Influence of ZIP14 (slc39A14) on intestinal zinc processing and barrier function

Abstract

ZIP14 is a zinc transport protein with high expression in the small intestine and liver. Zip14 is upregulated during endotoxemia and leads to increased liver zinc content and transient hypozinemia. Since body zinc status and inflammation are associated with changes in intestinal permeability, we hypothesized that ZIP14 may influence intestinal permeability. Wild-type (WT) and Zip14 knockout (KO) mice were used to determine ZIP14-associated intestinal zinc metabolism and effects on permeability. Fractionation of plasma membranes revealed that ZIP14 is localized to the basolateral membrane of enterocytes. Studies utilizing (65)Zn administered by subcutaneous injection revealed greater zinc accumulation in the SI of Zip14 KO mice compared with WT mice. Isolation of endosomes confirmed the presence of ZIP14. Quantification of endosomal zinc concentration by FluoZin-3AM fluorescence demonstrated that zinc is trapped in endosomes of Zip14 KO mice. Intestinal permeability assessed both by plasma FITC-dextran following gavage and by serum endotoxin content was greater in Zip14 KO mice. Threonine phosphorylation of the tight junction protein occludin, which is necessary for tight junction assembly, was reduced in KO mice. Claudin 1 and 2, known to have an inverse relationship in regards to tight junction integrity, reflected impaired barrier function in KO jejunum. These data suggest involvement of ZIP14 in providing zinc for a regulatory role needed for maintenance of the intestinal barrier. In conclusion, ZIP14 is a basolaterally localized protein in enterocytes and is involved in endosomal trafficking of zinc and is necessary for proper maintenance of intestinal tight junctions.

Keywords: endosomes; endotoxemia; intestinal permeability; mucosal defense; zinc transporter.

Fig. 1.

Zip14 is regionally expressed in mouse intestine and is localized to the basolateral membrane of enterocytes. The intestines of wild-type (WT) mice were separated into duodenum (Duo), jejunum (Jej), ileum (Ile), and colon (Col). In addition, villar (Vil) and crypt (Crp) epithelial cells from the entire intestine were separated by a mechanical method and collected by centrifugation. A: Zip14 mRNA was measured by quantitative PCR (qPCR) with values normalized to TATA binding protein (TBP). Data are expressed as means ± SD (n = 3 mice). B: cellular proteins were examined for ZIP14 expression by Western blot analysis with mZIP14 and β-actin (ACTB) as the loading control. C: immunofluorescence showing localization of ZIP13 (red) at the basolateral membrane of enterocytes of the jejunum of a WT mouse. The mZIP14 antibody as used in (B, D–F) was used to obtain this image. D: mucosa from jejunal tissue was collected from WT mice, and cytoplasmic (Cyt) and total membrane (Mem) fractions were isolated by use of NE-PER reagents. Proteins were analyzed by Western blot as above, plus Na⁺-K⁺-ATPase, and SGLT1 as positive controls for membrane localization. E: total small intestine from WT mice were subjected to apical surface biotinylation and purified by use of streptavidin-conjugated Sepharose beads. The pellet fraction (apical) and supernatant (basolateral/cytoplasmic; Cyt/Bas) were obtained and the proteins were analyzed as in D. SGLT1 served as the apical membrane marker and Na⁺-K⁺-ATPase served as a basolateral membrane marker. F: Caco-2 cells grown to 21-day postconfluence on Transwell plates were subjected to biotinylation in the upper well (apical compartment) and fractionated and analyzed as in E, except hZIP14 was used. Western blots are representative blots from multiple experiments.

Fig. 2.

Zip14 knockout (KO) mice display increased intestinal zinc concentrations via accumulation of systemic zinc. A: intestinal mucosa was obtained from the jejunum of WT and Zip14 KO mice. Abundance of ZIP14 was analyzed by Western blot with mZIP14 with ACTB as the loading control. B: the zinc concentration of jejunal mucosa was measured by absorption spectrophotometry. C: abundance of Mt-1 mRNA in jejunal total RNA. D and E: WT and Zip14 KO mice were fasted overnight then each mouse received an subcutaneous injection of 3 μCi of ⁶⁵Zn. The mice were killed 3 h later. The ⁶⁵Zn content of intestinal tissue and amount of ⁶⁵Zn secreted into the lumen were measured. ⁶⁵Zn was measured and the percentage of jejunal ⁶⁵Zn uptake was calculated as percentage of the total ⁶⁵Zn in the dose administered. F: nonheme iron concentration of jejunal mucosa was determined colorimetrically. Data are expressed as means ± SD (n = 5) (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 3.

Intestinal cell endosomes accumulate zinc with deletion of Zip14. Crude endosomal (CE) fractions were isolated by sucrose gradient centrifugation. A: endosomes were isolated from jejunal mucosa of WT and KO mice. Proteins from CE and total jejunal proteins (TP) were analyzed by Western blot with mZIP14. RAB11 and EEA1 were used as endosomal markers and GPR39, a plasma membrane marker, served as a negative control. B: endosomes from Caco-2 cells were isolated and analyzed as in A. RFU, relative fluorescence units. C: endosomes from WT and KO mice, isolated as in A, were incubated with the intracellular zinc-fluorophore FluoZin-3AM to detect labile zinc. Fluorescence was measured after 1 h. Data are expressed as relative fluorescence and are expressed as means ± SD (n = 3 separate preparations) (***P < 0.001).

Fig. 4.

Intestinal permeability increases with the loss of ZIP14 (KO). A and B: WT mice were injected with LPS (2 mg/kg ip) and were killed 3, 6, and 18 h later. Jejunal and liver tissues were obtained. Intestine and liver total lysates from WT mice were analyzed by Western blot with ACTB (jejunum) or tubulin (liver) as the loading control. C: relative cytokine expression in WT and KO jejunum. The qPCR data were normalized to TBP. Means are expressed as means ± SD (n = 6 mice). D: WT and KO mice were fasted overnight. The mice were administered FITC-dextran by gavage 1 h before being killed. Plasma was prepared and fluorescence was measured. Data were normalized to body weight and expressed as means ± SD (n = 10 mice). E: WT and KO mice were traditionally housed for 4 wk after weaning. Serum was prepared after the mice were killed and endotoxin levels were measured via a spectrophotometric assay. Data are expressed as means ± SD (n = 5 mice) (*P < 0.05; **P < 0.005; ***P < 0.001).

Fig. 5.

ZIP14 deletion (KO) produces differential expression of specific tight junction proteins. Jejunal tissue from WT and Zip14 KO mice was used. A: Western blot analysis of PKCζ, OCLN, and proteins with ACTB as the loading control along with analysis of immunoprecipitated phospho-threonine OCLN (pT-OCLN) with IgG as the loading control (n = 3 mice per treatment). B: CLDN1 and CLDN2 Western blot with ACTB as the loading control (n = 3 mice per treatment). C: representative immunohistochemistry of intestinal CLDN1 and CLDN2 from WT and KO mice.

Fig. 6.

Proposed model for the ZIP14-mediated influence on TJ function and intestinal permeability. ZIP14 is localized to the basolateral membrane and transports endogenous zinc (blue) into enterocytes. A: in WT mice after endocytosis, ZIP14 functions to release vesicular zinc into the cytoplasm where it acts to maintain OCLN phosphorylation. B: in ZIP14 KO mice, zinc is accumulated in vesicles, and there is loss of a gain in CLDN2 and OCLN phosphorylation at the TJ. Changes in these proteins could contribute to loss of TJ barrier function and lead to increased paracellular movement of enteric endotoxins (red) into systemic circulation. Blue represents Zn²⁺ and red represents enteric endotoxin.