Hepatic ZIP14-Mediated Zinc Transport Is Required for Adaptation to Endoplasmic Reticulum Stress

Hepatic ZIP14-mediated zinc transport is required for PNAS PLUS adaptation to endoplasmic reticulum stress

Min-Hyun Kima, Tolunay B. Aydemira, Jinhee Kima, and Robert J. Cousinsa,b,1

aFood Science and Human Nutrition Department, Center for Nutritional Sciences, College of Agricultural and Life Sciences, University of Florida, Gainesville, FL 32611; and bDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32611

Edited by Patrick J. Stover, Cornell University, Ithaca, NY, and approved June 13, 2017 (received for review March 9, 2017) Extensive endoplasmic reticulum (ER) stress damages the liver, maladaptation against ER stress. Sustained apoptosis from ER causing apoptosis and steatosis despite the activation of the un- stress influences numerous pathological conditions, including folded protein response (UPR). Restriction of zinc from cells can diabetes and obesity (8, 13). In the liver, apoptosis disrupts ho- induce ER stress, indicating that zinc is essential to maintain normal meostasis of lipid regulation, causing hepatic steatosis (14). ER function. However, a role for zinc during hepatic ER stress is Zinc is an essential mineral required for normal cellular func- largely unknown despite important roles in metabolic disorders, tions playing catalytic, structural, and regulatory roles (15). To including obesity and nonalcoholic liver disease. We have explored a maintain zinc homeostasis, mammalian cells use 24 known zinc role for the metal transporter ZIP14 during pharmacologically and transporters that tightly control the trafficking of zinc in and out of − − high-fat diet–induced ER stress using Zip14 / (KO) mice, which ex- cells and subcellular organelles. These transporters are within two hibit impaired hepatic zinc uptake. Here, we report that ZIP14- families: ZnT (Zinc Transporter; SLC30) and ZIP (Zrt-, Irt-like mediated hepatic zinc uptake is critical for adaptation to ER stress, protein; SLC39) (16–18). Physiological stimuli have been shown to preventing sustained apoptosis and steatosis. Impaired hepatic zinc regulate the expression and function of some of these transporters. uptake in Zip14 KO mice during ER stress coincides with greater Ablation of some of these transporters results in zinc dyshomeo- expression of proapoptotic proteins. ER stress-induced Zip14 KO mice stasis and metabolic defects. Ablation of Zip14 (Slc39a14) in mice show greater levels of hepatic steatosis due to higher expression of leads to a phenotype that includes endotoxemia, hyperinsulinemia, genes involved in de novo fatty acid synthesis, which are suppressed increased body fat, and impaired hepatic insulin receptor traf- in ER stress-induced WT mice. During ER stress, the UPR-activated ficking (19–21). Zinc and zinc transporters have been implicated α transcription factors ATF4 and ATF6 transcriptionally up-regulate in ER stress and the UPR. Zinc deficiency may induce or exac- Zip14 expression. We propose ZIP14 mediates zinc transport into erbate ER stress and apoptosis. In yeast and some mammalian hepatocytes to inhibit protein-tyrosine phosphatase 1B (PTP1B) ac- cells, the UPR was activated by zinc restriction (22, 23). A rat tivity, which acts to suppress apoptosis and steatosis associated with Zip14 model of alcoholic liver disease created by zinc deficiency was hepatic ER stress. KO mice showed greater hepatic PTP1B ac- shown to trigger ER stress-induced apoptosis (24). We reported tivity during ER stress. These results show the importance of zinc that consumption of a zinc-deficient diet by mice exacerbated ER trafficking and functional ZIP14 transporter activity for adaptation stress-induced apoptosis and hepatic steatosis during TM-induced to ER stress associated with chronic metabolic disorders. ER stress (25). Those recent studies showed that zinc can modulate the proapoptotic p-eIF2α/ATF4/CHOP pathway during ER unfolded protein response | p-eIF2α/ATF4/CHOP pathway | apoptosis | stress. A number of zinc transporters have also been associated steatosis | zinc metabolism with ER stress and the UPR. Administration of TM altered expression of numerous zinc transporter genes in mouse liver, in- he endoplasmic reticulum (ER) is a cellular organelle where cluding ZnT3, ZnT5, ZnT7, Zip13,andZip14 (23). In other Tappropriate folding, assembly, and modification of proteins vertebrate cells, knockdown (KD) of ZnT3, ZnT5, ZnT7,and occur (1). Normal ER function can be compromised by pharmacological stimuli, such as tunicamycin (TM) or thapsigargin (2, 3), Significance and by physiological stimuli, such as a high-fat diet (HFD), viral infection, oxidative stress, or chronic alcohol consumption (4–8). Perturbed ER function is collectively defined as ER stress. When Unresolved endoplasmic reticulum (ER) stress corresponds with ER stress occurs, mammalian cells activate the unfolded protein various chronic diseases, such as hepatic steatosis and diabetes. response (UPR), which comprises three discrete signaling path- Although cellular zinc deficiency has been implicated in causing ER stress, the effect of disturbed zinc homeostasis on hepatic ways: the activating transcription factor 6 (ATF6) branch, the ER stress and a role for zinc during stress are unclear. This study inositol-requiring enzyme 1 (IRE1) branch, and the dsRNA- reveals that ER stress increases hepatic zinc accumulation via activated protein kinase R-like ER kinase (PERK) branch (9). enhanced expression of metal transporter ZIP14. Unfolded These pathways act to relieve the protein burden of the ER by protein response-activated transcription factors ATF4 and reducing translation through mRNA decay (10) and by enhancing ATF6α regulate Zip14 expression in hepatocytes. During ER protein-folding capacity through the expression of ER chaperones, stress, ZIP14-mediated zinc transport is critical for preventing including 78-kDa glucose-regulated protein (GRP78/BiP) and 94- prolonged apoptotic cell death and steatosis, thus leading to kDa glucose-regulated protein (GRP94) (11). Cells survive if ER

hepatic cellular adaptation to ER stress. These results highlight SCIENCES function is recovered through the activation of these adaptation the importance of normal zinc transport for adaptation to ER

pathways. However, if components of the UPR are compromised stress and to reduce disease risk. APPLIED BIOLOGICAL or ER stress is too severe, the UPR leads cells to apoptotic cell death via PERK-mediated phosphorylation of eukaryotic trans- Author contributions: M.-H.K. and R.J.C. designed research; M.-H.K., T.B.A., and J.K. performed lation initiation factor 2α (eIF2α) (12). Phosphorylated eIF2α research; M.-H.K., T.B.A., and R.J.C. analyzed data; and M.-H.K. and R.J.C. wrote the paper. (p-eIF2α) selectively enhances translation of activating transcrip- The authors declare no conflict of interest. tion factor 4 (ATF4) mRNA, which leads to up-regulation of C/EBP- This article is a PNAS Direct Submission. homologous protein (CHOP), a transcription factor that induces 1To whom correspondence should be addressed. Email: [email protected]. expression of apoptosis-associated components. Thus, enhanced This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. activation of the p-eIF2α/ATF4/CHOP pathway is a hallmark of 1073/pnas.1704012114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1704012114 PNAS | Published online July 3, 2017 | E5805–E5814 Downloaded by guest on September 30, 2021 Zip13 induced or potentiated ER stress (26–28). The role for by ER stress: the pancreas, kidney, and white adipose tissue (Fig. ZIP14 in ER stress has not been defined. S1 B–D; WT). Therefore, the liver was our focus of further ex- In the present study, we explored a role of ZIP14 during periments. Because zinc homeostasis is maintained by zinc trans- − − pharmacologically, HFD-induced ER stress using a Zip14 / (KO) porter activity, we examined expression of 24 known zinc mouse model, which exhibits impaired hepatic zinc uptake. We transporter genes in the liver. The primer/probe sequences of genes report that during UPR activation, ATF4 and ATF6α transcrip- are provided in Table S1. Among ZIP family transporters, Zip14 tionally regulate Zip14 expression and that ZIP14-mediated hepatic expression was most highly up-regulated by TM (∼8.2-fold) (Fig. zinc accumulation is critical for adaptation to ER stress by pre- 1D). Among ZnT family transporters, ZnT3 mRNA expression was venting sustained apoptosis and steatosis. We propose that ZIP14- markedly increased after administration of TM (∼15-fold) (Fig. mediated zinc transport suppresses protein-tyrosine phosphatase 1B 1E). However, ZnT3 has a low hepatic abundance (16), and was (PTP1B) activity, which may contribute to overcoming ER stress. not further evaluated. ZIP14 mRNA and protein peaked at 12 h after administration of TM (Fig. 1 F and G), which coincided with Results maximum zinc accumulation (Fig. 1A). The increased hepatic zinc TM Administration Alters Hepatic Zinc Homeostasis and Zinc Transporter concentration and ZIP14 expression were also observed in mice Expression, Particularly ZIP14. First, to define the effect of ER stress injected with another ER stress inducer, thapsigargin, an inhibitor + on zinc homeostasis, TM, a potent ER stress inducer, was in- of ER Ca2 -ATPase (Fig. S2 A and B). Because liver tissue is jected i.p. into mice to induce systemic ER stress. TM is a composed of multiple cell types, we used human hepatoma compound that blocks N-glycosylation of newly synthesized HepG2 cells to support the in vivo studies. To measure total cel- protein. As measured by atomic absorption spectrophotome- lular zinc level in HepG2 hepatocytes, cell lysates were incubated try, TM-injected mice showed significantly higher hepatic with the zinc fluorophore FluoZin3-AM. Intensity of fluorescence zinc concentrations, along with hypozincemia, compared with represents the total cellular labile zinc concentration. Consistent vehicle-injected mice (Fig. 1 A and C). At 12 h after administra- with data from the liver of TM-treated mice, TM treatment sig- tion, liver zinc concentrations of the TM group were ∼15% nificantly increased fluorescence (∼2.1-fold after 12 h) (Fig. 1H), higher than liver zinc concentrations of the control group. Ad- which coincided with increased ZIP14 expression (Fig. 1I). ministration of 65Zn by gavage confirmed markedly increased zinc uptake by the liver after administration of TM (∼1.6-fold) (Fig. Zip14 KO Mice Display Impaired Hepatic Zinc Uptake and Higher 1B). No difference in zinc uptake was observed 12 h after ad- Apoptosis During TM-Induced ER Stress. Next, we examined a po- ministration of TM in tissues that are known to be highly affected tential role of ZIP14 during ER stress because increased hepatic

Fig. 1. TM-mediated ER stress increases hepatic zinc uptake and ZIP14. Hepatic Zn concentration (A) and 65Zn uptake (B) in mice (n = 3–4) are shown after administration of TM (2 mg/kg) or vehicle. (C) Serum Zn concentration in mice (n = 3–4) 18 h after administration of TM (2 mg/kg) or vehicle. Relative expression of members of the ZIP family (D) and ZnT family transporter (E) genes in the liver of mice (n = 3–4) are shown after administration of TM (2 mg/kg) or vehicle for 12 h. Time-dependent expression of Zip14 mRNA (n = 3–4) (F) and immunoblot analysis of ZIP14 and markers of ER stress (G) in liver lysates (n = 3–4, pooled samples) are shown after administration of TM or vehicle for the indicated times. (H) Total cellular Zn concentrations in HepG2 cells were determined by measurement of fluorescence after incubation with FluoZin3-AM (5 μM) following treatment with TM (1 μg/mL) or vehicle (n = 5). RFU, relative fluorescent unit. (I) Immunoblot analysis of ZIP14 and markers of ER stress in lysates of HepG2 cells after TM (1 μg/mL) or vehicle treatment. All data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Cont, control.

E5806 | www.pnas.org/cgi/doi/10.1073/pnas.1704012114 Kim et al. Downloaded by guest on September 30, 2021 ZIP14 expression and zinc accumulation after TM coincided with GRP94 than WT mice (∼0.6-fold) (Fig. 2D), which may indicate PNAS PLUS a marked reduction of CHOP expression (Fig. 1G). To test the an impaired protein folding function. hypothesis that ZIP14-mediated zinc uptake is critical to suppress To test if there is a direct effect of zinc in the HepG2 hepa- − − apoptosis, we used conventional Zip14 / (KO) mice (Fig. 2A). tocytes, we knocked down Zip14 with siRNA and then supple- During steady-state conditions, the liver of Zip14 KO mice did not mented zinc acetate along with pyrithione, a zinc ionophore. show any indices of UPR activation (Fig. S3). Following TM, Pyrithione was added to improve zinc access under Zip14 KD Zip14 KO mice exhibited less significant change in hepatic zinc conditions. The efficiency of Zip14 KD using siRNA transfection ∼ A Zip14 uptake, whereas WT mice displayed significant zinc accumulation was 90% (Fig. S4 ). TM-treated KD cells showed an ∼ (Fig. 2B). Measurement of radioactivity after 65Zn administration 24% lower cellular zinc level compared with TM-treated con- A by gavage also showed impaired zinc uptake in Zip14 KO mice trol cells, as measured by FluoZin3-AM (Fig. 3 , first four bars). during ER stress (Fig. 2C). Expression of UPR pathway compo- To establish an optimal level of zinc supplementation, we treated α cells with zinc acetate, ranging from 2.5 to 20 μM. A maximal nents, including proapoptotic pathway proteins (p-eIF2 ,ATF4, Zip14 and CHOP) and adaptation pathway proteins (BiP and GRP94), level of cellular zinc was obtained with TM-treated KD cells when 5 μM zinc acetate was added along with pyrithione was examined. WT mice showed a marked reduction of proa- (Fig. 3A). Thus, 5 μM zinc acetate was used subsequently to test poptotic protein expression 24 h after administration of TM, the effects of zinc supplementation. Higher expression of ATF4 whereas Zip14 KO mice displayed sustained expression (Fig. 2D). (∼2.6-fold) and CHOP (∼1.8-fold) and lower expression of TUNEL assays of liver sections showed a significantly greater ∼ Zip14 Zip14 ∼ GRP94 ( 0.4-fold) were observed in KD cells after TM number of TUNEL-positive cells in KO mice ( 2.3-fold), treatment (Fig. 3B, lanes 3 and 4). In response to TM, zinc- indicating that the KO mice had a greater level of apoptosis after supplemented Zip14 KD cells expressed markedly reduced E TM administration (Fig. 2 ). Serum alanine aminotransferase ATF4 (∼34%) and CHOP (∼50%) proteins compared with non– activity, a common marker for liver damage, was also greater zinc-supplemented Zip14 KD cells (Fig. 3B, lanes 4 and 6). In Zip14 ∼ in KO mice after administration of TM ( 2.5-fold) (Fig. addition, expression of GRP94 was increased (∼1.6-fold) after 2F). Furthermore, Zip14 KO mice expressed significantly less zinc supplementation of the cells. TM treatment increased p- eIF2α expression, which was reduced after zinc supplementation in Zip14 KD cells (Fig. 3B). The lower cell viability shown in TM-treated Zip14 KD cells (Fig. 3C) was eliminated by zinc supplementation (Fig. 3D).

Zip14 KO Mice Show a Greater Level of Hepatic Steatosis During TM- Induced ER Stress. Prolonged ER stress in the liver has been linked to the occurrence of hepatic steatosis. Greater levels of lipid droplet accumulation in Zip14 KO mice were observed with H&E staining of the liver sections after administration of TM (∼1.9-fold) (Fig. 4A). Quantification of triglyceride (TG) accumulation in the liver supported this observation (Fig. 4B). Be- cause hepatic lipid homeostasis is mainly maintained by four mechanisms, namely, de novo fatty acid (FA) synthesis, FA oxidation, FA uptake into the liver, and lipoprotein secretion, we measured expression of genes involved in these pathways in the Zip14 KO mice to elucidate which mechanism(s) led to severe steatosis. With TM administration, genes involved in de novo FA synthesis, such as Srebp1c, Acc, Fasn, and Scd1, were significantly suppressed in WT mice by ∼60%, ∼85%, and ∼74%, respectively (Fig. 4C). In contrast, expression of Srebp1c, Acc, Fasn, and Scd1 in TM-treated Zip14 KO mice was ∼2.5-fold, ∼2.2-fold, ∼3.2- fold, and ∼fourfold greater, respectively, than in TM-treated WT mice, indicating that FA synthesis in Zip14 KO mice was enhanced during the stress. In the same setting, there were no significant differences in expression of genes involved in FA oxidation, FA uptake, and lipoprotein secretion (Fig. 4D).

HFD-Mediated ER Stress Is Exacerbated in Zip14 KO Mice. Feeding an HFD has been used to trigger ER stress in rodents and provides a model with high physiological relevance (8). Thus, we analyzed in- Fig. 2. ZIP14 KO mice show greater ER stress-induced apoptosis. (A) Immu- − − noblot analysis of ZIP14 from liver lysates of WT and Zip14 / (KO) mice 12 h dicesofERstressafterfeedingmicewithanHFD(60kcal%fat)or after administration of TM (2 mg/kg) or vehicle. Hepatic Zn concentration chow diet (12 kcal% fat). After 16 wk, body weights of HFD-fed (B)and65Zn uptake (C)inWTandZip14 KO mice (n = 3–4) are shown after mice increased 10.2 ± 2.6 g, whereas chow-fed mice gained 2.8 ± SCIENCES administration of TM (2 mg/kg) or vehicle. (D) Immunoblot analysis of ER stress 0.2 g. Both genotypes gained weight at the same rate during HFD = – markers from liver lysates of WT and Zip14 KO mice (n 3 4, pooled samples) feeding. WT mice showed enhanced ZIP14 expression after the APPLIED BIOLOGICAL after administration of TM (2 mg/kg). Individual blots (24 h after TM, n = 4) HFD (Fig. 5 A and B). Hepatic zinc concentrations in HFD-fed WT were quantified using digital densitometry to determine relative protein mice were ∼17% higher compared with chow-fed WT mice, in- abundance. (E) Representative images of TUNEL assays of liver sections of WT dicating that an HFD increases zinc uptake (Fig. 5C). However, zinc and Zip14 KO mice 24 h after administration of TM (2 mg/kg) or vehicle. levels in HFD-fed Zip14 KO mice were unchanged, indicating that TUNEL-positive cells in fields were quantified. (Magnification: 40×.) (Scale bars: μ ZIP14 facilitates hepatic zinc accumulation. Although Zip9 mRNA 25 m.) (F) Serum alanine aminotransferase (ALT) activity of WT and Zip14 KO A mice 24 h after administration of TM (2 mg/kg) or vehicle (n = 3–4). n.d., not expression was also increased by the HFD (Fig. 5 ), its hepatic detected. All data are represented as mean ± SD. *P < 0.05. Labeled means abundance is known to be low (16); thus, it is unlikely that without a common letter differ significantly (P < 0.05). ZIP9 contributes markedly to hepatic zinc uptake. This conclusion

Kim et al. PNAS | Published online July 3, 2017 | E5807 Downloaded by guest on September 30, 2021 Fig. 3. Supplementation with zinc rescues ER stress-induced apoptosis in Zip14 KD hepatocytes. (A) Total cellular Zn concentrations were determined by measurement of fluorescence after incubation with FluoZin3-AM (5 μM). HepG2 cells were treated with the indicated dose of zinc acetate and/or pyrithione (50 μM) for 30 min, which was followed by TM (1 μg/mL) or vehicle treatment for 12 h. (B) Immunoblot analysis of ZIP14 and ER stress markers from cell lysates. Zip14 siRNA- transfected or control siRNA-transfected HepG2 cells were incubated for 30 min with zinc acetate (5 μM) and pyrithione (50 μM), which was followed by a 24-h incubation with TM (1 μg/mL) or vehicle. Individual blots (lanes 3–6, n = 3) were quantified using digital densitometry to determine relative protein abundance. (C and D) Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (C) Zip14 siRNA-transfected or control siRNA-transfected HepG2 cells were treated with TM (1 μg/mL) for 1 d or 2 d. (D) Cells were pretreated with zinc acetate (5 μM) and pyrithione (50 μM) for 30 min before TM (1 μg/mL) treatment for 18 h. All data are represented as mean ± SD. *P < 0.05. Labeled means without a common letter differ significantly (P < 0.05). Csi, control siRNA.

is also supported by showing that hepatic zinc does not increase in response to the HFD (Fig. 5D). However, HFD-fed Zip14 KO due to HFD consumption in Zip14 KO mice (Fig. 5C). Both gen- mice expressed greater levels of proapoptotic signatures, including otypes showed elevated expression of UPR components, including p-eIF2α, ATF4, and CHOP, compared with HFD-fed WT mice, p-eIF2α,ATF4,CHOP,BiP,andGPR94,indicatingUPRactivation indicating that ablation of Zip14 worsens ER stress-associated

Fig. 4. Zip14 KO mice exhibit a greater level of hepatic TG accumulation after TM-induced ER stress. (A) Representative images of H&E-stained liver sections of WT and Zip14 KO mice 24 h after administration of TM (2 mg/kg) or vehicle. The lipid droplet area in the field was measured. (Magnification: 10×.) (Scale bars: 100 μm.) (B) Liver TG levels of WT and Zip14 KO mice were measured 24 h after administration of TM (2 mg/kg) or vehicle (n = 3–4). Relative expression of genes that regulate FA synthesis (C)andFAβ-oxidation, FA uptake, and lipoprotein secretion (D)weremeasuredinliversofWTandZip14 KO mice 12 h after administration of TM (2 mg/kg) or vehicle (n = 3–4). All data are represented as mean ± SD. Labeled means without a common letter differ significantly (P < 0.05).

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Fig. 5. HFD-fed Zip14 KO mice show greater hepatic ER stress-induced apoptosis and TG accumulation. Mice were fed the HFD or a chow diet for 16 wk. (A) Relative gene expression of members of the ZIP family transporter in WT mice (n = 4). (B) Immunoblot analysis of ZIP14 from liver lysates of WT and KO mice. (C) Hepatic Zn concentration of WT and Zip14 KO mice (n = 4). (D) Immunoblot analysis of ER stress markers from liver lysates of WT and KO mice (n = 4, pooled samples used). Individual blots (HFD, n = 4) were quantified using digital densitometry to determine relative protein abundance. (E) Liver TG levels were quantified in WT and Zip14 KO mice (n = 4). (F) Relative expression of genes that regulate FA synthesis were measured in livers of WT and Zip14 KO mice (n = 4). All data are represented as mean ± SD. *P < 0.05. Labeled means without a common letter differ significantly (P < 0.05).

apoptosis in this setting. Expression of ATF4 and CHOP was cantly greater in Zip14 KO mouse liver (∼1.7-fold) (Fig. 6D). The ∼3.7-fold and ∼3.2-fold greater, respectively, in Zip14 KO mice. same pattern was observed in HFD-fed mice. PTP1B activity was Of particular note is the similarity of these results to the results significantly greater in the liver of Zip14 KO mice compared with attained with TM-induced ER stress. Specifically, the HFD-fed WT mice (∼1.5-fold), although the amount of protein expression Zip14 KO mice expressed a lower level of GRP94 than HFD- was not different (Fig. 6 E and F). In HepG2 hepatocytes, Zip14 fed WT mice (∼0.6-fold). Hepatic TG accumulation after the siRNA KD did not alter PTP1B protein levels (Fig. 6G). In HFD tended to be higher in Zip14 KO mice (Fig. 5E). Moreover, contrast, PTP1B activity was increased in the hepatocytes follow- this observation coincided with significantly higher mRNA ex- ing Zip14 siRNA KD and TM treatment, but it was significantly pression of Srebp1c (∼1.7-fold), Fasn (∼1.8-fold), and Scd1 (∼1.9- reduced with zinc supplementation (Fig. 6H). These data dem- fold) (Fig. 5F), suggesting a greater level of FA synthesis led to onstrate a direct effect of zinc on PTP1B activity in this setting. more hepatic TG accumulation in the Zip14 KO mice. Collectively, these data suggest that normal hepatocytes suppress PTP1B activity during ER stress by regulating cellular zinc avail- Increased PTP1B Activity Is Observed in Zip14 KO Mice During ER ability via ZIP14 induction. Impaired zinc uptake in the KO mice Stress. As a possible mechanism underlying the increased ER or by siRNA KD disturbs these events. A model underlying stress in Zip14 KO mice, we focused on PTP1B, an enzyme that ZIP14-mediated adaptation and defense against ER stress is has been implicated in ER stress. PTP1B expression was in- presented in Fig. 6I. creased in ER stress induced by TM and the HFD, and deletion of the protein in vivo and in vitro significantly reduced ER stress- Zip14 Is Transcriptionally Regulated by ATF4 and ATF6α During TM associated apoptosis and steatosis (29–31). Because zinc is a Treatment. Because we observed an induction of Zip14 during known inhibitor of PTP1B activity (32, 33), we hypothesized that TM- and HFD-induced ER stress, our next focus was to identify Zip14 KO would exhibit increased PTP1B activity during ER transcription factor(s) that regulate Zip14. For efficient gene stress due to impaired zinc uptake, and thus disrupt adaptation manipulation, we performed in vitro experiments using HepG2 to ER stress. Consistent with previous reports (30, 31), expres- hepatocytes. During TM treatment, Zip14 mRNA was increased sion of proapoptotic proteins was significantly decreased in TM- in a dose-dependent manner, and treatment with actinomycin D, SCIENCES Ptp1b treated KD cells compared with TM-treated control cells a transcription inhibitor, suppressed induction, indicating APPLIED BIOLOGICAL (Fig. 6A). ATF4 and CHOP expression was reduced by ∼42% that Zip14 induction by TM is regulated at the transcrip- and ∼30%, respectively, in the Ptp1b KD condition. This finding tional level (Fig. 7A). Transcriptional regulation was sup- was supported by markedly higher cell viability in Ptp1b KD cells ported by the time-dependent increases in Zip14 mRNA and after TM challenge (Fig. 6B). At steady state, Zip14 KO mice heterogeneous nuclear RNA. Both exhibited similar expression expressed less PTP1B protein than WT mice (∼0.6-fold), but patterns following TM treatment (Fig. 7B). Global gene expression PTP1B activity was comparable (Fig. 6 C and D). Following TM screening data provided clues regarding the potential Zip14-regu- administration, hepatic PTP1B expression was increased in both lating transcription factors ATF4 and ATF6α (34, 35). ATF4 and genotypes (Fig. 6C, TM). However, PTP1B activity was signifi- ATF6α have been shown to have a strong binding affinity to the

Kim et al. PNAS | Published online July 3, 2017 | E5809 Downloaded by guest on September 30, 2021 Fig. 6. ZIP14 is required to suppress hepatic PTP1B activity after TM administration and the HFD. Immunoblot analysis of PTP1B and ER stress markers (A)and measurement of cell viability using the MTT assay (B) in HepG2 hepatocytes transfected with Ptp1b siRNA or control siRNA are shown. Cells were treated with TM (1 μg/mL) or vehicle for 24 h. In A, individual blots (TM, n = 3) were quantified using digital densitometry to determine relative protein abundance. Immunoblot analysis of PTP1B protein (C) and measurement of PTP1B activity (D) in livers of WT and Zip14 KO mice are shown 12 h after administration of TM (2 mg/kg) or vehicle (n = 3–4, pooled samples used for C). Analysis of PTP1B protein (E) and measurement of PTP1B activity (F) in livers of WT and Zip14 KO mice fed with HFD or chow for 16 wk (n = 4, pooled samples used for E) are shown. Immunoblot analysis of PTP1B protein (G) and measurement of PTP1B activity (H) in HepG2 hepatocytes transfected with Zip14 siRNA or control siRNA are shown. Cells were pretreated with zinc acetate (5 μM) and pyrithione (50 μM) for 30 min before TM (1 μg/mL) treatment for 12 h. (I) Proposed model for ZIP14-mediated zinc transport and inhibition of PTP1B activity. All data are represented as mean ± SD. Labeled means without a common letter differ significantly (P < 0.05).

cAMP-response element (CRE) sequence [TGACGT(C/A)(G/A)] due to increased expression and higher binding affinity and ATF6α (Fig. 7C). Matinspector software analysis revealed that the Zip14 then binds to the promoter after ATF4 expression is decreased. promoter has a potential binding site for both ATF4 and ATF6α at −94 to −89, which matches with a core motif of CRE, and was Discussion conserved in mice and humans (Fig. 7D). To test the expression of In this report, we demonstrate that TM- and HFD-induced ER Zip14 under ATF4-KD or ATF6α-KD conditions, HepG2 hepato- stress triggers ZIP14-mediated zinc accumulation in mouse liver. cytes were transfected with either Atf4 or Atf6α siRNA (Fig. S4 B and Both routes to ER stress yield remarkably comparable results C). KD of Atf4 resulted in significantly reduced Zip14 induction at (Figs. 2 and 5). This event is critical for adaptation to ER stress by preventing prolonged apoptosis and steatosis, possibly via both 6 h and 24 h after TM treatment (Fig. 7E), whereas KD of Atf6α inhibition of PTP1B activity. During TM-induced ER stress, reduced induction only at 24 h after TM treatment (Fig. 7G). To Zip14 up-regulation in HepG2 hepatocytes is modulated at the ensure actual binding of transcription factors to the potential binding Zip14 transcriptional level by the UPR-activated transcription factors site of the promoter, we conducted ChIP-PCR. Detection of ATF4 and ATF6α. These findings are summarized in Fig. 8. DNA enrichment revealed high ATF4 DNA binding 6 h after TM During ER stress, cellular metabolism is largely altered by F treatment. Binding was reduced thereafter (Fig. 7 ). However, en- activation of UPR pathways in an effort to restore ER homeo- hanced binding of ATF6α was only detected 24 h after TM treatment stasis. This activation includes enhancing expression of proteins (Fig. 7H). Western blotting showed that ATF4 expression was in- that help the ER folding function, such as ER chaperones, and duced until 12 h after TM treatment, and was then decreased at 24 h reducing translation of mRNA to reduce cellular protein burden (Fig. 7I). This finding suggests a time-dependent regulation by (11). Several lines of evidence indicate that ER stress also alters ATF4 and ATF6α inwhichATF4firstbindstotheZip14 promoter the metabolism of some metals. For example, mice injected with

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Fig. 7. Zip14 is transcriptionally regulated by ATF4 and ATF6α in a time-dependent manner during TM treatment. (A) Relative expression of Zip14 mRNA in HepG2 cells treated with TM (1 μg/mL) and/or actinomycin D (Act D; 2 μg/mL) for 12 h. (B) Relative expression of Zip14 mRNA and heterogeneous nuclear RNA (hnRNA) in HepG2 cells treated with TM (1 μg/mL) or vehicle. (C) Consensus motif of CRE and binding motifs of ATF4 and ATF6. (D) Sequence of mouse and human Zip14 promoter regions (from −120 to +1). Identical nucleotides are indicated by an asterisk. The TGACG sequence (from −94 to −89) is marked by a box. Relative expression of Zip14 mRNA in TM-treated HepG2 cells (1 μg/mL) after transfection with control siRNA, Atf4 siRNA (E), or Atf6α siRNA (G) is shown. Enrichment of DNA bound to ATF4 antibody (F) or ATF6α antibody (H) was measured by quantitative real-time PCR after ChIP assays in TM-treated HepG2 cells (1 μg/mL). Nonspecific rabbit IgG antibody was used as a negative control. (I) Immunoblot analysis of ATF4 and full-length and cleaved ATF6α in TM-treated HepG2 cells (1 μg/mL). All data are represented as mean ± SD. *P < 0.05, **P < 0.01.

TM have been shown to exhibit hypoferremia and iron seques- D–F). Phenotypic changes in Zip14 KO mice, such as hepatic tration in the spleen and liver (36). Regarding zinc metabolism zinc accumulation and steatosis, are remarkably similar in those and ER stress adaptation, it has been shown recently in mice that two models. However, the magnitude of change is relatively TM administration alters hepatic gene expression of multiple marginal in the HFD model compared with TM model. This zinc transporters (23). That observation is supported by our difference may be caused by the chronic vs. acute nature of these present study, in which ZIP14-mediated hepatic zinc uptake was two models of ER stress. The HFD is a well-known model of the observed, along with hypozincemia, after TM and thapsigargin metabolic syndrome, causing insulin resistance or glucose in- treatments (Fig. 1 A–C and Fig. S2A), demonstrating that acute sensitivity (37). Our recent report has shown that HFD-fed ER stress induction alters zinc metabolism. We focused on the (6 wk) Zip14 KO mice do not develop a disturbed metabolic liver because no difference in zinc uptake was observed in the phenotype, such as hepatic insulin resistance or glucose in- pancreas, kidney or white adipose tissue, which are known to be sensitivity (20), indicating that the impaired stress adaptation of highly affected by ER stress (Fig. S1 B–D). Zip14 KO mice is probably not influenced by glucose stress, SCIENCES

We also examined ER stress with an HFD model, which is which is a known cause of ER stress (38). APPLIED BIOLOGICAL more physiologically relevant. It was previously demonstrated Our first question regarding the zinc, ZIP14, and ER stress that 16 wk of HFD feeding to mice induces ER stress in the liver relationship was whether the increased hepatic zinc uptake after (8). The HFD also produced ZIP14-mediated hepatic zinc ac- ER stress would be a factor in stress adaptation. In the sense that cumulation in WT mice, whereas Zip14 KO mice did not accu- ER function is dependent on zinc availability to provide zinc for mulate zinc during that time period (Fig. 5C). The impaired incorporation into and folding of zinc metalloproteins (39), in- hepatic zinc uptake of Zip14 KO mice possibly produced a creased zinc may play a role in the restoration of ER homeo- greater signature of ER stress-induced apoptosis and steatosis, stasis. In support of that notion, it has been shown that a supply which is consistent with the TM-induced ER stress model (Fig. 5 of zinc to the early secretory pathway via ZnT5, ZnT6, and

Kim et al. PNAS | Published online July 3, 2017 | E5811 Downloaded by guest on September 30, 2021 Based on these reports, we hypothesized that ER stress-induced mice increase hepatic ZIP14 expression to enhance zinc uptake for suppression of PTP1B activity. Zip14 KO mice showed significantly elevated PTP1B activity, whereas activity in WT mice remained unchanged after TM- and HFD-induced ER stress (Fig. 6 D and F), possibly due to impaired hepatic zinc uptake. In vitro, a direct effect of zinc on PTP1B activity inhibition was demonstrated using zinc supplementation, where the greater PTP1B activity in TM-treated Zip14 KD cells was reversed by zinc supplementation (Fig. 6H). These data are consistent with our previous study, where increased PTP1B activity was observed in mice fed a zinc-deficient diet compared with mice fed a zinc- adequate diet during TM challenge (25). Disrupted hepatic lipid homeostasis is another hallmark of unresolved ER stress. This defect hasbeenobservedincellswith Fig. 8. Role of ZIP14-mediated zinc transport in ER stress adaptation. Based compromised UPR function. Genetic ablations of UPR components, on the data in this report, we propose a cycle where ER stress sequentially including ATF6α,IRE1α,andeIF2α, result in the development of increases expression of ATF4 and ATF6α. The transcription factors increase hepatic steatosis (14, 54, 55). Loss of ATF4 increased free cholesterol transcription of Zip14, leading to increased ZIP14 in hepatocytes. Enhanced in rodent liver (35), and liver-specific deletion of XBP1 produced transporter activity increases intracellular zinc concentration, leading to inhibition of PTP1B activity. hypocholesterolemia and hypotriglyceridemia (56). Similar to these observations, the liver of Zip14 KO mice showed potentiated TG accumulation after TM administration due to enhanced FA synthesis ZnT7 is essential for helping protein folding via zinc incorporation (Fig. 4). Zinc-mediated PTP1B activity suppression may mechanis- − − into alkaline phosphatases, which are zinc metalloproteins (40, 41). tically explain these data because liver-specific Ptp1b / mice showed The importance of an adequate zinc supply for protein folding was diminished hepatic FA synthesis and TG storage during HFD feed- illustrated when TM-induced ER stress was exacerbated during ing (30), which is consistent with the ER stress-induced TG accu- − − − − zinc-deficient conditions in ZnT5 / /ZnT7 / cells (26). In contrast, mulation we showed in Zip14 KO mice. some metals may exhibit toxicity when their cellular concentrations UPR activation induces a variety of genes involved in protein are elevated and can aggravate processes such as ER stress. For folding, degradation, and trafficking to restore ER homeostasis via example, excessive accumulation of manganese (42–44), cadmium transcription factors, such as ATF6α, ATF4, and XBP-1 (12). We (45–47), and fluoride (48) induces ER stress and UPR activation. propose that one arm of UPR pathway activation is to modulate Here, we found that ZIP14-mediated zinc uptake was critical for zinc trafficking, based on our finding that Zip14 is a transcriptional adaptation to ER stress, because Zip14 KO mice displayed greater target of ATF4 and ATF6α (Fig. 7 E–H). ATF4 and ATF6α have a apoptosis (Figs. 2 D and E and 5D). The demonstration of im- significant binding affinity for the CRE consensus sequence (57–59). Zip14 paired GRP94 expression in ER stress-induced KO mice We found that the Zip14 promoter has a CRE-like sequence, and D D (Figs. 2 and 5 ) supports the notion that GRP94 is a critical demonstrated here that both ATF4 and ATF6α bound to the CRE- factor that supports recovery of ER homeostasis. Although like sequence after TM treatment in a time-dependent manner, – ZIP14 can contribute to uptake of manganese and non transferrin- leading to Zip14 mRNA increases through increased transcription bound iron under certain circumstances (49, 50), the hepatic (Fig. 7). Our data agree with the observations from global tran- concentration of these metals after TM administration was scriptional profiling, which showed significantly reduced Zip14 Zip14 E F − − comparable between WT and KO mice (Fig. S1 and ). mRNA levels after TM treatment in liver-specific Atf4 / mice (35) Zip14 − − In support, in vitro supplementation with zinc rescued KD and in Atf6α / fibroblasts (34). ATF4 and ATF6α regulation of HepG2 hepatocytes from ER stress-induced apoptosis (Fig. 3 B Zip14 suggests that these events lead to increased cellular zinc and D), demonstrating a direct effect of zinc. Because prolonged availability through ZIP14-mediated zinc transport. Of note is that apoptosis causes many pathogenic disorders, suppression of the the Zip14 promoter lacks the sequence required for the liver- p-eIF2α/ATF4/CHOP pathway can be a therapeutic target. Our specific transcription factor CREBH, which is needed for in- data collectively demonstrate that ZIP14-mediated hepatic zinc duction of hepcidin by ER stress (36). Limited evidence indicates uptake also influences this pathway and provides a mechanism that UPR components regulate expression of other zinc transporter by which apoptosis is suppressed. – hZnT5 Our next focus was to elucidate a direct target for zinc. Here, genes. XBP-1 mediated up-regulation during ER stress ZnT5−/− we propose that zinc influences the p-eIF2α/ATF4/CHOP occurs via direct binding to that promoter (26). In addition, / ZnT7−/− pathway through suppression of the activity of PTP1B. Various cells displayed an exacerbated ER stress response (26). Zip7 Zip13 ZnT3 pathways, including the pathways for insulin and leptin signaling, Similarly, KD of , ,and in cells induced ER stress are regulated by PTP1B, and dysregulation of PTP1B activity was and higher cell death, indicating the importance of normal zinc demonstrated to contribute to the pathogenesis of various dis- homeostasis for ER stress adaptation (27, 28, 60). eases, such as cancer and diabetes (51). Therefore, there has In conclusion, we demonstrate that ZIP14-mediated hepatic been a significant effort to develop a PTP1B inhibitor as a zinc uptake plays an important role in suppressing ER stress- drug target (52). PTP1B has been implicated in ER stress (53). induced apoptosis and hepatic steatosis (Fig. 8). ZIP14 displayed a − − Liver-specific Ptp1b / mice showed decreased activation of the protective effect against ER stress by influencing the proapoptotic p-eIF2α/ATF4/CHOP pathway, along with reduced indices of p-eIF2α/ATF4/CHOP pathway and de novo FA synthesis through metabolic syndrome, during TM- and HFD-induced ER stress zinc-mediated inhibition of PTP1B activity. Deletion of Zip14 (30, 31). Consequently, our data (Fig. 6 A and B) are consistent produced a phenotype presenting with hepatic lipid accumulation with the data from TM-treated Ptp1b KD hepatocytes. Zinc is during ER stress. In addition, we demonstrated that Zip14 is a known inhibitor of PTP1B through physical binding to the transcriptionally regulated by ATF4 and ATF6α. The present enzyme (32). We previously reported that overexpression of findings document the importance of functional zinc transporter ZIP14 in AML12 hepatocytes suppressed PTP1B activity (33). for adaptation to ER stress.

E5812 | www.pnas.org/cgi/doi/10.1073/pnas.1704012114 Kim et al. Downloaded by guest on September 30, 2021 Materials and Methods Western Blot Analysis. Tissue samples or cells were homogenized in radio- PNAS PLUS Mice and Diets. Development and characterization of the murine Zip14−/− (KO) immunoprecipitation assay lysis buffer (Santa Cruz Biotechnology) supple- mice have been described previously (19). A colony of Zip14+/− heterozygotes on mented with protease and phosphatase inhibitors (Thermo Fisher) using + + the C57BL/6/129S5 background was used to produce KO and Zip14 / (WT) mice. Bullet Blender (Next Advance). Proteins were then separated by SDS/PAGE The mice were backcrossed on a mixed 129S5 and C57BL/6 background. Mice had and transferred to a nitrocellulose membrane. Polyclonal rabbit antibody free access to a chow diet (Teklad 7912; Harlan) and tap water, and were against ZIP14 was raised in-house as described previously (61). Purchased maintained on a 12-h light/dark cycle. The same response to treatments was seen antibodies were BiP, CHOP, and phosphorylated eIF2α (Ser52) (all from Santa – in both male and female mice in pilot experiments. Young adult (8 16 wk of age) Cruz Biotechnology); ATF4, GRP94, and PTP1B (all from Cell Signaling); ATF6 male WT and KO mice were used for TM experiments. To model HFD-induced ER (Novus Bio); and tubulin (Abcam). Immunoreactivity was visualized by ECL stress, WT and KO mice were fed either the chow diet (17 kcal% fat) containing reagents (Thermo Fisher). Immunoblots for ATF4, CHOP, and GRP94 were 63 mg of zinc per kilogram as ZnO or an HFD (60 kcal% fat, D12492; Research quantified using densitometry. Diets) containing 39 mg of zinc per kilogram as ZnCO3 for 16 wk.

Statistical Analyses. Results are expressed as mean ± SD. Statistical signifi- Treatments. ER stress was induced by i.p. administration of TM (Sigma) or thapsigargin (Sigma) dissolved in 1% DMSO/150 mM glucose at 2 mg/kg of body cance was analyzed using GraphPad Prism 6 (GraphPad Software). Com- ’ weight (TM) or 1 mg/kg of body weight (thapsigargin), respectively. To assess zinc parisons between two groups were conducted by the Student s t test, and absorption and tissue distribution, 65Zn (2 μCi; PerkinElmer) was given to mice by comparisons among multiple groups were conducted using ANOVA fol- oral gavage 3 h before euthanasia. Accumulated 65Zn in tissue and plasma was lowed by a Tukey’s test. Statistical significance was set at P < 0.05. measured by gamma scintillation spectrometry. Mice were anesthetized by iso- flurane for injections and euthanasia by cardiac puncture. All mice were killed ACKNOWLEDGMENTS. This research was supported by NIH Grant R01DK94244 between 9:00 AM and 10:00 AM. Collected tissues were snap-frozen in liquid and the Boston Family Endowment of the University of Florida Foundation (to nitrogen and stored at −80 °C. All animal protocols were approved by the R.J.C.). M.-H.K. is an Alumni Graduate Fellow of the College of Agricultural and University of Florida Institutional Animal Care and Use Committee. Life Sciences.

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