MOLECULAR CARCINOGENESIS3:335-343 (1990)

Retinoid-Enhanced Gap Junctional Communication Is Achieved by - Increased Levels of Connexin 43 mRNA and Protein Michael Rogers,' John M. Berestecky: Mohammad 2. Hossain, Huiming Guo, Ranjana Kadle, Bruce 1. Nicholson, and John 5. Bertram3 Basic Science Program, Cancer Research Center of Hawaii, University of Hawaii, Honolulu, Hawaii (MR, JMB, MZH, HG, JSB) and Department of Biological Sciences, SUNY-Buffalo, Amherst, New York (RK, BJN)

Natural and synthetic retinoids are potent inhibitors of experimental carcinogenesis in animals and cause reversion o f premalignant lesions in humans. In t h e model C3H 10T1/2 cell system, retinoids enhance postconfluent growth control, reversibly inhibit carcinogen-induced transformation, and enhance gap junctional intercellular communication. These effects are highly correlated. 10T1/2 cells were found t o express low levels of connexin 43, a gap junctional protein first found in the heart. After treatment of confluent 10T1/2 cells with the synthetic retinoid tetrahydrotetramethylnapthalenylpropenylbenzoic acid (TTNPB), levels o f connexin 43 mRNA and protein increased within 6 h of treatment, while elevation of junctional communication was detected within 12-18 h. The maximally effective concentration of lTNPB (lo-' M)caused an approximate 10-fold elevation of connexin 43 gene transcripts after 72 h. Indirect immunofluorescence microscopy using a polyclonal antibody t o the synthetic C-terminal region of connexin 43 demonstrated that lTNPB induced many fluorescent plaques in regions o f cell-cell contact. These results provide a molecular basis for the retinoid-enhanced junctional communication in 10T1/2 cells. It is proposed that one action o f retinoids is t o modulate the intercellular transfer of signal molecules. These could mediate many of the physiological actions of retinoids on growth control and carcinogenesis. Key words: Cell-cell communication, cancer chemoprevention, vitamin A INTRODUCTION Vitamin A is required for vision, for normal differentiation of many tissues, for normal growth of the organism, and for fertility in both sexes. Apart from the role of 11cis retinal in the visual process, the mode of action of vitamin A is poorly understood a t the molecular level. Vitamin A is supplied to tissues as retinol and metabolized to retinoic acid. This metabolite appears to be the effector of growth and differentiation 11I. Natural and synthetic forms of vitamin A capable of maintaining normal growth and differentiation are called retinoids. In this study, we utilize one of these, tetrahydrotetramethylnapthalenylpropenylbenzoic acid (nNPB), a chemically stable benzoic analogue of retinoic acid 121. The recent discovery of a family of nuclear retinoic acid receptors (RARs) with strong homology to the steroid and thyroid binding proteins suggests that retinoids function by regulating gene transcription [3,4,5]. In addition to maintaining normal differentiation of epithelial tissues, retinoids can also correct abnormal differentiation. As such they have found wide use in dermatology [6] and are being evaluated as preventive agents in human cancer 171. In animal models, retinoids are potent inhibitors of carcinogen-inducedneoplasia at several anatomic sites [8] and will inhibit chemically induced neoplastic transformation in the well-characterized C3H 1OT1/2 mouse cell line (91. 0 1990 WILEY-LISS, INC.

A recently discovered action of retinoids, which may be relevant to inhibition of transformation in 10T1/2 cells, is their ability to upregulate homologousgap junctional communication in 1OT1/2 cells, in carcinogen-initiatedcultures, and in neoplastically transformed derivatives [10,1 11. Enhanced communication was statisticallyassociated with a reduced confluent saturation density, an in vitro manifestation of growth control, and the ability of retinoids to inhibit neoplastic transformation in carcinogen-exposed cultures. These results provided further evidence that gap junctions can serve as conduits for growth control signals as originally proposed by Loewenstein [ 121. It had previously been demonstrated that nontransformed 10T1/2 cells normally not in communication with 'Present address: Department of Biochemistry, Bowman Gray School of Medicine, Winston-Salem, NC. 'Present address: Kapiolani Community College, Honolulu, HI. 3Corresponding author: Basic Science Program, Cancer Research Center of Hawaii, University of Hawaii, 1236 Lauhala Street, Honolulu, HI 96813. Abbreviations: CAMP, cyclic adenosine monophosphate; dCTP. deoxycytidine triphosphate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; RAR, retinoic acid receptor; RARE, retinoic acid responsive element; RSA. rabbit serum albumin; SDS, sodium dodecyl sulfate; SET, 0.01 5 M NaCI, 0.002 M EDTA. 0.03 M Tris, pH 7.4; SSC, standard saline citrate; TBS, 10 mM Tris-HCI, pH 7.4, 0.9% NaCI; TPA, 12-0-tetradecanoylphorbol-13-acetate; TTNPB, tetrahydrotetramethylnapthalenylpropenylbenzoic acid.

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neoplastic cells can nevertheless cause reversible growth arrest of neoplastic 1OT1/2 cells under conditions where Aeterologousjunctional communication is increased by cyclic adenosine monophosphate (CAMP) [I31. Retinoids, for reasons not yet apparent, block this increased heterologous communication between normal and neoplastic lOTli2 cells, yet enhance homologous communication when tested against each cell type separately. In all cases examined, enhanced conditions of heterologous or homologous communication are associated with enhanced growth control and vice versa. It is proposed that junctional transfer of growth regulatory signals plays a major role in the control of proliferation in density-inhibited cells [I0,131. We now show that this enhanced communication is in whole or in part driven by synthesis of connexin 43. This protein, first identified as a major component of the intercalated disc of rat heart [14,15], is a member of a family of connexin molecules that differ primarily in the composition and length of their cytoplasmic domains [ I 61. The current work establishes retinoids as the first compounds found to modulate the expression of connexin 43. MATERIALS AND METHODS Materials Oligolabeling kit was obtained from Pharmacia (Piscataway, NJ); RNA ladder and formamide were purchased from Bethesda Research Laboratories (Gaithersburg, MD); 14C molecular weight markers and '2s1-labeled protein A (70-100 pCi/pg) were obtained from New England Nuclear (Boston, MA); [ ~ ~ ~ ~ P l d e o x y c y t itriphosphate dine (dCTP)(300 Ciimmol) was acquired from ICN Biomedicals Inc. (Costa Mesa, CA); and FITC-conjugated goat antirabbit IgG, F(ab') fragment, as well as all basic chemicals, were obtained from Sigma Chemical Co. (St. Louis, MO). nNPB was a gift from Hoffmann-La Roche (Nutley, NJ). Dr. E. Beyer, Harvard, generously supplied the G2A cDNA clone of connexin 43 [16]. cDNA probes for sea urchin ribosomal RNA [I71 were obtained from Dr. T. Humphreys, University of Hawaii. Cell Culture C3H 1OTli2 cells were passaged weekly in Eagle's basal medium with 25 pg/mL gentamicin and 5% fetal calf serum (Hyclone Laboratories Inc., Logan, UT) as previously described [18]. For experiments, lo5 cells were seeded in 150-mm culture dishes in 20 mL medium and were grown to confluence for 1 wk. They were then refed with medium and, 3 d later, stimulated with TTNPB in 40 p L acetone or with acetone as control. The cultures were harvested at the times indicated according to the procedures stated below. Measurement of Junctional Intercellular Communication Immediately prior to cell harvest, two dishes of each treatment group were assessed for gap junctional communication by microinjection of 10% Lucifer yellow CH in 0.33 M CiCl as previously described [ 1 I]. The number of communicating cells (i.e., number of fluorescent cells sur-

rounding the dye-injected cell) was quantitated by a blinded observer 10 min after dye injection. Northern Blotting and RNNcDNA Hybridization Total RNA isolation, agarose gel electrophoresis, cDNA isolation, and labeling were performed as described in Maniatis et al. [I91, except where noted. The hybridization procedure was carried out according to Fregien et al. [20]. At the times indicated, total RNA was isolated from about 2 x 1O7 cells/time point. Cultures were washed once in cold phosphate-buffered saline (PBS)/I 0 m M EDTA and scraped from the dish into the same solution; the cell pellet was then dissolved in guanidine isothiocyanate buffer. This cell lysate was overlayered onto cesium chloride sohtion and centrifuged at 174,000 g for 21 h at 20°C. The RNA-containing pellet was then brought into solution and extracted with phenokhloroform, followed by a cold ethanol precipitation. The resulting pellet was washed twice in 70% ethanol, vacuum dried, and dissolved in 20 p L sterile water. Quantitation and purity of the total RNA was determined by UV spectroscopy at 260/280 nm. Ten micrograms of the isolated total RNA was run on 1YO agarose/l6.5% formaldehyde gels using an RNA ladder for molecular weight determination. After electrophoresis, the RNA was partially hydrolyzed in 50 m M NaOH/lO m M NaCl for 45 min, neutralized in 0.2 M Tris, pH 7.4, for45 min, and then presoaked in 20 x SSC (1 x SSC, 0.15 M NaCI, 0.015 M sodium citrate, pH 7.0) for 60 min. The gel was then inverted and blotted overnight onto a 0.22-pm nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH) by capillary action using 20 x SSC buffer for transfer. After transfer, the blot was washed gently in 20 x SSC and vacuum dried a t 80°C for 2 h. The northern blot was hybridized to the 1.5-kb G2A cDNA clone of connexin 43. The blot was prewashed three times, using4 x SET(1 x SET, 0.01 5 M NaCI, 0.002 M EDTA, 0.03 M Tris,pH 7.4) and 50% formamide, with 10% Denhardt's solution/O. 1 % sodium dodecyl sulfate (SDS)/O. 1YO sodium pyrophosphate/lO pg/mL polyadenylicacidi250 pg/ mL salmon sperm DNA as blocking agents. The blot was hybridized using similar solutions as above except for the addition of 100 pg/mL salmon sperm DNA, 5% Denhardt's solution, 10% dextran sulfate, and 1O6 cpm/mL of a boiled [32P]dCTP-labeledconnexin 43 cDNA at 42°C overnight. After five washes with 4 x SET/O. 1YO SDYO.1 YO sodium pyrophosphate at 55T, two high-stringency washes were doneusing0.5 x SET/O.I YOsodiumpyrophosphateat 65°C. Excess SDS was removed using 0.5 x SET at room temperature. The blot was exposed to Kodak X-Omat AR film at - 70°C for 48 h using double intensifying screens. Antibody Production Based on the predicted amino acid sequence of connexin 43 [ 161, a 15-mer peptide corresponding to the C-terminal domain (positions 368-382) was synthesized on an Applied Bioscience solid phase synthesizer. This region is believed to be located in the cytoplasm, and computer searches revealed no homology with other known proteins. An N-terminal cysteine was added to allow con-

RETlNOlDSAND CONNEXlN 43 SYNTHESIS

jugation to rabbit serum albumin (RSA). Rabbits were immunized a total of three times with the peptide-RSA conjugate, and antipeptide antibody titers were monitored by radioimmunoassay.This antibody recognizes heart and fibroblast connexin 43 proteins that are also recognized by other polyclonal connexin 43 antibodies [21] and stained the intercalated disc of rat heart as demonstrated by indirect immunofluorescenceessentially performed as described below (data not shown). A detailed report on the synthesis and characterizationof this antibody will be presented elsewhere. lmmunoprecipitation Homogenates were solubilized in 1'70 SDS in 10 mM Tris-HCI, pH 7.4, containing 2 mM phenylmethylsulfonyl fluoride (PMSF). After dilution to a final concentration of 0.5% SDS, samples were incubated with affinity-purified N-terminal antibody to connexin 43 [22] at a dilution of 1 :40 in TBS (10 mM Tris-HCI, pH 7.4, and 0.9% NaCI) with 2% Triton X-100 and 4% Tween 20. After 16 h at room temperature, the connexin 43-antibody complex was precipitated by incubating at room temperature for 3 h with 30 pL of 8 pg/mL protein A-Sepharose (Sigma).After three washes with TBS containing 0.5% Tween 20, bound material was eluted in 15pL of 1YO SDS in 10 mM Tris-HCI, pH 7.4, and incubated at 37°C for 4 h with or without 1 unit of calf intestinal alkaline phosphatase (Promega Corp., Madison, WI) in a total reaction volume of 15 pL. For some samples, alkaline phosphatase was incubated with 1 mM vanadate for 30 min at 37°C before addition to immunoprecipitated gap junction protein. The samples were then subjected to SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions and to western blotting. Protein Electrophoresis and Western Blotting Preparation of cell lysates Cells grown to confluence in 150-mmculture dishes were washed twice with PBS containing 1 mM NaF and 1 mM PMSF and scraped off into the same buffer. The cells were then pelleted by centrifugation; the resulting cell pellets were dissolved in lysis buffer (1% NP 40,0.05 M iodoacetamide, 10 mM PMSF, 1 mM EDTA, 1 p M leupeptin, 2 pg/mL aprotinin, 0.7 pg/mL pepstatin in borate buffer, pH 8.0) at 2.5 x 10' cells/mL for 1 h at 4°C. Lysateswere clarified by centrifugation and assayed for protein (bicinchoninic acid (BCA) method; Pierce Chemical Co., Rockford, IL). Electrophoresis and western blotting

Equal protein concentrations of cell lysates were mixed 1 : 1 with 2x SDS sample buffer with or without 10% 2-mercaptoethanol, and the unheated samples were electrophoresed on 10% polyacrylamide gels (231. The gels were subsequently electroblotted onto lmmobilon (Millipore Corp., Bedford, MA), then blocked with 5% nonfat dry milk in PBS (blotto).The blots were treated for 1 h with immune or preimmuneserum diluted 1 : 100 in blotto: borate buffer (1 :2), washed in borate buffer, treated with '251-labeled protein A (1 O6 cpm/mL in blotto: borate buffer), extensively washed again, and mounted for autoradiography.

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lndir e d lmmunof luorescence Microscopy 1OT1/2 cells were grown on plastic slides (Permanox; Nunc Inc., Naperville, IL) then treated postconfluence for 4 d with TTNPB. Slides were first fixed by briefly washing in warm PBS, then immersed in dry - 20°C methanol for 2 min, transferred to 4°C PBS, then blocked for 1 h in a solution of PBS, 5% bovine serum albumin (BSA), and 0.2% sodium azide, pH 7.5. The primary rabbit anticonnexin 43 antibody was applied a t 1 :30 dilution in blocking solution at 4°C for 30 min. Slides were rinsed in cold PBS and washed for 20 min at 4°C with high-salt PBS (PBS 0.5 M NaCI), rinsed in PBS, then exposed to the FITC-conjugated second antibody (goat antirabbit IgG F(ab') fragment) in blocking buffer at 4°C for 30 min. Slides were washed as above and mounted in 50% glycerol in PBS, pH 8.0, containing 100 pglmLpphenylenediamine HCI 1241 to retard photobleaching. The coverslips were sealed with clear nail polish and were stable for 4-6 d at 4°C. Epifluorescent microscopy was performed using a Zeiss Axioplan microscope and photographed on Kodak TMAX 3200 film.

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RESULTS Expression of Connexin 43 Transcripts

Northern blots of total RNA from control and TTNPBstimulated 10T1/2 cells were probed with cDNA for connexins 21, 32, and 43. Under high stringency conditions, a 3.1-kb band hybridized with the connexin 43 cDNA; other cDNAs failed to hybridize (data not shown). Connexin 43 thus appears to be the only member of the gap junction family of genes so far characterized that is expressed in 1 OT1/2 cells. We have previously reported that junctional communication is enhanced by natural and synthetic retinoids and that this increase is detectable within 12-18 h of treatment [10,11]. To determine if this was associated with increased levels of connexin 43 gene products or, alternately, with increased permeability of existing junctional channels, we performed northern and western blotting on 1OT1/2 cells at various times after stimulation with graded doses of TTNPB. TTNPB was used because it was the most potent of a series of retinoids in its ability to inhibit transformation and enhance communication ( 1 11 and because retinoicacid itself is rapidly metabolized by 1OT1/2 cells [25]. Time Course of Connexin 43 mRNA Induction Northern blots of total RNA from 10T1/2 cells demonstrated that connexin 43 mRNA was elevated within 6 h of treatment with 10 - 8 M TTNPB and that levels continued to rise over the 72-h duration of the experiment (Figure 1). In three independent experiments, an 8- to 15-fold increase of the 3.1-kb transcript over basal unstimulated levels was measured by digital image analysis. Rehybridization of blots to cDNA of ribosomal RNA revealed a variation between key lanes of only about 20% in hybridization to this probe. Since it was shown that most of the effect of retinoids on cell-cell communication is reversible within 6 h of removal of nNP8 [lo], the reversibility of the mes-

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Figure 1. Time course of TTNPB-induced increase in connexin 43 mRNA. Confluent 10T1/2 cells were stimulated with M TTNPB for the times indicated. 10 k g total mRNA was loaded per lane, electrophoresed, and transferred to nitrocellulose by blotting. RNAkDNA hybridization using the 32P-labeledcDNA clone of connexin 43 [16] was done at 42°C; posthybridization washesincluded twostringentwashesof 0.5 x SETat 65°C. Lane 1, acetone, 6 h; lane 2, lTNPB, 6 h; lane 3, TrNPB, 12 h; lane 4, TTNPB, 24 h; lane 5, acetone, 48 h; lane 6, TTNPB, 48 h; lane 7, acetone, 72 h; lane 8, lTNPB, 72 h. This experiment is a representative sample of three experiments. Rehybridization of the blot to the cDNA of ribosomal RNA followed by digital image analysis demonstrated approximately equal hybridization in key lanes; for example, lanes 3, 4, and 6 gave a mean transmission value of 54.1 1+_ 6.1, while lane 5 (TTNPB control) had a value of 63.2 arbitrary transmission units.

sage induction was investigated.Within 6 h of the removal of TTNPB, the elevated level of gap junction mRNA had decreased to below detectable levels (Figure 2). The northern blot shown in Figure 1 was quantitated by digital densitometry and arbitrary transmission values plotted together with the junctional communication data obtained from the same cultures (Figure 3). Increased levels of connexin 43 mRNA clearly preceded increases in communication. In more extensive time course studies, we had been unable to detect significant increases in junctional communication prior to 12 h posttreatment with TTNPB [ 10,l I]. Western Blotting

Western blotting of lysates of confluent 1OT1/2 cells was performed under reducing and nonreducing conditions. This was found necessary because the immunoreactivity

Figure 2. Connexin 43 mRNA levels decrease rapidly after removal of lTNPB. Confluent 10T1/2 cultures were treated with lTNPB lo-' M for 96 h or with acetone as control. In half of the cultures, retinoid-containing medium was removed and replaced with fresh medium with or without TTNPB l o - * M; the other cultures were untouched. After 6 h, cultures were harvested; total RNA isolated; and equal amounts of RNA electrophoresed, blotted, and hybridized against labeled connexin 43 cDNA. M; Lane 3, Lane 1, control, acetone treated; lane 2, 'ITNPB TTNPB l o v 8 M then retreated with TTNPB lo-' M for 6 h; Lane 4, TTNPB lo-' M then treated with acetone for 6 h. The reduction in hybridization in lane 3 versus lane 2 is consistent with previous observations that fresh culture medium causes transient reduction in dye transfer.

Figure 3. Comparative time courses for induction of connexin 43 mRNA and functional gap junctions by lTNPB in confluent lOT1/2 cells. The autoradiograph of the northern blot shown in Figure 1 was quantitated by digital image analysis and plotted in arbitary units of transmission. Communication data was obtained from dye injection into 10 celldgroup immediately prior to harvesting for RNA isolation. Because of problems of protein aggregation, western blots were not analyzed. Comparable results were observed in three separate experiments. Closed and open triangles, 3.1-kb band intensities on northern blots, treated and control, respectively; closed and open squares, mean number of fluorescent cells surrounding a single dyeinjected cell, treated and control, respectively.

of connexin 43 to the polyclonal antibody to the C-terminal region of this protein was found to be sensitive to heat and denaturation. In control cultures (Figure 4, lanes 1 and 6), run under reducing conditions, immunoreactive bands were seen at approximately 43 and 45 kDa. Under nonreducing conditions (Figure 5A, lane l ) , bands in regions of 74 and 79 kDa and 107 and 126 kDa were detected and are presumed aggregates of connexin 43 [ 2 6 ] . Upon reduction of the same lysates, the 107- and 126-kDa bands disappeared (Figure 58). Bands in the 70-kDa range were strongly decreased in intensity but were not consistently absent, indicating incomplete reduction. In these control cultures light labeling at 43 kDa was observed. Heating to 50°C in reducing buffer resulted in loss of these 70-kDa bands together with major reductions in the 43- to 45-kDa

Figure 4. Time course of TrNPB-induced increase in connexin M for the 43. Confluent cultures were treated with TTNPB times indicated. Cell lysates were reduced prior to electrophoresis, equal protein amounts applied t o each lane, and western blots performed. lmmunoblotting with preimmune serum gave no signal. Lane 1, 0 h control; lane 2, 6 h TTNPB; lane 3, 24 h TTNPB; lane 4, 48 h TTNPB; lane 5, 96 h TTNPB; lane 6, 96 h control. The bands in the 70-kDa region (lanes 3 and 4) are believed to represent dimers of connexin 43 (see Figure 5). PosiDa) are shown. tions of molecular weight markers ( x

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sulted in loss of sensitivity such that increased levels of M connexin 43 were only detected at lo-’ and TNPB. In these retinoid-inducedcultures, the immunoreactive bands were generally observed as doublets.

Figure 5. Dose response for induction of connexin 43 by TTNPB. Confluent cultures of 1011/2 cells were treated with lTNPB or acetone vehicle for 96 h. Equal amounts of total cell protein were electrophoresed without reduction (Figure 5A, upper panel), or after reduction with 5% 2-mercaptoethanol for 5 min at 25°C (Figure 58, lower panel), and western blotting wasperformed. Lane 1, acetonecontrol; lane 2, TTNPB lO-’OM; lane 3, lTNPB lo-’ M; lane 4, lTNPB lo-’ M. lmmunoblotting with preimmune serum did not give a signal (data not shown). Note the aggregation of connexin 43 into probable dimers and tetramers under nonreducing conditions (Figure 5A) and the incomplete conversion t o monomers under reducing conditions. More vigorous denaturation at 50°C reduced the immunoreactivity of this antigen. Positions of molecular weight markers (x Da) are shown.

Multiple lmmunoreactive Bands of Connexin 43 Induced by Retinoids Represent Phosphorylated Forms o f the Protein 10T1/2 cells exhibit two clearly distinct immunoreactive bands at apparent molecular masses of 43 and 45 kDa, when electrophoresed under reducing Conditions. As described above, similar doublets were seen under nonreducing conditions a t higher molecular masses. To determine if this heterogeneity is due to protein phosphorylation, lysates of TNPB-treated 10T1/2 cells were immunoprecipitated; a portion of the immunoprecipitated cells was then subjected first to phosphatase digestion and then to electrophoresis under reducing Conditions. As seen in Figure 6, TNPB-treated cultures demonstrated three immunoreactive bands: a weak band a t 43 kDa and two stronger bands at approximately 44 and 45 kDa. Phosphatase digestion resulted in a single strong band at 43 kDa, parallel to the weak band seen in nondigested controls. Inhibition of phosphatase action by vanadate inhibited this band shift. These data indicate that a major portion of retinoid-induced connexin 43 becomes phosphorylated in 10T1/2 cells. lmmunofluorescent Localization o f Connexin 43 1OT1/2 cells were seeded onto krmanox plastic slides for fluorescent microscopysince conventionalplastic dishes

bands of the gel (data not shown). This variability in obtaining complete disaggregation of connexin 43 may be in part responsible for variations in relative intensities of the immunoreactivedoublets seen in control and treated blots.

-55 -43

Time Course Studies of Connexin 43 Induction Within 6 h of stimulation with M TNPB, increases in immunolabeling of connexin 43 were detected. This labeling increased progressively over the 96-h duration of the experiment (Figure 4). The control cultures also marginally increased their content of connexin 43 over this time period, which is consistent with similar small increases in junctional communicationwith time that have previously been noted [ 1 11. Dose-Response Studies Confluent cultures of 10T1/2 cells were treated with lo-’, or lo-’’ M TTNPB or with acetone control for 96 h. Cell lysates were then immunoblotted after SDSPAGE run under nonreducing (Figure 5A) or reducing (Figure 55) conditions. This range of TNPB concentration spans the minimum to maximum biological response range for induction of gap junctional communication and suppression of neoplastic transformation over treatment periods of 7 and 28 d, respectively [ l l ] . Under nonreducing conditions, a progressive increase in immunolabeling was seen over the entire dose range; reduction of lysates re-

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Figure 6. Effects of phosphatase digestion on connexin 43. Lysates from cells treated with TTNPB l o - * M for 96 h were immunoprecipitated with N-terminal connexin 43 antibody and proteins incubated with or without alkaline phosphatase. In some samples, phosphatase action was inhibited with 1 mM vanadate. Resulting digests were electrophoresedand immunoblotted. Lane 1, TTNPB treated; lane 2, lTNPB treated + alkaline phosphatase; lane 3, TTNPB treated + alkaline phosphatase + 1 mM vanadate. Molecular weight markers of 18, 24, 36, 43, 55, and 95 kDa were used to calibrate this gel. Calculated molecular masses of immunoreactivebands based on semilog plots of these markers were about 43-45 kDa for the lower and upper bands. Based on these calculations, the 43-kDa marker shown ran anomalously slowly on this gel.

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RETINOIDSAND CONNEXIN43 SYNTHESIS

(on which cells were grown for all the biological and moIPcular studies) autofluoresce and obscure the FlTC signal and 10T1/2 cells, when grown on glass, have a morphology quite distinct from that on plastic. When fixed and stained with antibodies to the C-terminal domain of connexin 43, control untreated cells showed occasional small fluorescent plaques localized in regions of cell-cell contact (Figure 7A). After exposure to 10 - 8 M TTNPB for 96 h, these plaques had dramatically increased in number and size, such that the region of intercellular contact with neighboring cells was clearly defined (Figure 7C). This region is poorly visualized by phase optics because of the highly flattened cytoplasm of 1OT1/2 cells, particularly after treatment with retinoids. Surveys of many thousands of cells exposed to M TTNPB demonstrated that all cells were surrounded by fluorescent plaques and would be expected to be part of a junctional network as predicted by the microinjection studies [ l 11. Cells treated with lo-' M TTNPB (Figure 7E) appeared to possess more numerous junctional plaques, but these plaques were smaller than those observed in cells receiving 10 - 8 M TTNPB (Figure 7C). Fluorescent plaques were limited to regions of cell-cell contact, not just peripheral regions, as was demonstrated in late logarithmic growth phase cells which had not yet formed a confluent monolayer (Figure 7G.H) DISCUSSION

Retinoidshave previously been demonstrated to upregulate homologous junctional communication in normal, chemically initiated, and transformed 10T1/2 cells [10,1 I]. We now show that this enhanced communication is associated with major increases in connexin 43, which in turn is driven by increases in message for this protein. The response of 1OTli2 cells to retinoids at the molecular level reflects their behavioral responses to retinoids (i.e., alterations in saturation density, gap junctional transfer, and the suppression of neoplastic transformation) since all of these effects are reversible in 1OT1/2 cells and follow a similar dose-response relationship [9,10,1 I ] . The causal linkage between increased cellular levels of connexin 43 and increased junctional communication is suggested by the following evidence: (1) connexin 43 is a major protein associated with the region of the intercalated disc of rat heart concerned with junctional signal transfer [22]; (2) microinjection of connexin 43 mRNA into frog oocytes confers gap junctional competence [27]; (3) connexin 43 shares homology with a family of known gap junctional proteins [16]; (4) increases in connexin 43 detected by western blotting immediately preceded observed retinoid-induced increases in dye transfer (Figure 3); (5) retinoid-induced

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connexin 43 became associated with plaques in regions of cell-cell contact (Figure 7). The increase in connexin 43 mRNA could be due to message stabilization or transcriptional activation. Our data cannot distinguish between these alternatives. Nor do we know if this represents a direct action of retinoids or requires prior activation of other genes. While there are several reports of retinoids altering mRNA levels [28,29], these most probably reflect secondary changes induced during retinoid-stimulated differentiation or are a consequence of correcting a state of retinoid deficiency [30]. There is clear evidence for direct transcriptional activation by retinoids of two genes: tissue transglutaminase in macrophages 1311and laminin in F9 teratocarcinoma cells [5]. In macrophages, increases in mRNA occurred within 15 min of stimulation, a far more rapid time course than for the induction of connexin 43 mRNA reported here. These differences could reflect differences in cell type or in mechanism. In the case of laminin gene activation in F9 cells 151 and granulocytic differentiation of HL60 cells [321, there is evidence that retinoic acid acts by binding to a nuclear RAR, which in turn interacts with genomic retinoic acid responsive elements (RAREs). The receptor itself, and the DNA responsive element to which it binds, are closely related to the steroid, thyroid, and vitamin D receptors and their respective responsive elements [3]. At least four RARs have been described with distinct tissue-specific expression [33,34], each potentially interacting with unique RAREs. This, coupled to interactions with thyroid responsive elements [35], provides a likely mechanistic basis for the multiple effects of retinoids in diverse tissues. lmmunoreactive connexin 43 was found to migrate on SDS polyacrylamide gels as a doublet in both reduced and nonreduced conditions (Figure 5). While both bands increased in intensity after retinoid treatment, the upper band, which we show to be a phosphorylated form of connexin 43 (Figure 6), generally exhibited the most dramatic increase. Connexin 43 doublets have also been seen in intact tissues and in cultured vole cells where the upper band has also been shown to be a phosphorylated form of this protein [21]. The functional significance of the phosphorylation of connexin 43 is at present unclear. In the heart, the upper band is predominant; in this organ, gap junctions are clearly required to be in their open state. In the brain, the lower band predominates (R. Kadle and B.J. Nicholson, submitted for publication); unfortunately, however, the physiological state of gap junctions in the brain is not known. In 1OT1/2 cells, the extensivejunctional communication seen in response to retinoids and the extensive phosphorylation of connexin 43 in retinoid-treated

Figure 7. Connexin 43 is localized in regions of cell-cell contact. 10T1/2 cells were grown on Permanox culture slides and treated with TTNPB or acetone solvent for 96 h. Cultures were then fixed and labeled first with connexin 43 antiserum, then with FITC-labeled goat antirabbit IgG F(ab')* fragment. Panels A and B. acetone control. fluorescent and remective Dhase image; panels C and D, TTNPB M,fluoresce'ntand iespective phase image; panels E and F, TTNPB lo-' M fluorescent and respective phase image; panels G and H, late logarithmic growth

Dhase cells treated with lo-' M lTNPB. fluorescent and resDechve phase image; panels I and J, higher magnification views of regions of cell-cell contact in cultures treated with lo-' and lo-' M TTNPB, respectively. In all treated cultures, the brightest fluorescence was observed in regions of cell-cell contact, regions which are Doorlv imaaed bv Dhase contrast microscoDv. The lack of such fluoresc6nt plaqueiin peripheralregions not'contacting other cells (arrows) can be seen in logarithmically growing cells (panelsGand H). Magnificationof panelsA-H x 750; I-J x 2000.

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cells both suggest that open gap junctions consist of phosphorylated proteins. The potential modulatory effects of phosphorylation of connexins are not understood; however, both A-kinase [36] and elevation of cAMP levels [13] have been shown to stimulate junctional communication. Conversely, the ability of pp60"-'" to inhibit junctional communication in vole cells has been associated with the phosphorylation of tyrosine residues on connexin 43 of the cells 1211. The observation that the upregulation of gap junctional communication induced by retinoids in 10T1/2 cells is strongly correlated with enhanced growth control [ 101and suppression of the neoplastic transformation of initiated cells [II ] suggests a functional relationship between these phenomena. In an extension of our observation that normal cells can reversibly suppress the growth of neoplastic cells when these cell types are induced to communicate across junctions in response to cAMP [I 31, we have hypothesized that similar junctional transfer of signal molecules results in the suppression of neoplastic transformation of carcinogen-initiated cells. Initiated cells, which occupy a position intermediate between normal and malignant in the carcinogenic process, can communicate extensively with nontransformed 1OTI /2 cells. Furthermore, retinoids upregulate junctional communication in both cell types. Thus, in mixtures of normal and initiated cells which are produced after exposure of normal cells to a carcinogen, retinoid treatment would place these initiated cells within an expanded network of communicating cells. Consistent with this model, just as transformed cells become growth arrested when placed in junctional communication with growth-arrested normal cells [I31, initiated cellsare inhibited from progressing to neoplasia under two circumstances: (1) if distributed in small colonies in close contact with surrounding normal cells [37] or (2) when distributed in large colonies where close contact is denied, if the network of communicating cells is expanded by retinoids [ I I]. Conversely the tumor promotor 12-0-tetradecanoylophorbol13-acetate(TPA), which blocks communication, inhibits the retinoid-induced suppression of transformation [II]. Both the identity of the postulated signal($ and the question of whether proliferation and transformation are influenced by the same signal($ are completely unresolved. The constraints of gap junctional pore size and the expectation that signals should not be membrane permeable predicts a water-soluble, charged ion or molecule of less than about 1000 Da. For example, cAMP [38], inosrtol triphosphate, and Ca2+ [39] have been shown to traverse gap junctions and to possess these physicochemicalproperties. Regulation of gap junctional communication by retinoids in mesenchymal cells (and in other cell types expressing connexin 43) in the intact animal could help explain certain physiologic and pharmacologic effects of retinoids in addition to their role in cancer chemoprevention. For example, it is known that gap junctional communication is developmentallyregulated during embryogenesis[401, that disruption of communication in hydra leads to abnormalities 1411, that a retinoic acid gradient determines pattern formation in the chick limb bud [42], and that retinoids

are teratogenic [43]. Our results suggest that in addition to direct effects of retinoids on genes controlling growth, differentiation, and development, retinoids could affect these functions indirectly by modulating the intercellular transfer of regulatory signals. A C K NOW LE DGMENTS This work was supported by grants CA 39947 (JB) and CA 48049 (BN) from the National Institutes of Health and by a fellowship from the American Heart Association (RK). We wish to thank Drs. T. Humphreys and G. Dolecki for guidance with northern blot techniques; Dr. E . Beyer for plasmid G2A; Melissa Churley, Karin Kohara, and Erick Cooke for technical assistance; and Linda Kuriyama for word processing Received July 3 1, 1990; revised September 7, 1990; accepted September 11, 1990.

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