Interleukin-1 Induces Transcription of Keratin K6 in Human Epidermal Keratinocytes
2001; Elsevier BV; Volume: 116; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.2001.01249.x
ISSN1523-1747
AutoresMayumi Komine, Laxmi Rao, Irwin M. Freedberg, Marcia Simon, Vladana Milisavljevic, Miroslav Blumenberg,
Tópico(s)Cellular Mechanics and Interactions
ResumoKeratinocytes respond to injury by releasing the proinflammatory cytokine interleukin-1, which serves as the initial ‘‘alarm signal’' to surrounding cells. Among the consequences of interleukin-1 release is the production of additional cytokines and their receptors by keratinocytes and other cells in the skin. Here we describe an additional effect of interleukin-1 on keratinocytes, namely the alteration in the keratinocyte cytoskeleton in the form of the induction of keratin 6 expression. Keratin 6 is a marker of hyperproliferative, activated keratinocytes, found in wound healing, psoriasis, and other inflammatory disorders. Skin biopsies in organ culture treated with interleukin-1 express keratin 6 in all suprabasal layers of the epidermis, throughout the tissue. In cultured epidermal keratinocytes, the induction of keratin 6 is time and concentration dependent. Importantly, only confluent keratinocytes respond to interleukin-1, subconfluent cultures do not. In the cells starved of growth factors, epidermal growth factor or tumor necrosis factor-α, if added simultaneously with interleukin-1, they synergistically augment the effects of interleukin-1. Using DNA-mediated cell transfection, we analyzed the molecular mechanisms regulating the keratin 6 induction by interleukin-1, and found that the induction occurs at the transcriptional level. We used a series of deletions and point mutations to identify the interleukin-1 responsive DNA element in the keratin 6 promoter, and determined that it contains a complex of C/EBP binding sites. The transcription factor C/EBPβ binds this element in vitro, and the binding is augmented by pretreatment of the cells with interleukin-1. The interleukin-1 responsive element is clearly distinct from the epidermal growth factor responsive one, which means that the proinflammatory and proliferative signals independently regulate the expression of keratin 6. Thus, interleukin-1 initiates keratinocyte activation not only by triggering additional signaling events, but also by inducing directly the synthesis of keratin 6 in epidermal keratinocytes, and thus changing the composition of their cytoskeleton. Keratinocytes respond to injury by releasing the proinflammatory cytokine interleukin-1, which serves as the initial ‘‘alarm signal’' to surrounding cells. Among the consequences of interleukin-1 release is the production of additional cytokines and their receptors by keratinocytes and other cells in the skin. Here we describe an additional effect of interleukin-1 on keratinocytes, namely the alteration in the keratinocyte cytoskeleton in the form of the induction of keratin 6 expression. Keratin 6 is a marker of hyperproliferative, activated keratinocytes, found in wound healing, psoriasis, and other inflammatory disorders. Skin biopsies in organ culture treated with interleukin-1 express keratin 6 in all suprabasal layers of the epidermis, throughout the tissue. In cultured epidermal keratinocytes, the induction of keratin 6 is time and concentration dependent. Importantly, only confluent keratinocytes respond to interleukin-1, subconfluent cultures do not. In the cells starved of growth factors, epidermal growth factor or tumor necrosis factor-α, if added simultaneously with interleukin-1, they synergistically augment the effects of interleukin-1. Using DNA-mediated cell transfection, we analyzed the molecular mechanisms regulating the keratin 6 induction by interleukin-1, and found that the induction occurs at the transcriptional level. We used a series of deletions and point mutations to identify the interleukin-1 responsive DNA element in the keratin 6 promoter, and determined that it contains a complex of C/EBP binding sites. The transcription factor C/EBPβ binds this element in vitro, and the binding is augmented by pretreatment of the cells with interleukin-1. The interleukin-1 responsive element is clearly distinct from the epidermal growth factor responsive one, which means that the proinflammatory and proliferative signals independently regulate the expression of keratin 6. Thus, interleukin-1 initiates keratinocyte activation not only by triggering additional signaling events, but also by inducing directly the synthesis of keratin 6 in epidermal keratinocytes, and thus changing the composition of their cytoskeleton. epidermal growth factor receptor keratin 6 protein kinase A protein kinase C The epidermis, a multilayered tissue made of and by keratinocytes, provides our first line of defense from external insults. It has been appreciated for a long time that epidermis passively protects the host from desiccation, chemical damage, and physical damage, such as mechanical injury or ultraviolet light. More recently, protection through active immunologic events was demonstrated, when it was realized that keratinocytes, when damaged, actively initiate inflammatory processes by releasing a system of signals consisting of cytokines and growth factors (Barker et al., 1991Barker J.N. Mitra R.S. Griffiths C.E. Dixit V.M. Nickoloff B.J. Keratinocytes as initiators of inflammation.Lancet. 1991; 337: 211-214Abstract PubMed Scopus (609) Google Scholar;Ollier, 1992Ollier S. Cutaneous responses–-a window on inflammatory processes.Clin Exp Allergy. 1992; 22: 3-6Crossref PubMed Scopus (4) Google Scholar). The most likely candidate for a signal that initiates such inflammation is the cytokine interleukin (IL)-1 (Groves et al., 1996Groves R.W. Rauschmayr T. Nakamura K. Sarkar S. Williams I.R. Kupper T.S. Inflammatory and hyperproliferative skin disease in mice that express elevated levels of the IL-1 receptor (type I) on epidermal keratinocytes. Evidence that IL-1-inducible secondary cytokines produced by keratinocytes in vivo can cause skin disease.J Clin Invest. 1996; 98: 336-344Crossref PubMed Scopus (77) Google Scholar). IL-1, in both the IL-1α and the IL-1β form, is present in healthy epidermis, but because it is cytoplasmic, IL-1 is unavailable to its receptor. When keratinocytes are damaged, however, the pre-stored IL-1 is released and it serves as a paracrine signal to fibroblasts to produce prostaglandins and collagenase, to endothelial cells to express selectins, as well as to lymphocytes, leading to their chemotaxis toward the site of injury (Dinarello and Wolff, 1993Dinarello C.A. Wolff S.M. The role of interleukin-1 in disease.N Engl J Med. 1993; 328: 106-113Crossref PubMed Scopus (915) Google Scholar). In addition, IL-1 serves as the autocrine signal to the surrounding undamaged keratinocytes, stimulating them to become activated (Kupper, 1990Kupper T.S. Role of epidermal cytokines.in: Oppenheim J.J. Shevach E.M. Immunophysiology the Role of Cells and Cytokines in Immunity and Inflammation. Oxford University Press, London1990: 285-305Google Scholar). Activated keratinocytes are migratory and hyperproliferative, producing a cornucopia of growth factors and cytokines that function in the inflammatory and wound healing processes. Among the products of keratinocytes activated by IL-1 are IL-3, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-α, and transforming growth factor-α. The production of IL-1 is further augmented by IL-1, TNF-α, and transforming growth factor-α, which can, in a series of interconnected autocrine loops, ‘‘conspire’' to keep keratinocytes in an activated state (Kupper and Groves, 1995Kupper T.S. Groves R.W. The interleukin-1 axis and cutaneous inflammation.J Invest Dermatol. 1995; 105: 62S-66SCrossref PubMed Scopus (151) Google Scholar). The IL-1 signal originates at the cell surface receptor, IL-1R type I, which avidly binds IL-1α, IL-1β, and IL-1ra, a receptor antagonist. A soluble form of the receptor, sIL-1R, competes for IL-1, attenuating the signal. Another high-affinity receptor, IL-1R type II, apparently does not transduce a signal and may actually be a ‘‘decoy’' receptor, also attenuating the effects of IL-1 (Sims et al., 1993Sims J.E. Gayle M.A. Slack J.L. et al.Interleukin 1 signaling occurs exclusively via the type I receptor.Proc Natl Acad Sci USA. 1993; 90: 6155-6159Crossref PubMed Scopus (536) Google Scholar). Apparently, keratinocytes have developed elaborate strategies both to respond to and to protect themselves from IL-1 (Mizutani et al., 1991Mizutani H. Black R. Kupper T.S. Human keratinocytes produce but do not process pro-interleukin-1 (IL-1) beta. Different strategies of IL-1 production and processing in monocytes and keratinocytes.J Clin Invest. 1991; 87: 1066-1071Crossref PubMed Scopus (172) Google Scholar;Kupper and Groves, 1995Kupper T.S. Groves R.W. The interleukin-1 axis and cutaneous inflammation.J Invest Dermatol. 1995; 105: 62S-66SCrossref PubMed Scopus (151) Google Scholar). Among the molecular effects of IL-1 is activation of signal transducing kinases that activate transcription factors AP1 and nuclear factor (NF) κB (Brasier et al., 1990Brasier A. Ron D. Tate J. Habener J. A family of constitutive C/EBP-like DNA binding proteins attenuate the IL-1 alpha induced, NF kappa B mediated trans-activation of the angiotensinogen gene acute-phase response element.EMBO J. 1990; 9: 3933-3944Crossref PubMed Scopus (125) Google Scholar;Novak et al., 1990Novak T.J. Chen D. Rothenberg E.V. 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Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8.Proc Natl Acad Sci USA. 1993; 90: 10193-10197Crossref PubMed Scopus (856) Google Scholar;Stein and Baldwin, 1993Stein B. Baldwin A. Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-kappa B.Mol Cell Biol. 1993; 13: 7191-7198Crossref PubMed Google Scholar;Klampfer et al., 1994Klampfer L. Lee T.H. Hsu W. Vilcek J. Chen-Kiang S. NF-IL6 and AP-1 cooperatively modulate the activation of the TSG-6 gene by tumor necrosis factor alpha and interleukin-1.Mol Cell Biol. 1994; 14: 6561-6569Crossref PubMed Scopus (69) Google Scholar;Sells et al., 1995Sells S.F. Muthukumar S. Sukhatme V.P. Crist S.A. Rangnekar V.M. The zinc finger transcription factor EGR-1 impedes interleukin-1-inducible tumor growth arrest.Mol Cell Biol. 1995; 15: 682-692Crossref PubMed Google Scholar). In various cell types this flurry of transcriptional activation results in the induction of expression of several proteins, such as collagenase, stromelysin, angiotensinogen, and transcription factors Fos and Jun, in addition to the growth factors and cytokines mentioned above. IL-1 causes keratinocyte activation in epidermis. Among the markers of activated keratinocytes is keratin K6 (Tomic-Canic et al., 1998Tomic-Canic M. Komine M. Freedberg I.M. Blumenberg M. Epidermal signal transduction and transcription factor activation in activated keratinocytes.J Dermatol Sci. 1998; 17: 167-181https://doi.org/10.1016/s0923-1811(98)00016-4Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Keratins constitute a large family of cytoskeletal proteins, differentially expressed in various epithelial cells (Schweizer, 1993Schweizer J. Murine epidermal keratins.in: Darmon M. Blumenberg M. Molecular Biology of the Skin: the Keratinocyte. Academic Press, Inc., New York1993: 33-72Crossref Google Scholar). They belong to two families, acidic and basic, are usually expressed in pairs and the keratin filaments are obligate heteropolymers of at least one member from each family. The basal layer of the epidermis produces keratins K5 and K14, whereas in the differentiating, suprabasal layers K1, K2e, and K10 are produced. Healthy interfollicular epidermis does not contain K6; however, it is found in the outer root sheath of the hair follicle, the nail bed, oral epithelia, and some other tissues. In epidermal wound healing, psoriasis, carcinomas, and other pathologic states, keratin K6 is expressed together with K16 or K17. Mutations in K6 keratin gene cause pachyonychia congenita (Bowden et al., 1995Bowden P.E. Haley J.L. Kansky A. Rothnagel J.A. Jones D.O. Turner R.J. Mutation of a type II keratin gene (K6a) in pachyonychia congenita.Nature Genet. 1995; 10: 363-365Crossref PubMed Scopus (204) Google Scholar). Our previous results have shown that extracellular signals that cause keratinocyte proliferation, specifically epidermal growth factor (EGF) and transforming growth factor-α, can induce the expression of K6 and have characterized the transcription factors that bind its promoter (Bernerd et al., 1993Bernerd F. Magnaldo T. Freedberg I.M. Blumenberg M. Expression of the carcinoma-associated keratin K6 and the role of AP-1 proto-oncoproteins.Gene Expression. 1993; 3: 187-199PubMed Google Scholar;Jiang et al., 1993Jiang C.K. Magnaldo T. Ohtsuki M. Freedberg I.M. Bernerd F. Blumenberg M. Epidermal growth factor and transforming growth factor alpha specifically induce the activation- and hyperproliferation-associated keratins 6 and 16.Proc Natl Acad Sci USA. 1993; 90: 6786-6790Crossref PubMed Scopus (161) Google Scholar). Because IL-1 is an important initiator of keratinocyte activation, we decided to determine whether IL-1 directly affects keratin gene expression. If so, that would mean that IL-1 not only induces the downstream signaling events, but also directly causes cytoskeletal changes characteristic of activated keratinocytes. We found that addition of IL-1 to organ culture of skin biopsies specifically induces the expression of K6. A large collection of DNA constructs containing keratin gene promoters was used to transfect cultures of human epidermal keratinocytes. Addition of IL-1 to the medium specifically increased the activity of the K6 promoter. Other keratin gene promoters tested were not affected. Focusing on the K6 promoter sequence, we have identified the DNA segment that responds to the IL-1 signal. The segment contains several C/EBP consensus sites and binds purified C/EBPβ protein in vitro. These results show that IL-1, an initiator of keratinocyte activation, directly induces the expression of keratin K6 in epidermis and thereby causes changes in the keratinocyte cytoskeleton. Additionally, our results begin to characterize the molecular signal transduction pathways along which IL-1 regulates gene expression in epidermis. The plasmids containing keratin promoters, the segments of the K6 promoter and the control plasmids pRSVZ have been described previously (Jiang et al., 1993Jiang C.K. Magnaldo T. Ohtsuki M. Freedberg I.M. Bernerd F. Blumenberg M. Epidermal growth factor and transforming growth factor alpha specifically induce the activation- and hyperproliferation-associated keratins 6 and 16.Proc Natl Acad Sci USA. 1993; 90: 6786-6790Crossref PubMed Scopus (161) Google Scholar,Jiang et al., 1994Jiang C.K. Flanagan S. Ohtsuki M. Shuai K. Freedberg I.M. Blumenberg M. Disease-activated transcription factor: allergic reactions in human skin cause nuclear transcription of STAT-91 and induce synthesis of keratin K17.Mol Cell Biol. 1994; 14: 4759-4769Crossref PubMed Scopus (66) Google Scholar;Ma et al., 1997Ma S. Rao L. Freedberg I.M. Blumenberg M. Transcriptional control of K5, K6, K14, and K17 keratin genes by AP-1 and NF-kappaB family members.Gene Expression. 1997; 6: 361-370PubMed Google Scholar). The plasmid containing the IL-8 promoter was a gift from J. Vilcek (NYU School of Medicine, New York, NY), the one expressing the EGF receptor (EGFR) gene from J. Schlessinger (NYU School of Medicine, New York, NY), and the one expressing C/EBPβ from S. Cheng (Cornell University). To clone the responsive element into a heterologous vector, we amplified the DNA using polymerase chain reaction (PCR). The synthesized DNA contained the C/EBP sites bracketed by restriction sites that facilitate cloning and were inserted into the enhancer trap TK-CAT vector (Promega, Madison, WI), using its BamHI cloning site. The CAT activity of the resulting plasmid was compared with that of the parental control plasmid, TK-CAT. The Kaplan Comprehensive Cancer Center Core Facility confirmed the sequences of the DNA inserted into the pGCAT vector using the dideoxy plasmid sequencing method. All DNA used in transfections were purified using the Magic Megapreps DNA purification system (Promega). HeLa cells were maintained in Dulbecco's minimal essential medium supplemented with 10% bovine serum, and transfected using modified calcium phosphate precipitation procedure (Jiang et al., 1991Jiang C.K. Connolly D. Blumenberg M. Comparison of methods for transfection of human epidermal keratinocytes.J Invest Dermatol. 1991; 97: 969-973Abstract Full Text PDF PubMed Google Scholar). Each transfection contained either 10 μg of K6 CAT per dish, or 15 μg of K6 deletions and mutants per dish, together with 3 μg of RSVZ construct per dish. The cells were then incubated with or without IL-1α in combination with various inhibitors as indicated. In all experiments we used the IL-1α form, except where specifically indicated. The sources of cytokines and inhibitors were Immunex (Seattle, WA) for IL-1α, IL-1 neutralizing antibody and antibodies reacting with the IL-1 receptor; Sigma (St Louis, MO) for cyclic adenosine monophosphate, forskolin, insulin, diacylglycerol, and bisindolylmaleimide; Intergen (Purchase, NY) for TNF-α U.B.I. (Lake Placid, NY) for interferon-γ and IL-1β R&D Systems (Minneapolis, MN) for EGF and phospholipase A. Eighteen to 48 hours after transfection cells were washed twice with phosphate-buffered saline and harvested by scraping. The cell disruption by repeated freeze-thaw cycles, as well as CAT and β-galactosidase assays have also been described (Jiang et al., 1991Jiang C.K. Connolly D. Blumenberg M. Comparison of methods for transfection of human epidermal keratinocytes.J Invest Dermatol. 1991; 97: 969-973Abstract Full Text PDF PubMed Google Scholar). In addition to using the functional assay, we measured the CAT protein using a CAT enzyme-linked immunosorbent assay kit purchased from Boehringer-Mannheim (Indianapolis, IN). Briefly, 96-well plates precoated with anti-CAT antibody were incubated 1 h with samples and standards, washed five times, incubated with anti-CAT conjugate for 1 h, washed five times again, incubated again with anti-digoxigenin conjugated with peroxidase and developed with chromogenic substrate with digoxigenin for 30 min. The optical density of each well was measured with a spectrophotometer using a 405 nm filter. These experiments were performed using duplicate plates with a 490 nm filter for reference. All CAT values were normalized for transfection efficiency by calculating the ratio of CAT activity to β-galactosidase in each transfected plate. Each transfection experiment was separately performed three or more times, with each data point resulting from duplicate or triplicate transfections. Normal human foreskin epidermal keratinocytes were first cultured using 3T3 feeder layers and then frozen in Liquid N2, as described (Simon and Green, 1984Simon M. Green H. Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte.Cell. 1984; 36: 827-834Abstract Full Text PDF PubMed Scopus (183) Google Scholar;Randolph and Simon, 1994Randolph R.K. Simon M. Characterization of retinol metabolism in cultured human epidermal keratinocytes.J Biol Chem. 1994; 268: 9198-9205Abstract Full Text PDF Google Scholar). Once thawed, the keratinocytes were grown without feeder cells in KGM, a defined serum-free keratinocyte medium supplemented with insulin, epidermal growth factor and bovine pituitary extract (keratinocyte-SFM, GIBCO, San Diego, CA). Cells were expanded through two 1:4 passages before transfection and usually transfected 1 d after they reached 100% confluence. Some 16–24 h before transfection, the cells were transferred into keratinocyte basal medium (KBM). KBM is the same defined medium, but without the bovine pituitary extract, EGF, insulin, hydrocortisone, or thyroid hormone. Transfections using polybrene with dimethyl sulfoxide shock were performed as previously described (Jiang et al., 1991Jiang C.K. Connolly D. Blumenberg M. Comparison of methods for transfection of human epidermal keratinocytes.J Invest Dermatol. 1991; 97: 969-973Abstract Full Text PDF PubMed Google Scholar). Keratinocytes were grown to confluence in KGM, the medium was switched to KBM for 16 h and then the cells were either left untreated or treated with 50 ng per ml of IL-1. After 40 min, cells from two 100 mm dishes were harvested by scraping, collected by centrifugation, washed with phosphate-buffered saline and resuspended in 100 μl of buffer containing 20 mM HEPES (pH 7.8), 450 mM NaCl, 0.4 mM ethylenediamine tetraacetic acid, 0.5 mM dithiothreitol, 25% glycerol and 0.5 mM phenylmethylsulfonyl fluoride. The cells were broken by freeze-thawing in liquid N2 three times and centrifuged at 4°C to remove debris. The protein concentration was determined and the extracts aliquoted and stored at -70°C until use. The extracts were thawed only once, on ice, immediately prior to use. The probe was labeled using the Klenow fragment (Boehringer-Mannheim) and [α32P]deoxycytidine triphosphate, 50 μCi (Amersham, Piscataway, NJ) per reaction, and purified by gel filtration using Sephadex G50 columns (Chroma-Spin, Clontech, Palo Alto, CA). The probes were annealed double-stranded DNA NFκB consensus GCC- TGGGAAAGTCCCCTCAACT and K6 promoter ATTTTGCCCTGC- CTAAAGGAAGCGAAAAATGCAATCTCGGTATTTCATAACTTT- TGTAATAATGC. Approximately 5 μg of the extract was initially incubated for 15 min on ice with or without excess of unlabeled competitor, in the presence of 1.5 μg of dIdC, a nonspecific competitor (Stratagene, La Jolla, CA), in a final volume of 25 μl. The binding buffer contained 20 mM Tris–HCl pH 7.6, 5 mM MgCl2, 100 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 2% polyvinyl alcohol, and 0.1 mM ethylenediamine tetraacetic acid. 32P-labeled oligonucleotide probe (30,000 cpm) was added and the incubation continued for an additional 30 min on ice. The free and the protein-bound DNA were separated on 5% polyacrylamide gels (acrylamide/bisacrylamide = 29 : 1). The gels were prerun for 30 min in 1 × Tris-borate pH 7, EDTA 1 mM (TBE) buffer and then run for 2–2.5 h at 125 V. The gels were transferred on to filter paper, dried and exposed to X-ray film (X-omat, Kodak, Rochester, NY) at - 70 °C for 24–48 h with screen intensifiers. To obtain purified C/EBPβ protein we used the plasmid expressing GST-tagged C/EBPβ (Hsu and Chen-Kiang, 1993Hsu W. Chen-Kiang S. Convergent regulation of NF-IL6 and October-1 synthesis by interleukin-6 and retinoic acid signaling in embryonal carcinoma cells.Mol Cell Biol. 1993; 13: 2515-2523Crossref PubMed Scopus (37) Google Scholar) to transform BL21(DE3) Escherichia coli (US Biologicals, Swampscott, MA). The bacteria were grown in Luria broth (LB) with Ampicilin to an OD600 of 0.8 and induced with 1 mM isopropyl thiogalactose for 3 h. Subsequently, we used the GST-bulk purification kit that includes glutathione-Sepharose-4B and followed the procedures recommended by the manufacturer (Pharmacia, Uppsala, Sweden). We prepared un-tagged protein using thrombin to remove the GST tag, but found the tagged and the native proteins to have indistinguishable properties in gel shift assays. The yield and purity of the proteins were assessed using standard sodium dodecyl sulfate polyacrylamide gels. The protein was incubated with the labeled probe, and the gels were run, dried, and exposed as described above. Pieces of normal human skin were obtained immediately after surgery. They were cut into pieces approximately 5 mm3, and incubated in KBM with or without IL-1α (50 ng per ml, Intergen), in a humidified incubator at 37°C for 24 h. The biopsies were mounted in tissue Tec OCT compound (Sakura Finetek, Torrance, CA) and immediately frozen in liquid nitrogen. Sections, 4–6 μm thick, were obtained with a cryostat (Miles Laboratories, Tarrytown, NY), fixed with methanol/acetone for 10 min and incubated with anti-keratin K6 antibody (Progen Biotechnik GMBH, Heidelberg, Germany) as a primary antibody at 4°C overnight. The sections were washed with phosphate-buffered saline three times and treated with peroxidase-conjugated anti-mouse IgG secondary antibody (Vecstatin ABC-mouse IgG kit from Vector Laboratories, Burlingame, CA), at room temperature for 1 h. The samples were washed again with phosphate-buffered saline, and incubated with ABC complex (Vector Laboratories) at room temperature for 1 h and treated with 3,3′-diaminobenzidine-tetrahydrochloride (Dojindo, Kumamoto, Japan) and 0.01% H2O2 as a substrate in Tris pH 7.6 for 2 min. The samples were observed and photographed under a light microscope (Microphot-FXA, Nikon, Melville, NY). Explanted skin samples were incubated with or without IL-1 for 16 h, harvested, and total RNA were isolated utilizing RNeasy RNA extraction kit from Qiagen (Santa Clarita, CA). Either 5 or 50 μg of RNA were subjected to reverse transcription–PCR, with the Access reverse transcription–PCR system from Promega. We used commercial primers for G3PDH (Clontech), and the K6 keratin primers are as follows: K6 reverse transcription–PCR Reverse TGACTTGTCCAACGCCTTCG; K6 reverse transcription–PCR Forward GAGATCGACCACGTCAAGAAGC. The PCR products were subjected to agarose gel electrophoresis, visualized with ethidium bromide (Sigma) with a transilluminator from UVP (Upland, CA) and photographed with a photographing unit from Polaroid (Germany). The densities of bands were quantified utilizing an image scanner (GT-9000 from Epson, Tokyo, Japan). Keratinocytes grown in vitro produce copious amounts of K6 protein and mRNA. It was, therefore, difficult to demonstrate an increase in K6 protein levels in response to IL-1 (not shown). On the other hand, healthy interfollicular epidermis does not contain K6, and therefore the induction of K6 expression by IL-1 could be easily observed in organ culture (Figure 1). To demonstrate the induction of K6 by IL-1 in epidermal keratinocytes, we took advantage of a new ‘‘nearly in vivo’' experimental system that uses organ culture of human skin samples that would be otherwise discarded during surgery (Mutasim et al., 1993Mutasim D.F. Vaughan A. Supapannachart N. Farooqui J. Skin explant culture: a reliable method for detecting pemphigoid antibodies in pemphigoid sera that are negative by standard immunofluorescence and immunoblotting.J Invest Dermatol. 1993; 101: 624-627Crossref PubMed Scopus (7) Google Scholar;Dickinson et al., 1994Dickinson A.M. Sviland L. Hamilton P.J. et al.Cytokine involvement in predicting clinical graft-versus-host disease in allogeneic bone marrow transplant recipients.Bone Marrow Transplant. 1994; 13: 65-70PubMed Google Scholar;Bhora et al., 1995Bhora F.Y. Dunkin B.J. Batzri S. Aly H.M. Bass B.L. Sidawy A.N. Harmon J.W. Effect of growth factors on cell proliferation and epithelialization in human skin.J Surg Res. 1995; 59: 236-244https://doi.org/10.1006/jsre.1995.1160Abstract Full Text PDF PubMed Scopus (136) Google Scholar;Stoll et al., 1997Stoll S. Garner W. Elder J. Heparin-binding ligands mediate autocrine epidermal growth factor receptor activation in skin organ culture.J Clin Invest. 1997; 100: 1271-1281Crossref PubMed Scopus (99) Google Scholar). We obtained 3–5 mm diameter biopsies of human skin and placed them in KBM culture medium. IL-1 was added to the experimental samples, whereas the control samples were incubated without IL-1. After 24 h the biopsies were removed from the medium, frozen, sectioned, and the presence of K6 determined using specific antibodies. IL-1 induced the expression of high amounts of K6 keratin within the 24 h period (Figure 1). As a control we used antibody detecting keratin K17, this protein was not induced by IL-1 (data not shown). Without IL-1 the presence of keratin K6 was detected in the perifollicular and eccrine epithelia, where K6 is normally seen. The K6 staining was present in all suprabasal epidermal layers. The staining was intense throughout the treated samples, suggesting that IL-1 entered the explants from the top, through the stratum corneum. We note, however, that at the edges of the biopsies after 24 h a weak presence of K6 can be detected even in the absence of added IL-1. This induction of K6 is presumably due to the release of the endogenous IL-1 by the peripheral keratinocytes damaged during the surgical procedure, and can be prevented with antibodies neutralizing IL-1 (Figure 1). To determine whether the induction of K6 occurs at the mRNA level, we used quantitative reverse transcription–PCR on IL-1-treated and IL-1-untreated samples. As the internal control we used GAPDH mRNA. As shown in Figure 2, the GAPDH mRNA levels did not change in response to the IL-1 treatment; in contrast, we saw a robust induction of the K6 mRNA in the IL-1-treated samples. Because the regulation of keratin gene expression occurs primarily at the level of transcription (Lu et al., 1994Lu B.
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