Differential Expression of Connexins During Stratification of Human Keratinocytes
2000; Elsevier BV; Volume: 115; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.2000.00043.x
ISSN1523-1747
AutoresLudovic Wiszniewski, Alain Limat, Jean‐Hilaire Saurat, Paolo Meda, Denis Salomon,
Tópico(s)Heat shock proteins research
ResumoTo assess whether gap junctions and connexins change during keratinocyte differentiation, we have studied epidermal equivalents obtained in organotypic cultures of keratinocytes from the outer root sheath of human hair follicles. These reconstituted tissues exhibit a number of differentiation and proliferation markers of human epidermis, including gap junctions, connexins, and K6 and Ki67 proteins. Immunostaining and northern blots showed that gap junctions of the epidermal equivalents were made of Cx26 and Cx43. Cx26 was expressed in all keratinocyte layers, throughout the development of the epidermal equivalents. In contrast, Cx43 was initially observed only in the basal layer of keratinocytes and became detectable in the stratum spinosum and granulosum only after the epidermal equivalents had thickened. The levels of Cx26 and its transcript markedly increased as a function of stratification of the epidermal equivalents, whereas those of Cx43 remained almost constant. Microinjection of Lucifer Yellow into individual keratinocytes showed that gap junctions were similarly permeable at all stages of development of the epidermal equivalents. The data show that epidermal equivalents (i) feature a pattern of connexins typical of an actively renewing human interfollicular epidermis, and (ii) provide a model that reproduces the tridimensional organization of intact epidermis and that is amenable for experimentally testing the function of junctional communication between human keratinocytes. To assess whether gap junctions and connexins change during keratinocyte differentiation, we have studied epidermal equivalents obtained in organotypic cultures of keratinocytes from the outer root sheath of human hair follicles. These reconstituted tissues exhibit a number of differentiation and proliferation markers of human epidermis, including gap junctions, connexins, and K6 and Ki67 proteins. Immunostaining and northern blots showed that gap junctions of the epidermal equivalents were made of Cx26 and Cx43. Cx26 was expressed in all keratinocyte layers, throughout the development of the epidermal equivalents. In contrast, Cx43 was initially observed only in the basal layer of keratinocytes and became detectable in the stratum spinosum and granulosum only after the epidermal equivalents had thickened. The levels of Cx26 and its transcript markedly increased as a function of stratification of the epidermal equivalents, whereas those of Cx43 remained almost constant. Microinjection of Lucifer Yellow into individual keratinocytes showed that gap junctions were similarly permeable at all stages of development of the epidermal equivalents. The data show that epidermal equivalents (i) feature a pattern of connexins typical of an actively renewing human interfollicular epidermis, and (ii) provide a model that reproduces the tridimensional organization of intact epidermis and that is amenable for experimentally testing the function of junctional communication between human keratinocytes. In complex organisms, several mechanisms of communication assemble cells into functionally coordinated multicellular units, which are thought to play an important role in maintaining tissue homeostasis. One mechanism of cell-to-cell communication operates via direct exchanges of ions and molecules through highly permeable membrane channels located at gap junctions (Kumar and Gilula, 1996Kumar N.M. Gilula N.B. The gap junction communication channel.Cell. 1996; 84: 381-388Abstract Full Text Full Text PDF PubMed Scopus (1583) Google Scholar;Werner, 1998Werner R. Gap Junctions. ed. IOS Press, Amsterdam1998Google Scholar). These channels are made of a family of nonglycosylated integral membrane proteins, referred to as connexins (Cx) (Kumar and Gilula, 1996Kumar N.M. Gilula N.B. The gap junction communication channel.Cell. 1996; 84: 381-388Abstract Full Text Full Text PDF PubMed Scopus (1583) Google Scholar;Werner, 1998Werner R. Gap Junctions. ed. IOS Press, Amsterdam1998Google Scholar). The gap junctions of human keratinocytes comprise mostly Cx43, which is abundantly expressed within interfollicular epidermis (Guo et al., 1992Guo H. Acevedo P. Parsa F.D. Bertram J.S. Gap-junctional protein connexin 43 is expressed in dermis and epidermis of human skin: differential modulation by retinoids.J Invest Dermatol. 1992; 99: 460-467Abstract Full Text PDF PubMed Google Scholar;Salomon et al., 1993Salomon D. Masgrau E. Vischer S. Chanson M. Saurat J.H. Spray D. Meda P. Gap-junction proteins and communication in human epidermis.Prog Cell Res. 1993; 3: 225-231Google Scholar,Salomon et al., 1994Salomon D. Masgrau E. Vischer S. et al.Topography of mammalian connexins in human skin.J Invest Dermatol. 1994; 103: 240-247Crossref PubMed Scopus (121) Google Scholar), and Cx26, which is codistributed with Cx43 in skin adnexae (Salomon et al., 1993Salomon D. Masgrau E. Vischer S. Chanson M. Saurat J.H. Spray D. Meda P. Gap-junction proteins and communication in human epidermis.Prog Cell Res. 1993; 3: 225-231Google Scholar,Salomon et al., 1994Salomon D. Masgrau E. Vischer S. et al.Topography of mammalian connexins in human skin.J Invest Dermatol. 1994; 103: 240-247Crossref PubMed Scopus (121) Google Scholar). Even though the role of gap junctions in epidermal homeostasis remains to be demonstrated, it is assumed that gap-junction-mediated communication is involved in the regulation of keratinocyte growth and differentiation (Salomon et al., 1993Salomon D. Masgrau E. Vischer S. Chanson M. Saurat J.H. Spray D. Meda P. Gap-junction proteins and communication in human epidermis.Prog Cell Res. 1993; 3: 225-231Google Scholar,Salomon et al., 1994Salomon D. Masgrau E. Vischer S. et al.Topography of mammalian connexins in human skin.J Invest Dermatol. 1994; 103: 240-247Crossref PubMed Scopus (121) Google Scholar;Lucke et al., 1999Lucke T. Choudhry R. Thom R. Selmer I.S. Burden A.D. Hodgins M.B. Upregulation of connexin 26 is a feature of keratinocyte differentiation in hyperproliferative epidermis, vaginal epithelium, and buccal epithelium.J Invest Dermatol. 1999; 112: 354-361https://doi.org/10.1046/j.1523-1747.1999.00512.xCrossref PubMed Scopus (129) Google Scholar). In this perspective, the differential expression of Cx43 and Cx26 within interfollicular epidermis and epidermal adnexae may be related to the specific differentiation program that keratinocytes undergo in these two different skin regions (Salomon et al., 1994Salomon D. Masgrau E. Vischer S. et al.Topography of mammalian connexins in human skin.J Invest Dermatol. 1994; 103: 240-247Crossref PubMed Scopus (121) Google Scholar). Consistent with this hypothesis are the observations that events leading to in vivo changes in the proliferation and/or state of differentiation of epidermis, e.g., chronic treatment with topical retinoic acid (Guo et al., 1992Guo H. Acevedo P. Parsa F.D. Bertram J.S. Gap-junctional protein connexin 43 is expressed in dermis and epidermis of human skin: differential modulation by retinoids.J Invest Dermatol. 1992; 99: 460-467Abstract Full Text PDF PubMed Google Scholar;Masgrau-Peya et al., 1997Masgrau-Peya E. Salomon D. Saurat J.H. Meda P. In Vivo Modulation of Connexins 43 and 26 of Human Epidermis by Topical Retinoic Acid Treatment.J Histochem Cytochem. 1997; 45: 1207-1215Crossref PubMed Scopus (48) Google Scholar), psoriatic lesions (Rivas et al., 1997Rivas M.V. Jarvis E.D. Morisaki S. Carbonaro H. Gottlieb A.B. Krueger J.G. Identification of aberrantly regulated genes in diseased skin using the cDNA differential display technique.J Invest Dermatol. 1997; 108: 188-194Crossref PubMed Scopus (62) Google Scholar;Labarthe et al., 1998Labarthe M.P. Bosco D. Saurat J.H. Meda P. Salomon D. Upregulation of connexin 26 between keratinocytes of psoriatic lesions.J Invest Dermatol. 1998; 111: 72-76Crossref PubMed Scopus (95) Google Scholar;Lucke et al., 1999Lucke T. Choudhry R. Thom R. Selmer I.S. Burden A.D. Hodgins M.B. Upregulation of connexin 26 is a feature of keratinocyte differentiation in hyperproliferative epidermis, vaginal epithelium, and buccal epithelium.J Invest Dermatol. 1999; 112: 354-361https://doi.org/10.1046/j.1523-1747.1999.00512.xCrossref PubMed Scopus (129) Google Scholar), or skin carcinomas (Wilgenbus et al., 1992Wilgenbus K.K. Kirkpatrick C.J. Knuechel R. Willecke K. Traub O. Expression of Cx26, Cx32 and Cx43 gap junction proteins in normal and neoplastic human tissues.Int J Cancer. 1992; 51: 522-529Crossref PubMed Scopus (220) Google Scholar), are associated with marked changes in the expression of connexins, particularly of Cx26. Upregulation of Cx26 was also observed in normal, non-hyperproliferative tissues, such as vaginal and buccal epithelia, suggesting that if a Cx26 increase accompanied conditions of keratinocyte hyperproliferation it was also required for keratinocyte differentiation (Lucke et al., 1999Lucke T. Choudhry R. Thom R. Selmer I.S. Burden A.D. Hodgins M.B. Upregulation of connexin 26 is a feature of keratinocyte differentiation in hyperproliferative epidermis, vaginal epithelium, and buccal epithelium.J Invest Dermatol. 1999; 112: 354-361https://doi.org/10.1046/j.1523-1747.1999.00512.xCrossref PubMed Scopus (129) Google Scholar). Direct experimental testing of why and how Cx26 is upregulated in an activated epidermis requires a model that can reproduce in vitro the in situ organization of this tissue. We have previously shown that, when cocultured with fibroblasts in a tridimensional system, keratinocytes derived from the outer root sheath (ORS) of human hair follicles develop epidermal equivalents, which display histologic and biochemical characteristics close to those of an activated human epidermis (Limat et al., 1996Limat A. Mauri D. Hunziker T. Successful treatment of chronic leg ulcers with epidermal equivalents generated from cultured autologous outer root sheath cells.J Invest Dermatol. 1996; 107: 128-135Crossref PubMed Scopus (91) Google Scholar,Limat et al., 1999Limat A. Salomon D. Carraux P. Saurat J.H. Hunziker T. Human melanocytes grown in epidermal equivalents transfer their melanin to follicular outer root sheath keratinocytes.Arch Dermatol Res. 1999; 291: 325-332https://doi.org/10.1007/s004030050417Crossref PubMed Scopus (16) Google Scholar). Here, we have studied these tridimensional epidermal equivalents to assess the presence of connexins, gap junctions, and cell coupling, and to follow the changes of these parameters as a function of keratinocyte stratification. Human ORS keratinocytes were cultured out of anagen hair follicles explanted from four healthy volunteers, in a Dulbecco's modified Eagle's medium (DMEM)/F12 (3:1) medium, supplemented with 10% fetal bovine serum, epidermal growth factor, hydrocortisone, choleratoxin, adenine, and triiodothyronine (all from Sigma, St. Louis, MO) and 1.5 mM Ca2+ (Limat et al., 1996Limat A. Mauri D. Hunziker T. Successful treatment of chronic leg ulcers with epidermal equivalents generated from cultured autologous outer root sheath cells.J Invest Dermatol. 1996; 107: 128-135Crossref PubMed Scopus (91) Google Scholar,Limat et al., 1999Limat A. Salomon D. Carraux P. Saurat J.H. Hunziker T. Human melanocytes grown in epidermal equivalents transfer their melanin to follicular outer root sheath keratinocytes.Arch Dermatol Res. 1999; 291: 325-332https://doi.org/10.1007/s004030050417Crossref PubMed Scopus (16) Google Scholar). Human dermal fibroblasts were obtained from skin explants of healthy individuals and cultured in DMEM supplemented with 10% fetal bovine serum (Limat et al., 1996Limat A. Mauri D. Hunziker T. Successful treatment of chronic leg ulcers with epidermal equivalents generated from cultured autologous outer root sheath cells.J Invest Dermatol. 1996; 107: 128-135Crossref PubMed Scopus (91) Google Scholar,Limat et al., 1999Limat A. Salomon D. Carraux P. Saurat J.H. Hunziker T. Human melanocytes grown in epidermal equivalents transfer their melanin to follicular outer root sheath keratinocytes.Arch Dermatol Res. 1999; 291: 325-332https://doi.org/10.1007/s004030050417Crossref PubMed Scopus (16) Google Scholar). ORS keratinocytes (first subculture) were plated at a density of 5 × 105 cells per cm2 in cell culture inserts (Transwell 3413, Corning Costar, Cambridge, MA) that were coated with 5 × 104 human dermal fibroblasts on the undersurface of their microporous membrane (Limat et al., 1996Limat A. Mauri D. Hunziker T. Successful treatment of chronic leg ulcers with epidermal equivalents generated from cultured autologous outer root sheath cells.J Invest Dermatol. 1996; 107: 128-135Crossref PubMed Scopus (91) Google Scholar). Culture medium was the same as for primary cultures. After 48 h (this time is thereafter referred to as day 0), the medium inside the inserts was aspirated to expose the cells to air. Seven days later, the cells were switched to KGM (Clonetics, San Diego, CA) containing 0.5 μg per ml hydrocortisone, 5 μg perml insulin, 5 ng per ml epidermal growth factor, 50 μg per ml gentamycin sulfate, and 1.5 mM Ca2+, a switch that we have found in repeated experiments to favor the differentiation of epidermal equivalents. Thereafter, the medium was changed three times per week, exactly as reported byLimat et al., 1999Limat A. Salomon D. Carraux P. Saurat J.H. Hunziker T. Human melanocytes grown in epidermal equivalents transfer their melanin to follicular outer root sheath keratinocytes.Arch Dermatol Res. 1999; 291: 325-332https://doi.org/10.1007/s004030050417Crossref PubMed Scopus (16) Google Scholar. For this study, four independent experiments were performed, each using keratinocytes from a different donor. In each experiment, keratinocytes were studied by the entire set of approaches described below, at each of the following time points: 2, 4, 7, and 14 d after exposure of the cells to the air–liquid interface. For conventional histology, epidermal equivalents were fixed in a Dubosq/Brazil solution and further processed according to standard procedures. For transmission electron microscopy, epidermal equivalents were fixed in 3% glutaraldehyde buffered with 0.1 M phosphate buffer (pH 7.4), embedded in Araldite (Serva, Heidelberg, Germany), cut, and contrasted with uranyl acetate and lead citrate, according to standard procedures. For freeze-fracture electron microscopy, the glutaraldehyde-fixed specimens were cryoprotected in 30% glycerol and frozen in liquid nitrogen. Photographs were taken with a Philips CM 10 or EM 300 electron microscope. For indirect immunofluorescence, epidermal equivalents were excised from the insert dish with a scalpel blade, embedded in Tissue-Tech OCT Compound (Miles, Elkhart, IN), frozen, and kept at -80°C until further processing. Sections 5 μm thick were cut with a cryomicrotome (CM 3050 Leica, Heidelberg, Germany), collected on silane-treated slides, and exposed for 3 min to -20°C acetone. All slides were first rinsed in cold (4°C) phosphate-buffered saline (PBS), blocked for 30 min with PBS supplemented with 2% bovine serum albumin, and incubated for 2 h at room temperature with one of the following antibodies: (i) affinity-purified rabbit polyclonal antibodies against residues 108–122 of liver Cx26, diluted 1:200 (Goliger and Paul, 1994Goliger J.A. Paul D.L. Expression of gap junction proteins Cx26, Cx31.1, Cx37, and Cx43 in developing and mature rat epidermis.Dev Dyn. 1994; 200: 1-13Crossref PubMed Scopus (133) Google Scholar); (ii) mouse monoclonal antibodies against a segment of the cytoplasmic loop of Cx26 (Zymed Laboratory, San Francisco, CA), diluted 1:100 (Masgrau-Peya et al., 1997Masgrau-Peya E. Salomon D. Saurat J.H. Meda P. In Vivo Modulation of Connexins 43 and 26 of Human Epidermis by Topical Retinoic Acid Treatment.J Histochem Cytochem. 1997; 45: 1207-1215Crossref PubMed Scopus (48) Google Scholar); (iii) affinity-purified rabbit polyclonal antibodies against residues 314–322 of heart Cx43, diluted 1:400 (Fishman et al., 1990Fishman G.I. Spray D.C. Leinwand L.A. Molecular characterization and functional expression of the human cardiac gap junction channel.J Cell Biol. 1990; 111: 589-598Crossref PubMed Scopus (157) Google Scholar); (iv) mouse monoclonal antibodies against 19 amino acids of the carboxy terminal portion of Cx43, diluted 1:1000 (Cat. No. 03–6900, Zymed Laboratories, South San Francisco, CA) [this is the original monoclonal antibody to Cx43 commercialized by Zymed, which recognizes both phosphorylated and nonphosphorylated forms of Cx43 (Kasper et al., 1996Kasper M. Traub O. Reimann T. Bjermer L. Grossmann H. Muller M. Wenzel K.W. Upregulation of gap junction protein connexin43 in alveolar epithelial cells of rats with radiation-induced pulmonary fibrosis.Histochem Cell Biol. 1996; 106: 419-424https://doi.org/10.1007/s004180050059Crossref PubMed Scopus (35) Google Scholar; Vozzi, Dupond, and Meda, unpublished)]; (v) mouse monoclonal antibodies against involucrin (Sigma), diluted 1:50; (vi) mouse monoclonal antibodies against cytokeratin K10 (Sigma), diluted 1:800; (vii) mouse monoclonal antibodies against human profilaggrin/filaggrin (BTI, Stoughton, MA), diluted 1:100; (viii) mouse monoclonal antibodies against human cytokeratin 6 (Novocastra Laboratories, Newcastle upon Tyne, U.K.), diluted 1:20. Sections were then rinsed in PBS and incubated for 60 min at room temperature with fluorescein-conjugated antirat, antirabbit, or antimouse antibodies, whichever applicable, diluted 1:400. After further rinsing, sections were stained with a 0.03% Evans' blue solution, covered with 0.02% paraphenylenediamine in PBS-glycerol (1:2 vol:vol), and photographed with an Axioplan microscope (Zeiss, Oberkochen, Germany) fitted with filters for fluorescein detection. As positive controls, we used cryosections of normal human skin obtained from healthy volunteers who had given informed consent. In accordance with the guidelines of our institutional committee for clinical investigation, keratome samples were obtained from either neck, under intradermal anesthesia by 1% xylocaine plus epinephrine, or breast and abdomen during reduction surgery (Salomon et al., 1994Salomon D. Masgrau E. Vischer S. et al.Topography of mammalian connexins in human skin.J Invest Dermatol. 1994; 103: 240-247Crossref PubMed Scopus (121) Google Scholar;Masgrau-Peya et al., 1997Masgrau-Peya E. Salomon D. Saurat J.H. Meda P. In Vivo Modulation of Connexins 43 and 26 of Human Epidermis by Topical Retinoic Acid Treatment.J Histochem Cytochem. 1997; 45: 1207-1215Crossref PubMed Scopus (48) Google Scholar;Labarthe et al., 1998Labarthe M.P. Bosco D. Saurat J.H. Meda P. Salomon D. Upregulation of connexin 26 between keratinocytes of psoriatic lesions.J Invest Dermatol. 1998; 111: 72-76Crossref PubMed Scopus (95) Google Scholar). In all cases, samples were rapidly frozen by immersion in 2-methylbutane (Merck, Basel, Switzerland), which was cooled in liquid nitrogen, and stored at -80°C until cryostat sectioning. In these control tissues, the distribution of Cx26, which was restricted to hair follicles and ducts of eccrine sweat glands, Cx43, involucrin, K10, fillagrin, and K6 were as previously reported in normal human samples (Tyner and Fuchs, 1986Tyner A.L. Fuchs E.E. Evidence for posttranscriptional regulation of the keratins expressed during hyperproliferation and malignant transformation in human epidermis.J Cell Biol. 1986; 103: 1945-1955Crossref PubMed Scopus (122) Google Scholar;Ebling et al., 1992Ebling F.J.G. Eayd R.A. Leigh I.M. Anatomy and organisation of human skin.in: Champion R.H. Burton J.L. Ebling F.J.G. Textbook of Dermatology. 5th edn. Blackwell Scientific Publications, Oxford1992: 49-123Google Scholar;Guo et al., 1992Guo H. Acevedo P. Parsa F.D. Bertram J.S. Gap-junctional protein connexin 43 is expressed in dermis and epidermis of human skin: differential modulation by retinoids.J Invest Dermatol. 1992; 99: 460-467Abstract Full Text PDF PubMed Google Scholar;Salomon et al., 1994Salomon D. Masgrau E. Vischer S. et al.Topography of mammalian connexins in human skin.J Invest Dermatol. 1994; 103: 240-247Crossref PubMed Scopus (121) Google Scholar;Limat et al., 1996Limat A. Mauri D. Hunziker T. Successful treatment of chronic leg ulcers with epidermal equivalents generated from cultured autologous outer root sheath cells.J Invest Dermatol. 1996; 107: 128-135Crossref PubMed Scopus (91) Google Scholar;Masgrau-Peya et al., 1997Masgrau-Peya E. Salomon D. Saurat J.H. Meda P. In Vivo Modulation of Connexins 43 and 26 of Human Epidermis by Topical Retinoic Acid Treatment.J Histochem Cytochem. 1997; 45: 1207-1215Crossref PubMed Scopus (48) Google Scholar;Labarthe et al., 1998Labarthe M.P. Bosco D. Saurat J.H. Meda P. Salomon D. Upregulation of connexin 26 between keratinocytes of psoriatic lesions.J Invest Dermatol. 1998; 111: 72-76Crossref PubMed Scopus (95) Google Scholar). Fragments of the very same samples of normal skin and of the epidermal equivalents prepared in these experiments were also fixed in formol and embedded in paraffin. Sections 7 μm thick were deparaffinized, rehydrated, boiled three times for 5 min in a 0.01M citrate buffer (pH 6) within a microwawe, cooled at room temperature, rinsed in PBS, and incubated 2h at room temperature with a rabbit serum against human Ki67 antigen, diluted 1:50 (Dako, Glostrup, Denmark). Sections were then reacted 60 min with a biotinylated goat antirabbit IgG, diluted 1:100 (Jackson Immunoresearch Laboratories, West Grove, PA), and incubated 60 min with a fluorescein-conjugated streptavidin, diluted 1:100 (Jackson Immunoresearch Laboratories). The distribution of Ki67 in normal skin was as previously reported (Ralfkiaer et al., 1986Ralfkiaer E. Wantzin G.L. Stein H. Mason D.Y. Photosensitive dermatitis with actinic reticuloid syndrome: an immunohistological study of the cutaneous infiltrate.Br J Dermatol. 1986; 114: 47-56Crossref PubMed Scopus (25) Google Scholar;Lucke et al., 1999Lucke T. Choudhry R. Thom R. Selmer I.S. Burden A.D. Hodgins M.B. Upregulation of connexin 26 is a feature of keratinocyte differentiation in hyperproliferative epidermis, vaginal epithelium, and buccal epithelium.J Invest Dermatol. 1999; 112: 354-361https://doi.org/10.1046/j.1523-1747.1999.00512.xCrossref PubMed Scopus (129) Google Scholar). cDNAs containing the entire coding region of Cx43 (Fishman et al., 1990Fishman G.I. Spray D.C. Leinwand L.A. Molecular characterization and functional expression of the human cardiac gap junction channel.J Cell Biol. 1990; 111: 589-598Crossref PubMed Scopus (157) Google Scholar) and Cx26 (Lee et al., 1992Lee S.W. Tomasetto C. Paul D. Keyomarsi K. Sager R. Transcriptional downregulation of gap-junction proteins blocks junctional communication in human mammary tumor cell lines.J Cell Biol. 1992; 118: 1213-1221Crossref PubMed Scopus (228) Google Scholar) were subcloned in plasmid Bluescript II KS and linearized with Xho I and EcoR I (for Cx43) and Hind III and EcoR I (for Cx26) restriction site enzymes (Boehringer Mannheim, Germany), as previously described (Salomon et al., 1994Salomon D. Masgrau E. Vischer S. et al.Topography of mammalian connexins in human skin.J Invest Dermatol. 1994; 103: 240-247Crossref PubMed Scopus (121) Google Scholar). Connexin antisense probes were synthesized by in vitro transcription (Meda et al., 1993Meda P. Pepper M.S. Traub O. et al.Differential expression of gap junction connexins in endocrine and exocrine glands.Endocrinology. 1993; 133: 2371-2378Crossref PubMed Scopus (146) Google Scholar). Skin samples and epidermal equivalents were homogenized in 2.5 ml 0.1 M Tris-HCl, pH 7.4, containing 2 M β-mercaptoethanol and 4 M guanidium thiocyanate. After addition of solid CsCl (0.4 g per ml), the homogenate was layered on a 2 ml cushion of 5.7 M CsCl and 0.1 M ethylenediamine tetraacetic acid (EDTA) (pH 7.4) and centrifuged for 20 h at 35,000 rpm and 20°C. Pelleted RNA was resuspended in 10 mM Tris-HCl, pH 8.1, supplemented with mM EDTA and 0.1% sodium dodecyl sulfate (SDS), extracted twice with phenol-chloroform, precipitated in ethanol, and resuspended in water. For northern blots, total cellular RNA was denatured with 1 M glyoxal in 0.01 M phosphate buffer containing 50% dimethylsulfoxide, separated by electrophoresis in a 1.2% agarose gel (5 μg total cellular RNA per lane), and transferred overnight onto nylon membranes (Hybond N; Amersham International). Filters were exposed for 30 s to 302 nm light, stained with methylene blue, and prehybridized for 2 h at 65°C in a 50% formamide solution buffered with 0.05 M Pipes (pH 6.8) and supplemented with 2mM EDTA, 0.1% SDS, 0.1 mg per ml salmon sperm DNA, 0.8 M NaCl, and 2.5 × Denhardt's solution. Filters were hybridized for 18 h at 65°C with 1.5 × 106 cpm per ml of one of the 32P-labeled probes mentioned above, washed twice at 65°C in 3 × sodium citrate/chloride buffer (SSC) and 2 × Denhardt's solution, then three times at 70°C in 0.2 × SSC, 0.1% SDS, and 0.1% sodium pyrophosphate, and eventually exposed to XAR-5 film (Eastman Kodak, Rochester, NY), between intensifying screens, at -80°C for 1–6 d. Epidermal equivalents were split from the insert membrane by a 30 min exposure to 1.2 U per ml dispase II (Boehringer Mannheim) and rinsed three times with PBS. They were then placed upside down on 4 μl Tissucol Duo (Immuno, Vienna, Austria), which was freshly prepared by mixing equal volumes of fibrinogen and a 1:100 dilution of thrombin. After 5 min, the insert membrane was removed with fine tweezers and the epidermal equivalents were covered with culture medium. Permeability of junctional channels was evaluated by impaling individual cells with a glass microelectrode (150–200 MΩ) filled with HEPES-buffered (pH 7.2) 150 mM LiCl that contained 4% Lucifer Yellow CH (Sigma) (Salomon et al., 1988Salomon D. Saurat J.H. Meda P. Cell-to-cell communication within intact human skin.J Clin Invest. 1988; 82: 248-254Crossref PubMed Scopus (79) Google Scholar). The microelectrode was connected to a pulse generator for passing current and recording membrane potentials. After successful cell impalement, 0.1 nA negative square pulses of 900 ms and 0.5 Hz frequency were applied for 2–15 min. At the end of the injections, the epidermal equivalents were fixed in 4% paraformaldehyde in PBS and photographed under fluorescence illumination. Injected areas (about six per culture time) were further scanned using a coulour chilled 3CCD video camera (Hamamatsu C5810) and scored for the number of cells containing Lucifer Yellow, including the injected one, by two independent investigators. Results were expressed as both mean +SEM and median values. When monolayers of ORS keratinocytes were exposed to the air–liquid interface (day 0), cells began to form a stratified epithelium. After 2 d, this epithelium was two to four layers thick and already comprised flattened cells at the air surface Figure 1a. Two days later, the epithelium showed a basal layer of columnar cells and eight to ten layers of polygonal cells that became flattened and parakeratotic close to the air surface Figure 1b. Between days 4 and 7, the number of cell layers almost doubled. Thus, the epithelium comprised a prominent stratum spinosum, a faint stratum granulosum, and an orthokeratotic layer of moderate thickness Figure 1c. In 14-d-old epidermal equivalents, the keratinocyte organization was comparable to that observed in human epidermis, i.e., comprised a layer of small, basal cells, several layers of progressively flattening spinous cells, two to three layers of granular keratinocytes, and several layers of orthokeratotic cells that detached at the tissue surface Figure 1d. At this stage, several markers of keratinocyte differentiation could be immunolocalized in the epithelium. Thus, involucrin was seen in several layers of suprabasal cells, starting from the mid-stratum spinosum Figure 2b, and fillagrin decorated upper spinous and granular cells Figure 2c. Cytokeratin K10 was found expressed in almost all suprabasal cells Figure 2d. At the ultrastructural level, keratinocytes of 14-d-old epidermal equivalents contained abundant bundles of intermediate filaments and were connected by numerous desmosomes and gap junctions Figure 3.Figure 3Gap junction plaques form in epidermal equivalents. (A) At day 14, suprabasal cells of epidermal equivalents showed several ultrastructural characteristics of differentiated keratinocytes, including bundles of intermediate keratin filaments (arrows), desmosomes (black arrowhead), and areas of close membrane apposition (white arrowheads). The higher magnification view of one such area (rectangle) illustrates the pentalaminar, slightly striated appearance of two adjacent cell membranes at a gap junction (inset). (B) Freeze-fracture showed that the plasma membrane of keratinocytes contained aggregates of uniformly large particles (on one fracture face, p) and pits (on the other fracture face, E), typical of gap junction plaques (white arrowheads). Smaller aggregates of heterogeneous particles are characteristic of desmosomes (black arrowheads). Scale bar: (A) 15 μm; (inset) 160 nm; (B) 270 nm.View Large Image Figure ViewerDownload (PPT) Immunolabeling of the same cultures showed that keratinocytes also expressed K6 and Ki67, two protein markers of active proliferation, throughout the development of the epidermal equivalents. Thus, antibodies to K6, which stained the epidermis of psoriatic plaques but not of control human skin (not shown), revealed abundant levels of this cytokeratin in the cytoplasm of all basal and suprabasal keratinocytes, from day 2 onwards Figure 4. Similarly, antibodies to Ki67, which revealed numerous proliferating keratinocytes in the epidermis of psoriatic plaques but rare dividing cells in control human skin (not shown), showed clust
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