IUBMB
Life, 57(10): 661 – 669, October 2005
Critical Review Zinc Deficiency-Induced Cell Death Michael S. Clegg1, Lynn A. Hanna1, Brad J. Niles1, Tony Y. Momma1 and Carl L. Keen1,2 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-k B 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; signal transduction; IGF; caspase; NF-kB; nutrition; p53; AKT; ERK; reactive nitrogen species; reactive oxygen species.
INTRODUCTION It is known that zinc deficiency is teratogenic in experimental 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 metalloproteinases) or an inhibitor (e.g., caspases) (6). As an activator of Received 4 May 2005; accepted 18 July 2005 Address correspondence to: Michael S. Clegg MBA/PhD, Department of Nutrition, University of California at Davis, One Shields Ave, Davis, CA 95616, USA. Tel: þ1 530 752 4658. E-mail:
[email protected] ISSN 1521-6543 print/ISSN 1521-6551 online Ó 2005 IUBMB DOI: 10.1080/15216540500264554
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 inhibition of apoptosis (e.g., IAP-2), and it is a constituent of numerous proteins participating in proteosome-mediated protein 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 interrelated 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 metallothionein, 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
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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 CELL PROLIFERATION 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 primarily 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 deficiencyinduced 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 pathways 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 supplementation, 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
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 (p 5 0.05). Note: This model utilizes medium with fetal bovine serum (FBS) depleted of zinc by short dialysis against an extracellular 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 medium ¼ 0.5 mM zinc). As a control, D medium was supplemented with exogenous zinc (S medium ¼ 50 mM zinc), and thus, D and S media differed only by their final concentrations of zinc (7).
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)ethylenediamine (TPEN) (14). These authors suggest that 50mM TPEN 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
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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 concentrations. 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.
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) Densitometry analysis of phosphorylated protein to total protein for each protein is indicated. Note: Cells cultured in undialyzed FBS/DMEM medium (C ¼ 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.
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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 transcription 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 hypophosphorylation 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
Table 1 Effects of Zn deficiency on p53 levels, DNA binding, and transactivation of pro-apoptotic gene BAX and cell cycle regulatory genes Gadd45 and p21waf1/cip1 support a role for p53 in zinc deficiency-induced cell death Ref
p53 mRNA
(20) (21) (22) (23) (24) (25)
" " " $ "
a
Nuclear p53
p53 EMSA or transactivation of reportersb
c
Bax mRNA
Gadd 45 mRNAc
p21waf1/cip1 mRNAc
# " $ $ $ "
"
$
$ $ "
"
" ¼ increase, $ ¼ no change, # ¼ decrease 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.
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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 AND INACTIVATION 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 methodologies for the following illustrations are published elsewhere (7, 27, 28). (A&B) 3T3 cells were cultured in 7zinc (0.5 mM zinc) 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. (10N ¼ primary necrosis, 20N ¼ secondary necrosis, AP ¼ 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 mm intervals, 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) or 7zinc (0.5 mg/g diet, panel H) 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, and H, all structures that are derivatives of neural crest cells. (S, somites; H, heart; B, branchial arches; Op, optic cup; Ot, otic vesicle).
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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 autocatalytic activation of initiator caspase-9 and cleavage of effector procaspase-3. The activated caspase-3 cleaves fulllength PKC-d, generating a 40 kDa fragment lacking its regulatory domain. The truncated PKC-d (tPKC-d) localizes to the mitochondria and nucleus, amplifying the caspase cascade (30). Central to MOMP activation are the prosurvival (e.g., BCL-2, BCL-XL) and pro-apoptotic (e.g., BAX, BAD) BCL-2 family members. Under normal conditions, proapoptotic 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 tPKCd 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 preT 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 apoptosis 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 zincdeficient animals can substantially protect pre-B cells (35), underscoring the point that zinc deficiency-induced cell death
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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 mechanisms 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 20 70 -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
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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 IkB family 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
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 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 groups at each specific time point. The S and C groups did not significantly differ at any time point.
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-kB complex levels in the cytosol increased, NF-kB binding to
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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
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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.). REFERENCES
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.
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