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Zinc deficiency affects the STAT1/3 signaling pathways in part through redox-mediated mechanisms

Zinc deficiency affects the development of the central nervous system (CNS) through mechanisms only partially understood. We previously showed that zinc deficiency causes CNS oxidative stress, damaging microtubules and impairing protein nuclear shuttling. STAT1 and STAT3 transcription factors, which require nuclear import for their functions, play major roles in CNS development. Thus, we investigated whether zinc deficiency disrupts STAT1 and STAT3 signaling pathways in the developing fetal CNS, characterizing the involvement of oxidative stress and the cytoskeleton in the adverse effects. Maternal (gestation day 0–19) marginal zinc deficiency (MZD) reduced STAT1 and STAT3 tyrosine phosphorylation and their nuclear translocation in the embryonic day 19 (E19) rat brain. Similar effects were observed in zinc depleted IMR-32 neuroblastoma cells, with an associated decrease in STAT1- and STAT3-dependent gene transactivation. Zinc deficiency caused oxidative stress (increased 4-hydroxynonenal-protein adducts) in E19 brain and IMR-32 cells, which was prevented in cells by supplementation with 0.5 mM α-lipoic acid (LA). In zinc depleted IMR-32 cells, the low tyrosine phosphorylation of STAT1, but not that of STAT3, recovered upon incubation with LA. STAT1 and STAT3 nuclear transports were also restored by LA. Accordingly, chemical disruption of the cytoskeleton partially reduced STAT1 and STAT3 nuclear levels. In summary, the redox-dependent tyrosine phosphorylation, and oxidant-mediated disruption of the cytoskeleton are involved in the deleterious effects of zinc deficit on STAT1 and STAT3 activation and nuclear translocation. Therefore, disruption of the STAT1 and STAT3 signaling pathways may in part explain the deleterious effects of maternal MZD on fetal brain development.


Introduction
Zinc is an essential nutrient that plays many critical biological roles in all tissues, including the central nervous system (CNS) [1]. Although severe zinc deficiency is relatively rare in human populations, moderate zinc deficiency is frequent throughout the world [2]. Approximately 82% of women worldwide have been reported to have insufficient zinc intake during pregnancy, and this could reach up to a 100% in developing countries [2]. Maternal zinc deficiency is associated with poor fetal growth and alters neurobehavioral development both in humans [3][4][5][6] and in animal models [7,8]. Marginal zinc deficiency (MZD) during gestation is not teratogenic but affects brain signaling and gene expression [9,10], impacting learning, memory and behavior later in life [11][12][13].
Zinc deficiency affects redox-regulated signals [14] that modulate cellular processes (proliferation, survival, apoptosis, differentiation, migration) central to CNS development. In neuronal cells, zinc deficiency causes an increased oxidant production via the NMDA receptor-dependent activation of NADPH oxidase and nitric oxide synthase [15]. One major associated consequence is the oxidation of tubulin thiols leading to impaired microtubule assembly [16]. In zinc depleted rat primary neuronal cultures and human IMR-32 neuroblastoma cells we observed that activated transcription factors NF-κB and NFAT fail to translocate into the nucleus given that their transport requires functional microtubules [9,[17][18][19]. We propose that the oxidant-mediated and zinc deficiency-induced alterations in cytoskeleton dynamics could also disrupt the signal transducers and activators of transcription STAT1 and STAT3, which also require nuclear translocation to regulate transcription. STAT1 and STAT3 play major roles in CNS development. STAT1 and STAT3 are involved in the modulation of cell apoptosis, proliferation, survival and differentiation [20][21][22] a selective signaling defect in response to interferon type I and II, causing high sensitivity to infection by microbial pathogens and viruses [23,24], disruption in neuronal connectivity and deficit in social behaviors [25]. STAT1 is expressed in different areas of the developing and adult rat CNS, and its expression increases from postnatal day 0 through adulthood [26][27][28]. In contrast, STAT3 is widely expressed in the CNS during development, mostly at ventricular areas, where neuronal proliferation and differentiation take place [29,30]. STAT3 knockout mice are embryonic lethal [31], stressing its critical role during development. In the CNS, STAT3 is involved in neural progenitor cell proliferation [32], migration [33] and fate decisions [34].
The JAK/STAT pathway is activated by a variety of ligands including cytokines and growth factors [35][36][37][38][39]. Initially, ligandreceptor binding causes dimerization of the receptor subunits. In the step of signal propagation, the cytosolic domains of the receptor subunits associate to JAK tyrosine kinases. The two proximate JAKs trans-phosphorylate each other, and subsequently phosphorylate the receptor and the substrate STATs (tyrosine-701 and tyrosine-705 for STAT1 and STAT3, respectively). STATs reside in a latent state in the cytosol, upon tyrosine phosphorylation they switch to an activated state triggering homo-or/and hetero-dimerization through interactions of their SH2 domains. This leads to STAT1 and STAT3 nuclear translocation, where they bind to specific sequences in the promoter region of target genes [40].
The potential involvement of zinc and redox mechanisms on STAT1 and STAT3 modulation, as well as the impact of zinc deficiency on STAT1 and STAT3 signalings during CNS development are unknown. Given the critical roles of STAT1 and STAT3 in the CNS, this study investigated whether zinc deficiency can affect STAT1 and STAT3 signalings in the E19 rat brain. The potential involvement of zinc deficiency-associated oxidative stress and tubulin disruption on STAT1 and STAT3 nuclear transport was characterized in IMR-32 neuroblastoma cells. We show that zinc deficiency impairs STAT1 and STAT3 signalings in part through oxidative stress-associated decreased tyrosine phosphorylation and disruption of STAT1 and STAT3 nuclear shuttling.

Animals and animal care
All procedures were in agreement with standards for the care of laboratory animals as outlined in the NIH Guide for the Care and Use of Laboratory Animals. All procedures were administered under the auspices of the Animal Resource Services of the University of California, Davis, which is accredited by the American Association for the Accreditation of Laboratory Animal Care, and adhere to the International Guiding Principles for Biomedical Research Involving Animals.
Experimental protocols were approved before implementation by the University of California, Davis Animal Use and Care Administrative Advisory Committee, and were administered through the Office of the Campus Veterinarian.
Adult Sprague-Dawley rats (Charles River, Wilmington, MA) (200-225 g) were housed individually in suspended stainless steel cages in a temperature (22-23°C) and photoperiod (12 h light/dark)-controlled room. An egg white protein based with adequate zinc (25 µg zinc/g) was the standard control diet [41]. Animals were fed the control diet for one week before breeding. Males and females were caged together overnight and the following morning the presence of a sperm plug confirmed a successful breeding. On gestation day 0 (approximate embryonic day 0; E0), rats (6 animals/group) were divided into two groups and fed one of the following diets until gestation day 19 (E19): a control diet ad libitum (25 μg zinc/g diet, C) or a diet containing a marginally zinc-deficient concentration (10 μg zinc/g diet, MZD). Food intake was recorded daily, and body weight was measured at 5-d intervals. On gestation day 19, dams were anesthetized with isoflurane (2 mg/kg body weight), and laparotomies were performed. The gravid uterus was removed, and fetuses were collected. Fetuses were examined for gross structural malformations and weighed. Fetal brains were excised and rinsed in ice-cold PBS, and meninges were removed before weighing.
As previously described [9], a marginally zinc deficient diet imposed from gestation day 0 through 19 did not affect food intake, maternal or fetal outcomes (Supplemental Table 1).
Zinc depleted FBS was prepared as previously described [42]. The zinc depleted FBS was diluted with complex medium to a final concentration of 3 mg protein/ml to match the protein concentration of the control non-depleted media. The zinc concentration of the depleted medium was 1.5 µM (1.5Zn). An aliquot of the zinc -depleted medium was supplemented with ZnCl 2 to obtain a final concentration of 15 μM (15Zn). Cells were grown in the control complex medium containing 10% (v/v) non-chelated FBS until confluence, after which the medium was removed and replaced by control medium or media containing 1.5 or 15 µM zinc incubated simultaneously without or with, 0.5 μM Vb, 0.5 μM Col, 0.5 μM Cyt D or 0.5 mM LA. Cells were harvested after 24 h in the different experimental conditions.
All samples were stored at −80°C until further determinations. Protein concentration was determined [45] immediately before starting the corresponding assays.

Electrophoretic mobility shift assay (EMSA)
Oligonucleotides containing the consensus sequence of STAT1 (5′-CAT GTT ATG CAT ATT CCT GTA AGT G-3′) or STAT3 (5′-GAT CCT TCT GGG AAT TCC TAG ATC-3′) were end-labeled with ( 32 P) ATP using T4 polynucleotide kinase and purified using Chroma Spin-10 columns. Samples were incubated with labeled oligonucleotide (20,000-30,000 cpm) for 20 min at room temperature in 1X binding buffer (5X binding buffer: 50 mM Tris-HCl buffer, pH 7.5, containing 20% (v/v) glycerol, 5 mM MgCl 2 , 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl and 0.25 mg/ml poly(dI-dC)). Products were separated by electrophoresis in a 6% (w/v) non-denaturing polyacrylamide gel Gestational MZD affects DNA binding and content of STAT1 and STAT3 in nuclear fractions isolated from E19 brains. From gestation day 0 through 19, dams were fed ad libitum control (C) or MZD diets. Nuclear and cytosolic fractions were prepared from E19 brains as described in the Materials and Methods section. (A) EMSA for STAT1 and STAT3 in nuclear and cytosolic fractions. To determine the specificity of each transcription factor-DNA complex, a control nuclear fraction was incubated in the presence of a 100-fold molar excess of unlabeled oligonucleotide containing the consensus sequence for either the specific (S) or an unspecific (U) transcription factor before the binding assay. Bands were quantified and the ratio nuclear/cytosolic DNA binding (NF/CF) was calculated. (B) Western blots for pY 701 -STAT1, pS 727 -STAT1, and STAT1 (left); and pY 705 -STAT3, pS 727 -STAT3, and STAT3 (right); and hnRNP as the nuclear housekeeping protein. After quantification, results were expressed as pY 701 -STAT1/STAT1, pS 727 -STAT1/STAT1 and STAT1/hnRNP (bottom left), or pY 705 -STAT3/STAT3, pS 727 -STAT3/STAT3, and STAT3/hnRNP (bottom right). Results were normalized to control values and are shown as means ± S.E.M. of 6 animals per group. *, ** Significantly different compared to C groups (P < 0.05 and P < 0.01, respectively, one-way ANOVA). using 0.5X TBE (Tris/borate 45 mM, EDTA 1 mM) as the running buffer. Gels were dried and the radioactivity quantitated in a Phosphoimager 840 (Amersham Pharmacia Biotech. Inc., Piscataway, NJ).

Transfections
IMR-32 cells were seeded in 6 well plates. After 24 h in culture, cells (2.5×10 6 cells) were transfected with Lipofect-AMINE™ 2000 according to the manufacturer's protocols (Invitrogen Life Technologies, Carlsbad, CA, USA). A vector expressing β-galactosidase (2 μg of DNA) as an internal control for transfection efficiency was cotransfected with STAT1-LUC or STAT3-LUC plasmids (1 μg of DNA). After 24 h of transfection, cells were treated with control, 1.5Zn or 15Zn media. Treated cells were harvested 24 h later and lysed. β-galactosidase and luciferase activities were determined following the manufacturer's protocol (Promega, Madison, WI).

Immunohistochemistry
The E19 brains were dissected out and fixed in 4% (w/v) paraformaldehyde in PBS (pH 7.2) overnight. Tissues were washed twice with PBS, incubated in 15% (w/v) sucrose in PBS for 24 h, and subsequently in 30% (w/v) sucrose for 3 d, after which brains were submerged in Cryoplast (Biopack, Buenos Aires, Argentina), frozen, cut into 18-µm coronal sections on a Leica CM 1850 cryotome (Leica Microsystems, Nussloch, Germany) and mounted on positively charged slides. Sections were processed for antigen retrieval by incubation in 10 mM sodium citrate buffer (pH 6.0) containing 0.05% (v/v) Tween 20 for 10 min at 95°C, washed twice with PBS and then blocked for 45 min in 1% (v/v) donkey serum, 0.1% (v/v) Triton X-100 in 0.1 M PBS and incubated overnight at 4°C with 4-HNE (1:100) primary antibody in blocking solution. Sections were then washed with PBS and incubated for 2 h at room temperature with a biotinylated secondary antibody diluted in blocking buffer (Vector Laboratories, Burlingame, CA). To visualize the antigen-antibody complex, the secondary antibodies were labeled with avidin-horseradish peroxidase for 2 h at room temperature followed by exposure to 3,3-diaminobenzene (DAB; Vector Laboratories, Burlingame, CA) for 3 min. Slides were washed, dehydrated, and cover slips were mounted with Permount (Fischer Sci, Hampton, NH). Two sections per animal (one from each litter, n=3/ group) were imaged on an Olympus BX50 epifluorescence microscope (Tokyo, Japan).

Statistical analysis
One-way analysis of variance (ANOVA) test, followed by Fisher's PLSD (protected least-squares difference) test and correlations, were performed using the routines available in Statview 5.0 (SAS Institute, Cary, NC, USA.). A p value < 0.05 was considered statistically significant. Values are given as means ± S.E.M. The litter was considered the statistical unit.

Gestational MZD affects the modulation and nuclear translocation of STAT1 and STAT3 in the E19 brain
In the E19 brain we initially measured an early event in STAT1/3 activation, the phosphorylation of tyrosine residues (Tyr701 and 705 for STAT1 and STAT3, respectively) by Western blot (Fig. 1). STAT1 and STAT3 tyrosine phosphorylation levels were lower (31% and 68%, respectively) in MZD fetal brains compared to controls (Fig. 1A and  B). Total STAT1 content was 33% higher, while a trend (p= 0.054) for lower total STAT3 was found in MZD fetal brains compared to controls. STAT1 and STAT3 were also phosphorylated in serine residues (Ser727). In MZD E19 brains serine phosphorylation levels were 48% higher or unchanged for STAT1 and STAT3, respectively, compared to controls ( Fig. 1A and B).
We next investigated the effects of gestational MZD on STAT1 and STAT3 nuclear transport in E19 brains. Initially, STAT1-and STAT3-DNA binding was measured in E19 brain nuclear fractions by EMSA. Ratios of nuclear/cytosolic STAT1-and STAT3-DNA binding were significantly lower in MZD E19 brains than in controls (82% and 84%, respectively) ( Fig. 2A). To further investigate if the nuclear transport of STAT1 and STAT3 could be affected by gestational MZD, we measured by Western blot the total content of STAT1 and STAT3 in nuclear fractions. The nuclear content of total STAT1 and STAT3 was lower (43% and 40%, respectively) in MZD fetal brains compared to controls (Fig. 2B). Levels of tyrosine phosphorylation for STAT1 and STAT3 in nuclear fractions were similar in both groups. Serine phosphorylation level of STAT1 was 2.3-fold higher, while that of STAT3 was similar, in the nuclear fraction from MZD E19 brains compared to controls (Fig. 2B). Alterations in the nuclear transport of STAT1 and STAT3 as a consequence of zinc deficiency are consistent with our previous findings for two other transcription factors, NF-κB and NFAT [9].

Zinc deficiency impairs STAT1 and STAT3 phosphorylation, DNA binding, and transactivating activity in human IMR-32 neuroblastoma cells
To further investigate the mechanisms underlying zinc deficiencyinduced impaired STAT1 and STAT3 regulation, we used human IMR-32 neuroblastoma cells. In this cell line we previously showed that zinc deficit promotes increased oxidant production, causes tubulin oxidation, alters polymerization and consequently inhibits NF-κB and NFAT nuclear import [17,19]. Cells were incubated for 24 h in control, zincdepleted (1.5Zn), or zinc-repleted (15 μM zinc, 15Zn) medium. Consistent with results in the E19 brain, STAT1 showed 48% lower and 300% higher tyrosine and serine phosphorylation, respectively, in zinc deficient compared to control cells (Fig. 3A). Total STAT1 protein content was 39% higher in 1.5Zn than in control cells (Fig. 3A). STAT3 tyrosine phosphorylation, was significantly lower (39%) in zinc-deficient than in control cells (Fig. 3B). Unlike findings for STAT1, total STAT3 and serine phosphorylation levels were not affected by zinc depletion.
STAT1-and STAT3-DNA binding was measured by EMSA in total cell extracts and in nuclear fractions. The ratio nuclear/total STAT1and STAT3-DNA binding was significantly lower (50% and 43%, respectively) in cells cultured in zinc depleted medium compared to those incubated in control or 15Zn medium (Fig. 4A). We next measured the STAT1/3 transactivation activity using a reporter gene assay. After 24 h incubation in control, 1.5Zn or 15Zn medium, STAT1and STAT3-driven luciferase activity (corrected for β-galactosidase activity) was 34% and 41% lower, respectively, in cells incubated in 1.5Zn medium than in control or 15Zn medium (Fig. 4B). Consistent with the DNA binding results, STAT1-and STAT3-mediated luciferase activity was recovered by repletion of 1.5Zn medium with 15 μM zinc (15Zn).

Zinc deficiency disrupts STAT1 and STAT3 nuclear translocation and phosphorylations in IMR-32 cells
We next evaluated the effects of zinc deficiency on STAT1 and STAT3 nuclear translocation using Western blot. Total STAT1 and STAT3 nuclear content were 34% and 18% lower in zinc deficient compared to control cells (Fig. 5A and B) Similar to results for the E19 brain, in IMR-32 cells zinc deficiency did not affect nuclear STAT1 tyrosine phosphorylation but caused higher 62% serine phosphorylation levels compared to controls (Fig. 5A). Nuclear STAT3 tyrosineand serine-phosphorylation levels were 53% and 31% lower in nuclear fractions from zinc-deficient than in control cells (Fig. 5B). 3.4. Zinc deficiency causes oxidative stress which is involved in the disruption of STAT1 and STAT3 nuclear translocation 4-hydroxynonenal (4-HNE) is a product of lipid oxidation that can diffuse and modify cellular components, including proteins. A potential condition of oxidative stress was assessed in E19 brains and IMR-32 cells by measuring 4-HNE-protein adducts. In the E19 brain, areas of stronger 4-HNE staining were identified by IHC to be mainly localized in the brain cortex (Fig. 6A). HNE-protein adducts were observed in the E19 MZD cortex, while they were mostly absent in controls. 4-HNE-protein adducts were evaluated by Western blot in IMR-32 cells incubated in control or 1.5Zn media for 24 h without or with supplementation with 0.5 mM LA. The 4-HNE staining of a band corresponding to a 50 kDa polypep- Fig. 7. α-Lipoic acid prevents zinc deficiency-induced impaired STAT1 and STAT3 nuclear import. Nuclear fraction and cytosolic fraction were isolated from IMR-32 cells incubated for 24 h in control (C), or zinc depleted (1.5Zn) medium with or without supplementation with 0.5mM α-lipoic acid (LA). Western blots for: (A) STAT1 and hnRNP as housekeeping protein in the nuclear fraction (top left), and STAT1 and β-actin as housekeeping protein in the cytosolic fraction (top right), and band quantification expressed as the ratio of total STAT1 nuclear/cytosolic (bottom); (B) STAT3 and hnRNP in the nuclear fraction (top left) and STAT3 and β-actin in the cytosolic fraction (top right), and band quantification expressed as the ratio total STAT3 nuclear/cytosolic fractions (bottom). Results were normalized to controls values, and shown as means ± S.E.M. of three independent experiments. *, ** Significantly different compared to C groups (P < 0.05 and P < 0.01, respectively, one-way ANOVA). tide in the Western blot was estimated to be 2.5-fold higher in 1.5Zn cells compared to controls, which was not observed upon coincubation with 0.5 mM LA (Fig. 6B).
We previously observed that co-incubation of cells in zincdepleted medium with LA prevented tubulin thiol oxidation and its impaired polymerization, restoring NF-κB nuclear transport [16]. Thus, we next investigated the effects of LA on STAT1 and STAT3 nuclear transport, by incubating IMR-32 cells in control or 1.5 Zn medium with or without supplementation with 0.5 mM LA. The total STAT1 and STAT3 protein levels in nuclear and cytosolic fractions were measured by Western blot. Ratios of nuclear/ cytosolic STAT1 and STAT3 were significantly lower (64%, and 59%, respectively) in zinc deficient cells than in controls, and this was prevented by co-incubation with LA ( Fig. 7A and B).

Cytoskeleton disruptors impair STAT1 and STAT3 nuclear translocation
Based on the above findings, we investigated whether alterations to the cytoskeleton (i.e. tubulin and actin) polymerization could affect the nuclear translocation of STAT1 and STAT3 in IMR-32 cells. The actions of disrupting agents of tubulin (vinblastine and colchicine) and actin (cytochalasin D) polymerization on STAT1-and STAT3-DNA binding were investigated in nuclear fractions from IMR-32 cells incubated for 24 h with or without the inhibitors. The ratio nuclear/ total STAT1-DNA binding was lower (36%, 22%, and 31%) in cells incubated with 0.5 μM vinblastine, colchicine and cytochalasin D, respectively, compared to controls (Fig. 8A). Similarly, vinblastine and cytochalasin D caused 27% and 26% reductions in the ratio nuclear/total STAT3-DNA binding, respectively, while no significant differences were observed for colchicine (Fig. 8B).
3.6. LA prevents zinc deficiency-induced impaired tyrosine phosphorylation of STAT1 but not STAT3 We next investigated if oxidative stress could be involved in the zinc deficiency-mediated disruption of the upstream STAT1 and STAT3 tyrosine phosphorylation. IMR-32 cells were incubated for 24 h in control or 1.5Zn medium with or without 0.5 mM LA, and tyrosine phosphorylation was analyzed in total cell fractions by Western blot. The low STAT1 tyrosine-phosphorylation level observed in cells incubated in 1.5Zn medium was prevented by simultaneous incubation with LA (Fig. 9A). However, LA failed to prevent the increase in STAT1 total protein expression caused by zinc deficiency (Fig. 9A). LA also failed to prevent the decrease of tyrosine-phosphorylated STAT3 levels in zinc deficient cells (Fig. 9B). As expected, zinc supplementation (15 μM) prevented the decrease of both STAT1 and STAT3 tyrosine phosphorylation in the zinc deficient cells (95 ± 9 and 109 ± 2% in 15Zn cells compared to control (100%), respectively (data not shown)). Zinc deficiency-induced increase in STAT1 serine phosphorylation, on the other hand, was not prevented by co-incubation with LA (Fig. 9A).

Discussion
STAT1 and STAT3 play crucial roles in key developmental events; the deregulation caused by suboptimal gestational zinc nutrition could explain in part the deleterious effects on offspring brain development. This study provides novel evidence that zinc deficits both in vivo and in vitro disrupt STAT1 and STAT3 signaling pathways. This is evidenced by altered STAT1 and STAT3 phosphorylation patterns and decreased nuclear shuttling, which are in part due to zinc deficiency-induced oxidative stress.
Phosphorylation of STAT1 and STAT3 on serine residues [46] have multiple but still poorly defined functional consequences [47][48][49]. We observed that STAT1 serine phosphorylation levels were high, while STAT3 serine phosphorylation remained unchanged by zinc deficiency in both rat fetal brain and human IMR-32 cells. Zinc deficiencyinduced oxidative stress leads to the activation of the redox-sensitive mitogen activated kinase p38 [9,50,51], which could explain the higher phosphorylation of STAT1 at Ser727 [52]. This was ruled out by the finding that, although LA inhibits zinc deficiency-induced p38 activation [50] it did not prevent the increase in STAT1 serine phosphorylation levels. Phosphorylation of STAT1 and STAT3 on tyrosine residues is involved in dimerization and nuclear translocation. STAT1 is phosphorylated in tyrosine by Jak2, which also phosphorylates microtubules facilitating the binding of STAT1 to tubulin for subsequent nuclear transport [53]. On the other hand, LA had no effect on the low STAT3 tyrosine phosphorylation levels in zinc deficient cells. A potential candidate that could explain STAT3 lower tyrosine levels is PTP1B, the STAT3 tyrosine phosphatase. PTP1B is regulated by both cell redox and zinc status [14]. Oxidation of a cysteine to sulfenylamide in the PTP1B catalytic domain inhibits the activity of the enzyme [54,55], while zinc per se inhibits PTP1B [56] which would lead to its activation in conditions of zinc deficits. This could explain the LAinsensitive decreased STAT3 tyrosine phosphorylation, and its recovery experiments. *, **, *** Significantly different compared to C groups (P < 0.05, P < 0.01, and P < 0.001, respectively, one-way ANOVA). upon zinc repletion. Further studies are needed to assess the participation of PTP1B, the differential responses of STAT1 and STAT3 phosphorylation to LA and their redox regulation in neuronal cells.
Tyrosine phosphorylation leads to STAT1 dimerization, which reveals a nuclear localization signal that is recognized by nuclear specific carriers (importins) for subsequent nuclear import [47][48][49]. While STAT3 tyrosine phosphorylation facilitates the dimer nuclear/ cytoplasmic shuttling [57], STAT3 nuclear translocation can also occur independently of tyrosine phosphorylation [49,58]. LA restored both STAT1 and STAT3 nuclear transport in zinc deficient IMR-32 cells, but only prevented STAT1 decreased tyrosine phosphorylation. This suggests that under a condition of decreased zinc availability, the nuclear transport is the central event in the disruption of STAT1 and STAT3 activation and the subsequent STAT1-and STAT3-driven transactivating activity by zinc deficiency. As previously described [53,[59][60][61], we observed that the cytoskeleton is required for STAT1 and STAT3 nuclear import in neuronal cells. Both in IMR-32 neuroblastoma cells and in fetal brain, zinc deficiency alters tubulin polymerization dynamics via oxidative stress-induced oxidation of tubulin thiols [16,18]. We previously showed that alterations in tubulin polymerization underlies zinc deficiency-associated impaired nuclear translocation of transcription factors NF-κB and NFAT in fetal rat brain, rat cortical neurons, differentiated PC12 cells and proliferating IMR-32 cells [16][17][18]. In fact, reduction of tubulin disulfides upon treatment with LA restored tubulin assembly and NF-κB nuclear translocation in zinc deficient IMR-32 cells [16]. In this study, we observed that zinc deficiency is associated with the accumulation of 4-HNE adducts in E19 rat brain and IMR-32 cells. 4-HNE has been shown to form adducts with tubulin involving Lys residues, which leads to the formation of aggregates and to impaired polymerization [62]. Findings that: i) under identical experimental conditions to those used in the current work LA prevented zinc deficiency induced tubulin oxidation and altered polymerization [16], ii) LA prevented zinc deficiency-associated decrease in nuclear STAT1 and STAT3, iii) the requirement of microtubules for STAT1 and STAT3 nuclear transport [53,[59][60][61]; suggest that tubulin oxidation contributes to the impaired nuclear import of STAT1 and STAT3 under conditions of zinc deficits. Current results also suggest the involvement of tubulin and actin in STAT1 and STAT3 nuclear transport. Recent observations of lower STAT3 binding to both tubulin and actin in the zinc deficient E19 brain (unpublished results) grant future research on the role of both actin and tubulin redox status on STAT3 and STAT1 modulation under conditions of decreased zinc availability.
In summary, a low zinc availability affected STAT1 and STAT3 modulation in both E19 rat brain and human neuroblastoma IMR-32 cells. Zinc deficiency caused oxidative modification of proteins, altered patterns of STAT1 and STAT3 phosphorylation, impaired nuclear translocation, DNA binding, and trans-activating activity. The data presented herein suggest a role for zinc deficiency-induced oxidative stress on the impaired tyrosine phosphorylation and nuclear shuttling of STAT1 and STAT3. The latter, together with the previously shown impaired nuclear transport of transcription factors NFAT and NF-κB, stresses the relevance of tubulin oxidation and altered cytoskeleton dynamics as central events in zinc deficiency-associated pathophysiology. Given the major and different roles that STAT1 and STAT3 play in the nervous system, their impaired modulation could in part explain the deleterious effects of gestational marginal zinc deficiency on early brain development.