Role of PKC and MAP kinase in EGF- and TPA-induced connexin43 phosphorylation and inhibition of gap junction intercellular communication in rat liver epithelial cells
Edgar Rivedal,1 and
Hanne Opsahl
Department of Environmental and Occupational Cancer, Institute for Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo, Norway
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Abstract
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Gap junction intercellular communication (GJIC) is involved in the regulation of many cellular processes. The gap junction channels are made up of connexins and the flow of polar low molecular weight molecules through these channels is inhibited by several groups of substances, such as tumour promoters and growth factors. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), chlordane and the growth factor epidermal growth factor (EGF) are potent inhibitors of GJIC in several cell types, including the rat liver epithelial cell line IAR6.1. The induced inhibition of communication by TPA and EGF in IAR6.1 cells is associated with hyperphosphorylation of connexin43, the connexin responsible for GJIC. Two enzyme inhibitors, PD98059, a specific inhibitor of MEK kinase, and GF109203X, a selective inhibitor of protein kinase C (PKC), were used to study the signalling pathways involved in the effect of EGF and TPA on GJIC, with the following conclusions. The inhibition of cell communication in IAR6.1 cells by EGF is likely to be mediated by direct phosphorylation of connexin43 by MAP kinase. TPA blocks GJIC mainly by the direct action of PKC, but also partly through cross-talk with the MAP kinase pathway. Connexin43 hyperphosphorylation induced by TPA is, as for EGF, mediated through MAP kinase, while PKC seems to block GJIC either through other substrates or induces a type of connexin43 phosphorylation that causes no significant electrophoresis mobility shift.
Abbreviations: Cx43, connexin43; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulphoxide; EGF, epidermal growth factor; GJIC, gap junction intercellular communication; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol 13-acetate.
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Introduction
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Exchange of low molecular weight substances through pores located in gap junctions is an important way for cells to regulate homeostasis, proliferation and differentiation (1) and maintain homogeneous behaviour and function (24). The role of gap junction intercellular communication (GJIC) in carcinogenesis is mainly based on findings of: inhibitory effects of various tumour promoters and non-genotoxic carcinogens on communication (58); aberrant communication observed in tumour cells (3); reconstitution of normal growth control; loss of tumourigenicity in parallel with induced GJIC in tumour cells following transfection with connexin genes (911).
GJIC is mediated by passive diffusion of polar molecules with molecular weights of <11.5 kDa through pores made up of two hexameric connexin protein structures, called connexons (12,13). The extent of GJIC is a direct result of the number and functionality of these pores (14). The finding of a large family of pore-forming connexin proteins has indicated intricate regulatory mechanisms for this type of exchange of information between cells (3,15). Long-term regulation may include rates of connexin transcription and mRNA stability, resulting in altered overall levels of connexin protein. The formation and efficiency of incorporation of functional connexons into the plasma membrane and docking of these structures to functional hemi-channels in neighbouring cells are also important determining factors for the level of cell communication (1618).
A large number of substances have been shown to influence GJIC (7,1921). Epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) have been shown to affect GJIC in a number of cell types, although the molecular mechanism is not fully understood (2225). EGF stimulates cell proliferation through binding to a cellular receptor and thereby initiating signalling pathways, the most important probably being activation of one of the MAP kinase pathways. EGF has been reported to inhibit GJIC in several cell types and this inhibition has been associated with activation of the ERK1/2 MAP kinase pathway followed by phosphorylation of connexin protein (24,26). The phorbol ester TPA is a classical inhibitor of cell communication in most cells studied. Its ability to inhibit communication has been associated with the activation of protein kinase C (PKC) (27). The ability of PKC to phosphorylate connexin43 (Cx43) in a cell-free system has pointed to this as a likely mechanism for the inhibitory effect of TPA on GJIC (28,29). Several reports have, however, demonstrated a lack of association between a block in communication and connexin hyper-phosphorylation and questioned the role of direct connexin phosphorylation by PKC in inhibition of GJIC (22,30). The aim of this study was to further investigate the role of the signalling pathways activated by EGF and TPA in the inhibitory effects of these substances on GJIC in the rat liver epithelial cell line IAR6.1.
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Materials and methods
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Cells and test substances
The rat liver epithelial cell line IAR6.1 was obtained from the International Agency for Research on Cancer (Lyon, France). The cells were originally isolated from normal inbred BDVI rats and treated twice weekly with dimethylnitrosamine (31,32). The cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco BRL Life Technologies, Inchinnan, UK). TPA and EGF were purchased from Sigma (St Louis, MO) and chlordane from Sulpelco (Bellefonte, PA). TPA and chlordane were dissolved in dimethyl sulphoxide (DMSO) and EGF in phosphate-buffered saline (PBS). The DMSO concentration in medium during exposure was 0.1% (v/v) or lower for all concentrations of test substance.
Determination of GJIC by scrape loading
Quantitative scrape loading was performed as previously described (33). Cells (5x105) were plated onto 60 mm Petri dishes (Costar, Cambridge, MA) 2 days before the experiment. To reduce the risk of cell detachment from the dish during the scrape loading procedure the growth medium was replaced with DMEM with 1% fetal bovine serum on day 1. Before scrape loading the confluent cell layer was washed twice with PBS. Two millilitres of 0.05% (w/v) Lucifer Yellow (Sigma) dissolved in PBS without Ca2+ and Mg2+ were added to the cell monolayer, which was cut 56 times with a surgical scalpel. After 3.5 min following scraping the Lucifer Yellow solution was removed, the dish rinsed four times with PBS, fixed in 4% formalin and mounted with a glass coverslip prior to acquiring digital monochrome images by means of a COHU 4912 CCD camera (COHU Inc., San Diego, CA) and a Scion LG-3 frame grabber card (Scion Corp., Frederick, MD). Analysis was done using the public domain program NIH Image (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/) and Microsoft Excel. The levels of GJIC were determined as distance of diffusion of the dye away from the scalpel cut. By microinjection experiments exposure of these cell types to 30 µM chlordane for 1 h was shown to result in a complete block of GJIC. Thus, the fluorescent cells following such exposure have obtained the dye directly through the scrape process and were used to define zero GJIC.
Western blotting
Cells were seeded and treated as for the scrape loading experiments. Following exposure as indicated, the dishes were washed with PBS, the cells scraped into 500 µl of SDS electrophoresis sample buffer (10 mM Tris, pH 6.8, 15% w/v glycerol, 3% w/v SDS, 0.01% w/v bromophenol blue and 5% v/v 2-mercaptoethanol) and sonicated in a Branson Sonifier. The extract was heated for 5 min at 95°C and 6 µl was used for electrophoresis and western blotting as described (25). The anti-Cx43 antiserum was made in rabbits injected with a synthetic peptide consisting of the 20 C-terminal amino acids of Cx43 (22). The blotting membranes were developed with 4-chloro-1-naphtol as described by the supplier (Bio-Rad) for the anti-Cx43 antiserum and with enhanced chemiluminescence as described by the supplier (Amersham) for the anti-MAP kinase antibody.
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Results
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EGF, TPA, chlordane and GJIC in IAR6.1 cells
The effects of EGF, TPA and chlordane on GJIC in IAR6.1 cells are shown in Figure 1
. Figure 1A
shows that exposure to 30 µM chlordane resulted in a rapid decrease in GJIC. As early as 5 min after exposure, 80% inhibition of communication was observed, while complete inhibition of communication was obtained after 12 h. TPA at 100 ng/ml blocked communication by ~50% after 5 min and, as for chlordane, completely blocked communication after 12 h. EGF has been observed to induce different effects on GJIC in different cell types (23,25,34). In IAR6.1 cells exposure to 100 ng/ml EGF resulted in a maximum inhibitory effect of ~50%. As for TPA and chlordane, this maximum effect was obtained after 12 h exposure.
Prolonged exposure for more than 2 h to TPA resulted in re-occurrence of cell communication and after 7 h continuous exposure communication was almost back to the control level. This re-occurrence has been shown to be associated with down-regulation of PKC based on the finding that cells became refractory to further effects of TPA on GJIC (28,35). A similar re-appearance of communication at 4 and 7 h incubation was also observed for EGF, while only a small increase in communication was observed after 7 h exposure to chlordane (Figure 1A
).
The effects of 1 h exposure to different concentrations EGF, TPA and chlordane is shown in Figure 1B
. Both TPA and chlordane were able to block communication completely, TPA at concentrations above 10 ng/ml and chlordane above 10 µM. Only a partial inhibitory effect (3040%) of EGF on cell communication was observed at concentrations above 3 ng/ml.
Connexin phosphorylation in IAR6.1 cells
IAR6.1 cells have been shown to contain Cx43, but not connexin26 or connexin32 (36). Cx43 is therefore assumed to be the main connexin in this cell type and is responsible for GJIC. Several substances with ability to affect GJIC have been shown to induce changes in the phosphorylation pattern of Cx43 visualized on western blots (17,23,27). To what extent this alteration in phosphorylation pattern is directly responsible for the induced inhibition of GJIC has been studied for several different cell types, and different results have been obtained (22,37,38). No significant induction of Cx43 phosphorylation was observed in Syrian hamster embryo fibroblasts in response to TPA exposure (22), while Cx43 in IAR6.1 cells was extensively phosphorylated (Figure 2
).

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Fig. 2. Effect of TPA, EGF and chlordane on Cx43 phosphorylation pattern in IAR6.1 cells. The cells were exposed to different concentrations and for different lengths of time to the substances prior to western blotting for Cx43. (A) Exposure to different concentrations of TPA for 30 min. (B) Exposure to 100 ng/ml TPA for from 5 min to 6 h. (C) Exposure to different concentrations of EGF for 15 min. (D) Exposure to 100 ng/ml EGF for from 5 min to 6 h. (E) Exposure to 30 µM chlordane for from 5 min to 6 h.
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The effect of TPA, EGF and chlordane on the Cx43 phosphorylation pattern in IAR6.1 cells is shown in Figure 2
. Western blots of extracts from unexposed IAR6.1 cells probed with an antibody against Cx43 resulted mainly in three bands. These bands are normally referred to as P0, P1 and P2; P0 indicating the non-phosphorylated form of Cx43 and P1/P2 two different phosphorylated forms (17). This is concluded from the finding that alkaline phosphatase treatment results in disappearance of the P1/P2 bands and an increase in the P0 band (17,22). In some blots of IAR6.1 cells the P0 band appears to split into two bands. Exposure of IAR6.1 cells to 10 and 100 ng/ml TPA for 30 min resulted in a shift to the higher molecular weight P2 band, while the P0 and P1 bands were weakened (Figure 2A
). In addition, some faint bands with even higher molecular weight appeared. The induced phosphorylation may be associated with the effect on cell communication, since 10 and 100 ng/ml TPA showed strong effects on communication, while 1 ng/ml did not (Figure 1B
). Figure 2B
shows the effect of different lengths of exposure to 100 ng/ml TPA. Altered phosphorylation of Cx43 is observed from ~15 min exposure to TPA. Between 30 min and 2 h exposure essentially one band of Cx43 with a molecular weight similar to that of P2 was observed. When the cells were exposed for 4 h or more the original band pattern of Cx43 returned, concomitant with re-occurrence of cell communication. Thus, both with regard to doseresponse characteristics and time course, there is an association between altered Cx43 phosphorylation pattern and degree of functional cell communication.
Association between communication and phosphorylation of Cx43 also seems to be the case when exposing IAR6.1 cells to EGF. EGF showed less dramatic effects on cell communication than TPA, which is paralleled by a lesser effect of EGF on the Cx43 phosphorylation pattern (Figure 2C and D
). Hyper-phosphorylation is observed especially at 10 and 100 ng/ml and 15 and 30 min exposure. The association between GJIC and Cx43 phosphorylation is not, however, present when IAR6.1 cells were exposed to chlordane. Exposure of the cells to 30 µM chlordane for increasing lengths of time resulted in no or little induction of Cx43 hyper-phosphorylation. In the experiment shown in Figure 2E
some increase in the P2 band may be indicated after 15 min exposure, while in other experiments this effect was not seen. On the other hand, an apparent shift to lower molecular weight connexin bands was observed after prolonged exposure to chlordane (4 and 7 h), as previously reported (38). This effect is not likely to be involved in the down-regulation of communication, which for this compound also occurred within minutes of exposure (Figure 1A
).
Two different signalling pathways are often associated with the effect of TPA and EGF. TPA is assumed to mainly mediate its cellular effects through activation of PKC (27), while the effect of EGF to a large extent has been shown to be mediated through one of the MAP kinase pathways (24). In order to shed light on the possible involvement of these pathways in the effects of EGF and TPA on GJIC, and also the possible role of Cx43 phosphorylation on functional cell communication, two enzyme inhibitors were used.
Since no specific inhibitor of MAP kinase is available, PD98059, a specific inhibitor of MEK1, which directly activates the MAP kinases ERK1/2, was used (39,40). GF109203X (bis-indolylmaleimide 1) is a potent and selective PKC inhibitor (PKC
, ßI, ßII,
,
and
isozyme inhibition) (41,42).
Figure 3A
shows the effect of increasing concentrations of PD58059 on the inhibitory effects of TPA and EGF on GJIC in IAR6.1 cells. The cells were pre-exposed to the inhibitor for 5 min prior to co-exposure to TPA and inhibitor or EGF and inhibitor for 1 h. The MEK inhibitor was obviously able to counteract the inhibitory effect of EGF on cell communication, while little or no effect on the inhibitory effect induced by TPA was observed. On the other hand, the PKC inhibitor GF109203X counteracted the inhibitory effect of TPA on cell communication in IAR6.1 cells, as shown in Figure 3B
, while the PKC inhibitor was unable to affect the EGF-induced inhibition of cell com- munication.
Figure 4
shows the effect of 10 µM PKC inhibitor GF109203X and 50 µM MEK inhibitor PD98059 on inhibition of GJIC in IAR6.1 cells by different concentrations of TPA, EGF and chlordane. The inhibiting effect of TPA is completely reversed by GF109203X and partly by PD98059 (Figure 4A
); the effect of EGF is unaffected by GF109203X and completely abolished by PD98059 (Figure 4B
); none of the inhibitors had any significant influence on the inhibition induced by chlordane (Figure 4C
).
The effect of the two kinase inhibitors on induction of Cx43 phosphorylation is shown in Figure 5
. As expected, and in accordance with the communication data in Figures 3 and 4
, the MEK inhibitor PD98059 reversed the induction of Cx43 phosphorylation by EGF (Figure 5A
). On the other hand, and also in accordance with GJIC, the PKC inhibitor GF109203X had no apparent effect on EGF-induced phophorylation of Cx43 (Figure 5B
). Interestingly, although the MEK inhibitor PD98059 showed no effect on the inhibitory effect of TPA on cell communication, it reversed the induction of Cx43 phosphorylation by TPA, as shown by the western blot in Figure 5C
. PD98059 at 50 µM completely reversed the TPA-induced hyper-phosphorylation of Cx43, while having little or no effect on cell communication (Figure 3A
). On the other hand, the PKC inhibitor GF109203X, shown to almost completely abolish the effect of TPA on GJIC (Figure 3B
), had little or no effect on TPA-induced hyper-phosphorylation of Cx43, as shown in Figure 5D
.

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Fig. 5. Effect of different concentrations of the MEK inhibitor PD98059 and the PKC inhibitor GF109203X on TPA (100 ng/ml)- and EGF (100 ng/ml)-induced phosphorylation of Cx43 in IAR6.1 cells. The cells were exposed for 15 min to combinations of: (A) EGF + PD98059; (B) EGF + GF109203X; (C) TPA + PD98059; (D) TPA + GF109203X. Following exposure cell extracts were prepared and run on western blots for Cx43 as described in Materials and methods.
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Thus, the PKC inhibitor GF109203X had little effect on TPA-induced Cx43 phosphorylation but a strong effect on TPA-induced inhibition of communication. The opposite, however, was observed for the MEK inhibitor PD98059; there was little effect on TPA-induced inhibition of communication but a strong effect on TPA-induced Cx43 phosphorylation. This may indicate that TPA induces inhibition of GJIC partly through PKC and partly through MAP kinase and that it is the MAP kinase pathway that is responsible for phosphorylation of Cx43. The data indicate that PKC induces a block in GJIC through a mechanism that does not cause apparent Cx43 phosphorylation or induces phosphorylation of Cx43 that does not show up in western blots.
Figure 6
shows the effects of EGF, TPA and chlordane on activation of the ERK1/2 MAP kinase pathway. Activation was visualized by western blotting using an antibody recognizing the phosphorylated and activated ERK1/2 MAP kinase (New England Biolabs). Studies have shown a correlation between MAP kinase activity and levels of phospho-MAP kinase identified by these antibodies (43). Figure 6A and B
show some activation of MAP kinase at 1 ng/ml EGF or TPA and strong activation at 10 and 100 ng/ml. This is in accordance with the concentrations inducing both connexin phosphorylation and inhibition of cell communication. As seen in Figure 6D and E
, activation of MAP kinase is very rapid and is already maximal after 5 min exposure to TPA or EGF. Activation is transient for both compounds; for TPA some of the ERK1 enzyme is still activated after 2 h, but inactive after 4 and 6 h exposure. The abolition of activated MAP kinase is thus associated with re-occurrence of non-phosphorylated Cx43, as well as reconstituted communication. This is most clearly observed for TPA, since the effects are stronger and therefore better visualized. The same association may, however, also be observed for EGF. Interestingly, chlordane is also observed to slightly activate MAP kinase (Figure 6C
). This small effect may be related to the slight induction of the Cx43 P2 band observed in Figure 2E
. The effect seems, however, not to be of any significant importance in the inhibitory effect of chlordane on GJIC, because the MEK inhibitor PD98059 is unable to counteract the inhibitory effect of chlordane on cell communication (Figure 4C
).

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Fig. 6. Dose-dependent activation of p42 MAP kinase in IAR6.1 cells by 15 min exposure to: (A) TPA; (B) EGF; (C) chlordane. Time-dependent activation of p42 MAP kinase by 100 ng/ml TPA (D) and by 100 ng/ml EGF (E).
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Figure 7
shows that the MEK inhibitor PD98059 was able to block MAP kinase activation induced by EGF and TPA (Figure 7A and C
). The protein kinase C inhibitor GF109203X blocked part of the MAP kinase activation induced by TPA (Figure 7B
), but less than the MEK inhibitor. On the other hand, the PKC inhibitor was, as expected, unable to block EGF-induced MAP kinase activation. Thus, the data in Figures 6 and 7
are all in accordance with the previously demonstrated ability of EGF to activate the ERK1/2 MAP kinase cascade and cross-talk between activated PKC and this pathway (44,45).

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Fig. 7. Effect of different concentrations of the MEK inhibitor PD98059 and the PKC inhibitor GF109203X on induced activation of p42 MAP kinase in IAR6.1 cells by TPA (100 ng/ml) and EGF (100 ng/ml). The cells were exposed for 15 min to combinations of: (A) TPA + PD98059; (B) TPA + GF109203X; (C) EGF + PD98059; (D) EGF + GF109203X.
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Discussion
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In addition to regulation of connexin synthesis through transcriptional control, it is also demonstrated that other and more rapid mechanisms are involved in the regulation of GJIC, i.e. altered subcellular localization and post-translational modification of connexins (46,47). Cx43 is, like many other connexins, a phosphoprotein and phosphorylation of this protein has been associated with both channel functionality and induced down-regulation of communication (27,48). Several phosphorylation sites have been identified in Cx43 and cells have been shown to become insensitive to GJIC-altering agents when these are altered to non-phosphorylatable residues (26,4951). The association between down-regulation of communication and Cx43 phosphorylation is, however, not always present and may seem to be cell dependent (22,38). It has, therefore, been difficult to establish a general role for Cx43 phosphorylation in the down-regulation of communication. Cross-talk between different signalling pathways has also made it difficult to determine the exact enzyme or signalling pathway responsible for the induced connexin phosphorylation (26,52).
Growth factors have been found to reduce GJIC and it has been suggested that there exists a reverse relationship between GJIC capacity and rate of proliferation (53,54). EGF is, however, observed to possess different effects on communication in different cell types. In some cells EGF is a potent inhibitor of communication (55), while in a human kidney epithelial cell line (K7) EGF has little or no inhibitory effect, but on the contrary induces enhancement of cell communication (25). In the present rat liver epithelial cell line IAR6.1 EGF induces a maximum of ~50% inhibition of GJIC. EGF has previously been shown to induce phosphorylation of Cx43 through activation of a MAP kinase cascade (ERK1/2) and it has been suggested that this phosphorylation is responsible for down-regulation of communication by EGF (24,26).
TPA is assumed to mediate most of its many effects on cells through activation of PKC (56) and has been shown to induce phosphorylation of Cx43 in association with down-regulation of communication (27). However, there exist examples of cell types where TPA is able to down-regulate communication without any significant change in the Cx43 phosphorylation pattern in western blots (22). Lampe et al. (57) have reported that a specific site in Cx43, Ser368, is a major site of PKC phosphorylation and is involved in TPA-induced inhibition of GJIC and alterations in single channel behaviour in two different cell types, T51B rat liver epithelial cells and cx43/ transfected fibroblasts. It is, however, not known whether Cx43 phosphorylated on Ser368 is mobility shifted and the authors state that TPA could still cause a Cx43 mobility shift in cells containing phosphorylated S368A.
PKC has been shown to activate a MAP kinase (ERK1/2) pathway through activation of RAF, demonstrating cross-talk between PKC and the MAP kinase signalling pathway (44,45). This implies the possibility that connexin phosphorylation as well as inhibition of GJIC by PKC activators could be mediated through MAP kinase. Platelet-derived growth factor (PDGF) has been shown to block GJIC and induce hyperphosphorylation of Cx43 in T51B rat liver epithelial cells expressing wild-type PDGF receptor ß (52). Hossain et al. (58) concluded further that PKC plays a crucial role in PDGF-induced Cx43 phosphorylation and down-regulation of GJIC in these cells. Unlike EGF (34), PDGF does not block GJIC in cells pre-treated with TPA to down-regulate PKC (58). It was, however, observed that PD98059, a specific inhibitor of MEK1 activation, eliminated the PDGF-induced block of GJIC and Cx43 hyperphosphorylation, suggesting that these effects may be caused by interference with MAPK activation. The authors raised the possibility that TPA could exert its GJIC inhibitory effects by activating MAPK and indicated that they had obtained data in support of this, showing that TPA was unable to block GJIC and phosphorylate Cx43 in cells pre-treated with PD98059 (58).
The data presented here show an association between Cx43 phosphorylation and inhibition of communication by both EGF and TPA. This association is evident both in concentrationresponse curves as well as exposure time effects. It is, however, important to realise that the data suggest but do not prove a direct relationship between Cx43 hyperphosphorylation and inhibition of GJIC. The variability of this association in different cell types may be taken as an argument against this being a causal relationship.
It is evident that TPA is a more potent inhibitor of communication as well as inducer of Cx43 hyper-phosphorylation than EGF, also in agreement with an association between phosphorylation and a decrease in communication. The data indicate an association between MAP kinase activation and connexin phosphorylation, suggesting that connexin phosphorylation induced by TPA is mediated through PKC-induced activation of the MAP kinase pathway and not by direct phosphorylation by PKC. This interpretation is based on experiments using the MEK inhibitor PD98059 and PKC inhibitor GF109203X. The MEK inhibitor was capable of preventing the induced hyper-phosphorylation of Cx43 by both EGF and TPA, while the PKC inhibitor had no effect on EGF-induced phosphorylation and only a limited effect on TPA-induced phosphorylation. Notably, the blocking effect of the PKC inhibitor on TPA-induced Cx43 phosphorylation is substantially less than that of the MEK inhibitor. The MEK inhibitor was also able to completely abolish the inhibitory effect of EGF on communication, but had little or no effect on TPA-induced inhibition of communication. On the contrary, the PKC inhibitor was able to abolish TPA-induced inhibition of communication, but had little or no effect on EGF-induced inhibition. Similarly, the MEK inhibitor was shown to fully suppress EGF-induced activation of MAP kinase, while the PKC inhibitor has no effect. For TPA-induced activation of MAP kinase, the PKC inhibitor shows a weak inhibiting effect, but significantly less than that of the MEK inhibitor. This is what would be expected in an amplifying cascade where PKC activation results in a downstream activation of MAP kinase.
The data are summarized in Table I
and suggest that hyper-phosphorylation of Cx43 induced by both EGF and TPA in IAR6.1 cells is mediated through MAP kinase activation, possibly by the ERK1/2 enzyme itself. There is no indication that any of the phosphorylated Cx43 forms visualized by western blotting occur as a result of direct phosphorylation by PKC. On the other hand, the data indicate that the major part of inhibition of cell communication induced by TPA is mediated by PKC itself, but without association with the type of Cx43 phosphorylation that is visualized on western blots. This could indicate a role for sites on connexin that do not result in gel mobility changes of connexin when phosphorylated or the possible role of other substrates than connexin in inhibition of cell communication, as suggested by Hossain et al. (59) in studies of the effect of PDGF and TPA on T51B rat liver epithelial cells. They reported a lack of correlation between activation of MAPK, Cx43 phosphorylation and GJIC blockade, suggesting the involvement of additional components.
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Notes
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1 To whom correspondence should be addressed
Email: edgar.rivedal{at}labmed.uio.no 
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Acknowledgments
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The excellent technical assistance of A. Nordahl and R. Skibakk is gratefully acknowledged. The work was supported by the Norwegian Cancer Society.
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Received December 27, 2000;
revised June 11, 2001;
accepted June 15, 2001.