Reduced AKT activation accompanied with high TP53 expression is implicated in the impaired hematogenesis in Ziegler-Huang syndrome and the Znt7 null mice partially recapitulates the human disease linked to pancytopenia

Liping Huang, Steven T Nguyen, Zhongyue Yang, Catherine P Kirschke, Clément Prouteau, Marie-Christine Copin, Dominique Bonneau, Odile Blanchet, Coralie Mallebranche, Isabelle Pellier, et al.

<figure><img src="image_1.png" /></figure>

To cite this version:

Liping Huang, Steven T Nguyen, Zhongyue Yang, Catherine P Kirschke, Clément Prouteau, et al. Reduced AKT activation accompanied with high TP53 expression is implicated in the impaired hematogenesis in Ziegler-Huang syndrome and the Znt7 null mice partially recapitulates the human disease linked to pancytopenia. Journal of Trace Elements in Medicine and Biology, 2025, 89, pp.127658. 10.1016/j.jtemb.2025.127658. hal-05460338

HAL Id: hal-05460338

https://nantes-universite.hal.science/hal-05460338v1

Submitted on 15 Jan 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

# Reduced AKT activation accompanied with high TP53 expression is implicated in the impaired hematogenesis in Ziegler-Huang syndrome and the Znt7 null mice partially recapitulates the human disease linked to pancytopenia

Liping Huang<sup>a,b,c,*</sup> <sup>□</sup>, Steven T. Nguyen<sup>a</sup>, Zhongyue Yang<sup>b,1</sup>, Catherine P. Kirschke<sup>a</sup>,

Clément Prouteau<sup>d</sup>, Marie-Christine Copin<sup>e</sup>, Dominique Bonneau<sup>d</sup>, Odile Blanchet<sup>f,g</sup>,

Coralie Mallebranche<sup>g</sup>, Isabelle Pellier<sup>g</sup>, Régis Coutant<sup>i,j</sup>, Charline Miot<sup>g,h</sup>, Alban Ziegler<sup>d,k,**</sup>

## ARTICLE INFO

### Keywords:

Ziegler-Huang Syndrome
SLC30A7
ZNT7
AKT
TP53
bone marrow failure 8

## ABSTRACT

**Background:** Inherited bone marrow failure (IBMF) is a life-threatening condition. Excessive expression of TP53 induces cell cycle arrest and apoptosis of hematopoietic cells in individuals with IBMF. We recently discovered two pathogenic variants, NM_001144884:c.21dup;p.(Asp8ArgfsTer3) and NM_001144884:c.842 + 15 T > C, in ZNT7 associated with IBMF (Ziegler-Huang Syndrome; BMF8). However, the pathophysiologic mechanism of IBMF caused by ZNT7 mutations remained unknown.

**Method:** We investigated TP53 expression and the activation of its upstream regulator, AKT, in cell lines from affected individuals. We rescued the wild-type phenotype of AKT activation via transduction of wild-type ZNT7 into patient's fibroblasts. We performed fluorescence microscopy to assess co-expression patterns of ZNT7 with hematopoietic cell markers in different human and mouse bone marrow cell types. Finally, we evaluated the hematological features of Znt7 deficient mice.

**Results:** The growth of patient's EBV-transformed B (B-EBV) lymphoblasts was impaired. We observed excessive expression of TP53 in the patient's B-EBV lymphoblasts accompanied by a significant decrease in AKT activation. Importantly, overexpression of wild-type ZNT7 in patient's fibroblasts rescued the activation of the AKT pathway by insulin. Additionally, human ZNT7 was expressed in myeloid and lymphoid lineage cells, whereas mouse Znt7 was mainly expressed in the nucleated hematopoietic cells in the respective bone marrow. Despite these differences, we observed progressive cytopenia in Znt7KO mice, partially recapitulating BMF8 in humans.

**Abbreviations:** ZNT, SLC30A; ZIP, SLC39A; IBMF, inherited bone marrow failure; ER, endothelium reticulum; OMIM, online mendelian inheritance in man; SRP72, signal recognition particle 72; ERCC6L2, excision repair 6 like 2; DNAJC21, DNAJ heat shock protein member C21; MYSM1, Myb like SWIRM and MPN-containing protein 1; TP53, tumor protein P53; MDM4, mouse double minute 4; ADH5, alcohol dehydrogenase 5; ALDH2, aldehyde dehydrogenase 2; AKT, protein kinase B serine/threonine kinase; ACTB, beta-actin; STAT3, signal transducer and activator of transcription 3.

* Corresponding author at: USDA/ARS/Western Human Nutrition Research Center, 430 West Health Sciences Drive, Davis, CA 95616, USA.

** Corresponding author at: Department of Genetics, University Hospital of Angers, 4 rue Larrey, Angers Cedex 9, 49333, France.

E-mail addresses: lphuang99@yahoo.com (L. Huang), ziegler.a@chu-toulouse.fr (A. Ziegler).

¹ Current address: Zhongyue Yang, Center for Clinical Sciences Research Atrium, Stanford Medicine, 269 Campus Dr., California 94305, USA

<footnote><sup>a</sup> USDA/ARS/Western Human Nutrition Research Center, 430 West Health Sciences Drive, Davis, CA 95616, USA</footnote>

<footnote><sup>b</sup> Graduate Group of Nutritional Biology, Department of Nutrition, University of California at Davis, One Shields Ave, Davis, CA 95616, USA</footnote>

<footnote><sup>c</sup> Integrative Genetics and Genomics, University of California at Davis, One Shields Ave, Davis, CA 95616, USA</footnote>

<footnote><sup>d</sup> Department of Genetics, University Hospital of Angers, 4 rue Larrey, Cedex 9, Angers 49333, France</footnote>

<footnote><sup>e</sup> Department of Pathology, University Hospital of Angers, Angers 49333, France</footnote>

<footnote><sup>f</sup> Biobank Resource Center, CHU Angers, University Hospital of Angers, 4 rue Larrey, Cedex 9, BB-0033-00038, 49333, France</footnote>

<footnote><sup>g</sup> University Angers Nantes Université CHU Angers INSERM CNRS CRC12NA SFR ICAT, Cedex 9, Angers 49333, France</footnote>

<footnote><sup>h</sup> Immunology and Allergology Laboratory, University Hospital of Angers, 4 rue Larrey, Cedex 9, Angers 49333, France</footnote>

<footnote><sup>i</sup> Department of Pediatric Endocrinology, University Hospital of Angers, Cedex 9, Angers 49333, France</footnote>

<footnote><sup>j</sup> Reference Center for Rare Pituitary Diseases, University of Hospital of Angers, Cedex 9, Angers 49333, France</footnote>

<footnote><sup>k</sup> Department of Medical Genetics, University Hospital of Toulouse, Toulouse 31100, France</footnote>
Conclusion: Excessive expression of TP53 and down-regulation of AKT activation induced by ZNT7 deficiency might impair cell survival, which may contribute to the pathophysiology of bone marrow failure in affected individuals with BMF8.

# 1. Introduction

Zinc is a vital micronutrient within most living organisms for development and growth [1]. It plays a crucial role in biological and physiological processes either as a cofactor or a regulator in DNA replication, hormone synthesis and secretion, signaling transduction, and gene expression [2]. Thus, a low zinc status impairs the function of vital physiological systems of the human body, such as the immune system, the central nervous system, the gastrointestinal tract, and the oxidative system via DNA damage, apoptosis, and oxidative stress [3-5].

Cellular zinc homeostasis is maintained by zinc transporters belonging to two families, namely ZNT (SLC30A) and ZIP (SLC39A) along with other small molecules, such as metallothioneins. The ZNT family has 10 members (SLC30A1-10, aka ZNT1-10) responsible for reducing cytoplasmic zinc concentrations by export of zinc out of the cell or by sequestration of zinc into intracellular organelles or vesicles while the ZIP family members (SLC39A1-14, aka ZIP1-14) do just the opposite to increase the cytoplasm zinc levels by zinc uptake or moving zinc out the storage [6,7]. The expression of these zinc transporters varies significantly in different tissues and cell types allowing for specialized functions conducted in different tissues or organs. For example, ZNT8 is predominantly expressed in β-cells of the pancreatic islet supporting insulin crystallization and glucose-stimulated insulin secretion in the pancreas [8]. Another example is the ZIP4 gene that is almost exclusively expressed in the epithelium of the small intestine responsible for dietary zinc absorption [9]. Hence, genetic disorders caused by mutations in the human zinc transporters present with significant variations in clinical signs. To date, ten genetic disorders caused by mutations in the ZNT and ZIP members have been reported (Table 1), including ZNT2 [10], ZNT7 [11], ZNT9 [12], ZNT10 [13,14], ZIP4 [9], ZIP5 [15], ZIP7 [16], ZIP8 [17,18], ZIP13 [19], and ZIP14 [20]. The phenotypic variability of these genetic disorders suggest that each zinc transporter is uniquely required for a specific function in tissue or cell development and organogenesis.

We have previously reported that ZNT7 is a zinc transporter regulating accumulation of zinc in the Golgi apparatus as well as the vesicular compartment in the cell [21]. ZNT7 acts as a Zn⁺⁺/H⁺ antiporter to move zinc ions from the cytoplasm to the Golgi lumen for zinc incorporation into proteins or acting as a cofactor for enzymatic activities [22,23]. The expression of ZNT7 protein can be detected in a variety of tissues or cells, including the gastrointestinal tract, bone marrow [11], immune cells [24], prostate [25], adipocytes [26], skeletal muscle [27], and testis [11,28]. Znt7 knockout mice (Znt7KO) are mildly zinc deficient accompanied with slower growth and are susceptible to diet-induced insulin resistance [27,29,30]. Znt7 deficiency also increases risk of the onset of prostate cancer and subsequent metastasis in mice [25]. In humans, individuals with Ziegler-Huang Syndrome/BMF8 who carry mutations in ZNT7 showed mild serum zinc deficiency and growth and puberty delay accompanied with delayed bone age and testicular hypoplasia. These characteristics are comparable to those of children presenting with dietary zinc deficiency [1,11,31]. Notably, BMF8 patients exhibited pancytopenia due to bone marrow failure [11], features not previously reported with dietary zinc deficiency. While impaired immune function is a common feature of zinc deficiency resulting from inadequate intake [32] and observed in the Znt7KO mouse model [29], bone marrow failure represents a novel finding of the zinc role in hematopoiesis in humans.

Inherited bone marrow failure (IBMF) is a life-threatening condition, especially when it occurs in the pediatric age [32]. Seven known inherited BMF syndromes (BMFS) with genetically defined entities have

been recorded in OMIM (https://omim.org/; Jan. 16, 2025). These BMFS are 1) BMFS1 (mutations in SRP72 affecting targeting secretory proteins to the ER) [33]; 2) BMFS2 (mutations in ERCC6L2 affecting DNA repair) [34]; 3) BMFS3 (mutations in DNAJC21 affecting ribosomal biogenesis) [34]; 4) BMFS4 (mutations in MYSM1 affecting DNA repair) [35]; 5) BMFS5 (mutations in TP53 affecting DNA repair and apoptosis) [36]; 6) BMFS6 (mutations in MDM4, a TP53 suppressor) [37]; 7) BMFS7 (digenic mutations in ADH5 and ALDH2 affecting DNA replication) [38], and 8) BMFS8 (mutations in ZNT7 affecting cellular zinc homeostasis) [11]. Moreover, the pathogenic causes for other genetic diseases involved in single cytopenia, such as Fanconi anemia (FA), dyskeratosis congenita (DC), Shwachman-Diamond syndrome (SDS), Diamond-Blackfan anemia (DBA), congenital dyserythropoietic anemias (CDAs), severe congenital neutropenia (SCN), and congenital amegakaryocytic thrombocytopenia (CAMT), have been demonstrated having defects in DNA repair (FA), telomere stability (DC), ribosome biogenesis (SDS and DBA), vesicular functions (CDAs & SCN), and thrombopoietin receptor deficiency (CAMT). Taken together, defects in DNA repair/replication and ribosomal biogenesis are the major causes for inherited

<table><caption>**Table 1**
Genetic disorders of human SLC30A and SLC39A proteins.</caption><thead><tr><th>Gene name</th><th>Disease name</th><th>Clinical presentation</th><th>OMIM® ID*</th></tr></thead><tbody><tr><td>SLC30A2/ ZNT2</td><td>Transient neonatal zinc deficiency</td><td>Transient neonatal zinc deficiency</td><td>608118</td></tr><tr><td>SLC30A7/ ZNT7</td><td>Ziegler-Huang Syndrome/Bone marrow failure 8</td><td>Mild zinc deficiency, pancytopenia, bone marrow failure, stunted growth, and hypogonadism</td><td>611149</td></tr><tr><td>SLC30A9/ ZNT9</td><td>Birk-Landau-Perez Syndrome</td><td>Movement disorder, renal impairment, developmental regression, and oculomotor abnormalities</td><td>604604</td></tr><tr><td>SLC30A10/ ZNT10</td><td>Hypermanganesemia with dystonia 1</td><td>Hypermanganesemia, dystonia, and chronic liver disease</td><td>613280</td></tr><tr><td>SLC39A4</td><td>Acrodermatitis enteropathica</td><td>Zinc deficiency</td><td>607059</td></tr><tr><td>SLC39A5</td><td>Myopia 24</td><td>High myopia</td><td>615946</td></tr><tr><td>SLC39A7</td><td>Agammaglobulinemia 9</td><td>Immunodeficiency associated with agammaglobulinemia and absent circulating B cells.</td><td>619693</td></tr><tr><td>SLC39A8</td><td>Congenital disorder of glycosylation, type IIn</td><td>Profound psychomotor retardation with delayed head control, severe hypotonia, inability to walk, variable ability to sit independently, and profound intellectual disability</td><td>616721</td></tr><tr><td>SLC39A13</td><td>Ehlers-Danlos syndrome, spondylodysplastic type, 3</td><td>Postnatal growth retardation, moderate short stature, protuberant eyes with bluish sclerae, hands with finely wrinkled palms, atrophy of the thenar muscles, and tapering fingers</td><td>612350</td></tr><tr><td>SLC39A14</td><td>Hypermanganesemia with dystonia 2</td><td>Hypermanganesemia with progressive dystonia and other neurologic abnormalities.</td><td>617013</td></tr></tbody></table>

<footnote>* Online Mendelian Inheritance in Man®</footnote>
disorders of hematopoiesis in humans [39,40].

Increased TP53 expression or activity in hematopoietic stem cells is strongly associated with bone marrow failure syndromes [41]. For example, BMFS5 is caused by mutations in TP53 that lead to a gain-function variant of TP53 in hematopoietic stem cells [42]. Another example is BMFS6 which is caused by mutations in MDM4 resulting in an increase of TP53 expression in hematopoietic progenitor cells due to lack of binding of the negative regulator of MDM4 to TP53 [28]. Moreover, constantly excessive expression of TP53 is often observed in hematopoietic cells associated with Fanconi anemia [43,44], dyskeratosis congenita [45], Shwachman-Diamond syndrome [46-48], or Diamond-Blackfan anemia [42], leading to excessive cell cycle arrest and apoptosis in the bone marrow.

It is well established that, under normal conditions, the expression level of TP53 is tightly regulated by the insulin-like growth factors/AKT signaling pathway that maintains the concentration or activity of TP53 at a relatively low level to allow cells to survive [49]. We previously demonstrated that Znt7KO mice were highly susceptible to diet-induced insulin resistance due to impaired activation of AKT in muscle cells and adipocytes [26,27,30]. Hence, we hypothesized that lack of or significantly reduced ZNT7 expression in hematopoietic progenitor cells might impair the AKT signaling pathway resulting in excessive expression of TP53 and subsequent depletion of bone marrow cells. In this study, we used different cell lines from patients with Ziegler-Huang syndrome/BMF8 and parental controls to establish a causative link between ZNT7 expression and the activation of AKT signal, as well as TP53 expression. Additionally, we demonstrated that, in the human bone marrow, ZNT7 was expressed in all hematopoietic progenitor cells producing white and red blood cells and platelets [11] while, in the mouse bone marrow, its expression was limited to progenitor cells developing into lymphocytes, monocytes, and neutrophils.

## 2. Methods and materials

### 2.1. Ethical approval

The affected individuals (Proband 1 and Proband 2) were brothers from a French family. The brothers and their parents were evaluated by a clinical geneticist, a pediatric hematologist, and an endocrinologist at the University Hospital of Angers, Angers, France. The study was approved by the Ethics Committee of the University Hospital of Angers (CB 2014/03), Angers, France. Informed consent was obtained directly from the participants (parents) and parents/legal guardians of the two affected individuals who were under the age of 18.

All animal experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Experimental Animals, and animal protocols were approved by the Institutional Animal Care and Use Committee of the University of California, Davis (protocol number: 23125).

### 2.2. Epstein-Barr virus-transformed B lymphocytes

The establishment of Epstein-Barr virus-transformed B (B-EBV) lymphoblastoid cell lines of Proband 1 (P1) and the patient mother as well as the stable ZNT7-KD and vector control Raji cell lines were described previously [11,24]. B lymphocytes were maintained as a suspension culture in the RPMI 1640 complete medium containing 20% FBS, 5 mM GlutaMax™, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 U/mL streptomycin (Gibco by Thermo Fisher Scientific, Waltham, MA) at 37°C in 5% CO₂. For B-EBV lymphocyte growth curve assays, the lymphocytes from P1 and the mother were counted using a TC20™ automated Cell counter (Bio-Rad Laboratories, Hercules, CA) and diluted to 2 ×10⁶ in 10 mL complete RPMI medium (Day 0) and grown for 7 days. Cells were then counted from days 2 to 7 of the growth period. The experiments were repeated three times each was in triplicate. For Western blot analyses, B-EBV lymphocytes from P1 and the

mother were seeded to 2 ×10⁶ in 10 mL complete RPMI medium, cultured for 3 days, and then harvested. For insulin stimulation assays, 2 ×10⁶ cells were preincubated in 2 mL RPMI medium without fetal bovine serum at 37 °C for 4 h. Cells were then stimulated with 0 or 10 nM insulin at 37 °C for 10 min followed by adding 5 mL ice-cold 1x PBS and pelleted. Cells were washed twice with ice-cold 1x PBS and pelleted for Western blot analysis.

### 2.3. Culture of primary fibroblasts and Lenti ZNT7 viral particle transduction

The establishment of primary fibroblast cell line from Proband 2 (P2) was described previously [11]. The WI-38 human fibroblast cell line was obtained from the American Type Culture Collection (Cat. # CCL-75, Manassas, VA). All fibroblasts were maintained in a fibroblast growth medium 2 (PromoCell GmbH, Sickingenstr, Germany) containing fetal calf serum (2% v/v), basic fibroblast growth factor (recombinant human at 1 ng/mL), insulin (recombinant human) at 5 µg/mL, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C in 5% CO₂.

Lentiviral particles containing a human ZNT7 open reading frame (ORF) with a Myc-DDK tag to the C-terminal end of the ZNT7 sequence were purchased from OriGene (Rockville, MD; Cat.# RC209198L4V) along with the vector control lentiviral particles (Cat.# PS100093V). An optimal puromycin concentration for drug selection of transduced P2 fibroblasts was performed by plating cells in a 12-well plate to 70-80% confluent and cultured for 2 days. Puromycin was diluted in the culture medium at the concentration at 0, 0.1, 0.25, 0.5, 0.75, and 1 µg/mL. The optimal puromycin concentration was determined as 0.25 µg/mL at which complete cell death was observed after 4 days.

The lentiviral transduction was achieved by plating P2 fibroblasts in duplicate in a 6-well cell culture plate at ~70% and incubated at 37 °C for 2 days following change of a fresh medium containing 8 µg/mL polybrene. The solution for lentiviral particles (either ZNT7-Myc-DDK ORF or the vector) was added at 0.25 MOI (multiplicity of infection) and cells were incubated at 37 °C overnight. Next day, the viral containing medium was replaced with fresh medium without polybrene and then cells were incubated for two more days at 37 °C. Puromycin at 0.25 µg/mL was added to cells to select resistant cells. The control cells with control vector lentiviral infection were used as positive controls for the puromycin selection success. The polyclonal puromycin resistant cells (ZNT7-OE or Vector control) were selected after 4-5 days of drug treatment. Cells were then expanded and maintained in the puromycin-free culture medium. Total RNA or protein lysate were purified from the established P2 fibroblast cell lines (2 polyclonal cell lines for each ZNT7-OE and Vector control) to verify the expression of the ectopic ZNT7-Myc-DDK fusion transcripts by quantitative RT-PCR or fusion protein by Western blot analysis using an anti-Myc antibody (9E10) (Santa Cruz Biotechnology, Inc. Dallas, TX) [21].

### 2.4. Total RNA isolation, cDNA synthesis, and quantitative PCR

Total RNA was purified from cultured P2 fibroblast cell lines (ZNT7-OE and Vector control) using an Aurum™ Total RNA Mini Kit including a DNase treatment according to the manufacturer's instructions (Bio-Rad Laboratories). RNA concentrations were determined by a Nanodrop instrument (Thermo Fisher Scientific). cDNA was synthesized from 100 ng of total RNA using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). For quantitative PCR analysis, synthesized cDNAs were diluted 5-fold with double-distilled water, and 2 µL was used in a SYBR green-based PCR using a PowerUp™ SYBR™ Green Master Mix (Applied Biosystems™ by Thermo Fisher Scientific) containing 2 µL of diluted cDNA sample, 1 µL of 5 pmol/µL forward and reverse primers for the target or housekeeping gene. Quantitative PCR was performed on a Quantstudio™ 7 flex real-time PCR system (Thermo Fisher Scientific). The RT-PCR was performed three times, and each quantitative PCR was run in triplicate. The average crossing point value
(Ct) was used for relative expression calculations [50]. The expression of the target genes was normalized to the expression of the housekeeping gene, HPRT1. The primer sequences are given in Table 2. All primers were synthesized by Life Technologies by Thermo Fisher Scientific (Carlsbad, CA).

## 2.5. Western blot analysis

Cell pellets were lysed in MPER™ protein lysis buffer containing 1 x Halt™ protease and phosphatase inhibitor (Thermo Fisher Scientific) and centrifuged at 18,400 g at 4 °C for 10 min to recover supernatant. The protein concentration was determined using the BCA method (Bio-Rad) and then mixed with 4 x loading buffer containing 10% β-mercaptoethanol (v/v; Bio-Rad). Samples were denatured through boiling for 5 min and then centrifuged at 18,400 at 4 °C for 10 min. The samples were subjected to electrophoresis on 10% or 4-20% Mini-PROTEAN TGX Stain-free Precast Gels (Bio-Rad). Blots were obtained from the Trans-blot Turbo packs (0.2 µm PVDF) via a Trans-Blot Turbo Transfer System (Bio-Rad) and blocked in 5% bovine serum albumin in 1 X PBS (Thermo Fisher Scientific). After incubation with primary and secondary antibodies, the blots were washed in 1X TBST and targeted proteins were visualized using chemiluminescence (ECL) and a ChemiDoc™ Imaging System as described by the manufacturer (Bio-Rad). Antibodies used in this study included a rabbit anti-phosphor-AKT (S473) antibody (Cat# 9271), a rabbit anti-AKT antibody (Cat# 9272), a rabbit anti-p53 antibody (Cat# 9282), a rabbit anti-phosphor-STAT3 (Y705) (D3A7) (Cat# 9145), a rabbit anti-STAT3 (D1B2J) antibody (Cat# 30835), and a mouse anti-ACTB (8H10D10) antibody (Cat# 3700). All antibodies were purchased from Cell Signaling Technology® (Danvers, MA) and were diluted to 1:1000 except the anti-ACTB that was diluted to 1:20000-25000. Horseradish peroxidase (HRP) secondary antibodies were used at 1:3000-1:6000 (Cell Signaling Technology).

## 2.6. Immunofluorescence staining

Formalin-fixed and paraffin-embedded (FFPE) normal human bone marrow tissue sections (HuFPT181) were purchased from TissueArray.

com (US Biomax, Derwood, MD). The bone marrow sections were collected from a 35-year-old male. The mouse sternum bones were dissected at necropsy and placed in 4% paraformaldehyde solution made in 1x PBS, pH 7.4 overnight. Preparation of paraffin embedded tissue, deparaffinization, rehydration, and subsequent sectioning (3-5 µm), and mounting were performed as previously described [51,52]. Immunofluorescence staining for P2-Vec and ZNT7-OE fibroblasts using a monoclonal anti-Myc antibody (9E10) (Santa Cruz Biotechnology, Inc.) was described previously [21]. The antibodies for the bone marrow cell markers were purchased from companies as follows: an anti-human/mouse myeloperoxidase (MPO) polyclonal goat antibody (Cat# AF3667; 1:40 dilution) was purchased from R&D Systems (Minneapolis, MN). An anti-CD4 (GK1.5) antibody (Cat# 14-0041-82; 1:10 dilution), an anti-human/mouse/rat CD19 monoclonal antibody (6OMP31) (Cat# 14-0194-82; 1:50 dilution), an anti-human/mouse CD71 (OKT9) monoclonal antibody (Cat# 14-0719-82; 1:50 dilution), an anti-mouse CD34 (RAM34) monoclonal antibody (Cat# 14-0341-82; 1:100 dilution), and an anti-mouse CD45 (30-F11) monoclonal antibody (Cat# 12-0451-82; 1:50 in dilution) were purchased from Thermo Fisher Scientific. The polyclonal anti-ZNT7 antibody was used in 1:750 dilution as described previously [11,21]. All antibodies were diluted in 1X PBS containing 2% mouse and 2% goat sera for immunofluorescence staining. Alexa Fluor™ 488 or Alexa Fluor™ 594-conjugated secondary antibodies were purchased from Thermo Fisher Scientific and diluted at 1:200-250 according to the manufacturer's instructions. Nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA). Photomicrographs were obtained using an EVOS FL Auto Imaging System (Life Technologies by Thermo Fisher Scientific).

## 2.7. Mice and diets

All mice were housed in a temperature-controlled room at 22-24 °C with a 12-h light:dark cycle. Heterozygous breeders (Znt7+/−) were fed a standard laboratory chow diet (CD, Laboratory Rodent Diet 5001, Lab-Diet, Brentwood, MO) and double-distilled water at ad libitum. Pups were genotyped at 2 weeks old and weaned at 3 weeks old. Mice were humanely euthanized in compliance with all institutional and national

<table><caption>**Table 2**

Primers used in the gene expression study.</caption><thead><tr><th>Gene name</th><th>Forward primer sequence (5' → 3')</th><th>Reverse primer sequence (5' → 3')</th><th>PrimerBank ID</th></tr></thead><tbody><tr><td>ZNT7*</td><td>CTCTACGGCATCTGGAGCA</td><td>TGCCAGTCCAGCCAAAAT</td><td>N.A.</td></tr><tr><td>ZNT7-Myc-DDK*</td><td>ATTCTCATGATGGCCCGTCC</td><td>ACCTTATCGTCGTCATCCTTGT</td><td>N.A.</td></tr><tr><td>ZNT1</td><td>CTCGCGTTAAGAGCACCCG</td><td>CAATTTCAGCCCGTTGGAGTT</td><td>52352802c1</td></tr><tr><td>ZNT2</td><td>CGGCAATCCGGTCATACAC</td><td>AGATGGCAGAGGCTACATACAG</td><td>52352806c1</td></tr><tr><td>ZNT3</td><td>AGCCTGCGTTTGAAGAGTCTC</td><td>GCTGTGTGCCAGATACCCG</td><td>52630414c1</td></tr><tr><td>ZNT4</td><td>TGGAAGCGCCTCAAATCTATG</td><td>CAGTAAGGAATCATCGTCGGC</td><td>52630415c1</td></tr><tr><td>ZNT5</td><td>ACCAAACACCAGTGGATCAAAA</td><td>CAGCAAAGTCCTTAGTGGTCC</td><td>20070323a1</td></tr><tr><td>ZNT6</td><td>AGGAAACCTAGCCCTGTCTATT</td><td>CAAAAAGCGTTCTGCACTTTCT</td><td>301898445c1</td></tr><tr><td>ZNT8</td><td>TGAGTCACGCCTATGCCAAGTG</td><td>CTGGTCAGGTCAATTAAGAGGTG</td><td>289803012c1</td></tr><tr><td>ZNT9</td><td>AGGGAGTATGGCTCAAAGTACA</td><td>GGGACTTCGTCGTCTGATTTTTC</td><td>57164947c1</td></tr><tr><td>ZNT10</td><td>ACCGTGTTCGCAAACGTAG</td><td>TCACATGCAAAAGTACACCTCTCTG</td><td>52351207c1</td></tr><tr><td>ZIP1</td><td>TGAGCCTAGTAAGCTGTTTCGC</td><td>CAGGGCCTCATCTATGGCA</td><td>34147669c1</td></tr><tr><td>ZIP2</td><td>CTGCTGGAGGATCGACAGTG</td><td>CACAAGCCCCTTATGAGCCA</td><td>7657699a1</td></tr><tr><td>ZIP3</td><td>TGCTCCCCGTGAAGATCATC</td><td>TCCAAAGGTGTTGCAGAGAGA</td><td>47080102c1</td></tr><tr><td>ZIP4</td><td>CGAGGTCGCCTATGACGCTG</td><td>CACTCAGGCATACCGTGTCC</td><td>115430254c1</td></tr><tr><td>ZIP5</td><td>GGTCCCCAGTGAGTCATCTG</td><td>GCCAGGTAATGGTTCTGCTCC</td><td>206597538c1</td></tr><tr><td>ZIP6</td><td>ATGCAAGTCACCACCATAGTCA</td><td>ACGTGGAATCAAAATAGGCACT</td><td>153252200c1</td></tr><tr><td>ZIP7</td><td>GGACACGCTCACAGTCATACA</td><td>CTCCTCGCCTCTTCTGAACC</td><td>117553607c1</td></tr><tr><td>ZIP8</td><td>ATGCTACCCAAATAACCAGCTC</td><td>ACAGGAATCCATATCCCCAAACT</td><td>205830404c1</td></tr><tr><td>ZIP9</td><td>CATGCTGGCTTAGAGCGGAAT</td><td>TGGTGCTGCCAATGCAAAGA</td><td>356460994c1</td></tr><tr><td>ZIP10</td><td>TTTCACTCACATAACCACCAGC</td><td>GTGATGACGTAGGCGGTGATT</td><td>187936948c1</td></tr><tr><td>ZIP11</td><td>CAGCTCTCGTGTTCGTATTCTC</td><td>TCAGCCAAGTAGACAAAAGCC</td><td>229577417c1</td></tr><tr><td>ZIP12</td><td>TTCTTCTACTCAGCCGTGTGTTTT</td><td>GTCACCAGCAGAGAGAACCT</td><td>223633938c1</td></tr><tr><td>ZIP13</td><td>TCAGCGGCTACCTCAACCT</td><td>AGGAGCCCGATCTTCTTGCT</td><td>291490667c1</td></tr><tr><td>ZIP14</td><td>GAGGCTCACGCTTCATCCC</td><td>CCCTCGCCATACCGATGTATTA</td><td>205830421c1</td></tr><tr><td>ACTB*</td><td>GTCACCAACTGGGACGACAT</td><td>GAGGCGTACAGGGATAGCAC</td><td>N.A.</td></tr><tr><td>HPRT1*</td><td>CCTGGCGTCGTGATTAGTGA</td><td>CGAGCAAGACGTTCAGTCCT</td><td>N.A.</td></tr></tbody></table>

<footnote>* Primers were designed using a Primer Designing Tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/).</footnote>
guidelines for the care and use of laboratory animals, under our
approved protocols. Mice were first anesthetized with 100 mg/kg ke-
tamine/10 mg/kg xylazine, and deep anesthesia was confirmed by the
absence of the pedal reflex followed by cervical dislocation to ensure
swift and humane euthanization. At necropsy, sternum bones were
isolated and prepared for immunocytochemistry and
immunohistochemistry.

2.8. Genotyping

Genotyping was performed as described previously using tail clips
[21]. Briefly, genomic DNA was isolated from mouse tail tissue using a
DNeasy Tissue Kit (Qiagen, Valencia, CA). PCR conditions and primer
pairs used for genotyping were described previously [21].

2.9. Decalcification and immunohistochemical analysis

The mouse sternum bone was dissected from wild-type mice (WT,
C57BL/6 J, 8 weeks) and placed in 4% buffered formalin solution
(Thermo Fisher Scientific) for 1-3 days. The tissue was then rinsed three
times with 1 X PBS, pH 7.4. Decalcification was achieved by incubating
the specimen in 8% 1:1 hydrochloric acid/formic acid solution twenty
times of the volume of the specimen for 48 h at room temperature (RT)
followed by one time solution change and incubation for additional
24 h. The specimen was then placed into an embedding cassette and
rinsed under running tap water for 30 min. Acid in the tissue was then
neutralized by immersing the tissue in 5 mL of decalcification solution
which was then neutralized the solution with dropwise ammonium
hydroxide solution until the pH of the solution was neutral to pH paper
followed by adding 5 mL of saturated ammonium oxalate. After shaking
briefly, the specimen was allowed to set in the solution for 30 min at RT
followed by rinsing the tissue with running tap water for 30 min. The
decalcified tissue was stored in 70% ethanol until use. Further pro-
cessing, including paraffin embedding, deparaffinization, rehydration,
subsequent sectioning (5 µm), and mounting were described previously
[51,52]. The normal human bone marrow section (HuFPT181) was
purchased from US Biomax. The human sections were baked at 60 °C for
60 min before processing for staining. The anti-ZnT7 antibody was
diluted at 1:500-750 in 1X PBS containing 2% mouse and 2% goat sera.
A secondary biotinylated goat anti-rabbit antibody (Vector Labora-
tories) was diluted at 1:200-250 in 1× PBS containing 2% goat serum.
Immunoperoxidase staining was accomplished using a Vectastain ABC
kit and DAB substrate (Vector Laboratories). The immunoreactivity was
developed in the tissue sections using a VECTASTAIN elite ABC kit and a
HRP substrate ImmPACT DAB kit (Vector Laboratories) that produced
brown deposits in the cells. Sections were counterstained with hema-
toxylin. Photomicrographs were obtained using an EVOS FL Auto Im-
aging System (Life Technologies by Thermo Fisher Scientific).

2.10. Hematological analysis

Non-fasting blood (50 µL) was collected from Znt7KO and WT littermates at 4, 8, 14, and 18 weeks of age for complete blood counts by the Comparative Pathology Laboratory, Veterinary Medicine, University of California Davis (Davis, CA) at 8:00-9:00 in the morning via the submandibular vein into EDTA-coated blood collection tubes. A blood smear section was done by placing a small drop of blood (<10 µL) on a glass slide and spread it into a thin layer. The slide was then fixed with methanol for 10-30 s at RT followed by Wright and Giemsa staining. Morphologies of blood cells were examined using an EVOS AMEP 4696 lens (100X), and photomicrographs were obtained using an EVOS FL Auto Imaging System (Life Technologies by Thermo Fisher Scientific).

2.11. Histological analysis

Paraffin-embedded sternum bone marrow sections of Znt7KO mice

and their WT littermates at 4, 14, and 18 weeks of age were processed as
described above (Immunofluorescence staining). Sections were stained
with hematoxylin and eosin (H&E). Photomicrographs were obtained
using an EVOS FL Auto Imaging System (Life Technologies by Thermo
Fisher Scientific).

2.12. Statistical analysis

Results are presented as mean ± SEM. Student t-test was used to identify statistical differences between genotypic groups. Differences were significant at *p* < 0.05.

3. Results

3.1. Impaired growth of B-EBV lymphocytes from the patient carrying mutations in ZNT7

We previously reported that two pathogenic variants, NM_001144884:c.21dup; p.(Asp8ArgfsTer3) and NM_001144884: c.842+15 T>C, in the human ZNT7 gene were associated with pancytopenia and bone marrow failure among other symptoms, including growth delay and testicular hypoplasia in the affected individuals [11]. To investigate the underlying mechanisms associated with bone marrow failure in this new genetic disease, we analyzed the growth of established B-EBV lymphoblast cell lines generated from the affected patient (P1) and the proband's healthy mother carrying the c.21dup mutation of ZNT7 in the heterozygous state [11]. Growth of the P1 B-EBV lymphocytes in culture was significantly reduced compared to the mother cells over time, with a 61.7% decrease after 7 days (p < 0.001) (Fig. 1A). Moreover, we noted that the growth delay of P1 B-EBV cells was apparent as early as day 2 with P1 cells growing 41% slower than the mother cells (p < 0.001).

3.2. Increased TP53 expression and attenuated activation of AKT in P1 B-EBV lymphocytes

Increased TP53 expression or activity in hematopoietic stem cells is strongly associated with bone marrow failure syndromes [42,45-47,53]. For example, BMFS5 is caused by heterozygous mutations in TP53 that lead to a gain-function variant of TP53 in hematopoietic stem cells due to a truncation of TP53 at the C-terminal end [36]. Another example is BMFS6 that is caused by mutations in MDM4 resulting in an increase of TP53 expression in cells due to lack of binding of the negative regulator of MDM4 to TP53 [37]. Other bone marrow failure syndromes caused by excessive expression of TP53 are Fanconi anemia, Shwachman-Diamond, Diamond-Blackfan, and dyskeratosis congenita [42,43,45,46]. Activation of the AKT signaling pathway by insulin-like growth factors or insulin is a major regulatory signal for cell survival via ubiquitination of TP53 and subsequent degradation of the protein [49] (Fig. 1B). Importantly, our previous studies suggested that depletion of Znt7 expression could lead to significant reduction in the activation of AKT by insulin in mouse primary myoblasts [30] and adipocytes [26].

Hence, we investigated whether the expression of TP53 was elevated
in patient B-EBV lymphocytes and whether AKT activation was altered
due to mutations in ZNT7. Remarkably, we observed that TP53 was
highly expressed in patient's cells accompanied by a strongly decreased
expression of phosphorylated AKT when compared to the mother cells
(Fig. 1C). The total AKT levels were not significantly different between
P1 and mother cells, suggesting that the decreased phosphorylation of
AKT was related to an impaired AKT activation in P1 cells rather than a
reduced AKT expression. To further confirm that the activation of AKT
was impaired in P1 cells, we stimulated P1 cells with 10 nM insulin at
37 °C for 10 min after 4 h preincubation in the medium without fetal
bovine serum. Insulin stimulation had little to no effect on the activation
of AKT in P1 B-EBV cells whereas the insulin-stimulated activation of
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Effect of mutations in the human ZNT7 gene on the growth of B-EBV lymphocytes and the activation of AKT. A. Growth of B-EBV lymphocytes from Proband 1 (P1) and the mother control. Data are shown as the mean ± S.E. (n = 3). Data were obtained from three independent experiments, and each experiment was performed triplicate. B. Overview of the PI3K/AKT signaling pathway and its downstream effectors on cell proliferation and apoptosis. Binding of insulin or insulin-like growth factors, such as IGF1, to its receptor activates the PI3K/AKT signaling pathway that subsequently stimulates the phosphorylation of MDM2/MDM4 and STAT3 leading to cell proliferation and survival. Binding of p-MDM2, an E3 ubiquitin ligase, causes TP53 to be ubiquitinated and degraded resulting in an inhibition of TP53-mediated apoptosis. C. A representative blot of Western blot analysis with antibodies against TP53, p-AKT (Ser473), total AKT, and ACTB. D. Activation of AKT by insulin. E. Phosphorylation of STAT3. A representative blot of Western blot analysis with antibodies against phosphorylated STAT3, total STAT3, and ACTB. F. A representative blot of Western blot analysis with antibodies against p-AKT (Ser473), total AKT, and ACTB using protein lysates from Raji B lymphocytes treated with 0 or 10 nM insulin in cells with or without ZNT7 knockdown. ACTB serves as the loading control for all assays. Western blot assays were performed using protein lysate obtained from at least two independent experiments. Vec, vector control; KD, ZNT7 knockdown.</figcaption></figure>

AKT was evident in the mother cells with an increased phosphorylation of AKT (Fig. 1D). Moreover, we confirmed the negative impact of ZNT7 on the activation of AKT in Raji B lymphocytes with ZNT7 expression reduced by gene knockdown (ZNT7-KD) [24], when compared to the vector control (Fig. 1F).

As AKT activation stimulates STAT3 phosphorylation leading to cell survival [54], we evaluated the phosphorylated STAT3 and total STAT3 levels in P1 and mother B-EBV cells. As expected, the phosphorylated STAT3 level was reduced in P1 cells compared to the mother cells (Fig. 1E). Taken together, these results highlight a diminished activation
of the AKT signaling pathway and subsequent downstream dysregula-
tion of AKT targets, including increased TP53 expression and decreased
STAT3 phosphorylation in patient's B-EBV lymphocytes. These subse-
quent dysregulations might lead to reduced cell survival, explaining the
growth delay of the patient's B-EBV cells.

### 3.3. Overexpression of ZNT7 in Proband 2 fibroblasts rescues the responsiveness of AKT phosphorylation to insulin stimulation

We next investigated whether the impaired AKT phosphorylation in response to insulin stimulation in P1 B-EBV lymphocytes could also be detected in primary fibroblasts isolated from Proband 2 (P2, who carries both pathogenic ZNT7 variants). P2 fibroblasts and WI-38 control fibroblasts were stimulated with 0, 10, or 100 nM insulin for 10 min and

<figure><img src="image_6.png" /></figure>

**Fig. 2.** Overexpression of wild-type ZNT7 rescued the activation of AKT by insulin in Proband 2 (P2) fibroblasts. A. Activation of AKT by insulin. B. Quantitative RT-PCR analysis of ectopic overexpression of WT ZNT7 in P2 fibroblasts (ZNT7-OE). The ectopic ZNT7 protein was expressed as a fusion protein with a Myc-DDK tag fused at the C-terminal of ZNT7. Three primer sets were used: 1) specifically detecting all available ZNT7 mRNA; 2) specifically detecting the ZNT7-Myc-DDK fusion mRNA; and 3) specifically detecting ACTB, a housekeeping gene. C. Western blot analysis of the expression of ZNT7-Myc-DDK fusion protein in ZNT7-OE fibroblasts. The fusion protein was detected using an anti-Myc monoclonal antibody. The protein size markers are shown. D. Immunofluorescent detection of the ZNT7-Myc-DDK fusion protein in P2 fibroblasts using an anti-Myc monoclonal antibody. The vector (Vec) was used as a control. Representative microphotographs of the stained ZNT7-Myc-DDK (green) are shown. DAPI was used to stain the nuclei in the vector control cells to indicate cells stained negatively for the anti-Myc antibody. Scale bar = 25 µm. E. Activation of AKT by insulin. ACTB serves as the loading control for all Western blot assays. Western blot assays were performed using protein lysate obtained from at least two independent experiments.
AKT activation was assessed. Our results showed that the basal phosphorylated AKT level in P2 cells was greatly lower than the control cells (Fig. 2A). Noticeably, insulin stimulation had no apparent effect on the AKT activation in the WI-38 control cells nor the P2 cells even with an insulin stimulation at 100 nM (Fig. 2A). On the other hand, we observed that the expression of the total AKT was comparable between P2 and WI-38 cells (Fig. 2A).

To further explore the connection between ZNT7 deficiency and the AKT activation in patient cells, we established P2 fibroblast cell lines expressing a wild-type ZNT7 protein with a Myc-DDK tag at the C-terminal end of the protein (ZNT7-OE). Meanwhile, we established a vector control cell line transduced with a control empty vector (Vec). We then examined mRNA expression of ZNT7 using different primer sets that could detect either the endogenously expressed ZNT7 or the ectopic expressed ZNT7-Myc-DDK fusion transcripts. As expected, ZNT7-Myc-DDK fusion transcripts were readily detected in ZNT7-OE cells but were absent in the vector control cells (Fig. 2B). The results also revealed that the total ZNT7 mRNA expression (including endogenous ZNT7 and ZNT7-Myc-DDK transcripts) increased in ZNT7-OE cells compared to vector control cells. We next examined the expression of the ZNT7-Myc-

DDK fusion protein in ZNT7-OE cells using an antibody against the Myc tag epitope. As shown in Fig. 2C, the ZNT7-Myc-DDK fusion protein was readily visualized at around 50 kDa, consistent with the size of human ZNT7 protein reported previously [24].

ZNT7 protein is localized to the Golgi apparatus in cells where it transports zinc from the cytoplasm to the Golgi apparatus [21]. Therefore, we investigated whether the ZNT7-Myc-DDK fusion protein was localized to the Golgi apparatus through immunofluorescence staining on ZNT7-OE cells. As shown in Fig. 2D, in the ZNT7-OE cells, the anti-Myc antibody-stained protein (green color) clustered in the perinuclear region of the cell, suggesting that ZNT7-Myc-DDK fusion protein was localized to the Golgi apparatus. On the other hand, in the vector control cells, no ZNT7-Myc-DDK fusion protein was detected by the anti-Myc antibody.

Furthermore, we showed that over-expression of ZNT7-Myc-DDK was able to rescue the response to insulin-stimulated AKT activation in P2 fibroblasts (Fig. 2E). Remarkably, we observed that over-expression of ZNT7-Myc-DDK could also increase the expression of total AKT in P2 fibroblasts compared to the vector control in both basal and insulin-stimulated states (Fig. 2E). Taken together, these data show

A

<figure><img src="image_7.png" /></figure>

B

<figure><img src="image_8.png" /></figure>

**Fig. 3.** mRNA expression of the members of ZNT and ZIP zinc transporter families in B-EBV lymphocytes from P1 and the mother. A. Relative mRNA expression of the members of the ZNT family by quantitative RT-PCR. B. Relative mRNA expression of the members of the ZIP family by quantitative RT-PCR. Total RNA was purified from the B-EBV lymphocytes and then converted to cDNA. The target gene expression was normalized to the expression of HPRT1 (hypoxanthine phosphoribosyltransferase 1). Values are Mean ± SEM; n = 3. An unpaired and parametric Student t-test was used for analysis. Little to no mRNA expression was detected for ZNT2, ZNT3, ZNT8, ZNT10, ZIP2, ZIP4, ZIP5, and ZIP12 in B-EBV lymphocytes from both P1 and mother.
that ZNT7 deficiency has a direct effect on the AKT activation.

### 3.4. mRNA expression of members of ZNT and ZIP zinc transporter families

We further evaluated whether ZNT7 deficiency would trigger any compensatory activation or suppression of gene transcription of other zinc transporters, including the members of the ZNT and the ZIP families in P1 B-EBV lymphocytes. We could detect mRNA transcripts in 5 ZNT genes (ZNT1, ZNT4, ZNT5, ZNT6, and ZNT9) (Fig. 3A) and 11 ZIP genes (ZIP1, ZIP3, ZIP6, ZIP7, ZIP8, ZIP9, ZIP10, ZIP11, ZIP13, and ZIP14).

(Fig. 3B). Among the zinc transporter genes expressed in P1 and mother cells, we did not observe significant differences in transcriptional levels between P1 and mother cells, except for ZNT4 with the expression trending toward to be about 2-fold increase in P1 cells compared to mother cells. However, it did not reach statistical significance (p = 0.06) (Fig. 3A). Altogether, these results suggest that ZNT7 deficiency is not compensated by regulating gene expression of other zinc transporters.

<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Immunofluorescent co-staining of ZNT7 with hematopoietic cell markers in the human bone marrow. ZNT7 was co-stained with CD34 (lymphohematopoietic cells), MPO (myeloid lineage cells), and CD71 (erythroid precursors), CD4 (T lymphocytes), and CD19 (B lymphocytes). We detected that the expression of ZNT7 (green color) in hematopoietic precursor cells were completely overlapped with the hematopoietic markers (red color) including CD34, CD71, CD4, and CD19 (yellow/orange color in the merged micrographs). However, only a small fraction of MPO-positive cells was co-expressed with ZNT7. Representative microphotographs are shown. MPO, myeloperoxidase. Scale bar = 25 µm.</figcaption></figure>
### 3.5. Co-expression of ZNT7 with hematopoietic markers of progenitor cells in the human bone marrow

The affected individuals with Ziegler-Huang Syndrome/BMF8 suffered from pancytopenia due to bone marrow failure, suggesting that ZNT7 plays a crucial role in bone marrow stem cell development. Therefore, we assessed ZNT7 expression in common myeloid progenitors that then develop into megakaryocytes and erythrocytes, and myeloblasts that develop into monocytes, neutrophils, basophils, and eosinophils, as well as common lymphoid progenitors that develop into T and B lymphocytes and NK cells. We studied the co-expression of ZNT7 with hematopoietic progenitor cell markers, including CD34, a marker for early progenitor cells including myeloid progenitors and lymphoid progenitors [55], MPO (myeloperoxidase), a marker for early myeloid cell lineage [56], CD71, a marker for erythroid precursor cells [55], CD4, a marker for T lymphocytes [55], and CD19, a marker for B cell precursors and mature B cells [55]. We found that besides MPO that displayed a partial co-staining pattern with ZNT7, other hematopoietic cell markers were completely co-expressed with ZNT7 (Fig. 4), confirming that ZNT7 is expressed in myeloid and lymphoid lineage cells in the human bone marrow.

### 3.6. Znt7 knockout mice partially recapitulates the hematological features of human disease related to ZNT7 deficiency

We have developed a Znt7 knockout mouse model (Znt7KO) to study the effects of Znt7 deficiency on body zinc status and pathophysiology of growth and metabolism. We found that Znt7KO mice were weighed less than the WT littermates due to zinc deficiency and were susceptible to diet-induced insulin resistance due to a decreased activation of AKT by insulin in muscle and adipose tissues [26,30]. In the current study, we confirmed again that Znt7KO mice were significantly smaller than the WT littermates (Fig. 5A) [29]. Examination of peripheral blood lymphocytes, leukocytes, erythrocytes, and platelets by a Wright-Giemsa staining did not reveal any significant signs of abnormalities in blood cell morphologies between Znt7KO and WT littermates (Fig. 5B). The hematoxylin and eosin staining study of the sternum bone marrow revealed similar characteristics of the bone marrow in terms of overall health, tissue structure, and cellularity of the marrow between Znt7KO and WT littermates (Fig. 5C).

To examine whether Znt7 deficiency was associated with cytopenia in mice, we performed complete blood counts in Znt7KO mice and compared them to the WT littermates at ages 4, 8, 14, and 18 weeks. Our results demonstrated that Znt7KO mice had significantly lower total white cell counts starting at 4-week-old ($p < 0.05$), worsening with age ($p < 0.01$) (Fig. 6A). More precisely, Znt7KO mice had decreased the number of total lymphocytes throughout the examined age compared to the WT controls (Fig. 6B). Znt7KO mice also had reduced the numbers of monocytes and neutrophils occurring at 8 weeks of age (Fig. 6C & D). Moreover, we detected that Znt7KO mice suffered from anemia at 18 weeks of age evident by the reductions in the red blood cell numbers, hemoglobin concentrations, and hematocrit (Fig. 6F-H). However, no difference was detected in the platelet counts between Znt7KO and WT littermates at all ages examined (Fig. 6E). Overall, these results highlight progressive cytopenias in Znt7KO mice, partially recapitulating the progressive pancytopenia related to bone marrow failure observed in BMF8 patients.

We next compared Znt7 protein expression between the mouse and human bone marrows. As shown in Fig. 7A, the human ZNT7 was clearly expressed in all bone marrow cells including megakaryocytes whereas the mouse ZnT7 was only expressed in a fraction of granular bone marrow cells. Co-staining of mouse ZnT7 with hematopoietic cell markers indicated that the mouse ZnT7 was partially co-stained with Cd34, a marker for myeloid and lymphoid progenitors [55] and Cd45, a marker for all nucleated hematopoietic cells including immune cells [57]. However, ZnT7 was not co-expressed with Cd71 in erythroid

precursor cells [55] as well as not co-expressed with Mpo, a marker for early myeloid cell lineage [56]. Together, these results suggest that the mouse ZnT7 protein is predominantly expressed in nucleated hematopoietic cells in the bone marrow, but not the erythroid and myeloid cell lineages.

### 4. Discussion

Inherited bone marrow failure syndromes in humans are a heterogeneous group of life-threatening genetic diseases due to inadequately producing circulating blood cells [32]. The excessive expression or persistent activation of TP53, driven by the mutations in TP53 [36,37, 42,47] or in genes encoding proteins that directly or indirectly influence TP53 expression or activity [35,43,45,46,53,58], has been implicated in the development of bone marrow failure. In this study, we demonstrate that pathogenic biallelic variants in ZNT7 cause an excessive expression of TP53 in B-EBV lymphoblasts from an affected individual with Ziegler-Huang Syndrome/BMF8. The activation of AKT, an upstream key regulatory signaling protein of TP53, was impaired by the ZNT7 mutations. Introduction of wild-type ZNT7 into fibroblasts of the affected individual greatly improved the AKT activation. Thus, we demonstrate that ZNT7 plays an important role in regulating AKT activity. Thereby, ZNT7 may likely influence TP53 expression levels indirectly through regulation of the activation of AKT, which was probably achieved by regulating zinc concentrations in the Golgi apparatus and its network [21,22]. Our data also demonstrates that ZNT7 is expressed in hematopoietic progenitor cells responsible for developing into circulating neutrophils, erythrocytes, lymphocytes, and platelets in the human bone marrow. This finding suggests that ZNT7 deficiency in bone marrow progenitor cells may lead to pancytopenia in affected individuals [11].

The IGF1 (insulin-like growth factor 1) is a growth factor that binds to its receptor on the cell surface to activate the PI3K/AKT signal pathway by phosphorylation. One of the major functions of the activated AKT is to control the expression or activities of downstream target proteins that regulate cell proliferation, growth, and survival, including TP53 and its negative regulator MDM4 in bone marrow progenitor cells [37,59]. Activated AKT leads to the phosphorylation of MDM4 resulting in translocation of the protein to the nucleus and binding to the transcriptional activation domain of TP53, which inhibits the transcriptional activity of TP53 for its targets and promotes cell survival [37]. The binding of MDM4 to TP53 facilitates the ubiquitination of TP53 through a MDM4/MDM2 complex in the cytoplasm, ultimately leading to TP53 degradation in proteasomes [37].

It has been shown that phosphorylation of Thr308 in AKT by PDK1 (phosphoinositide-dependent kinase 1) and phosphorylation of Ser473 by MTORC2 (mechanistic target of rapamycin complex 2) leads to cell survival and cell cycle progression [59]. Deactivation of AKT is facilitated by several phosphatases, including PP2A (protein phosphatase 2A), PHLPP (PH domain and leucine rich repeat protein phosphatases), and PTEN (phosphatase and tensin homolog deleted on chromosome 10) [60–63]. Studies have also shown that zinc positively impacts the PI3K/AKT signaling pathway through various mechanisms, including enhancing kinase expression levels, facilitating kinase-substrate binding, modulating the cellular redox balance, and, most notably, down-regulating phosphatase activities [63–67]. On the other hand, zinc deficiency has an opposite effect on the PI3K/AKT signaling pathway negatively affecting cell survival [68,69].

PP2A is encoded by the PPP2CA gene, a key regulator of the AKT activation as it binds to AKT and directly dephosphorylates AKT, resulting in an inhibition of the PI3K-AKT signaling pathway [60,61]. PP2A is a heterotrimeric protein phosphatase consisting of a structural A subunit, a regulatory B subunit, and a catalytic C subunit [60]. Xiong et al. showed that zinc can directly inhibit PP2A activity by binding to the catalytic domain of the C subunit (PP2Ac) and preventing the enzyme from conducting dephosphorylation function [61]. Manganese
<figure><img src="image_10.png" /><figcaption>**Fig. 5.** Mouse Znt7KO and WT mice and features of mouse peripheral blood and bone marrow cells. A. A representative 18-week-old male Znt7KO mouse and a WT littermate. The knockout mouse was smaller than the control as observed previously [29]. B. Morphological comparison of peripheral white blood cells (neutrophils, lymphocytes, monocytes, eosinophils, and basophiles) and red blood cells (erythrocytes) as well as platelets between Znt7KO and WT mice. Wright-Giemsa staining. Magnification, x100. C. H&E staining of the sternum bone marrow of Znt7KO and WT mice. The sternum was harvested for staining at 4, 14, and 18 weeks of age. The nuclei of bone marrow cells were stained blue with hematoxylin and the cytoplasm were stained pink with eosin. The red * indicates megakaryocytes in the bone marrow. Scale = 20 µm. Znt7KO, Znt7 knockout; WT, wild-type.</figcaption></figure>
A

<figure><img src="image_11.png" /></figure>

B

<figure><img src="image_12.png" /></figure>

C

<figure><img src="image_13.png" /></figure>

D

<figure><img src="image_14.png" /></figure>

E

<figure><img src="image_15.png" /></figure>

F

<figure><img src="image_16.png" /></figure>

G

<figure><img src="image_17.png" /></figure>

H

<figure><img src="image_18.png" /><figcaption>**Fig. 6.** Complete blood counts (CBC) using fresh blood collected from Znt7KO and WT literatures at ages of 4, 8, 14, 18 weeks. A. Numbers of white blood cells (WBCs). B. Numbers of lymphocytes. C. Numbers of monocytes. D. Numbers of neutrophils. E. Numbers of platelets. F. Numbers of red blood cells (RBCs). G. Hemoglobin concentrations in RBCs. H. The percentage of RBCs in the blood. Data are shown as Mean ± SEM; n = 6 pairs at 4-week-old; n = 8 pairs at 8-week-old; n = 8 pairs at 14-week-old; n = 4 pairs at 18-week-old. \*, *p* < 0.05; \*,\*, *p* < 0.01; \*,\*,\*, *p* > 0.001. Student t-test was used for analysis.</figcaption></figure>
A

<figure><img src="image_19.png" /></figure>

B

Mouse sternum bone marrow

<figure><img src="image_20.png" /></figure>

(caption on next page)
**Fig. 7.** The expression of ZNT7 in human and mouse bone marrows. A. Representative microphotographs of immunohistochemical staining of ZNT7 are shown. In the human bone marrow, the ZNT7 protein (brown color) was detected in a mix of cell types covering all areas of the microphotograph except the red blood cells within the blood vessels indicated as *. Fat drops are shown as the empty space in a circle. Solid black arrows indicate megakaryocytes. In the mouse sternum bone marrow, only a fraction of cells was stained positive for ZnT7. No fat droplets were found. Scale bar = 50 µm. B. Immunofluorescent co-staining of the mouse ZnT7 with hematopoietic cell markers in the sternum bone marrow. ZnT7 was co-stained with Cd34 (lymphohematopoietic cells), Cd45 (nucleated hematopoietic cells), Mpo (myeloid lineage cells), and Cd71 (erythroid precursors). We detected most hematopoietic cells co-expressed ZnT7 with Cd34 or Cd45 (open arrows in the merged images). However, a small fraction of cells positive for Cd34 or Cd45 did not co-express with ZnT7 (solid arrows). Furthermore, no obvious co-expression of ZnT7 with Mpo or Cd71 was detected. Mpo, myeloperoxidase. Scale bar = 20 µm.

is a crucial cofactor for the PP2A enzymatic activity that directly binds to the active site of the catalytic subunit. It is plausible that in the normal physiological condition, zinc may compete with manganese for binding to the active site of PP2Ac that negatively regulates PP2A activity. Whereas in a zinc deficient condition this competition weakens favoring manganese binding to the active site of PP2Ac enabling the enzyme to efficiently dephosphorylate AKT [66]. Moreover, a study from Gao et. al indicate that PHLPP is capable of directly dephosphorylating Ser473 of AKT triggering apoptosis [62]. PHLPP includes two isoforms, PHLPP1 and PHLPP2. The two isoforms have a similar protein structure containing a PP2c phosphatase domain, a series of leucine-rich repeats, a putative Ras association domain, a pleckstrin homology domain, and a C-terminal PDZ ligand domain. Like PP2A, PHLPP also requires manganese as a cofactor bound to the PP2c domain for enzymatic activities. Thus, it is likely that a decreased activation of AKT observed in the cells of affected individuals with Ziegler-Huang Syndrome/BFM8 was induced by a disruption of zinc supply, due to ZNT7 deficiency, in the Golgi apparatus or the Golgi network where manganese is incorporated into the PP2c domain of the phosphatases, which enhances the availability of active PP2A and/or PHLPP in the patients' cells triggering apoptosis and suppressing cell growth. Future research is needed to identify which phosphatase is the target of ZNT7 that regulates the activity of the AKT signaling pathway, and to study the mechanisms underlying the preservation of the balance between zinc and manganese liable pools affecting signal transduction activities in cells.

The major function of PTEN is to act as a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase that specifically dephosphorylates phosphatidylinositol (3,4,5)-trisphosphate (PIP₃) to phosphatidylinositol (4,5)-trisphosphate (PIP₂) resulting in a reduced PDK1 activation and subsequent reduced AKT activation followed by increased apoptosis and an inhibition of cell cycle progression via TP53 [49]. The PTEN protein is comprised of two domains, a catalytic domain and a C2 structure domain that binds to phospholipid membrane tethering the catalytic site to the membrane-bound PIP3 to dephosphorylate it [70]. The association of zinc and PTEN activity was elucidated by Wu et al. indicating that zinc could induce PTEN protein degradation through ubiquitin-associated proteolytic process in human airway epithelia cells and rat lungs [65]. Additionally, studies from Plum et al. suggest that zinc may bind to two cysteine residues at 71 and 124 within the catalytic site of PTEN inhibiting its enzymatic activity in T lymphocytes [63]. Moreover, Han et al. showed that cellular zinc levels were associated with the activities of PTEN and the AKT and MDM2 activation as well as p53 levels in the nucleus [69]. Based on these reports and the results from the current study, we speculate that a reduction in cellular zinc accumulation caused by ZNT7 deficiency may result in an inhibition of proteolytic degradation of PTEN leading to down-regulation of the AKT signaling pathway, and subsequently increased apoptosis and inhibited cell cycle progression via increased TP53 levels [5].

It is worth noting that increased PTEN or decreased AKT activities can also induce cell apoptosis via Ca⁺⁺ ion release from the ER store and subsequent transfer to the mitochondria through the inhibition of the activity of IP3R (inositol 1,4,5-trisphosphate receptor type 3) [71,72]. Increased concentrations of Ca⁺⁺ ions in the mitochondria promote pro-apoptotic factors being released enabling apoptosis [73]. Thus, it is possible that reduced cell survival observed in the proband with ZNT7 deficiency could be triggered by alterations of both zinc and calcium signals in the cell. Future studies are warranted to confirm this theory.

In response to a variety of physiological or physiopathological signals, such as growth, inflammation, or DNA damage, AKT can also be activated by many other kinases, including tyrosine kinases and serine/threonine kinases independent of the classical PI3K-dependent pathway. For example, in response to growth factor stimulations, receptor tyrosine kinases, such as IGF1R (insulin-like growth factor 1 receptor) and EGFR (epidermal growth factor receptor) tyrosine kinases, can activate AKT by activating mTORC2 (mTOR complex 2) that directly phosphorylates AKT at Ser473 [74]. In addition, TBK1 (TANK-binding kinase 1), a serine/threonine protein kinase, regulates cell proliferation and apoptosis in the PI3K-independent pathway by directly phosphorylating AKT at Thr308 [75]. Moreover, Src family kinases can activate AKT at Tyr315 and Tyr326, promoting AKT stability and preventing apoptosis [76]. Together, AKT activation involves serine/threonine kinases and tyrosine kinases in both PI3K-dependent and PI3K-independent pathways, which are essential for cell survival, growth, and proliferation.

A balanced activity of TP53 in cells, such as hematopoietic progenitor cells, is critical for maintaining genomic stability and regulating apoptosis, cell cycle progression, as well as senescence. Reduced TP53 expression, caused by mutations in its DNA-binding domain, is linked to the development of various cancers. These mutations impair TP53's ability to bind specific promoter sequences of its target genes, leading to disrupted gene transcription during DNA repair and cell cycle progression, thereby promoting cancer cell growth [49]. On the other hand, TP53 also plays a central regulatory role in pathogenesis of bone marrow failure due to excessively expressed TP53 promoting cell cycle arrest, senescence, and apoptosis [36,45,77]. Excessive TP53 expression has been identified as a pathogenic factor in cytopenia and bone marrow failure syndromes, particularly in many inherited bone marrow failure disorders. Mutations occurring at the C-terminal end of TP53, resulting in a loss of the C-terminal amino acids, where the protein stability domain resides, often lead to significantly increased TP53 stability and enhanced DNA-binding capability [36,41,42,45]. This, in turn, causes stronger transactivation of TP53 to its target genes, more effective cell cycle arrest in response to DNA damage, and heightened senescence and apoptosis that are frequently observed in the cases of inherited bone marrow failure syndromes [41]. Moreover, genetic mutations in genes that encode proteins regulating 1) TP53 mRNA expression, such as PARN (dyskeratosis congenita) [78]; 2) ubiquitin-mediated TP53 degradation, such as MDM4 (bone marrow failure syndrome 6) [37]; or 3) defects in ribosomal RNA biogenesis or telomeres shortening triggering TP53 expression, such as DNAJC21 (bone marrow failure syndrome 3) [34], are all implicated in the development of bone marrow failure and cytopenia in humans. Given the reduced AKT activation and subsequent overexpression of TP53 in patient's B-EBV lymphocytes with ZNT7 mutations while the patient's heterozygous carrier mother is relatively unaffected, it is reasonable to suggest that excessive expression of TP53 may likely be implicated in bone marrow failure and cytopenia observed in patients with Ziegler-Huang Syndrome/BMF8.

It has been reported previously that zinc deficiency impaired hematopoietic progenitor cells partly via TP53 activation-induced apoptosis supporting the results from this study [79]. Other studies also showed that TP53 regulates some zinc transporter expressions, such as ZNT1 and ZIP14 to maintain intracellular zinc homeostasis [80,81]. However, in this study, we did not observe this regulatory phenomenon, at least, at the mRNA levels in the patient's B-EBV lymphocytes compared to the control cells from the patient's mother. Nevertheless, it
is important to point out that mRNA levels do not always correlate with protein levels due to many reasons, such as different turnover rates of mRNAs and proteins, post-translation modifications affecting protein detectability, and the availability of antibodies suitable for protein quantification. Thus, future studies are warranted to illustrate the responses of other zinc transporters at protein levels as well as at the post-translational modification levels in cells with or without ZNT7 mutations.

It is worth noting that TP53 is a zinc dependent transcription factor with a single zinc ion bound to the DNA binding domain to facilitate DNA binding in the target promoters for transcriptional regulation. It has also been reported that zinc supplementation is beneficial for suppressing tumor growth via reactivating TP53 in TP53-deficient cancers [82]. Interestingly, in the current study, we found that TP53 protein levels were not significantly changed by zinc treatments at various levels in the patient B-EBV lymphocytes deficient for ZNT7 (Suppl. Fig. 1), indicating the importance of ZNT7 for zinc levels in the Golgi apparatus.

Interestingly, in mice, Znt7 deficiency negatively affected the numbers of circulating neutrophils, lymphocytes, and monocytes without affecting red blood cells (at early age) or platelets. However, it is worth noting that adult Znt7KO mice at 18 weeks of age started to display anemia with reduced red cell counts and lower hemoglobin, as well as lower hematocrit. But we have no evidence indicating that mouse ZnT7 is involved in platelet development in the mouse bone marrow, nor that it has a negative effect on the numbers of circulating platelets. These data suggest, although ZNT7 is critical for mature blood cell production, the mouse does not perfectly replicate the human disorder likely because of the differences in the ZNT7 expression pattern in hematopoietic progenitor cells between mice and humans.

In the patient's B-EBV lymphocytes, ZNT7 deficiency could trigger genetic compensation [83], leading to a less severe phenotype by upregulating or downregulating the expression or function of other zinc transporters to maintain zinc homeostasis in cells. It has been reported that zinc concentrations in the Golgi apparatus are preserved by ZNT7 and ZIP7 proteins [16,21,84,85]. Since the function of ZIP7 is to remove zinc out from the Golgi apparatus to the cytoplasm, a function just opposite to ZNT7 [85], we expected that the expression of ZIP7 would be downregulated to preserve zinc levels in the organelle. While the current study did not reveal changes in mRNA expression of ZIP7 between P1 and mother cells, it is crucial to concede that the possibility of changes in ZIP7 protein levels, its cellular localization patterns, or its function in terms of the phosphorylation status, independent of transcription, remains. Future studies are needed to explore the implications of ZIP7 in the development of bone marrow failure.

In conclusion, our studies highlight the critical role of ZNT7 in the activation of AKT and control of TP53 expression in bone marrow cell development. We also show that the regulatory role of the human and mouse ZnT7 in the activation of AKT is conserved [26,30], but the differences in expression patterns explain the variations in clinical manifestation between the two species.

## Ethics declarations

### Contributions

LH conceived and designed molecular and cellular work as well as animal work for this study. She established and maintained transformed B lymphoblast cell lines. She also established and maintained P2 fibroblast cell lines overexpressing wild-type ZNT7 in the cells. She also maintained other cell lines in this study. She participated in experimental work obtaining and analyzing the data, creating figures and tables, drafting the manuscript, and finally approving the manuscript. AZ participated in generating hypotheses for the study. He also participated in editing and finally approving the manuscript. STN conducted Western blot analyses and edited the manuscript. ZY conducted RNA purification and quantitative RT-PCR; participated in editing manuscript;

maintained the mouse colony; collected experimental mice and performed genotyping. CPK performed total RNA isolation and Western blot analyses. She also carried out immunohistochemistry and immunocytochemistry. CPK participated in editing the manuscript. CP contributed to managing clinical samples and clinical data collection. He also participated in editing manuscripts. CM and IP conducted hematological investigations of patients and participated in management of the two probands. RC carried out the endocrinological investigation of patients and participated in management for the two probands. MCC performed bone marrow histological analyses of patients. DB participated in data acquisition, funding, and manuscript editing. OB participated in the human sample biobank registry and manuscript editing.

### CRediT authorship contribution statement

**Bonneau Dominique:** Writing – review & editing, Funding acquisition, Data curation. **Blanchet Odile:** Resources. **Copin Marie-Christine:** Writing – review & editing, Data curation. **Coutant Régis:** Writing – review & editing, Investigation. **Miot Charline:** Writing – review & editing, Investigation. **Huang Liping:** Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Mallebranche Coralie:** Writing – review & editing. **Pellier Isabelle:** Writing – review & editing, Investigation. **Kirschke Catherine P.:** Writing – review & editing, Supervision, Resources, Methodology, Investigation, Data curation. **Prouteau Clément:** Writing – review & editing, Resources. **Ziegler Alban:** Writing – review & editing, Resources, Investigation, Data curation. **Nguyen Steven T.:** Writing – review & editing, Visualization, Data curation. **Yang Zhonggyue:** Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation.

### Consent for publication

All authors consent for publication.

### Ethics approval

All animal experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Experimental Animals and animal protocols were approved by the Institutional Animal Care and Use Committee of the University of California, Davis. Animal protocol # 23125.

## Funding and additional information

This work was supported by USDA/ARS/Western Human Nutrition Research Center project funds (2032-51000-005-00D) to Dr. Liping Huang. This work was also supported by the University Hospital Angers, Angers, France and by GROUPAMA foundation for rare disease. The USDA is an equal opportunity provider and employer. The content is solely the responsibility of the authors and does not necessarily represent the official views of the USDA.

## Author statement

All authors have edited and approved the final revision of the submitted manuscript.

## Declaration of Competing Interest

All authors declare that they have no competing interests.

## Acknowledgements

We thank USDA/ARS/Western Human Nutrition Research Center for the general support for this research. We thank the French family for
their participation in the study.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jtemb.2025.127658.

## Data Availability

Datasets generated during the current study are available from Dr. Liping Huang (USDA/ARS/Western Human Nutrition Research Center, lphuang99@yahoo.com) upon reasonable request.

## References

[1] A.S. Prasad, A. Miale, Jr, Z. Farid, H.H. Sandstead, A.R. Schulert, Zinc metabolism in patients with the syndrome of iron deficiency anemia, hepatosplenomegaly, dwarfism, and hypogadism, J. Lab. Clin. Med. 61 (1963) 537-549.

[2] J. Frausto da Silva, R. Williams. The Biological Chemistry of the Elements, Second Edition, Oxford University Press, New York, NY, 2001.

[3] Y. Song, S.W. Leonard, M.G. Traber, E. Ho, Z. Zinc deficiency affects DNA damage, oxidative stress, antioxidant defenses, and DNA repair in rats, J. Nutr. 139 (2009) 1626-1631, https://doi.org/10.3945/jn.109.106369.

[4] D. Wilson, G. Varigos, M.L. Ackland, Apoptosis may underlie the pathology of zinc-deficient skin, Immunol. Cell Biol. 84 (2006) 28-37, https://doi.org/10.1111/j.1440-1711.2005.01391.x.

[5] R.S. Corniola, N.M. Tassabehji, J. Hare, G. Sharma, C.W. Levenson, Zinc deficiency impairs neuronal precursor cell proliferation and induces apoptosis via p53-mediated mechanisms, Brain Res. 1237 (2008) 52-61, https://doi.org/10.1016/j.brainres.2008.08.040.

[6] L. Huang, S. Tepaamorndech, The SLC30 family of zinc transporters - a review of current understanding of their biological and pathophysiological roles, Mol. Asp. Med. 34 (2013) 548-560, https://doi.org/10.1016/j.mam.2012.05.008.

[7] J. Jeong, D.J. Eide, The SLC39 family of zinc transporters, Mol. Asp. Med. 34 (2013) 612-619, https://doi.org/10.1016/j.mam.2012.05.011.

[8] F. Chimienti, S. Devergnas, A. Favier, M. Seve, Identification and cloning of a beta-cell-specific zinc transporter, Znt-8, localized into insulin secretory granules, Diabetes 53 (2004) 2330-2337, https://doi.org/10.2337/diabetes.53.9.2330.

[9] K. Wang, B. Zhou, Y.M. Kuo, J. Zemansky, J. Gitschier, A novel member of a zinc transporter family is defective in acrodermatitis enteropathica, Am. J. Hum. Genet 71 (2002) 66-73, https://doi.org/10.1086/341125.

[10] W. Chowanadisai, B. Lonnerdal, S.L. Kelleher, Identification of a mutation in SLC30A2 (ZnT-2) in women with low milk zinc concentration that results in transient neonatal zinc deficiency, J. Biol. Chem. 281 (2006) 39699-39707, https://doi.org/10.1074/jbc.M605821200.

[11] L. Huang, Z. Yang, C.P. Kirschke, C. Prouteau, M.C. Copin, D. Bonneau, et al., Identification of novel compound heterozygous variants in the SLC30A7 (ZNT7) gene in two French brothers with stunted growth, testicular hypoplasia and bone marrow failure, Hum. Mol. Genet 32 (2023) 2016-2031, https://doi.org/10.1093/hmg/hda033.

[12] D.B.D. Steel, F.R. Danti, M. Abunada, B. Kamien, S. Malhotra, M. Topf, et al., Clinical phenotype in individuals with birk-landau-perez syndrome associated with biallelic SLC30A9 pathogenic variants, Neurology 100 (2023) e2214-e2223, https://doi.org/10.1212/WNL.0000000000207241.

[13] M. Quadri, A. Federico, T. Zhao, G.J. Breedveld, C. Battisti, C. Delnooz, et al., Mutations in SLC30A10 cause parkinsonism and dystonia with hypermanganesemia, polycythemia, and chronic liver disease, Am. J. Hum. Genet 90 (2012) 467-477, https://doi.org/10.1016/j.ajhg.2012.01.017.

[14] K, Tuschl, P.T. Clayton, S.M. Gospe Jr., S. Gulab, S. Ibrahim, P. Singhi, et al., Syndrome of hepatic cirrhosis, dystonia, polycythemia, and hypermanganesemia caused by mutations in SLC30A10, a manganese transporter in man, Am. J. Hum. Genet 90 (2012) 457-466, https://doi.org/10.1016/j.ajhg.2012.01.018.

[15] H. Guo, X. Jin, T. Zhu, T. Wang, P. Tong, L. Tian, et al., SLC39A5 mutations interfering with the BMP/TGF-beta pathway in non-syndromic high myopia, J. Med. Genet 51 (2014) 518-525, https://doi.org/10.1136/jmedgenet-2014-102351.

[16] C. Anzilotti, D.J. Swan, B. Boisson, M. Deobagkar-Lele, C. Oliveira, P. Chabosseau, et al., An essential role for the Zn(2+) transporter ZIP7 in B cell development, Nat. Immunol. 20 (2019) 350-361, https://doi.org/10.1038/s41590-018-0295-8.

[17] K.M. Boycott, C.L. Beaulieu, K.D. Kernohan, O.H. Gebril, A. Mhanni, A.E. Chudley, et al., Autosomal-recessive intellectual disability with cerebellar atrophy syndrome caused by mutation of the manganese and zinc transporter gene SLC39A8, Am. J. Hum. Genet 97 (2015) 886-893, https://doi.org/10.1016/j.ajhg.2015.11.002.

[18] J.H. Park, M. Hogrebe, M. Gruneberg, I. DuChesne, A.L. von der Heiden, J. Reunert, et al., SLC39A8 deficiency: a disorder of manganese transport and glycosylation, Am. J. Hum. Genet 97 (2015) 894-903, https://doi.org/10.1016/j.ajhg.2015.11.003.

[19] C. Giunta, N.H. Elcioglu, B. Albrecht, G. Eich, C. Chambaz, A.R. Janecke, et al., Spondylocheiro dysplastic form of the Ehlers-Danlos syndrome–an autosomal-recessive entity caused by mutations in the zinc transporter gene SLC39A13, Am. J. Hum. Genet 82 (2008) 1290-1305, https://doi.org/10.1016/j.ajhg.2008.05.001.

[20] K. Tuschl, E. Meyer, L.E. Valdivia, N. Zhao, C. Dadswell, A. Abdul-Sada, et al., Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia, Nat. Commun. 7 (2016) 11601, https://doi.org/10.1038/ncomms11601.

[21] C.P. Kirschke, L. Huang, ZnT7, a novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus, J. Biol. Chem. 278 (2003) 4096-4102, https://doi.org/10.1074/jbc.M207644200.

[22] H.B. Bui, S. Watanabe, N. Nomura, K. Liu, T. Uemura, M. Inoue, et al., Cryo-EM structures of human zinc transporter ZnT7 reveal the mechanism of Zn(2+) uptake into the Golgi apparatus, Nat. Commun. 14 (2023) 4770, https://doi.org/10.1038/s41467-023-04521-5.

[23] H. Yuasa, N. Morino, T. Wagatsuma, M. Munekane, S. Ueda, M. Matsunaga, et al., ZNT5-6 and ZNT7 play an integral role in protein N-glycosylation by supplying Zn (2+) to Golgi alpha-mannosidase II, J. Biol. Chem. 300 (2024) 107378, https://doi.org/10.1016/j.jbc.2024.107378.

[24] S. Tepaamorndech, P. Oort, C.P. Kirschke, Y. Cai, L. Huang, ZNT7 binds to CD40 and influences CD154-triggered p38 MAPK activity in B lymphocytes-a possible regulatory mechanism for zinc in immune function, FEBS Open Bio 7 (2017) 675-690, https://doi.org/10.1002/2211-5463.12211.

[25] S. Tepaamorndech, L. Huang, C.P. Kirschke, A null-mutation in the *Znt7* gene accelerates prostate tumor formation in a transgenic adenocarcinoma mouse prostate model, Cancer Lett. 308 (2011) 33-42, https://doi.org/10.1016/j.canlet.2011.04.011.

[26] S. Tepaamorndech, C.P. Kirschke, T.L. Pedersen, W.R. Keyes, J.W. Newman, L. Huang, Zinc transporter 7 deficiency affects lipid synthesis in adipocytes by inhibiting insulin-dependent Akt activation and glucose uptake, FEBS J. 283 (2016) 378-394, https://doi.org/10.1111/febs.13582.

[27] L. Huang, S. Tepaamorndech, C.P. Kirschke, J.W. Newman, W.R. Keyes, T. L. Pedersen, et al., Aberrant fatty acid metabolism in skeletal muscle contributes to insulin resistance in zinc transporter 7 (*znt7*)-knockout mice, J. Biol. Chem. 293 (2018) 7549-7563, https://doi.org/10.1074/jbc.M117.817692.

[28] Q. Chu, Z.H. Chi, X. Zhang, D. Liang, X. Wang, Y. Zhao, et al., A potential role for zinc transporter 7 in testosterone synthesis in mouse Leydig tumor cells, Int J. Mol. Med 37 (2016) 1619-1626, https://doi.org/10.3892/ijmm.2016.2576.

[29] L. Huang, Y.Y. Yu, C.P. Kirschke, E.R. Gertz, K.K. Lloyd, Znt7 (Slc30a7)-deficient mice display reduced body zinc status and body fat accumulation, J. Biol. Chem. 282 (2007) 37053-37063, https://doi.org/10.1074/jbc.M706631200.

[30] L. Huang, C.P. Kirschke, Y.A. Lay, L.B. Levy, D.E. Lamirande, P.H. Zhang, Znt7-null mice are more susceptible to diet-induced glucose intolerance and insulin resistance, J. Biol. Chem. 287 (2012) 33883-33896, https://doi.org/10.1074/jbc.M111.309666.

[31] A.S. Prasad, A. Miale, Jr, Z. Farid, H.H. Sandstead, A.R. Schulert, W.J. Darby, Biochemical studies on dwarfism, hypogonadism, and anemia, Arch. Intern. Med. 111 (1963) 407-428, https://doi.org/10.1001/archinte.1963.03620280007003.

[32] A. Shimamura, B.P. Alter, Pathophysiology and management of inherited bone marrow failure syndromes, Blood Rev. 24 (2010) 101-122, https://doi.org/10.1016/j.blr.2010.03.002.

[33] M. Kirwan, A.J. Walne, V. Plagnol, M. Velangi, A. Ho, U. Hossain, et al., Exome sequencing identifies autosomal-dominant SRP72 mutations associated with familial aplasia and myelodysplasia, Am. J. Hum. Genet 90 (2012) 888-892, https://doi.org/10.1016/j.ajhg.2012.03.020.

[34] H. Tummala, A.J. Walne, M. Williams, M. Bockett, L. Collopby, S. Cardoso, et al., DNAJC21 mutations link a cancer-prone bone marrow failure syndrome to corruption in 60S ribosome subunit maturation, Am. J. Hum. Genet 99 (2016) 115-124, https://doi.org/10.1016/j.ajhg.2016.05.002.

[35] A. Alsultan, H.E. Shamseldin, M.E. Osman, M. Aljabri, F.S. Alkuraya, MYSM1 is mutated in a family with transient transfusion-dependent anemia, mild thrombocytopenia, and low NK- and B-cell counts, Blood 122 (2013) 3844-3845, https://doi.org/10.1182/blood-2013-09-527127.

[36] T. Toki, K. Yoshida, R. Wang, S. Nakamura, T. Maekawa, K. Goi, et al., De novo mutations activating germline tp53 in an inherited bone-marrow-failure syndrome, Am. J. Hum. Genet 103 (2018) 440-447, https://doi.org/10.1016/j.ajhg.2018.07.020.

[37] E. Toufekchian, V. Lejour, R. Durand, N. Giri, I. Draskovic, B. Bardot, et al., Germline mutation of MDM4, a major p53 regulator, in a familial syndrome of defective telomere maintenance, Sci. Adv. 6 (2020) eaay3511, https://doi.org/10.1126/sciadv.aay3511.

[38] Y. Oka, M. Hamada, Y. Nakazawa, H. Muramatsu, Y. Okuno, K. Higasa, et al., Digenic mutations in ALDH2 and ADH5 impair formaldehyde clearance and cause a multisystem disorder, AMED syndrome, Sci. Adv. 6 (2020), https://doi.org/10.1126/sciadv.abd7197.

[39] M. Park, Overview of inherited bone marrow failure syndromes, Blood Res 57 (2022) 49-54, https://doi.org/10.5045/br.2022.2022012.

[40] I. Roberts, I.S. Dokal, Inherited bone marrow failure syndromes, in: J. Firth, C. Conlon, T. Cox (Eds.), Inherited bone marrow failure syndromes, Oxford University Press, Oxford, 2020, pp. 5326-5336.

[41] J. Rakotopare, F. Toledo, p53 in the molecular circuitry of bone marrow failure syndromes, Int. J. Mol. Sci. 24 (2023), https://doi.org/10.3390/ijms241914940.

[42] D. Fedorova, G. Ovsyanikova, M. Kurnikova, A. Pavlova, T. Konyukhova, A. Pshonkin, et al., De novo TP53 germline activating mutations in two patients with the phenotype mimicking Diamond-Blackfan anemia, Pediatr. Blood Cancer 69 (2022) e29558, https://doi.org/10.1002/pbc.29558.

[43] R. Ceccaldi, K. Parmar, E. Mouly, M. Delord, J.M. Kim, M. Regairaz, et al., Bone marrow failure in Fanconi anemia is triggered by an exacerbated p53/p21 DNA damage response that impairs hematopoietic stem and progenitor cells, Cell Stem Cell 11 (2012) 36-49, https://doi.org/10.1016/j.stem.2012.05.013.
[44] X. Li, A.F. Wilson, W. Du, Q. Pang, Cell-cycle-specific function of p53 in fanconi anemia hematopoietic stem and progenitor cell proliferation, Stem Cell Rep. 10 (2018) 339-346, https://doi.org/10.1016/j.stemcr.2017.12.006.

[45] W.C. Fok, E.L.O. Niero, C. Dege, K.A. Brenner, C.M. Sturgeon, L.F.Z. Batista, p53 Mediates definitive human hematopoiesis in dyskeratosis congenita, Stem Cell Rep. 9 (2017) 409-418, https://doi.org/10.1016/j.stemcr.2017.06.015.

[46] M.T. Elghetany, B.P. Alter, p53 protein overexpression in bone marrow biopsies of patients with Shwachman-Diamond syndrome has a prevalence similar to that of patients with refractory anemia, Arch. Pathol. Lab. Med. 126 (2002) 452-455, https://doi.org/10.5858/2002-126-0452-PPOIBM.

[47] J. Donadieu, F. Delhommeau, TP53 mutations: the dawn of Shwachman clones, Blood 131 (2018) 376-377, https://doi.org/10.1182/blood-2017-11-815431.

[48] J. Xia, C.A. Miller, J. Baty, A. Ramesh, M.R.M. Jotte, R.S. Fulton, et al., Somatic mutations and clonal hematopoiesis in congenital neutropenia, Blood 131 (2018) 408-416, https://doi.org/10.1182/blood-2017-08-801985.

[49] H. Wang, M. Guo, H. Wei, Y. Chen, Targeting p53 pathways: mechanisms, structures, and advances in therapy, Signal Transduct. Target Ther. 8 (2023) 92, https://doi.org/10.1038/s41392-023-01347-1.

[50] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods 25 (2001) 402-408, https://doi.org/10.1006/meth.2001.1262.

[51] Y.Y. Yu, C.P. Kirschte, L. Huang, Immunohistochemical analysis of ZnT1, 4, 5, 6, and 7 in the mouse gastrointestinal tract, J. Histochem. Cytochem. 55 (2007) 223-234, https://doi.org/10.1369/jhc.6A7032.2006.

[52] Y. Cai, C.P. Kirschte, L. Huang, SLC30A family expression in the pancreatic islets of humans and mice: cellular localization in the beta-cells, J. Mol. Histol. 49 (2018) 133-145, https://doi.org/10.1007/s10735-017-9753-0.

[53] S. Dutt, A. Narla, K. Lin, A. Mullally, N. Abayasekara, C. Megerdichian, et al., Haploinsufficiency for ribosomal protein genes causes selective activation of p53 in human erythroid progenitor cells, Blood 117 (2011) 2567-2576, https://doi.org/10.1182/blood-2010-07-295238.

[54] Z. Sun, Q. Jiang, B. Gao, X. Zhang, L. Bu, L. Wang, et al., AKT Blocks SIK1-mediated repression of STAT3 to promote breast tumorigenesis, Cancer Res 83 (2023) 1264-1279, https://doi.org/10.1158/0008-5472.CAN-22-3407.

[55] F. Naeim, Principles of Immunophenotyping: F. Naeim (Eds.), Princ. Immunophenotyping, Acad. Press, Oxf. (2008) 27-55.

[56] W.M. Nauseef, I. Olsson, K. Arnljots, Biosynthesis and processing of myeloperoxidase-a marker for myeloid cell differentiation, Eur. J. Haematol. 40 (1988) 97-110, https://doi.org/10.1111/j.1600-0609.1988.tb00805.x.

[57] S. Dornan, Z. Sebestyen, J. Gamble, P. Nagy, A. Bodnar, L. Alldridge, et al., Differential association of CD45 isoforms with CD4 and CD8 regulates the actions of specific pools of p56lck tyrosine kinase in T cell antigen receptor signal transduction, J. Biol. Chem. 277 (2002) 1912-1918, https://doi.org/10.1074/jbc.M108386200.

[58] D.M. Townsley, B. Dumitriu, N.S. Young, Bone marrow failure and the telomeropathies, Blood 124 (2014) 2775-2783, https://doi.org/10.1182/blood-2014-05-526285.

[59] B.D. Manning, L.C. Cantley, AKT/PKB signaling: navigating downstream, Cell 129 (2007) 1261-1274, https://doi.org/10.1016/j.cell.2007.06.009.

[60] U.S. Cho, W. Xu, Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme, Nature 445 (2007) 53-57, https://doi.org/10.1038/nature05351.

[61] C. Letourneux, G. Rocher, F. Porteu, B56-containing PP2A dephosphorylate ERK and their activity is controlled by the early gene IEX-1 and ERK, EMBO J. 25 (2006) 727-738, https://doi.org/10.1038/sj.emboj.7600980.

[62] T. Gao, F. Furnari, A.C. Newton, PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth, Mol. Cell 18 (2005) 13-24, https://doi.org/10.1016/j.molcel.2005.03.008.

[63] L.M. Plum, A. Brieger, G. Engelhardt, S. Hebel, A. Nessel, M. Arlt, et al., PTEN-inhibition by zinc ions augments interleukin-2-mediated Akt phosphorylation, Metallomics 6 (2014) 1277-1287, https://doi.org/10.1039/c3mt00197k.

[64] D. Liang, L. Xiang, M. Yang, X. Zhang, B. Guo, Y. Chen, et al., ZnT7 can protect MC3T3-E1 cells from oxidative stress-induced apoptosis via PI3K/Akt and MAPK/ERK signaling pathways, Cell Signal 25 (2013) 1126-1135, https://doi.org/10.1016/j.cellsig.2013.02.003.

[65] W. Wu, X. Wang, W. Zhang, W. Reed, J.M. Samet, Y.E. Whang, et al., Zinc-induced PTEN protein degradation through the proteasome pathway in human airway epithelial cells, J. Biol. Chem. 278 (2003) 28258-28263, https://doi.org/10.1074/jbc.M303318200.

[66] M. Taniguchi, A. Fukunaka, M. Hagihara, K. Watanabe, S. Kamino, T. Kambe, et al., Essential role of the zinc transporter ZIP9/SLC39A9 in regulating the activations of Akt and Erk in B-cell receptor signaling pathway in DT40 cells, PLoS One 8 (2013) e58022, https://doi.org/10.1371/journal.pone.0058022.

[67] K.J. Anson, G.A. Corbet, A.E. Palmer, Zn(2+) influx activates ERK and Akt signaling pathways, Proc. Natl. Acad. Sci. USA 118 (2021), https://doi.org/10.1073/pnas.2015786118.

[68] Y. Zhao, Y. Tan, J. Dai, B. Wang, B. Li, L. Guo, et al., Zinc deficiency exacerbates diabetic down-regulation of Akt expression and function in the testis: essential roles of PTEN, PTP1B and TRB3, J. Nutr. Biochem 23 (2012) 1018-1026, https://doi.org/10.1016/j.jnutbio.2011.05.011.

[69] C.T. Han, N.W. Schoene, K.Y. Lei, Influence of zinc deficiency on Akt-Mdm2-p53 and Akt-p21 signaling axes in normal and malignant human prostate cells, Am. J. Physiol. Cell Physiol. 297 (2009) C1188-C1199, https://doi.org/10.1152/apjcell.00042.2009.

[70] J.O. Lee, H. Yang, M.M. Georgescu, A. Di Cristofano, T. Maehama, Y. Shi, et al., Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association, Cell 99 (1999) 323-334, https://doi.org/10.1016/s0092-8674(00)81663-3.

[71] M. Bittremieux, J.B. Parys, P. Pinton, G. Bultynck, ER functions of oncogenes and tumor suppressors: Modulators of intracellular Ca(2+) signaling, Biochim Biophys Acta 1863 (2016) 1364-1378, https://doi.org/10.1016/j.bbamcr.2016.01.002.

[72] A.J. Eustace, M.J. Lee, G. Colley, J. Roban, T. Downing, P.J. Buchanan, Aberrant calcium signalling downstream of mutations in TP53 and the PI3K/AKT pathway genes promotes disease progression and therapy resistance in triple negative breast cancer, Cancer Drug Resist 5 (2022) 560-576, https://doi.org/10.20517/cdr.2022.41.

[73] S. Marchi, A. Rimessi, C. Giorgi, C. Baldini, L. Ferroni, R. Rizzuto, et al., Akt kinase reducing endoplasmic reticulum Ca2+ release protects cells from Ca2+-dependent apoptotic stimuli, Biochem Biophys. Res Commun. 375 (2008) 501-505, https://doi.org/10.1016/j.bbrc.2008.07.153.

[74] S.F. Moore, R.W. Hunter, I. Hers, mTORC2 protein complex-mediated Akt (Protein Kinase B) Serine 473 Phosphorylation is not required for Akt1 activity in human platelets corrected, J. Biol. Chem. 286 (2011) 24553-24560, https://doi.org/10.1074/jbc.M110.202341.

[75] Y.H. Ou, M. Torres, R. Ram, E. Formstecher, C. Roland, T. Cheng, et al., TBK1 directly engages Akt/PKB survival signaling to support oncogenic transformation, Mol. Cell 41 (2011) 458-470, https://doi.org/10.1016/j.molcel.2011.01.019.

[76] R. Chen, O. Kim, J. Yang, K. Sato, K.M. Eisenmann, J. McCarthy, et al., Regulation of Akt/PKB activation by tyrosine phosphorylation, J. Biol. Chem. 276 (2001) 31858-31862, https://doi.org/10.1074/jbc.C100271200.

[77] E. Ho, C. Courtemanche, B.N. Ames, Zinc deficiency induces oxidative DNA damage and increases p53 expression in human lung fibroblasts, J. Nutr. 133 (2003) 2543-2548, https://doi.org/10.1093/jn/133.8.2543.

[78] S. Dhanraj, S.M. Gunja, A.P. Deveau, M. Nissbeck, B. Boonyawat, A.J. Coombs, et al., Bone marrow failure and developmental delay caused by mutations in poly(A)-specific ribonuclease (PARN), J. Med. Genet 52 (2015) 738-748, https://doi.org/10.1136/jmedgenet-2015-103292.

[79] R. Seth, R.S. Corniola, S.D. Gower-Winter, T.J. Morgan, Jr, B. Bishop, C. W. Levenson, Zinc deficiency induces apoptosis via mitochondrial p53- and caspase-dependent pathways in human neuronal precursor cells, J. Trace Elem. Med. Biol. 30 (2015) 59-65, https://doi.org/10.1016/j.jtemb.2014.10.010.

[80] N. Zhao, A.S. Zhang, A.M. Wortham, S. Jue, M.D. Knutson, C.A. Enns, The Tumor Suppressor, P53, Decreases the Metal Transporter, ZIP14, Nutrients 9 (2017), https://doi.org/10.3390/nu9121335.

[81] B. Bri克, X. Yu, D. Carpizo, A Nov. Funct. p53: Regul. zinc Homeost. (February 11 2025), https://sites.rutgers.edu/nb-senior-exhibits/partners/honors-college/briken-a-novel-function-of-p53-regulation-of-zinc-homeostasis/.

[82] X. Yu, S. Kogan, Y. Chen, A.T. Tsang, T. Withers, H. Lin, et al., Zinc metallochaperones reactivate mutant p53 using an ON/OFF switch mechanism: a new paradigm in cancer therapeutics, Clin. Cancer Res 24 (2018) 4505-4517, https://doi.org/10.1158/1078-0432.CCR-18-0822.

[83] A. Rossi, Z. Kontarakis, C. Gerri, H. Nolte, S. Holper, M. Kruger, et al., Genetic compensation induced by deleterious mutations but not gene knockdowns, Nature 524 (2015) 230-233, https://doi.org/10.1038/nature14580.

[84] E. Tuncay, C.V. Bitirim, Y. Olgar, A. Durak, G.A. Rutter, B. Turan, Zn(2+)-transporters ZIP7 and ZnT7 play important role in progression of cardiac dysfunction via affecting sarco(endo)plasmic reticulum-mitochondria coupling in hyperglycemic cardiomyocytes, Mitochondrion 44 (2019) 41-52, https://doi.org/10.1016/j.mito.2017.12.011.

[85] L. Huang, C.P. Kirschte, Y. Zhang, Y.Y. Yu, The ZIP7 gene (Slc39a7) encodes a zinc transporter involved in zinc homeostasis of the Golgi apparatus, J. Biol. Chem. 280 (2005) 15456-15463, https://doi.org/10.1074/jbc.M412188200.