ORIGINAL ARTICLE

An Acrodermatitis Enteropathica-Associated
Zn Transporter, ZIP4, Regulates Human
Epidermal Homeostasis
Bum-Ho Bin1, Jinhyuk Bhin2, Nan-Hyung Kim3, Su-Hyon Lee4, Haeng-Sun Jung4, Juyeon Seo1,
Dae-Kyum Kim5,6, Daehee Hwang7, Toshiyuki Fukada8, Ai-Young Lee3, Tae Ryong Lee1 and
Eun-Gyung Cho1

Acrodermatitis enteropathica is an autosomal recessive disorder characterized by scaly eczematous dermatosis

accompanied by alopecia and diarrhea. Various mutations in the SLC39A4 gene (ZIP4), which encodes a zinc
transporter, are responsible for this disorder. However, the molecular mechanism underlying the involvement
of ZIP4 in the pathogenesis of this condition has yet to be established. In this study, we report the role of ZIP4
in human epidermis. ZIP4 is predominantly expressed in human keratinocytes, and its expression is dramati-
cally reduced on epidermal differentiation. ZIP4 knockdown in human keratinocytes down-regulates zinc (Zn)
levels and the transcriptional activity of a key epidermal Zn-binding protein, DNp63, and dysregulates
epidermal differentiation in a reconstituted human skin model, resulting in the appearance of proliferating
keratinocytes even in the uppermost layers of the skin. We verified that, among the amino acid residues in its
Zn-binding motif, Cys205 is critical for the processing and nuclear distribution of DNp63 and, therefore,
Zn-dependent transcriptional activity. Our results suggest that ZIP4 is essential for maintaining human
epidermal homeostasis through the regulation of Zn-dependent DNp63 activity and can provide insight
into the molecular mechanisms responsible for the cutaneous symptoms observed in Acrodermatitis
enteropathica patients.
Journal of Investigative Dermatology (2017) 137, 874e883; doi:10.1016/j.jid.2016.11.028

INTRODUCTION intracellular organelles, respectively (Jeong and Eide,

Zinc (Zn) is an essential metal ion found in living organ-
isms that is estimated to bind approximately 3,000 proteins
in vivo, which corresponds to approximately 10% of the
human proteome (Kimura and Kambe, 2016). Intracellular
Zn homeostasis is systemically regulated by Zrt- and
Irt-like proteins (ZIP; encoded by SLC39A), and Zn
transporters (ZnT; encoded by SLC30A) (Fukada and
Kambe, 2011; Taylor and Nicholson, 2003). ZIP and ZnT
direct Zn across biological membranes, that is, into the
cytosol from extracellular spaces or intracellular organ-
elles and from the cytosol to extracellular spaces or into
1
Basic Research & Innovation Division, R&D Unit, AmorePacific
Corporation, Yongin, Republic of Korea; 2Department of Chemical
Engineering, POSTECH, Pohang, Republic of Korea; 3Department of
Dermatology, Dongguk University Ilsan Hospital, Goyang, Republic of
Korea; 4Bio Solution Corporation, Seoul, Republic of Korea; 5Donnelly
Centre, Departments of Molecular Genetics and Computer Science,
University of Toronto, Toronto, Ontario, Canada; 6Lunenfeld-Tanenbaum
Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada;
7
Department of New Biology and Center for Plant Aging Research, Institute
for Basic Science, DGIST, Daegu, Republic of Korea; and 8Faculty of
Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan
Correspondence: Eun-Gyung Cho and Tae Ryong Lee, Yongin, Gyeonggi-do
446-729, Republic of Korea. E-mail: egcho@amorepacific.com or trlee@
amorepacific.com
Abbreviations: AE, acrodermatitis enteropathica; MT, metallothionein; ZIP,
Zrt- and Irt-like proteins
Received 4 July 2016; revised 24 October 2016; accepted 16 November
2016; accepted manuscript published online 7 December 2016; corrected
proof published online 24 February 2017
2013; Tian et al., 2014).
At least 14 ZIPand 9 ZnT transporters have been identified in
mammalian genomes (Fukada and Kambe, 2011; Taylor and
Nicholson, 2003). Each transporter shows a different expres-
sion pattern with respect to tissue distribution, cellular local-
ization, and responsiveness to stimuli (Kimura and Kambe,
2016), suggesting a unique and pivotal role of each trans-
porter under specific circumstances. Fourteen ZIP family
members are further divided into four subfamilies, LIV-1, gufA,
and ZIP I and II (Kambe and Andrews, 2009). Of these, LIV-1
family members are observed only in eukaryotic cells,
implying that this family plays an important role as a primary
Zn transporter in eukaryotes (Taylor and Nicholson, 2003).
Null mouse studies of each LIV-1 member have determined
that ZIP10 regulates antiapoptotic signaling and B-cell antigen
receptor signaling by modulating caspase-3 and CD45R
phosphatase activities, respectively (Hojyo et al., 2014; Miyai
et al., 2014). ZIP13 is highly expressed in connective tissue and
regulates the nuclear translocation of the Zn-finger transcrip-
tion factor SMAD for collagen production, resulting in growth
retardation and skeletal and connective tissue abnormalities in
ZIP13-null mice (Bin et al., 2011; Bin et al., 2014a; Bin et al.,
2014b; Fukada et al., 2008). Considering the unique charac-
teristics of individual ZIP knockout mice, there are likely no
redundancies among them, and these members play essential
physiological roles in mammals.
ZIP4 is mutated at various sites in rare recessive genetic
disorders in humans, including acrodermatitis enteropathica
(AE), which is caused by insufficient Zn absorption in the

874 Journal of Investigative Dermatology (2017), Volume 137 ª 2016 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.

intestine and characterized by severe dermatosis symptoms,
including scaly eczematous, psoriasiform skin, diarrhea, and
alopecia (Kambe and Andrews, 2009; Wang et al., 2004;
Wang et al., 2002). ZIP4 is known to be expressed at the
apical surface of enterocytes in the intestine to regulate Zn up-
take in mice (Kambe and Andrews, 2009; Wanget al., 2002).
Homozygous ZIP4 knockout mice are embryonic lethal during
early development, and heterozygous offspring are hypersen-
sitive to Zn deficiency, displaying developmental defects, such
as exencephalia, anophthalmia, and growth retardation
(Dufner-Beattie et al., 2007). Although ZIP4 is highly associated
with AE pathogenesis in humans, the molecular mechanisms
underlying the regulation of ZIP4 in human are unknown. This
study describes the function of ZIP4 in human skin. We found
that ZIP4 is essential for epidermal differentiation in vitro and in
a reconstituted human skin model.
RESULTS
ZIP4, a Zn transporter in human epidermis, regulates
epidermal Zn homeostasis
To investigate whether Zn is involved in human epidermal
homeostasis, we examined the distribution of Zn in human
skin by staining for the Zn scavenging and responding protein
metallothionein (MT) (Coyle et al., 2002). In murine skin, MT
and Zn are enriched in proliferative epidermal keratinocytes
(Hanada et al., 1998; Iwata et al., 1999; Karasawa et al.,
1991). As expected, the expression of MT was enriched in
the basal layer of human skin epidermis (Figure 1a), implying
that Zn levels are higher in hyperplastic and less differenti-
ated layers than in differentiated layers. Zn levels in cultured
human primary keratinocytes were consistently high under
growth conditions but were remarkably reduced on differ-
entiation (1.8 mM Ca2þ treatment for 4 days) when measured
using the Zn-selective indicator FluoZin-3 (Figure 1b). These
data correspond well with a previous report that Zn levels are
higher in many progenitor and premature cells than in
differentiated cells (Kitamura et al., 2006; Miyai et al., 2014)
and suggest that Zn may be involved in epidermal homeo-
stasis by regulating differentiation. To examine any significant
effects caused by Zn deficiency, we treated primary human
keratinocytes with Zn-depleted media, in which Zn was
chelated by N,N,N0 ,N0 -tetrakis (2-pyridylmethyl) ethylenedi-
amine (TPEN), a well-known high-affinity Zn chelator. Recent
data found that TPEN treatment could induce Zn deficiency
in cultured cells (Homma et al., 2013). Although Zn has a
high binding affinity for Zn-binding proteins and organic
compounds, Zn can move from protein to protein or other
molecules within cells (Gupta et al., 2014; Lu and Fu, 2007).
Under Zn-deficient conditions (TPEN treatment), primary
human keratinocytes exhibited abnormal morphology
compared with nontreated control cells (Figure 1c), and
cotreatment with TPEN and several metal ions other than Zn
could inhibit the proper growth of primary human keratino-
cytes (Figure 1c). Because the Zn distribution inside and
outside of cells or organelles is dependent on Zn transporters,
we examined the expression of multiple Zn transporters in
human epidermal keratinocytes. In human keratinocytes,
ZIP4 was predominantly detected (Figure 1d), suggesting its
involvement in the formation of human epidermal cells. The
mRNA and protein levels of ZIP4 were remarkably lowered
B-H Bin et al.
ZIP4 Regulates Epidermal Homeostasis
upon the differentiation of human keratinocytes (Figure 1d
and e), and Zn levels were severely reduced after ZIP4
knockdown (KD) by specific shRNA against ZIP4 in undif-
ferentiated keratinocytes (Figure 1f and g). The relative
Zn-FluoZin-3 complex intensity was analyzed using ImageJ
software (NIH, Bethesda, MD; http://rsbweb.nih.gov/ij/
download.html) (Figure 1g, bottom). To confirm that ZIP4
KD reduces the intracellular Zn level specifically, we intro-
duced specific siRNA against ZIP4 (siZIP4) or scrambled
control siRNA (siControl) into keratinocytes for 3 days, and
inductively coupled plasma mass spectrometry analysis was
performed. We found that ZIP4 KD reduced the intracellular
Zn level (Figure 1h). These data suggest that ZIP4 is involved
in regulating Zn levels in the human epidermis; therefore, we
focused on verifying the physiological role of ZIP4 in
epidermal homeostasis.
ZIP4 knockdown disturbs epidermis formation in a
reconstituted human skin model
To investigate the role of ZIP4 in the differentiation of human
epidermis, we introduced siZIP4 or siControl into keratino-
cytes and then generated a reconstituted human skin model.
The knockdown efficiency of siZIP4 was confirmed with
quantitative reverse transcriptase-PCR in real time and Western
blotting (Figure 2a and b). In the siControl-treated reconstituted
model, the epidermis was properly constructed, exhibiting
condensed and stratified layers (Figure 2c; siControl). How-
ever, siZIP4-treated epidermis was improperly differentiated
in its upper layers, displaying countable nuclei (inset magni-
fied) even in the uppermost layer and sparse meshlike
morphology in the suprabasal layers (Figure 2c; siZIP4). In an
siZIP4-treated skin model, the expression patterns of epidermal
differentiation markers, such as filaggrin, involucrin, and
keratin 14, were altered: filaggrin levels decreased, involucrin
levels increased, and keratin 14 levels decreased (Figure 2d),
indicating that the epidermis was abnormally formed when
ZIP4 was knocked down. To determine whether the nuclei
observed in the upper layers of the siZIP4-treated reconstituted
skin model are caused by the presence of proliferating kerati-
nocytes, we stained the epidermis with antibodies against
proliferating cell nuclear antigen to identify replicating cells
and found that proliferating keratinocytes existed in the upper
layers (Figure 2e; siZIP4), which was not observed in normal
epidermal differentiation (Fig. 2e; siControl). Consistent
with these results, we also observed the effects of ZIP4 KD in
human primary keratinocytes, including abnormal differenti-
ation characterized by the existence of nuclei in ZIP4
KD-transfected cells under calcium-induced differentiation
conditions (Figure 2f; siZIP4, Differentiated).
Zn and ZIP4 are essential for functional p63 activity during
epidermal differentiation
Based on our results showing high levels of Zn in the basal
layer (Figure 1a) and the existence of proliferating cells in the
upper layer of the siZIP4-treated reconstituted skin model
(Figure 2e), we hypothesized that protein activities requiring
Zn in the basal layer might be involved in epidermal differ-
entiation and, therefore, regulated by ZIP4. Various tran-
scription factors involved in epidermal formation require Zn
(Koster and Roop, 2007). Of these factors, p63 is an essential
Zn-binding transcription factor orchestrating epidermal
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 B-H Bin et al.
ZIP4 Regulates Epidermal Homeostasis

Figure 1. ZIP4, a major Zn a b

transporter in human epidermis,
regulates epidermal Zn homeostasis.
(a) Expression of the MT protein in
human epidermis. Scale bars ¼ 50
mm. (b) Intracellular Zn levels were
seen by FluoZin-3 staining. Scale
bars ¼ 50 mm. (c) Human epidermal
keratinocytes were incubated with
conditional medium containing either
TPEN (5 mM) alone or TPEN (5 mM)
with the indicated metal compounds
(each 5 mM) for 72 hours. Scale bars ¼
100 mm. (d) The expression of Zn
transporters was analyzed with
c d
quantitative reverse transcriptase-PCR. Human epidermal
*P < 0.05. (e) Expression levels of the keratinocyte
400 *

Relative transcript level

ZIP4 protein. (f) Expression levels of
the ZIP4 protein in cells harboring 300
either control shRNA or shZIP4. (g)
Intracellular Zn levels were seen by 200
FluoZin-3 staining. Relative intensity
(bottom). Scale bars ¼ 50 mm; 100
*P < 0.05. (h) Measurement of Zn
level with inductively coupled plasma 0

3
P4

P5

P8
atomic emission spectroscopy.

P1

P1

P1
ZI

ZI

ZI
ZI

ZI

ZI
***P < 0.005.

Undifferntiated
Differentiated

e g h

development, as demonstrated in p63 null mice, which exhibit
significant epidermal malformations (Koster et al., 2007;
Koster et al., 2004; Yang et al., 1999). Many pathogenic mu-
tations have also been found in a Zn-binding motif of p63
(Chen et al., 2011; Yue et al., 2005). Therefore, we examined
whether ZIP4 KD affects the expression and activity of p63.
When siZIP4-treated reconstituted human epidermis was
stained with an anti-p63 antibody, the p63-positive layers were
found to be thinner (Figure 3a, asterisks) in siZIP4-treated
epidermis than in siControl-treated epidermis, and the p63
signal was enriched along the basement membrane (Figure 3a,
inset magnified). We also found that ZIP4 KD significantly
reduced the transcriptional activity of p63 (Figure 3b). In
addition, ZIP4 KD reduced the expression of the p63 target
genes adenosine deaminase (ADA), cyclin D3 (CCND3), and
conserved helix-loop-helix ubiquitous kinase (CHUK) in
human epidermal keratinocytes (Figure 3c). The expression of
these genes was also reduced under Zn-deficient conditions
achieved by TPEN treatment but recovered with Zn repletion
(Figure 3d), indicating that the regulatory effect of ZIP4 KD on
p63 activity was phenocopied with Zn deficiency. The alter-
ations in filaggrin and involucrin expression caused by ZIP4
KD (Figure 2d) were also reproduced by Zn deficiency and
restored by Zn repletion with the recovery of TP63 and ZIP4
expression (Supplementary Figure S1 online). Overall, these
results indicate that both Zn and ZIP4 are directly involved in
the regulation of p63 activity and, therefore, epidermal
differentiation in human skin.

876 Journal of Investigative Dermatology (2017), Volume 137


Cys205 in a Zn-binding motif is critical for the Zn-dependent
functional activity of DNp63
In addition to the down-regulation of p63 activity, we found
that the distribution of endogenous p63 within the nucleus was
altered after treatment with shZIP4 or TPEN (Supplementary
Figure S2 online). To investigate the direct involvement of Zn
in the distribution of p63, we replaced three cysteine residues
and one histidine residue in a conserved Zn-binding motif
B-H Bin et al.
ZIP4 Regulates Epidermal Homeostasis
Figure 2. ZIP4 knockdown disturbs
the formation of the epidermis in a
reconstituted human skin model.
(a, b) Human epidermal keratinocytes
were treated with siControl or siZIP4
for 3 days. The expression of ZIP4 was
analyzed with quantitative reverse
transcriptase-PCR (a) or with western
blot analysis (b). (c) ZIP4 KD disturbs
epidermal stratification in a human
skin equivalent model. Scale bars ¼
400 mm. (d) ZIP4 KD reduces the
expression of filaggrin, involucrin, and
keratin 14 in a human skin equivalent
model. Scale bars ¼ 400 mm. (e)
Immunohistochemistry for
proliferating cell nuclear antigen
reveals that ZIP4 KD disturbs the
proper formation of the epidermis.
Scale bars ¼ 100 mm. (f) Human
epidermal keratinocytes were treated
with siRNA for 48 hours and then
cultured with medium containing 1.8
mM Ca2þ for 4 days. Cells were
visualized with an inverted
microscope. Boxed areas are
magnified. Scale bars ¼ 50 mm.
within DNp63, a major form of p63 found in human epidermis,
with serine residues (DNp63Mut-4S) (Figure 4a) and examined
the distribution and activity of DNp63 after overexpressing
wild-type or mutant constructs in HeLa cells. DNp63 is known
to regulate stimuli-responsive genes after being processed by
proteases upon treatment with doxorubicin (Jeon et al., 2012;
Sayan et al., 2007). Wild-type DNp63 (DNp63WT) was prop-
erly processed by doxorubicin treatment at moderate
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 B-H Bin et al.
ZIP4 Regulates Epidermal Homeostasis

Figure 3. Zn and ZIP4 are essential

for p63 activity during epidermal
differentiation. (a) Immunohisto-
chemical staining for p63 found that
ZIP4 KD disturbs the proper formation
of the epidermis. Scale bars ¼ 100
mm; Vertical bar indicates epidermal
thickness. Asterisk indicates
suprabasal layer. (b) H1299 cells
stably expressing the DNp63 protein
were transfected with siZIP4. Forty-
eight hours after transfection, the cells
were further transfected with PG13-
Luc for 24 hours, and a luciferase
assay was performed. ***P < 0.001.
(c) The expression levels of ADA,
CCND3, and CHUK in human
epidermal keratinocytes were
analyzed with quantitative reverse
transcriptase-PCR. *P < 0.05. (d)
Human epidermal keratinocytes
were treated with 5 mM TPEN in
combination with 5 mM ZnSO4 for
24 hours, after which mRNA was
isolated. The expression levels of
each gene in human epidermal
keratinocytes were analyzed with
quantitative reverse transcriptase-PCR.
***P < 0.001.

concentrations (0.1w0.4 mM), leading to the disappearance of
full-length DNp63 (Figure 4b, DNp63WT). However, treatment
at a cytotoxic concentration of 0.8 mM led to the accumulation
of DNp63 (Figure 4b, DNp63WT), implying that a structural
change affects the protease-dependent processing of DNp63.
Compared with the wild-type protein, the level of mutant
DNp63 protein (DNp63Mut-4S), which harbors four serine
residues, was sustained even at low concentrations of doxo-
rubicin, leading to remarkable accumulation at higher
concentrations (Figure 4b, DNp63Mut-4S). We also observed
immunocytochemically that DNp63Mut-4S was distributed
differently from the wild-type protein in the nucleus, localizing
in certain areas but not in promyelocytic leukemia protein-
positive regions (Figure 4c; Supplementary Figure S3 online).
These data imply that the DNp63 protein, which has mutations
in its Zn-binding motif, underwent structural changes and that
this mutation was directly related to impaired DNp63 function
due to changes in protein processing and nuclear distribution.
Among the four amino acids in the conserved Zn-binding
motif, the His208, Cys269, and Cys273 residues were identi-
fied to be mutated to Tyr in several genetic malformation
syndromes (Chen et al., 2011; Yue et al., 2005). However, the
involvement of Cys205 has not been associated with human
disease. To investigate whether Cys205 is involved in the
observed alteration in DNp63 processing and distribution, we
introduced single or multiple mutations in the Zn-binding
motifs of full-length p63 (TAp63) or DNp63 (Figure 4d,
Mut-1S, Mut-2S, and Mut-3S) and analyzed the effects of these
mutations. Interestingly, a single mutation replacing Cys205
with Ser (C205S) (DNp63Mut-1S), rather than multiple muta-
tions, was sufficient to induce the altered distribution and
doxorubicin-induced accumulation of DNp63 (Figure 4e,

878 Journal of Investigative Dermatology (2017), Volume 137

 B-H Bin et al.
ZIP4 Regulates Epidermal Homeostasis

a b c Figure 4. Cys205 in the Zn-binding

motif is critical for the Zn-dependent
functional activity of DNp63. (a)
Models of the DNp63Mut-4S protein. (b)
TAp63WT
The expression of DNp63Mut-4S in
ΔNp63WT
CPNH CNSSC
HeLa cells was analyzed by western
ΔNp63Mut-4S blotting 24 hours after DOX
SPNS SNSSCS incubation. (c) Nuclear localization of
the DNp63Mut-4S protein. Scale bars ¼
25 mm. (d) Models of the mutated p63
proteins. (e) The nuclear localization
d e of mutant p63 proteins. Scale bars ¼
25 mm. (f) Expression of the C205S
(DNp63Mut-1S) mutant protein. (g)
TAp63Mut-1S H1299 cells were transfected with
ΔNp63Mut-1S each DNp63 construct and PG13-Luc
(C205S) SPNH CNSSC for 24 hours, after which a luciferase
assay was performed. ***P < 0.001.
TAp63Mut-2S
(h) Posttranslational degradation of
ΔNp63Mut-2S
SPNS CNSSC DNp63WT under Zn deficiency. 293T
cells were analyzed after 10 mM CHX
TAp63Mut-3S treatment in combination with 2 mM
ΔNp63Mut-3S TPEN for 24 hours. The relative
SPNS SNSSC expression level (right). Data are
representative of two independent
experiments.

f g

h 1.2
Relative expression level

0.9

0.6

0.3 DMSO
TPEN
0
0 2 4

lower panel; Figure 4f) and significantly decreased the tran-
scriptional activity of DNp63 (Figure 4g). These results suggest
that Cys205 is a critical site for the Zn-dependent processing,
distribution, and transcriptional activity of DNp63. Interest-
ingly, the distribution of DNp63, but not TAp63, was signifi-
cantly affected by mutations in its Zn-binding motif (Figure 4e),
despite the fact that this Zn-binding motif was identical in the
two proteins (Figure 4d). Finally, we analyzed the post-
translational degradation of DNp63WT under Zn-deficient
conditions. The results showed that TPEN treatment inhibited
the degradation of DNp63WT, which is consistent with the
results observed when the Zn-binding motif was mutated
(Figure 4f and h), supporting the hypothesis that Zn deficiency
could inhibit the proper proteolysis of DNp63.
ZIP4 is enriched in the hyperproliferative rete ridge area of
psoriatic skin
Considering our result that ZIP4 is essential for epidermal
homeostasis via its role in the regulation of DNp63 activity
(Figures 2e4), we assumed that ZIP4 might be enriched in
the basal layer, in which p63 is supposed to be tightly
regulated. In human skin specimens, the ZIP4 signal was
distributed in the epidermis, with immunohistochemical
analysis using an anti-ZIP4 antibody, which found a
condensed signal at the basement membrane (Figure 5a,
arrowheads). ZIP4 expression was also found to be increased
in the basal layer (in the rete ridges) of psoriatic skin
epidermis (Figure 5b, arrowheads), where keratinocytes
proliferate extensively and cause one of the dominant

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 B-H Bin et al.
ZIP4 Regulates Epidermal Homeostasis

Figure 5. ZIP4 is enriched in the hyperproliferative rete ridge area of psoriatic skin. (a) Normal areas (N) and (b) psoriatic skin lesions (P) in human skin
specimens obtained from two psoriasis patients (#1 and #2) were stained with anti-ZIP4 antibody (green); nuclei were stained with DAPI (blue). Arrowheads
indicate the apparent expression of ZIP4. The insets are magnified. Scale bars ¼ 400 mm.

characteristics of psoriasis, hyperplasia (Mason et al., 2013; DISCUSSION

Spuls et al., 2010; Thielen et al., 2005). These data suggest
that ZIP4 is enriched in the human epidermis, especially in
the basal layer, in which keratinocytes reside and proliferate,
that Zn-dependent DNp63 regulates epidermal differentia-
tion, and that the overstimulation of ZIP4 may be involved in
the progression of psoriasis.
We found that, within the Zn transporter ZIP family, the
AE-causative ZIP4 is dominantly expressed in human epidermis.
Similar to the expression pattern of a Zn-induced protein, MT,
ZIP4 expression is enriched in the basal layer of the human
epidermis, in which the Zn-binding master transcription factor
DNp63 is expressed, to orchestrate epidermal homeostasis.

880 Journal of Investigative Dermatology (2017), Volume 137


There are two variants of p63, a full-length protein called
TAp63 and a variant called DNp63 that lacks the N-terminal
transactivation domain (McKeon, 2004). In adult mice, the
expression of TAp63 is extremely low, and the results of a
study with mice lacking TAp63 in the keratin 14epositive
basal layer found that TAp63 is not crucial for the formation
of epidermis (Su et al., 2009). Instead, DNp63 is the primary
form expressed in the adult epidermis and is involved in
epidermal morphogenesis (Koster and Roop, 2007). There-
fore, the relationship between DNp63 and Zn is important in
adult epidermal homeostasis. Zn deficiency induces a
mutant-like conformation in superoxide dismutase 1, which
binds to Derlin-1, thus initiating the homeostatic endo-
plasmic reticulum stress response (Homma et al., 2013).
When epithelial-mesenchymal transition occurs, the zinc
transporter ZIP6 is essential for the nuclear localization of the
zinc-finger protein SNAIL, a master regulator of epithelial-
mesenchymal transition (Yamashita et al., 2004). The loss of
ZIP13 induces the mislocalization of the zinc-binding protein
SMAD (Fukadaet al., 2008). In a similar way, Zn availability
via ZIP4 may be essential for DNp63 function and the proper
cellular distribution and conformation of DNp63. We found
that mutations in the Zn-binding motif of DNp63 inhibit
protease-dependent processing and induce abnormal nuclear
distribution. In contrast with DNp63, full-length TAp63 does
not exhibit changes in its nuclear distribution when muta-
tions are present in its Zn-binding motif, suggesting different
sensitivities to Zn level or a compensatory role of the N-ter-
minal transactivation domain in Zn-deficient conditions. In
the Zn-binding motif of DNp63, we identified Cys205 as an
essential residue for Zn-dependent DNp63 regulation.
Despite its obvious contribution to DNp63 regulation, only
Cys205 mutations have not been reported in association with
human disease and pathogenesis (Chenet al., 2011), sug-
gesting that mutations at Cys205 may cause embryonic
lethality.
Considering the importance of Zn availability via the ZIP4
transporter to DNp63 activity in the human epidermis, we
propose a pathogenic mechanism in which the cutaneous
manifestations of AE such as scaly, psoriasiform, or mildly
eczematous eruptions can be interpreted on a molecular level.
In normal skin, Zn in the basal layer is transported to cells via
ZIP4 and sufficiently supplied to proteins, including DNp63,
which are essential for epidermal differentiation. Thus, differ-
entiation is properly regulated. In contrast, in patients with AE,
we propose that DNp63 is Zn deficient due to mutations in
ZIP4 and is less functional because of changes in conformation
and localization (Supplementary Figure S4 online).
Human epidermal keratinocytes exhibit high Zn levels
when undifferentiated or in the basal layer, but Zn levels
dramatically decrease upon cellular or epidermal differenti-
ation, that is, in the suprabasal layers. Undifferentiated ker-
atinocytes in the basal layer are thought to undergo
continuous cell division to achieve epidermal homeostasis
(Blanpain and Fuchs, 2009), implying that these cells require
more Zn than differentiated cells. Proliferating cells actively
synthesize DNA and RNA, and the polymerases involved in
each process possess Zn-binding sites and require Zn to
properly interact with DNA or other proteins (Petrucco and
Percudani, 2008). Including the polymerases that synthesize
B-H Bin et al.
ZIP4 Regulates Epidermal Homeostasis
DNA and RNA, approximately 5% to 10% of all cellular
proteins are known to bind Zn (Andreini et al., 2006). Like-
wise, Zn deficiency due to ZIP4 mutations or deletions can
affect the activity and stability of diverse Zn-binding tran-
scription factors and enzymes, leading to alterations in
physiological processes.
Because homozygous null mice exhibit embryonic
lethality (Dufner-Beattie et al., 2007), ZIP4 is known to be
essential for murine embryogenesis and physiological
development. In accordance with the indispensable impor-
tance of ZIP4 in mice, many mutations in the ZIP4 sequence
have been identified in human patients with rare autosomal
recessive genetic disorders associated with AE (Kambe and
Andrews, 2009; Nakano et al., 2003). ZIP4 mediates the
influx of dietary Zn into the intestinal brush border of both
humans and mice (Kambe and Andrews, 2009; Wanget al.,
2004; Wanget al., 2002). However, in the skin, the expres-
sion of ZIP4 and its contribution to epidermal homeostasis
seem to be different in humans and mice. ZIP4 is dominantly
expressed in human epidermal keratinocytes, but it is barely
expressed in murine epidermal keratinocytes (Inoue et al.,
2014). Human epidermis is distinct from murine epidermis
in several ways: (i) human epidermis is composed of multiple
layers and is a thick epidermis, (ii) human epidermis pos-
sesses widespread rete ridge structures; in which keratino-
cytes actively proliferate to maintain epidermal homeostasis;
and thus (iii) human keratinocytes may require more Zn than
murine keratinocytes. Therefore, as an essential Zn trans-
porter in human epidermis, ZIP4 is an appropriate target for
the regulation of Zn-dependent epidermal homeostasis, and
studies of this molecule will provide insights into the patho-
genesis of AE and its various cutaneous symptoms.
We show that an essential Zn transporter in human
epidermis, ZIP4, governs Zn-dependent epidermal homeo-
stasis by regulating the transcriptional activity of a key ecto-
dermal Zn-binding transcription factor, DNp63. Our study
may provide insight to use in the interpretation of patholog-
ical conditions such as psoriasis or dermatitis in AE patients.
MATERIALS AND METHODS
Cell culture and materials
Normal human keratinocytes (Cascade Biologics, Portland, OR)
were maintained in keratinocyte growth (KGM) GOLD medium
(Lonza, Basel, Switzerland). To make conditional medium, the so-
dium form of Chelex-100 (Bio-Rad, Hercules, CA) was incubated (10
g/500 ml) with KGM GOLD for 1 hour at room temperature. HeLa
and H1299 cells were obtained from the Korean Cell Line Bank
(KCLB, Seoul, Korea). FluoZin-3 (Life Technologies, Carlsbad, CA),
TPEN (Sigma, St. Louis, MO), doxorubicin (Sigma), and cyclohexi-
mide (Sigma) were dissolved in DMSO. pcDNA4-HisMax constructs
expressing either TAp63a or DNp63a were generous gifts from Dr
Chin Ha Jung at Seoul National University, South Korea.
Reconstituted human skin model
A human skin epidermal equivalent was reconstructed as previously
described (Jung et al., 2014). Briefly, primary human keratinocytes
were seeded on a 12-mm Millicell (Millipore, Billerica, MA) and
incubated for 7 days until confluence. Next, epithelial differentiation
was induced by air-liquid interface culture for 10 days with 3T3
www.jidonline.org 881
 B-H Bin et al.
ZIP4 Regulates Epidermal Homeostasis
feeder layers. For the siZIP4-treated human skin model, ZIP4 siRNA
was added to the culture every 5 days.
Human skin specimens
This study was approved by the Institutional Review Board of
Dongguk University Ilsan Hospital and was conducted according to
the principles described in the Declaration of Helsinki. Five patients
(female-to-male ratio, 3:2; age, 21e68 years, with a mean of
45.4 years) diagnosed with psoriasis were enrolled in this study after
providing written informed consent. Psoriasis skin specimens were
biopsied with a 3-mm-diameter punch for immunohistochemical
analysis. Skin specimens from three healthy individuals (females;
age, 53e67, with a mean of 60 years) were used as controls.
siRNA and shRNA transfection
Human epidermal keratinocytes were transiently transfected with
100 nM ON-TARGETplus SMARTpool siRNA (Thermo Fisher
Scientific, Waltham, MA) using Lipofectamine RNAiMAX (Life
Technologies). To obtain shRNA-expressing cells, a ZIP4 shRNA
plasmid was constructed using the ZIP4 sequence (50 -ATGTCAGGA
GCGGGTCTTGC-30 ). After transfection with this plasmid, selection
was performed for 2 weeks with 200e1,000 mg/ml Zeocin (Geno-
lution, Seoul, Korea), as previously described (Bin et al., 2015).
Immunostaining
Cells were cultured on Lab-Tek chamber slides (Nunc, Penfield, NY),
fixed with 4% paraformaldehyde in phosphate buffered saline, per-
meabilized with 0.1% Triton X-100 in phosphate buffered saline con-
taining 1% BSA for 5 min, and incubated with anti-p63 antibody
(Abcam, Cambridge, United Kingdom) or anti-promyelocytic leukemia
protein antibody (Sigma). For immunohistochemistry, epidermal
specimens were fixed in 4% paraformaldehyde and embedded in
paraffin (Bin et al., 2016). After blocking with 3% BSA, sections were
incubated first with primary antibodies to MT (1:100) (Abcam), p63
(1:100), (Santa Cruz Biotechnology, Dallas, Texas), proliferating cell
nuclear antigen (Dako), keratin 14 (1:100) (Abcam), involucrin (1:100)
(NeoMarkers, Fremont, CA), filaggrin (1:100) (Novocastra Laboratories,
Newcastle, United Kingdom), and ZIP4 (Abcam).
Western blot analysis
Cells were lysed with RIPA lysis buffer (Sigma) containing protease
inhibitors (Sigma). Protein concentrations were determined using
bicinchoninic acid assays, and samples were resolved with SDS-
PAGE, transferred to a polyvinylidene difluoride membrane, and
probed with each indicated antibody.
Quantitative real-time PCR
Total RNA was isolated using TRIzol reagent (Life Technologies) and
reverse transcribed into cDNA using ReverTra Ace (Toyobo, Osaka,
Japan). cDNA from K38 mouse keratinocytes was purchased
(Sigma). Gene expression analyses were performed using TaqMan
Universal Master Mix and TaqMan Gene Expression assays (Applied
Biosystems, Foster City, CA).
Measurement of Zn level
To quantify the cellular Zn level in ZIP4-KD cells, 5  106 cells were
subjected to a modified acid deproteinizing method (Nomoto, 1987)
and then analyzed with inductively coupled plasma-atomic emis-
sion spectrometry as previously described (Bin et al., 2014a).
Luciferase assay
To analyze the effect of ZIP4 function on DNp63 activity, a luciferase
assay was performed based on the rationale described in previous
882 Journal of Investigative Dermatology (2017), Volume 137
reports (Jeon et al., 2012; Jeong et al., 2012). Briefly, H1299 cells
were seeded in 12-well plates and then transfected with PG13-Luc
and with a b-galactosidase-expressing plasmid to normalize trans-
fection efficiency. The luciferase assay was performed as described
by the manufacturer (Promega, Madison, WI).
Statistical analysis
Two-tailed t tests were used to analyze differences between two
groups. P-values less than 0.05 were considered statistically
significant.
CONFLICT OF INTEREST
B-HB, JS, TRL, and E-GC are all employees of Amorepacific Corporation. The
other authors declare that they have no conflicts of interest with regard to the
content of this article.
ACKNOWLEDGMENTS
A-YL was supported by a National Research Foundation of Korea
(NRF) grant funded by the Korean government (MSIP) (No. NRF-
2014R1A2A2A09051812).
AUTHOR CONTRIBUTIONS
B-HB, JB, N-HK, S-HL, H-SJ, JS, D-KK, DH, TF, A-YL, TRL, and E-GC
conceived and designed the experiments. B-HB, JB, S-HL, H-SJ, and JS per-
formed the experiments. B-HB, JB, D-KK, DH, A-YL, TRL, and E-GC analyzed
the data. B-HB, JB, TF, TRL, and E-GC wrote the paper.
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online version of the paper at www.
jidonline.org, and at http://dx.doi.org/10.1016/j.jid.2016.11.028.
REFERENCES
Andreini C, Banci L, Bertini I, Rosato A. Counting the zinc-proteins encoded
in the human genome. J Proteome Res 2006;5:196e201.
Bin BH, Bhin J, Yang SH, Shin M, Nam YJ, Choi DH, et al. Membrane-
associated transporter protein (MATP) regulates melanosomal pH and in-
fluences tyrosinase activity. PloS One 2015;10:e0129273.
Bin BH, Fukada T, Hosaka T, Yamasaki S, Ohashi W, Hojyo S, et al.
Biochemical characterization of human ZIP13 protein: a homo-dimerized
zinc transporter involved in the spondylocheiro dysplastic Ehlers-Danlos
syndrome. J Biol Chem 2011;286:40255e65.
Bin BH, Hojyo S, Hosaka T, Bhin J, Kano H, Miyai T, et al. Molecular path-
ogenesis of spondylocheirodysplastic Ehlers-Danlos syndrome caused by
mutant ZIP13 proteins. EMBO Mol Med 2014a;6:1028e42.
Bin BH, Hojyo S, Ryong Lee T, Fukada T. Spondylocheirodysplastic Ehlers-
Danlos syndrome (SCD-EDS) and the mutant zinc transporter ZIP13.
Rare Dis 2014b;2:e974982.
Bin BH, Kim DK, Kim NH, Choi EJ, Bhin J, Kim ST, et al. Fibronectin-
containing extracellular vesicles protect melanocytes against ultraviolet
radiation-induced cytotoxicity. J Invest Dermatol 2016;136:957e66.
Blanpain C, Fuchs E. Epidermal homeostasis: a balancing act of stem cells in
the skin. Nat Rev Mol Cell Biol 2009;10:207e17.
Chen C, Gorlatova N, Kelman Z, Herzberg O. Structures of p63 DNA binding
domain in complexes with half-site and with spacer-containing full
response elements. Proc Natl Acad Sci USA 2011;108:6456e61.
Coyle P, Philcox JC, Carey LC, Rofe AM. Metallothionein: the multipurpose
protein. Cell Mol Life Sci 2002;59(4):627e47.
Dufner-Beattie J, Weaver BP, Geiser J, Bilgen M, Larson M, Xu W, et al. The
mouse acrodermatitis enteropathica gene Slc39a4 (Zip4) is essential for
early development and heterozygosity causes hypersensitivity to zinc
deficiency. Hum Mol Genet 2007;16:1391e9.
Fukada T, Civic N, Furuichi T, Shimoda S, Mishima K, Higashiyama H, et al.
The zinc transporter SLC39A13/ZIP13 is required for connective tissue
development; its involvement in BMP/TGF-beta signaling pathways. PloS
One 2008;3:e3642.
Fukada T, Kambe T. Molecular and genetic features of zinc transporters in
physiology and pathogenesis. Metallomics 2011;3:662e74.

Gupta S, Chai J, Cheng J, D’Mello R, Chance MR, Fu D. Visualizing the ki-
netic power stroke that drives proton-coupled zinc(II) transport. Nature
2014;512:101e4.
Hanada K, Sawamura D, Hashimoto I, Kida K, Naganuma A. Epidermal
proliferation of the skin in metallothionein-null mice. J Invest Dermatol
1998;110:259e62.
Hojyo S, Miyai T, Fujishiro H, Kawamura M, Yasuda T, Hijikata A, et al. Zinc
transporter SLC39A10/ZIP10 controls humoral immunity by modulating B-
cell receptor signal strength. Proc Natl Acad Sci USA 2014;111:11786e91.
Homma K, Fujisawa T, Tsuburaya N, Yamaguchi N, Kadowaki H, Takeda K,
et al. SOD1 as a molecular switch for initiating the homeostatic ER stress
response under zinc deficiency. Mol Cell 2013;52:75e86.
Inoue Y, Hasegawa S, Ban S, Yamada T, Date Y, Mizutani H, et al. ZIP2
protein, a zinc transporter, is associated with keratinocyte differentiation.
J Biol Chem 2014;289:21451e62.
Iwata M, Takebayashi T, Ohta H, Alcalde RE, Itano Y, Matsumura T. Zinc
accumulation and metallothionein gene expression in the proliferating
epidermis during wound healing in mouse skin. Histochem Cell Biol
1999;112:283e90.
Jeon YJ, Jo MG, Yoo HM, Hong SH, Park JM, Ka SH, et al. Chemosensitivity is
controlled by p63 modification with ubiquitin-like protein ISG15. J Clin
Invest 2012;122:2622e36.
Jeong J, Eide DJ. The SLC39 family of zinc transporters. Mol Aspects Med
2013;34:612e9.
Jeong J, Walker JM, Wang F, Park JG, Palmer AE, Giunta C, et al. Promotion of
vesicular zinc efflux by ZIP13 and its implications for spondylocheiro
dysplastic Ehlers-Danlos syndrome. Proc Natl Acad Sci USA 2012;109:
E3530e8.
Jung KM, Lee SH, Jang WH, Jung HS, Heo Y, Park YH, et al. KeraSkin-VM: a
novel reconstructed human epidermis model for skin irritation tests. Tox-
icol In Vitro 2014;28:742e50.
Kambe T, Andrews GK. Novel proteolytic processing of the ectodomain of
the zinc transporter ZIP4 (SLC39A4) during zinc deficiency is inhibited
by acrodermatitis enteropathica mutations. Mol Cell Biol 2009;29:
129e39.
Karasawa M, Nishimura N, Nishimura H, Tohyama C, Hashiba H, Kuroki T.
Localization of metallothionein in hair follicles of normal skin and the
basal cell layer of hyperplastic epidermis: possible association with cell
proliferation. J Invest Dermatol 1991;97:97e100.
Kimura T, Kambe T. The functions of metallothionein and ZIP and ZnT
transporters: an overview and perspective. Int J Mol Sci 2016;17.
Kitamura H, Morikawa H, Kamon H, Iguchi M, Hojyo S, Fukada T, et al. Toll-
like receptor-mediated regulation of zinc homeostasis influences dendritic
cell function. Nat Immunol 2006;7:971e7.
Koster MI, Dai D, Marinari B, Sano Y, Costanzo A, Karin M, et al. p63 induces
key target genes required for epidermal morphogenesis. Proc Natl Acad Sci
USA 2007;104:3255e60.
Koster MI, Kim S, Mills AA, DeMayo FJ, Roop DR. p63 is the molecular switch for
initiation of an epithelial stratification program. Genes Dev 2004;18:126e31.
B-H Bin et al.
ZIP4 Regulates Epidermal Homeostasis
Koster MI, Roop DR. Mechanisms regulating epithelial stratification. Annu
Rev Cell Dev Biol 2007;23:93e113.
Lu M, Fu D. Structure of the zinc transporter YiiP. Science 2007;317:1746e8.
Mason AR, Mason JM, Cork MJ, Hancock H, Dooley G. Topical treatments for
chronic plaque psoriasis of the scalp: a systematic review. Br J Dermatol
2013;169:519e27.
McKeon F. p63 and the epithelial stem cell: more than status quo? Genes Dev
2004;18:465e9.
Miyai T, Hojyo S, Ikawa T, Kawamura M, Irie T, Ogura H, et al. Zinc trans-
porter SLC39A10/ZIP10 facilitates antiapoptotic signaling during early B-
cell development. Proc Natl Acad Sci USA 2014;111:11780e5.
Nakano A, Nakano H, Nomura K, Toyomaki Y, Hanada K. Novel SLC39A4
mutations in acrodermatitis enteropathica. J Invest Dermatol 2003;120:
963e6.
Nomoto S. A simplified method for trace metal analysis of biological mate-
rials. J UOEH 1987;9 Suppl:111e22.
Petrucco S, Percudani R. Structural recognition of DNA by poly(ADP-ribose)
polymerase-like zinc finger families. FEBS J 2008;275:883e93.
Sayan BS, Sayan AE, Yang AL, Aqeilan RI, Candi E, Cohen GM, et al. Cleavage
of the transactivation-inhibitory domain of p63 by caspases enhances
apoptosis. Proc Natl Acad Sci USA 2007;104:10871e6.
Spuls PI, Lecluse LL, Poulsen ML, Bos JD, Stern RS, Nijsten T. How good are
clinical severity and outcome measures for psoriasis?: quantitative evalu-
ation in a systematic review. J Invest Dermatol 2010;130:933e43.
Su X, Paris M, Gi YJ, Tsai KY, Cho MS, Lin YL, et al. TAp63 prevents premature aging
by promoting adult stem cell maintenance. Cell stem cell 2009;5:64e75.
Taylor KM, Nicholson RI. The LZT proteins; the LIV-1 subfamily of zinc
transporters. Biochim Biophys Acta 2003;1611:16e30.
Thielen AM, Kuenzli S, Saurat JH. Cutaneous adverse events of biological therapy
for psoriasis: review of the literature. Dermatology 2005;211:209e17.
Tian X, Zheng Y, Li Y, Shen Z, Tao L, Dou X, et al. Psychological stress induced
zinc accumulation and up-regulation of ZIP14 and metallothionein in rat
liver. BMC Gastroenterol 2014;14:32.
Wang F, Kim BE, Dufner-Beattie J, Petris MJ, Andrews G, Eide DJ. Acro-
dermatitis enteropathica mutations affect transport activity, localization
and zinc-responsive trafficking of the mouse ZIP4 zinc transporter. Hum
Mol Genet 2004;13:563e71.
Wang K, Zhou B, Kuo YM, Zemansky J, Gitschier J. A novel member of a zinc
transporter family is defective in acrodermatitis enteropathica. Am J Hum
Genet 2002;71:66e73.
Yamashita S, Miyagi C, Fukada T, Kagara N, Che YS, Hirano T. Zinc trans-
porter LIVI controls epithelial-mesenchymal transition in zebrafish gastrula
organizer. Nature 2004;429:298e302.
Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, et al. p63 is
essential for regenerative proliferation in limb, craniofacial and epithelial
development. Nature 1999;398:714e8.
Yue P, Li Z, Moult J. Loss of protein structure stability as a major causative
factor in monogenic disease. J Mol Biol 2005;353:459e73.
www.jidonline.org 883