Epigenetic regulation of gap junctional intercellular communication: More than a way to keep cells quiet?

Biochimica et Biophysica Acta 1795 (2009) 53–61

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a c a n

Review

Epigenetic regulation of gap junctional intercellular communication: More than a way to keep cells quiet? Mathieu Vinken a,⁎,1, Evelien De Rop a, Elke Decrock b,2, Elke De Vuyst b, Luc Leybaert b, Tamara Vanhaecke a,1, Vera Rogiers a a b

Department of Toxicology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium Department of Physiology and Pathophysiology, Faculty of Medicine and Health Sciences, Ghent University, De Pintelaan 185, B-9000 Ghent, Belgium

a r t i c l e

i n f o

Article history: Received 15 May 2008 Received in revised form 14 August 2008 Accepted 18 August 2008 Available online 29 August 2008 Keywords: Gap junction Connexin Histone acetylation DNA methylation MicroRNA

a b s t r a c t The establishment of gap junctional intercellular communication is a prerequisite for appropriate control of tissue homeostasis. Gap junctions consist of connexin proteins, whereby a myriad of factors govern the connexin life cycle. At the transcriptional level, most attention has yet been paid to the classical cis/trans machinery (i.e. the interaction between transcription factors and regulatory elements in connexin gene promoter regions) as a gatekeeper of connexin expression. In the last few years, it has become clear that epigenetic processes are also essentially involved in connexin gene transcription. Major determinants of the epigenome include histone modifications and DNA methylation, and recently, microRNA species have also been described as key regulators of the epigenetic machinery. In the present paper, the emerging roles of epigenetic events in the control of connexin expression, and consequently of gap junctional intercellular communication, are reviewed. Besides an updated theoretical background concerning gap junctions and epigenetic phenomena, we provide an in-depth overview of their interrelationship and we demonstrate the clinical relevance of the topic. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . Gap junctions: key players in homeostatic control . . . . 2.1. Structure . . . . . . . . . . . . . . . . . . . . 2.2. Function . . . . . . . . . . . . . . . . . . . . 2.3. Regulation . . . . . . . . . . . . . . . . . . . Epigenetics: key mechanisms in transcriptional control . . 3.1. Introduction. . . . . . . . . . . . . . . . . . . 3.2. Histone modifications . . . . . . . . . . . . . . 3.3. DNA methylation . . . . . . . . . . . . . . . . 3.4. MiRNA-related control of the epigenetic machinery Epigenetic mechanisms in charge of connexin expression . 4.1. Histone modifications . . . . . . . . . . . . . . 4.2. DNA methylation . . . . . . . . . . . . . . . . 4.3. MiRNA-related control of connexin expression . .

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Abbreviations: 4-Me2N-BAVAH, 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxamide; 4-PB, 4-phenylbutyrate; AP1, activator protein 1; ATP, adenosine trisphosphate; BRMS1, breast cancer metastasis suppressor 1; cAMP, cyclic adenosine monophosphate; CBP, CREB-binding protein; CL, cytoplasmic loop; CpG, cytosine–guanine; CT, cytoplasmic carboxy tail; Cx, connexin; DNMT, DNA methyltransferase; DNMTi, DNA methyltransferase inhibitor(s); EL1–2, extracellular loop 1–2; ERK1/2, extracellular signal-regulated kinase 1/2; GJIC, gap junctional intercellular communication; HAT, histone acetyltransferase; HDAC(s), histone deacetylase(s); HDACi, histone deacetylase inhibitor(s); HMBA, hexamethylene bisacetamide; IP3, inositol trisphosphate; MAPK, mitogen-activated protein kinase; MBP, methylated DNA-binding protein(s); MeCP2, methyl-CpG-binding protein 2; miRNA(s), microRNA(s); mRNA(s), messenger RNA(s); NaB, sodium butyrate; NAD+, nicotinamide adenine dinucleotide; ncRNA(s), non-coding RNA(s); NT, cytoplasmic amino tail; PKA, protein kinase A; PKC, protein kinase C; pre-miRNA (s), precursor microRNA(s); pri-miRNA, primary microRNA; REST, RE-1 silencing transcription factor; RISC, RNA-induced silencing complex; SAHA, suberoylanilide hydroxamic acid; siRNA, small interfering RNA; Sp1, specificity protein 1; TM1–4, membrane-spanning domain 1–4; TSA, Trichostatin A; UTR(s), untranslated region(s) ⁎ Corresponding author. E-mail address: [email protected] (M. Vinken). 1 M.V. and T.V. are postdoctoral research fellows of the Fund for Scientific Research Flanders (FWO-Vlaanderen), Belgium. 2 E.D. is a doctoral research fellow of the Fund for Scientific Research Flanders (FWO-Vlaanderen), Belgium. 0304-419X/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.bbcan.2008.08.002

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5. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

1. Introduction The maintenance of tissue homeostasis relies on the wellorchestrated interplay between extracellular, intracellular and intercellular communication networks. Direct communication between adjacent cells mainly occurs through vast arrays of intercellular channels, called “gap junctions”, which are formed by members of the connexin family [1–4]. Numerous factors are known to drive physiological gap junction production and activity. Connexin gene transcription is ruled by specific sets of transcription factors [5]. Structural chromatin modifications are also known to alter transcriptional activity. Such epigenetic mechanisms have gained particular attention in a clinical context. Indeed, a major hallmark of cancer includes the drastically altered gene expression patterns, whereby oncogenes and tumor suppressor genes are typically induced and inactivated, respectively. Epigenetic processes play pivotal roles in the silencing of tumor suppressor genes [6,7]. The anticipated anti-tumor properties of connexin genes suggest that they may also be subject to such regulation [8,9]. In the current paper, we provide a state of the art picture of the current knowledge concerning the involvement of epigenetic mechanisms in connexin expression and function. 2. Gap junctions: key players in homeostatic control 2.1. Structure Gap junctions, formerly known as nexus or maculae communicantes, are organized in plaques at the cell membrane surface and arise from the interaction of 2 connexons (hemichannels) from neighboring cells. Connexons, in turn, are built up by 6 connexin (Cx) proteins (Fig. 1). More than 20 mammalian connexin paralogues have been characterized, and they are named after their molecular weight [2–4,10,11]. They are expressed in a cell-specific way, with Cx43 being the most abundant connexin species [12]. Connexins share a common structure, consisting of 1 cytoplasmic carboxy tail (CT), 4 membrane-spanning domains (TM), 2 extracellular loops (EL), 1 cytoplasmic loop (CL) and 1 cytoplasmic amino tail (NT) (Fig. 1) [2–4,10,11]. Variety between connexins is mainly due to structural differences within the cytoplasmic areas [13]. Diversity also exists at the level of connexons, which can be composed of either 6 identical connexin subunits (“homomeric” connexons) or more than 1 connexin species (“heteromeric” connexons). This “connexin code” becomes even more complicated when considering gap junction architecture, as these channels consist of 2 heteromeric connexons (“heteromeric” gap junctions), 2 similar homomeric connexons (“homotypic” gap junctions), or 2 different homomeric connexons (“heterotypic” gap junctions) [14–16]. 2.2. Function Gap junctions provide a pathway for communication between adjacent cells, about 180 Å in length and 15 Å in diameter [17]. The flux of molecules through gap junctions, called “gap junctional intercellular communication” (GJIC), includes the passive flux of small and hydrophilic substances, such as cyclic adenosine monophosphate (cAMP), inositol trisphosphate (IP3) and ions (Ca2+) [18]. Although this seems a rather general route for exchange of essential metabolites between cells, GJIC is quite specific and the biophysical properties of a given gap junction type are determined by the nature of the composing connexin species (i.e. the connexin code) [14,18]. By

doing so, GJIC is considered as a key mechanism in the control of tissue homeostasis. Numerous studies have actually demonstrated determinant roles of gap junctions in the occurrence of cell proliferation, cellular differentiation and apoptotic cell death [2,4,9,19–21]. Recent findings also point to GJIC-independent functions of connexins and connexons in these processes. Indeed, connexins as such can trigger gene expression, whereas gap junction hemichannels provide a pathway for the extracellular release of essential homeostasis regulators, like adenosine trisphosphate (ATP) [2,11,22–24]. Not surprisingly, connexins and their channels are frequently impaired upon disruption of the homeostatic balance. During cancer, for instance, physiological connexin expression patterns are abrogated, which typically burgeons into the drastic loss of GJIC [8–10]. 2.3. Regulation A plethora of regulatory mechanisms govern the connexin life cycle and GJIC. Short-term GJIC control, so-called “gating”, is mediated by a number of factors, including transmembrane voltage, and Ca2+ ions and H+ ions [14]. Among all gating mechanisms, phosphorylation has been most extensively studied. With the exception of Cx26, all connexins are phosphoproteins. Regulation of GJIC by connexin phosphorylation is quite complex, as the outcome of this posttranslational modification is both connexin-inherent and kinase-specific [25,26]. This is particularly true for Cx43, which can be phosphorylated by protein kinase A (PKA), protein kinase C (PKC) and members of the mitogen-activated protein kinase (MAPK) family, to name a few [26,27]. Long-term control of GJIC mainly concerns regulation at the transcriptional level of connexin expression. The structure of connexin genes is rather simple, namely a first exon containing the 5′untranslated region (UTR), which is separated by an intron of varying length from a second exon, bearing the complete coding sequence, and the 3′-UTR [5,12,28]. Yet, 2 major exceptions to this common gene structure have been reported. First, the coding region can be interrupted by introns, such as in the case of Cx36 and Cx57. Second, different 5′-UTRs can be spliced in a consecutive and/or alternate manner (e.g. Cx32) [5,12,28,29]. Connexin gene promoters contain several binding sites for both ubiquitous transcription factors, like activator protein 1 (AP1) and specificity protein 1 (Sp1), and tissuespecific transcription factors [5]. In addition, epigenetic mechanisms are also essentially involved in connexin gene transcription, as will be outlined in the following sections. 3. Epigenetics: key mechanisms in transcriptional control 3.1. Introduction A central dogma in molecular biology is that gene expression is governed by transcription factors which interact with specific DNA sequences, a process known as cis/trans regulation. In the last decades, however, it has become more than clear that other processes are also essentially involved in transcriptional control. These mechanisms are commonly referred to as epigenetic events and can be defined as mitotically and meiotically heritable changes in gene expression that are not coded in the DNA sequence itself [30]. Thus far, 2 major epigenetic regulatory mechanisms have been characterized, namely histone modifications and DNA methylation. Recently, microRNA (miRNA) species have been described as crucial regulators of epigenetic events [30–39]. For this reason, miRNAs are considered

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histones (H2A, H2B, H3, and H4) around which approximately 147 base pairs of DNA are wrapped [35,39–41]. The N-terminal tails of the core histones, protruding from the nucleosomal surface, contain a number of conserved amino acid residues, including lysine, arginine and serine. They interact with the poly-anionic backbone of the DNA, resulting in a compact chromatin structure [32,38,40–42]. Histone tails, particulary those of histones H3 and H4, are subject to a vast array of covalent modifications, including methylation, acetylation, phosphorylation, ubiquitination, sumoylation, biotinylation, ADP ribosylation, deimination and proline isomerization [38,40]. Most of the posttranslational histone modifications are dynamic and their interplay is believed to formulate a complex code that regulates gene transcription, mitotic condensation of chromatin and DNA repair [31,38–40]. Acetylation is among the best understood components of the “histone code”. The primary sites of histone acetylation are ɛamino groups in the positively charged lysine residues. A number of histone acetyltransferase (HAT) complexes mediate the addition of an acetyl group from acetyl-coenzyme A, resulting in the neutralization of the positive charge and thus the loosening of histone–DNA contacts [43]. This process, in turn, promotes decondensation of the chromatin, thereby facilitating the accessibility of the transcriptional machinery to the DNA. The inverse reaction is catalyzed by histone deacetylase (HDAC) enzymes and is frequently associated with transcriptional repression [38–40,43]. At present, 18 mammalian HDACs have been identified, which have been assigned to 3 classes according to their homology to yeast HDACs, type of catalytic site and cellular localization. An exception is made for HDAC11, which displays too low sequence similarity to be grouped into these subfamilies [44]. Class I (yeast Rpd3-like) HDACs (HDAC1-3, 8) are found in the nucleus of most cell types and participate in the deacetylation of histones and non-histone proteins [32,45]. Class II (yeast HDA1-like) HDACs (HDAC4–7, 9, 10) shuttle between the nuclear and the cytoplasmic compartments and serve many functions, whether or not in relation to histones [45]. Class III (Sir2-like) HDACs or sirtuins (SIRT1–7) are present at several cellular localizations, including the nucleus, and are involved in chromatin silencing, cellular metabolism and ageing. In contrast to their class I and II counterparts, sirtuins require nicotinamide adenine dinucleotide (NAD+) for their catalytic activity and are not sensitive to typical HDAC inhibitors (HDACi), like Trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) [43,46]. 3.3. DNA methylation

Fig. 1. Molecular architecture of gap junctions. Gap junctions are grouped in plaques at the membrane surface, and are composed of 12 connexin proteins, organized as 2 hexameric connexons (hemichannels). The connexin structure consists of 4 membranespanning domains (TM1–4), 2 extracellular loops (EL1–2), 1 cytoplasmic loop (CL), 1 cytoplasmic amino tail (NT) and 1 cytoplasmic carboxy tail (CT).

as parts of the epigenetic machinery, and will therefore be described together with the classical epigenetic mechanisms in the following sections. 3.2. Histone modifications The nucleosome constitutes the basic structural unit of the eukaryotic genome and is composed of an octamer of pairs of 4 core

The target molecules for methylation in the DNA are cytosine bases in cytosine–guanine (CpG) dinucleotides. In the vast majority of the genome, CpG dinucleotides can be found in a ratio of 1 per 80 base pairs, and up to 70% of these sites are methylated. Higher frequencies of CpG dinucleotides are found in so-called “CpG islands” that comprise 1–2% of the genome. CpG islands are typically present in gene promoter regions and are usually in an unmethylated status [47–50]. DNA methylation includes the addition a methyl group from S-adenosyl methionine to the carbon-5 position of cytosine and is controlled by DNA methyltransferase (DNMT) enzymes. De novo methylation of previously unmethylated CpG dinucleotides is preferentially performed by DNMT3a and DNMT3b, whereas DNMT1 is crucial for maintaining established methylation patterns upon cell division [39,51]. As such, DNA methylation plays a key role in maintaining gene silencing that is necessary for tissue-specific and developmentspecific gene expression [32]. The negative correlation between the DNA methylation status and gene expression may be mediated by methylated DNA-binding proteins (MBP). Among the latter, methylCpG-binding protein 2 (MeCP2) is frequently concentrated at hypermethylated CpG dinucleotides, where it interacts with HDAC enzymes that trigger chromatin condensation and gene silencing [39,52]. However, MeCP2 binding not always correlates with gene promoter

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and Argonaute proteins was also reported in human cells [64]. Furthermore, DNMTs as well as HDACs are direct targets for miRNAs in animal cells [65,66]. Vice versa, classical epigenetic mechanisms control miRNA expression in human cells, since many of them are upregulated by simultaneous treatment with decitabine and the HDACi 4-phenylbutyrate (4-PB) [36,67,68].

methylation, and might even be associated with induction of transcriptional activity [53]. This could also explain why only a subset of genes are synergistically activated by combining HDACi with typical DNMT inhibitors (DNMTi), such as decitabine [54–56]. 3.4. MiRNA-related control of the epigenetic machinery

4. Epigenetic mechanisms in charge of connexin expression

About 25% of the genome is attributed to a repository of regulatory elements, including non-coding RNA (ncRNA) genes [57]. Increasing evidence shows that ncRNAs, and more specifically miRNAs, are implicated in a variety of epigenetic mechanisms [31,58]. In the nucleus, miRNA genes are transcribed by RNA polymerase II, yielding long primary miRNA (pri-miRNA) transcripts, which are subsequently cleaved by the nuclear microprocessor complex to 70-nucleotide hairpins, known as precursor miRNAs (pre-miRNAs). The latter are transported to the cytoplasm by exportin-5, where they are processed by the endonuclease Dicer to 22-nucleotide duplexes of mature miRNA. These duplexes are loaded into the RNA-induced silencing complex (RISC), where the Argonaute protein Ago2 mediates elimination of one of the miRNA strands. The remaining strand guides RISC to target messenger RNAs (mRNAs) that have miRNA complementary sites in the 3′-UTR. RISC then suppresses translation, cleaves or degrades the mRNA, depending on the degree of mRNA–miRNA complementarity [34,36,59,60]. At least a thousand miRNAs have been predicted to operate in humans, and they regulate about 30% of all protein-coding genes [60]. Although their actions are primarily located at the posttranscriptional level, they also affect gene expression at the transcriptional stage, where they interfere with epigenetic mechanisms [61,62]. In plants, Dicer-associated Argonaute proteins were found to be involved in both DNA methylation and histone methylation [63]. A similar link between histone methylation

4.1. Histone modifications The majority of evidence that demonstrates the involvement of histone modifications, and histone acetylation in particular, in the control of connexin expression, comes from work with HDACi (Table 1). Several reports have described the inductive effects of these epigenetic modifiers on GJIC in a broad variety of experimental settings [69–78], and in most cases, this is directly linked to increased connexin production [69,71–76]. Their specific outcome, however, depends on the cellular context, the nature of the connexin species, as well as on the epigenetic agent in question. For instance, TSA induces Cx36 expression in mouse pancreatic cell lines but not in mouse fibroblasts, neuronal cells and pituitary cells [79]. On the other hand, TSA downregulates Cx43 production, but does not affect Cx26 and Cx32 in human liver cancer cells [80]. Very few studies have actually addressed the molecular mechanisms that underlie the effects of HDACi on connexin expression. Hypothetically, HDACi could affect connexin production indirectly, by altering the transcriptional activity of genes which in turn regulate connexin expression. Most evidence, however, shows that connexin genes as such are directly targeted by HDACi. Hernandez and group found that the TSA-mediated induction of Cx43 in human prostate cancer cells depends on the recruitment of

Table 1 Effects of histone deacetylase inhibitors on connexin expression Model Human neural progenitor cells NGC-407 HNSC.100 Human kB nasopharyngeal tumor cells Human kB nasopharyngeal tumor cells Human prostate carcinoma cells DU145 PC3 LNCaP Human glioblastoma cells LN18 U87 Human glioblastoma cells Human peritoneal mesothelial cells Human peritoneal mesothelial cells Human Huh7 liver cancer cells Human normal prostate epithelial cells Rat glioma cells C6 9L Rat C6 glioma cells Rat CC531 colon cancer cells Rat WB-F344 cells (ras-transformed) Rat primary hepatocytes Rat primary hepatocytes Mouse pancreas cells αTC1-p βTC3 Mouse AtT20 pituitary corticotrophic cells Mouse SN56 neuronal cells Mouse LMTK− fibroblasts Mouse embryonic stem cells

HDACi

Upregulation

4-PB TSA/4-PB 4-PB/NaB TSA

Cx432 Cx432 Cx432

TSA TSA TSA

Cx431,2 Cx431,2 Cx431,2

NaB NaB 4-PB HMBA SAHA TSA TSA NaB NaB 4-PB NaB SAHA TSA 4-Me2N-BAVAH TSA TSA TSA TSA TSA TSA

Downregulation

No effect

Reference

Cx432

[73] [73] [117] [117] [72] [72] [72]

Cx432 Cx432 2

Cx43 Cx431,2 Cx431,2 Cx431

Cx321/Cx261

Cx431,2 Cx432 Cx432

Cx262 Cx432/Cx262

[118] [118] [69] [119] [76] [77] [78]

Cx431

[79] [79] [79] [79] [79] [88]

Cx432 Cx431 Cx431,2 Cx432/Cx322 Cx322

[118] [118] [71] [74,75] [76] [80] [72]

Cx361 Cx361 Cx361 Cx361 Cx361

(1 = mRNA level; 2 = protein level; 4-Me2N-BAVAH, 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxamide; 4-PB, 4-phenylbutyrate; Cx, connexin; HDACi, histone deacetylase inhibitor; HMBA, hexamethylene bisacetamide; NaB, sodium butyrate; SAHA, suberoylanilide hydroxamic acid; TSA, Trichostatin A).

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p300/CREB-binding protein (CBP), a transcriptional coactivator displaying HAT activity, and the transcription factors AP1 and Sp1 to the Cx43 gene promoter. This was accompanied by hyperacetylation of histones H4 surrounding the AP1- and Sp1-responsive gene elements [72]. Similarly, SAHA caused accumulation of acetylated histones H3 and H4 in the Cx43 gene locus, which was associated with enhancement of Cx43 expression and GJIC between human peritoneal mesothelial cells [76]. Cx36 expression in pancreatic cells is controlled by the RE-1 silencing transcription (REST) factor, a transcriptional repressor that contains 2 independently acting HDAC-recruiting repression domains [81,82]. Active Cx36 production in these cells was evidenced by the presence of trimethylated lysine 4 residues in histone H4 near its gene promoter, an epigenetic marker of actively transcribed genes, and could be induced by TSA [79]. Little is known about the identity of the specific HDAC enzymes that are involved in connexin expression. Upon transfection, the breast cancer metastasis suppressor 1 (BRMS1) protein localizes in the cell nucleus, and restores GJIC in human breast cancer cells [83–85] and in melanoma cells [86]. In the former case, this coincides with elevated Cx43 mRNA levels and simultaneous decreased Cx32 gene transcription [83,85]. Later on, it was found that BRMS1 interacts with the large mSin3 HDAC complex, which contains both HDAC1 and HDAC2, but also forms smaller complexes with HDAC1 [87]. Specific deletion of HDAC1 as well as exposure to TSA decreased Cx43 mRNA levels in mouse embryonic stem cells. Loss of HDAC1 drastically increased trimethylation of lysine 9 residues in histone H3 surrounding the Cx43 gene promoter region, an epigenetic marker of silenced genes, and only slightly reduced histone H3 and H4 acetylation. Thus, the Cx43 gene requires both HDAC1 presence and activity for its transcription, but histones H3 and H4 are merely minor targets in this regulatory process [88]. Interestingly, HDACi can also elicit effects on connexin expression levels, other than the transcriptional one. Research from our laboratory showed that TSA and its structural analogue 5-(4-dimethylaminobenzoyl) aminovaleric acid hydroxamide (4-Me2N-BAVAH) both enhance GJIC between primary cultured hepatocytes, a finding which was associated with differential effects on the Cx26, Cx32 and Cx43 protein contents, but not with alterations in the corresponding mRNA amounts [77,78]. Furthermore, HDACi also interfere with posttranslational connexin control, as they both increase [71,74–76] and decrease [73] the abundance of phosphorylated Cx43 isoforms. Resveratrol, an activator of Sir2 orthologues, induces GJIC in human U-87MG glioblastoma cells [89] as well as between WB-F344 cells pretreated with GJIC-reducing compounds [90–92]. This occurred independently of changes in Cx43 mRNA levels [91] and protein content [89,91], but was allied with an altered Cx43 phosphorylation status [89–91]. Likewise, the HDACi sodium butyrate (NaB) prevents tumor promoter-mediated inhibition GJIC via extracellular signal-regulated kinase 1/2 (ERK1/2) inactivation, whilst TSA restored experimentally elicited GJIC reduction and Cx43 hyperphosphorylation by preventing p38 MAPK in WB-F344 cells [93]. 4.2. DNA methylation DNA methylation, like other epigenetic mechanisms, is frequently studied in a clinical context. Hypermethylation in the promoter region of (tumor suppressor) genes is typically linked to transcriptional silencing [39]. This also holds true for connexins, which have repeatedly been demonstrated to possess potent anti-tumor properties by inducing cell cycle arrests, differentiation and apoptosis in neoplastic cells [2,9,10]. In fact, abrogation of connexin expression (e.g. Cx26, Cx32 and Cx43) is associated with the accumulation of methylated CpG dinucleotides in the connexin gene promoter in a plethora of malignant cells, including cultured human lung cancer cells [94], primary human renal carcinoma cells [95,96], cultured human esophageal cancer cells [97], cultured human breast cancer cells [98], cultured human nasopharyngeal cancer cells [99], cultured [100] and primary [101,102] rat liver cancer cells, and primary rat lung

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cancer cells [103]. Shimizu and group showed that disturbances in the DNA methylation patterns in the Cx26 gene are particularly observed in the early phase of hepatocarcinogenesis, induced by a cholinedeficient L-amino acid defined diet in rats, and correlates with elevated DNMT1 mRNA levels [101]. Inhibition of DNMT enzymes by decitabine indeed counteracts repression of connexin gene transcription [94,95,97,99,100,104–106] and triggers GJIC [95,99] in a variety of cancer cell lines, although this occurs in a cell type-dependent and connexin-specific fashion (Table 2) [95,97,100]. The functional link between DNA hypermethylation and impairment of connexin production is, however, not entirely clear. Aberrant binding of transcription factors to the methylated connexin gene promoter regions may be responsible for the poor connexin expression in cancer cells. In this respect, Chen and co-workers found that the decreased Cx43 gene transcription in primary human non-small cell lung cancer cells is accompanied by DNA methylation and is simultaneously related to reduced binding of AP1 to the Cx43 gene promoter [107]. Furthermore, methylated CpG dinucleotides are preferentially located in the Sp1 binding sites of the Cx26 gene promoter and the Cx32 gene promoter in cultured and primary human breast cancer cells [106], and cultured rat liver cancer cells [100], respectively. 4.3. MiRNA-related control of connexin expression MiRNAs have started to enter the connexin arena only very recently. Thus far, they have only been found to control connexin production at the posttranscriptional level, and therefore, they cannot yet be considered as true epigenetic regulators of connexin

Table 2 Effects of DNA methyltransferase inhibitors on connexin expression Model

DNMTi

Human lung carcinoma cells H2170 decitabine H226 decitabine Human Caki-2 renal decitabine carcinoma cells Human Caki-1 renal decitabine carcinoma cells Human HK-2 renal tubular decitabine cells Human esophageal cancer cells TE-1 decitabine TE-2 decitabine TE-3 decitabine TE-8 decitabine TE-9 decitabine Human MDA-MB-453 decitabine breast cancer cells Human breast cancer cells MCF-7 decitabine MCF-10 decitabine MDA-MB231 decitabine BT-20 decitabine T47-D decitabine Human cervical decitabine adenocarcinoma cells decitabine Human CNE-1 nasopharyngeal cancer cells Rat WB-F344 liver decitabine epithelial cells Mouse pancreas cells αTC1-p 5-azacytidine βTC3 5-azacytidine Mouse AtT20 pituitary 5-azacytidine corticotrophic cells Mouse SN56 neuronal cells 5-azacytidine Mouse LMTK− fibroblasts 5-azacytidine

Upregulation

No effect

Cx261 Cx261 Cx321,2

Reference [94] [94] [95]

Cx321

[104] 1,2

Cx32

[95]

Cx261/Cx431 Cx261/Cx431 Cx261/Cx431 Cx261/Cx431 Cx261

[97] [97] [97] [97] [97] [106]

Cx261 Cx261 Cx261 Cx261 Cx261 Cx432

[98] [98] [98] [98] [98] [105]

Cx431,2

[99]

Cx431 Cx261

Cx431

Cx321

[100]

Cx361 Cx361 Cx361

[79] [79] [79]

Cx361 Cx361

[79] [79]

(1 = mRNA level; 2 = protein level; Cx, connexin; DNMTi, DNA methyltransferase inhibitor).

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expression. Kim and group showed that the muscle-specific miRNAs miR-206, miR-1 and miR-133 are induced during in vitro differentiation of C2C12 myoblast cells into multinucleate myotubes. During this process, Cx43 mRNA levels decline, whereby Cx43 is a direct negative target for miR-206 [108]. It was further shown that regulation of Cx43 by miR-206 and miR-1 occurs through direct binding to the 3′-UTR of the Cx43 mRNA transcript [109,110]. The biological relevance of Cx43 downregulation during myogenesis is not fully understood. It is thought that, despite the requirement for Cx43 during the initial phases of myogenesis, the subsequent downregulation of Cx43depending GJIC is important in generating insulated muscle fibers that are singly innervated for fine motor control [109]. 5. Conclusions and perspectives Virtually all aspects of the cellular life cycle are regulated by chemical signals that are intercellularly exchanged via gap junctions. Hence, GJIC is considered as a central control platform in the maintenance of the homeostatic balance [2,4,9,19–21]. A strict and well-coordinated regulation is compulsory for appropriate GJIC functioning. Management of GJIC over the long term mainly occurs at the level of connexin expression. Many efforts have yet been focused on the elucidation of the cis/trans machinery that drives connexin gene transcription [5]. Increasing evidence also points to the critical involvement of epigenetic phenomena in this process (Fig. 2). Classical epigenetic mechanisms like histone acetylation and DNA methylation indeed are essential determinants of connexin expression [3,5]. Recently, miRNA-associated mechanisms have been implicated in gap junction biogenesis, by affecting connexin production at the

posttranscriptional level [108–110]. In addition to their emerging roles in mediating posttranscriptional gene silencing, miRNAs act in concert with histone modifications and DNA methylation at the transcriptional stage [36,63,64,67,68]. It remains to be established whether miRNAs also interfere with these classical epigenetic mechanisms in the regulation of connexin expression. Histone modifications and DNA methylation are also directly connected with less acknowledged components of the epigenetic circuit, including ATP-dependent chromatin remodelling systems [6,39]. In this respect, a major challenge lies ahead in deciphering the global epigenetic codes that determine connexin expression. A number of innovative tools are now available that will allow clarification of the epigenetic crosstalk in connexin control, including isotype-specific epigenetic modifiers [111], small interfering RNA (siRNA) duplexes [112], and miRNA inhibitors (“antagomirs”) [113,114]. The knowledge that will be gained from these experiments will shed more light on the biological importance of epigenetic actions during the establishment of tissuespecific and development-specific connexin expression patterns. In the last few years, a new type of gap junction-forming proteins has been characterized, the “pannexins”, which also display patterned distribution [11]. It is tempting to speculate that epigenetic phenomena also contribute to this process. Although it seems obvious that epigenetic mechanisms are master regulators of physiological gene expression programs, they have been mainly studied in pathophysiological conditions [6,7,37]. Indeed, impairment of the epigenetic machinery during cancer triggers the silencing of tumor suppressor genes, including connexins [8,9]. Upregulation of connexin expression may therefore represent an attractive anti-cancer therapy [4,9]. Epigenetic modulators of gene

Fig. 2. Epigenetic mechanisms involved in connexin expression. At least 2 epigenetic mechanisms control connexin expression, namely histone acetylation and DNA methylation. MiRNAs were found to direct connexin production at the posttranscriptional level. Since miRNAs interfere with classical epigenetic mechanisms, they are considered as parts of the epigenetic machinery. Their clear involvement in the epigenetic control of connexin expression, however, yet remains to be demonstrated. (Ac, acetyl group; CH3, methyl group; mRNA, messenger RNA; miRNA, microRNA; RISC, RNA-induced silencing complex).

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expression seem very promising in this context. On many occasions, prototypical HDACi and DNMTi have been shown to strongly induce connexin expression in tumor cells (Tables 1 and 2). It should be stressed, however, that the therapeutic use of most of these agents is rather limited due to various reasons, including low potency, metabolic instability and the occurrence of toxic side effects. The design of specific epigenetic modifiers with more favourable pharmaco-toxicological profiles has therefore become a central research topic in many pharmaceutical companies worldwide [115,116]. Furthermore, epigenetic therapy can be implemented into other anti-cancer approaches. The HDACi 4-PB, for instance, was found to greatly enhance the efficiency of the herpes simplex virus thymidine kinase/ganciclovir therapy by inducing Cx43 expression [69]. It is expected that further exploration of such “epigenetic connexin therapies” will open new perspectives for the future clinical management of cancer. Acknowledgements This work was supported by the grants from the Fund of Scientific Research Flanders (FWO-Vlaanderen), Belgium, and the Research Council of the Vrije Universiteit Brussel (OZR-VUB). Special thanks go the European FP6 projects carcinoGENOMICS (PL-037712) and LIINTOP (STREP-037499).

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