Inhibition of Gap Junctional Intercellular Communication by Toxic Metals

Sep 24, 2010 - As metals are ubiquitous in the environment, humans are continuously exposed to them. Although some metallic ions play key roles in hum...
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Inhibition of Gap Junctional Intercellular Communication by Toxic Metals Mathieu Vinken,* Liesbeth Ceelen, Tamara Vanhaecke, and Vera Rogiers Department of Toxicology, Faculty of Medicine and Pharmacy, Vrije UniVersiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium ReceiVed May 25, 2010

As metals are ubiquitous in the environment, humans are continuously exposed to them. Although some metallic ions play key roles in human physiology, most metals are redundant and are actually hazardous to humans. A frequent event in the toxicological cascade triggered by nonessential metals concerns the abrogation of cellular signaling mediated by gap junctions. This paper provides a literature overview of the documented effects of mercury, cadmium, arsenic, aluminum, lead, nickel, vanadium, and indium on gap junctional intercellular communication. Whenever available, particular attention is paid to the mechanistic basis of this deleterious biological outcome, which may involve the gap junction activity level or may arise from modifications in the expression of the gap junction building stones, the connexins. Contents 1. Introduction 2. Gap Junctional Intercellular Communication 2.1. Structural Aspects 2.2. Functional Aspects 2.3. Regulatory Aspects 2.4. Pathophysiological Aspects 3. Effects of Toxic Metals on Gap Junctions 3.1. Mercury 3.2. Cadmium 3.3. Arsenic 3.4. Aluminum 3.5. Lead 3.6. Nickel 3.7. Vanadium 3.8. Indium 4. Conclusions and Perspectives

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1. Introduction As much as 80% of the normally occurring elements described in the periodic system are metals. Some of them, such as zinc and chromium, are vital to the mammalian organism as they fulfill clear-cut functions in a wide range of critical physiological processes. Inherent to this pivotal task, pathologies may develop in cases of deficiencies or overloads of these essential metals. Other metals have no physiological function and may cause noxious effects in the organism by binding to macromolecules or by activating or inactivating cellular processes otherwise controlled by essential metals. This typically burgeons into a multitude of adverse reactions that become manifested in several organ systems (1-4). Although a plethora of mechanisms contribute to their toxicity, modified cellular signaling seems to be a typical event in this detrimental process. In this work, the involvement of gap junctional intercellular communication in the toxicity triggered by nonessential metals * To whom correspondence should be addressed: Department of Toxicology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. Telephone: +32.2.477.45.87. Fax: +32 0.2.477.45.82. E-mail: [email protected].

is reviewed. In the first part, key features of gap junctions with respect to their structure, function, and (dys)regulation are outlined. In the second part, the harmful outcome of nonessential metals in gap junctions is discussed, whereby the main focus is put on the molecular mechanisms that underlie these pernicious effects.

2. Gap Junctional Intercellular Communication 2.1. Structural Aspects. Morphologically, gap junctions appear as plaques at the cell plasma membrane surface and arise from the interaction of two hemichannels (connexons) of adjacent cells, which in turn are composed of six connexin (Cx) units. The connexin family comprises as many as twenty isoforms in mammals. They share a similar molecular architecture, consisting of four membrane-spanning domains, two extracellular loops, one intracellular loop, one cytoplasmic N-terminal tail, and one C-terminal tail (Figure 1). Connexins are named after their molecular weight and are expressed in a tissue-specific and even cell-specific manner (5-9). In the kidney, Cx40 is abundantly expressed in the juxtaglomerular apparatus, whereas both Cx37 and Cx40 are strongly produced by endothelial cells of the preglomerular vessels (10). In the brain, Cx36 is mainly present in neurons and Cx43 is prominently found in astrocytes (11). In the liver, hepatocytes produce Cx32, while non-parenchymal liver cells preferentially harbor Cx43 (8). Cx43 also is the major connexin present in lung epithelium, though Cx32 is uniquely expressed in type II alveolar epithelial cells (12). In the past few years, a second set of gap junction-related proteins has been characterized, the pannexins, which are structurally similar to members of the connexin family. At present, three pannexins have been identified in humans and rodents, and they mainly occur in a hemichannel configuration (5, 13). 2.2. Functional Aspects. Gap junctional intercellular communication (GJIC)1 denotes the passive intercellular diffusion of small and hydrophilic substances, including glucose, glutamate, 1 Abbreviations: Cx, connexin; GJIC, gap junctional intercellular communication; ATP, adenosine triphosphate.

10.1021/tx100276f  2010 American Chemical Society Published on Web 09/24/2010

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Figure 1. Molecular architecture of gap junctions. Gap junctions are grouped in plaques at the cell plasma membrane surface of two apposed cells and are composed of twelve connexin proteins, organized as two hexameric hemichannels. The connexin protein is organized as four membranespanning domains (TM), two extracellular loops (EL), one cytoplasmic loop (CL), one cytoplasmic amino tail (NT), and one cytoplasmic carboxy tail (CT).

glutathione, cyclic adenosine monophosphate, adenosine triphosphate (ATP), inositol trisphosphate, and ions (e.g., Ca2+, K+, and Na+) (14). The biophysical permeation characteristics of these substances rely on the nature of the connexin species that form the gap junction. For instance, ATP passes significantly better through Cx43-based gap junctions than through channels composed of Cx32 (15). As numerous physiological processes are driven by these substances, GJIC is considered as a key mechanism in the maintenance of tissue homeostasis (5-9). Recent findings also point to GJIC-independent functions of connexins in the control of cell growth and cell death. Indeed, connexin proteins as such can directly or indirectly modulate the expression of critical homeostasis regulators, such as caspases and cyclins, by affecting their gene transcription or by influencing factors that control this process (5, 9). Connexin hemichannels, on the other hand, foresee a pathway for communication between the intracellular compartment and the extracellular environment. The substances that travel through connexin hemichannels are very similar to those that are intercellularly exchanged via gap junctions, namely, ATP, nicotinamide adenine dinucleotide, glutamate, glutathione, and prostaglandins (5, 9, 16). Unlike their full channel counterparts, however, connexin hemichannels seem to preferentially play a role in pathophysiological processes. Similar functions have been attributed to pannexin hemichannels (5). 2.3. Regulatory Aspects. A myriad of mechanisms governs connexin metabolism and GJIC. Short-term GJIC control, called gating, is mediated by a number of factors such as transmembrane voltage, pH, and Ca2+ ions, yet phosphorylation, mainly occurring at the C-terminal connexin tail, has received most attention as a gating system. With the exception of Cx26, all connexins are phosphoproteins, whereby the outcome of this posttranslational modification depends on both the connexin and the kinase type (5-9). Gap junction gating is directly or indirectly controlled by the numerous proteins that physically interact with connexins, including junctional proteins (e.g., zonula occludens 2), cytoskeletal proteins (e.g., actin), trafficking regulators (e.g., tumor susceptibility gene 101), posttranslational modifiers (e.g., protein kinase C), and growth regulators (e.g., discs large homologue 1) (17, 18). Regulation of GJIC over the long-term basically concerns peritranscriptional control of connexin expression. The structure of most connexin genes is

rather simple and consists of a first exon, containing the 5′untranslated region that is separated by an intron from a second exon, bearing the complete coding sequence and the 3′untranslated region (19, 20). Epigenetic mechanisms, like histone acetylation and DNA methylation, predominate the pretranscriptional platform of connexin expression. Most evidence in this respect comes from experiments using chemicals that directly interfere with epigenetic processes. However, very little is known about the identity of the actual epigenetic enzymes that control connexin expression (6). Connexin gene transcription as such is ruled by conventional cis/trans actions, involving both ubiquitous (e.g., activator protein 1) and tissue-specific (e.g., hepatocyte nuclear factor 1) transcription factors. In recent years, microRNA species have also been identified as master regulators of connexin expression at the posttranscriptional level (6). Cx43 mRNA, for instance, has been characterized as a direct negative target for microRNA miR-206 (21-23). 2.4. Pathophysiological Aspects. Given its key role in the maintenance of homeostasis, it is not surprising that GJIC is frequently involved during dysregulation of this critical balance, as in the case of toxicity and carcinogenesis (7, 24). Indeed, it has been reported on numerous occasions that gap junction activity is drastically downregulated among cancer cells as well as between malignant cells and their healthy neighbors (25-28). Furthermore, gap junctions are targeted by several tumor promoters and epigenetic carcinogens (7, 29). This typically burgeons into the enhancement of proliferative activity at the expense of cell death, and thus into tumorigenesis (26-28). The molecular mechanisms that underpin GJIC abrogation upon carcinogenesis are manifold but do not involve connexin gene mutagenesis. Instead, changes in epigenetic regulation and subsequent silencing of connexin production are often observed in cancer cells. Carcinogenic effects may also occur at more downstream levels, such as the aberrant trafficking and localization of connexin proteins (27, 28). Curiously, downregulated connexins in cancer are sometimes replaced by other connexin species, regardless of whether they are physiologically present in the tissue in question. In cancerous hepatocytes, for example, the level of Cx32 expression is greatly reduced, whereas Cx43 becomes detectable (8, 30, 31). Such connexin switching events are not fully understood. In any event, the typical downregulation of GJIC in cancer not only renders gap junctions

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interesting targets for clinical therapy (32, 33) but also suggests its application as a biomarker during the in vitro screening of (nongenotoxic) carcinogens (7, 34).

3. Effects of Toxic Metals on Gap Junctions 3.1. Mercury. Mercury is an extremely rare element in the earth’s crust that has been classified as a transition metal. As such, mercury exists in three species, namely, elemental (metallic) mercury, inorganic mercury (e.g., mercuric chloride), and organic mercury (e.g., methylmercury). Metallic mercury (IARC class 3) naturally occurs in liquid form at room temperature and quickly turns to vapor when heated. Principal sources of human exposure include dental amalgam and thermometers. Evaporated elemental mercury is easily absorbed by the lungs and accumulates in the brain, liver, and kidney. It induces a broad variety of effects, such as dyspnea, tremor, and polyneuropathy. A similar outcome in the central and peripheral nervous system, including cerebellar ataxia, dysarthtria, constriction of the visual fields, and loss of hearing, also becomes manifested upon chronic exposure to methylmercury (IARC class 2B), which may occur as a result of food (e.g., fish) contamination. Mercuric chloride (IARC class 3), on the other hand, which has been used for many years in skin creams, teething powders, and germicidal soaps, mainly targets the kidney during long-term exposure (1, 4, 35-37). As such, it has been shown that mercuric chloride decreases the extent of GJIC in cultures of canine tubular epithelial kidney cells. This is preceded by inhibition of glutathione peroxidase and to a lesser extent of catalase, two enzymes that degrade hydrogen peroxide. The deteriorated antioxidant cellular defense is therefore thought to indirectly lead to the abolishment of GJIC (35). Organic mercury also inhibits renal GJIC in vitro. Methylmercury indeed causes dysfunction of gap junctions in cultures of primary rat renal proximal epithelial cells, which results from an increase in the intracellular Ca2+ concentration, a process known to directly induce gap junction closure (38). Methylmercury also abrogates GJIC by a similar mechanism in cultured rat osteoblast-like cells (39). Both methylmercury and mercuric chloride negatively affect gap junction functionality in cultures of human keratinocytes. This is equally associated with reduced intracellular levels of interleukin 1β and tumor necrosis factor R. The mechanistic interrelationship between both events is yet unclear, but the events are thought to be linked by the occurrence of an oxidative stress response (40-43). 3.2. Cadmium. Cadmium is a relatively rare transition metal that has several industrial applications, such as the production of nickel-cadmium batteries. Exposure to the general population is low and occurs through food and water. Cigarette smoke is actually a major source of cadmium. Cadmium preferentially accumulates in the liver and kidney and has a half-life of up to twenty years. Acute and chronic exposure to cadmium mainly leads to renal tubular damage (1, 4, 44-47). In fact, treatment of cultured primary rat renal proximal epithelial cells with cadmium dichloride (IARC class 1) strongly counteracts GJIC, which is a direct result of an increase in the intracellular Ca2+ concentration (44). The liver is also a major target for cadmium toxicity, whereby chronic liver toxicity is manifested as granulomatous inflammation, cell proliferation, nodular hyperplasia, and apoptosis (1, 4, 44-47). Upon administration of cadmium dichloride to mice, a time- and concentration-dependent reduction in the level of GJIC in the liver is observed. This is accompanied by decreased Cx26 and Cx32 immunoreactivities. Cadmium dichloride also disrupts the actin cytoskeleton, which may be the actual cause of GJIC abrogation (45). Likewise,

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cadmium dichloride downregulates gap junction functionality and Cx43 expression in a rat epithelial cell line, a process that is thought to involve protein kinase C (48). Cadmium dichloride also reduces Cx43 production in cultured mouse Sertoli cells (49). Furthermore, short-term exposure (i.e., hour range) of mouse embryonic fibroblasts to cadmium dichloride decreases Cx32 protein levels and simultaneously enhances phosphorylation of Cx43. When longer exposure regimes (i.e., week range) are used, connexin levels tend to increase, though Cx43 becomes located in the perinuclear region (50). 3.3. Arsenic. Arsenic, a metalloid that is highly abundant in the earth’s crust, has been widely distributed in the environment by human activities, including mining and manufacturing. Arsenic compounds are found in organic and inorganic forms, and they exist in two different oxidation states, namely, as arsenite and as arsenate, with the former being more toxic than the latter. Chronic exposure to arsenic (e.g., via contaminated drinking water) causes a variety of neurological disorders and diabetes mellitus and exacerbates atherosclerotic plaque formation, which in turn may lead to heart attack and stroke (4, 51, 52). With respect to the latter, it has been reported that treatment of cultured human aortic endothelial cells with arsenic trioxide (IARC class 1) results in the reduced production of nitric oxide and endothelial nitric oxide synthase, being indicators of endothelial function, as well as in inhibition of gap junction functionality. The latter is associated with downregulation of Cx43 expression both at the transcriptional level and at the translational level. The decrease in Cx43 production can be prevented by protease inhibitors, thus suggesting the involvement of Cx43 protein degradation in the deleterious effects of arsenic trioxide on gap junctions. These in vitro findings have been partly confirmed by a subsequent in vivo study, because administration of arsenic trioxide to rats resulted in reduced immunoreactivity to Cx37, Cx40, and Cx43 in aortic endothelial cells (53). 3.4. Aluminum. Aluminum is one of the most abundant metals in the earth’s crust. In nature, aluminum is present as a trivalent cation, most of it being associated with silicate and forming water-insoluble complexes. Aluminum is widely used in human daily life, whereby consumption occurs through food and drinking water. Aluminum does not fulfill any biological function but instead accumulates in tissues and triggers toxicity. In animal experiments, aluminum intoxication shows similarities to the pathology observed in neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (4, 54, 55). This type of negative outcome is also observed at the gap junction level, because aluminum acetate reduces the level of GJIC in cultures of primary rat astroglial cells. This is caused by impaired trafficking of the Cx43 protein from the Golgi complex to the cell plasma membrane surface in cell prolongations, which in turn is due to destruction of the actin cytoskeleton (56). 3.5. Lead. Lead is found in nature as a divalent cation, mainly forming stable complexes with sulfur. It is a metal without a known biological function in humans, though it can damage various systems in the organism, including the hematopoietic, renal, skeletal, and central nervous systems, with the latter being the primary target. Human exposure to lead occurs via food, drinking water, and soil (1, 4, 55). Lead acetate (IARC class 3) was reported to inhibit GJIC in a hamster lung fibroblast cell line, though the mechanisms underlying this observation remain to be elucidated (57). 3.6. Nickel. Nickel is naturally occurring in the earth’s crust and is widely applied in industry with other metals to form alloys

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Chem. Res. Toxicol., Vol. 23, No. 12, 2010 1865 Table 1. Effects of Toxic Metals on Gap Junctional Intercellular Communication cellular system

compound

concentration

GJIC

mechanism

primary rat osteoblast-like cells primary human keratinocytes Chinese hamster V79 cells Madin-Darby canine kidney cells primary rat renal epithelial cells primary rat osteoblast-like cells primary human keratinocytes primary human keratinocytes

Hg (elemental) HgCl2 HgCl2 HgCl2 CH3Hg CH3Hg CH3Hg (CH3)2Hg

Mercury 5 µM 0.01 µM 2-3 µM 0.1-50 µM 30 µM 1-10 µM 0.25 µM 250-500 µM

∼ V V V V V V ∼

mouse liver rat liver WB-F344 epithelial cells primary rat renal epithelial cells Chinese hamster V79 cells Chinese hamster V79 cells Syrian hamster embryo cells

CdCl2 CdCl2 CdCl2 CdCl2 Cd(NO3)2 Cd(C2H3O2)2

Cadmium 5-60 µM/kga 0.03-200 µMb 100 µM 1-6 µM 1.06 µM 110 µM

V V V ∼ V V

V Cx26/Cx32 expression V Cx43 expression v intracellular Ca2+ concentration

45 48 44 57 59 58

human aortic endothelial cells Chinese hamster V79 cells

As2O3 H3AsO4

Arsenic 0.05-5 µM 5-10 µM

V V

V Cx43 expression

53 57

primary rat astroglial cells

C9H15AlO9

340 µM

V

V Cx43 trafficking

56

oxidative stress V glutathione peroxidase/catalase activity v intracellular Ca2+ concentration v intracellular Ca2+ concentration

refrence 39 40-43 57 35 38 39 40 40

Aluminum Lead primary rat osteoblast-like cells Chinese hamster V79 cells Syrian hamster embryo cells primary rat astroglial cells primary human keratinocytes

Pb (elemental) Pb(C2H3O2)2 Pb(C2H3O2)2 Pb(C2H3O2)2 Pb(C2H3O2)2

5 µM 50-500 µM 2600 µM 0.1-1 µM not specified

∼ V ∼ ∼ ∼

39 57 58 76 40

Syrian hamster embryo cells Chinese hamster V79 cells

NiSO4 NiCl2

Nickel 500-3500 µM 75 µM

V V

58 57

Chinese hamster V79 cells Syrian hamster embryo cells Syrian hamster embryo cells Syrian hamster embryo cells Syrian hamster embryo cells Chinese hamster V79 cells

Na3VO4 Na3VO4 Na3VO3 VOSO4·5H2O NH4VO3 VOCl2

Vanadium 5.4-21.6 µM 19 µM 1-1000 µM 1-1000 µM 1-1000 µM not specified

V ∼ ∼ ∼ ∼ ∼

59 60 77 77 77 59

primary rat hepatocytes

InCl3

Indium 100-1000 µM

V

61

a

b

In vivo study with the dose expressed per killigram of body weight. Depending on the time frame of application.

used in the production of coins, jewelry, stainless steel, and nickel-cadmium batteries. The most important route of human exposure to nickel is inhalation. Besides pulmonary disorders, nickel also causes cardiovascular and renal diseases (4, 52). In line with its toxicity profile, nickel dichloride (IARC class 1) was demonstrated to inhibit GJIC in cultures of hamster lung fibroblasts (57). Similarly, nickel sulfate caused gap junction closure in cultured hamster embryo cells by a yet unknown molecular action (58). 3.7. Vanadium. Vanadium is a transition metal, existing in a number of valence states, with very little biological significance. Upon inhalation, vanadium compounds can cause severe damage to the respiratory tract, which might result into bronchitis and asthmatoid symptoms (2, 4). Not surprisingly, sodium orthovanadate inhibits GJIC in cultures of hamster lung fibroblasts (59). Most likely, this is an indirect consequence of its acknowledged inhibitory actions on phosphatases, which in turn may disrupt gap junction phosphorylation patterns and hence GJIC (60). 3.8. Indium. Indium is a rare metal that is used for the surface protection of metals and alloys. It has been shown that indium compounds, especially when inhaled, cause hemorrhagic lesions, and inflammatory and degenerative changes in the lung, kidney, heart, and liver in rodents (2, 61). Abolishment of GJIC might be related to indium toxicity, because indium trichloride

was found to directly inhibit gap junction activity in primary cultures or rat hepatocytes in a concentration-dependent manner (62).

4. Conclusions and Perspectives Human exposure to metals arises from both environmental and anthropogenic sources. Upon chronic contact or accumulation in the organism, metals, especially belonging to the nonessential group, cause a variety of deleterious effects. As a matter of fact, the majority of toxic metals have the ability to ultimately elicit carcinogenesis in animals and humans. Many metals indeed behave as genotoxicants in vitro by directly or indirectly interacting with DNA. They also induce carcinogenicity via a number of nongenotoxic pathways (63), among which the abolishment of cellular contacts is a prominent mechanism. Cadmium, for instance, is known to disrupt adherens junctions by displacing Ca2+ ions in its cadherin building stones (64-67). As addressed in this work, communicative cell junctions represent another class of molecular targets for toxic metals (Table 1). A variety of mechanisms hereby seem to underlie the abrogation of GJIC, implying both the most upper regulatory levels of connexin expression and the downstream platform of posttranslational control of gap junction functionality. In most cases discussed, the GJIC block occurs before the actual onset

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of the homeostatic imbalance (35, 38, 44), which could indicate that gap junction inhibition has to be considered as an early marker of metal-induced toxicity. It is, however, not always clear how disruption of gap junctions is connected to metalinduced pathology. Microarrays and other “-omics” techniques are useful tools in this respect (68). Thus, genome-wide analysis of kidneys of rats that have been treated with mercuric chloride identified lowered levels of Cx26 mRNA production as a potential biomarker for the prediction of its nephrotoxicity (69). Interestingly, a handful of reports have revealed that the adverse outcome of metals is not restricted to gap junctions as such but may also involve hemichannels. In this context, the essential metal zinc was reported to counteract the activity of hemichannels composed of either connexins (70-72) or pannexins (73) in different cellular in vitro systems. Similarly, cadmium dichloride triggers closure of hemichannels formed by Cx32 chimera in Xenopus laeVis oocytes (74). The contribution of hemichannels to the pathophysiological sequelae of metals has been further demonstrated in a recent study by Bhabra and colleagues (75). Using an elegant in vitro setting, it was shown that cobalt-chromium nanoparticles indirectly cause DNA damage in human fibroblasts by affecting the cellular traffic of ATP through gap junctions, connexons, and pannexin-based hemichannels. The research field of these hemichannels is, however, still in its infancy, mainly because of the ubiquitous lack of exploratory agents that allow unequivocal discrimination either from their full channel counterparts, as in the case of connexins, or from other channel types, especially holding true for pannexins. We can anticipate that more insight will be gained into the exact participation of each of these cellular signaling pathways in the toxicity caused by metallic ions upon introduction of appropriate experimental instruments. Acknowledgment. This work was supported by grants from the Research Council of the Vrije Universiteit Brussel (OZRVUB), the Fund for Scientific Research Flanders (FWOVlaanderen), and the European Union (FP6 Project carcinoGENOMICS). M.V. and T.V. are postdoctoral research fellows of the Fund for Scientific Research Flanders (FWO-Vlaanderen), Belgium. Supporting Information Available: Additional data. This material is available free of charge via the Internet at http:// pubs.acs.org.

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