Zinc deficiency promotes endothelin secretion and endothelial cell migration through nuclear hypoxia-inducible factor-1 translocation.

Zinc is involved in the expression and function of various transcription factors, including the hypoxia-inducible factor-1 (HIF-1). HIF-1 and its target gene endothelin-1 (ET-1) are activated by intermittent hypoxia (IH), one of the main consequences of obstructive sleep apnea (OSA), and both play a key role in the cardiovascular consequences of IH. Because OSA and IH are associated with zinc deficiency, we investigated the effect of zinc deficiency caused by chelation on the HIF-1/ET-1 pathway and its functional consequences in endothelial cells. Primary human microvascular endothelial cells (HMVEC) were incubated with submicromolar doses of the zinc-specific membrane-permeable chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylene diamine (TPEN, 0.5 µM) or ET-1 (0.01 µM) with or without bosentan, a dual ET-1-receptor antagonist. HIF-1α expression was silenced by transfection with specific siRNA. Nuclear HIF-1 content was assessed by immunofluorescence microscopy and Western blot. Migratory capacity of HMVEC was evaluated with a wound-healing scratch assay. Zinc chelation by TPEN exposure induced the translocation of the cytosolic HIF-1α subunit of HIF-1 to the nucleus as well as an HIF-1-mediated ET-1 secretion by HMVEC. Incubation with either TPEN or ET-1 increased endothelial wound-healing capacity. Both HIF-1α silencing or bosentan abolished this effect. Altogether, these results suggest that zinc deficiency upregulates ET-1 signaling through HIF-1 activation and stimulates endothelial cell migration, suggesting an important role of zinc in the vascular consequences of IH and OSA mediated by HIF-1-ET- signaling.

Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

Zinc, an essential micronutrient, has a cancer preventive role. Zinc deficiency has been shown to contribute to the progression of esophageal cancer. Orai1, a store-operated Ca2+ entry (SOCE) channel, was previously reported to be highly expressed in tumor tissues removed from patients with esophageal squamous cell carcinoma (ESCC) with poor prognosis, and elevation of its expression contributes to both hyperactive intracellular Ca2+ oscillations and fast cell proliferation in human ESCC cells. However, the molecular basis of cancer preventive functions of zinc and its association with Orai1-mediated cell proliferation remains unknown. The present study shows that zinc supplementation significantly inhibits proliferation of ESCC cell lines and that the effect of zinc is reversible with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, a specific Zn2+ chelator, whereas nontumorigenic esophageal epithelial cells are significantly less sensitive to zinc treatment. Fluorescence live cell imaging revealed that extracellular Zn2+ exerted rapid inhibitory effects on Orai1-mediated SOCE and on intracellular Ca2+ oscillations in the ESCC cells. Knockdown of Orai1 or expression of Orai1 mutants with compromised zinc binding significantly diminished sensitivity of the cancer cells to zinc treatment in both SOCE and cell proliferation analyses. These data suggest that zinc may inhibit cell proliferation of esophageal cancer cells through Orai1-mediated intracellular Ca2+ oscillations and reveal a possible molecular basis for zinc-induced cancer prevention and Orai1-SOCE signaling pathway in cancer cells.-Choi, S., Cui, C., Luo, Y., Kim, S.-H., Ko, J.-K., Huo, X., Ma, J., Fu, L.-W., Souza, R. F., Korichneva, I., Pan, Z. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

Zinc transporters and dysregulated channels in cancers.

As a nutritionally essential metal ion, zinc (Zn) not only constitutes a structural element for more than 3000 proteins but also plays important regulatory functions in cellular signal transduction. Zn homeostasis is tightly controlled by regulating the flux of Zn across cell membranes through specific transporters, i.e. ZnT and ZIP family proteins. Zn deficiency and malfunction of Zn transporters have been associated with many chronic diseases including cancer. However, the mechanisms underlying Zn regulatory functions in cellular signaling and their impact on the pathogenesis and progression of cancers remain largely unknown. In addition to these acknowledged multifunctions, Zn modulates a wide range of ion channels that in turn may also play an important role in cancer biology. The goal of this review is to propose how zinc deficiency, through modified Zn homeostasis, transporter activity and the putative regulatory function of Zn can influence ion channel activity, and thereby contribute to carcinogenesis and tumorigenesis. This review intends to stimulate interest in, and support for research into the understanding of Zn-modulated channels in cancers, and to search for novel biomarkers facilitating effective clinical stratification of high risk cancer patients as well as improved prevention and therapy in this emerging field.

Recent Advances in Oncogenic Roles of the TRPM7 Chanzyme.

Transient Receptor Potential Melastatin-related 7 (TRPM7) is a non-selective cation channel fused with a functional kinase domain. Physiologically, TRPM7 channel is involved in magnesium homeostasis, cell survival and gastrulation. The channel part is responsible for calcium, magnesium, and metal trace entries. Cation current through TRPM7 channel is inhibited by both intracellular magnesium and magnesium complexed with nucleotides. In parallel, the kinase is able to phosphorylate cytoskeleton proteins like myosin chain regulating cell tension and motility. Moreover, TRPM7 kinase domain can be cleaved by caspase and participates to apoptosis signaling. Importantly, TRPM7 channel expression is aberrant in numerous cancers including breast, glioblastoma, nasopharynx, ovarian, and pancreatic. Moreover, TRPM7 high expression is an independent biomarker of poor outcome in breast cancer. Pharmacological modulation or silencing of TRPM7 strongly affects proliferation, adhesion, migration or invasion in cancer cell lines. Nevertheless, it is still not clear by which mechanism TRPM7 channels may disturb cancer cell hallmarks. In the present review, we will discuss the role of TRPM7 channels in malignancies. In particular, we will distinguish the role of cation signaling from kinase function in order to better understand how TRPM7 channels may play a central role in cancer progression. We will also discuss the recent advances in pharmacological blockers of TRPM7 and their potential use for cancer therapy.

Endoplasmic reticulum stress as a novel inducer of hypoxia inducible factor-1 activity: its role in the susceptibility to myocardial ischemia-reperfusion induced by chronic intermittent hypoxia.

BACKGROUND: Obstructive sleep apnea (OSA) is a highly prevalent disease and a risk factor for myocardial infarction expansion in humans. Intermittent hypoxia (IH) is known to be the most important OSA feature in terms of cardiovascular morbi-mortality. Since ER stress and HIF-1 are known to be involved in cardiomyocyte life or death, this study investigates the role of ER stress on HIF-1 activation in myocardial susceptibility to ischemia-reperfusion (I/R) induced by IH. METHODS: C57Bl6J, HIF-1α(+/-) and their respective control mice were exposed to 14 days of IH (21-5% FiO2, 60 scycle, 8h/day). Myocardial inter-organelle calcium exchanges, ER stress and HIF-1 activity were investigated and in vivo I/R was performed to measure infarct size. In additional groups, tauroursodeoxycholic acid (TUDCA, 75 mg·kg(-1)), an ER stress inhibitor, was administered daily during exposure. RESULTS: In C57Bl6J mice, chronic IH induced an increase in ER-Ca(2+) content, ER stress markers and HIF-1 activity, associated with an enhanced infarct size (33.7 ± 9.4 vs. 61.0 ± 5.6% in N and IH, respectively, p<0.05). IH failed to increase infarct size in HIF-1α deficient mice (42.4 ± 2.7 and 24.7 ± 3.4% N and IH, respectively). Finally, TUDCA totally abolished the IH-induced increase in HIF-1 activity (1.3 ± 0.04 vs. 0.14 ± 0.02 fold increase in IH vs. IH-TUDCA respectively, p<0.0001) and in infarct size (55.5 ± 7.6 vs. 49.9 ± 3.0 in N-TUDCA and IH-TUDCA, respectively). CONCLUSION: This novel regulatory mechanism of HIF-1 activity by ER stress should be considered as a potential diagnostic tool for cardiovascular complications in OSA patients as well as a therapeutic target to limit myocardial ischemic damage.

Zinc Binding to MG53 Protein Facilitates Repair of Injury to Cell Membranes.

Zinc is an essential trace element that participates in a wide range of biological functions, including wound healing. Although Zn(2+) deficiency has been linked to compromised wound healing and tissue repair in human diseases, the molecular mechanisms underlying Zn(2+)-mediated tissue repair remain unknown. Our previous studies established that MG53, a TRIM (tripartite motif) family protein, is an essential component of the cell membrane repair machinery. Domain homology analysis revealed that MG53 contains two Zn(2+)-binding motifs. Here, we show that Zn(2+) binding to MG53 is indispensable to assembly of the cell membrane repair machinery. Live cell imaging illustrated that Zn(2+) entry from extracellular space is essential for translocation of MG53-containing vesicles to the acute membrane injury sites for formation of a repair patch. The effect of Zn(2+) on membrane repair is abolished in mg53(-/-) muscle fibers, suggesting that MG53 functions as a potential target for Zn(2+) during membrane repair. Mutagenesis studies suggested that both RING and B-box motifs of MG53 constitute Zn(2+)-binding domains that contribute to MG53-mediated membrane repair. Overall, this study establishes a base for Zn(2+) interaction with MG53 in protection against injury to the cell membrane.

TRPM7 involvement in cancer: a potential prognostic factor.

Calcium (Ca(2+)) and magnesium (Mg(2+)) are important metal elements that regulate a variety of cellular processes such as proliferation, migration, and apoptosis, in cancer cells. Among the ionic channels mediating intracellular entry, the transient receptor potential melastatin type 7 (TRPM7) channel is of particular interest, it being a non-selective, cationic channel mediating both Ca(2+) and Mg(2+) influx. TRPM7 is highly expressed in a number of human cancer tissues and cell lines. In this review, we summarise current knowledge on the physiological role of the dual function TRPM7 chanzyme, the potential application of TRPM7 as a diagnostic and prognostic marker of cancer progression with respect to clinical and pathological characteristics, and the molecular mechanisms implicated in cancerogenesis that specifically involve Ca(2+) and Mg(2+) influx through TRPM7 or kinase activity and interaction with cytoskeletal proteins.

Atorvastatin protects against deleterious cardiovascular consequences induced by chronic intermittent hypoxia.

Chronic intermittent hypoxia (IH), a major component of obstructive sleep apnea (OSA), contributes to the high risk of cardiovascular morbidity. We have previously demonstrated that IH-induced oxidative stress is involved in the hypertension and in the hypersensitivity to myocardial infarction. However, the mechanisms underlying these cardiovascular alterations are still unclear, as well as the role of potential protective treatment. Atorvastatin has pleiotropic actions, including increasing nitric oxide (NO) bioavailability and reducing inflammation and oxidative damage. The aim of this study was to evaluate the beneficial effect of a two time course of this treatment against the deleterious cardiovascular consequences of IH. Rats were divided into two groups subjected to chronic IH or normoxic (N) exposure. IH consisted of repetitive one-minute cycles (with only 30 s of a 5% inspired O2 fraction) and was applied for eight hours during daytime, for 14 (simultaneous protocol) or 28 d (delayed protocol). Atorvastatin (10 mg/kg/ d) or its vehicle was administered during the 14 d simultaneous protocol or the last 14 d of the delayed protocol. For both protocols, systolic arterial pressure was significantly increased by 14 d IH exposure. Atorvastatin prevented this deleterious effect in the simultaneous protocol. Carotid artery compliance and endothelial function were significantly altered after 28 d but not after 14 d of IH exposure. Delayed atorvastatin administration preserved these vascular parameters. IH also increased hypersensitivity to myocardial infarction after 14 d exposure, and atorvastatin abolished this deleterious effect. IH also enhanced cardiac NADPH expression and decreased aortic superoxide dismutase activity after 14 d exposure. Atorvastatin significantly restored these activities. In conclusion, whereas IH rapidly increased blood pressure, myocardial infarction hypersensitivity and oxidative stress, compliance, endothelial function and the structural wall of the carotid artery were only altered after a longer IH exposure. Atorvastatin prevented all these deleterious cardiovascular effects, leading to a potentially novel pharmacological therapeutic strategy for OSA syndrome.

Zinc and cardiovascular disease.

Zinc is a vital element in maintaining the normal structure and physiology of cells. The fact that it has an important role in states of cardiovascular diseases has been studied and described by several research groups. It appears to have protective effects in coronary artery disease and cardiomyopathy. Intracellular zinc plays a critical role in the redox signaling pathway, whereby certain triggers such as ischemia and infarction lead to release of zinc from proteins and cause myocardial damage. In such states, replenishing with zinc has been shown to improve cardiac function and prevent further damage. Thus, the area of zinc homeostasis is emerging in cardiovascular disease research. The goal of this report is to review the current knowledge and suggest further avenues of research.

Free zinc ions outside a narrow concentration range are toxic to a variety of cells in vitro.

The zinc(II) ion has recently been implicated in a number of novel functions and pathologies in loci as diverse as the brain, retina, small intestine, prostate, heart, pancreas, and immune system. Zinc ions are a required nutrient but elevated concentrations are known to kill cells in vitro. Paradoxical observations regarding zinc's effects have appeared frequently in the literature, and often their physiological relevance is unclear. We found that for PC-12, HeLa and HT-29 cell lines as well as primary cultures of cardiac myocytes and neurons in vitro in differing media, approximately 5 nmol/L free zinc (pZn = 8.3, where pZn is defined as--log(10) [free Zn(2+)]) produced apparently healthy cells, but 20-fold higher or (in one case) lower concentrations were usually harmful as judged by multiple criteria. These results indicate that (1) the free zinc ion levels of media should be controlled with a metal ion buffer; (2) adding zinc or strong zinc ligands to an insufficiently buffered medium may lead to unpredictably low or high free zinc levels that are often harmful to cells; and (3) it is generally desirable to measure free zinc ion levels due to the presence of contaminating zinc in many biochemicals and unknown buffering capacity of many media.

Protective role of intracellular zinc in myocardial ischemia/reperfusion is associated with preservation of protein kinase C isoforms.

The recent discovery of zinc signals and their essential role in the redox signaling network implies that zinc homeostasis and the function of zinc-containing proteins are probably altered as a result of oxidative stress, suggesting new targets for pharmacological intervention. We hypothesized that the level of intracellular labile zinc is changed in hearts subjected to ischemia/reperfusion (I/R) and investigated whether the maintenance of myocardial zinc status protected heart functions. Using fluorescent imaging, we demonstrated decreased levels of labile zinc in the I/R hearts. Phorbol 12-myristate 13-acetate, a known trigger of zinc release, liberated zinc ions in control hearts but failed to produce any increase in zinc levels in the I/R rat hearts. Adding the zinc ionophore pyrithione at reperfusion improved myocardial recovery up to 100% and reduced the incidence of arrhythmias more than 2-fold. This effect was dose-dependent, and high concentrations of zinc were toxic. Adding membrane-impermeable zinc chloride was ineffective. Hearts from rats receiving zinc pyrithione supplements in their diet fully recovered from I/R. The recovery was associated with the prevention of degradation of the two protein kinase C isoforms, delta and epsilon, during I/R. In conclusion, our results suggest a protective role of intracellular zinc in myocardial recovery from oxidative stress imposed by I/R. The data support the potential clinical use of zinc ionophores in the settings of acute redox stress in the heart.

Zinc dynamics in the myocardial redox signaling network.

Zinc plays a vital role in various cellular functions. Zinc deprivation is associated with severe disorders related to growth, maturation, and stress responses. In the heart, zinc affects differentiation and regeneration of cardiac muscle, cardiac conductance, acute stress responses, and recovery of heart transplants. Recent discoveries of the molecular players in zinc homeostasis revealed that the amount of intracellular free zinc is tightly controlled on the level of uptake, intracellular sequestration, redistribution, storage, and elimination, consequently creating a narrow window of optimal zinc concentration in the cells. Most of intracellular zinc is bound to numerous structural and regulatory proteins, with metabolically active, labile zinc present in picoto nanomolar concentrations. The central position of zinc in the redox signaling network is built on its unique chemical nature. The redox inert zinc creates a redox active environment when it binds to a sulfur ligand. The reversible oxidation of the sulfur ligand is coupled to the reversible zinc release from the protein, thereby executing the task of so-called protein "redox zinc switch." Clearly, the impairment of zinc homeostasis will have far reaching physiological consequences.

Redox regulation of cardiac protein kinase C.

The two extremes of redox stress imposed on cardiac tissue under ischemia and reperfusion change the redox potential of the cells and affect numerous redox-sensitive molecules, including the ones involved in intracellular communication. Protein kinase C (PKC), a key signalling kinase, is one of those subject to redox control. Activation of PKC by oxidation represents a new paradigm of the alternate signalling principle. Reactive oxygen species act directly on PKC, releasing chelated Zn(2+) ions from the zinc finger of the regulatory domain. Zn(2+) release from PKC by oxidative stress has been shown at the level of isolated protein fragments, PKC immune complexes and single cells. Zn(2+) movements have been further characterized in cryosections prepared from adult rat hearts subjected to in vivo stress by global ischemia followed by reperfusion. The morphology of labile zinc in cardiac tissue and zinc release following PKC stimulation with lipid activator are described. The studies lead to an unexpected and intriguing result, suggesting that in addition to serving a structural function, Zn(2+) ions are likely to play a dynamic regulatory role in PKC. The cysteine-rich domains of the serine/threonine kinases are identified as redox sensors. Thus, being an integrated composite of redox signalling systems, free Zn(2+) reflects the protein redox status and serves as a valid biomarker of stressed tissue and its capacity to respond to stimuli.

Regulation of the cardiac mitochondrial membrane potential by retinoids.

Cardiomyocytes suffering irreversible damage under oxidative stress during ischemia activate their suicide program. Mitochondria play a key role in this process, while they themselves are subject to regulation by a number of signaling pathways. We demonstrate here that retinoids influence mitochondrial function in cardiomyocytes. Depending on their chemical nature, retinoids can either ameliorate or exacerbate stress-related damage. Thus, vitamin A, retinol, was protective because retinol deprivation enhanced oxidative damage, as indicated by rapid loss of mitochondrial membrane potential. Supplementation with a physiological concentration of retinol reversed this effect. Anhydroretinol (AR), a known antagonist, which works by displacing retinol from the common binding sites on serine/threonine kinases, also caused mitochondrial membrane depolarization. The AR effect was both Ca(2+)-dependent and cyclosporin-sensitive, suggesting an upstream signaling mechanism rather than direct membrane effect. Our results agree with a model where retinol supports mitochondrial integrity by enabling upstream signaling processes. The consequences of disrupting these processes by AR are opening of the permeability transition pore, release of cytochrome c, and activation of the suicide program.

Zinc release from protein kinase C as the common event during activation by lipid second messenger or reactive oxygen.

Zinc is a structural component of many regulatory molecules including transcription factors and signaling molecules. We report that two alternate signaling pathways of protein kinase C (PKC) activation involving either the lipid second messengers (diacylglycerol and its mimetics, the phorbol esters) or reactive oxygen converge at the zinc finger of the regulatory domain. They all trigger the release of zinc ions. An increase in intracellular free Zn(2+) was observed by confocal microscopy in intact cells treated with phorbol ester or by mild oxidation. The source of liberated Zn(2+) was traced to PKC and particularly the zinc finger domains. The activated form of native PKCalpha contained significantly less Zn(2+) than the resting form. Furthermore, purified recombinant PKC protein fragments shed stoichiometric amounts of Zn(2+) upon reaction with diacylglycerol, phorbol ester, or reactive oxygen in vitro. Our results offer new insight into the regulation of PKC. Far from cementing rigid structures, zinc actually is the linchpin that orchestrates dynamic changes in response to specific signals, allowing kinase activity to be turned on or off.

Activation of c-Raf kinase by ultraviolet light. Regulation by retinoids.

The present study highlights retinoids as modulators of c-Raf kinase activation by UV light. Whereas a number of retinoids, including retinol, 14-hydroxyretroretinol, anhydroretinol (AR), and retinoic acid bound the c-Raf cysteine-rich domain (CRD) with equal affinity in vitro as well as in vivo, they displayed different, even opposing, effects on UV-mediated kinase activation; retinol and 14-hydroxyretroretinol augmented responses, whereas retinoic acid and AR were inhibitory. Oxidation of thiol groups of cysteines by reactive oxygen, generated during UV irradiation, was the primary event in c-Raf activation, causing the release of zinc ions and, by inference, a change in CRD structure. Retinoids modulated these oxidation events directly: retinol enhanced, whereas AR suppressed, zinc release, precisely mirroring the retinoid effects on c-Raf kinase activation. Oxidation of c-Raf was not sufficient for kinase activation, productive interaction with Ras being mandatory. Further, canonical tyrosine phosphorylation and the action of phosphatase were essential for optimal c-Raf kinase competence. Thus, retinoids bound c-Raf with high affinity, priming the molecule for UV/reactive oxygen species-mediated changes of the CRD that set off GTP-Ras interaction and, in context with an appropriate phosphorylation pattern, lead to full phosphotransferase capacity.