Labile Zinc Research Articles

Optical Monitoring of Labile Zinc inside Metastatic Cells with Plasmonic Chemonanosensors.

We report the development and characterization of an optical nanosensor for the detection of labile zinc in biological environments. The readout is based on surface-enhanced Raman scattering promoted by a Raman reporter conjugated to the inner plasmonic cavity of hollow silica nanocapsules. We quantify Zn2+ by obtaining the ratio between a Zn2+-sensitive band and a Zn2+-insensitive band. The Raman reporter measures within the range from 10-5 to 10-11 M and exhibits a limit of detection of 10-11.72 M. The nanosensor discriminates between intracellular and extracellular Zn2+ concentrations.

Fluorescent protein-based Zn2+ sensors reveal distinct responses of aerobic and anaerobic Escherichia coli cultures to excess Zn2+

Zinc ions are required by all known organisms. Maintaining zinc homeostasis by preventing toxic overload while ensuring sufficient acquisition for cellular functions is crucial for survival and growth of bacteria. Bacteria, however, frequently encounter and must survive in various environments. During infection in host animals, for example, bacteria are exposed to acidic conditions in the stomach and anaerobic conditions in the intestines, but the effects of oxygen on zinc homeostasis in E. coli have not been well-studied. Previously, we reported a flavin-binding fluorescent protein-based zinc sensor, CreiLOVN41C, which can respond to changes in labile Zn2+ levels in bacteria under both aerobic and anaerobic conditions. Here, we combined the use of CreiLOVN41C with established oxygen-dependent fluorescent protein-based sensors, inductively coupled plasma-mass spectrometry (ICP-MS), and growth curves to evaluate how oxygen levels affect zinc uptake in E. coli. ICP-MS results showed that cells grown aerobically with added zinc acquired more zinc, but no additional zinc was accumulated when cells were grown anaerobically. Using oxygen-independent CreiLOVN41C and the oxygen-dependent ZapCY series of sensors, intracellular labile zinc was detected in E. coli grown with varied zinc under varied conditions. Although little to no endogenous zinc was detected by any sensor in E. coli cells grown with up to 2 mM added zinc, CreiLOVN41C revealed that when Zn2+ was added and detected by cells in real-time, anaerobic cells required more Zn2+ to similarly saturate the sensor. Overall, this work reveals that zinc uptake in E. coli is impacted by oxygen levels during cell growth.

Cellular zinc status alters chromatin accessibility and binding of p53 to DNA.

Zn2+ is an essential metal required by approximately 850 human transcription factors. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Using ATAC-seq to profile regions of accessible chromatin coupled with transcription factor enrichment analysis, we examined how increases and decreases in the labile zinc pool affect chromatin accessibility and transcription factor enrichment. We found 685 transcription factor motifs were differentially enriched, corresponding to 507 unique transcription factors. The pattern of perturbation and the types of transcription factors were notably different at promoters versus intergenic regions, with zinc-finger transcription factors strongly enriched in intergenic regions in elevated Zn2+ To test whether ATAC-seq and transcription factor enrichment analysis predictions correlate with changes in transcription factor binding, we used ChIP-qPCR to profile six p53 binding sites. We found that for five of the six targets, p53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc alter chromatin accessibility and transcription factor binding to DNA.

Dietary Zinc Supplementation in Steers Modulates Labile Zinc Concentration and Zinc Transporter Gene Expression in Circulating Immune Cells.

Zinc (Zn) is critical for immune function, and marginal Zn deficiency in calves can lead to suboptimal growth and increased disease susceptibility. However, in contrast to other trace minerals such as copper, tissue concentrations of Zn do not change readily in conditions of supplementation or marginal deficiency. Therefore, the evaluation of Zn status remains challenging. Zinc transporters are essential for maintaining intracellular Zn homeostasis, and their expression may indicate changes in Zn status in the animal. Here, we investigated the effects of dietary Zn supplementation on labile Zn concentration and Zn transporter gene expression in circulating immune cells isolated from feedlot steers. Eighteen Angus crossbred steers (261 ± 14 kg) were blocked by body weight and randomly assigned to two dietary treatments: a control diet (58 mg Zn/kg DM, no supplemental Zn) or control plus 150 mg Zn/kg DM (HiZn; 207 mg Zn/kg DM total). After 33 days, Zn supplementation increased labile Zn concentrations (as FluoZin-3 fluorescence) in monocytes, granulocytes, and CD4 T cells (P < 0.05) but had the opposite effect on CD8 and γδ T cells (P < 0.05). Zn transporter gene expression was analyzed on purified immune cell populations collected on days 27 or 28. ZIP11 and ZnT1 gene expression was lower (P < 0.05) in CD4 T cells from HiZn compared to controls. Expression of ZIP6 in CD8 T cells (P = 0.02) and ZnT7 in B cells (P = 0.01) was upregulated in HiZn, while ZnT9 tended (P = 0.06) to increase in B cells from HiZn. These results suggest dietary Zn concentration affects both circulating immune cell Zn concentrations and Zn transporter gene expression in healthy steers.

Cochlear zinc signaling dysregulation is associated with noise-induced hearing loss, and zinc chelation enhances cochlear recovery

Exposure to loud noise triggers sensory organ damage and degeneration that, in turn, leads to hearing loss. Despite the troublesome impact of noise-induced hearing loss (NIHL) in individuals and societies, treatment strategies that protect and restore hearing are few and insufficient. As such, identification and mechanistic understanding of the signaling pathways involved in NIHL are required. Biological zinc is mostly bound to proteins, where it plays major structural or catalytic roles; however, there is also a pool of unbound, mobile (labile) zinc. Labile zinc is mostly found in vesicles in secretory tissues, where it is released and plays a critical signaling role. In the brain, labile zinc fine-tunes neurotransmission and sensory processing. However, injury-induced dysregulation of labile zinc signaling contributes to neurodegeneration. Here, we tested whether zinc dysregulation occurs and contributes to NIHL in mice. We found that ZnT3, the vesicular zinc transporter responsible for loading zinc into vesicles, is expressed in cochlear hair cells and the spiral limbus, with labile zinc also present in the same areas. Soon after noise trauma, ZnT3 and zinc levels are significantly increased, and their subcellular localization is vastly altered. Disruption of zinc signaling, either via ZnT3 deletion or pharmacological zinc chelation, mitigated NIHL, as evidenced by enhanced auditory brainstem responses, distortion product otoacoustic emissions, and number of hair cell synapses. These data reveal that noise-induced zinc dysregulation is associated with cochlear dysfunction and recovery after NIHL, and point to zinc chelation as a potential treatment for mitigating NIHL.

Zinc depletion induces JNK/p38 phosphorylation and suppresses Akt/mTOR expression in acute promyelocytic NB4 cells

BackgroundMyeloid leukemia is associated with reduced serum zinc and increased intracellular zinc. Our previous studies found that zinc depletion by TPEN induced apoptosis with PML-RARα oncoprotein degradation in acute promyelocytic NB4 cells. The effect of zinc homeostasis on intracellular signaling pathways in myeloid leukemia cells remains unclear. ObjectiveThis study examined how zinc homeostasis affected MAPK and Akt/mTOR pathways in NB4 cells. MethodsWe used western blotting to detect the activation of p38 MAPK, JNK, ERK1/2, and Akt/mTOR pathways in NB4 cells stimulated with the zinc chelator TPEN. Whether the effects of TPEN on these pathways could be reversed by zinc or the nitric oxide donor sodium nitroprusside (SNP) was further explored by western blotting. We used Zinpyr-1 staining to assess the role of SNP on labile zinc levels in NB4 cells treated with TPEN. In additional, we evaluated expressional correlations between the zinc-binding protein Metallothionein-2A (MT2A) and genes related to MAPKs and Akt/mTOR pathways in acute myeloid leukemia (AML) based on the TCGA database. ResultsZinc depletion by TPEN activated p38 and JNK phosphorylation in NB4 cells, whereas ERK1/2 phosphorylation was increased first and then decreased. The protein expression levels of Akt and mTOR were downregulated by TPEN. The nitric oxide donor SNP promotes zinc release in NB4 cells under zinc depletion conditions. We further found that the effects of zinc depletion on MAPK and Akt/mTOR pathways in NB4 cells can be reversed by exogenous zinc supplementation or treatment with the nitric oxide donor SNP. By bioinformatics analyses based on the TCGA database, we demonstrated that MT2A expression was negatively correlated with the expression of JNK, and was positively correlated with the expression of ERK1 and Akt in AML. ConclusionOur findings indicate that zinc plays a critical role in leukemia cells and help understanding how zinc depletion induces apoptosis.

Human cells experience a Zn2+ pulse in early G1

Zinc is an essential micronutrient required for all domains of life. Cells maintain zinc homeostasis using a network of transporters, buffers, and transcription factors. Zinc is required for mammalian cell proliferation, and zinc homeostasis is remodeled during the cell cycle, but whether labile zinc changes in naturally cycling cells has not been established. We use genetically encoded fluorescent reporters, long-term time-lapse imaging, and computational tools to track labile zinc over the cell cycle in response to changes in growth media zinc and knockdown of the zinc-regulatory transcription factor MTF-1. Cells experience a pulse of labile zinc in early G1, whose magnitude varies with zinc in growth media. Knockdown of MTF-1 increases labile zinc and the zinc pulse. Our results suggest that cells need a minimum zinc pulse to proliferate and that if labile zinc levels are too high, cells pause proliferation until labile cellular zinc is lowered.

Intracellular Zinc Trafficking during Crotalus atrox Venom Wound Development.

In this study, we examined zinc trafficking in human umbilical vein endothelial cells (HUVEC) stimulated with Crotalus atrox (CA venom) snake venom. We utilized MTS cytotoxicity assays to monitor the cytotoxic range of CA venom. HUVEC monolayers stimulated with 10 µg/mL CA venom for 3 h displayed cellular retraction, which coincided with 53.0 ± 6.5 percent viability. In contrast, venom concentrations of 100 µg/mL produced a complete disruption of cellular adherence and viability decreased to 36.6 ± 1.0. The zinc probe Fluozin-3AM was used to detect intracellular zinc in non-stimulated controls, HUVEC stimulated with 10 µg/mL CA venom or HUVEC preincubated with TPEN for 2 h then stimulated with 10 µg/mL CA venom. Fluorescent intensity analysis returned values of 1434.3 ± 197.4 for CA venom demonstrating an increase of about two orders of magnitude in labile zinc compared to non-stimulated controls. Endothelial response to CA venom induced a 96.1 ± 3.0- and 4.4 ± 0.41-fold increase in metallothionein 1X (MT1X) and metallothionein 2A (MT2A) gene expression. Zinc chelation during CA venom stimulation significantly increased cell viability, suggesting that the maintenance of zinc homeostasis during envenomation injury improves cell survival.

Investigation on the Toxicity of Nanoparticle Mixture in Rainbow Trout Juveniles

The environmental impacts of nanoparticle mixtures in the aquatic environment is not well understood. The purpose of this study examined the sub-lethal toxicity of low concentrations (ug/L range) of selected nanoparticles alone and in mixtures in juvenile trout. Fish were exposed to to individual and two environmentally relevant mixtures of silver (nAg), copper oxide (nCuO) and cerium oxide (nCeO) nanoparticles for 96 h at 15 °C. After the exposure period, fish were depurated overnight and tissue levels in Ag, Ce, Cu and Zn were determined along with a suite of effects biomarkers such as oxidative stress/inflammation, denatured protein tagging (ubiquitin), DNA strand breaks (genotoxicity) and acetylcholinesterase (AChE) activity. The data showed that these nanoparticles behaved as suspended matter but were nevertheless bioavailable for fish with bioconcentration factors of 6, 8 and 2 for nAg, nCeO and nCuO respectively. Only nCuO alone increased malonaldehyde (lipid peroxidation) contents but all nanoparticles increased DNA damage, protein-ubiquitin labeling, and decreased AChE activity. Globally, the toxicity of nCeO and nCuO was generally stronger than nAg, and antagonist effects were found in the mixtures. The interactions involved in these antagonisms are not well understood but do not involve the liberation of free ions and labile zinc in tissues. In conclusion, the bioavailability and toxicity of these nanoparticles are influenced by mixtures of nanoparticles, which is likely to occur in contaminated environments.

Natural organic matter source, concentration, and pH influences the toxicity of zinc to a freshwater microalga

Zinc is a contaminant of concern in aquatic environments and is a known toxicant to many aquatic organisms. Dissolved organic matter (DOM) is a toxicity modifying factor for zinc and is an important water chemistry parameter. This study investigated the influence of DOM concentration, source, and water pH on the chronic toxicity of zinc to a freshwater microalga, Chlorella sp. The influence of DOM on zinc toxicity was dependent on both concentration and source. In the absence of DOM, the 72-h EC50 was 112 μg Zn.L−1. In the presence of a DOM high in fulvic-like components, zinc toxicity was either slightly decreased (<4-fold increase in EC10s across 15 mg C.L−1 range) or unchanged (minimal difference in EC50s). In the presence of a DOM high in humic-like (aromatic and high molecular weight) components, zinc toxicity was slightly decreased at the EC10 level and strongly increased at the EC50 level. The influence of pH on zinc toxicity was dependent on the source of DOM present in the water. In the presence of DOM high in humic-like components pH did not influence toxicity. In the presence of DOM high in fulvic-like components, pH had a significant effect on EC50 values. Labile zinc (measured by diffusive gradients in thin-films) followed linear relationships with dissolved zinc but could not explain the changes in observed toxicity, with similar DGT-labile zinc relationships shown for the two DOMs despite each DOM influencing toxicity differently. This indicates changes in toxicity may be unrelated to changes in zinc lability. The results suggest that increased toxicity of zinc in the presence of DOM may be due to direct uptake of Zn-DOM complexes. This study highlights the importance of considering DOM source and characteristics when incorporating DOM into water quality guidelines through bioavailability models.

Zinc dynamics regulate early ovarian follicle development

Zinc fluctuations regulate key steps in late oocyte and preimplantation embryo development; however, roles for zinc in preceding stages in early ovarian follicle development, when cooperative interactions exist between the oocyte and somatic cells, are unknown. To understand the roles of zinc during early follicle development, we applied single cell X-ray fluorescence microscopy, a radioactive zinc tracer, and a labile zinc probe to measure zinc in individual mouse oocytes and associated somatic cells within early follicles. Here, we report a significant stage-specific increase and compartmental redistribution in oocyte zinc content upon the initiation of early follicle growth. The increase in zinc correlates with the increased expression of specific zinc transporters, including two that are essential in oocyte maturation. While oocytes in follicles exhibit high tolerance to pronounced changes in zinc availability, somatic survival and proliferation are significantly more sensitive to zinc chelation or supplementation. Finally, transcriptomic, proteomic, and zinc loading analyses reveal enrichment of zinc targets in the ubiquitination pathway. Overall, these results demonstrate that distinct cell type-specific zinc regulations are required for follicle growth and indicate that physiological fluctuation in the localization and availability of this inorganic cofactor has fundamental functions in early gamete development.

Rational Design of Ratiometric Fluorescent Probe for Zn2+ Imaging under Oxidative Stress in Cells

Zn2+ is a vital ion for most of the physiological processes in the human body, and it usually has a mutual effect with oxidative stress that often occurs in pathological tissues. Detecting fluctuation of Zn2+ level in cells undergoing oxidative stress could be beneficial to understanding the relationship between them. Herein, a ratiometric fluorescent Zn2+ probe was rationally designed. The wavelength corresponding to the maximum fluorescence intensity bathometrically shifted from 620 nm to 650 nm after coordinating with Zn2+. The intensity ratio of two fluorescence channels changed significantly in cells treated by oxidative stress inducers. It was shown from the results that the labile zinc level was generally elevated under oxidative stress stimulated by various inducers.

Single molecule microscopy to profile the effect of zinc status on transcription factor dynamics

The regulation of transcription is a complex process that involves binding of transcription factors (TFs) to specific sequences, recruitment of cofactors and chromatin remodelers, assembly of the pre-initiation complex and recruitment of RNA polymerase II. Increasing evidence suggests that TFs are highly dynamic and interact only transiently with DNA. Single molecule microscopy techniques are powerful approaches for tracking individual TF molecules as they diffuse in the nucleus and interact with DNA. Here we employ multifocus microscopy and highly inclined laminated optical sheet microscopy to track TF dynamics in response to perturbations in labile zinc inside cells. We sought to define whether zinc-dependent TFs sense changes in the labile zinc pool by determining whether their dynamics and DNA binding can be modulated by zinc. We used fluorescently tagged versions of the glucocorticoid receptor (GR), with two C4 zinc finger domains, and CCCTC-binding factor (CTCF), with eleven C2H2 zinc finger domains. We found that GR was largely insensitive to perturbations of zinc, whereas CTCF was significantly affected by zinc depletion and its dwell time was affected by zinc elevation. These results indicate that at least some transcription factors are sensitive to zinc dynamics, revealing a potential new layer of transcriptional regulation.

Assessement of the heavy metal contamination of soils in Kondopoga and Kostomuksha (Republic of Karelia)

Surveys were carried out in Kondopoga and Kostomuksha, where the main industries are pulp-and-paper making and iron ore mining and concentration, respectively. Samples were taken from the top 0–10 cm soil layer in sites belonging to different land use categories and the content of heavy metals (Pb, Cu, Zn, Ni, Co, Cr, Mn) in the samples was determined by atomic absorption spectrophotometry (Aqua Regia extract). In addition, subsidiary soil pits were made for sampling at 0–5, 5–10, and 10–20 cm depths. These samples were analyzed for physico-chemical properties and heavy metal content. The results showed a relatively low level of heavy metal pollution of soil in the mentioned towns. Surveys in Kondopoga revealed limited general-use areas containing some elements in concentrations exceeding Russian national maximum allowable (MAC) and tentative allowable (TAC) levels (Pb – 6x MAC, Cu – 9x TAC, Zn – 16x TAC), and with labile copper and zinc concentrations up to 2x–3x MAC. Soils of Kostomuksha exhibited relatively low levels of the analyzed heavy metals with concentrations generally not exceeding the regional background in all land use categories. It was only occasionally that pollutant concentrations (Pb, Ni, Cu, Zn) reached 1–2x MAC/TAC. The resultant data can be used in the monitoring of urban soils and for working out recommendations for environmental protection.

Pyruvate Prevents Dopaminergic Neurodegeneration and Motor Deficits in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the selective loss of dopamine(DA)rgic neurons in the substantia nigra of the midbrain, and primarily causes motor symptoms. While the pathological cause of PD remains uncertain, oxidative damage, neuroinflammation, and energy metabolic perturbation have been implicated. Pyruvate has been shown neuroprotective in animal models for many neurological disorders, presumably owing to its potent anti-oxidative, anti-inflammatory, and energy metabolic properties. We therefore investigated whether exogenous pyruvate could also protect nigral DA neurons from degeneration and reverse the associated motor deficits in an animal model of PD using the DA neuron-specific toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP (20mg/kg) was injected four times every 2h into the peritoneum of mice, which resulted in a massive loss of DA neurons as well as an increase in neuronal death and cytosolic labile zinc overload. There were rises in inflammatory and oxidative responses, a drop in the striatal DA level, and the emergence of PD-related motor deficits. In comparison, when sodium pyruvate was administered intraperitoneally at a daily dose of 250mg/kg for 7days starting 2h after the final MPTP treatment, significant relief in the MPTP-induced neuropathology, neurodegeneration, DA depletion, and motor symptoms was observed. Equiosmolar dose of NaCl had no neuroprotective effect, and lower doses of sodium pyruvate did not have any statistically significant effects. These findings suggest that pyruvate has therapeutic potential for the treatment of PD and related neurodegenerative diseases.

Neuronal signalling of zinc: from detection and modulation to function.

Zinc is an essential trace element that stabilizes protein structures and allosterically modulates a plethora of enzymes, ion channels and neurotransmitter receptors. Labile zinc (Zn2+) acts as an intracellular and intercellular signalling molecule in response to various stimuli, which is especially important in the central nervous system. Zincergic neurons, characterized by Zn2+ deposits in synaptic vesicles and presynaptic Zn2+ release, are found in the cortex, hippocampus, amygdala, olfactory bulb and spinal cord. To provide an overview of synaptic Zn2+ and intracellular Zn2+ signalling in neurons, the present paper summarizes the fluorescent sensors used to detect Zn2+ signals, the cellular mechanisms regulating the generation and buffering of Zn2+ signals, as well as the current perspectives on their pleiotropic effects on phosphorylation signalling, synapse formation, synaptic plasticity, as well as sensory and cognitive function.

Enrichment and delivery of bioavailable zinc by microalgae polyphosphate nanoparticles

Nanoscale delivery systems have gained a surge of interest in improving the stability, food compatibility, and bioavailability of essential minerals. Biogenic polyphosphate nanoparticles from microalgae have a great potential to enrich and deliver mineral nutrients. Here, zinc-enriched polyphosphate nanoparticles (ZnPNPs) with a zinc/calcium molar ratio of around 0.2 were obtained from Synechococcus sp. PCC 7002 supplemented with 3.75 mg/L zinc in the growth medium, and displayed small size and anionic nature with a monomer diameter of around 18 nm and a zeta potential of around −36 mV. Zinquin fluorescence measurements unveiled that ZnPNPs were well absorbed and readily incorporated into labile zinc pool by polarized Caco-2 cells in vitro. ZnPNPs showed good resistance to phytate's inhibition on zinc transportation across ligated mouse small intestine ex vivo at the phytate × calcium/zinc molar ratio as seen in baked whole wheat bread, and by treating with NaN3, amiloride, low temperature, chloroquine, and wortmanin, ZnPNPs transportation was found to invoke macropinocytic internalization, lysosomal degradation, and transcytosis. Oral pharmacokinetics and liver and skeletal muscle retention of zinc in rats revealed good in vivo zinc bioavailability of ZnPNPs under high dietary phytate conditions. Overall, ZnPNPs are attractive for zinc supplementation/fortification to populations on phytate-rich diets.

Cardiovascular Diseases and Zinc.

Zinc is structurally and functionally essential for more than 300 enzymes and 2000 transcription factors in human body. Intracellular labile zinc is the metabolically effective zinc and tiny changes in its concentrations significantly affect the intracellular signaling and enzymatic responses. Zinc is crucial for the embrionic and fetal development of heart. Therefore, it is shown to be related with a variety of congenital heart defects. It is involved in epithelial-to-mesenchymal transformation including endocardial cushion development, which is necessary for atrioventricular septation as well as the morphogenesis of heart valves. In atherosclerosis, monocyte endothelial adhesion, and diapedesis, activation and transformation into macrophages and forming foam cells by the ingestion of oxidized LDL are monocyte related steps which need zinc. Intracellular zinc increases intracellular calcium through a variety of pathways and furthermore, zinc itself can work as a second messenger as calcium. These demonstrate the significance of intracellular zinc in heart failure and arterial hypertension. However, extracellular zinc has an opposite effect by blocking calcium channels, explaining decreased serum zinc levels, contrary to the increased cardiomyocyte and erythrocyte zinc levels in hypertensive subjects. These and other data in the literature demonstrate that zinc has important roles in healthy and diseased cardiovascular system but zinc-cardiovascular system relationship is so complex that, it has not been explained in all means. In this article, we try to review some of the available knowledge about this complex relationship.

Dynamic zinc fluxes regulate meiotic progression in Caenorhabditis elegans†.

Zinc influx and efflux events are essential for meiotic progression in oocytes of several mammalian and amphibian species, but it is less clear whether this evolutionary conservation of zinc signals is also important in late-stage germline development in invertebrates. Using quantitative, single cell elemental mapping methods, we find that Caenorhabditis elegans oocytes undergo significant stage-dependent fluctuations in total zinc content, rising by over sevenfold from Prophase I through the beginning of mitotic divisions in the embryo. Live imaging of the rapid cell cycle progression in C. elegans enables us to follow changes in labile zinc pools across meiosis and mitosis in single embryo. We find a dynamic increase in labile zinc prior to fertilization that then decreases from Anaphase II through pronuclear fusion and relocalizes to the eggshell. Disruption of these zinc fluxes blocks extrusion of the second polar body, leading to a range of mitotic defects. We conclude that spatial temporal zinc fluxes are necessary for meiotic progression in C. elegans and are a conserved feature of germ cell development in a broad cross section of metazoa.

Therapeutic targeting of the USP2-E2F4 axis inhibits autophagic machinery essential for zinc homeostasis in cancer progression

ABSTRACT Macroautophagy/autophagy is a conserved cellular process associated with tumorigenesis and aggressiveness, while mechanisms regulating expression of autophagic machinery genes in cancers still remain elusive. Herein, we identified E2F4 (E2F transcription factor 4) as a novel transcriptional activator of cytoprotective autophagy crucial for zinc homeostasis in cancer cells. Gain- and loss-of-function studies showed that E2F4 promoted autophagy in a cell cycle-dependent manner, resulting in facilitated degradation of MT (metallothionein) proteins, elevated distribution of Zn2+ within autophagosomes, decreased labile intracellular zinc ions, and increased growth, invasion, and metastasis of gastric cancer cells. Mechanistically, E2F4 directly regulated the transcription of ATG2A (autophagy related 2A) and ULK2 (unc-51 like autophagy activating kinase 2), leading to autophagic degradation of MT1E, MT1M, and MT1X, while USP2 (ubiquitin specific peptidase 2) stabilized E2F4 protein to induce its transactivation via physical interaction and deubiquitination in cancer cells. Rescue experiments revealed that USP2 harbored oncogenic properties via E2F4-facilitated autophagy and zinc homeostasis. Emetine, a small chemical inhibitor of autophagy, was able to block interaction between UPS2 and E2F4, increase labile intracellular zinc ions, and suppress tumorigenesis and aggressiveness. In clinical gastric cancer specimens, both USP2 and E2F4 were upregulated and associated with poor outcome of patients. These findings indicate that therapeutic targeting of the USP2-E2F4 axis inhibits autophagic machinery essential for zinc homeostasis in cancer progression. Abbreviations: 3-MA: 3-methyladenine; ANOVA: analysis of variance; ATG2A: autophagy related 2A; ATG5: autophagy related 5; ATP: adenosine triphosphate; BECN1: beclin 1; BiFC: bimolecular fluorescence complementation; CCND1: cyclin D1; CDK: cyclin dependent kinase; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; Co-IP: co-immunoprecipitation; DAPI: 4’,6-diamidino-2-phenylindole; E2F4: E2F transcription factor 4; eATP: extracellular adenosine triphosphate; EBSS: Earle’s balanced salt solution; FP: first progression; FRET: fluorescence resonance energy transfer; FUCCI: fluorescent ubiquitination-based cell cycle indicator; GFP: green fluorescent protein; GST: glutathione S-transferase; HA: hemagglutinin; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MDM2: MDM2 proto-oncogene; MKI67/Ki-67: marker of proliferation Ki-67; MT: metallothionein; MT1E: metallothionein 1E; MT1M: metallothionein 1M; MT1X: metallothionein 1X; MTT: 3-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide; OS: overall survival; PECAM1/CD31: platelet and endothelial cell adhesion molecule 1; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; qPCR: quantitative PCR; RFP: red fluorescent protein; SQSTM1/p62: sequestosome 1; UBXN1: UBX domain protein 1; Ub: ubiquitin; ULK2: unc-51 like autophagy activating kinase 2; USP14: ubiquitin specific peptidase 14; USP2: ubiquitin specific peptidase 2; USP5: ubiquitin specific peptidase 5; USP7: ubiquitin specific peptidase 7; ZnCl2: zinc chloride.