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. 2012 Jun;142(7):1536-46.e5.
doi: 10.1053/j.gastro.2012.02.046. Epub 2012 Feb 25.

The zinc transporter Zip14 influences c-Met phosphorylation and hepatocyte proliferation during liver regeneration in mice

Tolunay Beker Aydemir  1 , Harry S Sitren, Robert J Cousins
Affiliations

The zinc transporter Zip14 influences c-Met phosphorylation and hepatocyte proliferation during liver regeneration in mice

Tolunay Beker Aydemir et al. Gastroenterology. 2012 Jun.

Abstract

Background & aims: Zinc homeostasis in cells is maintained through tight regulation of zinc influx, efflux, and distribution to intracellular organelles by zinc transporters. The Zrt-Irt-like protein (ZIP) transporters facilitate zinc influx to the cytosol. Expression of the ZIP family member Zip14 can be induced by inflammatory cytokines, which also initiate liver regeneration. Hepatocyte proliferation is required for liver regeneration. Zinc regulates cell proliferation, tissue growth, and many mitogenic signaling pathways; we investigated its role in hepatocytes.

Methods: Wild-type and Zip14(-/-) mice that underwent partial hepatectomy (70% of liver removed) were used as models of liver regeneration. We also analyzed AML12 hepatocytes that overexpressed Zip14. Proliferation was assessed with proliferating cell nuclear antigen, CD1, and Ki67 markers and along with assays of zinc content was related to protein tyrosine phosphatase 1B (PTP1B) and extracellular signal-regulated kinase 1/2 signaling.

Results: Zip14 was up-regulated and hepatic zinc content increased during liver regeneration. Increased hepatic zinc inhibited activity of the phosphatase PTP1B and increased phosphorylation of c-Met, which promoted hepatocyte proliferation. AML12 cells that overexpressed Zip14 increased in zinc content and proliferation; PTP1B was inhibited and phosphorylation of c-Met increased. The increases in hepatic levels of zinc and hepatocyte proliferation that occurred following partial hepatectomy were not observed in Zip14(-/-) mice.

Conclusions: The transporter Zip14 mediates hepatic uptake of zinc during liver regeneration and for hepatocyte proliferation. These findings indicate that zinc transporter activity regulates liver tissue growth by sequestering zinc. Reagents that regulate ZIP14 activity might be developed as therapeutics to promote liver regeneration in patients with chronic liver disease.

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Conflict of interest statement

Conflicts of interest

The authors disclose no conflicts.

Figures

Figure 1
Figure 1
Changes in proliferation markers, Zip14 and hepatic zinc in response to PHx over 48 hours. (A) Liver was collected from PHx and sham-operated mice. Quantitative PCR and Western blot analysis of CD1 and PCNA expression. (B) Total zinc concentrations in liver and serum. (C) Quantitative PCR analysis of ZIP transporters. (D) Quantitative PCR and Western blot analysis showing Zip14 expression after PHx. Values are means ± SD (n = 5 for PHx group and n = 3 for sham-operated group). *P < .05, **P < .01, ***P < .0001.
Figure 2
Figure 2
Dietary zinc content influences serum ALT activity and liver PCNA expression. (A) Serum ALT activity for 48 hours after PHx. (B) Serum ALT activity of mice fed a low (ZnD), adequate (ZnA), or high (ZnH) level of dietary zinc for 1 week with blood obtained 24 hours after PHx. (C) PCNA expression as measured by quantitative PCR and Western blot analysis in liver from mice in B. (D) Liver/body weight ratio measurements. Values are means ± SD (n = 5 for PHx and n =3 for sham groups).
Figure 3
Figure 3
Zinc treatment of AML12 hepatocytes influences proliferation in response to HGF stimulation. (A) Following 20 hours of serum starvation, AML12 hepatocytes were pre-treated with a combination of pyrithione and 8 μmol/L zinc and then total zinc concentrations were measured. (B–D) Cells were incubated with zinc before addition of HGF. After 48 hours, cells were harvested and (B and C) PCNA expression and (D) BrdU incorporation were measured. Values are means ± SD (n = 3–5).
Figure 4
Figure 4
Comparison of Zip14 up-regulation with the intracellular zinc concentration and proliferation of AML12 hepatocytes. Macrophage CM was obtained from LPS-treated RAW cells. (A) AML12 hepatocytes were incubated with CM for 2 hours. Cells were harvested, and Zip14 expression was measured by quantitative PCR and Western blot analysis. The same samples were used to measure (B) metallothionein mRNA and (C) PCNA mRNA and protein. (B) Total zinc concentration of AML12 hepatocytes was measured. Values are means ± SD (n =3–9). Values with a different superscript are statistically different (P < .05 to .001).
Figure 5
Figure 5
Effect of zinc on PTP1B activity and c-Met pathway. AML12 hepatocytes were treated with either pyrithione or 8 μmol/L zinc, along with (A) phosphatase inhibitors for 30 minutes or (B) macrophage CM for 2 hours. Cells were harvested and total membrane fractions were incubated with phosphosubstrate and inorganic phosphate release was measured. (C) AML12 hepatocytes were treated with either 2 different concentrations of zinc or CM for 2 hours. Cells were harvested and total cell lysates were used for the detection of indicated proteins by Western blot analysis. Values are means ± SD (n = 3) (B). Values with a different superscript are statistically different (P < .05 to .001).
Figure 6
Figure 6
Zip14 overexpression in AML12 hepatocytes influences proliferation and the c-Met signaling pathway. AML12 hepatocytes were transfected with either pCMV Sport-Zip14 or Zip14 small interfering RNA and then serum starved for 20 hours. Next, cells were incubated either with HGF and BrdU or HGF alone for 24 hours. (A, B, and E) Cells were harvested and used to measure Zip14 and PCNA expression or BrdU incorporation. (C) After 96 hours of Zip14 transient transfection, total cell membranes were isolated and used for assay of phosphatase activity. (D and F) Cells were serum starved in the last 20 hours of transfection and then were incubated with HGF for 30 minutes. Total cell lysates were used for Western blot analysis. Values are means ± SD (n = 3–5).
Figure 7
Figure 7
Zip14−/− reduces hepatic zinc accumulation and proliferation in response to PHx. The PHx was performed on WT and Zip14−/− mice. (A) Zip14 protein expression and (B) liver zinc concentration were measured. (C) Liver CD1, PCNA protein, and Ki67 staining were measured by Western blot and immunohistochemistry, respectively. Quantification of Ki67-positive cells was performed by digital imaging. (D) Serum ALT measurements. (E) Liver/body weight ratio measurements. Values are means ± SD (n = 4). (F) Schematic representation of involvement of zinc and Zip14 in LR. Kupffer and innate immune system cells produce IL-6, which up-regulates Zip14 at the priming step of LR. Zip14 up-regulation leads to an increase in hepatic zinc concentration. Increased hepatic zinc inhibits PTP1B activity and thus enhances hepatocyte proliferation by increased c-Met–ERK1/2 phosphorylation.
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