Modulation of gap junctional intercellular communication by EGFin human kidney epithelial cells
Edgar Rivedal2, Steen Mollerup1, Aage Haugen1 andGunnhild Vikhamar
Laboratory for Environmental and Occupational Cancer, Institute for CancerResearch, The Norwegian Radium Hospital, N-0310 Oslo and 'Departmentof Toxicology, National Institute of Occupational Health, PO Box 8149Dep, N-0033 Oslo, Norway2To whom correspondence should be addressed
Modulation of gap junctional intercellular communication(GJ1C) was studied in a multistep model of human renalepithelial carcinogenesis. We report that the majority ofprimary human kidney epithelial cells (NHKE) grown fromfetal kidney explants did not communicate through gapjunctions. Communication could, however, be observedwithin a subpopulation of the cells. Ni(II)-immortalizedcells (IHKE) showed GJIC at a level of 10-20 communicat-ing cells, but with heterogeneous regions on the dish,with regard to both communication and distribution ofconnexin43. The heterogeneity was less pronounced in aras-transfected tumourigenic cell line (THKE), which alsoshowed communication of -10-20 dye-coupled cells. Com-munication within the IHKE sub-clone K7 decreased from55 dye-coupled cells communicating on day 1 after seedingto -13 in cells grown for 4 days. Daily change of growthmedium reduced the decrease in GJIC. EGF enhancedcommunication following a lag period which depended ondays in culture. The largest increase in GJIC was observedin 2-day-old cultures, where the number of communicatingcells in some experiments increased from -45 to 130 dye-coupled cells 4 h following change to medium with EGF. Theinduction was concentration dependent and communicationwas enhanced gradually between 2 and 7 h after exposureto EGF. A 15 min pulse of EGF was sufficient to inducethe GJIC increase if the total incubation period wasunchanged. Cycloheximide completely blocked the EGF-induced enhancement of communication, while actinomycinD had no effect. EGF exposure resulted in an increase inthe cellular level of connexin43 protein in parallel with theenhancement in communication. Together, these resultsindicate that the EGF-induced enhancement of GJIC inhuman kidney epithelial cells was mediated through trans-lational control of connexin43 expression.
Introduction
Direct exchange of molecules between the cytoplasm ofneighbouring cells takes place through channels located in gapjunctions and has been shown to be an important way in which
cells communicate and regulate several vital functions, suchas homeostasis, proliferation and differentiation (1-4). Thegap junction channels are made up of two opposing hexamericprotein structures, connexons, through which polar moleculesof mol. wt < 1-1.5 kDa may pass. A family of pore proteins,connexins, have been identified (5-8). Aberrant control ofgap junctional intercellular communication (GJIC*) has beenindicated to play an important role in cancer development (9-17). Tumour cells often have reduced gapjunctional communic-ating capacity (18), among themselves or with their normalsurrounding neighbour cells (18,19). GJIC has also been foundto decrease with increased metastatic potential (20), while theinduced expression of connexin protein in non-communicatingtransformed cells results in decreased growth potential, as wellas a normalized phenotype (21-23). Different types of tumourpromoting compounds have been shown to inhibit GJIC in vitro(24-27) and in vivo (28,29). This may take place through post-translational modification, such as phosphorylation (30-34),or by a decrease in connexin gene expression (35).
The proliferation of normal cells is tightly controlled by theinterplay of growth inducing (growth factors) and growthinhibitory (tumour suppressors) factors. Tumour cells arecharacterized by having abrogated normal growth regulatorycontrol (14,18,36). Many growth factors induce cellular prolif-eration and differentiation through plasma membrane tyrosinekinase receptors, thereby activating a cascade of events medi-ated through intracellular signalling proteins (37). Severalgrowth factors have been shown to affect GJIC, although themechanism for this is largely unclear (38,39). Epidermalgrowth factor (EGF) induces mitogenesis in most cells ofepithelial origin through binding to the protein tyrosine kinaseEGF receptor (EGF-R) (40). The activation of tyrosine kinasereceptors could be expected to influence GJIC through a directtyrosine phosphorylation of connexin proteins. This seems,however, not to be the case, and for EGF the inhibitory effecton GJIC has been associated with induction of phosphorylationof connexin43 on serine residues. On the other hand, tyrosinephosphorylation of connexin43 seems to be involved inppoV'^-induced inhibition of communication (41-43).
It is of importance to obtain more knowledge on themechanism of regulation of GJIC in different cell types tounderstand its role in cell behaviour. In the present work wehave studied GJIC in primary normal human kidney epithelialcells (NHKE) and cell lines derived thereof. The immortalizedand tumourigenic human kidney epithelial cell lines (IHKEand THKE) have been shown to have abrogated normal growthcontrol and to express elevated numbers of EGF-R (44). Inaddition to stimulation of growth in these cells (44,45), EGFis also a potent modulator of GJIC. Here we present evidencethat control of connexin43 protein expression is involved inEGF-mediated enhancement of GJIC.
Materials and methodsChemicalsEGF, 12-0-tetradecanoylphorbol-13-acetate (TPA) and Lucifer Yellow CHwere purchased from Sigma Chemical Co. (St Louis, MO).
Fig. 1. Compartmentalization of GJ1C in primary human embryonic kidneycells. Lucifer Yellow was microinjected into two different single cells(marked 1 and 2) and its spreading to neighbouring cells observed 10 minlater (A). Lucifer Yellow in cell no. 2 did not spread to the neighbouringcells, indicating no GJIC. From cell no. 1 Lucifer Yellow spread toneighbouring cells, but only to cells with the clonal appearance of slightlyaltered morphology. (B) Phase contrast micrograph of (A).
Cell cultures
NHKE cells were obtained by explant outgrowth culture (45). Treatment ofkidjiey explains with NiSC>4 during long-term culture resulted in immortalized,non-tumourigenic cell lines (IHKE) (45) and transfection of IHKE cells withthe v-Ha-ros oncogene resulted in tumourigenic (THKE) cells (46). Thekidney epithelial cell lines were cultured in Dulbecco's modified Eagle'smedium/Ham's F12 (1:1) supplemented with insulin (5 |ig/ml), transferrin(5 ng/ml), hydrocortsone (36 ng/ml) and sodium selinite (5 ng/ml). NHKEcells required in addition 10 ng/ml EGF and 5% fetal bovine serum (FBS),while IHKE, THKE and K7 cells showed optimal growth at 1% FBS andwere normally grown without EGF. The cell lines were maintained at 37°Cin humidified air containing 5% CO2.
Cell—cell communication
Intercellular communication was measured as spreading of microinjectedLucifer Yellow (10% w/v in 0.33 M lithium chloride) to neighbouring cellsin a monolayer. A Narishige (Tokyo, Japan) micromanipulator mounted on aNikon Diaphot microscope was used. The cells were exposed as indicated inthe figures and the test compound was present during dye transfer. The numberof dye-coupled cells was counted 10 min following injection using fluorescenceequipment attached to the same microscope.
ImmunofluorescenceCells were grown in 60 mm Petri dishes. The dishes were washed three timeswith phosphate-buffered saline (PBS) and fixed with cold (-20°C) methanolfor 30 min. The dishes were incubated with 5% (w/v) dry milk in PBS for1 h and then overnight at 4°C with a conncxin43 anti-peptide rabbit antibodydiluted 1:1000 in PBS with 1% dry milk. The antibody was raised in rabbitsagainst a synthetic peptide identical to amino acids 363-381 of the C-terminalpan of connexin43 conjugated to keyhole limpet haemocyanin. After washing
Fig. 2. Immunofluorescence of connexin43 in IHKE cells (see Materials andmethods). Three areas on the same dish with different patterns ofconnexin43 staining. (A) Punctate staining across the cell. (B) Area withlack of connexin43 punctate staining. (C) Punctate staining in nuclearmembrane.
four times with PBS the cells were incubated for 1 h at room temperaturewith FTTC-conjugated anti-rabbit IgG (BioRad) diluted 1:1000 in PBS with1% dry milk. The dishes were washed four times in PBS, mounted in Tris,pH 8.5, containing 9% Mowiol 4-88 (Hoechst, Frankfurt, Germany) and 20%glycerol and examined under a fluorescence microscope. Micrographs weretaken at a magnification of X400.
Western blottingThe cells were exposed to EGF for the indicated time. The dishes werewashed twice with PBS and the cells scraped into SDS electrophoresis samplebuffer without mercaptoethanol (10 mM Tris, pH 7.8, 1 mM EDTA, 3% SDS,5% glycerol) and sonicated three times for 5 s each in a Branson Sonifer. Theextract was heated for 5 min at 95°C and protein concentration determinedusing BioRad Detergent Compatible Protein Assay. Mercaptoethanol andbromophenol blue were added to 5% and 0.05% respectively and 5 ng proteinwere run per lane on 8% polyacrylamide electrophoresis gels (BioRad MiniProtean System) and blotted onto nitrocellulose filters. The filters were blocked
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Fig. 3. GJIC in K7 cells at different times (days) following seeding. • ,Cells were seeded at different densities (1.5-9X105 cells/dish), to give aconfluent monolayer on the day of measuring communication. Medium wasnot changed during the experiment. O, Conditions as in (A) but medium(DMEM/Ham's F12 with 1% FBS) was changed every day. *P < 0.01,Student /-test, n > 10, relative to day 1. Error bars = SD.
in 5% (w/v) dry milk in PBS and incubated for 1 h at room temperature inrabbit anti-connexin43 antibody diluted 1:1000 in PBS with 19b dry milk.After washing four times with 0.1% Tween in PBS the blots were incubatedfor 1 h at room temperature with horseradish peroxidase (HRP)-conjugatedanti-rabbit IgG antibody (BioRad, CA) diluted 1:1000 in PBS with 1% drymilk. The washing was repeated and the blots stained using 4-chloro-l-naphthol as HRP substrate. Densitometnc analysis of the blots was performedon a Macintosh computer using the public domain NTH Image program.
Results
Human kidney-derived epithelial cells are heterogeneous withregard to communicationInterestingly, the majority of the primary cells growing out ofexplants of normal embryonic kidney tissue did not communic-ate through gap junctions. The microinjected Lucifer Yellowwas in most cases confined to the injected cell and did notspread to neighbouring cells. Some exceptions to this did,however, occur and spreading of Lucifer Yellow indicatinggap junctional communication took place in certain areas onthe dish. Figure 1 shows that communicating cells consistedof clones with a slightly different morphology. The enhancedGJIC seemed to be confined to these cell clones and shut offtowards surrounding cells. This might indicate the presence ofdifferent cell types or that GJIC could be influenced by thegrowth of human kidney epithelial cells.
The nickel-immortalized cell line IHKE showed GJIC of~ 10-20 communicating cells following Lucifer Yellow micro-injection. Many of the cells injected did not, however, commun-icate at all with neighbouring cells, indicating a significantheterogeneity with regard to GJIC among the cells. Theheterogeneous communication was reflected in a variableamount and distribution of connexin43, as visualized byimmunofluorescense. In some areas on the dish the cellsshowed strong histochemical punctate staining for connexin43(Figure 2A), while other areas were devoid of connexin43
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Fig. 4. Effect of EGF/FBS on GJIC in K7 cells. Cell communication wasmeasured at different intervals following change to medium (DMEM/Ham'sF12 1:1) with 1% FBS and 100 ng/ml EGF in cells on different daysfollowing seeding. The cells were seeded at a density to give a confluentmonolayer on the day of Lucifer Yellow microinjection. A, 1 day old cellcultures; A, 2 day old cell cultures; • . 3 day old cell cultures; q, 4 day oldcell cultures. *P > 0.01, Student Mest, n > 10, relative to untreatedcontrol. Error bars = SD.
staining (Figure 2B). In yet other areas there was strongconnexin43 staining in what appeared to be the nuclearmembrane (Figure 2C). This heterogeneiety was less pro-nounced among the ras-transfected malignant (THKE) cells.These cells communicated among themselves at a similarlevel of 10-20 dye-coupled cells following microinjection ofLucifer Yellow.
Based on the observed heterogeneous communication andvariable connexin43 distribution we realized the need for amore homogeneous cell population and attempted this throughsubcloning of the IHKE cells. The cells were seeded at lowdensity for colony formation and colonies were picked byplacing a cylinder over individual colonies and adding trypsinto the inside of the cylinder. The 10 cell lines obtained weretested for connexin43 immunofluorescense and GJIC. Allcloned cell lines were found to contain connexin43 and showedsubstantial GJIC. Although the different clones appearedhomogeneous with regard to amount and distribution of con-nexin^, this differed between the different cell lines. In someclones connexin43 was mainly spread throughout the cytoplasmand in the Golgi area, while in others the connexin proteinwas situated more in cell-cell boundaries. The level of GJICwas clearly related to the amount and distribution of connexin43staining, since cells with higher amounts and more staining incell-cell boundaries showed more extensive GJIC, as judgedby microinjection of Lucifer Yellow (data not shown).
The cell line K7, cloned from the original IHKE cells, waschosen to study the effect of growth parameters on GJIC. Thiscell line expressed an intermediate amount of connexin43,mainly distributed away from cell-cell boundaries. Differenttypes of connexin have been found to be expressed in kidneywhile established cell lines tend to express only connexin43(4,7). Using RT-PCR we have found that the K7 cell lineexpresses connexin43 but not connexin32 (data not shown).
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The cells showed a high capacity for GJIC, but communicationdecreased when the cells were left on the dish for a few days.Twenty four hours after seeding ~55 cells communicated. Thisnumber dropped to ~13 in cells grown for 4 days, when thenumber of cells seeded was adjusted to give a confluentmonolayer on the day of injection (Figure 3). The decrease inGJIC following incubation was smaller when the cells wereseeded at higher density to give confluence after 1 day ofincubation (data not shown). This indicates that the time periodafter the cells reached confluence was not the only criticalfactor, but that possibly depletion of nutrients or growthstimulatory components in the medium affected the commun-ication capability of the cells. This was further supported bythe finding that change of growth medium every day partlyprevented the decrease in GJIC (Figure 3).
The K7 cells were normally grown in medium containing1% FBS and without EGF. Medium change to fresh mediumcontaining 1% FBS and 100 ng/ml EGF resulted in a largeincrease in communication following a lag period (Figure 4).The length of this lag period depended on days in culture, i.e.if the medium was changed 1 or 2 days following seeding,the enhancement in GJIC occurred after 4 h incubation, whilecells grown in culture for 3-4 days required longer incubationfor GJIC enhancement to appear. In 24 h cultures GJICincreased from ~40 to 85 communicating cells after 4 hincubation, while 4-day-old cultures shifted from <10 to ~70communicating cells in 18 h. The largest increase in GJICwas observed in cells cultured for 2 days, where the numberof communicating cells in some experiments increased from~45 to >100 4 h following change to medium with EGF.Figure 5 shows representative micrographs of microinjectedLucifer Yellow in 2-day-old K7 cell cultures without mediumchange (Figure 5A), 4 h exposure to 100 ng/ml EGF (Figure5B) and 1 h exposure to 0.1 ug/ml 12-O-tetradecanoylphorbol-13-acetate (TPA. The tumour promoter TPA was a potentinhibitor of GJIC in these cells (Figure 5C), as in most othercell types.
EGF is the essential component for enhancement of GJIC inK7 cells
Figure 6 shows that EGF was the essential compound forenhancement of GJIC in human kidney epithelial cells. Thisexperiment was performed on K7 cells after 2 days in culture.Four hours incubation in growth medium with 1 mg/ml bovineserum albumin (BSA) or 1% FBS had no effect on cellcommunication. There was no significant difference in theenhancement of GJIC induced by EGF alone and by EGF incombination with FBS, demonstrating that EGF is the essentialcomponent and that there seemed to be no cooperative orsynergistic effect of growth factors or other components inFBS. This experiment does not, however, rule out the possibilityof potentiating effects of FBS at lower and sub-maximal effectconcentrations of EGF. The presence of EGF was not neededthroughout the incubation period. Fifteen minutes incubationwith 100 ng/ml EGF followed by medium containing 1% FBSfor the rest of the 4 h incubation period resulted in the sameenhancement of GJIC as did the presence of EGF for thewhole period (Figure 6). This indicates that the responseinduced by the EGF pulse was sufficient for GJIC enhancementin these cells.
Figure 7A shows that incubation with 1 ng/ml EGF for 4 hhad no effect in GJIC, but that 10 ng/ml or higher EGFconcentrations strongly enhanced communication in K7 cells,
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which correlated with the EGF concentration (~3 ng/ml)needed to induce maximum DNA synthesis and proliferationin IHKE cells (unpublished results). Incubation of K7 cellswith 100 ng/ml EGF for 1 h or less had no effect on GJIC(Figure 7B). Communication, however, increased graduallybetween 2 and 7 h exposure to EGF. At 18 h incubation thelevel of communication was lower than at 4 and 7 h, but wasstill elevated compared with cells without medium change(data not shown).EGF-induced enhancement of GJIC may be mediated throughtranslational regulationExposure of K7 cells for 4 h to actinomycin D (1 (ig/ml) hadno effect on GJIC and very little effect on the EGF-induced
Fig. 5. GJIC in immortalized K7 cells. Spreading of microinjected LuciferYellow. (A) Unexposed 2-day-old cell cultures. (B) Two-day-old cellcultures exposed to 100 ng/ml EGF for 4 h. (C) Cells exposed for 1 h to0.1 ng/ml TPA.
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enhancement of communication, indicating that the enhance-ment was not mediated through transcription (Figure 8).Cyclo-heximide (10 ug/ml), however, completely blocked the EGF-induced enhancement of communication when present 30 minprior to and during the 4 h incubation with EGF, indicatingthat the EGF-induced enhancement of GJIC was mediatedthrough translational regulation (Figure 8).
Immunoblotting of the K7 cells for connexin43 revealedmainly two bands, equivalent to the non-phosphorylated formPo and the phosphorylated form Pl. Exposure to EGF resultedin the transient appearance of a smear of bands with higher
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Fig. 6. Effect of EGF and FBS on GJIC in 2-day-old K7 cell cultures.Communication was measured 4 h following change to growth mediumwith the following components: 1, No medium change; 2, 1 mg/ml BSA; 3,1% FBS; 4, 1 mg/ml BSA + 100 ng/ml EGF; 5, 1% FBS + 100 ng/mlEGF; 6, 15 min exposure to 100 ng/ml EGF and 3 h 45 min exposure to1% FBS. *P > 0.01, Student f-test, n > 10, relative to 1. Error bars = SD.
molecular weight, including one with a molecular weightsimilar to the phosphorylated connexin43 form P2. The P2smear appeared after a few minutes incubation with EGF, butdisappeared during the first hour (data not shown). The relativeamount of total connexin43 protein in the K7 cells followingdifferent times of exposure to EGF was determined by densito-metric scanning of immunoblots. Figure 9 shows the integrateddensity of the connexin43 bands following EGF exposure.Connexin43 protein level increased in a time-dependent man-ner. Enhancement of the connexin43 level was observed after1 h, reaching a maximum level at 2-7 h exposure. Thisenhancement in amount of gap junction protein precedes theenhancement in cell communication, which could indicate thetime needed following protein synthesis to make the gapjunction channels functional. Together with the responseobtained with cycloheximide, this indicates that the enhance-ment of GJIC induced by EGF was caused by a translationalup-regulation of connexin43 synthesis. As shown in Figure 9,the total cellular protein was not significantly different fromthat of unexposed cells at any time point up to 7 h after EGFexposure. Immunohistochemical staining for connexin43 gaveno indication of a change in connexin localization followingEGF exposure (data not shown).
Discussion
Carcinogenesis is known to be a multistep process involvingan initial irreversible genetic change (initiation) followed bythe clonal expansion of the initiated cells (promotion) and thelater acquisition of additional genetic alterations resulting inmalignancy (progression). Initiation and progression seems tobe driven by mutagenic insults, while the promotion stage isreversible and associated with selective proliferation. GJIChas been demonstrated to be of significant importance inmany biological processes, including regulation of growth anddifferentiation (3,4,18,47). The finding that many tumourigeniccells have aberrant GJIC and that many tumour promoters,oncogene products and growth factors inhibit communicationsupport the hypothesis that this type of communication is of
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Fig. 7. Cell communication in 2-day-old cell cultures measured after (A) 4 h incubation in medium with different concentrations of EGF (+ 1 mg/ml BSA);(B) different lengths of incubation in medium containing 100 ng/ml EGF (+ 1 mg/ml BSA). *P < 0.01, Student r-test, n > 10, relative to no EGF. Errorbars = SD.
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1 2 3 4 5 6Fig. 8. Effect of actinomycin D and cycloheximide on the EGF-inducedenhancement of GJIC. Two-day-old cell cultures were incubated for 4 hwith 100 ng/ml EGF (+ 1 mg/ml BSA) with or without actinomycin D orcycloheximide. 1, No medium change; 2, 100 ng/ml EGF; 3, 10 Hg/mlcycloheximide; 4, 100 ng/ml EGF + 10 )ig/ml cycloheximide; 5, 1 (ig/mlactinomycin D; 6, 100 ng/ml EGF + 1 |ig/ml actinomycin D. *P > 0.01,Student (-test, n > 10, relative to 1. Error bars = SD.
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importance in carcinogenesis (14,48,49). Tumour promotingsubstances are believed to mediate their effect through induc-tion of clonal expansion of the initiated cell population.Inhibition of GJIC is one of several changes seen in cells afterexposure to tumour promoters and may take place throughdifferent mechanisms involving transcriptional or translationaldown-regulation of gap junction proteins or by modificationof intracellular control mechanisms involved in the formationof functional gap junctions (29,50-52). The down-regulationof communication between preneoplastic cells and their normalsurrounding cells may allow the preneoplastic cells to clonallyexpand by selective proliferation (18,19,53).
There are several lines of evidence to link GJIC to growthregulatory signals. In many instances the enhancement of
communication has been related to inhibition of cellular growthand, conversely, reduced communication with stimulation ofgrowth (4,18,36). EGF decreased GJIC in normal humankeratinocytes following 24 h treatment and enhanced DNAsynthesis and cell proliferation (54). On the other hand, forcedhaemopoietic stem cell division was found to cause a 100-foldincrease in connexin43 expression. The level of connexin43expression was also 80-fold higher in normal adult haemo-poietic tissue than in neonatal marrow, where fewer cells aredividing. This suggested that GJIC in these cells may be apositive signal for cell division (55). Interleukins 1 and 6 havealso been shown to be involved in the induced enhancementof connexin43 in human aortic wall cells (56). At the sametime, the incorporation of connexin genes into the genome intumourigenic and communication-deficient cell lines has beenshown to up-regulate communication and normalize the growthpattern (10,57-59). Decreased synthesis of growth factorscombined with increased levels of growth inhibitors have beenindicated as being responsible for reduced proliferation capacityin rat glioma cells transfected to overexpress connexin43 (60).A number of growth factors and oncogene products have beenshown to influence GJIC, but their effects seem to varybetween different cell types. The growth factors EGF, PDGFand bFGF interact with and activate receptor proteins withtyrosine kinase activity. Thus, it might be anticipated that theactivation of these receptors would result in a direct tyrosinephosphorylation of connexin proteins and thereby influencegap junction permeability. This seems, however, not to be thecase. The inhibitory effect of EGF on GJIC has been associatedwith activation of MAP kinase and elevated phosphorylationof connexin43 on serine residues (61). This is unlike the casefor yy&)v~src, where the induced inhibition of communicationwas associated with tyrosine phosphorylation of connexin43(41^3).
The study of human carcinogenesis has been hampered bythe fact that human cells are largely refractory to in vitrotransformation (62). The development of a multistep model ofhuman renal epithelial carcinogenesis in vitro, consistingof primary (NHKE), Ni(II)-immortalized (IHKE) and ray-transformed (THKE) human kidney epithelial cells has beendescribed. Normal human kidney epithelial cells have beenimmortalized by nickel(II) (45) and further neoplasticallytransformed with the v-Ha-ras oncogene (46). This modeloffers a human cell system where cellular changes duringcarcinogenesis may be studied. GJIC in the IHKE-derived K7cells decreased with time in culture. Communication on day4 following seeding of the cells was reduced to ~20% of thecommunication on day 1. The drop in communication wassignificantly smaller if the growth medium was changed everyday. GJIC has also been found to decrease in senescent humanumbilical vein endothelial cells in parallel with decreasedconnexin43 mRNA and protein, while EGF decreased GJICin young cells but showed no effect in senescent cells (63).Cell communication in primary rat hepatocytes was also foundto decrease rapidly and even disappear within 12-24 h inculture, depending on type of growth medium. Glucagonand linoleic acid delayed this decrease in communication.Treatment of the cells showing decreased communication withglycosaminoglycans or proteoglycans, but not with hormonesor growth factors, resulted in synthesis of connexin proteinand re-occurrence of communication (64). In Madin Derbycanine kidney (MDCK) cells, GJIC was found to be dramatic-ally reduced after confluence (65). Exposure of the present
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human kidney epithelial cells to EGF at different concentrationsfor from 5 min to 20 h was never found to decrease communica-tion. In contrast, EGF caused a strong increase in GJIC inthese cells. The time needed to increase communication was,however, dependent on days in culture of the cells. One- or2-day-old cells showed increased communication after 2-4 hexposure to EGF, while older cells needed a longer timefollowing EGF exposure for enhancement to occur. It was,however, demonstrated that a 15 min pulse of EGF wassufficient to induce enhancement of GJIC if the total incubationperiod was unchanged. This is in contrast to the observationthat EGF induction of mitosis requires the continuous presenceof the ligand for up to 8 h (66). In addition, we have shownthat EGF-induced protein tyrosine phosphorylation in HIKEcells was dependent on the presence of EGF throughout theincubation period (unpublished data). Early passage normalhuman fetal kidney epithelial cells have previously also beenshown to be heterogeneous with regard to growth potentialand deficiency in GJIC (67). A fat storing cell line (CFSC) alsoshowed heterogeneous expression of connexin43, proliferationand extracelluar matrix components (68).
GJIC has been shown to be modulated through geneticchanges such as mutation (69,70) and transcriptional regulation(13,50,71), as well as post-translational changes (52,72) inconnexins. The present data indicate that the enhancement ofGJIC by EGF is mediated through enhanced translation ofconnexin43. To our knowledge, this is the first report to showincreased GJIC in response to EGF. The transient duration ofthe enhancing effect of EGF on GJIC may be the reason whysuch enhancement has not been observed in other cell types.It is, however, a general impression from the literature thatgrowth factors may have opposing effects on GJIC in differentcell types. Further studies are needed to fully understand thecontrol of GJIC in human renal epithelial cells and its role ingrowth regulation and carcinogenesis.
AcknowledgementsThis study was supported by the Norwegian Cancer Society and a grant fromEU programme CT93-0860.
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Received on January 17, 1996; revised on July 8, 1996; accepted on July26. 1996
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