In Vitro Cell. Dev. Biol.--Animal 31:738-740, November 1995 © 1995 Society for In Vitro Biology 1071-2690/95 $05.00+0.00

Letter to the Editor TIGHT JUNCTIONS AND MUCIN mRNA IN BEAS-2B CELLS

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Dear Editor: The BEAS-2B cell line has been used as a model for human airway epithelial cells in several recent studies (5,8,9,14). This cell line was derived from normal human bronchial epithelial cells immortalized using an SV40-adenovirus 12 hybrid virus (12). We investigated the expression by BEAS cultures of tight junction formation and mucin mRNA. Because the morphologic appearance of the cultures appeared to change over multiple passages, we investigated these functions in both "intermediate" (Passages 34-72) and "late" (Passages 80-125) cultures. Our results suggest that BEAS cell cultures have significant transepithelial resistances under some culture conditions, and express human tracheobronchial-specific mucin mRNA. These findings appear to be influenced by passage number and by the concentration of calcium in cell culture media. BEAS cells ($6 subclone, originally obtained at Passage 34) were a gift from Dr. C. C. Harris of NIH. Cells were maintained on plastic tissue culture dishes in serum-free, partially hormonally defined media (KGM, Clonetics Corp., San Diego, CA) with a calcium concentration of 0.15 mM. Cells were seeded onto collagen-coated filters (Transwell-COL, Costar, Cambridge, MA; 24 mm diameter; 3.0 gm pore size) at a density of 1 × 10~ cells per filter. After seeding, cells were maintained for 2 d in KGM. Cells were then switched into either KGM alone, or KGM supplemented with CaClz (1.3 mM) and/or another differentiation-promoting factor, retinoic acid (3 × 10 -l° M; Biofluids, Rockville, MD). At time points with peak resistance for each condition, wells were processed for histologic examination by light microscopy and examination of freeze-fracture replicas by transmission electron microscopy. Differences in appearance were noted between intermediate- and late-passage BEAS cultures. Intermediate-passage layers showed marked variations in thickness, ranging from single-cell in some areas to many cells thick in others; phase-contrast microscopy showed elongated cells with occasional mounds of cells. Late-passage cells formed more uniform layers that were roughly two cells thick, with a cobblestone morphology under phase contrast. Freezefracture electron micrographs from cell layers with higher transepithelial resistance revealed tight junction networks. The complexity of the junctions varied from a single strand to multiple strands. Tight junction number and complexity appeared to increase with increasing transepithelial resistance, but because the relationship between the structure and function of junctional complexes is unclear (6), we made no attempt to quantify these trends. Serial measurements of BEAS cell layers were made to establish the time course and relative magnitude of transepithelial resistance, as a functional indicator of tight junction expression, under various culture conditions. Measurements were made using a pair of handheld Ag/AgC1 electrodes connected to a portable volt-ohmmeter (EVOM; World Precision Instruments, West Haven, CT). The resis-

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FIG. 1. Time course of development of transepithelial resistance (measured by EVOM) in late-passage (Passages 84-124) BEAS cells under various culture conditions. All cells were seeded at confluent density onto collagencoated filters in unsupplemented KGM and changed to "differentiation" media at 2 d (arrow). Open circles, solid line = late-passage, KGM + retinoic acid + calcium; closed circles, dotted line = late-passage, KGM + calcium; closed circles, solid line = late-passage, KGM. Each point represents mean _+ SEM for n = 12-18 wells (KGM + calcium + retinoic acid), n = 3-8 wells (KGM + calcium) or n = 9-13 wells (KGM alone). * = P < 0.05 vs. cultures in KGM alone for each time point (ANOVAwith Bonferroni multiple comparison posttests). For clarity, error bars are omitted for Day 2.

tance of a filter without cells in identical media was subtracted from that of each experimental well to determine the resistance of the epithelial cell layer. Apical and basolateral media were replaced (1.5 ml apically and 2.6 ml basolaterally) and plates rewarmed to 37 ° C just prior to measurements to insure as little variation in media volume and temperature as possible. Intermediate (Passages 34--66) BEAS did not achieve maximum resistance of greater than 100 ohm-cmz under any of the conditions. In contrast, late (Passages 84-124) cultures averaged a maximum resistance of 271 ohm-cm2 in KGM alone, with a marked increase when Ca2+e was increased to 1.3 raM. The time course of change in resistance in late-passage cultures is shown in Fig. 1. Significant increases in resistance were observed 2-3 days after changing to media containing 1.3 mM Ca 2+ (Days 4-5 after seeding onto filters) in late-passage euhures. Maximum resistance was usually reached 5-7 d after seeding on filters, and 3-5 d after changing to high-Ca2+e media. Retinoie acid did not increase resistance significantly over controls at concentrations from 0.3 nM to 30 nM. To cheek the accuracy of EVOM measurements, transepithelial resistance was also measured in a number of late-passage BEAS cultures in a temperature and pH-controlled Ussing chamber with uniform current density. Cultures were maintained at 37 ° C with a heating jacket, and both apical and basolateral surfaces were continuously perfused with a Krebs bicarbonate Ringer solution with 1.1

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BEAS-2B CELL TIGHT JUNCTIONS/MUCIN mM CaC12 and pH of 7.4. The cultures were clamped to 0 mV, and transepithelial resistance was calculated from the changes of clamp current induced by 1.0 mV voltage pulses and Ohm's law. Five to seven days after seeding on filters, resistance measured in the Ussing chamber averaged 68 + 5% (n = 11) of resistance measured on the identical wells several hours prior using the EVOM system. In Ussing chamber measurements, resistance in high Ca2+e was 723 + 40 ohm-cm2 (n = 7), compared to 168 + 30 ohm-cm2 in controls (n = 3). Transepithelial potential differences of all wells were < 2 mV (apical negative). Increased Ca2+e had no effect on cell number (4.2 + 0.2 × 106 cells/filter, vs. 4.2 + 0.5 × 106 cells/filter in controls; n = 3), while EVOM measurements on the same wells demonstrated a significant increase in resistance induced by high Ca2÷e (496 _+ 23 ohm-cm 2, vs. 276 + 9 ohm-cm2 for controls; n = 3; P = 0.005). However, there were differences between cell numbers in intermediate- and late-passage cultures at similar time points. On Day 7 after seeding on filters at 1 × 106 cells/well, Passage 54 cell layers contained 2.0 _+ 0.1 × 106 cells per well (n = 9), whereas Passage 128 layers had 4.2 _+ 0.2 × 106 cells per well (n = 9). This difference between intermediate and late passage cell numbers was likely due to differences in proliferation rates, because attachment efficiency after seeding onto filters was the same for both (96.7 + 0.4%, intermediate; 97.2 + 0.4%, late; n = 5). The 1.3 mM Ca2+e-induced resistance in late-passage BEAS is comparable to values reported in human airway epithelial cells in primary culture (17). Measurements of resistance made with the epithelial volt-ohmmeter (EVOM) were consistently higher than conventional Ussing chamber measurements on the same cell layers, but values obtained using these two methods correlated well, suggesting that the EVOM device provides a reasonable estimate of resistance that may be used to test cultures serially. Transepithelial resistances of 400--600 ohm-cm2 measured 6 d after changing confluent cultures on collagen-coated filters to high-Ca2÷e media, as measured using the EVOM device, have persisted in BEAS cultures up to passage numbers greater than 200. Our finding in Ussing experiments of transepithelial potential differences of less than 2 mV, however, suggests that relatively little ion transport occurs in this cell line under these culture conditions. The effects of CaZ÷e on tight junctions in epithelial cell cultures have been well described previously (13). The time course we observed for development of maximum resistance by BEAS cultures (Fig. 1) was similar to that reported for a toad kidney epithelial cell line (10) and suggests that synthesis of tight junction proteins may be induced by increased CaZ+e. The mechanism of calcium-induced transepitheliat resistance in our late passage BEAS cultures remains unclear at present, but expression of mRNA for ZO-1, a protein associated with tight junction formation (16), did not appear to increase in response to passage number or high Ca2+e (data not shown). Recently, genes coding for mucin polypeptides designated MUC1, MUC2, and MUC5 (Th46 and Th47) have been cloned and found to be expressed in human airway tissue (1-3,7). In separate studies, we have found that of these sequences, Th46/Th47 is predominant in cultured and freshly isolated human nasal and bronchial epithelium (4). Using semiquantitative PCR, we comlSared BEAS culture MUC1, MUC2, Th46, and Th47 mRNA abundance under differentiation conditions as described above. BEAS cell RNA was isolated by guanidinium isothiocyanate extraction followed by CsCl gradient centrifugation. 0.1 p.g of RNA for each condition was reverse transcribed

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FIG. 2. Relative expression of mucin mRNAs encoding MUC2, TH46, and TH47 sequences, under culture conditions described in the text. Purified RNA from BEAS cultures was reverse transcribed, then 0.1 ktg RNA amplified using PCR, followed by slot blots with radiolabeled oligonucleotides complementary to mucin sequences. Data are expressed as cpm mucin × 10 6. Each bar represents mean + SEM (n = 4). * = P < 0.05 vs. Passage 72, 0.15 mM calcium (ANOVA).

(MMLV reverse transcriptase, GIBCO BRL, Gaithersburg, MD) and the resulting cDNA amplified by PCR as described in detail elsewhere (4). Optical density of bands representing GAPDH, as an internal standard sequence for RNA input into reverse transcription, did not vary significantly with passage or calcium (passage 72, low calcium = 0.39 + 0.12; passage 72, high calcium = 0.43 + 0.03; passage 123, low calcium = 0.47 + 0.08; passage 123, high calcium = 0.49 + 0.05). Amplification was carried out to 36 cycles for the human tracheobronchial mucin sequences MUC1, MUC2, Th46, and Th47. MUC1 was easily detectable in BEAS cells but showed no passageor calcium-related trends in mRNA abundance. Hybridization of MUC2, Th46, and Th47 probes under the various passage and calcium conditions is shown in Fig. 2. In contrast to the effects we noted on transepithelial resistance, we did not observe statistically significant calcium-induced increases in mucin mRNA expression in later passage BEAS cultures, although there was a tendency for 1.3 mM calcium to be associated with increased abundance of MUC2 at both passages and Th46 at intermediate-passage (Fig. 2). Retinoic acid, at the concentrations used in these experiments (3 X 10- ~0M), did not appear to influence mucin mRNA abundance. In conclusion, significantly increased transepithelial resistance could be induced by 1.3 mM Ca2+e in late but not intermediate passage BEAS cell line cultures, suggesting the selection over time of a subpopulation of cells that is more highly differentiated with regard to tight junction formation. The factors responsible for this selection are unclear, but might include differential expression of growth-factor receptors or different susceptibility to unknown stresses in culture. The presence of variation in morphology at intermediate passage suggested that the cultures were heterogeneous at that stage, and differences in growth rate between subpopulations may have led to gradual elimination of slower-growing cells from the culture. The increased cell numbers in late-passage cultures favor this hypothesis. Our observations may be important for investigations of polarized airway epithelial processes that employ the BEAS cell line. Although the biologic significance of changes in quantities of mucin mRNAs for the function of mucus is currently unclear, the finding of MUC mRNA in BEAS cells may also allow use of this cell line for studies of regulation of mucin-related gene expression by airway cells.

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NOAH ET AL. ACKNOWLEDGMENTS

We thank Drs. J. Lechner and C. Harris for providing the BEAS cell line. This work was supported by grants from the Cystic Fibrosis Foundation (CFF/ I 256); the North Carolina American Lung Association's Thomas H. Davis Award; The University of North Carolina Center for Environmental Medicine and Lung Biology (Cooperative Agreement #CR817643, U.S. Environmental Protection Agency); and the National Heart, Lung, and Blood Institute (SCOR grant #HL19171). The research described in this article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

REFERENCES 1. An, G.; Luo, G.; Wu, R. Expression of MUC2 gene is down-regulated by vitamin A at the transcriptional level in vitro in tracheobronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 10:546-551; 1994. 2. Aubert, J.-P.; Porchet, N.; Crepin, M., et al. Evidence for different human tracheobronchial mucin peptides deduced from nucleotide cDNA sequences. Am. J. Respir. Cell Mol. Biol. 5:178-185; 1991. 3. Audie, J. P.; Janin, A.; Porchet, N., et al. Expression of human mucin genes in respiratory, digestive, and reproductive tracts ascertained by in situ hybridization. J. Histochem. Cytochem. 41:1479-1485; 1993. 4. Cazares, L. H.; Reed, W.; McKinnon, K. P., et al. Characterization of mucin gene expression in human airway ceils. Am. J. Respir. Crit. Care Med. 149:A28; 1994. 5. Devlin, R. B.; McKinnon, K. P.; Noah, T. L., et al. Cytokine and fibronectin production by human alveolar macrophages and airway epithelial cells exposed to ozone in vitro. Am. J. Physiol.: Lung Cell. Mol. Physiol.; 266:L612-L619, 1994. 6. Gonzalez-Mariscal, L.; Chavez de Ramirez, B.; Lazaro, A., et al. Establishment of tight junctions between cells from different animal species and different sealing capacities. J. Membr. Biol. 107:43-56; 1989. 7. Hollingsworth, M. A.; Batra, S. K.; Qi, W.-N., et al. MUC1 mucin mRNA expression in cultured human nasal and bronchial cells. Am. J. Respir. Cell Mol. Biol. 6:516-520; 1992. 8. McDonald, R. J.; St. George, J. A.; Pan, L. C., et al. Neutrophil adherence to airway epithelium is reduced by antibodies to the leukocyte CD11/ CD18 complex. Inflammation 17:145-151; 1993. 9. Noah, T. L.; Paradiso, A. M.; Madden, M. C., et al. The response of a human bronchial epithelial cell line to histamine: Intracellular calcium changes and extracellular release of inflammatory mediators. Am. J. Respir. Cell Mol. Biol. 5:484-492; 1991. 10. Perkins, F. M.; Handler, J. S. Transport properties of toad kidney epithelia in culture. Am. J. Physiol. 241:C154-C159; 1981.

11. Pillai, S.; Bikle, D. D.; Manciati, M. L., et al. Calcium regulation of growth and differentiation of normal human keratinocytes: Modulation of differentiation competence by stages of growth and extracellular calcium. J. Cell. Physiol. 143:294--302; 1990. 12. Reddel, R. R.; Ke, Y.; Gerwin, B. I., et al. Transformation of human bronchial epithelial ceils by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res. 48:1904--1909; 1988. 13. Simons, K.; Fuller, S. D. Cell surface polarity in epithelia. Annu. Rev. Cell Biol. 1:243-288; 1985. 14. Stark, J. M.; van Egmond, A. W.; Zimmerman, J. J., et al. Detection of enhanced neutrophil adhesion to parainfluenza-infected airway epithelial cells using a modified myeloperoxidase assay in a microtiter format. J. Virol. Methods 40:225-242; 1992. 15. Steiger, D.; Fahy, J.; Boushey, H., et al. Use of mucin antibodies and cDNA probes to quantify hypersecretion in vivo in human airways. Am. J. Respir. Cell Mol. Biol. 10:538-545; 1994. 16. Stevenson, B. R.; Siciliano, J. D.; Mooseker, M. S., et al. Identification of ZO-I: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J. Cell Biol. 103:755-766; 1986. 17. Widdicombe, J. H.; Coleman, D. L.; Finkbeiner, W. E., et al. Electrical properties of monolayers cultured from cells of human tracheal mueosa. J; Appl. Physiol. 58:1729-1735; 1985. Terry L. Noah 1 James R. Yankaskas Johnny L. Carson Todd M. Gambling

Lisa H. Cazares Karen P. McKinnon Robert B. Devlin

Department of Pediatrics (T. L. N., J. L. C., T. M. E.) and Medicine (J. R. Y.) and the Center for Environmental Medicine and Lung Biology University of Noah Carolina School of Medicine (T. L. N., J. L. C., T. H. G., L. H. C.); TRC Alliance Corporation (K. P. M.) Chapel Hill, Noah Carolina 27514; Health Effects Research Laboratory U.S. Environmental Protection Agency (R. B. D.) Research Triangle Park, Noah Carolina 27711. (Received 12 December 1994) 1To whom correspondence should be addressed at 635 Burnett-Womack Building, CB#7220, Chapel Hill, North Carolina 27599-7220.