Inhibition of Gap Junctional Intercellular Communication byNoncoplanar Polychlorinated Biphenyls: Inhibitory Potencies and
Screening for Potential Mode(s) of Action
Miroslav Machala,*,1 Ludek Blaha,*,† Jan Vondracek,*,‡ James E. Trosko,§ Jacob Scott,§ and Brad L. Upham§
*Veterinary Research Institute, Department of Chemistry and Toxicology, Hudcova 70, 62132 Brno, Czech Republic; †RECETOX, Masaryk University,Brno, Czech Republic; ‡Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic; and §Department of Pediatrics &
Human Development, and the National Food Safety & Toxicology Center, Michigan State University, East Lansing, Michigan 48824
Received June 2, 2003; accepted July 25, 2003
Polychlorinated biphenyls (PCBs), a structurally diverse groupof environmental pollutants, are effective promoters in two-stagecancer models, which implies that epigenetic mechanisms areinvolved. Inhibition of gap junctional intercellular communication(GJIC) belongs among critical epigenetic events of tumor promo-tion. We determined the relative potencies of a series of environ-mentally relevant PCB congeners to inhibit GJIC in vitro in a ratliver epithelial cell line with pluripotent oval cell characteristics.The nonplanar PCBs were potent inhibitors of GJIC, whereas thecoplanar PCBs did not inhibit GJIC. We then compared theeffects of the coplanar PCB 126 (3,3�,4,4�,5-pentachlorobiphenyl)and the noncoplanar PCB 153 (2,2�,4,4�,5,5�-hexachlorobiphenyl)with effects of two model GJIC inhibitors, a tumor promoter12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermalgrowth factor (EGF). In contrast to TPA or EGF, PCB 153 eliciteda long-term downregulation of GJIC (up to 48 h). Using Westernblot analysis with phospho-specific antibodies, it was found thatPCB 153, and not PCB 126, activated mitogen-activated proteinkinases ERK1/2; however in contrast to TPA and EGF, thisactivation was observed at the time points subsequent to GJICinhibition. Moreover, blocking of ERK1/2 activation did not pre-vent the GJIC inhibition induced by PCB 153. Therefore, addi-tional intracellular signaling pathways potentially involved in thedownregulation of GJIC by PCBs were screened by using specificchemical probes inhibiting serine/threonine kinases, tyrosine ki-nases, and phospholipases. The inhibition of diacylglycerol lipasepartially blocked and the selective inhibition of Src kinases andphosphatidylcholine-specific phospholipase C (PC-PLC) com-pletely blocked the inhibitory effects of the noncoplanar PCB onGJIC, indicating that PC-PLC or sphingomyelinase and Src mightbe upstream regulators of noncoplanar PCB-induced inhibition ofGJIC.
Polychlorinated biphenyls (PCBs) are a group of structurallydiverse and persistent environmental pollutants, widely distrib-uted as complex mixtures. Non-ortho-substituted coplanarPCBs have been shown to elicit a set of adverse effectsassociated with the activation of the aryl hydrocarbon receptor(AhR), resulting in liver damage, thymus atrophy, skin lesions,a wasting syndrome, and tumor promotion (van den Berg et al.,1998). Di-ortho-substituted PCBs, which tend to be noncopla-nar structures of the biphenyl molecule that does not signifi-cantly activate AhR, exhibit a different spectrum of toxicmodes of action, such as modulation of steroid hormone andCa2�-induced intracellular signaling, and have been linked toneurotoxicity, immunotoxicity, endocrine disruption, and tu-mor promotion (Brouwer et al., 1999; Dean et al., 2002;Hansen, 1998; Robertson and Hansen, 2001). Such toxic ef-fects suggest interactions with different cellular componentsother than AhR; however, exact modes of action of noncopla-nar PCBs still remain unclear. Mechanistic studies are ex-tremely important because of an urgent need to estimate con-tributions of noncoplanar congeners to overall PCB toxicity(Hansen, 1998). In the currently accepted toxic equivalencyfactors approach, which is based exclusively on AhR-mediatedtoxicity data, the more prevalent di-ortho-substituted PCBshave zero toxic potencies (van den Berg et al., 1998), despitetheir known toxicity.
Widespread distribution and persistence of PCBs led tostudies on their potential role in carcinogenesis (Silberhorn etal., 1990). Evidence that exposure to PCBs, as well as poly-brominated biphenyls (PBBs), leads to mutations has beenambiguous and controversial (Stapleton et al., 2001; Tsushi-moto et al., 1983), but numerous studies indicate that PCBsand PBBs act as tumor promoters (Jensen et al., 1982; Safe,1989; Silberhorn et al., 1990; van der Plas et al., 2000). Theseresults imply that nongenotoxic (epigenetic) mechanisms areinvolved in their carcinogenic potential. Although coplanarPCBs are supposed to play an important role in tumor-promot-ing effects of PCB mixtures, such as Aroclor 1260, throughAhR activation, the noncoplanar fraction of this mixture alsocontributed significantly to its tumor-promotion potential (van
1 To whom correspondence should be addressed at Dept. Chemistry andToxicology, Veterinary Research Institute, Hudcova 70, 62132 Brno, CzechRepublic. Fax: �420-541321241. E-mail: [email protected].
der Plas et al., 2000). Therefore, understanding the underlyingmechanisms of the nongenotoxic tumorigenic potential of thenoncoplanar PCBs is essential in establishing a more accurateassessment of risk that environmentally relevant PCBs pose tohuman health.
Three critical epigenetic events are needed at the promo-tional stages of cancer: one being the removal of an initiatedcell from the suppression of growth by neighboring cellsthrough the intercellular transfer of signal transductants via gapjunctions; the second being the activation of a mitogenic path-way such as the extracellular receptor kinase (ERK) class ofmitogen-activated protein kinases (MAPK); and the third is theinhibition of apoptosis. The downregulation of gap junctionalintercellular communication (GJIC) by tumor-promoting com-pounds is considered to be a critical step in the removal of aninitiated cell from the growth suppression of neighboring cells(Ruch and Trosko, 2001; Yamasaki, 1996). Therefore, theinhibition of GJIC can be assumed to be a representativemarker of tumor-promoting potency for a given compound(Rosenkrantz et al., 2000). PBBs were the first polyhaloge-nated biphenyls shown to inhibit GJIC (Trosko et al., 1981),and a subsequent study demonstrated that the chlorinated iso-mers of the biphenyls inhibited GJIC in a manner similar totheir brominated analogues (Tsushimoto et al., 1983). Numer-ous studies have since demonstrated that several PCBs caninhibit GJIC, both in vivo (Bager et al., 1997; Dean et al.,2002; Haag-Gronlund et al., 1998; van der Plas et al., 2000)and in vitro in rat liver epithelial cells, mouse and rat hepato-cytes, human keratinocytes, and normal human breast epithe-lial cells (Hemming et al., 1991; Kang et al., 1996; Ruch andKlaunig, 1986; Swierenga et al., 1990). Although relativepotencies of a number of PCBs to inhibit GJIC have beenreported (Hemming et al., 1991), there are still many PCBcongeners occurring in the environment, where this importantinformation is missing.
The mechanism by which PCBs inhibit GJIC is not known.In general, the intracellular signals involved in acute inhibitionof GJIC are still poorly understood; only the mechanisms ofprototypical GJIC inhibitors, such as 12-O-tetradecanoylphor-bol-13-acetate (TPA) or epidermal growth factor (EGF), havebeen studied in some detail (Lau et al., 1992; Matesic et al.,1994; Rivedal and Opsahl, 2001; Warn-Cramer et al., 1998).EGF-induced inhibition of GJIC has been associated with theactivation of the membrane-bound EGF receptor (EGFR)–Ras–Raf–MEK–ERK1/2 signal transduction cascade, andTPA-induced inhibition of GJIC has been associated with theactivation of protein kinase C (PKC) followed by the conse-quent activation of ERK1/2. Pretreatment of cells with specificMAPK or PKC inhibitors has been demonstrated to preventEGF or TPA-induced inhibition of GJIC (Husoy et al., 2001;Rivedal and Opsahl, 2001; Ruch et al., 2001). While theinhibition of GJIC by either TPA or EGF are MEK-dependent,the activation of MEK and ERK alone is insufficient for theinhibition of GJIC (Hossain et al., 1999; Rummel et al., 1999).Furthermore, not all compounds, such as polycyclic aromatic
hydrocarbons, inhibit GJIC through MEK-dependent path-ways, even though these compounds can activate MAPK(Rummel et al., 1999). Besides protein kinase activation, phos-pholipase C (PLC) (Upham et al., 2003) and phospholipase A2(PLA2) (ref. Wang and Loch-Caruso, 2002) have been impli-cated in the mechanism of GJIC inhibition. Involvement ofthese signaling pathways in PCB-induced inhibition of GJIChas yet to be determined.
The aims of the present study were to determine relativepotencies of a series of environmentally relevant PCB conge-ners to inhibit GJIC in vitro in a rat liver epithelial cell linewith pluripotent oval cell characteristics. We then comparedthe effects of the coplanar PCB 126 (3,3�,4,4�,5-pentachloro-biphenyl) and the noncoplanar PCB 153 (2,2�,4,4�,5,5�-hexa-chlorobiphenyl) on the phosphorylation of ERK1/2. Finally,we used specific chemical probes that inhibit protein kinasesand phospholipases to screen the intracellular signaling path-ways potentially involved in the downregulation of GJIC bynoncoplanar PCBs and compared these results with effects ofprototypical GJIC inhibitors.
MATERIALS AND METHODS
Chemicals. PCB congeners were purchased from Promochem, with ex-ception of PCB18, which was a generous gift from Dr. Hansen (University ofIllinois, Urbana, IL), and PCBs 70 and 74 obtained from Dr. Lehmler (Uni-versity of Iowa, Iowa City, IA). Chemical inhibitors U0126, H-89, LY294002,AG 825, AG 879, D609, ET-18-0CH3, MJ-33, and MAFP were from Calbio-chem (San Diego, CA); SB203,580, PP2, and AACOCF3 from Alexis (Carls-bad, CA); forskolin, RHC 80267, and BEL (bromoethanol lactone) fromBiomol (Hamburg, Germany). TPA, EGF, lucifer yellow, dimethylsulfoxide(DMSO), formaldehyde, GF109203X (bis-indoleylmaleimide), and otherchemical modulators of cell signaling were supplied by Sigma-Aldrich(Prague, Czech Republic). All chemicals used in the study were of the highestavailable purity.
Cell culture and treatment with chemicals. WB-F344 rat liver epithelialcells (Tsao et al., 1984) were cultured in modified Eagle’s Minimum EssentialMedium (Sigma-Aldrich, Prague, Czech Republic), supplemented with pyru-vate (110 mg/l), 10 mM HEPES, and 5% fetal bovine serum (Sigma-Aldrich,Prague, Czech Republic). Confluent cells, grown in 24-well plates, wereexposed to various concentrations of individual PCB congeners (up to 100�M) or solvent (DMSO, 0.1%, v/v) for 30 min. For the study of acute andsustained downregulation of GJIC, prolonged 24- or 48-h exposure to TPA,coplanar PCB 126, or noncoplanar di-ortho-chlorinated PCB 153 were used.
Pretreatment with modulators of intracellular signal transduction path-ways. The model tumor promoter, TPA, and endogenous peptide, EGF, wereused as prototypical inhibitors of GJIC. Modes of action of GJIC inhibitorswere studied in the cells pretreated for 20 min (if not indicated otherwise) withselective inhibitors or activators of intracellular protein kinases or phospho-lipases followed by the 30-min exposure to a compound under study (PCB 153,TPA, or EGF). Tyrosine kinases were inhibited by 30-min pretreatment with50 �M genistein, 10 �M AG1478, 50 �M AG829, 5 �M AG879, or 50 �MPP2, selective inhibitors of EGF receptor (EGFR), Neu/ErbB-2, nerve growthfactor receptor, and Src kinases, respectively. Inhibition of MEK1/2, p38MAPK, and phosphoinositol triphosphate kinase (PI3 K) were performed by a30-minute preincubation with 20 �M U0126, 10 �M SB203,580, and 25 �MLY294002, respectively. Activation of PKCs was blocked by 30-min pretreat-ment with 5 �M GF109203X, a general inhibitor of PKCs, or by a 24-hpretreatment with 20 nM TPA, inducing PKC depletion. Protein kinase A(PKA) was inhibited by treating the WB cells with 10 �M H-89 and activatedby 5 mM 8-bromo-cAMP or 40 �M forskolin (30-min pretreatment). Several
PLA2 and PLC inhibitors were used to test the involvement of phospho-lipase(s) in inhibition of GJIC after exposure to PCB153. Methyl arachido-nyl fluorophosphonate (MAFP) and arachidonyl trifluoromethylketone(AACOCF3), inhibitors of IV4A-PLA2 and VIA-PLA2, were used in 1.25 and10 �M concentrations, respectively; 2.5 �M bromoenol lactone (BEL), a specificinhibitor of VI-PLA2, 10 �M MJ33 inhibiting preferentially acidic iPLA2, 5�M p-bromophenacyl bromide (BrPhBr), which selectively inhibits secretoryPLA2 isoenzymes, 2.5 �M U73122 and 40 �M ET-18-OCH3, two inhibitorsof phosphatidylinositol-specific PLC (PI-PLC), 40 �M xanthogenate tricyclo-decan-9-yl (D609), which blocks phosphatidylcholine-specific PLC (PC-PLC)and sphingomyelinase (SMase) activities, and 20 �M RHC 80267, an inhibitorof diacylglycerol lipase, were used to selectively inhibit major known intra-cellular protein kinase and phospholipase signal transduction segments.
Inhibitor concentrations were selected according to previously publisheddata on the use of specific inhibitors in various in vitro systems, including ratliver oval cells. Several inhibitors, namely PP2, genistein, and H-89, weretested at least at three different concentrations. The chemical inhibitors them-selves did not affect GJIC, with exception of AG879, U73122, BrPhBr, BEL,MJ33, and MAFP, which reduced GJIC at the lowest effective concentrations15, 5, 10, 5, 20, and 5 �M, respectively.
GJIC inhibition assay. After exposure, a modified protocol of scrape-loading/dye transfer technique (Blaha et al., 2002; El Fouly et al., 1987) wasused to assess in vitro modulations of GJIC. The cells were washed twice byphosphate-buffered saline solution (PBS), fluorescent dye was added (luciferyellow, 0.05% w/v in PBS), and the cells were scraped using a surgical steelblade. After 2 min of the dye diffusion between the adjacent cells via gapjunctions, the cells were washed by PBS and fixed with 4% (v/v) formalde-hyde. The ratio of the gap-junctional dye transfer from the scrape line wasmeasured with an epifluorescence microscope (Nikon Inc., Japan). At leastthree independent experiments were carried out in duplicates; at least threescrapes per well were evaluated. Cytotoxicity was measured by a conventionalneutral red release assay (Balls et al., 1991). No apparent toxic effects of PCBswere observed within concentrations and exposure periods under study unlessstated otherwise in the text.
Western blotting. Cells were grown to the same confluency as for theSL/DT assay and then deprived of serum for 18–24 h to synchronize the cellsand to reduce the background levels of ERK activity. PCB 153 inhibited GJICat the same dose and time as in serum sufficient cells. Extraction and SDS–PAGE separation of proteins was performed according to the method ofRummel et al. (1999). The protein concentration was determined with BioRadDC protein kit (BioRad, Hercules, CA), and 15 �g of protein was loaded foreach sample. The equal loading was verified by staining the blots with PonceauS. Phosphorylated ERK 1 and ERK 2 were detected with a 1:2000 dilution ofanti-phospho-ERK polyclonal antibodies for 24 h (New England Biolabs,Beverly, MA). The protein-primary antibody complex was probed with a1:1000 dilution of HRP-conjugated anti-rabbit antibodies (Amersham LifeScience Products, Arlington Heights, IL) for 1 h. The ERK protein bands weredetected using the Super Signal chemiluminesence detection kit (Pierce Corp.,Arlington Heights, IL) and ECL Hyperfine X-ray film.
Statistical data analysis. The ratio of GJIC inhibition related to the neg-ative control was evaluated and expressed in % (fraction of control, FOC).Nonparametric statistical methods were used for the data analyses. Kruskal-Wallis ANOVA followed by the Mann-Whitney test were used for the assess-ment of significance, and p values of less than 0.05 were considered statisti-cally significant. Inhibition potency of a xenobiotic was expressed as aconcentration causing 50% inhibition of GJIC (IC50); the IC50 values weredetermined from individual experiments by logit regression; relative error ofestimate did not exceed 15%.
RESULTS
Acute Inhibition of GJIC by PCBs
The scrape load–dye transfer assay was used to measureGJIC in the WB-F344 rat epithelial cells, and the inhibitory
effect of the noncoplanar PCB 153 and the lack of an inhibitoryeffect by the coplanar PCB 126 are shown in the fluorescentmicrographs of Figure 1A. Inhibition of GJIC by PCB153occurred within 15 min (Fig. 1B). The acute GJIC inhibitorypotencies were determined for a series of 37 total environmen-tally occurring PCB congeners (Table 1). The IC50 values ofthe inhibiting compounds ranged within one order of magni-tude (mostly between 10 and 25 �M), the result correspondingto previous findings with PCBs (Hemmings et al., 1991) orother organic environmental pollutants, e.g., DDT (Ren et al.,1998; Ruch et al., 1994; Warngard et al., 1989), lindane(Leibold and Schwarz, 1993) or polycyclic aromatic hydrocar-bons (Blaha et al., 2002; Upham et al., 1998). The noncoplanartri- to hexachlorobiphenyls with chlorine substitutions at theortho-position, such as PCB 153, were found to be potentinhibitors of GJIC. The most potent di-ortho-substituted PCB47 elicited inhibiton of GJIC with IC50 being 10.1 �M. Alsomono-ortho-chlorinated congeners showed similar potency toinhibit GJIC in the rat liver epithelial cells. High-molecular-weight hepta- and octachlorinated congeners and the non-orthosubstituted PCBs elicited minimal or zero inhibition in theWB-F344 cells (Table 1, Figs. 1 and 2).
Inhibition Effects of PCB153 and PCB126 after ProlongedExposure
We next investigated inhibition of GJIC by di-ortho-substi-tuted noncoplanar PCB 153, coplanar PCB 126, or TPA duringprolonged 24-h or 48-h exposure (Fig. 3). The transient inhib-itory effect of the TPA and the recovery of GJIC after theprolonged exposure was observed as previously reported (Renet al., 1998; Rivedal and Opsahl, 2001). On the other hand,PCB 153 induced a long-term downregulation of gap junctionfunction between adjacent cells after 24 and 48 h within thesame concentration range as was found after the acute 30-mintreatment. Coplanar PCB 126 had no significant inhibitoryeffect on GJIC after 30 min, 24 h, or 48 h. Cytotoxicity wasobserved only after prolonged exposure to PCB 126 at thehighest concentration (100 �M).
Coplanar versus Noncoplanar PCB Effects on MAPK
Two prototypical PCB congeners, Nos. 126 and 153, wereselected for the study. The coplanar PCB 126 did not alter thephosphorylation of ERK1/2 as compared to the DMSO vehicle(Fig. 4), although slight differences in the phosphorylationstatus of ERK proteins were observed even with the vehiclecontrol, probably due to high sensitivity of WB-F344 cells tomanipulation (unpublished data). In contrast, the noncoplanarPCB 153 significantly activated ERK1/2 at 60 and 80 min (Fig.4). Activation occurred after inhibition of GJIC, which issimilar to the effects of polycyclic aromatic hydrocarbons(Rummel et al., 1999). In contrast to TPA, no apparent hyper-phosphorylation of connexin43, which is a principal constitu-ent of gap junction channels in WB-F344 cells, was observed
after PCB 153 treatment (data not shown). These and the aboveresults indicate that inhibition of GJIC by PCB153 is indepen-dent of MAPK and differs from the reported mechanisms ofTPA and EGF action (Hossain et al., 1999; Rivedal and Op-sahl, 2001; Warn-Cramer et al., 1998).
Screening Mode(s) of Action of Inhibitors of GJIC
A combination of pretreatment with several selective inhib-itors or other modulators of protein kinases and phospho-lipases, and exposure to EGF, TPA, or PCB 153 followed bythe determination of GJIC was used to compare possiblemode(s) of action of noncoplanar PCB with those of TPA andEGF (see Tables 2 and 3). The major components of intracel-lular signal transduction pathways involved in acute inhibitionof prototypical compounds TPA or EGF have been previouslyreported. EGF blocks GJIC by the direct activation of EGFreceptor and consequent activation of MEK1/2–ERK1/2 sig-naling pathway; TPA inhibits GJIC via the activation of PKCand by indirect activation of MEK1/2–ERK1/2 (Rivedal andOpsahl, 2001; Ruch et al., 2001). Our study confirmed thesefindings; the pretreatment of the cells with U0126, a specificinhibitor of MEK1/2, completely prevented the effect of bothreference compounds. In addition, the pretreatment of the cellswith AG1478, a selective inhibitor of membrane EGF receptor,prevented the inhibitory effect of the EGF on GJIC, while
inhibition of PKC by GF109203X, as well as PKC depletion byprolonged 24-h pretreatment with TPA, prevented the down-regulation of GJIC after TPA exposure (Table 2). However,neither U0126 nor GF109203X blocked PCB 153-inducedinhibition of GJIC (Table 2). Therefore, effects of inhibitors/activators of several additional protein kinases were inves-tigated. These included receptor tyrosine kinase ErbB-2 (re-ported to be a target of organochlorine pesticides in somecellular models; see Hatakeyama and Matsumura, 1999;Tessier and Matsumura, 2001), nerve growth factor receptor,src tyrosine kinase, phosphoinositol 3-kinase (PI3-K), p38mitogen-activated protein kinase, or PKA.
Genistein, a nonspecific inhibitor of protein tyrosine kinases,significantly, although not completely, blocked the inhibitionof GJIC (Fig. 5). None of other chemical inhibitors of proteinkinases was able to block inhibition of GJIC after PCB 153treatment with the exception of PP2, a selective inhibitor ofSrc, and H-89, a compound reported to be a potent inhibitor ofPKA (Table 2). However, pretreatment of the WB-F344 cellswith specific PKA activators, forskolin or 8-Br-cAMP, did notcause inhibition of GJIC; therefore, a possible role for PKA insignal transduction leading to downregulation of GJIC was notconfirmed. These data seem to suggest that PCB congenersoperate by a different mechanism than the prototypical GJICinhibitors TPA and EGF. Neither the growth factor receptor
FIG. 1. Fluorescent microphoto-graphs of results of scrape-loading dyetransfer assay in PCB126- and PCB153-treated WB-F344 cells (A), and imagesdocumenting a lack of GJIC recovery inPCB 153-treated cells (B).
tyrosine kinases under study (ERK1/2, p38, PI3-K, PKA) norGF106203X-sensitive PKCs are probably involved in down-regulation of GJIC observed after PCB 153 treatment in theWB-F344 cells.
PCBs or other organochlorines have been previously shown
to activate PLA2 or PLC in rat neutrophils and several othercell types (Shin et al., 2002; Tithof et al., 1997; Wang andLoch-Caruso, 2002). Therefore, in this study a series of selec-tive inhibitors of PLA2 isoenzymes, PI-PLC, and PC-PLCwere investigated for potential effects on downregulation ofGJIC by PCB 153 or TPA (Table 3). PLA2 inhibitors did notprevent but rather caused a slight increase in TPA- and PCB153-induced inhibition of GJIC. Similarly, two PI-PLC inhib-itors, U73122 and ET-18-OCH3, had no effect on GJIC inhi-bition. On the other hand, an inhibitor of PC-PLC, sphingo-myelin synthase and SMase, D-609, prevented inhibition ofGJIC. RHC 80267, a specific inhibitor of DAG lipase, anenzyme operating downstream of PLC isozymes, partiallyblocked the inhibition of GJIC by PCB 153 (Table 3). Inter-estingly, these compounds also blocked TPA inhibition ofGJIC when concentrations of TPA was lower than 10 nM(Table 2).
DISCUSSION
Imbalance in tissue homeostasis due to disruption of cell-to-cell communication has been linked to growth and devel-
FIG. 2. Dose-dependent inhibition of GJIC in WB-F344 after 30 minexposure to coplanar PCB 126 and noncoplanar PCB 153.
FIG. 3. The effect of TPA, PCB153, and PCB126 on GJIC in WB-F344cells after 30-min, 24-h, and 48-h exposure periods.
TABLE 1Effects of 37 Individual PCB Congeners on GJIC in
WB-F344 Cells Regarding the Structure and the Number ofortho-Substituents
opmental diseases, such as cancer. Although there are manycritical molecular events involved in maintaining homeostasis,considerable data accumulated over the last two decades indi-cate that intercellular communication through gap junctionsplays an important role (Ruch and Trosko, 2001). Numerousstudies have linked the interruption of GJIC, by either onco-genes or tumor promoters, with cancer, indicating that GJICmight be an important, albeit insufficient step of tumor pro-motion (Ruch et al., 2001). In addition to the removal of aninitiated cell from growth suppression, mitogenic signal trans-duction pathways are also required for tumorigenesis. There-fore it is not surprising that PCBs were also shown to induceMAP kinase signal transduction pathways in our cell system,as well as activation of transcription factors such as AhR,NFkB, AP-1, or oxidative stress in other cell systems (re-viewed in Glaubert et al., 2001), although their modes ofmitogenic action are not sufficiently characterized. Both copla-nar and noncoplanar PCB congeners have been shown to elicithepatic tumor-promoting effects in rats (Buchmann et al.,1986; Dean et al., 2002; Glaubert et al., 2001), however at leastone ortho-chlorine appeared to be essential for eliciting the
inhibition of GJIC in rat liver epithelial cells (Hemming et al.,1991). Mono-ortho-substituted PCB114 and also PCB153 in-hibited GJIC in human liver cells and keratinocytes, whilecoplanar PCB77 elicited no inhibition of GJIC (Swierenga etal., 1990). On the other hand, coplanar PCBs inhibited GJIC inmouse hepatoma cell line Hepa-1, and the AhR might beinvolved in this effect (De Haan et al., 1994).
Apparent relationships between the structure and the acuteinhibition of GJIC by PCB congeners were also found in thepresent study. The noncoplanar tri- to hexachlorobiphenylswith chlorine substitutions at the ortho-position, such asPCB153, were found to be potent inhibitors of GJIC, and themono-ortho-chlorinated congeners showed similar potency toinhibit GJIC in the rat liver epithelial cells (Table 1). Impor-tantly, the inhibition of GJIC by prototypical noncoplanarPCB153 was not transient and lasted as long as 48 h (Fig. 2).The short time period of 15 min required for inhibition of GJICindicated that posttranslational modification of the gap junc-tions was the most probable effect. On the other hand, high-molecular-weight hepta- and octachlorinated congeners elic-ited minimal or no inhibition. The non-ortho-substituted PCBs,which are potent AhR inducers, such as PCB126, had noinhibitory effect on GJIC in the WB-F344 cells, even afterprolonged exposure (Fig. 2).
These findings confirmed previously reported effects of sev-eral noncoplanar, mono-ortho-substituted and coplanar PCBsin the same rat liver epithelial cell line (Hemming et al., 1991).We have identified in the present study a number of environ-mentally relevant PCB congeners that are also potent GJICinhibitors, including PCBs 18, 47, 74, 114, 149, 163, 180, and187 as well as other PCB congeners Nos. 110, 119, 123, 129,
FIG. 4. The effect of 50 �M PCB126 and PCB153 on the phosphorylationof ERK1/2 at various exposure times (C, untreated cells; V, vehicle-treatedcells; P44, ERK 1 protein; P42, ERK 2 protein); 15 �g of protein wasuniformly loaded per sample.
TABLE 2Effects of Protein Kinase Modulators on Partial or Total Acute Inhibition of GJIC by PCB 153, TPA, or EGF
Note. Effects expressed as % dye transfer; mean of at least three independent experiments, SD � 15%. The numbers in bold type indicate a significantprevention of GJIC inhibition; GPCRs, G protein-coupled receptors; n.d., not determined; see Materials and Methods for details on treatment and otherabbreviations.
aSpecific inhibition of enzymatic activity if not stated otherwise
157 occurring in the environment at significant concentrations(Rose et al., 2002; Hansen, 1998).
The difference between the effects of coplanar PCB conge-ners on the liver epithelial (oval-like) cells used in our systemand hepatocyte-type cells (De Haan et al., 1994) might berelated to the fact that the two cell types express differentconnexin proteins. While the oval cells express connexin 43,mature hepatocytes express connexin 26 and 32 as their pre-dominant gap junction protein (Zhang and Thorgeirsson,1994). AhR ligands, such as 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD), are known to decrease GJIC in isolated hepa-tocytes, and this effect is probably related to AhR-controlleddownregulation of connexin32 mRNA (Baker et al., 1995;Herrmann et al., 2002). Cell context has been also suggested toplay a decisive role in effects of potential GJIC inhibitors,based on the fact that cell-specific, rather than connexin-spe-cific inhibition by TPA, p,p�-DDT, or phenobarbital has beenobserved (Ren et al., 1998). Thus, while noncoplanar PCBs
could inhibit GJIC in oval stem-like liver epithelial cells (thisstudy), hepatocytes might be a target of inhibitory action ofcoplanar PCBs (De Haan et al., 1994). An importance of thecellular context for inhibition of GJIC by noncoplanar andcoplanar PCBs is supported by the fact that coplanar PCBs didnot affect GJIC in the rat liver epithelial cells WB-F344,although inducible AhR is probably present in these cells. Thepresence of functional AhR and P450-dependent monooxygen-ase activities in rat liver epithelial cells has been questioned(Herrmann et al., 2002; Schrenk et al., 1991). However, it hasbeen shown that WB-F344 cells contain dioxin-inducibleEROD activity (Kohle et al., 1999), and we have found thatboth TCDD and PAHs, known as AhR ligands, are able toinduce CYP1A-dependent activity in this cellular model,which was inhibited by a specific inhibitor, �-naphthoflavone(unpublished data). This seems to suggest a presence of func-tional AhR in this cellular model. However, a possible role ofchaperon complexes of AhR and other transcription factors
TABLE 3Effects of Phospholipase and DAG Lipase Inhibitors on Partial or Total Acute Inhibition of GJIC by PCB153, TPA or EGF
Note. Effects expressed as % dye transfer; mean of at least three independent experiments, SD � 15%. The numbers in bold type indicate a significantprevention of GJIC inhibition; SM synthase, sphingomyelin synthase; SMase, sphingomyelinase; n.d., not determined; see Materials and Methods for details ontreatment and other abbreviations.
FIG. 5. Effects of selected proteinkinase inhibitors on the acute downregu-lation of GJIC by PCB 153, TPA, andEGF.
such as CAR, which is known to be activated by noncoplanarPCBs, has not been currently studied in immediate intracellularevents (Pascussi et al., 2003).
Although the inhibitory effects of noncoplanar PCBs onGJIC have been reported from various in vitro and in vivosystems, the mechanism(s) of their action is still unknown. Theset of selective chemical inhibitors of protein kinases andphospholipases was used to identify the regulatory mechanismsof intracellular signaling pathways responsible for inhibition ofGJIC. Noncoplanar PCB 153, as well as model prototypicalinhibitors of GJIC, TPA, and EGF were selected for thiscomparative study. The participation of the EGF receptor–Ras–Raf–MEK1/2–ERK 1
2 signal transduction pathway in in-hibition of GJIC by EGF is well documented (Kanemitsu andLau, 1993; Rivedal and Opsahl, 2001; Ruch et al., 2001;Warn-Cramer et al., 1998), and our data were consistent withthis model. It is believed that TPA blocks GJIC mainly by adirect action of PKC, but also partly through cross-talk with theERK1/2 pathway (Oh et al., 1991; Ren et al., 1998; Rivedaland Opsahl, 2001). In this study, the specific inhibitor of EGFR(AG1478) blocked inhibition of GJIC after treatment withEGF; the general inhibitor of conventional and novel PKCs,GF109203X, prevented the GJIC inhibition after exposure toTPA. The MEK inhibitor U0126 prevented inhibition of GJICcaused by both TPA and EGF (Table 2).
From our data, it seems evident that a prototypical nonco-planar PCB 153 operates by a mode of action independent ofERK1/2 or PKC activation. Although PCB 153 activatedERK1/2, the phosphorylated forms of ERK1/2 were detectedsubsequently to inhibition of GJIC. This sequence of events issimilar to the effects of polycyclic aromatic hydrocarbons(Rummel et al., 1999). Moreover, a specific inhibitor ofERK1/2 activation, U0126, did not affect the inhibitory po-tency of PCB 153. No apparent hyperphosphorylation of con-nexin43, which is a principal constituent of gap junction chan-nels in WB-F344 cells and becomes phosphorylated after EGFor TPA treatment, was induced by PCB 153 (data not shown).These results indicate that inhibition of GJIC by PCB 153 isindependent of ERK1/2. Similarly, inhibitors of other proteinkinases, including PI3K, p38, and the general inhibitor of PKC,did not modulate the inhibition of GJIC by PCB 153. Incontrast, the inhibitory effect of PCB 153 was partially blockedby genistein, a nonspecific tyrosine kinase inhibitor, and astrong prevention was found also after pretreatment with a highconcentration of PP2, an inhibitor of Src tyrosine kinase. Thisseems to suggest an involvement of tyrosine kinase in effectsof PCB 153, which remains to be specified. However, pretreat-ment with specific inhibitors of selected receptor tyrosine ki-nases, EGFR, ErbB2/Neu, and NGFR, did not prevent GJICinhibition by PCB 153 (Table 2).
H-89, a widely used inhibitor of intracellular PKA, blockedinhibition of GJIC after treatment with PCB 153. However inour study, the activation of PKA by forskolin or the brominatedanalog of cyclic adenosine monophosphate (8-Br-cAMP) wasnot associated with inhibition of GJIC (Table 2). Therefore, we
can conclude either that PKA was not involved in inhibition ofGJIC after treatment with PCB 153 and prevention by H-89was probably due to a broader inhibition specificity of H-89, orthat cAMP alone is insufficient to activate the appropriatecombination of signaling pathways needed to inhibit GJIC.Specificities of many inhibitors of protein kinases and probablyalso phospholipases have not been tested sufficiently (Davies etal., 2000), and it is necessary to be careful when the data frominhibitory studies are interpreted. For example, H-89 has beenreported to selectively antagonize beta adrenergic receptorsand other G protein-coupled receptors (Penn et al., 1999).Notably, G protein-coupled receptor agonists, such as lyso-phosphatidic acid, rapidly disrupt GJIC by activation of Srctyrosine kinase (Giepmans et al., 2001; Postma et al., 1998) orRas-Raf-MEK1/2-ERK1/2 pathway (Warn-Cramer et al.,1998).
Another important group of enzymes involved in intracellu-lar signaling are phospholipases and lipases, such as PLA2
isozymes, PI-PLC, PC-PLC or diacylglycerol (DAG)-lipase(Nozawa, 2002). PCBs have been reported to activate PLA2 orPLC in various cell types (Shin et al., 2002; Tithof et al., 1997;Wang and Loch-Caruso, 2002). In the present study, the in-hibitor of PC-PLC, D609, strongly blocked downregulation ofGJIC, while a series of PLA2 and PI-PLC inhibitors failed toprevent inhibition of GJIC by PCB 153 (Table 3). In addition,DAG lipase inhibitor RHC 80267 also partially blocked GJICinhibition induced by PCB 153, which suggests a potential rolefor DAG lipase products in the inhibitory mode(s) of action ofnoncoplanar PCB 153.
Unlike some bacterial analogues, the mammalian PC-PLChas not yet been characterized at the molecular and regulatorylevels. Therefore, the exact role of PC-PLC in signal transduc-tion remains to be defined, and all the published studies arebased on the use of D609, its putative inhibitor (Nozawa,2002). D609 is considered to be a specific inhibitor of PC-PLCactivity, since it has been reported not to interfere with activ-ities of PI-PLC, PLA2, or PLD (Amtmann, 1996). D609 hasbeen reported to also inhibit sphingomyelin synthase activity,which modulates DAG/ceramide ratio with resulting signifi-cant effects on proliferation and apoptosis (Luberto and Han-nun, 1998) and SMase activity. It is currently not knownwhether sphingomyelin synthase and PC-PLC activities aredue to the same or different enzymes (Ohanian and Ohanian,2001). Interestingly, a partial inhibition of GJIC by low TPAconcentrations was also prevented by H-89, PP2, D609, orRHC 80267, suggesting that low doses of TPA might operatethrough a slightly different mechanism than solely by thepreviously reported activation of phorbol ester-dependent PKCisoenzymes (Rivedal and Opsahl, 2001; Ruch et al., 2001).
In conclusion, a series of environmentally relevant mono-ortho- and di-ortho-substituted PCBs caused the inhibition ofGJIC in the rat liver epithelial (oval-like) cells; this cell linewas not a direct target of disruption of intercellular communi-cation by coplanar PCB congeners. In contrast to prototypicalinhibitors of GJIC, TPA, and EGF, noncoplanar PCB153 elic-
ited a potent inhibition of GJIC by a different mode of action,which probably does not involve activation of PKC or ERK1/2.Using selective chemical inhibitors of signal transduction path-ways revealed that PC-PLC, Src, and DAG lipase might beinvolved in the GJIC inhibitory effect of PCBs.
ACKNOWLEDGMENTS
This research was supported by the Czech Ministry of Agriculture (grant No.QC0194), the Grant Agency of the Czech Republic (No. 525/00/D101) toL. B., and the National Institute of Environmental Health Science Superfund(grant #P42 ES04911-07) to J.E.T.
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