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Critical Review
Zinc Deficiency-Induced Cell Death
Michael S. Clegg1, Lynn A. Hanna
1, Brad J. Niles
1, Tony Y. Momma
1and Carl L. Keen
1,2
Departments of 1Nutrition and 2Internal Medicine, University of California at Davis, Davis, California, USA
Summary
Zinc deficiency is characterized by an attenuation of growth
factor signaling pathways and an amplification of p53 pathways.
This outcome is facilitated by hypo-phosphorylation of AKT and
ERK secondary to zinc deficiency, which are permissive events to
the activation of the intrinsic cell death pathway. Low zinc
concentrations provide an environment that is also conducive to
the production of reactive oxygen/reactive nitrogen species (ROS/
RNS) and caspase activation. Additionally, during zinc deficiency
endogenous survival pathways such as NF-kB are inhibited in their
transactivation potential. The above factors contribute to the
irreversible commitment of the zinc deficient cell to death.
IUBMB Life, 57: 661–670, 2005
Keywords Zinc; zinc deficiency; apoptosis; growth factors; signaltransduction; IGF; caspase; NF-kB; nutrition; p53;AKT; ERK; reactive nitrogen species; reactive oxygenspecies.
INTRODUCTION
It is known that zinc deficiency is teratogenic in experi-
mental animals, and suboptimal zinc status is an etiologic
factor in human reproductive disorders and disease (1 – 2).
Indeed, animals consuming diets deficient in zinc display a
variety of maladies including stunted growth, reproductive
failure, teratology, immunoincompetence, cachexia and death.
The sequence of events underlying these defects is difficult to
decipher given the myriad of functions zinc serves in the body.
Querying the current Pfam data base for zinc yields an
impressive list of 173 protein family members (3). Zinc is an
integral component of proteins involved in cell structures (e.g.,
tubulin) (4, 5), and can be an activator (e.g., matrix metallopro-
teinases) or an inhibitor (e.g., caspases) (6). As an activator of
metalloproteinases, zinc is bound to three histidine residues in
the active site, while the fourth site is occupied by water,
allowing the Lewis acidity of this site to hydrolyze amide
bonds of protein substrates. Conversely, as an inhibitor of
caspases, zinc binding to an active site cysteine within a
conserved QACXG sequence most likely serves as the site of
inhibition.
Zinc is a component of protein domains that are essential
for binding DNA (e.g., p53), protein-protein interactions (e.g.,
protein kinase c (PKC)) (7), and protein – lipid interactions
(e.g., SARA). This essential element is a component of proteins
involved in ROS metabolism (e.g., CuZnSOD) and the inhi-
bition of apoptosis (e.g., IAP-2), and it is a constituent of
numerous proteins participating in proteosome-mediated pro-
tein turnover (e.g., E3 ligases). In many of these proteins, zinc is
associated with critical cysteine residues, which are sensitive to
the redox environment of the cell. These residues can undergo
post-translational modifications to become oxidized to cystine
or sulfenic acid, modified to mixed disulfides by S-thiolation
reactions (cysteine, glutathione) or S-nitrosolated (nitric oxide
(NO)). The outcome of these modifications is the loss of zinc,
which can be temporary or permanent, and leads to a gain, or
loss, of function of the specific affected protein.
Despite a plethora of essential functions, zinc’s roles in the
processes of cell proliferation and cell death deserve special
attention. The balance between these opposing, yet inter-
related forces ultimately governs the organism’s well being. In
this paper, we discuss how attenuation and amplification of
the mitogenic and cell death machinery, respectively, represent
mechanisms that underlie zinc deficiency-induced cell death.
Not discussed in this review, due to space constraints, are
studies demonstrating the protective effects of metallothio-
nein, a low molecular weight and cysteine-rich zinc binding
protein, which when induced by zinc, or recombinantly
overexpressed, protects cells against many physical and
chemical inducers of apoptosis (reviewed by Coyle et al. (8)).
Additionally, this review does not consider the role of zinc
transporters, whose levels in the cell can be impacted by
intracellular zinc concentrations and consequently may play
Address correspondence to: Michael S. Clegg MBA/PhD, Depart-ment of Nutrition, University of California at Davis, One ShieldsAve, Davis, CA 95616, USA. Tel: þ1 530 752 4658.E-mail: [email protected]
Received 4 May 2005; accepted 18 July 2005
IUBMBLife, 57(10): 661 – 669, October 2005
ISSN 1521-6543 print/ISSN 1521-6551 online � 2005 IUBMB
DOI: 10.1080/15216540500264554
an important role in protecting the cell against the damaging
effects of zinc loss (reviewed by Liuzzi and Cousins (9)).
ZINC DEFICIENCY-INDUCED ALTERATIONS IN CELLPROLIFERATION AND SURVIVAL PATHWAYS
Cell proliferation and cell survival pathways are intertwined
and both can be activated, or deactivated, by the addition or
withdrawal of protein growth factors. For example, insulin-like
growth factors, IGF-1 and IGF-2, function to increase growth,
promote differentiation, and inhibit apoptosis by acting pri-
marily through the IGF-1 receptor (IGF-1R). The actions of
IGFs are modulated by IGF Binding Proteins (IGFBPs) 1 – 6
that are present in the circulation and extracellular matrix, and
associated with cell surfaces. Rodents consuming zinc deficient
diets have low message and circulating levels of IGF-1, and
altered profiles of circulating IGFBPs (10). Paradoxically,
IGF-1 augmentation does not prevent the zinc deficiency-
induced reduction in growth (11). IGF-1 resistance may be a
consequence of the altered distribution of serum IGFBPs,
which affect the biological half-life of circulating IGF-1/2 and
sequester the IGF ligands from the IGF-1R (10).
Zinc deficiency may also disrupt signal transduction path-
ways after ligand binding. Numerous growth factor receptors,
such as IGF-1R, are receptor tyrosine kinases (RTKs). After
ligand binding, RTKs activate pro-survival and mitogenic
kinase pathways such as the RAS!ERK and PI3K!AKT
pathways. AKT and ERK are serine/threonine kinases that
phosphorylate and activate cell cycle machinery. Given that
zinc-deficient animals are growth refractory to IGF-1 supple-
mentation, we hypothesized that zinc-deficient animals and
cells would be refractory to other growth factors as well. 3T3
cells cultured in zinc-deficient medium (D medium) for 32 h
are characterized by a 50% reduction in cellular zinc (7) and a
90% reduction in cell number after 48 h (Fig. 1). Conversely,
cells cultured in zinc-deficient medium supplemented with zinc
(S medium) show a 2-fold increase in cell number during the
same period (Fig. 1). Others have demonstrated that 3T3 cells
cultured in zinc deficient medium up-regulate IGFBP-3, which
may in turn sequester IGF-1 and reduce IGF-1R signaling
(12). However, we observed that zinc-deficient cells are
unresponsive to LR3-IGF-1, a modified IGF-1 that activates
IGF-1R but does not interact with IGFBPs, as well as other
growth factors that were tested (Fig. 1). Importantly, cells
cultured in S medium respond to multiple growth factors as
indicated by enhanced cell growth (Fig. 1). Collectively, these
results suggest that a broad defect in RTK signaling occurs
during zinc deficiency.
Supporting this concept, 3T3 cells cultured in D medium
for 24 h display reduced phosphorylation of both AKT and
ERK proteins (Fig. 2). Changes in ERK phosphorylation as a
consequence of zinc deficiency have been noted earlier (13).
Importantly, IGF-1R, AKT, and ERK protein levels are
similar between the zinc deficient and control groups. This is
important, as activated caspases (see below) can efficiently
degrade these targets, making protein phosphorylation status
a moot point. In the work described above, we did not observe
a decrease in the phosphorylation of the upstream IGF-1R,
although this outcome has been reported for cells treated with
the chelator N, N, N’, N0-tetrakis(2-pyridylmethyl)ethylene-
diamine (TPEN) (14). These authors suggest that 50mMTPEN
can activate a protein tyrosine phosphatase (PTP) that
dephosphorylates the IGF-1R, perhaps by releasing the zinc
inhibition of the active site cysteine group (14). The different
outcomes between our study and that of Haase and Maret (14)
probably reflect the latter’s use of high concentrations of an
intracellular chelator, versus our use of a chelator-free zinc
deficient medium. TPEN has an affinity for zinc that far
Figure 1. 3T3 cells cultured in zinc deficient medium are
refractory to growth factor supplementation. 3T3 cells were
cultured for 48 h in either D (0.5 mM zinc) or S (50 mM zinc)
medium with or without growth factors, or growth factor
combinations at the following concentrations: 75 ng/ml IGF-1
or LR3-IGF-1, 10 ng/ml FGF, 0.65 mg/ml insulin, 25 ng/ml
PDGF. Cell numbers were determined by DNA assay. These
results are summarized from three separate experiments and
data were expressed as Mean+ SEM. Data were analyzed by
ANOVA, and post hoc differences were computed among
groups if a significant F value was obtained. Different lower
case letters indicate significantly different groups (p5 0.05).
Note: This model utilizes medium with fetal bovine serum
(FBS) depleted of zinc by short dialysis against an extra-
cellular chelator, followed by removal of the chelator-zinc
complex by exhaustive equilibrium dialysis. Zinc-deficient
medium was made from zinc depleted FBS diluted with
DMEM (D medium¼ 0.5 mM zinc). As a control, D medium
was supplemented with exogenous zinc (S medium¼ 50 mMzinc), and thus, D and S media differed only by their final
concentrations of zinc (7).
662 CLEGG ET AL.
exceeds even that of endogenous zinc binding proteins such as
metallothionein, and zinc finger transcription factors. Thus,
TPEN chelation may block attempts by the cell to adjust zinc
homeostasis in the face of declining intracellular zinc concen-
trations. A further complication in interpreting these studies is
that PTPs are also regulated by other factors including
oxidation, S-glutathionation, and S-nitrosylation, reactions
which can be influenced by intracellular zinc concentrations.
It is known that growth factor withdrawal, defective
growth factor signaling, and genotoxic stressors can trigger
cell cycle arrest and apoptosis. Several investigators have
noted cell cycle irregularities during zinc deficiency (13, 15 –17).
The tumor suppressor, p53, is activated by DNA damage
initiated by UV/g irradiation, hypoxia, chemotherapeutics,
and endogenous ROS production. p53 is a zinc finger trans-
cription factor that regulates both G1 and G2 checkpoints,
preventing cells with damaged DNA from proceeding with
new DNA synthesis or undergoing cell division. Zinc finger
transcription factors are logical targets for deactivation in vivo,
given that in vitro studies have demonstrated their sensitivity
to environments limited in zinc or high in ROS/RNS stress
((18, 19) and references within). p53 can induce cell cycle arrest
or apoptosis depending upon cellular context. Cell cycle
control is primarily accomplished by p53 transcriptional
activation of the p21waf1/cip1 gene, the protein product of
which can inhibit cyclin-D, cyclin dependent kinases (Cdk4, 6)
and cyclin D/Cdk2 complexes, resulting in the hypo-
phosphorylation of the retinoblastoma protein and G1 block.
Several investigators have examined whether zinc deficiency
induces changes in p53 mRNA levels, nuclear accumulation
of p53 protein, p53 DNA binding ability (measured by
electrophoretic mobility shift assay (EMSA)), and/or p53
transactivation potential (measured by reporter constructs or
target gene expression) (12, 20-25). Results of these studies are
summarized in Table 1. While the results are not completely
consistent across studies, no doubt due to differences in
chelation model and type of cell utilized, the final impression,
in our view, supports the concept of p53 activation occurring
during zinc deficiency. Could p53 activation be responsible for
the observed alterations in cell cycle and apoptosis during zinc
deficiency? Although p53 may contribute to these outcomes it
is not essential based on at least two observations: (1) zinc
deficiency-induced blocks in the G1 phase occur both in p53
dependent and independent cell lines (16, 17, 26), but zinc
deficiency-induced blocks in the G2/M phase do not appear to
Figure 2. Zinc deficiency results in deactivation of growth
factor pathways. (A) Synchronized 3T3 cells were cultured in
D (0.5 mM zinc), S (50 mM zinc) or C (undialyzed FBS
medium, 4 mM zinc) medium for 24 h and then subjected to
Western analysis with the indicated antibodies. (B) Densito-
metry analysis of phosphorylated protein to total protein for
each protein is indicated. Note: Cells cultured in undialyzed
FBS/DMEM medium (C¼ 4 mM zinc) served as an additional
control. Data are expressed as Mean+ SEM. Data from three
separate experiments were analyzed by ANOVA, and post hoc
differences were computed among groups if a significant F
value was obtained. * Indicates D is significantly different
from the S and C. The S and C groups did not significantly
differ from each other.
Table 1
Effects of Zn deficiency on p53 levels, DNA binding, and transactivation of pro-apoptotic gene BAX and cell cycleregulatory genes Gadd45 and p21waf1/cip1 support a role for p53 in zinc deficiency-induced cell death
Ref p53 mRNA Nuclear p53ap53 EMSA or transactivation
of reportersb Baxc mRNA
Gadd 45
mRNAcp21waf1/cip1
mRNAc
(20) #(21) " "(22) " $ $(23) " $(24) $ $ " $ $ "(25) " " "
"¼ increase,$¼no change, #¼decrease relative to control. aTranslocation of p53 protein to the nucleus. bMeasures of p53 DNA binding activity by
electromobility shift assay (EMSA) or expression of transfected reporter constructs. cDownstream targets of p53 activity.
ZINC DEFICIENCY-INDUCED CELL DEATH 663
occur (16, 26); and (2) caspase-3 activation and apoptosis
occur with similar kinetics in both p53 wild type and null cell
lines (7, 17). Thus, whether p53 is functional or not, alterations
in the cell cycle and the induction of apoptosis occur in similar
fashion during the course of zinc deficiency.
The decreased growth factor signaling through AKT and
ERK, and the likelihood of p53 activation suggest that
alterations in the proliferative machinery precede, and
sensitize, the cell to the forces of cell death.
ZINC DEFICIENCY-INDUCED CELL DEATH ANDINACTIVATION OF CELL SURVIVAL PATHWAYS
Cell death occurs primarily through either necrosis, a rapid
energy independent event normally associated with alterations
in the integrity of the plasma membrane, or apoptosis, an
energy dependent, evolutionarily conserved genetic program
that relies upon the activation of initiator and effector caspases.
Apoptosis occurs via two predominant pathways, an extrinsic
or receptor-mediated pathway exemplified by FasL/Fas/
caspase-8 interaction, or an intrinsic pathway characterized by
mitochondrial release of cytochrome c and caspase activation
as a consequence of physical or chemical stressors.
We have examined zinc deficiency-induced cell death at
different periods of development, both in vivo and in vitro.
In peri-implantation mouse embryos and in vitro cell models,
extra-cellular zinc deficiency results in cell death primarily by
apoptosis (Fig. 3) (7, 17, 27). Similarly, in whole animals,
Figure 3. Zinc deficiency-induced cell death. Detailed meth-
odologies for the following illustrations are published elsewhere
(7, 27, 28). (A&B) 3T3 cells were cultured in 7zinc (0.5 mMzinc) or þzinc (50 mM zinc) medium for 32 h and stained with
Hoechst (blue nuclei), propidium iodide (red nuclei), and
Annexin V-Alexa-488 (green plasma membrane) and imaged
with epi-fluorescent microscopy. (10N¼ primary necrosis,
20N¼ secondary necrosis, AP¼ apoptotic cell). Note in panel
B the false positive staining of a necrotic cell by Annexin-V.
(C&D) Peri-implantation mouse embryos cultured in zinc
adequate (þzinc (4 mM), panel C) or zinc deficient (7zinc (0.5
mM), panel D) medium for 144 h. Nuclei were stained with
Hoechst and imaged by epi-fluorescent microscopy (EB,
epiblast; EPC, ectoplacental cone; VE, visceral endoderm;
PE, parietal endoderm; TB, trophoblast giant cells, ICM, inner
cell mass). It can be seen that the þzinc peri-implantation
embryo (panel C) contains many more cells and differentiated
cell layers than the 7zinc embryo (panel D). (E&F) Cell death
assessed by TUNEL analysis and confocal microscopy is lower
in the þzinc peri-implantation embryo (panel E) than in the –
zinc embryo (panel F), despite the many more cells in the
former. Note: The embryos were optically sectioned at 5 mmintervals, and the peudo-colors indicate cell death occurring in
discreet cell layers. (G&H) Embryos were taken from rat dams
fed þzinc (30 mg/g diet, panel G) or7zinc (0.5 mg/g diet, panelH) at gestation day 10.5, and stained with nile blue sulfate to
label apoptotic cells. Labeling in Ot and Op show normal levels
of cell death in both treatments. However, 7zinc embryos
show excessive labeling in the S, B, andH, all structures that are
derivatives of neural crest cells. (S, somites; H, heart; B,
branchial arches; Op, optic cup; Ot, otic vesicle).
664 CLEGG ET AL.
fetuses from zinc-deficient rat dams are characterized by
inappropriate apoptosis in derivatives of neural crest cells such
as somites, branchial arches, and the heart (Fig. 3G) (28).
Many apoptotic stressors induce the intrinsic pathway of
apoptosis by causing cytochrome c release from mitochondria.
Loss of cytochrome c is a result of mitochondrial outer
membrane permeabilization (MOMP) (29). In many cases
MOMP is preceded by a decline in the inner mitochondrial
transmembrane potential (Dcm). Cytochrome c release into
the cytosol, and its recruitment into the apoptosome complex
(caspase-9 zymogen, Apaf-1, and dATP) lead to the auto-
catalytic activation of initiator caspase-9 and cleavage of
effector procaspase-3. The activated caspase-3 cleaves full-
length PKC-d, generating a 40 kDa fragment lacking its
regulatory domain. The truncated PKC-d (tPKC-d) localizesto the mitochondria and nucleus, amplifying the caspase
cascade (30). Central to MOMP activation are the pro-
survival (e.g., BCL-2, BCL-XL) and pro-apoptotic (e.g., BAX,
BAD) BCL-2 family members. Under normal conditions, pro-
apoptotic family members BAX and BAD are located in the
cytosol, while pro-survival members such as BCL-2 are
located in the outer mitochondrial membrane. A number of
apoptotic stimuli, including growth factor withdrawal, trigger
the dephosphorylation and cytoplasmic release of BAD, the
conformational activation of BAX, and the subsequent
migration of these proteins to the mitochondria. Both tPKC-
d and p53 appear to directly activate BAX (31, 32). BAD/
BCL-2 heterodimerization sequesters and reduces the effective
levels of BCL-2, and this condition results in BAX permeating
the outer and inner mitochondrial membranes resulting in a
loss of Dcm, release of cytochrome c, and activation of the
caspase cascade. AKT and ERK phosphorylate BAD,
facilitating its sequestration in the cytosol by 14-3-3 proteins.
Thus, AKT and ERK provide a central link between the
pathways of cell proliferation, survival and cell death. Given
the reduction in phosphorylation of AKT and ERK in zinc
deficient cells (Fig. 2), it is reasonable to speculate that BAD
translocation to the mitochondria occurs. Supporting the
concept that BAD/BAX mediated cell death is a characteristic
of zinc deficiency, King et al. (33) reported that zinc-deficient
mice are characterized by a preferential loss of pre-B and pre-
T cells. These cell types express low amounts of BCL-2 and
BCL-XL, while their more mature counterparts express high
levels of these pro-survival proteins, and accordingly are more
resistant to zinc deficiency-induced apoptosis (33). A caveat to
the concept that lowered cellular zinc is the driving force in the
observed apoptosis is the finding that serum from zinc
deficient animals often contains elevated glucocorticoid
concentrations. Glucocorticoids are potent inducers of apop-
tosis in pre-B and pre-T cells, and other cell types, perhaps via
their ability to down regulate p65 expression and interfere with
NF-kB DNA binding ((34) see below). Adrenalectomy of zinc-
deficient animals can substantially protect pre-B cells (35),
underscoring the point that zinc deficiency-induced cell death
in animals is a more complicated scenario than that found in
isolated cells, independent of the in vitro model used.
Most in vitro evidence supports a role for the intrinsic cell
death pathway in zinc deficiency-induced apoptosis. For
example, it has been reported that subsequent to a drop in
cellular zinc, Dcm decreases, which is followed by caspase-3
activation (17). This outcome is characterized by the
mitochondrial accumulation of the tPKC-d fragment during
effector caspase-3 activation (after 32 h of culture); if this
accumulation is blocked with rottlerin, a PKC-d inhibitor, cell
viability is greatly increased (7). However, Kolenko et al. (36),
using TPEN, found that cytochrome c release and caspase
activation preceded changes in Dcm. TPEN treatment of cells
in the 10 – 100mM range causes rapid activation of caspase-3
(within 1 h) (36, 37), with minimal activation of caspase-9 (37,
38). This outcome suggests that TPEN treatment bypasses the
normal activation of caspase-3 by caspase-9 via the intrinsic
pathway. Support for this concept comes from studies
suggesting that a small, latent proportion of caspase-3
normally exists in the active form (22, 37), but that it is held
in check by intracellular inhibitors such as zinc (6). Release of
this inhibition could trigger broad caspase-3 activation.
However, the role of intracellular zinc as an inhibitor of
caspases remains controversial and this concept has been
challenged by others (39). Another interpretation of the TPEN
data is that normal activation of the intrinsic pathway occurs
during TPEN-induced apoptosis, but the hierarchal activation
of the caspases is difficult to resolve given the short time frame
between the activation of initiator caspases and the subsequent
activation of effector caspases. Thus, zinc may function to
inhibit caspases upstream or downstream of their activation.
Finally, both Chimienti et al. (37) and Kolenko et al. (36)
found activation of effector caspase-8 during TPEN chelation,
but they attribute its activity to feedback activation by
caspase-3, rather than to an activation of the extrinsic cell
death pathway per se. However, this outcome might differ in
cell or animal models where the cell death is not so rapid and
thus the concept of zinc deficiency induced extrinsic cell death
warrants further study.
In addition to defects in growth factor signaling, and the
direct activation of caspases by zinc chelation, other mechan-
isms can contribute to zinc deficiency-induced apoptosis. One
common inducer of apoptosis is oxidative stress and the
associated iron accrual which occurs with zinc deficiency (40).
Iron can accumulate in protein sites vacated by zinc and
induce Fenton reactions that lead to the formation of ROS
that in turn can damage cellular macromolecules (20, 40).
Another source of ROS production are mitochondria (41),
which can produce and leak ROS as a consequence of
chemical and physical damage, NO inhibition, or by being
breeched by pore forming proteins such as BAX. The probe
2070-dichlorodihydrofluorcein diacetate (H2DCFDA) has been
used to monitor the progression of oxidative stress in
zinc deficient cells (20, 21, 40, 42). However, the probe also
ZINC DEFICIENCY-INDUCED CELL DEATH 665
cross-reacts with other reactive species such as NO generated
by cellular nitric oxide synthases (iNOS, mtNOS, eNOS,
nNOS). iNOS is up-regulated during zinc deficiency (43), and
at least one group has reported elevated nitrite production (an
indirect measure of NO) in zinc-deficient cells (20). Thus, RNS
can also be a source of the observed H2DCFDA fluorescence.
NO has a strong affinity for cysteines, and their reversible
modification by NO can affect the protein structure, catalytic
activity and DNA binding of many proteins involved in signal
transduction, oxidant defense, metabolism, transcriptional
regulation and apoptosis. Persistent elevations of cellular
NO, and its reaction with superoxide radical can lead to the
formation of peroxynitrite, which can oxidize numerous
biologically active molecules including glutathione.
Glutathione depletion by ROS, RNS, or by cellular export
has often been associated with the induction of apoptosis. The
addition of extracellular or intracellular metal chelators
directly to culture medium has been shown to deplete reduced
glutathione (GSH) levels (44, 45), while the addition of the
glutathione precursor, N-acetylcysteine (NAC), to cells
cultured in TPEN can inhibit broad based caspase activation
(38). Interestingly, we observed increased levels of GSH in 3T3
cells subjected to 32 h of zinc deficiency (Fig. 4B), although
oxidized glutathione (GSSG) levels seem to precede the rise in
GSH levels (Fig. 4A). However, when the levels of GSH and
GSSG were put into the context of the Nernst potential, the
overall redox state of zinc-deficient cells did not differ from
those of the two control groups (Fig. 4C). These data indicate
that if the cells have time to respond, they can adjust to the
zinc deficiency-induced oxidative stress, albeit, this outcome
still does not prevent caspase-3 activation and cell death. The
elevation in glutathione concentrations noted during zinc
deficiency are closely paralleled by similar findings in cells
deprived of protein growth factors (46), supporting the
concept that zinc deficiency results in altered growth factor
signaling pathways. However, further studies are needed to
examine if oxidative stress preferentially occurs in specific
cellular organelles (e.g., mitochondria), an outcome that
would not have been detected by the above analysis.
AKT and ERK phosphorylation of BAD represents an
endogenous survival pathway supported by growth factors.
Considered to be another endogenous survival pathway in
most cell types, the NF-kB pathway was found during studies
of TNF-a mediated cell death. NF-kB is a transcription factor
complex that is normally sequestered in the cytosol by the IkBfamily of proteins (inhibitor of kB), but traverses to the
nucleus when cells encounter oxidative stress or other
apoptotic stimuli. Nuclear NF-kB (homo and hetero dimers
of c-rel, RelA/p65, NF-kB1/p50, RelB, and NF-kB2/p52)promotes the transcription of several anti-apoptotic genes
(e.g., MnSOD, Bcl-2) and cell cycle regulatory genes
(e.g., p53). Persistent activation of NF-kB can result in
pathological conditions, and this activity can be attenuated
by zinc supplementation (47). Elevated ROS concentrations
can induce phosphorylation and proteosomal degradation of
IkBa and the cytosolic release of the NF-kB complex. It has
been reported during the course of zinc deficiency in IMR-32
and 3T3 cells, that while IkBa is degraded, and NF-kBcomplex levels in the cytosol increased, NF-kB binding to
Figure 4. Redox state of zinc-deficient cells. Synchronized 3T3
cells were cultured for the indicated time in D (0.5 mM zinc), S
(50 mM zinc), or C (undialyzed FBS medium, 4 mM zinc)
medium (7). Glutathione species were determined by reverse
phase HPLC utilizing electrochemical detection. Increases in
GSSG (A) preceded the increase in GSH levels (B). However,
when concentrations of the glutathione species were applied to
the Nernst equation, redox potential was similar among
groups (C). As positive or negative controls for oxidative
stress, cells were cultured for 24 h in 10 mM L-buthionine-
(S,R)-sulfoximine (BSO, depletes GSH levels) or 1 mM NAC
(augments GSH synthesis). Data from three separate experi-
ments were analyzed by ANOVA, and post hoc differences
were computed among groups if a significant F value was
obtained. * Indicates D is significantly different from the S and
C groups at each specific time point. The S and C groups did
not significantly differ at any time point.
666 CLEGG ET AL.
DNA and the ability of NF-kB to transactivate a reporter
construct is reduced (5,42). Similarly, in zinc-deficient animals,
it was reported that NF-kB EMSA activity is reduced (48). In
contrast to these findings, Ho and Ames (20) reported that
IkBa was not degraded during cellular zinc deficiency, nor was
the NF-kB complex increased in either the cytosol or the
nucleus. Both groups reported decreased affinity for NF-kB to
its consensus DNA sequence. One explanation of the different
findings is the likelihood that the magnitude of oxidative stress
differed among the studies.
In summary, zinc deficiency-induced cell death likely is
mediated through the intrinsic cell death pathway leading to
caspase-3 activation (Fig. 5). Zinc, by its ability to support
RTK signaling and directly interact with caspase-3, inhibits
both the processing and the activity of the enzyme. Ironically,
zinc deficiency-induced cell death is biochemically and
phenotypically similar in appearance to protein growth factor
withdrawal, yet, abundant growth factor stimulation of zinc
deficient cells fails to rescue them. This paradox can be
explained by diminution of RTK signaling, which results in
hypo-phosphorylation of AKT and ERK, two kinases
intimately involved in cell cycle and apoptosis regulation. At
some point, increased ROS/RNS during zinc deficiency leads
to DNA damage and activation of p53, which supports
inhibition of the cell cycle and induction of apoptosis.
Additionally, endogenous survival pathways supported by
growth factors and NF-kB are inactivated during zinc
deficiency, irreversibly committing the cell to death.
ACKNOWLEDGEMENTS
This work was supported by NIH grants HD01743 and
DK07355 (B.J.N. and T.Y.M.).
REFERENCES1. Shrimpton, R., Gross, R., Darnton-Hill, I., and Young, M. (2005)
Zinc deficiency: What are the most appropriate interventions? BMJ
330, 347 – 349.
2. Keen, C. L., Clegg, M. S., Hanna, L. A., Lanoue, L., Rogers, J. M.,
Daston, G. P., Oteiza, P., and Uriu-Adams, J. Y. (2003) The
plausibility of micronutrient deficiencies being a significant contribut-
ing factor to the occurrence of pregnancy complications. J. Nutr. 133,
1597S – 1605S.
3. Bateman, A., Coin, L., Durbin, R., Finn, R. D., Hollich, V., Griffiths-
Jones, S., Khanna, A., Marshall, M., Moxon, S., Sonnhammer, E. L.,
Studholme, D. J., Yeats, C., and Eddy, S. R. (2004) The Pfam protein
families database. Nucleic Acids Res. 32, D138 – 141.
4. Ho, L. H., Ratnaike, R. N., and Zalewski, P. D. (2000) Involvement
of intracellular labile zinc in suppression of DEVD-caspase activity in
human neuroblastoma cells. Biochem. Biophys. Res. Commun. 268,
148 – 154.
5. Mackenzie, G. G., Zago, M. P., Keen, C. L., and Oteiza, P. I. (2002)
Low intracellular zinc impairs the translocation of activated NF-
kappa B to the nuclei in human neuroblastoma IMR-32 cells. J. Biol.
Chem. 277, 34610 – 34617.
6. Perry, D. K., Smyth, M. J., Stennicke, H. R., Salvesen, G. S., Duriez,
P., Poirier, G. G., and Hannun, Y. A. (1997) Zinc is a potent inhibitor
of the apoptotic protease, caspase-3. A novel target for zinc in the
inhibition of apoptosis. J. Biol. Chem. 272, 18530 – 18533.
7. Chou, S. S., Clegg, M. S., Momma, T. Y., Niles, B. J., Duffy, J. Y.,
Daston, G. P., and Keen, C. L. (2004) Alterations in protein kinase C
activity and processing during zinc-deficiency-induced cell death.
Biochem. J. 383, 63 – 71.
8. Coyle, P., Philcox, J. C., Carey, L. C., and Rofe, A. M. (2002)
Metallothionein: The multipurpose protein. Cell. Mol. Life Sci. 59,
627 – 647.
9. Liuzzi, J. P., and Cousins, R. J. (2004) Mammalian zinc transporters.
Annu. Rev. Nutr. 24, 151 – 172.
10. Clegg, M. S., Keen, C. L., and Donovan, S. M. (1995) Zinc
deficiency-induced anorexia influences the distribution of serum
insulin-like growth factor-binding proteins in the rat. Metabolism
44, 1495 – 1501.
11. Ninh, N. X., Maiter, D., Verniers, J., Lause, P., Ketelslegers, J. M., and
Thissen, J. P. (1998) Failure of exogenous IGF-I to restore normal
growth in rats submitted to dietary zinc deprivation. J. Endocrinol. 159,
211 –217.
Figure 5. Postulated influence of zinc status on the balance
between RTK-mediated growth and cell death. Under zinc
adequate conditions (þZn), zinc will inhibit caspase activity,
and block iron accrual and cell death. In zinc deficiency (ZnD)
AKT and ERK signals are blocked downstream from ligand
binding to RTK’s, slowing cell cycle machinery and removing
blocks in apoptotic signals. Zinc deficiency is speculated to be
permissive to BAD translocation where it may reduce the
functional levels of BCL-2 in the mitochondria. p53 and
truncated PKC-d (tPKC-d) facilitate BAX translocation to the
mitochondria, causing reduced Dcm and MOMP. MOMP is
followed by release of cytochrome c (cyto c) into the cytosol,
which forms the apoptosome complex and activates caspase-3.
Zinc inhibits the processing of procaspase-3 and may inhibit
the activated enzyme. Fe accrual and aberrant mitochondrial
function during zinc deficiency result in the accumulation of
ROS, which damages DNA and activates p53. Zinc deficiency
is associated with elevated glucocorticoids (GC), which
compromise the transactivation potential of NF-kB and may
reduce its induction of survival genes.
ZINC DEFICIENCY-INDUCED CELL DEATH 667
12. MacDonald, R. S. (2000) The role of zinc in growth and cell
proliferation. J. Nutr. 130, 1500S – 1508S.
13. Lefebvre, D., Boney, C. M., Ketelslegers, J. M., and Thissen, J. P.
(1999) Inhibition of insulin-like growth factor-I mitogenic action by
zinc chelation is associated with a decreased mitogen-activated
protein kinase activation in RAT-1 fibroblasts. FEBS Lett. 449,
284 – 288.
14. Haase, H., and Maret, W. (2003) Intracellular zinc fluctuations
modulate protein tyrosine phosphatase activity in insulin/insulin-like
growth factor-1 signaling. Exp. Cell Res. 291, 289 – 298.
15. MacDonald, R. S., Wollard-Biddle, L. C., Browning, J. D., Thornton,
W. H., Jr, and O’Dell, B. L. (1998) Zinc deprivation of murine 3T3
cells by use of diethylenetrinitrilopentaacetate impairs DNA synthesis
upon stimulation with insulin-like growth factor-1 (IGF-1). J. Nutr.
128, 1600 – 1605.
16. Cui, L., Schoene, N. W., Zhu, L., Fanzo, J. C., Alshatwi, A., and Lei,
K. Y. (2002) Zinc depletion reduced Egr-1 and HNF-3beta expression
and apolipoprotein A-I promoter activity in Hep G2 cells. Am. J.
Physiol. Cell Physiol. 283, C623 – 630.
17. Duffy, J. Y., Miller, C. M., Rutschilling, G. L., Ridder, G. M., Clegg,
M. S., Keen, C. L., and Daston, G. P. (2001) A decrease in
intracellular zinc level precedes the detection of early indicators of
apoptosis in HL-60 cells. Apoptosis 6, 161 – 172.
18. Cobbs, C. S., Whisenhunt, T. R., Wesemann, D. R., Harkins, L. E.,
Van Meir, E. G., and Samanta, M. (2003) Inactivation of wild-type
p53 protein function by reactive oxygen and nitrogen species in
malignant glioma cells. Cancer Res. 63, 8670 – 8673.
19. Duffy, J. Y., Overmann, G. J., Keen, C. L., Clegg, M. S., and Daston,
G. P. (2004) Cardiac abnormalities induced by zinc deficiency are
associated with alterations in the expression of genes regulated by the
zinc-finger transcription factor GATA-4. Birth Defects Res. Part B
Dev. Reprod. Toxicol. 71, 102 – 109.
20. Ho, E., and Ames, B. N. (2002) Low intracellular zinc induces
oxidative DNA damage, disrupts p53, NFkappa B, and AP1 DNA
binding, and affects DNA repair in a rat glioma cell line. Proc. Natl.
Acad. Sci. USA 99, 16770 – 16775.
21. Ho, E., Courtemanche, C., and Ames, B. N. (2003) Zinc deficiency
induces oxidative DNA damage and increases p53 expression in
human lung fibroblasts. J. Nutr. 133, 2543 – 2548.
22. Fanzo, J. C., Reaves, S. K., Cui, L., Zhu, L., and Lei, K. Y. (2002) p53
protein and p21 mRNA levels and caspase-3 activity are altered by
zinc status in aortic endothelial cells. Am. J. Physiol. Cell Physiol. 283,
C631 – 638.
23. Reaves, S. K., Fanzo, J. C., Arima, K., Wu, J. Y., Wang, Y. R., and
Lei, K. Y. (2000) Expression of the p53 tumor suppressor gene is up-
regulated by depletion of intracellular zinc in HepG2 cells. J. Nutr.
130, 1688 – 1694.
24. Sun, Y., Bian, J., Wang, Y., and Jacobs, C. (1997) Activation of p53
transcriptional activity by 1,10-phenanthroline, a metal chelator and
redox sensitive compound. Oncogene 14, 385 – 393.
25. Fanzo, J. C., Reaves, S. K., Cui, L., Zhu, L., Wu, J. Y., Wang, Y. R.,
and Lei, K. Y. (2001) Zinc status affects p53, gadd45, and c-fos
expression and caspase-3 activity in human bronchial epithelial cells.
Am. J. Physiol. Cell Physiol. 281, C751 – 757.
26. Thornton, W. H., Jr., MacDonald, R. S., Wollard-Biddle, L. C.,
Browning, J. D., and O’Dell, B. L. (1998) Chelation of extracellular
zinc inhibits proliferation in 3T3 cells independent of insulin-like
growth factor-I receptor expression. Proc. Soc. Exp. Biol. Med. 219,
64 – 68.
27. Hanna, L. A., Clegg, M. S., Momma, T. Y., Daston, G. P., Rogers,
J. M., and Keen, C. L. (2003) Zinc influences the in vitro development
of peri-implantation mouse embryos. Birth Defects Res. Part A Clin.
Mol. Teratol. 67, 414 – 420.
28. Jankowski-Hennig, M. A., Clegg, M. S., Daston, G. P., Rogers, J. M.,
and Keen, C. L. (2000) Zinc-deficient rat embryos have increased
caspase 3-like activity and apoptosis. Biochem. Biophys. Res. Commun.
271, 250 – 256.
29. Green, D. R., and Kroemer, G. (2004) The pathophysiology of
mitochondrial cell death. Science 305, 626 – 629.
30. DeVries, T. A., Neville, M. C., and Reyland, M. E. (2002) Nuclear
import of PKCdelta is required for apoptosis: Identification of a novel
nuclear import sequence. EMBO J. 21, 6050 – 6060.
31. Chipuk, J. E., Kuwana, T., Bouchier-Hayes, L., Droin, N. M.,
Newmeyer, D. D., Schuler, M., and Green, D. R. (2004) Direct activa-
tion of Bax by p53 mediates mitochondrial membrane permeabiliza-
tion and apoptosis. Science 303, 1010 – 1014.
32. Sitailo, L. A., Tibudan, S. S., and Denning, M. F. (2004) Bax
activation and induction of apoptosis in human keratinocytes by the
protein kinase C delta catalytic domain. J. Invest. Dermatol. 123,
434 – 443.
33. King, L. E., Osati-Ashtiani, F., and Fraker, P. J. (2002) Apoptosis
plays a distinct role in the loss of precursor lymphocytes during zinc
deficiency in mice. J. Nutr. 132, 974 – 979.
34. Wang, W., Wykrzykowska, J., Johnson, T., Sen, R., and Sen, J. (1999)
A NF-kappa B/c-myc-dependent survival pathway is targeted by
corticosteroids in immature thymocytes. J. Immunol. 162, 314 – 322.
35. DePasquale-Jardieu, P., and Fraker, P. J. (1980) Further character-
ization of the role of corticosterone in the loss of humoral immunity in
zinc-deficient A/J mice as determined by adrenalectomy. J. Immunol.
124, 2650 – 2655.
36. Kolenko, V. M., Uzzo, R. G., Dulin, N., Hauzman, E., Bukowski, R.,
and Finke, J. H. (2001) Mechanism of apoptosis induced by
zinc deficiency in peripheral blood T lymphocytes. Apoptosis 6,
419 – 429.
37. Chimienti, F., Seve, M., Richard, S., Mathieu, J., and Favier, A.
(2001) Role of cellular zinc in programmed cell death: Temporal
relationship between zinc depletion, activation of caspases, and
cleavage of Sp family transcription factors. Biochem. Pharmacol. 62,
51 – 62.
38. Carter, J. E., Truong-Tran, A. Q., Grosser, D., Ho, L., Ruffin, R. E.,
and Zalewski, P. D. (2002) Involvement of redox events in caspase
activation in zinc-depleted airway epithelial cells. Biochem. Biophys.
Res. Commun. 297, 1062 – 1070.
39. Takahashi, A., Alnemri, E. S., Lazebnik, Y. A., Fernandes-Alnemri,
T., Litwack, G., Moir, R. D., Goldman, R. D., Poirier, G. G.,
Kaufmann, S. H., and Earnshaw, W. C. (1996) Cleavage of lamin A
by Mch2 alpha but not CPP32: Multiple interleukin 1 beta-converting
enzyme-related proteases with distinct substrate recognition properties
are active in apoptosis. Proc. Natl. Acad. Sci. USA 93, 8395 – 8400.
40. Mackenzie, G. G., Keen, C. L., and Oteiza, P. I. (2002) Zinc status of
human IMR-32 neuroblastoma cells influences their susceptibility to
iron-induced oxidative stress. Dev. Neurosci. 24, 125 – 133.
41. Cadenas, E. (2004) Mitochondrial free radical production and cell
signaling. Mol. Aspects Med. 25, 17 – 26.
42. Oteiza, P. I., Clegg, M. S., Zago, M. P., and Keen, C. L. (2000) Zinc
deficiency induces oxidative stress and AP-1 activation in 3T3 cells.
Free Radic. Biol. Med. 28, 1091 – 1099.
43. Cui, L., Takagi, Y., Wasa, M., Iiboshi, Y., Khan, J., Nezu, R., and
Okada, A. (1997) Induction of nitric oxide synthase in rat intestine by
interleukin-1alpha may explain diarrhea associated with zinc defi-
ciency. J. Nutr. 127, 1729 – 1736.
44. Nakatani, T., Tawaramoto, M., Opare Kennedy, D., Kojima, A.,
and Matsui-Yuasa, I. (2000) Apoptosis induced by chelation of
intracellular zinc is associated with depletion of cellular reduced
glutathione level in rat hepatocytes. Chem. Biol. Interact. 125, 151 –
163.
668 CLEGG ET AL.
45. Kojima-Yuasa, A., Ohkita, T., Yukami, K., Ichikawa, H., Takami,
N., Nakatani, T., Opare Kennedy, D., Nishiguchi, S., and Matsui-
Yuasa, I. (2003) Involvement of intracellular glutathione in zinc
deficiency-induced activation of hepatic stellate cells. Chem. Biol.
Interact. 146, 89 – 99.
46. Kirkland, R. A., and Franklin, J. L. (2001) Evidence for redox
regulation of cytochrome C release during programmed neuronal
death: Antioxidant effects of protein synthesis and caspase inhibition.
J. Neurosci. 21, 1949 – 1963.
47. Connell, P., Young, V. M., Toborek, M., Cohen, D. A., Barve, S.,
McClain, C. J., and Hennig, B. (1997) Zinc attenuates tumor necrosis
factor-mediated activation of transcription factors in endothelial cells.
J. Am. Coll. Nutr. 16, 411 – 417.
48. Oteiza, P. I., Clegg, M. S., and Keen, C. L. (2001) Short-term zinc
deficiency affects nuclear factor-kappaB nuclear binding activity in rat
testes. J. Nutr. 131, 21 – 26.
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