Anisomycin downregulates gap-junctional intercellular communication via the p38 MAP-kinase pathway

Gap junctions consist of plasma-membrane-spanning channels that permit the intercellular exchange of ions and low molecular weight molecules (Loewenstein, 1990). Gap-junctional intercellular communication (GJIC) is therefore believed to be involved in cell growth and differentiation; aberrant control might also play an important role in cancer development (Loewenstein, 1990; Trosko and Ruch, 1998). The mechanisms that regulate GJIC are not yet fully understood, although there is evidence that post-translational alterations of the connexins are involved (Musil and Goodenough, 1991; Musil and Goodenough, 1993; Trosko and Ruch, 1998). Connexin 43 (Cx43) is a widely expressed gap-junction protein found in many animal organs (Beyer et al., 1987; Dupont et al., 1991) and many recent investigations into the relationships between Cx43 phosphorylation and events in gap-junction assembly (channel gating) suggest that several protein kinases are capable of mediating both Cx43 phosphorylation and GJIC inhibition (Berthoud et al., 1993; Cho et al., 2002; Hossain et al., 1998a; Kanemitsu and Lau, 1993; Laird et al., 1995; Lau et al., 1992; Musil et al., 1990; Musil and Goodenough, 1991; Musil and Goodenough, 1993; Trosko and Ruch, 1998; Warn-Cramer et al., 1998; Warn-Cramer et al., 1996).

The mitogen-activated protein (MAP) kinase belongs to an important family of protein kinases that act by phosphorylating specific amino acids on their target substrates. Of the classic MAP kinases, extracellular signal-regulated protein kinases 1 and 2 (ERK) are especially well known and can be activated by various physiological stimuli, including some growth factors (Seger and Krebs, 1995). Previous investigations have shown that activation of ERK is an important element in the regulation of both GJIC and Cx43 (Berthoud et al., 1993; Hossain et al., 1998b; Hossain et al., 1999a; Kanemitsu and Lau, 1993; Lau et al., 1992; Ruch et al., 2001; Warn-Cramer et al., 1996; Warn-Cramer et al., 1998) but other studies investigating a range of ERK activators and inhibitors suggest that the correlation between ERK activation, Cx43 phosphorylation and GJIC inhibition is by no means perfect (Hii et al., 1995a; Hii et al., 1995b; Hossain et al., 1998a; Hossain et al., 1999a). Previous evidence (Hii et al., 1995b; Matesic et al., 1994) indicates that Cx43 hyperphosphorylation by ERK activation, as indicated by a reduction in the mobility [on sodium dodecyl sulfate (SDS) gels] of the resulting Cx43 derivatives, is one mechanism of GJIC disruption available to cells, but it seems likely that other mechanisms of disruption, not necessarily dependent on Cx43 hyperphosphorylation, can and do exist.

We believe that other kinases and/or cofactors might be involved in the process of GJIC disruption; it is even possible that GJIC blockage is dependent upon the coordinated action of different MAP kinases, including ERK. Other members of the MAP-kinase family might also be involved (Cano and Mahadevan, 1995). Of these, p38 MAP kinase is the most obvious candidate, if only because of its involvement in signal transduction pathways that work in parallel with ERK (with which it shares ∼50% sequence identity) (Tong et al., 1997). Given that these two signal transduction pathways overlap and `cross talk' (Cano and Mahadevan, 1995; Helliwell et al., 2000; Töröcsik and Szeberényi, 2000), it seems reasonable to postulate a role for both p38 MAP kinase and ERK in the downregulation of Cx43 and/or the disruption of GJIC. Two recent reports, one suggesting that p38 MAP kinase is involved in GJIC upregulation and a second suggesting its involvement in GJIC downregulation (Cho et al., 2002; Polontchouk et al., 2002), are difficult to interpret because different cell types were used. Such contradictory findings do little if anything to clarify the relationship between p38 MAP kinase and GJIC regulation, and we thus designed a new study that we hoped would allow us to determine whether p38 MAP kinase contributes to Cx43 phosphorylation and/or gap-junctional disruption and, if so, how.

As a first step, we decided to examine the effects of anisomycin on GJIC. This interesting compound is well known to act as a protein synthesis inhibitor and a pharmacologically specific activator of two distinct kinds of kinases, p38 MAP kinase and c-Jun N-terminal kinases (JNKs) (Barros et al., 1997; Cano et al., 1994; Cano and Mahadevan, 1995; Hazzalin et al., 1998; Kyriakis et al., 1994), both of which can be stimulated by a wide variety of stress stimuli, including DNA damage, heat and osmotic shock, cytokines, and protein synthesis inhibitors (Minden and Karin, 1997). We also designed experiments in which we made use of a specific inhibitor of p38 MAP kinase, known as SB203580 (Cuenda et al., 1995; Tong et al., 1997), and other kinds of cell-to-cell-junction-related proteins, including ZO-1, occludin, E-cadherin and β-catenin. We also performed total cell metabolic labelling with [³⁵S]-methionine in order to try and rule out any possibility that the observed effects of anisomycin resulted from activation of the JNK pathway or a more general inhibitory effect on protein synthesis. Our findings strongly favour the hypothesis that p38 MAP kinase plays an important role in the disruption of GJIC by reducing the total amounts of phosphorylated Cx43, which effect is possibly due to accelerated degradation of phosphorylated Cx43 in the cells, and reducing the number of Cx43 proteins observed at cellular borders.

The Fisher 344 rat-liver-derived epithelial cell line WB-F344 (Tsao et al., 1984) was cultured at 37°C in a 95% O₂, 5% CO₂ atmosphere in a modified Eagle's medium (MEM) supplemented with 7% foetal calf serum (FCS), 50 U ml^-1 penicillin and 50 μg ml^-1 streptomycin sulfate. Passage 8-21 cells were used in all experiments.

Anisomycin (2-p-methoxyphenylmethyl-3-acetoxy-4-hydroxypyrrolidine) and dimethyl sulfoxide (the vehicle for SB203580) were from Sigma (St Louis, MO) and SB203580 [4-(4-fluorophenyl)-2-(4-methylsulphonylphenyl)-5-(4-pyridyl) imidazole] was from Calbiochem (La Jolla, CA). PhosphoPlus p38 MAP kinase and SAPK/JNK Antibody Kit™ were from Cell Signaling Technology (Beverly, MA). Carboxyfluorescein diacetate (CFDA) was from Molecular Probes (Eugene, OR). Lab-Tek Chamber Slides™ were from Nalge Nunc International (Naperville, IL). Anti-Cx43 monoclonal antibody and Alexa-488-conjugated goat anti-mouse antibody were from Chemicon International (Temecula, CA) and Molecular Probes (Eugene, OR), respectively. Anti-ZO-1 and anti-occludin polyclonal antibodies (Zymed Laboratories, San Francisco, CA), and anti-E-cadherin (Transduction Laboratories, Lexington, KY), anti-β-catenin (Zymed Laboratories), anti-β-actin (Sigma) monoclonal antibodies were also used. SDS polyacrylamide-gel electrophoresis (SDS-PAGE) supplies and reagents for western blot analyses were from Bio-Rad (Richmond, CA). The enhanced chemiluminescence detection kit was from Renaissance Western Blot Chemiluminescence Reagent (NEN Life Science Products, Boston, MA). [³⁵S]-Methionine and [³²P]-orthophosphates were from Perkin Elmer Life and Analytical Sciences (Boston, MA).

WB-F344 cells seeded in dishes or slides were grown to approximately 80% confluence. The medium was replaced with fresh medium containing 0.2% FCS and the cells were then incubated for a further 48 hours. Cells were treated with 10 μg ml^-1 (10 mg ml^-1 stock in distilled water) anisomycin for 5, 30 60, 90, 120 and 180 minutes at 37°C. To block the p38 MAP kinase, cells were treated with 10 μM SB203580 (50 mM stock in dimethyl sulfoxide) for 30 minutes at 37°C before being exposed to anisomycin for 60 minutes. Controls for the experiments were treated with 0.02% dimethyl sulfoxide and/or distilled water. Aliquots of the treated cell suspensions were then used for measurements of GJIC, MAP kinase activation levels, Cx43 phosphorylation levels and immunofluorescence.

Our procedure was a modified version of the standard method for measuring GJIC by quantitative fluorescence recovery after photobleaching (FRAP) (Ogawa et al., 1999; Trosko et al., 2000; Wade et al., 1986). Assays were performed using an ACAS Ultima laser cytometer (Meridian Instruments, Okemos, MI). After selectively bleaching cells with a micro-laser beam, we followed the rate of transfer of CFDA from adjacent labelled cells back into bleached cells. Recovery of fluorescence was assessed at 1-minute intervals and recovery rates (RRs) were calculated as percentages of fluorescence recovered per minute. Anisomycin and/or SB203580 were not present in the medium during labelling of the cells with CFDA, photobleaching or calculation of RR. All calculated rates were corrected for the loss of fluorescence by unbleached control cells and the results expressed as the average percentage (mean±s.e.m.) recovery rate of treated cells relative to the recovery rate of untreated cells.

WB-F344 cells were cultured as previously described (Trosko et al., 2000) and the cells plated on a Lab-Tek Chamber Slide™ for anisomycin and/or SB203580 treatment. After treatment, the cells were washed twice in PBS and fixed in periodate-lysine-paraformaldehyde fixative for 30 minutes; they were then washed and permeabilized three times with 0.1% Triton-X-100/PBS (PBST), and incubated in 20% BlokAce (Dainihon Pharmaceuticals, Tokyo, Japan) for 1 hour. The next stage involved overnight incubation at 4°C in a 1:2000 dilution of anti-Cx43 monoclonal antibody (Chemicon) followed by three washes with PBST before incubation in Alexa-488-conjugated goat anti-mouse antibody (Molecular Probes) at a dilution of 1:2000 for 1 hour in dark conditions. The cells were then washed three times in PBST and once in PBS before being mounted in Gel/Mount (Biomeda, Foster City, CA) for examination in a Zeiss LSM 510 laser-scanning confocal microscope.

Cells grown to approximately 80% confluence in 10-cm dishes were treated with anisomycin, anisomycin plus SB203580, or SB203580 plus either distilled water or dimethyl sulfoxide as the vehicle control. At the end of the treatment period, the monolayers were rinsed three times with ice-cold PBS. Lysates were prepared with ice-cold lysis buffer containing 20 mM Tris-buffered saline (TBS), pH 7.5, 1% Triton X-100, 150 mM NaCl, 1 mM each of EDTA, EGTA, β-glycerophosphate, Na₃VO₄ and phenylmethyl-sulfonyl fluoride (PMSF), 2.5 mM sodium pyrophosphate, and 1 μM leupeptin, and then sonicated. The samples were diluted 1:5 in water and their protein concentrations determined using the DC protein assay™ (Bio-Rad). Samples (20 μg) of protein were then dissolved in Laemmli sample buffer, separated on 12.5% polyacrylamide gels, and transferred to polyvinilidene difluoride membranes (Bio-Rad) before determining their phosphorylated p38 MAP kinase and JNK levels according to the manufacturer's protocols for PhosphoPlus p38 (Thr180/Tyr182) and SAPK/JNK (Thr183/Tyr185) MAP Kinase Antibody Kit™ (Cell Signaling Technology) assays. The Cx43 content of the various samples was also determined by incubating the samples with an anti-Cx43 monoclonal antibody (Chemicon; diluted 1:2000), and later adding a horseradish-peroxidase-conjugated secondary antibody (diluted 1:2000; Amersham, Arlington Heights, IL) and an enhanced chemiluminesce detection reagent (NEN Life Science Products). The membrane was stripped and reprobed with anti-ZO-1 and anti-occludin polyclonal antibodies (Zymed, diluted 1:2000), and anti-E-cadherin (Transduction, 1:2000), anti-β-catenin (Zymed, 1:2000) and anti-β-actin (Sigma, 1:2000) monoclonal antibodies, respectively. The average control value was assigned an arbitrary value of 1 unit and relative band intensities were standardized to this arbitrary unit (AU).

To confirm that anisomycin acts as a protein synthesis inhibitor in vivo, metabolic labelling of WB-F344 cells was conducted. Cells were starved of methionine for 30 minutes at 37°C in methionine-free MEM. The medium was then replaced with fresh labelling medium containing [³⁵S]-methionine (0.1 mCi per 6 cm dish of cells) and incubation was continued for the final 60 minutes. Anisomycin and SB203580 were incubated for the last 60 or 90 minutes. The radioactive medium was removed after a pulse labelling and cells were rinsed with PBS and solubilized in cold RIPA buffer containing 2 mM sodium orthovanadate, 1 mM PMSF and 1% Triton-X-100. Cells were harvested and the DNA was sheared by drawing the lysate through a 26 G needle. After centrifugation, supernatant was collected and resuspended (1 μl) in 1 ml of scintillate followed by liquid scintillation spectrometry.

Radioimmunoprecipitation of Cx43 was performed as described above. Cells were starved of phosphates for 30 minutes at 37°C in phosphate-free MEM and supplemented with 0.2% dialysed FCS. The medium was then replaced with fresh labelling medium containing [³²P]-orthophosphate (0.1 mCi per 6 cm dish of cells) and preincubation was continued for 60 minutes. After the addition of anisomycin and/or SB203580, culture was continued in radioactive medium. Each treatment group was exposed to 0.02% dimethyl sulfoxide as vehicle alone for 90 minutes, anisomycin at 10 μg ml^-1 for 60 minutes, SB203580 plus anisomycin (pretreatment of SB203580 for 30 minutes, then co-treatment with anisomycin for an additional 60 minutes), or SB203580 alone for 90 minutes. Anisomycin and SB203580 were incubated for the last 60 or 90 minutes. The radioactive medium was removed after one pulse for three hours, and cells were rinsed with TBS and solubilized in cold RIPA buffer containing sodium orthovanadate (2 mM), 1 mM PMSF and 1% Triton-X-100. Cellular debris was concentrated by centrifugation (10 minutes, 20,400 g), and supernatant was collected and incubated with 20 μl of a 50% slurry of Protein-G/Sepharose CL-4B (Amersham Pharmacia Biotech, Uppsala, Sweden) and the samples were rotated for 1 hour at room temperature. The samples were then centrifuged at 20,400 g for 10 minutes and the supernatant was collected and incubated with 1 μg anti-Cx43 monoclonal antibody (Chemicon) for 1 hour, followed by addition of 20 μl of the 50% slurry of Protein-G/Sepharose CL-4B for 1 hour. The immunoprecipitates were washed four times with TBS and supplemented with Laemli sample buffer and boiled at 95°C for 5 minutes. Samples, derived from equal volume of cell lysates, were electrophoresed on 12.5% SDS gels and autoradiographed. Phosphorylated bands were analysed by autoradiography using Personal Molecular Imager FX (Bio-Rad).

Exposed films were scanned using a flatbed scanner and band density was analysed using NIH Image™.

Data were analysed using Statview II software™ (Apple Computer, Cupertino, CA). The two-tailed unpaired Student's t-test was used in comparisons of the anisomycin- or SB203580-treated cultures with control cultures; differences were considered significant at P<0.05. Results are expressed as the mean±s.e.m.

Typical digitized fluorescence images and plots of fluorescence recovery after photobleaching of untreated cells and cells treated with anisomycin for 60 minutes are shown in Fig. 1. The untreated cells recovered their fluorescence within 4 minutes, whereas the anisomycin treated cells did not.

To determine whether this anisomycin-induced effect is specifically related to the ability of anisomycin to activate p38 MAP kinase, we also examined the phosphorylation status of the other MAP kinase families. To do this, we assessed the activities of p38 MAP kinase and JNK by measuring the levels of their active phosphorylated forms using immunoblotting. Densitometric band analyses of the phosphorylated forms of p38 MAP kinase and JNK (p46/p54) are shown in Fig. 2. The time-course study shows that anisomycin treatment led to the phosphorylation of both p38 MAP kinase and JNK (Fig. 2A). Phosphorylation of p38 MAP kinase was not observed in the absence of anisomycin, a peak level being reached at 5 minutes and sustained until 60 minutes. Phosphorylated JNK levels increased much more slowly after anisomycin treatment, with a peak becoming evident at 30 minutes. The dose study shows that phosphorylated p38 MAP kinase and JNK increased at concentrations of more than 0.1 μg ml^-1, with peaks of 8.1 times the control at 10 μg ml^-1 (p38 MAP kinase) and 8.3 times the control at 1 μg ml^-1 (JNK) (Fig. 2B).

We therefore used the FRAP assay to measure the RR at each time point as a simple way of quantifying the effects of anisomycin treatment on GJIC. The results were consistent: the addition of anisomycin (10 μg ml^-1) to WB-F344 cells led to a time-dependent decrease in RR, in that there was a significant decrease to ∼80% of the control value at 5 minutes and then to ∼55% at 30 minutes and ∼50% at 60 minutes (this was the lowest level reached and was retained up to 180 minutes) (Fig. 3A). Cells that had been incubated for up to 60 minutes with the same concentration of anisomycin turned out to have near-normal RRs only 24 hours after the removal of the anisomycin (data not shown). To assess the dose effect of anisomycin, 60 minute assays were performed at various concentrations of anisomycin. There was no significant decrease at <0.1 μg ml^-1 but the RR decreased to about 75% of control at 1 μg ml^-1, with maximal effect at 10 μg ml^-1 (about 40% of control) (Fig. 3B). In order to assess effects of anisomycin concentrations on protein synthesis, we estimated the amount of proteins produced de novo using an [³⁵S]-methionine metabolic labelling assay (Fig. 3C). There was a significant decrease in the total protein level even at the lowest concentration (10 ng ml^-1) of anisomycin by which p38 MAP kinase could not be activated (Fig. 2B). Thus, we could not dissociate the inhibition of protein synthesis from the GJIC downregulation by using lower concentrations of anisomycin (around 25-50 ng ml^-1; socalled subinhibitory level) that appeared to have an ability to activate the p38 MAP kinase.

An examination of the profiles of the various forms of Cx43 before and after anisomycin treatment led to some interesting findings. Before treatment, we could detect three predominant forms of the Cx43 protein by SDS-PAGE; each form could be seen as a distinct immunoreactive band of between 41 and 43 kDa. The minor unphosphorylated form of Cx43 (P0) migrated fastest, whereas the more abundant phosphorylated forms (P1 and P2) migrated more slowly (Fig. 4A), as noted previously (Trosko and Ruch, 1998). After setting the total Cx43 level of control cells at 1 AU, we noted that the combined intensities of P0, P1 and P2 had reached their lowest point (0.46 AU) at 90 minutes and retained up to 180 minutes. Following the addition of anisomycin, the absolute amount of the phosphorylated forms of Cx43 (i.e. P1+P2), and also of P0, tended to decrease time dependently until 120 minutes, which coincided almost precisely with the disruption of GJIC (Fig. 3A). By contrast, none of ZO-1, occludin, E-cadherin and β-catenin showed any changes. The ratios of P1+P2 to P0 were assayed at intervals throughout the course of a 180-minute treatment period. Although the ratio increased about ninefold, reaching a peak at 60 minutes, it then decreased rapidly, only to increase again at 180 minutes (Fig. 4C). The disruption of GJIC that we were observing appeared to vary inversely with the absolute amounts of Cx43 phosphorylated forms P1+P2 and P0 that were present.

As expected, we were able to detect phosphorylation of p38 MAP kinase in anisomycin-treated cells not pretreated with SB203580, whereas cells treated with only SB203580 did not appear to contain any phosphorylated p38 MAP kinase whatsoever (Fig. 5A). The levels of p38 MAP kinase phosphorylation in normal cells were found to have increased about sixfold after anisomycin treatment, but only twofold or so in cells pretreated with SB203580 (Fig. 5B). Anisomycin treatment also led to significant increases (about fivefold) in JNK phosphorylation levels in normal cells. Although pretreatment with SB203580 led to somewhat smaller anisomycin-induced increases (about fourfold), there was every indication that SB203580 was far less effective at blocking JNK phosphorylation than it had been at blocking p38 MAP kinase phosphorylation. We found that cells pretreated with 10 μM SB203580 for 30 minutes were no longer subject to the anisomycin-induced reductions in GJIC experienced by cells pretreated with SB203580-free medium (Fig. 5C). To determine whether SB203580 could inhibit the anisomycin-induced suppression of protein synthesis, we examined the [³⁵S]-methionine contents of total cell lysates and found that SB203580 was not able to restore the level of protein synthesis to the control levels (Fig. 5D). Because the addition of SB203580 appeared to maintain the RR completely (Fig. 5C), it is most likely that the effect of anisomycin on GJIC was not dependent on the inhibition of protein synthesis.

The relationship between p38 MAP kinase phosphorylation/activation and the cellular distribution of Cx43 was assessed by indirect immunofluorescence with monoclonal antibodies in a confocal laser-scanning microscope. Cellular regions containing Cx43 protein molecules were readily identified as a series of brightly stained punctate maculae at the borders of unstimulated cells but significant quantities were located elsewhere (e.g. in a few small compartments adjacent to the nucleus – see the arrows in Fig. 6). By 60 minutes, there was much less Cx43 at most cell borders. These observations are consistent with the profile of anisomycin-induced GJIC loss as judged by FRAP assay results; moreover, the fact that Cx43 protein could still be detected close to cell nuclei after the addition of anisomycin seems to indicate that the anisomycin-induced decreases in GJIC were mostly a result of the loss of Cx43 molecules from cell borders, as opposed to losses from elsewhere in the cell. It is interesting that, as might have been predicted on the basis of our FRAP results, most of the losses of Cx43 molecules from the cell border regions detected in anisomycin-treated WB-F344 cells were not apparent in their SB203580-pretreated counterparts (Fig. 6).

Whole-cell lysates were prepared from cells exposed to SB203580 for 30 minutes before being incubated in the presence or absence of anisomycin. Equal amounts of protein (20 μg) were then extracted from each cell lysate and subjected to SDS-PAGE, immunoblotting and densitometric analyses (Fig. 7A,B). The cells that had been incubated with anisomycin for 60 minutes and, as a result, had lost half their capacity for GJIC turned out to have significantly reduced P1+P2 and P0 levels (Fig. 4). By contrast, cells exposed to 10 μM SB203580 before their anisomycin treatment seemed to lose very little of their P1 and P2. As expected, the P1 and P2 levels in cells exposed to SB203580 alone were virtually the same as those in the appropriate controls. Interestingly, pretreatment of cells with SB203580 did not seem to prevent anisomycin-induced loss of P0, despite their P1+P2 level appearing to be much the same in the presence and absence of anisomycin. We further analysed phosphorylation levels of Cx43 by [³²P]-orthophosphate metabolic labelling (Fig. 7C). Incorporated [³²P]-phosphates measured as relative radioactivity were augmented, despite the apparent decrease of phosphorylated forms of Cx43 at 60 minutes after anisomycin treatment. This implies that a Cx43 molecule incorporated more phosphates after anisomycin treatment than the untreated control. This phosphorylation augmentation could be almost completely inhibited by the addition of SB203580 (Fig. 7C), indicating that the excess phosphorylation caused by anisomycin is specific to activation of p38 MAP kinase.

To resolve the issue of whether p38 MAP kinase is involved in the post-translational regulation of GJIC, experiments were designed to examine whether anisomycin, an activator of p38 MAP kinase and an inhibitor to protein synthesis, could modulate GJIC in a diploid, non-tumorigenic rat liver epithelial cell line. We found that incubation with anisomycin led to marked reductions in GJIC in WB-F344 cells and that this reduction was accompanied by decreases of the phosphorylated Cx43 protein moieties with which WB-F344 cells are usually associated. The most obvious change involved significant reductions in the levels of the P1 and P2 forms of Cx43. These reductions were reflected in significant losses of Cx43 protein at cellular borders. Such findings are especially interesting in the light of suggestions by previous workers that activation of the ERK signalling pathway leads to hyperphosphorylation of Cx43 and that this in turn leads to reductions in GJIC (Hossain et al., 1998a; Hossain et al., 1999b; Kanemitsu and Lau, 1993; Warn-Cramer et al., 1998; Warn-Cramer et al., 1996).

We reported that GJIC levels are greatly reduced in human primary cultured cells by losses of phosphorylated Cx43 moieties (Ogawa et al., 2001). In the present study, we noted that the absolute levels of phosphorylated Cx43 molecules that we could detect were always significantly lower in anisomycin-treated cells whose GJIC levels had been reduced. This might mean that the decreases in GJIC that we observe in such circumstances are the result of decreases in intracellular levels of phosphorylated Cx43 moieties. Current understanding of gap junction assembly, stability and turnover is that the post-translational phosphorylation of Cx43, as shown by overall changes in species migration on SDS gels, is essential for the functioning of gap junction channels (Elvira et al., 1993; Hossain et al., 1998b; Musil et al., 1990). We therefore believe that there is an additional cause of GJIC disruption in WB-F344 cells: the rapid depletion of phosphorylated Cx43 by anisomycin and the subsequent selective regional losses of Cx43 moieties from cell borders. We found that anisomycin treatment could activate Cx43 phosphorylation and this effect was completely inhibited by the pretreatment with SB203580 (Fig. 7C). This result indicated that the anisomycin-induced activation of MAP kinase actually enhanced phosphorylation of Cx43, although it reduced the total amount of Cx43 protein as well as those of P1 and P2. This might indicate the possibility of increased phosphorylation per Cx43 molecule (Lau et al., 1992; Warn-Cramer et al., 1998; Warn-Cramer et al., 1996), although this conclusion needs further study, including the identification of phosphorylated sites. It has been suggested that the degradation of Cx43 depends on increased phosphorylation level of Cx43 (Girão and Pereira, 2003; Guan and Ruch, 1996). Thus, our interpretation of the present result is that MAP-kinase-activated Cx43 phosphorylation might promote the degradation of Cx43.

We found, as have several other groups (Barros et al., 1997; Cano et al., 1994; Cano and Mahadevan, 1995; Hazzalin et al., 1998; Kyriakis et al., 1994), that anisomycin treatment can lead to activation of both p38 MAP kinase and JNK, and that it almost certainly does so by increasing their phosphorylation levels and hence their capacity for enzymatic action. However, we also demonstrated that anisomycin-induced disruption of GJIC was completely blocked in cells pretreated with 10 μM SB203580 (a concentration that had virtually no effect on the anisomycin-induced phosphorylation of JNK). Given these observations, it seems unlikely that the JNK pathway will prove to play a significant role in the anisomycin-induced disruption of GJIC. Thus, even though anisomycin appears to be capable of provoking the phosphorylation – and hence activation – of JNK, it might well be that its ability to inhibit GJIC is due almost entirely to its ability to activate p38 MAP kinase.

In this study, we used a very high concentration (10 μg ml^-1) of anisomycin, primarily because this level was maximally effective as a GJIC inhibitor in WB-F344 cells (Fig. 2B). We were aware that anisomycin might also act as a translational inhibitor at such concentrations. A previous study (Mahadevan and Edwards, 1991) has shown that relatively low concentrations of anisomycin (around 25-50 ng ml^-1; so-called subinhibitory levels) can selectively activate the p38 MAP kinase without inhibiting protein synthesis. In our study, however, even a lower concentration of anisomycin (10 ng ml^-1) appeared to have an ability to inhibit protein synthesis, but this concentration could not activate p38 MAP kinase (Fig. 2B, Fig. 3C). This discrepancy might be due to the different cell lines used in the studies. In any event, GJIC could be maintained at the control level by SB203580 pretreatment without recovery of protein synthesis (Fig. 5D), indicating that the effect of anisomycin on GJIC was not dependent on the inhibition of protein synthesis. Our conclusion gained further support from our observation that SB203580 pretreatment of the cells appeared to be capable of preventing anisomycin-induced losses of the P1 or P2 (but not P0) forms of Cx43. Moreover, both before and after treatment in our experiments, we checked the effects on the intracellular levels of ZO-1, occludin, E-cadherin and β-catenin as reference proteins, which have short half-lives like that of Cx43. We did not detect changes in the levels of these reference proteins in any anisomycin treatment experiment.

Anisomycin is also a well known inducer of apoptosis in other cell lines (Polverino and Patterson, 1997; Stadheim and Kucera, 2002). Furthermore, the up- or downregulation of GJIC has been reported in the apoptotic process (Wilson et al., 2000). However, in our experiment, at least up to 120 minutes, anisomycin did not induce apoptosis in WB-F344 cells (data not shown).

A key finding of our present study is that pretreatment of anisomycin-exposed WB-F344 cells with SB203580 appeared to inhibit both the redistribution of Cx43 derivatives and to ameliorate or prevent the disruption of GJIC that anisomycin would otherwise have provoked. It is also important that the protection against GJIC disruption afforded by SB203580 pretreatment was, for all intents and purposes, complete. Given that SB203580 is reputed to be a highly specific inhibitor of p38 MAP kinase (Cuenda et al., 1995; Tong et al., 1997), it seems likely that the most important single event in the anisomycin-induced disruption of GJIC involves its ability to activate this particular kinase. It remains uncertain whether our interpretation will turn out to be an oversimplification, if only because WB-F344 cells pretreated with SB203580 appear to experience virtually no disruption of GJIC under circumstances in which the inhibition of p38 MAP kinase by SB203580 is not complete. Moreover, western blotting analysis revealed that SB203580 was capable of inhibiting JNK and that inhibition of p38 MAP kinase was incomplete. These observations are in accordance with a report that SB203580 is capable of inhibiting JNK when used at relatively high concentrations (Clerk and Sugden, 1998) and others that SB203580 only appears to exert its inhibitory effect on specific isoforms of p38 MAP kinase (Cuenda et al., 1995; Lee et al., 1994). Suggestions that the p38 MAP kinase and JNK pathways engage in cross talk in response to anisomycin might also be relevant (Töröcsik and Szeberényi, 2000). We are currently conducting additional experiments in the hope of obtaining a more thorough understanding of the mechanisms involved in GJIC inhibition by the p38 MAP kinase pathway.

We thank Baxter Limited Renal Division for their support in conducting this study. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and for Cancer Research from the Ministry of Health and Welfare of Japan, and by a grant from the Smoking Research Foundation. We thank E. Suzaki (Department of Histology and Cell Biology, Hiroshima University School of Medicine, Hiroshima, Japan) and K. Yamashita (Department of Anatomy and Developmental Biology, Hiroshima University School of Medicine, Hiroshima, Japan) for helpful advice.