Different Actions of Intracellular Zinc Transporters ZIP7 and ZIP13 Are Essential for Dermal Development

Abstract

Two mesenchymal zinc transporters, ZIP7 and ZIP13, play critical roles in dermal development. ZIP7 and ZIP13 are the closest among the conserved mammalian zinc transporters. However, whether their functions are complementary remains a controversial issue. In the present study, we found that the expression of ZIP13, but not ZIP7, is elevated by transforming growth factor beta (TGF-β) treatment, indicating that TGF-β-mediated ZIP13 amplification is crucial for collagen production during dermal development. Genome-wide gene expression analysis revealed that ~26% of genes are dependent on either ZIP7 or ZIP13, which is greater than the ~17% of genes dependent on both of them. ZIP7 depletion induces endoplasmic reticulum (ER) stress in mesenchymal stem cells, resulting in significant inhibition of fibrogenic differentiation. However, ZIP13 depletion does not induce ER stress. Though both ZIP7 and ZIP13 contain traditional ER signal peptides for their intracellular localization, their distributions are distinct. When ZIP7 and ZIP13 are coexpressed, their localizations are distinct; ZIP7 is located on the ER, but ZIP13 is located on both the ER and Golgi, indicating that only ZIP13 is a zinc gatekeeper on the Golgi. Our data illustrate that the different actions of ZIP7 and ZIP13 are crucial for dermal development.

Keywords: ZIP13; ZIP7; Zinc; browning; connective tissue; mesenchymal stem cells.

Figure 1

Transforming growth factor beta (TGF-β) induces ZIP13 expression. (A,B) mRNA expression levels in mouse embryo cDNA and (B) human mesenchymal stem cells (hMSCs) after 20 nM TGF-β treatment. Data are representative of three independent experiments (*** p < 0.005).

Figure 2

Genome-wide analysis revealed the distinct roles of ZIP7 and ZIP13. (A) Western blot analysis revealed that ZIP13 protein was successfully downregulated by treatment with ZIP13-targeting siRNA. (B) Scatter plots of averaged log2 intensities of expressed genes in wild-type (WT) versus ZIP7 knockdown (KD) and ZIP13-KD hMSCs. (C) Validation of identified genes whose expression was significantly changed after siRNA treatment by RT-qPCR analysis. Data are representative of three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.005). (D) Identification of differentially expressed genes (DEGs). Genes were screened for p-value < 0.05 and fold change > 0.4428 (95%). (E) Gene expression patterns determined by comparing DEGs obtained from ZIP7-KD and ZIP13-KD hMSCs. Seven clusters were obtained when the two sets of DEGs were compared. (F) Venn diagram based on DEGs obtained by comparing ZIP7-KD and ZIP13-KD hMSCs versus WT hMSCs. Genes with adjusted p-value < 0.05 and log2 fold change > 0.4428 (95% of randomized log2 fold change) are included. (G,H) Log2 fold changes in the expression of ZIP (SLC39) family members (G) and ZNT (SLC30) family members (H) are shown.

Figure 3

Functional analysis of ZIP7 and ZIP13. (A,B) Biological processes enriched in genes uniquely upregulated in either ZIP7-KD (cluster 2) or ZIP13-KD (cluster 4) hMSCs. (B) Biological processes enriched in genes uniquely downregulated in either ZIP7-KD (cluster 5) or ZIP13-KD (cluster 6) hMSCs. (C) Biological processes enriched in genes commonly downregulated in both ZIP7-KD and ZIP13-KD hMSCs (cluster 7). Gene Ontology Biological Process (GOBP) analysis was conducted with DAVID V6.8. Processes with a p-value < 0.05 and gene count > 2 were selected as representative GOBPs. The vertical red line indicates a cutoff p-value = 0.05.

Figure 4

ZIP7 is indispensable for the maintenance of mesenchymal stem cells. (A) mRNA expression levels of zinc transporters and ER stress-response genes in hMSCs were analyzed by RT-qPCR after four days of treatment with each siRNA. Data are representative of three independent experiments (*** p < 0.005). (B) Cell growth curves after treatment with each siRNA. Data are representative of three independent experiments (*** p < 0.005). (C) Only siZIP7 disturbed the differentiation of hMSCs toward fibrogenic and osteogenic lineages. hMSCs were differentiated by their culture in differentiation medium for three weeks. Each siRNA was applied every four days. Relative intensity was calculated with ImageJ software (http://rsbweb.nih.gov/ij/download.html). Data are representative of three independent experiments (* p < 0.05; *** p < 0.005).

Figure 5

Differences in regulatory functions of ZIP7 and ZIP13. (A) Signal peptides of ZIP7 and ZIP13 proteins with potential signal peptidase complex cleavage sites shown by arrows. Red: positively charged amino acids; green: small amino acids conserved in the ER signal peptide. (B) Cellular distribution of ZIP7 and ZIP13 in hMSCs. hMSCs were transfected with plasmids encoding V5-tagged ZIP7 and FLAG-tagged ZIP13. hMSCs were stained with anti-FLAG and anti-V5 antibodies. Inset is magnified. (C) hMSCs were transfected with plasmid encoding V5-tagged ZIP7 and stained with anti-V5 antibody and a Golgi tracker. Inset is magnified. (D) hMSCs were transfected with a plasmid encoding FLAG-tagged ZIP13 and stained with anti-FLAG antibody and either an ER tracker or anti-binding immunoprotein (BIP) antibody. Inset is magnified. White arrows indicate unmerged regions. (E) hMSCs were transfected with a plasmid encoding FLAG-tagged ZIP13 and stained with anti-FLAG antibody and either a Golgi tracker or anti-trans-Golgi network (TGN) antibody. Inset is magnified. Black arrows indicate merged regions.

Figure 6

Functional differences between ZIP7 and ZIP13. ZIP7 is involved in zinc homeostasis in the ER, where it supports classical ER functions such as protein folding and modification. ZIP13 is involved in zinc homeostasis in the Golgi and associated with collagen production. ZIP13 is also expressed in the ER; however, its functional association with the ER remains unknown. Purple dots indicate phosphate involved in receptor-mediated phosphorylation.