Kidney International, Vol. 57 (2000), pp. 1915–1926

PDGF regulates gap junction communication and connexin43phosphorylation by PI 3-kinase in mesangial cells

JIAN YAO, TETSUO MORIOKA, and TAKASHI OITE

Department of Cellular Physiology, Institute of Nephrology, Niigata University School of Medicine, Niigata, Japan

PDGF regulates gap junction communication and connexin43 ions, secondary messengers, and small signaling mole-phosphorylation by PI 3-kinase in mesangial cells. cules. Intercellular communication via the gap junction

Background. Gap junctional intercellular communication (GJIC) is thought to play an important role in the regula-(GJIC) plays an important role in the regulation of cell growth,tion of embryonic development, in the maintenance ofmigration, and differentiation. Ultrastructural and histochemi-tissue and organ homeostasis, and in the control of cellcal studies indicate the existence of a high density of gap junc-

tions among mesangial cells (MCs), but little is known about growth, migration, differentiation, and electric coupling.their regulation. Because of the close link between growth GJs are formed by specified proteins termed connexins,and GJIC, we examined how platelet-derived growth factor which are coded by a multigene family. Fourteen con-(PDGF) may affect GJIC in cultured MCs.

nexin molecules have been identified thus far. AmongMethods. MCs were exposed to PDGF in the presence orthem, connexin43 (Cx43) is expressed most abundantlyabsence of phosphatidylinositol 39 kinase (PI3K) inhibitors,

and GJIC was evaluated by the transfer of Lucifer yellow. The by a variety of cell types [reviewed in 1–4].gap junction protein connexin43 (Cx43) was examined by immu- In the glomerulus, the endocapillary region is a special-nohistochemistry, immunoprecipitation, and Western blot.

ized microvasculature composed of a capillary lumen,Results. The addition of PDGF into MC culture caused aendothelial cells, mesangial cells (MCs), and extracellu-rapid and transient inhibition of GJIC, with maximal inhibition

(80%) occurring 15 minutes after PDGF exposure and re- lar matrix, including glomerular basement membraneturning to control levels after 90 minutes. This action of PDGF (GBM). MCs form a tree-like branching network fromcould be largely prevented by pretreatment of MCs with the the hilar site to glomerular capillary loops and connectPI3K inhibitor LY294002. Immunochemical staining showed

with each other. An intriguing feature of the mesangiumthat PDGF did not alter the localization and distribution of Cx43.resides in the remarkably high density of GJs betweenImmunoprecipitation studies demonstrated that PDGF induced

a rapid and transient increase of tyrosine phosphorylation of both glomerular and extraglomerular MCs. This findingCx43 protein, which was dose dependent and in accordance was first reported in the rat kidney using freeze-fracturewith the time course of the disruption of GJIC. PDGF also techniques [5] and was subsequently confirmed by immu-elicited activation of extracellular signal-regulated kinase (ERK).

nohistochemistry and reverse transcription–polymeraseUsing two structurally unrelated PI3K inhibitors, wortmaninchain reaction (RT-PCR) [6–8]. Furthermore, culturedand LY294002, both tyrosine phosphorylation of Cx43 and

activation of ERK stimulated by PDGF were largely blocked. rat and human MCs were shown to express Cx43 at bothConclusion. These results suggest that PDGF abrogates the mRNA and the protein level [9]. Additionally, the

GJIC function in MCs via the PI3K-dependent signaling path- presence of GJIC between cultured rat MCs and its po-way. Disruption of GJIC by PDGF could be one mechanismtential role in the propagation of calcium waves wereby which PDGF modulates MC behavior. Participation of PI3Kreported by Ijima, Moore, and Goligorsky [10]. It hasin the regulation of GJIC demonstrates the complex coordina-

tion of molecular events that accompany MC mitogenesis. been suggested that GJ, acting as a sophisticated cellcommunication system, bridges each MC between thejuxtaglomerular region (extraglomerular mesangium)

Gap junctions (GJs) are clusters of transmembrane and the glomerular mesangium, and provides the mesan-channels that permit the direct intercellular exchange of gium with the characteristics of a functional syncytium

[5, 10, 11].Little is known about the exact role of GJIC in theKey words: mitogenesis, platelet-derived growth factor, gap junction,

cell development, phosphatidylinositol 39 kinase. mesangium. One of the generally accepted functions ofGJIC is the regulation of cell growth. ConsiderableReceived for publication August 12, 1999amounts of evidence support the viewpoint of a closeand in revised form November 23, 1999

Accepted for publication December 18, 1999 link between growth and GJIC. In most cases, an inverserelationship appears to exist between the growth and 2000 by the International Society of Nephrology

1915

Yao et al: PDGF regulates gap junction communication1916

expression of connexin/GJIC. For example, the number METHODSof GJs and the capacity for GJIC are reduced or absent Materialsin many types of neoplastic cells [12, 13], and transfection Lucifer yellow (LY), LY294002, phenylarsine oxideof connexin genes into communication-deficient tumor (PAO), wortmanin, horseradish peroxidase-conjugatedcells establishes GJIC and partially reverses the tumori- rabbit antimouse IgG, and Dulbecco’s modified Eagle’sgenic phenotype [14, 15]. Also, GJIC can be inhibited by medium (DMEM) were obtained from Sigma (St. Louis,mitogens, such as peptide growth factors [16–19], tumor MO, USA). PD98059 was from Biomol (Plymouth Meet-promoters and carcinogens [13, 20], and the src [21, 22] ing, PA, USA). Polyclonal rabbit anticonnexin 43 andand ras [23] oncogenes. Agents that transform cells, such recombinant protein G-sepharose were purchased fromas polyoma virus middle T antigen, SV40 transforming Zymed Labs (South San Francisco, CA, USA). Mono-

clonal anticonnexin 43 antibodies came from Chemicongenes and Ad E1 A, generally disrupt GJIC [24]. InInternational (Temecula, CA, USA). Phospho-p44/42contrast, GJIC among fibroblasts is enhanced by growthantibody and p44/42 MAPK antibody were from Newinhibitors, transforming growth factor-b [16], and reti-England BioLabs (Beverly, MA, USA). Recombinantnoids [25]. Collectively, this indirect evidence supportshuman PDGF-bb was obtained from Pepro Tech ECthe proposed role of GJIC in regulating cell proliferation,(London, UK). Immobilon PVDF membrane was fromby providing a means for the direct intercellular ex-Millipore (Bedford, MA, USA). Enhanced chemilumi-change of either positive or negative growth signals. Innescence reagents were obtained from Amersham (Ar-this context, GJIC may be an important mediator oflington Heights, IL, USA).pathological conditions characterized by abnormal MC

growth, such as mesangial proliferative glomerulone- Rat mesangial cell culturephritis. As a first step toward understanding the involve-

Mesangial cell isolation and culture were performedment of GJIC in MC proliferation, we examined GJIC as described previously [35]. In brief, the renal corticesin cultured MC in response to platelet-derived growth of male Wistar rats (150 g) were homogenized underfactor (PDGF), which is the most potent mitogen for sterile conditions and passed over three sieves with poreMCs and plays a critical role in the pathogenesis of mes- sizes of 200, 100, and 75 mmol/L. Glomeruli, which wereangial proliferative glomerulonephritis [26, 27]. retained on the 75 mmol/L sieve, were seeded in DMEM

Platelet-derived growth factor mediates its cellular containing 20% fetal calf serum (FCS), insulin (5 mg/mL),functions via activation of its receptor tyrosine kinase, penicillin (100 U/mL), and streptomycin (100 U/mL).followed by the recruitment and activation of several After three to four passages in DMEM containing 20%signaling molecules. These signaling molecules then initi- FCS, pure MC populations were obtained. MCs wereate specific signaling cascades, finally resulting in dis- characterized by the following criteria: positive immuno-

cytochemical staining with antibodies against Thy-1.1tinct physiological effects [28]. The mitogenic signal ofand smooth muscle a-actin. The absence of MC stainingPDGF in MCs has been reported to be mediated bywas documented for the following antigens: factor VIIIphosphatidylinositol 39 kinase (PI3K) and ras-raf mito-and cytokeratins 5 and 8. MCs were used for experimentsgen-activated protein kinase [MAPK or extracellular sig-at passages 5 to 20.nal-regulated kinase (ERK)]. Inhibition of PI3K blocks

PDGF-induced activation of MAPK, as well as MC pro-Measurement of gap junctional intercellular

liferation [29]. Because MAPK has also been considered communicationto play a central role in the regulation of GJIC through

Gap junctional intercellular communication was as-phosphorylating Cx43 on ser/thr residues in response tosessed by transfer of the membrane-impermeant fluo-a variety of growth factors, including PDGF [30–34], werescent dye LY after a single-cell microinjection with anspeculated that PI3K signaling might be an importantautomated microinjection system from Zeiss company

player, actively participating in PDGF-elicited regula- (Zeiss Oberkochen, Jena, Germany) [17]. Briefly, con-tion of GJIC in MCs. Therefore, the second goal of this fluent MCs in 3 cm dishes were starved for two days instudy was to test this hypothesis. 0.5% FCS-DMEM and then stimulated with PDGF in

In this article, we present data showing the presence the presence or absence of PI3K inhibitors for the peri-of GJIC in cultured MCs, and that it is subject to regula- ods of time indicated, and then cells were microinjectedtion by PDGF. We also looked at the influence of PDGF with a mixture of LY (10% dissolved in 0.33 mol/L lith-on localization, distribution, as well as phosphorylation ium chloride) and ethidium bromide (0.5 mg/mL; forof Cx43. Furthermore, the role of PI3K in the PDGF- nuclear staining) using a Zeiss-Eppendorff automatedinduced disruption of GJIC and tyrosine phosphoryla- microinjection system at pressures of 800 hectopascals

applied for 0.8 seconds. After microinjection, cells weretion of Cx43 was established.

Yao et al: PDGF regulates gap junction communication 1917

washed with phosphate-buffered saline (PBS), and intra- b-mercaptoethanol in 62.5 mmol/L Tris-HCl, pH 6.8, for30 minutes at 608C, and reprobed for Cx43, as describedcellular LY/ethidium bromide fluorescence was exam-

ined under a fluorescence microscope immediately there- previously in this article.after (total diffusion time, 3 to 5 min). The number of

Immunocytochemistrycells exhibiting dye labeling was counted.Immunocytochemical staining for Cx43 in cultured

Immunoprecipitation MCs was done as described previously [35]. MCs wereseeded onto eight-well chamber slide (Nunc, Naperville,Mesangial cells were seeded onto 10 cm culture plates

and allowed to grow in 20% FCS-DMEM until 90% con- IL, USA) and grown until subconfluence. The cells werestarved for two days in low-serum medium (0.5% FCS-fluence. Then MCs were starved in low serum DMEM

(0.5% FCS) for two days, before stimulating with differ- DMEM) before being exposed to PDGF, in the presenceor absence of PI3K inhibitors, for 30 minutes. The me-ent agents for various periods of time. The reaction was

terminated by washing cells rapidly with cold PBS at dium was then removed, and MCs were rinsed in PBSand fixed in paraformaldehyde (3%, 20 min 48C). Free48C. The cells were lyzed with 500 mL RIPA lysis buffer

[50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% aldehyde groups were blocked with ammonium chloride(50 mmol/L in PBS, 20 min, 48C). Cells were then perme-Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl

sulfate (SDS)] containing 25 mg/mL aprotinin, 2 mmol/L abilized with 1% Triton X-100. After blocking the non-specific binding with FCS (20 min, 208C), slides weresodium orthovanadate, 25 mg/mL leupeptin, 2 mmol/L

phenylmethylsulfonyl fluoride, and 50 mmol/L sodium incubated overnight with the anti-Cx43 antibody (diluted1:100 in 1% FCS in PBS, 48C) and washed with PBS,fluoride for 30 minutes on ice. Lysates were clarified by

centrifugation at 13,000 r.p.m. for 15 minutes at 48C, and and the secondary antibody (diluted in 1% FCS in PBS,378C) was added for two hours before final washing. Theprotein concentrations were determined using the Bio-

rad protein assay kit. Lysates were adjusted to equal slides were covered with Tris-buffered moviol, pH 8.6,and microscopy was performed using a Leitz Aristoplanprotein concentrations and volumes and precleared for

two hours at 48C by tumble incubation with protein microscope with a 1003 Planapo and 570 nm emissionfilter. MCs were photographed using Kodak T-MAXG-sepharose beads and 1 mg/mL of control mouse IgG.

The beads were sedimented by brief centrifugation, and (400 ASA) film (Eastman-Kodak, Rochester, NY, USA).Controls with inappropriate second or primary antibod-immunoprecipitations were performed by incubating the

precleared lysates with the designated antibodies (0.5 to ies did not exhibit significant immunostaining.1 mg/mL) together with protein G-sepharose for four

Statistical analysishours at 48C in a rotary mixer. The beads were washedfour times with ice-cold RIPA lysis buffer and then twice Statistical analyses were performed by unpaired, two-

tailed Student’s t-test. Data are presented as mean 6with the same buffer containing 500 mmol/L NaCl. Im-munoprecipitates were boiled in reducing SDS sample SD. P values of ,0.05 were considered as statistically

significant.buffer for five minutes and applied to the SDS gel.

Western blotsRESULTS

Equal amounts of cell lysates or immunoprecipitatesStaining for Cx43 in cultured rat MCswere separated in 10% SDS-polyacrylamide gels. The

separated proteins were electrotransferred to 0.4 mmol/L First, we confirmed the presence of the main GJ protein,Cx43, in the cultured MCs by histochemical staining ofpolyvinylidine difluoride membranes. The membranes

were blocked with 3% bovine serum albumin (BSA) in confluent MCs with an antibody recognizing the C-termi-nal tail of Cx43. Figure 1 demonstrates the characteristicPBS-0.1% Tween-20, pH 7.4, overnight at 48C. After

washing with PBS-0.1% Tween, membranes were incu- spotted Cx43 staining in the membranes of MCs at theregions of cell–cell contact, with little intracellular local-bated with antiphosphotyrosine antibody (1:1000) or

anti-Cx43 antibody (1:1000) or antiphosphorylated MAP ization.(1:1000) kinase antibody at room temperature for one

Inhibition of GJIC by PDGFhour. After extensive washing with three changes ofPBS-0.1% Tween 20, filters were incubated for one hour Platelet-derived growth factor has been reported to

inhibit GJIC in several cell lines [17, 18, 36]. To examinewith horseradish peroxidase-conjugated sheep antirabbitIgG or rabbit antimouse IgG at 1:10,000 dilution in whether PDGF influences GJIC in cultured MCs, the

transfer of LY was studied. Microinjection of this fluo-blocking buffer. After washing, immunoreactivity was de-tected by using the enhanced chemiluminescence (ECL) rescent dye into a single cell in a confluent MC culture

typically yielded a network of about 10 to 20 cells thatsystem. To assess the amount of Cx43 protein loaded,filters were then treated with 2% SDS, 100 mmol/L then became positive (Fig. 2 A–C). PDGF (10 ng/mL)

Yao et al: PDGF regulates gap junction communication1918

Fig. 3. Time course of platelet-derived growth factor (PDGF)-induceddisruption of gap junctional intercellular communication (GJIC) in

Fig. 1. Presence and localization of connexin43 (Cx43) in mesangial cultured MCs. Confluent MCs were treated with PDGF (10 ng/mL) forcell (MC) monolayers. Immunofluorescent staining of cultured MCs the indicated lengths of time. GJIC was assessed by Lucifer yellowwith monoclonal anti-Cx43 antibody. Note the characteristic spotted microinjection into single cell. Data points represent the mean 6 SDstaining of Cx43 at cell–cell contact areas (magnification 3400). of cells (N 5 10) to which Lucifer yellow was transferred from the

injected cell within three minutes. Similar results were obtained in twoadditional experiments.

Fig. 2. Lucifer yellow diffusion from microinjected MCs. Lucifer yellow was pressure injected, together with ethidium bromide, into a single MCin a monolayer. Lucifer yellow diffusion into adjacent cells was monitored over a three-minute time period. (Upper panels) MCs without pretreatmentwith PDGF. (Lower panels) MCs pretreated with PDGF for 15 minutes before injection. (A and D) Phase-contrast micrographs. (B and E) Luciferyellow fluorescence. (C and F) Ethidium bromide staining of the microinjected cell (magnification 3320). The arrow indicates the microinjected cell.

Yao et al: PDGF regulates gap junction communication 1919

caused a dramatic disruption of intercellular communica-tion in MCs after 15 to 30 minutes of treatment (Fig. 2D–F and Fig. 3). A detailed time-course study showedthat the inhibitory effect of PDGF on GJIC was alreadyobvious after 5 minutes of treatment and was maximalat 15 to 30 minutes; after that, the number of communi-cating cells gradually increased and by 90 minutes ap-proached normal levels (Fig. 3).

Prevention of PDGF-induced disruption of GJIC byPI3K inhibitor

Phosphatidylinositol 39-kinase has been demonstratedto be critical in mediating several aspects of PDGF ac-tions in MCs and also other cell lines [29, 37, 38]. Toexplore the potential role of PI3K signaling in the signal- Fig. 4. Prevention of PDGF-induced disruption of GJIC by PI3K in-ing processes involved in the PDGF-induced disruption hibitor LY294002. Confluent MCs were pretreated with either 100

mmol/L of LY294002 or were left untreated before exposure to 10of GJIC in MCs, we measured GJIC in MCs with orng/mL of PDGF for 15 minutes. GJIC was assessed by Lucifer yellowwithout pretreatment with the PI3K inhibitor LY294002 microinjection into a single cell. Data points represent the mean 6 SD

(100 mmol/L) before exposure to 10 ng/mL PDGF for of the number of cells (N 5 10) to which Lucifer yellow was transferredfrom the injected cells within three minutes of injection. *P , 0.01 vs.15 minutes. As shown in Figure 4, preincubation of MCscontrol; #P , 0.01 vs. PDGF alone. Similar results were obtained inwith LY294002 (100 mmol/L) for one hour almost com- two additional experiments.

pletely prevented the inhibition of GJIC caused byPDGF, while the inhibitor itself did not exert much in-fluence on GJIC, as compared with the control.

PI3 inhibitors block the MAPK activationPrevention of PDGF-induced tyrosine induced by PDGFphosphorylation of Cx43 by PI3K inhibitors

Previous studies in other cell lines have suggested thatThe rapid and transient disruption of GJIC by PDGF the disruption of GJIC elicited by PDGF was due to

might be due to the reduction or redistribution, as well serine phosphorylation of Cx43 following the activationas increased phosphorylation of the connexin proteins. of MAPK, rather than tyrosine phosphorylation [31, 36].The preventive effect of PI3K inhibitor prompted us Therefore, we examined the activation of MAPK (ERK)to examine these possible mechanisms in more detail. by PDGF and the influence of PI3K inhibitors on thisImmunochemical staining showed that there was no de- activation. To approach this question, we used a phos-tectable alteration of the localization of Cx43 protein in

photyrosyl-ERK1/ERK2–specific antibody that reactsMCs after exposure to PDGF (10 ng/mL) for 30 minutes,

only with the tyrosine-phosphorylated, active form ofeither with or without pretreatment of MCs with PI3KERK1/ERK2. As demonstrated in Figure 7A, PDGFinhibitor LY294002 (100 mmol/L; data not shown). Im-quickly induced activation of ERK, which peaked at fivemunoprecipitation of Cx43 with anti-Cx43 antibody andminutes and lasted for at least one hour. This action ofthen immunoblotting with an antityrosine phosphoryla-PDGF was dose dependent. The equal presence oftion antibody showed that PDGF induced a rapid tyro-ERK1/ERK2 protein was confirmed by using an anticon-sine phosphorylation of Cx43 protein, which increasedtrol ERK antibody (Fig. 7B). PI3K inhibitor, LY294002from 5 minutes and peaked at 15 minutes, and after 30and wortmanin could largely prevent the activation ofminutes, the phosphorylation level gradually decreasedboth ERK1 and ERK2 (Fig. 8).(Fig. 5A). These effects of PDGF were dose dependent

(Fig. 5B). Consistent with the data showing that the PI3Involvement of both tyrosine phosphorylation of Cx43inhibitor LY294002 prevented PDGF-induced disrup-and MAPK activation in the disruption of GJICtion of GJIC, the increased phosphotyrosine levels of

PI3K inhibitors could block both tyrosine phosphory-Cx43 induced by PDGF could also be largely blockedlation of Cx43 and MAPK activation. Then we askedby pretreatment of the cells with LY294002 (Fig. 6 A,whether both of them are really involved in mediatingB). The effects of LY294002 were dose dependent, andthe PDGF-induced disruption of GJIC in MCs. To deter-the concentration needed for 50% inhibition of tyrosinemine the role of tyrosine phosphorylation of Cx43 inphosphorylation elicited by PDGF was about 30 mmol/LGJIC, we examined the effect of a tyrosine phosphatase(Fig. 6 C, D). A structurally unrelated PI3K inhibitor,inhibitor, PAO, on GJIC. The addition of PAO (5wortmanin, also significantly inhibited PDGF-elicited ty-

rosine phosphorylation of Cx43 (data not shown). mmol/L) into MC culture caused a rapid and remarkable

Yao et al: PDGF regulates gap junction communication1920

Fig. 5. Time- and dose-dependent inductionof tyrosine phosphorylation of Cx43 proteinby PDGF in MCs. Confluent MCs were treatedwith PDGF (10 ng/mL) for the lengths of timeindicated (A) or with indicated concentrationsof PDGF (B) for 15 minutes. Cellular proteinswere then harvested and immunoprecipitatedwith anti-Cx43 antibody (A, monoclonal anti-Cx43; B, polyclonal rabbit anti-Cx43 anti-body). After SDS-polyacrylamide gel electro-phoresis and transfer to PVDF, blots wereprobed with antityrosine mAb PY20 (left) orstripped and reprobed with anti-Cx43 anti-body (right) to verify that the difference intyrosine phosphorylation of Cx43 was not dueto differences in the loading of proteins. Blotsshown are from one representative experi-ment from a series of three with similar results.

increment of tyrosine phosphorylation of Cx43 (Fig. 9). Platelet-derived growth factor-mediated interruptionPAO also induced an obvious reduction of GJIC, albeit of GJIC was previously reported in several cell lines [17,to a lesser extent compared with PDGF (Fig. 10). These 18, 36]; however, the effects of PDGF (or other peptideresults support the role of tyrosine phosphorylation of growth factors) on GJIC in MCs, as well as the underly-Cx43 in the disruption of GJIC. However, the much ing mechanisms and signal transduction pathways impli-more potent action of PDGF on the reduction of GJIC, cated, have not been fully examined so far. Here, weas compared with PAO (Fig. 10), suggests the presence reported several novel findings related to the regulationof additional mechanisms other than tyrosine phosphor- of GJIC by PDGF. First, PDGF-induced disruption ofylation. GJIC in MCs was accompanied by clearly increased tyro-

We then attempted to address whether MAPK is one sine phosphorylation of Cx43. Second, the disruption ofof the other effectors of PDGF-mediated closure of GJIC, as well as the increased tyrosine phosphorylationGJIC. For this purpose, inhibition of MAPK activation of Cx43 elicited by PDGF, was mediated by a PI3Kwas achieved by using the specific MEK inhibitor signal transduction pathway in MCs.PD98059. Pretreatment of MCs with the 100 mmol/L The addition of PDGF into MC culture resulted in aPD98059 almost completely blocked the PDGF-induced rapid and transient inhibition of GJIC. This result isactivation of MAPK, as demonstrated in Figure 11A. It not surprising considering that similar results have beenalso significantly prevented the PDGF-induced disrup- reported previously in other cell lines [17, 18, 36]. GJICtion of GJIC (Fig. 11B). activity is regulated in many ways at the transcriptional,

translational, and post-translational levels of connexins,which make up the GJs. The rapidity and reversibilityDISCUSSIONof PDGF action on the disruption of GJIC in MCs sug-Platelet-derived growth factor is the most potent mito-gest the closure of existing channels, rather than an alter-gen for MCs and plays an important role in both theation of rates of assembly or disassembly of the GJ. Consis-renal development and pathogenesis of glomerulone-tent with this hypothesis, we did not detect significantphritis [26, 27, 39, 40]. Because of the close link betweenchanges in the localization and distribution of Cx43 ingrowth and GJIC, we examined how PDGF may affectMCs after exposure to PDGF. We focused on Cx43 be-the GJIC in cultured MCs. We also examined the effectscause it is the only GJ protein reported to be expressedof PDGF on Cx43 localization, distribution, and phos-in MCs to date [9]. Alteration of its abundance and/orphorylation, as well as the signal transduction pathway

involved. properties is most likely to affect GJIC. Previous studies

Yao et al: PDGF regulates gap junction communication 1921

Fig. 6. Prevention of PDGF-induced tyrosine phosphorylation of Cx43 by PI3K inhibitors. (A) Confluent MCs were exposed to PDGF (10 ng/mL)for 15 minutes with or without the pretreatment with 100 mmol/L LY294002. Cellular proteins were then harvested and immunoprecipitated witha monoclonal anti-Cx43 antibody. After SDS-polyacrylamide gel electrophoresis and transfer to PVDF, blots were probed with antityrosine mAbPY20 (left) or stripped and reprobed with a monoclonal anti-Cx43 antibody (right) to verify that the difference in tyrosine phosphorylation ofCx43 was not due to differences of the proteins loaded. Similar results were obtained in two additional experiments. (B) Densitometric analysisof the Western blots. Results are expressed as arbitrary unit calculated from the ratio of the densitometric values of phosphotyrosine to Cx43protein for each treatment. (C) MCs were pretreated with or without the indicated concentrations of LY294002 before exposure to PDGF (10ng/mL) for 15 minutes. Phosphorylation of Cx43 was detected as described in this article. A similar result was obtained in an additional experiment.(D) Densitometric analysis of Western blot C. Results are expressed as percentage of control without pretreatment of LY294002.

relate the closure of gap junctional channels to the conformational change of the connexin molecule leadingto GJIC blockade has been suggested [45]. A previouschange of phosphorylation state of connexins. Most of

these connexin-phosphorylation studies were performed study has linked PDGF-elicited disruption of GJIC to theser/thr phosphorylation of Cx43 via PKC and MAPK-on cells expressing Cx43, which exists as a phosphopro-

tein and contains consensus phosphorylation sites for related mechanisms [31], but not to tyrosine phosphory-lation. Interestingly, in our system, we were able to detectseveral known protein kinases [30, 32, 41–43]. To date,

two types of phosphorylation of Cx43 leading to the a rapid and marked increase in tyrosine phosphorylationof Cx43 after exposure to PDGF, and we consider thisinhibition of GJIC have been reported. In communica-

tion-deficient src-transformed cells, Cx43 was phosphor- to be one of the major contributors to the PDGF-elicitedblockade of GJIC. This notion is supported by the follow-ylated at tyrosine residues [21, 44], whereas disruption of

GJIC by 12-O-tetradecanoylphorbol-13-acetate (TPA), ing evidence: (1) In accordance with the disruption ofGJIC, there was an increased level of tyrosine phosphor-epidermal growth factor (EGF), PDGF, and lysophos-

phatidic acid (LPA) were associated with ser/thr phos- ylation of Cx43. (2) Prevention of PDGF-induced disrup-tion of GJIC by PI3K inhibitor was accompanied byphorylation of Cx43, which is thought to be mediated

by MAPK and/or protein kinase C (PKC) activation [31, inhibition of tyrosine phosphorylation of Cx43. (3) Theinduction of tyrosine phosphorylation of Cx43 by phos-33, 34]. Consistent with this, MAPK is able to phosphory-

late Cx43 on ser/thr in vitro [30]. The precise mechanism phatase inhibitor PAO also resulted in an obvious inhibi-tion of GJIC. However, tyrosine phosphorylation ofof the junctional regulation by Cx43 phosphorylation

has yet to be described, although a postphosphorylation Cx43 could not be the sole factor responsible for the

Yao et al: PDGF regulates gap junction communication1922

Fig. 7. Activation of extracellular signal-reg-ulated kinase (ERK) by PDGF. ConfluentMCs were treated with PDGF (10 ng/mL) forthe indicated lengths of time (left) or with theindicated concentrations of PDGF (right) forfive minutes. Cellular proteins were then har-vested and analyzed by Western blot with ei-ther antiphosphorylated ERK (pERK) anti-body (A) or anticontrol MAPK antibody (B).Two additional experiments done with thesame schedule showed a similar result.

Fig. 8. PI3K inhibitors inhibit PDGF-inducedactivation of extracellular signal-regulated ki-nase (ERK). Confluent MCs were exposed toPDGF (10 ng/mL) for 10 minutes with or with-out pretreatment with 100 mmol/L LY294002(A) or the indicated concentrations of wort-manin (B). Cellular proteins were then har-vested and analyzed by Western blot with aphosphorylated ERK (pERK) antibody. Simi-lar results were obtained in three additionalexperiments.

Yao et al: PDGF regulates gap junction communication 1923

Fig. 10. Disruption of GJIC by the tyrosine phosphatase inhibitorPAO. Confluent MCs were treated with either PAO (5 mmol/L) orPDGF (10 ng/mL) for 15 minutes. GJIC was assessed by Lucifer yellowmicroinjection into a single cell. Data points represent the mean 6 SDof the number of cells (N 5 10) to which Lucifer yellow was transferredfrom the injected cells within three minutes of injection. *P , 0.01 vs.control. Similar results were obtained in an additional experiment.

not be excluded. Several recent studies showed that theCx43 phosphorylation alone was insufficient for mediat-ing the disruption of GJIC or even lack of correlationbetween Cx43 phosphorylation and GJIC in other celltypes [19, 47]. The reasons for this disparity are unknown

Fig. 9. Induction of tyrosine phosphorylation of Cx43 by tyrosine phos- at present. One hypothesis is that the disclosure of GJICphatase inhibitor PAO. Confluent MCs were either left untreated requires the complex interactions of Cx43 with other(lane 1) or treated with 5 mmol/L PAO (lane 2), 0.05% dimethyl sulfox-

unidentified accessory proteins [19, 47]. Thus, the exactide control (lane 3), or 10 ng/mL PDGF (lane 4) for 15 minutes. Cellularproteins were then harvested and immunoprecipitated with polyclonal mechanisms responsible for PDGF-induced disruptionrabbit anti-Cx43 antibody. After SDS-polyacrylamide gel electrophore- of GJIC in MCs remain to be established.sis and transfer to PVDF, blot was probed with antityrosine mAb PY20

The signal transduction pathways and key signal mole-(A) or stripped and reprobed with anti-Cx43 antibody to verify thatthe difference in tyrosine phosphorylation of Cx43 was not due to cules involved in PDGF-induced disruption of GJIC aredifferences in the loading of proteins (B). presently not fully clear. Following autophosphorylation

after binding with PDGF, PDGF receptor (PDGFr) re-cruits diverse signal transduction molecules, includingGTPase activating protein (GAP), phospholipase Cg1closure of GJIC induced by PDGF in MCs. The much(PLCg1), Src homology 2 (SH2) domain containing phos-more potent action of PDGF on GJIC, as comparedphotyrosine phosphatase-2 (SHP-2), and PI3K, whichwith PAO, suggests the existence of other mechanisms.then initiate diverse cascades of activities involving acti-Indeed, the contribution of ser/thr phosphorylation ofvation of protein kinases and phosphatases, finally lead-Cx43 to GJIC in our experimental system cannot being to biological responses such as cell growth, migration,ignored. PDGF has been reported to be able to induceand differentiation [28]. Since it has been demonstratedthe activation of both PKC and MAPK [28, 29, 46].that association of certain of these proteins with PDGFrExposure of rat MCs to PDGF resulted in a rapid activa-is capable of eliciting particular cell responses, elucida-tion of MAPK, as demonstrated by antiphosphotyrosyltion of the signal transduction pathway through whichERK antibodies. Furthermore, inhibition of MAPK acti-cell–cell communication is regulated is essential for un-vation using MEK inhibitor PD98059 significantly blockedderstanding the role of GJIC in the processes involved.PDGF-induced closure of GJIC. Thus, it is likely that

The results presented here demonstrate that the PI3Kboth tyrosine and ser/thr phosphorylation of Cx43 medi-inhibitor LY294002, at the concentrations reported toate the closure of GJIC induced by PDGF in MCs. Itbe effective for inhibition of PI3K activity [29, 37], dra-should be noted that although phosphorylation of Cx43matically prevents PDGF-induced disruption of GJICis believed to be causally linked with the disruption ofand tyrosine phosphorylation of Cx43, as well as theGJIC [21, 31, 33, 34, 43, 44], the participation of currently

unidentified components in the regulation of GJIC could activation of MAPK. This suggests that activation of

Yao et al: PDGF regulates gap junction communication1924

Fig. 11. Prevention of PDGF-induced acti-vation of MAPK and disruption of GJIC byMEK inhibitor PD98059. (A) Confluent MCswere exposed to PDGF (10 ng/mL) for 15minutes with or without pretreatment withPD98059. Cellular proteins were then har-vested and analyzed by Western blot with ei-ther antiphosphorylated ERK (pERK) anti-body (A, left) or anticontrol ERK antibody(A, right). (B) Confluent MCs were pretreatedwith 100 mmol/L of PD98059 for one hour orleft untreated before exposure to 10 ng/mL ofPDGF for 15 minutes. GJIC was assessed byLucifer yellow microinjection into a single cell.Data points represent the mean 6 SD of thenumber of cells (N 5 10 to 14) to which Luciferyellow was transferred from the injected cellswithin three minutes of injection. *P , 0.01vs. control; #P , 0.01 vs. PDGF-treated cells.Similar results were obtained in an additionalexperiment.

PI3K is necessary for PDGF-induced disruption of GJIC the PDGF-induced activation of PKC [49–51] and MAPK[29], two central ser/thr kinases implicated in the closureand phosphorylation of Cx43. It also indicates that PI3K

lies upstream in the signal transduction pathway linking of GJIC and phosphorylation of Cx43 on ser/thr residues.It could also interfere with events of tyrosine phosphory-the PDGF receptor to phosphorylation of Cx43. It is

worth mentioning that the ability of PDGF to activate lation of Cx43, as indicated in this article. Thus, modula-tion of PDGF-induced disruption of GJIC by PI3K inPI3K in MCs and other cells has been extensively docu-

mented [29, 37, 48]. Of note, although our results provide MCs could be due to the multiple effects of PI3K on avariety of signaling molecules. It is worth mentioningsupport for an important role of PI3K signaling in the

PDGF-induced blockade of GJIC in MCs, the participa- that Cx43 is not the only protein where phosphorylationof tyrosine residues, elicited by peptide growth factors,tion of other signaling pathways and molecules could

not be excluded. A recent publication by Hossain et al is mediated through the PI3K signaling transductionpathway. Tyrosine phosphorylation of three other impor-demonstrated that PDGF-induced disruption of GJIC in

T51B rat epithelial cells is mediated by multiple signaling tant signal and structural proteins, focal adhesion kinase(FAK), paxillin, and P130ca, by PDGF are also mediatedpathways and requires participation of multiple compo-

nents, such as SHP-2 and phospholipase Cg1 [19]. Inter- by PI3K [37, 52, 53]. Interestingly, all of them are poten-tial substrates of Src kinase. Furthermore, the associationestingly, consistent with our data, they also demonstrated

a partial involvement of PI3K in the PDGF-induced dis- of PDGF receptor with Src and activation of Src byPDGF have been previously reported [54, 55]. In addi-ruption of GJIC [19].

How does PI3K signaling influence GJIC and phos- tion, Src kinase is the only tyrosine kinase that has beendemonstrated to be able to interact directly with Cx43,phorylation of Cx43? This question remains to be ad-

dressed. PI3K has multiple effects on a variety of signal- through SH2 and SH3 domains, to phosphorylate Cx43on tyrosine residue, and to mediate the closure of GJICing molecules. It has been reported to be able to mediate

Yao et al: PDGF regulates gap junction communication 1925

11770594) from the Ministry of Education, Science, Sports and Culture,[42, 44, 56]. Src might play an important role in PDGF-Japan. The authors wish to thank Dr. S. Batsford, M.D., Institute of

elicited disruption of GJIC in MCs. In agreement with Medical Microbiology and Hygiene, Freiburg University, Germany,for critical review and suggestions for our manuscript, and Ms. K.this idea, a recent publication demonstrates that G-pro-Kamata for excellent technical assistance.tein–coupled receptors use an Src tyrosine kinase path-

way to transiently inhibit Cx43-based cell–cell communi- Reprint requests to Takashi Oite, M.D., Department of Cellular Phys-iology, Institute of Nephrology, 1-757 Asahimachi-dori, Niigata 951-cation [17].8510, Japan.

PI3K activity has been shown to increase in response E-mail: oite@med.niigata-u.ac.jpto a number of stimuli, such as peptide growth factorsand hormones, as well as in cells transformed by tyrosine APPENDIXkinase oncogenes and polyoma virus middle T antigen. Abbreviations used in this article are: Cx43, connexin43; DMEM,Although the precise biological functions of PI3K and Dulbecco’s modified Eagle’s medium; ECL, enhanced chemilumines-

cence; EGF, epidermal growth factor; ERK, extracellular signal-regu-its lipid products have not been clearly defined, they havelated kinase; FAK, focal adhesion kinase; FCS, fetal calf serum; GJIC,been implicated in various biological processes, such as gap junctional intercellular communication; JAG, juxtaglomerular appa-

cell growth, migration, transformation, and differentia- ratus; LPA, lysophosphatidic acid; LY, Lucifer yellow; MAPK, mitogen-activated protein kinase; MC, mesangial cell; PAO, phenylarsine oxide;tion [57, 58]. Because PI3K is critical for growth factor-PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor;induced mitogenesis [58], involvement of this enzyme in PI3K, phosphatidylinositol 39-kinase; PKC, protein kinase C; SDS, so-

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