestinal organoids after 10 days of culture (n = 3/group). Values are means ± SE; n, number of biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test.
Figure 5.
Figure 5.
Enterocyte-specific Zip14 deletion alters expression of genes needed for zinc trafficking. A: Zip (Slc39a) transporter mRNA expression in intestinal epithelial cells (IECs) from F/F and ΔIEC mice (n = 4/group). B: ZnT (Slc30a) transporter mRNA expression in IECs from F/F and ΔIEC mice (n = 4/ group). C: metallothionein 1 mRNA expression in IECs, colon, liver, lung, kidney, muscle, and spleen from F/F and ΔIEC mice (n = 3–5/group). D: serum zinc concentrations of F/F and ΔIEC mice (n = 5/group). E: zinc uptake by intestinal organoids from F/F and ΔIEC mice as measured with the zinc fluorophore, FluoZin-3-AM (n = 11 or 12/ group; 4 domes/mouse from 3 individual mice). E: concentrations of zinc, iron, and manganese in IECs from F/F and ΔIEC mice (n = 3 or 4/group). F: zinc uptake by intestinal organoids from F/F and ΔIEC mice as measured with the zinc fluorophore, FluoZin-3-AM (n = 11 or 12/group; 4 domes/mouse from 3 individual mice). G: expression of zinc chaperone Zng1 (Cbwd1) mRNA in IECs from F/F and ΔIEC mice (n = 4/group). Values are means ± SE; n, number of biological replicates. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant; Student’s t test.
Figure 6.
Figure 6.
Chromatin immunoprecipitation (ChIP) assays reveal differences in transcription factor binding to promoters of tight junction and intestinal homeostatic genes in intestinal epithelial cells (IECs) from mice with Zip14 ablation. A: NF-κβ binding of promoter sites for Nos2, Saa1, Saa3, and Il-18. B: FXR binding to Fabp6 promoter and c/EBPα and GATA4 binding to the Fabp1 promoter sites. C: STAT3 and NF-κβ binding to Cldn2 promoter. D: NF-κβ binding to Cldn8 promoter. E: comparative binding of CDX2 (caudal-related homeobox) to two sites of the Cldn-2 promoter and two sites of the Cldn-1 promoter. F: GATA4 binding to three sites of the Cldn2 promoter. qPCR was performed on individual chromatin samples from three mice (n = 3/group), and the enrichment was calculated relative to IgG or 2% input. Binding motifs and transcription binding sites were obtained from Transfac 7.0 and Jaspar. The qPCR analysis of the enrichment transcriptional factor elements in each promoter region was performed relative to the binding site. Values are means ± SE; n, number of biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; Student’s t test.
Figure 7.
Figure 7.
Zip14 ablation decreases histone deacetylase (HDAC) activity and regulates activity of histone H3 interaction in intestinal epithelial cells (IECs). A: intestinal HDAC activity in mucosal scrapings from proximal small intestine, isolated IECs, and total small intestine of Zip14F/F and Zip14ΔIEC mice (n = 4/group). B: Western analysis of ac-H3K9 to estimate total acetylation using lysates from intestinal mucosal scrapings of both genotypes (n = 3/group). C: activity of HDAC3 in IECs from Zip14F/Fand Zip14ΔIEC mice (n = 5/group). D: ChIP-qPCR of H3K4 binding to two separate sites of the Cldn1 and Cldn2 promoters. Data are expressed as percentage of total DNA input for H3K4me3 antibody relative to normal IgG (n = 3/group). E: ChIP-qPCR of H3K27 binding to two separate sites of Cldn1 and Cldn2 promoters. Data are expressed as percentage of total DNA input for H3K27me3 antibody relative to normal IgG (n = 3/group). F: ChIP-qPCR of H3K9 binding to the promoters of Cldn1 and Cldn2. Data are expressed as percentage of total DNA input for H3K9ac antibody relative to normal IgG (n = 3/group). Values are means ± SE; n, number of biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; Student’s t test.
Figure 8.
Figure 8.
Intestinal organoids derived from F/F or ΔIEC mice respond to zinc supplementation with reverses in Cldn1 and Cldn2 expression. A–C: qPCR analyses of specific tight junction genes using organoid RNA. Two groups of organoids were cultured for 10 days. One group was treated with 15 μM of zinc for the last 7 days (n = 3/group). Each organoid culture used Matrigel domes, 6,000 crypts/dome, and 4 domes/well derived from three individual mice from ΔIEC or F/F mice. Values are means ± SE; n, number of biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; one-way analysis of variance (ANOVA) was performed, followed by Tukey’s test.
Figure 9.
Figure 9.
Loss of Zip14 causes epigenetic modifications that disrupt intestinal homeostasis. Integrative model explaining how loss of Zip14-mediated zinc ion transport in intestinal epithelial cells alters histone deacetylase (HDAC) activity that influences tight junction gene expression through differential binding of transcription factors to specific promoters. ZnCh, zinc chaperone. A: intestinal epithelial cells (IECs) from control, F/F mice. Showing normal IL-18 and IL-33 expression and high abundance of Cldn1 and Alpi. B: enterocyte-specific Zip14 (Slc39a14) ablation leads to reduced HDAC activity, specific dysbiosis, decreased alkaline phosphatase activity, endotoxemia, downregulation of protective cytokine genes, greater production of Cldn2 and-8, altered zinc trafficking, and loss of intestinal homeostasis.
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