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Establishment of human corneal epithelial cells stably expressing human connexin43
Experimental Eye Research 90 (2010) 4–9
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Establishment of human corneal epithelial cells stably expressing human connexin43q Kazuhiro Kimura a, *, Shinichiro Teranishi b, Teruo Nishida b a b
Department of Ocular Pathophysiology, Yamaguchi University, Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi 755-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 June 2009 Accepted in revised form 3 September 2009 Available online 30 September 2009
Corneal epithelial cells communicate with each other through gap junctions. Whereas this property is retained in corneal epithelial cells in primary culture, it is often lost in immortalized epithelial cells. However, the life span of primary cultured corneal epithelial cells is short and the availability of human tissue for their preparation is limited. To examine the role of the gap-junction protein connexin43 (Cx43) in human corneal epithelial cells, we set out to establish an immortal human corneal epithelial cell line that stably expresses this protein. An expression vector encoding human Cx43 fused to enhanced green fluorescent protein (EGFP) was constructed and introduced by transfection into SV40-immortalized human corneal epithelial (HCE) cells. Stable transfectants were isolated by selection with the antibiotic G418. The expression and localization of the Cx43-EGFP fusion protein were examined by immunoblot analysis and fluorescence microscopy, respectively, and gap-junctional intercellular communication was monitored on the basis of dye coupling. HCE cells stably expressing Cx43-EGFP manifested intercellular dye transfer, whereas those stably expressing EGFP alone did not. Cx43-EGFP localized to the interfaces of neighboring cells. Stable expression of Cx43-EGFP in HCE cells did not affect the expression of keratins 3 and 12, which is a characteristic of corneal epithelial cells, but it did inhibit cell proliferation. We have established an HCE cell line that stably expresses human Cx43 and forms functional gap junctions. These cells may prove useful for studies of the role of gap junctions in the human corneal epithelium. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: corneal epithelial cell stable cell line gap junction connexin43
1. Introduction The corneal epithelium forms the anterior surface of the cornea and plays an important role in the maintenance of corneal transparency. It is a multilayered structure that sits on the epithelial basement membrane, and corneal epithelial cells are joined to each other by junctional structures including tight junctions and adherens junctions (Gipson, 1992; Scott et al., 1997; Suzuki et al., 2000; Ban et al., 2003). In addition, corneal epithelial cells form gap junctions, which consist of two hemichannels, one in each apposing cell membrane, and which mediate communication between cells (Dong et al., 1994; Wolosin et al., 2004; Shurman et al., 2005). Gap junctions contribute to the regulation of corneal epithelial cell functions such as cell growth, differentiation, adhesion, migration, and barrier formation (Suzuki et al., 2000; Kimura et al., 2008). Studies of the
q This study was supported in part by a grant (21791687) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. * Corresponding author. Tel.: þ81 836 22 2277; fax: þ81 836 22 2334. E-mail address: [email protected] (K. Kimura). 0014-4835/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2009.09.002
human corneal epithelium in vitro have been performed with primary cultured cells (Sun and Green, 1977; Ebato et al., 1987; Kahn et al., 1993). However, such cells become senescent after only a few passages in vitro and the availability of donor corneal tissue for their isolation is limited. As an alternative to primary cultured cells, several immortalized human corneal epithelial cell lines have been established and characterized (Kahn et al., 1993; Araki-Sasaki et al., 1995; Offord et al., 1999). One of the most widely studied of these cell lines is a line established by transformation of human corneal epithelial cells with simian virus 40 (SV40) (Araki-Sasaki et al., 1995). Gap junctions mediate direct communication among connected cells by allowing the exchange of small molecules or ions of <1 kDa including metabolites and second messengers (Revel et al., 1992; Laird, 1996). Gap junctions are composed of connexins, a family of integral membrane proteins that are expressed in a cell typespecific manner (Lo et al., 1996; Saez et al., 2005). Connexin43 (Cx43) is widely expressed among tissues and organs (Beyer et al., 1989; Christ, 2000). It is abundant in suprabasal cells of the corneal and limbal epithelia but not in basal cells of the limbal epithelium (Schlotzer-Schrehardt and Kruse, 2005; Chee et al., 2006; Wolosin, 2006). We also previously showed that the expression of Cx43 in
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basal cells of the corneal epithelium is increased in a rat model of diabetes (Wakuta et al., 2007). Furthermore, the expression of Cx43 is found to be altered at the leading edge of the epithelium during corneal wound healing (Suzuki et al., 2000). Gap-junctional intercellular communication (GJIC) is impaired in various immortalized epithelial cells (Mesnil, 2004; Vinken et al., 2006; Pointis et al., 2007). We have now examined the expression of Cx43 and GJIC in SV40-transformed human corneal epithelial (HCE) cells. Furthermore, to characterize the function of Cx43 in human corneal epithelial cells, we established a line of HCE cells that stably express human Cx43. 2. Methods 2.1. Materials A mixture of Dulbecco’s modified Eagle’s medium and nutrient mixture F-12 (DMEM/F-12) as well as fetal bovine serum, gentamicin, trypsin-EDTA, and Lipofectamine 2000 were obtained from Invitrogen-Gibco (Carlsbad, CA). Bovine insulin, cholera toxin, human recombinant epidermal growth factor, and a protease inhibitor cocktail were from Sigma–Aldrich (St. Louis, MO). Plastic six- or 24-well culture plates and 60- or 100-mm culture dishes were obtained from Corning (Corning, NY), glass-bottom dishes (35-mm) were from Iwaki (Chiba, Japan), and G418 was from Nakalai Tesque (Kyoto, Japan). Mouse monoclonal antibodies to Cx43 were obtained from Chemicon (Temecula, CA), those to green fluorescent protein (GFP) were from BD-Clontech (San Jose, CA), those to E-cadherin were from Santa Cruz Biotechnology (Santa Cruz, CA), those to actin were from Sigma– Aldrich (St. Louis, MO), and those to keratin 3 or to keratin 12 were from Millipore (Billerica, MA). Rabbit polyclonal antibodies to zonula occludens (ZO)-1 were from Invitrogen-Gibco. Trizol reagent, Lucifer yellow and rhodamine-phalloidin were obtained from Invitrogen (Carlsbad, CA). Horseradish peroxidase-conjugated goat secondary antibodies and ECL Plus detection reagents were from Amersham Biosciences (Little Chalfont, UK). The pEGFP-N1 vector was obtained from BD-Clontech, MMLV reverse transcriptase was from Promega (Madison, WI), and Pfu Taq polymerase, BamHI, KpnI, were from Stratagene (La Jolla, CA). 2.2. Cells and cell culture SV40-immortalized human corneal epithelial (HCE) cells (ArakiSasaki et al., 1995) were obtained from RIKEN Biosource Center (Tsukuba, Japan). They were passaged in supplemented hormonal epithelial medium (SHEM), which consists of DMEM/F-12 supplemented with 15% heat-inactivated fetal bovine serum, bovine insulin (5 mg/mL), cholera toxin (0.1 mg/mL), human recombinant epidermal growth factor (10 ng/mL), and gentamicin (40 mg/mL). Human corneal fibroblasts were isolated as described previously (Kumagai et al., 2000) from tissue left over after corneal transplantation surgery; the tissue was used in strict accordance with the tenets of the Declaration of Helsinki. 2.3. Cloning of human Cx43 cDNA and plasmid construction Total RNA was isolated from human corneal fibroblasts with the use of the Trizol reagent. A portion (1 mg) of the RNA was subjected to reverse transcription with MMLV reverse transcriptase, and the resulting cDNA was subjected to the polymerase chain reaction (PCR) with the primers 50 -GGGGTACCGCCACCATGGGTGACTGGAGCGCCTTAG-30 (forward) and 50 -CGGGATCCCCGATCTCCAGGTCATCAGG-30 (reverse) and with Pfu Taq polymerase in order to amplify the coding region for Cx43. PCR was performed for 30 cycles of incubation at 94 C for 1 min, 50 C for 1 min, and 72 C for 2 min. The PCR product
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was digested with BamHI and KpnI, subcloned into pEGFP-N1. The identity of the insert was confirmed by sequencing. 2.4. Transfection and establishment of a stable cell line HCE cells (2 105) were cultured in a 60-mm culture dish for 24 h before transient transfection with 2 mg of plasmid DNA combined with Lipofectamine 2000 in SHEM for 16–18 h. For stable transfection, HCE cells (2 105) were plated in a 100-mm culture dish, incubated overnight, and then transfected with 10 mg of plasmid DNA combined with Lipofectamine 2000 in SHEM for 16–18 h. The cells were then cultured in SHEM containing G418 (600 mg/mL), and stably transfected clones were isolated and expanded. For experiments, stably transfected clones were usually cultured in SHEM containing G418 (600 mg/mL). 2.5. Dye coupling HCE cells (3 105) plated in 35-mm glass-bottom dishes were monitored for GJIC with the use of Lucifer yellow as described previously (Hao et al., 2005). In brief, the cells were washed with Ca2þ- and Mg2þ-free phosphate-buffered saline [PBS()], and Lucifer yellow (10% in water) was injected into two widely separated cells with the use of a microinjector (Micromanipulator and Transinjector; Eppendorf, Hamburg, Germany). The cells were fixed with 3.7% formalin, observed with a laser confocal microscope (Axioscope; Carl Zeiss Meditec, Hallbergmoos, Germany), and photographed 5 min after dye injection. The number of cells containing the dye was counted, and GJIC activity was expressed as the mean number of cells coupled to each injected cell. 2.6. Fluorescence microscopy HCE cells (1 105) cultured on 12-mm cover glasses in 24-well plates were fixed for 15 min with ice-cold acetone, washed with PBS(), permeabilized for 5 min with 0.1% Triton X-100 in PBS(), and incubated for 1 h at room temperature with 1% bovine serum albumin in PBS(). They were then incubated for 1 h with rhodamine-phalloidin (1:200 dilution) in PBS() containing 1% bovine serum albumin before examination with a laser confocal microscope (LSM5, Zeiss). 2.7. Immunoblot analysis HCE cells (2 105) cultured in 60-mm dishes were lysed on ice in 0.5 mL of a solution containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5 mM NaF, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, and 1% protease inhibitor cocktail. The lysates were centrifuged at 15,000 g for 15 min at 4 C, and the resulting supernatants (20 mg of protein) were subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel. The separated proteins were transferred to a nitrocellulose membrane, which was then exposed to 5% skim milk for 1 h at room temperature before incubation for 1 h with various primary antibodies at a dilution of 1:1000 in washing buffer [20 mM Tris–HCl (pH 7.4), 5% skim milk, 0.1% Tween 20]. The membrane was washed in washing buffer, incubated for 1 h at room temperature with horseradish peroxidaseconjugated secondary antibodies (1:1000 dilution in washing buffer), washed again, incubated with ECL Plus detection reagents for 5 min, and then exposed to film. 2.8. Cell proliferation assay Cells (2 104) seeded on 12-well culture plates were cultured for various times, detached by exposure to trypsin-EDTA, and
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counted with the use of a Coulter Particle Counter (Beckman Coulter, CA, Fullerton). 2.9. Statistical analysis Quantitative data are presented as means SE. Differences were analyzed with Dunnett’s test. A P value of <0.05 was considered statistically significant. 3. Results 3.1. GJIC and Cx43 expression in HCE cells We first examined whether Cx43 is expressed in HCE cells by immunoblot analysis. Cx43 was not detected in lysates of HCE cells, whereas it was present in those of human corneal fibroblasts examined as a positive control (Fig. 1A). We next determined whether HCEs manifest GJIC by monitoring dye coupling with Lucifer yellow, an anionic dye that diffuses through most types of gap-junction channels. Dye injected into HCE cells did not diffuse into neighboring cells (Fig. 1B), indicating the absence of GJIC. These results thus suggested that HCE cells have lost the ability to form gap junctions as a result of the loss of Cx43 expression. 3.2. Transient expression Cx43-EGFP in HCE cells To examine the effects of Cx43 expression in HCE cells, we constructed an expression plasmid for human Cx43 fused at its COOHterminus to enhanced green fluorescent protein (EGFP). HCE cells were transiently transfected with the Cx43-EGFP vector or with the corresponding plasmid encoding EGFP alone (pEGFP-N1) as a control. Immunoblot analysis with antibodies to GFP revealed a major band corresponding to Cx43-EGFP (w70 kDa) in the cells transfected with the Cx43-EGFP vector as well as a band corresponding to EGFP (w30 kDa) in those transfected with pEGFP-N1 (Fig. 2A). Immunoblot analysis with antibodies to Cx43 also revealed the band corresponding to Cx43-EGFP (Fig. 2B). These results thus showed that HCE cells are competent to express the Cx43-EGFP fusion protein. 3.3. Establishment of an HCE cell line stably expressing Cx43-EGFP To establish a human corneal epithelial cell line stably expressing human Cx43, we transfected HCE cells with the Cx43EGFP vector (or with pEGFP-N1 as a control) and then subjected the cells to selection by culture in the presence of G418. We isolated several cell clones after culture of the cells for 2 weeks, and we
Fig. 2. Transient expression of Cx43-EGFP in HCE cells. Cells transiently transfected with an expression vector for Cx43-EGFP or for EGFP were lysed and subjected to immunoblot analysis with antibodies to GFP (A) or to Cx43 (B). Data are representative of three independent experiments.
selected one clone showing the highest level of Cx43-EGFP expression for subsequent characterization. Immunoblot analysis of cell lysates with antibodies to GFP revealed major bands of the expected sizes for Cx43-EGFP or EGFP (Fig. 3A). We next examined the distribution of Cx43-EGFP in HCE cells stably expressing the fusion protein. Fluorescence microscopy revealed signals corresponding to Cx43-EGFP at the interfaces of apposing cells as well as in association with the Golgi apparatus and in the perinuclear region (Fig. 3B). In contrast, in cells stably expressing EGFP, the corresponding signals were detected in association with the Golgi apparatus and in the perinuclear region but not at the cell surface. To determine whether HCE cells stably expressing Cx43-EGFP manifest GJIC, we examined dye coupling with Lucifer yellow. GJIC was apparent in HCE cells stably expressing Cx43-EGFP but not in those stably expressing EGFP (Fig. 4). Moreover, in contrast to those stably expressing EGFP, HCE cells stably expressing Cx43-EGFP exhibited a cobblestone-like morphology similar to that of welldifferentiated corneal epithelial cells (Fig. 4A). To investigate the possible effects of stable expression of Cx43-EGFP on the phenotype of HCE cells, we examined the expression of keratins 3 and 12, which are characteristic of differentiated corneal epithelial cells, as well as that of the adherens-junction protein E-cadherin and the tight-junction protein ZO-1. Immunoblot analysis revealed that HCE cells stably expressing Cx43-EGFP also expressed keratins 3 and 12, E-cadherin, and ZO-1 at levels similar to those apparent in nontransfected cells or in cells stably expressing EGFP (Fig. 5). Finally, we examined the effect of stable expression of Cx43EGFP on the proliferation of HCE cells. The number of HCE cells stably expressing Cx43-EGFP was significantly smaller than that of those expressing EGFP after culture for 3, 5, or 7 days (Fig. 6), suggesting that expression of Cx43-EGFP inhibits the proliferation of HCE cells. 4. Discussion
Fig. 1. Analysis of Cx43 expression and GJIC in HCE cells. (A) Lysates of HCE cells and human corneal fibroblasts (CFs) were subjected to immunoblot analysis with antibodies to Cx43. (B) HCE cells were cultured for 48 h to confluence, after which an individual cell was microinjected with Lucifer yellow. The cells were fixed after 30 min, and the injected cell and its neighbors were examined with a confocal microscope. All data are representative of three independent experiments.
We have shown that SV40-transformed human corneal epithelial (HCE) cells do not express Cx43 or manifest GJIC. We further showed that these cells were capable of expressing a fusion protein of human Cx43 tagged with EGFP by transient transfection as well as established HCE cells stably expressing this fusion protein. The stable expression of Cx43-EGFP in HCE cells conferred
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Fig. 3. Stable expression and distribution of Cx43-EGFP in stably transfected HCE cells. (A) Cells stably transfected with an expression vector for Cx43-EGFP or for EGFP were lysed and subjected to immunoblot analysis with antibodies to GFP. (B) Cells stably expressing Cx43-EGFP or EGFP were cultured to confluence, stained with rhodamine-phalloidin for detection of F-actin, and examined by fluorescence microscopy. Red and green fluorescence represents rhodamine-phalloidin and either EGFP or Cx43-EGFP, respectively. Arrows indicate Cx43-EGFP signals at the interfaces of adjacent cells. Data are representative of three independent experiments.
the ability to communicate via gap junctions, and the fusion protein was detected at the interfaces of neighboring cells. The stable expression of Cx43-EGFP did not affect the expression of keratin 3, keratin 12, E-cadherin, or ZO-1 in HCE cells, but it did inhibit the proliferation of these cells. We have thus generated a line of immortalized human corneal epithelial cells that stably express human Cx43 and form functional gap junctions. Our observation that Cx43-EGFP localizes to the interfaces of apposing HCE cells stably expressing the fusion protein is consistent with the previously observed distribution of Cx43-EGFP in keratinocytes, testicular epithelial cells, and cancer cells (Holm et al., 1999; Jordan et al., 1999; Paemeleire et al., 2000; Roger et al., 2004). We showed that HCE cells stably expressing Cx43-EGFP adopted a cobblestone-like appearance similar to that of corneal epithelial cells in situ, and that these cells continue to express keratin 3 and keratin 12, both of which are characteristic of differentiated corneal epithelial cells. Moreover, HCE cells stably expressing Cx43-EGFP manifested GJIC, as do corneal epithelial cells in primary culture (Hernandez Galindo et al., 2003). The morphology and other
phenotypic characteristics of HCE cells stably expressing Cx43-EGFP are thus consistent with those of normal corneal epithelial cells. SV40-transformed human corneal epithelial (HCE) cells are a wellcharacterized model system that has been adopted widely for studies of corneal epithelial cell activities such as proliferation, differentiation, adhesion, migration, and barrier formation (Chiambaretta et al., 2004; Kimura et al., 2006, 2008). We have now shown that, in contrast to primary cultured corneal epithelial cells (Anderson et al., 2002; Williams and Watsky, 2002, 2004), these cells do not express Cx43 or manifest GJIC. However, the life span of primary cultured corneal epithelial cells is short and the availability of human tissue for their isolation is limited. The HCE cell line stably expressing Cx43-EGFP established in the present study may thus provide a model system that is more similar to primary cultured human corneal epithelial cells than is the parent HCE cell line. Cx43-mediated GJIC has been associated with the regulation of cell proliferation and differentiation as well as with development (Mesnil, 2002; Oviedo-Orta and Howard Evans, 2004; Elias and Kriegstein, 2008; Kibschull et al., 2008). Connexins are also implicated in the
Fig. 4. GJIC in HCE cells stably expressing Cx43-EGFP. (A) Confluent HCE cells stably expressing Cx43-EGFP or EGFP were assayed for dye coupling with Lucifer yellow. Cells were fixed 5 min after dye injection and were examined with a confocal microscope. (B) Quantitation of GJIC in images similar to that shown in (A). Data are means SE for 3 injected cells in a representative experiment. Similar results were obtained in a total of three independent experiments. *P < 0.05 (ANOVA followed by Dunnett’s test).
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phosphorylation level of connexin43 (Koo et al., 1997). The mechanism of regulation for cell cycle by connexin43 in corneal epithelial cells remains to be determined. In conclusion, we have established a human corneal epithelial cell line that stably expresses Cx43-EGFP and forms functional gap junctions. Further study of these cells may provide a better understanding not only of the function of Cx43 in corneal epithelial cells but also of the biological properties of the corneal epithelium. Acknowledgments The authors thank Yasumiko Akamatsu and the staff of the Yamaguchi University Center for Gene Research for technical assistance. Fig. 5. Expression of keratin 3, keratin 12, E-cadherin, and ZO-1 in HCE cells stably expressing Cx43-EGFP. Cells stably transfected with vectors for Cx43-EGFP or for EGFP, or nontransfected cells, were lysed and subjected to immunoblot analysis with antibodies to keratin 3, keratin 12, E-cadherin, ZO-1, or actin (loading control). Data are representative of three independent experiments.
suppression of tumor growth (Mesnil, 2002; Naus, 2002; Leithe et al., 2006). Cx43 is expressed in basal and suprabasal epithelial cells in the cornea (Schlotzer-Schrehardt and Kruse, 2005; Chee et al., 2006; Wolosin, 2006). Limbal stem cells for the corneal epithelium have a higher proliferative potential than do peripheral and central corneal epithelial cells (Wolosin et al., 2004; Schlotzer-Schrehardt and Kruse, 2005; Revoltella et al., 2007). Whereas limbal basal epithelial cells were found not to express Cx43, suprabasal cells showed Cx43 immunoreactivity along the basal membrane (Wolosin et al., 2004; Chen et al., 2006). We have now shown that the stable expression of Cx43 in HCE cells suppressed cell proliferation. We also previously demonstrated that the expression of Cx43 in basal corneal epithelial cells is increased and that the proliferation of these cells is impaired in a rat model of diabetes (Wakuta et al., 2007). These various observations suggest that Cx43 play an important role in regulation of corneal epithelial cell growth. Moreover, Inhibition of gap junction increased the level of cyclins D1 and D3, responsible for the transition of cell cycles, in astrocytes (Tabernero et al., 2006). Gap-junctional communication during the progression of cell cycle correlated with the
8 EGFP
7 Cell number (105 cells)
Cx43-EGFP
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3 2 1 0
0
*
1 3 Time (days)
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Fig. 6. Effect of stable expression of Cx43-EGFP on the proliferation of HCE cells. Cells stably expressing Cx43-EGFP or EGFP were cultured for the indicated times in SHEM containing G418 (600 mg/mL), after which the cell number was determined. Data are means SE of triplicates from an experiment that was repeated a total of three times with similar results. *P < 0.05 versus the corresponding value for cells expressing EGFP (ANOVA followed by Dunnett’s test).
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Schlotzer-Schrehardt, U., Kruse, F.E., 2005. Identification and characterization of limbal stem cells. Exp. Eye Res. 81, 247–264. Scott, R.A., Lauweryns, B., Snead, D.M., Haynes, R.J., Mahida, Y., Dua, H.S., 1997. E-cadherin distribution and epithelial basement membrane characteristics of the normal human conjunctiva and cornea. Eye 11 (Pt 5), 607–612. Shurman, D.L., Glazewski, L., Gumpert, A., Zieske, J.D., Richard, G., 2005. In vivo and in vitro expression of connexins in the human corneal epithelium. Invest. Ophthalmol. Vis. Sci. 46, 1957–1965. Sun, T.T., Green, H., 1977. Cultured epithelial cells of cornea, conjunctiva and skin: absence of marked intrinsic divergence of their differentiated states. Nature 269, 489–493. Suzuki, K., Tanaka, T., Enoki, M., Nishida, T., 2000. Coordinated reassembly of the basement membrane and junctional proteins during corneal epithelial wound healing. Invest. Ophthalmol. Vis. Sci. 41, 2495–2500. Tabernero, A., Sanchez-Alvarez, R., Medina, J.M., 2006. Increased levels of cyclins D1 and D3 after inhibition of gap junctional communication in astrocytes. J. Neurochem. 96, 973–982. Vinken, M., Vanhaecke, T., Papeleu, P., Snykers, S., Henkens, T., Rogiers, V., 2006. Connexins and their channels in cell growth and cell death. Cell Signal. 18, 592–600. Wakuta, M., Morishige, N., Chikama, T., Seki, K., Nagano, T., Nishida, T., 2007. Delayed wound closure and phenotypic changes in corneal epithelium of the spontaneously diabetic Goto-Kakizaki rat. Invest. Ophthalmol. Vis. Sci. 48, 590–596. Williams, K., Watsky, M., 2002. Gap junctional communication in the human corneal endothelium and epithelium. Curr. Eye Res. 25, 29–36. Williams, K.K., Watsky, M.A., 2004. Bicarbonate promotes dye coupling in the epithelium and endothelium of the rabbit cornea. Curr. Eye Res. 28, 109–120. Wolosin, J.M., 2006. Cell markers and the side population phenotype in ocular surface epithelial stem cell characterization and isolation. Ocul. Surf. 4, 10–23. Wolosin, J.M., Budak, M.T., Akinci, M.A., 2004. Ocular surface epithelial and stem cell development. Int. J. Dev. Biol. 48, 981–991.
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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Establishment of human corneal epithelial cells stably expressing human connexin43q Kazuhiro Kimura a, *, Shinichiro Teranishi b, Teruo Nishida b a b
Department of Ocular Pathophysiology, Yamaguchi University, Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi 755-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 June 2009 Accepted in revised form 3 September 2009 Available online 30 September 2009
Corneal epithelial cells communicate with each other through gap junctions. Whereas this property is retained in corneal epithelial cells in primary culture, it is often lost in immortalized epithelial cells. However, the life span of primary cultured corneal epithelial cells is short and the availability of human tissue for their preparation is limited. To examine the role of the gap-junction protein connexin43 (Cx43) in human corneal epithelial cells, we set out to establish an immortal human corneal epithelial cell line that stably expresses this protein. An expression vector encoding human Cx43 fused to enhanced green fluorescent protein (EGFP) was constructed and introduced by transfection into SV40-immortalized human corneal epithelial (HCE) cells. Stable transfectants were isolated by selection with the antibiotic G418. The expression and localization of the Cx43-EGFP fusion protein were examined by immunoblot analysis and fluorescence microscopy, respectively, and gap-junctional intercellular communication was monitored on the basis of dye coupling. HCE cells stably expressing Cx43-EGFP manifested intercellular dye transfer, whereas those stably expressing EGFP alone did not. Cx43-EGFP localized to the interfaces of neighboring cells. Stable expression of Cx43-EGFP in HCE cells did not affect the expression of keratins 3 and 12, which is a characteristic of corneal epithelial cells, but it did inhibit cell proliferation. We have established an HCE cell line that stably expresses human Cx43 and forms functional gap junctions. These cells may prove useful for studies of the role of gap junctions in the human corneal epithelium. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: corneal epithelial cell stable cell line gap junction connexin43
1. Introduction The corneal epithelium forms the anterior surface of the cornea and plays an important role in the maintenance of corneal transparency. It is a multilayered structure that sits on the epithelial basement membrane, and corneal epithelial cells are joined to each other by junctional structures including tight junctions and adherens junctions (Gipson, 1992; Scott et al., 1997; Suzuki et al., 2000; Ban et al., 2003). In addition, corneal epithelial cells form gap junctions, which consist of two hemichannels, one in each apposing cell membrane, and which mediate communication between cells (Dong et al., 1994; Wolosin et al., 2004; Shurman et al., 2005). Gap junctions contribute to the regulation of corneal epithelial cell functions such as cell growth, differentiation, adhesion, migration, and barrier formation (Suzuki et al., 2000; Kimura et al., 2008). Studies of the
q This study was supported in part by a grant (21791687) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. * Corresponding author. Tel.: þ81 836 22 2277; fax: þ81 836 22 2334. E-mail address: [email protected] (K. Kimura). 0014-4835/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2009.09.002
human corneal epithelium in vitro have been performed with primary cultured cells (Sun and Green, 1977; Ebato et al., 1987; Kahn et al., 1993). However, such cells become senescent after only a few passages in vitro and the availability of donor corneal tissue for their isolation is limited. As an alternative to primary cultured cells, several immortalized human corneal epithelial cell lines have been established and characterized (Kahn et al., 1993; Araki-Sasaki et al., 1995; Offord et al., 1999). One of the most widely studied of these cell lines is a line established by transformation of human corneal epithelial cells with simian virus 40 (SV40) (Araki-Sasaki et al., 1995). Gap junctions mediate direct communication among connected cells by allowing the exchange of small molecules or ions of <1 kDa including metabolites and second messengers (Revel et al., 1992; Laird, 1996). Gap junctions are composed of connexins, a family of integral membrane proteins that are expressed in a cell typespecific manner (Lo et al., 1996; Saez et al., 2005). Connexin43 (Cx43) is widely expressed among tissues and organs (Beyer et al., 1989; Christ, 2000). It is abundant in suprabasal cells of the corneal and limbal epithelia but not in basal cells of the limbal epithelium (Schlotzer-Schrehardt and Kruse, 2005; Chee et al., 2006; Wolosin, 2006). We also previously showed that the expression of Cx43 in
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basal cells of the corneal epithelium is increased in a rat model of diabetes (Wakuta et al., 2007). Furthermore, the expression of Cx43 is found to be altered at the leading edge of the epithelium during corneal wound healing (Suzuki et al., 2000). Gap-junctional intercellular communication (GJIC) is impaired in various immortalized epithelial cells (Mesnil, 2004; Vinken et al., 2006; Pointis et al., 2007). We have now examined the expression of Cx43 and GJIC in SV40-transformed human corneal epithelial (HCE) cells. Furthermore, to characterize the function of Cx43 in human corneal epithelial cells, we established a line of HCE cells that stably express human Cx43. 2. Methods 2.1. Materials A mixture of Dulbecco’s modified Eagle’s medium and nutrient mixture F-12 (DMEM/F-12) as well as fetal bovine serum, gentamicin, trypsin-EDTA, and Lipofectamine 2000 were obtained from Invitrogen-Gibco (Carlsbad, CA). Bovine insulin, cholera toxin, human recombinant epidermal growth factor, and a protease inhibitor cocktail were from Sigma–Aldrich (St. Louis, MO). Plastic six- or 24-well culture plates and 60- or 100-mm culture dishes were obtained from Corning (Corning, NY), glass-bottom dishes (35-mm) were from Iwaki (Chiba, Japan), and G418 was from Nakalai Tesque (Kyoto, Japan). Mouse monoclonal antibodies to Cx43 were obtained from Chemicon (Temecula, CA), those to green fluorescent protein (GFP) were from BD-Clontech (San Jose, CA), those to E-cadherin were from Santa Cruz Biotechnology (Santa Cruz, CA), those to actin were from Sigma– Aldrich (St. Louis, MO), and those to keratin 3 or to keratin 12 were from Millipore (Billerica, MA). Rabbit polyclonal antibodies to zonula occludens (ZO)-1 were from Invitrogen-Gibco. Trizol reagent, Lucifer yellow and rhodamine-phalloidin were obtained from Invitrogen (Carlsbad, CA). Horseradish peroxidase-conjugated goat secondary antibodies and ECL Plus detection reagents were from Amersham Biosciences (Little Chalfont, UK). The pEGFP-N1 vector was obtained from BD-Clontech, MMLV reverse transcriptase was from Promega (Madison, WI), and Pfu Taq polymerase, BamHI, KpnI, were from Stratagene (La Jolla, CA). 2.2. Cells and cell culture SV40-immortalized human corneal epithelial (HCE) cells (ArakiSasaki et al., 1995) were obtained from RIKEN Biosource Center (Tsukuba, Japan). They were passaged in supplemented hormonal epithelial medium (SHEM), which consists of DMEM/F-12 supplemented with 15% heat-inactivated fetal bovine serum, bovine insulin (5 mg/mL), cholera toxin (0.1 mg/mL), human recombinant epidermal growth factor (10 ng/mL), and gentamicin (40 mg/mL). Human corneal fibroblasts were isolated as described previously (Kumagai et al., 2000) from tissue left over after corneal transplantation surgery; the tissue was used in strict accordance with the tenets of the Declaration of Helsinki. 2.3. Cloning of human Cx43 cDNA and plasmid construction Total RNA was isolated from human corneal fibroblasts with the use of the Trizol reagent. A portion (1 mg) of the RNA was subjected to reverse transcription with MMLV reverse transcriptase, and the resulting cDNA was subjected to the polymerase chain reaction (PCR) with the primers 50 -GGGGTACCGCCACCATGGGTGACTGGAGCGCCTTAG-30 (forward) and 50 -CGGGATCCCCGATCTCCAGGTCATCAGG-30 (reverse) and with Pfu Taq polymerase in order to amplify the coding region for Cx43. PCR was performed for 30 cycles of incubation at 94 C for 1 min, 50 C for 1 min, and 72 C for 2 min. The PCR product
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was digested with BamHI and KpnI, subcloned into pEGFP-N1. The identity of the insert was confirmed by sequencing. 2.4. Transfection and establishment of a stable cell line HCE cells (2 105) were cultured in a 60-mm culture dish for 24 h before transient transfection with 2 mg of plasmid DNA combined with Lipofectamine 2000 in SHEM for 16–18 h. For stable transfection, HCE cells (2 105) were plated in a 100-mm culture dish, incubated overnight, and then transfected with 10 mg of plasmid DNA combined with Lipofectamine 2000 in SHEM for 16–18 h. The cells were then cultured in SHEM containing G418 (600 mg/mL), and stably transfected clones were isolated and expanded. For experiments, stably transfected clones were usually cultured in SHEM containing G418 (600 mg/mL). 2.5. Dye coupling HCE cells (3 105) plated in 35-mm glass-bottom dishes were monitored for GJIC with the use of Lucifer yellow as described previously (Hao et al., 2005). In brief, the cells were washed with Ca2þ- and Mg2þ-free phosphate-buffered saline [PBS()], and Lucifer yellow (10% in water) was injected into two widely separated cells with the use of a microinjector (Micromanipulator and Transinjector; Eppendorf, Hamburg, Germany). The cells were fixed with 3.7% formalin, observed with a laser confocal microscope (Axioscope; Carl Zeiss Meditec, Hallbergmoos, Germany), and photographed 5 min after dye injection. The number of cells containing the dye was counted, and GJIC activity was expressed as the mean number of cells coupled to each injected cell. 2.6. Fluorescence microscopy HCE cells (1 105) cultured on 12-mm cover glasses in 24-well plates were fixed for 15 min with ice-cold acetone, washed with PBS(), permeabilized for 5 min with 0.1% Triton X-100 in PBS(), and incubated for 1 h at room temperature with 1% bovine serum albumin in PBS(). They were then incubated for 1 h with rhodamine-phalloidin (1:200 dilution) in PBS() containing 1% bovine serum albumin before examination with a laser confocal microscope (LSM5, Zeiss). 2.7. Immunoblot analysis HCE cells (2 105) cultured in 60-mm dishes were lysed on ice in 0.5 mL of a solution containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5 mM NaF, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, and 1% protease inhibitor cocktail. The lysates were centrifuged at 15,000 g for 15 min at 4 C, and the resulting supernatants (20 mg of protein) were subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel. The separated proteins were transferred to a nitrocellulose membrane, which was then exposed to 5% skim milk for 1 h at room temperature before incubation for 1 h with various primary antibodies at a dilution of 1:1000 in washing buffer [20 mM Tris–HCl (pH 7.4), 5% skim milk, 0.1% Tween 20]. The membrane was washed in washing buffer, incubated for 1 h at room temperature with horseradish peroxidaseconjugated secondary antibodies (1:1000 dilution in washing buffer), washed again, incubated with ECL Plus detection reagents for 5 min, and then exposed to film. 2.8. Cell proliferation assay Cells (2 104) seeded on 12-well culture plates were cultured for various times, detached by exposure to trypsin-EDTA, and
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counted with the use of a Coulter Particle Counter (Beckman Coulter, CA, Fullerton). 2.9. Statistical analysis Quantitative data are presented as means SE. Differences were analyzed with Dunnett’s test. A P value of <0.05 was considered statistically significant. 3. Results 3.1. GJIC and Cx43 expression in HCE cells We first examined whether Cx43 is expressed in HCE cells by immunoblot analysis. Cx43 was not detected in lysates of HCE cells, whereas it was present in those of human corneal fibroblasts examined as a positive control (Fig. 1A). We next determined whether HCEs manifest GJIC by monitoring dye coupling with Lucifer yellow, an anionic dye that diffuses through most types of gap-junction channels. Dye injected into HCE cells did not diffuse into neighboring cells (Fig. 1B), indicating the absence of GJIC. These results thus suggested that HCE cells have lost the ability to form gap junctions as a result of the loss of Cx43 expression. 3.2. Transient expression Cx43-EGFP in HCE cells To examine the effects of Cx43 expression in HCE cells, we constructed an expression plasmid for human Cx43 fused at its COOHterminus to enhanced green fluorescent protein (EGFP). HCE cells were transiently transfected with the Cx43-EGFP vector or with the corresponding plasmid encoding EGFP alone (pEGFP-N1) as a control. Immunoblot analysis with antibodies to GFP revealed a major band corresponding to Cx43-EGFP (w70 kDa) in the cells transfected with the Cx43-EGFP vector as well as a band corresponding to EGFP (w30 kDa) in those transfected with pEGFP-N1 (Fig. 2A). Immunoblot analysis with antibodies to Cx43 also revealed the band corresponding to Cx43-EGFP (Fig. 2B). These results thus showed that HCE cells are competent to express the Cx43-EGFP fusion protein. 3.3. Establishment of an HCE cell line stably expressing Cx43-EGFP To establish a human corneal epithelial cell line stably expressing human Cx43, we transfected HCE cells with the Cx43EGFP vector (or with pEGFP-N1 as a control) and then subjected the cells to selection by culture in the presence of G418. We isolated several cell clones after culture of the cells for 2 weeks, and we
Fig. 2. Transient expression of Cx43-EGFP in HCE cells. Cells transiently transfected with an expression vector for Cx43-EGFP or for EGFP were lysed and subjected to immunoblot analysis with antibodies to GFP (A) or to Cx43 (B). Data are representative of three independent experiments.
selected one clone showing the highest level of Cx43-EGFP expression for subsequent characterization. Immunoblot analysis of cell lysates with antibodies to GFP revealed major bands of the expected sizes for Cx43-EGFP or EGFP (Fig. 3A). We next examined the distribution of Cx43-EGFP in HCE cells stably expressing the fusion protein. Fluorescence microscopy revealed signals corresponding to Cx43-EGFP at the interfaces of apposing cells as well as in association with the Golgi apparatus and in the perinuclear region (Fig. 3B). In contrast, in cells stably expressing EGFP, the corresponding signals were detected in association with the Golgi apparatus and in the perinuclear region but not at the cell surface. To determine whether HCE cells stably expressing Cx43-EGFP manifest GJIC, we examined dye coupling with Lucifer yellow. GJIC was apparent in HCE cells stably expressing Cx43-EGFP but not in those stably expressing EGFP (Fig. 4). Moreover, in contrast to those stably expressing EGFP, HCE cells stably expressing Cx43-EGFP exhibited a cobblestone-like morphology similar to that of welldifferentiated corneal epithelial cells (Fig. 4A). To investigate the possible effects of stable expression of Cx43-EGFP on the phenotype of HCE cells, we examined the expression of keratins 3 and 12, which are characteristic of differentiated corneal epithelial cells, as well as that of the adherens-junction protein E-cadherin and the tight-junction protein ZO-1. Immunoblot analysis revealed that HCE cells stably expressing Cx43-EGFP also expressed keratins 3 and 12, E-cadherin, and ZO-1 at levels similar to those apparent in nontransfected cells or in cells stably expressing EGFP (Fig. 5). Finally, we examined the effect of stable expression of Cx43EGFP on the proliferation of HCE cells. The number of HCE cells stably expressing Cx43-EGFP was significantly smaller than that of those expressing EGFP after culture for 3, 5, or 7 days (Fig. 6), suggesting that expression of Cx43-EGFP inhibits the proliferation of HCE cells. 4. Discussion
Fig. 1. Analysis of Cx43 expression and GJIC in HCE cells. (A) Lysates of HCE cells and human corneal fibroblasts (CFs) were subjected to immunoblot analysis with antibodies to Cx43. (B) HCE cells were cultured for 48 h to confluence, after which an individual cell was microinjected with Lucifer yellow. The cells were fixed after 30 min, and the injected cell and its neighbors were examined with a confocal microscope. All data are representative of three independent experiments.
We have shown that SV40-transformed human corneal epithelial (HCE) cells do not express Cx43 or manifest GJIC. We further showed that these cells were capable of expressing a fusion protein of human Cx43 tagged with EGFP by transient transfection as well as established HCE cells stably expressing this fusion protein. The stable expression of Cx43-EGFP in HCE cells conferred
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Fig. 3. Stable expression and distribution of Cx43-EGFP in stably transfected HCE cells. (A) Cells stably transfected with an expression vector for Cx43-EGFP or for EGFP were lysed and subjected to immunoblot analysis with antibodies to GFP. (B) Cells stably expressing Cx43-EGFP or EGFP were cultured to confluence, stained with rhodamine-phalloidin for detection of F-actin, and examined by fluorescence microscopy. Red and green fluorescence represents rhodamine-phalloidin and either EGFP or Cx43-EGFP, respectively. Arrows indicate Cx43-EGFP signals at the interfaces of adjacent cells. Data are representative of three independent experiments.
the ability to communicate via gap junctions, and the fusion protein was detected at the interfaces of neighboring cells. The stable expression of Cx43-EGFP did not affect the expression of keratin 3, keratin 12, E-cadherin, or ZO-1 in HCE cells, but it did inhibit the proliferation of these cells. We have thus generated a line of immortalized human corneal epithelial cells that stably express human Cx43 and form functional gap junctions. Our observation that Cx43-EGFP localizes to the interfaces of apposing HCE cells stably expressing the fusion protein is consistent with the previously observed distribution of Cx43-EGFP in keratinocytes, testicular epithelial cells, and cancer cells (Holm et al., 1999; Jordan et al., 1999; Paemeleire et al., 2000; Roger et al., 2004). We showed that HCE cells stably expressing Cx43-EGFP adopted a cobblestone-like appearance similar to that of corneal epithelial cells in situ, and that these cells continue to express keratin 3 and keratin 12, both of which are characteristic of differentiated corneal epithelial cells. Moreover, HCE cells stably expressing Cx43-EGFP manifested GJIC, as do corneal epithelial cells in primary culture (Hernandez Galindo et al., 2003). The morphology and other
phenotypic characteristics of HCE cells stably expressing Cx43-EGFP are thus consistent with those of normal corneal epithelial cells. SV40-transformed human corneal epithelial (HCE) cells are a wellcharacterized model system that has been adopted widely for studies of corneal epithelial cell activities such as proliferation, differentiation, adhesion, migration, and barrier formation (Chiambaretta et al., 2004; Kimura et al., 2006, 2008). We have now shown that, in contrast to primary cultured corneal epithelial cells (Anderson et al., 2002; Williams and Watsky, 2002, 2004), these cells do not express Cx43 or manifest GJIC. However, the life span of primary cultured corneal epithelial cells is short and the availability of human tissue for their isolation is limited. The HCE cell line stably expressing Cx43-EGFP established in the present study may thus provide a model system that is more similar to primary cultured human corneal epithelial cells than is the parent HCE cell line. Cx43-mediated GJIC has been associated with the regulation of cell proliferation and differentiation as well as with development (Mesnil, 2002; Oviedo-Orta and Howard Evans, 2004; Elias and Kriegstein, 2008; Kibschull et al., 2008). Connexins are also implicated in the
Fig. 4. GJIC in HCE cells stably expressing Cx43-EGFP. (A) Confluent HCE cells stably expressing Cx43-EGFP or EGFP were assayed for dye coupling with Lucifer yellow. Cells were fixed 5 min after dye injection and were examined with a confocal microscope. (B) Quantitation of GJIC in images similar to that shown in (A). Data are means SE for 3 injected cells in a representative experiment. Similar results were obtained in a total of three independent experiments. *P < 0.05 (ANOVA followed by Dunnett’s test).
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phosphorylation level of connexin43 (Koo et al., 1997). The mechanism of regulation for cell cycle by connexin43 in corneal epithelial cells remains to be determined. In conclusion, we have established a human corneal epithelial cell line that stably expresses Cx43-EGFP and forms functional gap junctions. Further study of these cells may provide a better understanding not only of the function of Cx43 in corneal epithelial cells but also of the biological properties of the corneal epithelium. Acknowledgments The authors thank Yasumiko Akamatsu and the staff of the Yamaguchi University Center for Gene Research for technical assistance. Fig. 5. Expression of keratin 3, keratin 12, E-cadherin, and ZO-1 in HCE cells stably expressing Cx43-EGFP. Cells stably transfected with vectors for Cx43-EGFP or for EGFP, or nontransfected cells, were lysed and subjected to immunoblot analysis with antibodies to keratin 3, keratin 12, E-cadherin, ZO-1, or actin (loading control). Data are representative of three independent experiments.
suppression of tumor growth (Mesnil, 2002; Naus, 2002; Leithe et al., 2006). Cx43 is expressed in basal and suprabasal epithelial cells in the cornea (Schlotzer-Schrehardt and Kruse, 2005; Chee et al., 2006; Wolosin, 2006). Limbal stem cells for the corneal epithelium have a higher proliferative potential than do peripheral and central corneal epithelial cells (Wolosin et al., 2004; Schlotzer-Schrehardt and Kruse, 2005; Revoltella et al., 2007). Whereas limbal basal epithelial cells were found not to express Cx43, suprabasal cells showed Cx43 immunoreactivity along the basal membrane (Wolosin et al., 2004; Chen et al., 2006). We have now shown that the stable expression of Cx43 in HCE cells suppressed cell proliferation. We also previously demonstrated that the expression of Cx43 in basal corneal epithelial cells is increased and that the proliferation of these cells is impaired in a rat model of diabetes (Wakuta et al., 2007). These various observations suggest that Cx43 play an important role in regulation of corneal epithelial cell growth. Moreover, Inhibition of gap junction increased the level of cyclins D1 and D3, responsible for the transition of cell cycles, in astrocytes (Tabernero et al., 2006). Gap-junctional communication during the progression of cell cycle correlated with the
8 EGFP
7 Cell number (105 cells)
Cx43-EGFP
6 5 4
*
3 2 1 0
0
*
1 3 Time (days)
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Fig. 6. Effect of stable expression of Cx43-EGFP on the proliferation of HCE cells. Cells stably expressing Cx43-EGFP or EGFP were cultured for the indicated times in SHEM containing G418 (600 mg/mL), after which the cell number was determined. Data are means SE of triplicates from an experiment that was repeated a total of three times with similar results. *P < 0.05 versus the corresponding value for cells expressing EGFP (ANOVA followed by Dunnett’s test).
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