(CANCER RESEARCH 52, 5707-5712, October 15, 1992]

Carotenoids Up-Regulate Connexin43 Gene Expression Independent of TheirProvitamin A or Antioxidant Properties1

Li-Xin Zhang, Robert V. Cooney, and John S. Bertram2

Molecular Oncology Unit, Cancer Research Center of Hawaii. University of Hawaii, Honolulu, Hawaii 96813

ABSTRACT

Epidemiological evidence and studies in whole animals and cell culture have indicated that carotenoids have cancer chemopreventive action. In mouse C3H10T1/2 cells, this activity is highly correlated withthe ability of carotenoids to up-regulate gap junctional intercellularcommunication. Here, we report that in mouse cells, carotenoids increase the expression of connexM.ì,a gene that encodes a major gapjunction protein. This effect appears unrelated to their provitamin Aor antioxidant properties, since carotenoids with and without provitaminA activity increased levels of connexin43 mRNA and protein, whereasthe antioxidants methyl-bixin and a-tocopherol were inactive. Moreover, the active carotenoid canthaxanthin did not induce the vitaminA-inducible gene retinoic acid receptor-0. Connexln4i is the first caro-tenoid-inducible gene described in mammals. By indicating an additional pathway through which carotenoids function, these data providea mechanistic basis for cancer chemoprevention by carotenoids and maylead to a re-evaluation of carotenoid physiology.

INTRODUCTION

Carotenoids, yellow and red pigments found in vegetablesand fruits, are common components of the human diet. Accumulating evidence suggests that carotenoids, particularly 0-car-otene, have diverse beneficial roles in animals and humans.Among these are: provitamin A activity (1), cancer chemoprevention (2-4), stimulation of immune responses (5), suppression of atherosclerosis (6), and prevention of cataract formation(7). High dose ^-carotene is clinically approved for use inpatients with erythropoietic protoporphyria (8), is currentlyundergoing clinical trial as a cancer preventive agent (9), and,together with canthaxanthin, is a widely used, United StatesDepartment of Agriculture-approved, food coloring agent (10).

Until recently, biological activity of carotenoids in mammalswas considered limited to those with provitamin A activity.However, since Peto étal.(\ 1) in 1981 first raised the question,"Can dietary 0-carotene materially reduce human cancerrates?", the focus of carotenoid research has shifted to include

studies of their properties as antioxidants (12, 13). At present,therefore, 2 competing, but not mutually exclusive, hypothesesexist to explain carotenoid action in mammals: (a) conversionto retinoids, vitamin A-like compounds possessing multiplephysiological properties; and (b) activity of the intact moleculeas a lipid-phase antioxidant. In previous studies using carcinogen-treated C3H10T1/2 cells, we have confirmed that carotenoids can function as cancer preventive agents (14, 15) andhave demonstrated a novel action of carotenoids: that of up-regulating gap junctional communication (16). The ability ofcarotenoids to enhance communication was highly correlatedwith their ability to inhibit neoplastic transformation, however,

Received 4/23/92; accepted 8/3/92.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This research was supported by Grant BC686 from the American CancerSociety.

2 To whom requests for reprints should be addressed, at University of Hawaii atManoa, Cancer Research Center, Department of Molecular Oncology. 1236 Lau-hala Street, Honolulu, HI 96813.

their antioxidant capabilities were not. Neither activity wasassociated with the provitamin status of the various carotenoidstested (16).

Here, we explore the mechanism by which various carotenoids elevate junctional communication. Gap junctions arecomposed of transmembrane proteins called connexins, whichform a family of proteins (17). One member of this family,Cx43,3 is a major gap junctional protein in various mammalian

cells including 10T1/2 cells (18). The data indicate that carotenoids enhance gap junctional communication by increasingthe levels of Cx43 mRNA and protein. This effect appearsunrelated to their provitamin A activity or antioxidant properties.

MATERIALS AND METHODS

Chemicals. Lycopene and fi-carotene were obtained from SigmaChemical Co. (St. Louis, MO) and were 90 and 80% pure, respectively,as determined by high-performance liquid chromatography. Canthaxanthin and /rans-methyl-bixin were gifts from Hoffmann-LaRoche(Nutley, NJ), and were >99% and 90% pure, respectively. Because of itspurity and greater chemical stability, canthaxanthin was used for manyof the molecular studies. The synthetic benzoidal derivative of retinoicacid, TTNPB, was a kind gift from Hoffmann-LaRoche. a-Tocopherolwas purchased from Sigma.

Cell Culture. Mouse embryonic fibroblast C3H/10T1/2 cells werecultured in Eagle's basal medium with Earle's salts (GIBCO), supple

mented with 5% fetal bovine serum (Hyclone Laboratories) and 25jig/ml gentamicin sulfate, and were incubated at 37°Cin 5% COi in air

as described previously (15,19). Unless otherwise mentioned, cells wereseeded in plastic culture dishes (Falcon). When confluent, 1 week post-seeding, the medium was changed to 3% fetal bovine serum. Drugtreatment started with the first medium change unless otherwise indicated. Cell number was counted by Coulter counter. The murine embryonal teratocarcinoma F9 cell line was kindly supplied by Dr. Lorraine J. Gudas (Harvard) and cultured as described (20). Briefly, thecells were cultured in Dulbecco's modified Eagle's medium (Flow) con

taining 10% fetal calf serum (Hyclone Laboratories) and 25 *¿g/mlgentamicin sulfate and were incubated at 37°Cin 5% CO: ¡na¡r-Cells

were seeded in 150-mm plastic culture dishes. Since this transformedcell line rapidly lifts off from the culture dish after attaining confluence,it was treated on the second day after seeding.

Treatment. All carotenoids tested were delivered to cells using tet-rahydrofuran as solvent (99.9% with 0.025% butylated hydroxytoluene;purchased from Aldrich Chemical Co.) as described previously (15).TTNPB and a-tocopherol were dissolved in acetone unless indicated.The final concentration of acetone or tetrahydrofuran in the culturemedium was 0.5%.

Gap Junctional Communication Assay. Cell cultures were grown in60-mm dishes and treated with drugs after confluence. Gap junctionalcommunication was measured by microinjection of the fluorescenttracer dye Lucifer yellow CH (Sigma) as a 10% solution in 0.33 MLiCIas described previously (21). Micropipettes were prepared from glasscapillary tubes with the aid of a Flaming-Brown micropipette puller(Sutler Instrument Co., Novata, CA), and Lucifer yellow was microin-jected into a single cell with an Eppendorf microinjector. The total

3 The abbreviations used are: Cx43, connexin43; TTNPB. tetrahydrotetramethylnaphthalenyl propenylben/oic acid: SDS. sodium dodecyl sulfate: RAR, retinoicacid receptor.

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UP-REGULATION OF CONNEXIN43 GENE EXPRESSION BY CAROTENOIDS

number of fluorescent neighboring cells was counted 10 min after mi-croinjection and served as an index of gap junctional communication.The communicating networks were digitized using a video camera andstored for later analysis.

Isolation of Protein. Cell cultures were grown in 150-mm dishes andwere treated after reaching confluence. The cells were washed withice-cold phosphate-buffered saline containing 1 ITIMNaF and 1 imiphenylmethylsulfonyl fluoride. After centrifugation at 3,000 rpm for10 min, cell pellets were either frozen at -70°C for future analysis or

lysed in 30-50 ¡i\of lysis buffer (1% Nonidet P-40, 0.05 M iodoaceta-mide, 10 mM phenylmethylsulfonyl fluoride. 1 m\i EDTA, 1 MMleu-peptin. 2 ¿ig/mlaprotinin, 0.7 ¿tg/mlpepstatin in borate buffer, pH 8.0)for 1-2 h at 4°C.Lysates were then centrifuged at 10,000 x g for

15 min, and the supernatant was used as the source of Cx43. Proteincontent was measured with a commercial Protein Assay Reagent kit(Pierce Chemical Co., Rockford, IL) according to the manufacture's

instructions.Protein Electrophoresis and \Yestern Blotting. Cell lysates contain

ing an equal amount of protein (usually 35-50 Mg/lane) were mixed with2x SDS sample buffer containing 20% 0-mercaptoethanol. After atleast 15 min at room temperature, the samples were electrophoresed ona 10% SDS-polyacrylamide mini-gel (Bio-Rad, Richmond, CA) at 200V. A prestained protein standard was also loaded for indication of bothmolecular weight and transference efficiency. After electrophoresis.Western blots were performed as previously described using a rabbitpolyclonal antibody raised against a 15-mer synthetic polypeptide located in the C-terminal domain of rat heart Cx43 (22).

Immunofluorescent Detection of Gap Junctional Plaques. This analysis was essentially performed as previously described (22) using thesame polyclonal anti-Cx43 antibody as described above.

Isolation of Total RNA and Poly(A)' RNA. After treatment, cellswere harvested with cold phosphate-buffered saline containing 10 mstEDTA and pelleted by centrifugation at 3000 rpm for 10 min. The total RNA was isolated by using RNA isolation solvent RNAzol B kit(TM Cinna Scientific, Inc., Friendswood, TX). The poly(A+) RNA was

isolated from above total RNA by using PolyATtract mRNA isolationsystem III kit (Promega, Madison, WI).

Agarose Gel Electrophoresis and Northern Blotting. Total RNA(10 Mg)or poly(A+) RNA (4.5 jig) wrasseparated by 1% agarose/2.2 Mformaldehyde gel electrophoresis and blotted overnight onto a Hy-bond-N+ nylon membrane (Amersham, Oakville, Ontario, Canada).The cDNA probes were radiolabeled with [«'2P]dCTP (300 Ci/mmol)

using the Oligolabeling kit (Pharmacia, Piscataway, NJ). The G2AcDNA clone of rat heart Cx43 was a kind gift from Dr. E. Beyer,Harvard University (18). The cDNA clone of human RAR-0 was a giftfrom Dr. Chambón (23). The cDNA clone for glyceraldehyde-3-phos-phate dehydrogenase was from American Type Culture Collection. Thelabeled cDNA was purified from unincorporated radioactivity by passing it through a commercially available column (Schleicher andSchuell). The membranes were hybridized with labeled probes for 24 hat 42°Cfollowed by 2 stringent washes with 75 mM NaCl, 15 HIMTris,and l mM EDTA, pH 7.5/0.1% SDS/0.1% Na-PPi at 65°Cfor 20 min.

The final post-hybridization wash was with 600 mMNaCl, 120 mMTris,and 8 HIMEDTA, pH 7.5, at room temperature for 20 min followed byautoradiography with Kodak X-Omat AR film at -70°C using double

intensifying screens. In some experiments, membranes were reprobedafter first stripping off the radioactivity by boiling in 0.5% SDS for 1 h.The adequacy of stripping was confirmed autoradiographically. Membranes were reprobed as described above.

RESULTS

We have previously found that carotenoids enhance junctional communication in 10T1/2 cells and that this activity washighly correlated with their ability to inhibit carcinogen-induced neoplastic transformation in these cells (16). Here, wefurther explore the mechanism by which selected carotenoids(structures shown in Fig. 1) up-regulate junctional communication. As shown in Fig. 2A, a 7-day treatment of 10T1/2 cells

Lycopene

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Fig. 1. Structures of carotenoids tested.

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treatment (days)

B

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Cx43

0 0.3 1.0 3.0 10canthaxanthin (uM)

Fig. 2. Induction of Cx43 in IOT1/2 cells by carotenoids and TTNPB but nota-tocopherol. Confluent cells were treated with indicated drugs and then harvested. Protein isolation, electrophoresis, and Western blotting were performed asdescribed using a polyclonal antibody to the C-terminal region of Cx43 (22). A,cultures were treated with I0~8 M TTNPB for 3 days. I0~5 M fi-carotene andcanthaxanthin for 7 days, or IO"5 M lycopene and cr-tocopherol for 7 days withretreatment every 3 days. Lane I, solvent control; Lane 2, a-tocopherol; Lane 3,canthaxanthin: Lane 4. lycopene; Lane 5, /¿-carotene:Lane 6, TTNPB. Lanes 7and 8 show results of a separate experiment in which cultures received solventcontrol (Lane 7) or 10~5 M methyl-bixin for 7 days (Lane 8). B, time course.Cultures were treated with 10 * Mcanthaxanthin. C. dose response. Cultures weretreated with various concentrations of canthaxanthin for 7 days.

with 0-carotene, canthaxanthin, or lycopene at concentrationsof 10~5 M resulted in large increases in Cx43 levels in compar

ison with control solvent-treated cultures. This combination ofconcentration and time had previously been shown to cause adramatic increase in junctional communication as measured bydye micro-injection (Fig. 3) (16). In contrast, treatment withmethyl-bixin [a carotenoid also active in protecting from membrane oxidation (16)], or with a-tocopherol [a highly potentlipid-phase antioxidant (16)], did not alter Cx43 levels. In carotenoid- and in retinoid-treated cultures (included as positivecontrols), the immunoreactive bands were observed as doublets.It appears likely that in accordance with previous observationsin retinoid-treated 10T1/2 cells (22), the upper band represents

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UP-REGULATION OF CONNEXIN43 GENE EXPRESSION BY CAROTENOIDS

Fig. 3. Increased gap junctional communication is accompanied b\an increased number and size of immunofluorescent plaques in regions of cell-cell contact. Confluent cultures were treated with IO"5 M

canthaxanthin for 7 days. A and B, photomicrographs of dye transferA, solvent control; B, canthaxanthin (stars indicate the injected cell).Junctional permeability was assayed by microinjection of the flúorescent dye Lucifer yellow CH (10% in 0.33 M LiCI) into a single cell asdescribed (21). C-F, immunofluorescent detection of junctionalplaques induced by canthaxanthin. C and E, control, fluorescent andrespective phase image; D and F, canthaxanthin treated, fluorescentand respective phase image (arrows indicate junctional plaques).10T1/2 cells were seeded on Permanox plastic slides and treated as inA and B. Cultures were fixed and labeled with the same Cx4i antibod>as used in Western blotting, and then with FITC labeled goat antirabbit IgG F(ab)2 fragment as described (22).

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eza phosphorylated form of Cx43. In other cells, Cx43 phosphor-ylation on serine residues is associated with gap junctional competence (24). The inability of a-tocopherol or methyl-bixin toincrease Cx43 levels indicates that the increases observed in/3-carotene-, canthaxanthin-, and lycopene-treated cultures arenot related to their antioxidant properties, nor to their provi-tamin-A activities, since, of the 3 active compounds, only ^-carotene is known to be converted to retinoids in mammals (1).

The induction of Cx43 by canthaxanthin was found to betime- and dose-dependent (Fig. 2, B and C). When confluent10T1/2 cells were treated with 10~5 Mdrug, an increase in Cx43

level was detected within 2 days of treatment and progressivelyincreased thereafter. Induction of Cx43 preceded the previouslyreported time-course for induction of communication (16).Dose-response studies with canthaxanthin demonstrated that aconcentration of 3 x 10~7 M produced a minimally detectable

increase in Cx43 and that higher concentrations resulted inprogressively increased levels (Fig. 2C). This concentrationrange spans the minimum to maximum biological responserange for induction of gap junctional communication and inhi

bition of neoplastic transformation (15, 16). The increase in theCx43 level after canthaxanthin treatment was accompanied byan increase in junctional communication as detected by dye-transfer measurements (Fig. 3^4 and B), and by a dramaticincrease in immunoreactive plaques in regions of cell-cell contact as detected by immunofluorescent microscopy (Fig. 3, Cand D). Thus, carotenoid-induced Cx43 becomes localizedwhere junctional plaques are expected to form.

Total RNA from control and carotenoid-stimulated 10T1/2cells was analyzed by Northern blotting to determine whetherthe increased protein levels of Cx43 were the result of increasedlevels of Cx43 mRNA. Blots were probed with a full lengthCx43 cDNA cloned from rat heart (18). Under high stringencyconditions, a 3.1-kilobase band was detected (Fig. 4/Õ),which isthe reported transcript size of Cx43 (18). The steady-state levelof Cx43 mRNA was markedly elevated by /3-carotene, canthaxanthin, and to a lesser extent by lycopene. Induction of Cx43mRNA by canthaxanthin was detected within 1 day of treatment and increased progressively over the 7-day observationperiod (Fig. 4B). This increase preceded the induction of Cx43

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HP-REGULATION Of CO.\.\KXI.\43 GENE EXPRESSION BY CAROTENOIDS

1234

3.1kb

B

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123456Fig. 4. Induction of Cx43 mRNA levels by carotenoids. Total RNA was iso

lated by using RNAzol B kit (TM Cinna Scientific, Inc.) and hybridized to ratheart Cx43 cDNA (18). Agarose gel electrophoresis. Northern blotting, Cx43cDNA labeling, and hybridization were performed as described (22).. I. confluentcells were treated once with 10~5 Mcarotenoids for 7 days. Lane 1, solvent control;

Lane 2, d-carotene; Lane 3, canthaxanthin: Lane 4, lycopene. B, time course forinduction of Cx43 mRNA. Confluent cultures were treated with IO"5 M canthax

anthin and cells harvested at increasing times thereafter. Lanes 1-6, 0. 1, 2, 3, 4,and 7 days, respectively.

protein levels by about 1 day. These data indicate that enhancement of junctional communication by carotenoids is achievedby increasing the levels of Cx43 mRNA and protein, resultingin an increased number of junctional channels.

The ability of carotenoids to up-regulate expression of a gene,Cx43, also up-regulated by retinoids suggests that both actthrough a common mechanism. This concept is reinforced bythe structural similarities between carotenoids and retinoids[lycopene is the immediate precursor to /3-carotene in plants,whereas /3-carotene is a precursor to retinal and retinoic acid inmammals (1)]. To test this concept, we investigated the abilityof canthaxanthin to up-regulate expression of RAR-0, a geneknown to contain a retinoic acid responsive element in its promoter region (25) and whose expression can be up-regulated byretinoids in diverse cell types (26-29). We chose canthaxanthinfor these studies because of its chemical purity and its greaterchemical stability than ß-caroteneand lycopene (15), and because it has been widely used as a non-provitamin A carotenoid.As shown in Fig. 5, a 3-day treatment of 10T1/2 cells with thesynthetic retinoid TTNPB at 10~8 Mstrongly increased the level

of RAR-0 mRNA, whereas treatment with canthaxanthin at10~5 Mfor 7 days had no effect. This was also the case in murine

embryonic teratocarcinoma F9 cells, in which both TTNPB andcanthaxanthin elevated Cx43 mRNA levels, but only TTNPBinduced RAR-/3 mRNA (Fig. 5).

DISCUSSION

The demands to reduce human cancer morbidity and mortality place a heavy burden on prevention. Of over 1000 compounds tested, the retinoids and carotenoids have obtainedmost attention and possess great promise for cancer chemopre-vention (30). However, the high toxicity of retinoids greatlylimits their use (30). Although carotenoids have no known toxicity (31), their lower potency as chemopreventive agents mayalso limit their clinical usefulness. Several problems have hin

dered the further understanding of carotenoid physiology andpharmacology. Principal among these are: (a) lack of animalmodels that mimic humans; (b) difficulties in delivering thesehighly lipophilic compounds to cultured cells; and (c) uncertainties regarding the choice of biological endpoint to be studied. An additional problem is that /3-carotene, the most widelystudied carotenoid, is the major metabolic precursor of retinoids, compounds with multiple physiological functions, including cancer chemoprevention (1-16).

The present studies, by demonstrating overlapping biologicalactivities for some (cancer chemoprevention, Cx43 expression,and junctional communication) but not all (RAK-.-f induction)

of the actions of carotenoids and retinoids may have relevanceto the different clinical properties of these compounds. If, asseems likely, clinical toxicity of retinoids is related to activationof retinoic acid responsive elements in diverse retinoid-respon-sive genes, then administration of carotenoids (which do notactivate RAR-0; Fig. 5) in sufficiently high concentrationswould circumvent toxicity yet result in effective cancer chemoprevention. This analysis assumes that the enhanced junctionalcommunication induced by retinoids and carotenoids is causalto cancer chemoprevention: as discussed below, there is evidence obtained from studies in cultured cells to support thisstatement. Furthermore, in a recent clinical trial, all-fra/w-ret-inoic acid, when applied topically, was shown to up-regulateexpression of Cx43 in human epidermis (32), demonstratingthat this action of retinoids is not limited to cells in culture.

Multiple lines of evidence support the concept that gap junctions serve as conduits for the passage of growth regulatorymolecules between communicating cells. This topic has been

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Cx43

GAPDH

10T1/2 F9Fig. 5. Comparison between the effects of canthaxanthin and TTNPB on in

duction of Cx43 mRNA and RAR-/3 mRNA in 10T1/2 cells (left) and murineteratocarcinoma F9 cells (right). Confluent 10T1/2 cells were treated with 10~8 MTTNPB for 3 days or with I0~s M canthaxanthin (CTX) for 7 days and thenharvested. F9 cells were treated with IO 8 M TTNPB or 10~5 M canthaxanthin for

5 days then harvested. Total RNA was isolated as described in Fig. 4 and thenPoly(A)+ RNA extracted using the PolyATtract system III kit (Promega). Agar-

ose gel electrophoresis. Northern blotting, cDNA labeling, and hybridization wereperformed as described (22); 5.5 ng of poly(A)+ RNA from 10T1/2 cells and 4.5

Mgfrom F9 cells were loaded in each lane. The membranes were first probed withhuman RAR-0 cDNA (23), then with glyceraldehyde-3-phosphate dehydrogenasecDNA to normalize the amount of RNA loaded, and finally with Cx43 cDNA.The poor hybridization to Cx43 mRNA presumably reflects the extensive processing of these membranes.

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UP-REGULATION OF CONNEXM43 GENE EXPRESSION BY CAROTENOIDS

the subject of several reviews (33-36). In 10T1/2 cells, up-regulation of communication between homologous cells(i.e., normal:normal or transformedrtransformed) by retinoidscorrelates with enhanced growth control (21, 37, 38), conversely, tumor promoters both in vitro and in vivo cause rapiduncoupling of communicating cells (34-36). Tumor cells are ingeneral deficient in junctional communication (34), howevercommunication with normal 10T1/2 cells can be restored byelevation of intracellular cAMP: in this situation, tumor cellsbecome growth arrested in proportion to the increase in heter-ologous communication (21). Furthermore, over-expressionof Cx43 in transformed cells by gene transfection causes a partial reversion of the transformed phenotype (39). In contrast,induction of transformation by v-src is temporally associatedwith the phosphorylation of tyrosine residues on Cx43 andinhibition of communication (40). Finally, in the 10T1/2 modelsystem we have demonstrated a highly significant statisticalcorrelation between the ability of diverse retinoids and caro-

tenoids to inhibit chemically induced neoplastic transformationand their ability to stimulate junctional communication (16,21). These observations have led to the proposal that retinoidsand carotenoids inhibit transformation in the 10T1/2 cell system by placing carcinogen-initiated cells in extensive communication with surrounding noninitiated cells, thereby preventingtheir transformation (16, 21). Whether the antiproliferative action of this enhanced communication is adequate to explain thisinhibition is as yet unknown. The present studies provide amechanistic basis for enhanced communication induced bycarotenoids.

The evidence that both provitamin A and non-provitamin Acarotenoids increased levels of Cx43 mRNA and protein,whereas other antioxidants, such as a-tocopherol and the car-otenoid methyl-bixin, were ineffective, implies that induction ofCx43 gene expression by carotenoids is independent of theirprovitamin A or antioxidant properties. However, both retinoids and carotenoids reversibly inhibit the postinitiation phaseof carcinogenesis (14, 41), enhance junctional communication(Fig. 3) (16, 21), and increase Cx43 mRNA and protein levels(Fig. 2 and 4) (22). These remarkable similarities between carotenoids and retinoids suggest a common mechanism, perhapsinvolving a low rate of conversion of carotenoids into activeretinoids in cell culture by chemical oxidation (1) or other processes. This rate (should it occur in vivo) would be insufficientto support growth (the classical assay for vitamin A activity)since there is no evidence that canthaxanthin or lycopene can beconverted to retinoids in mammals (1). Furthermore, we hadpreviously been unable to detect the conversion of l4C-labeled^-carotene to retinoids in 10T1/2 cells (42). The recent discovery of high affinity (10~9 to 10~8 M) nuclear RARs (26, 43, 44)

raises the possibility that a very low level of conversion ofcarotenoids to retinoids (not detectable because of the low specific activity of available [l4C]-|3-carotene) could activate RARs.

However, the demonstration of a dissociation between the action of canthaxanthin and TTNPB on RAR-0 expression inboth 10T1/2 and F9 cells (Fig. 5) strongly indicates that theinduction of Cx43 gene expression by canthaxanthin is independent of conversion to retinoids capable of activating RARsand inducing expression of RAR-/3. We cannot rule out thepossibility that carotenoids or carotenoid metabolites are activating a novel receptor, perhaps one of the RXR family ofreceptors (45-47), with ligand binding affinity for retinoids andcarotenoids, capable of inducing Cx43 but not RAR-/3 geneexpression. The knowledge that these receptors activate distinct

classes of responsive elements and thus differentially activatedistinct genes could explain our results. It is of interest that9-m-retinoic acid has recently been reported to be a physiological ligand for RXR II (47); 9-m-/3-carotene is found in humanserum (48) and in preliminary studies was found to be morepotent than all-fra/is-ß-carotene in inhibiting transformationand stimulating communication in 10T1/2 cells.4. It is notknown whether 9-m-0-carotene can be converted to the corresponding retinoid or whether RXR responsive elements exist inthe promoter region of the Cx43 gene.

The present data demonstrate that certain carotenoids have ahitherto undetected ability to regulate gene expression unrelated to their activity as provitamin A carotenoids or antioxidants. Since the biological activity of carotenoids in mammalswas considered limited to these activities, these data indicatethat carotenoid physiology in mammals or the classification ofprovitamin A carotenoids needs to be re-evaluated.

We have recently extended these studies to human dermalfibroblasts in culture and find that, as in 10T1/2 cells, certaincarotenoids induce junctional communication and Cx43 geneexpression,5 demonstrating that this novel action of caro

tenoids is not limited to the 10T1/2 cell model system. It remains to be determined whether carotenoids can up-regulateCx43 expression in a relevant whole animal model.

The independence of carotenoid action on Cx43 expressionfrom their provitamin A or antioxidant activities may provideimportant insights into carotenoid physiology, and in view ofthe increasing clinical use of these compounds, their pharmacology. In this context, it should be noted that serum levels of/3-carotene that have been reported after high-dose administration to humans are comparable to the concentrations used inour cell culture studies (49).

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Gene ExpressionConnexin43Carotenoids Up-Regulate

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