TY - JOUR TI - Expression of CCAAT/enhancer binding proteins (C/EBP) is associated with squamous differentiation in epidermis and isolated primary keratinocytes and is altered in neoplasms AU - Oh, HS AU - Smart, RC T2 - JOURNAL OF INVESTIGATIVE DERMATOLOGY AB - The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that undergo sequential changes in gene expression during differentiation. CCAAT/enhancer binding proteins (C/EBP) are members of the bZIP family of DNA binding proteins/transcription factors. Northern analysis demonstrated that C/EBPα, C/EBPβ, and C/EBPδ mRNA are expressed in mouse epidermis and their mRNA levels were generally greater than those observed in other tissues known to express high levels of C/EBP. Western analysis of isolated epidermal cell nuclei demonstrated the presence of a 42 and 30 kDa C/EBPα protein and 35 kDa C/EBPβ protein. Immunohistochemical localization of C/EBPα and C/EBPβ in intact interfollicular epidermis revealed that C/EBPβ expression is exclusive to the nuclei of a three-cell cluster of suprabasal keratinocytes that is morphologically consistent with the central column of the epidermal proliferative unit, and that C/EBPα is expressed in the nuclei and cytoplasm of suprabasal keratinocytes and weakly expressed in a perinuclear manner in some basal keratinocytes. In squamous cell carcinomas the expression of C/EBPα and C/EBPβ was greatly diminished as both the intensity of nuclear staining and the number of cells expressing C/EBPα and C/EBPβ were reduced. In isolated primary mouse keratinocytes, calcium-induced differentiation was accompanied by specific temporal changes in the expression of C/EBPα, C/EBPβ, and C/EBPδ mRNA and C/EBPα and C/EBPβ protein. These results implicate a role for the C/EBP family in the regulation of genes involved in or specifically expressed during the process of squamous differentiation in epidermis. The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that undergo sequential changes in gene expression during differentiation. CCAAT/enhancer binding proteins (C/EBP) are members of the bZIP family of DNA binding proteins/transcription factors. Northern analysis demonstrated that C/EBPα, C/EBPβ, and C/EBPδ mRNA are expressed in mouse epidermis and their mRNA levels were generally greater than those observed in other tissues known to express high levels of C/EBP. Western analysis of isolated epidermal cell nuclei demonstrated the presence of a 42 and 30 kDa C/EBPα protein and 35 kDa C/EBPβ protein. Immunohistochemical localization of C/EBPα and C/EBPβ in intact interfollicular epidermis revealed that C/EBPβ expression is exclusive to the nuclei of a three-cell cluster of suprabasal keratinocytes that is morphologically consistent with the central column of the epidermal proliferative unit, and that C/EBPα is expressed in the nuclei and cytoplasm of suprabasal keratinocytes and weakly expressed in a perinuclear manner in some basal keratinocytes. In squamous cell carcinomas the expression of C/EBPα and C/EBPβ was greatly diminished as both the intensity of nuclear staining and the number of cells expressing C/EBPα and C/EBPβ were reduced. In isolated primary mouse keratinocytes, calcium-induced differentiation was accompanied by specific temporal changes in the expression of C/EBPα, C/EBPβ, and C/EBPδ mRNA and C/EBPα and C/EBPβ protein. These results implicate a role for the C/EBP family in the regulation of genes involved in or specifically expressed during the process of squamous differentiation in epidermis. enhancer binding protein epidermal proliferative unit The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that undergo a highly coordinated program of sequential changes in gene expression during differentiation from proliferating basal cells through morphologically distinct suprabasal cells, ending in the nonviable cornified stratum corneum (Watt, 1989Watt F.M. Terminal differentiation of epidermal keratinocytes.Curr Opin Cell Biol. 1989; 1: 1107-1115Crossref PubMed Scopus (214) Google Scholar; Fuchs, 1990Fuchs E. Epidermal differentiation: the bare essentials.J Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (568) Google Scholar). The expression of catalytic proteins such as transglutaminase (Thacher and Rice, 1985Thacher S.M. Rice R.H. Keratinocyte-specific transglutaminase of cultured human epidermal cells: relation to cross-linked envelope formation and terminal differentiation.Cell. 1985; 40: 685-695Abstract Full Text PDF PubMed Scopus (384) Google Scholar), assembly proteins such as filaggrin (Dale et al., 1985Dale B.A. Resing K.A. Lonsdale-Eccles J.D. Filaggrin: a keratin filament associated protein.Ann NY Acad Sci. 1985; 455: 330-342Crossref PubMed Scopus (131) Google Scholar), and structural proteins such as loricrin (Mehrel et al., 1990Mehrel T. Hohl D. Rothnagel J.A. et al.Identification of a major keratinocyte cell envelope protein, loricrin.Cell. 1990; 61: 1103-1112Abstract Full Text PDF PubMed Scopus (360) Google Scholar), keratins (Fuchs and Green, 1980Fuchs E. Green H. Changes in keratin gene expression during terminal differentiation of the keratinocyte.Cell. 1980; 19: 1033-1042Abstract Full Text PDF PubMed Scopus (779) Google Scholar), involucrin (Simon and Green, 1984Simon M. Green H. Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte cell.Cell. 1984; 36: 827-834Abstract Full Text PDF PubMed Scopus (183) Google Scholar), and cornifin-α/SPRR1 (Gibbs et al., 1993Gibbs S. Fijneman R. Wiegant J. van Kessel A.D. van De Putte P. Backendorf C. Molecular characterization and evolution of the SPRR family of keratinocyte differentiation markers encoding small proline-rich proteins.Genomics. 1993; 16: 630-637Crossref PubMed Scopus (176) Google Scholar; Owens et al., 1996Owens D.M. Zainal T.A. Jetten A.M. Smart R.C. Localization and expression of cornifin-α/SPRR1 in mouse epidermis, anagen hair follicles, and skin neoplasms.J Invest Dermatol. 1996; 106: 647-654Crossref PubMed Scopus (21) Google Scholar), is strictly regulated as a function of the stage of keratinocyte differentiation. The induction of certain differentiation responsive genes is tightly coupled with the repression of others during differentiation. In addition to undergoing a precisely defined program of differentiation, keratinocytes are a rich source of numerous cytokines such as IL-1α, IL-6, IL-7, IL-8, granulocyte macrophage-colony stimulating factor (GM-CSF), and tumor necrosis factor-α that can regulate the function of cells in skin (for review, see Matsue et al., 1992Matsue H. Cruz Jr., P.D. Bergstresser P.R. Takashima A. Cytokine expression by epidermal cell subpopulations.J Invest Dermatol. 1992; 99: 42S-45SAbstract Full Text PDF PubMed Scopus (77) Google Scholar). These epidermal cytokines can act as autocrine and paracrine factors that may play a role in amplifying responses of the skin to various stimuli such as chemicals and UV light. Although cytokine expression in keratinocytes and the coordinate and sequential alterations in specific genes involved in keratinocyte differentiation have been well characterized, the transcription factors that regulate these processes in keratinocytes are largely uncharacterized. CCAAT/enhancer binding protein α (C/EBPα) is a heat-stable transcription factor that recognizes consensus sequence (5′-ATTGCGCAAT-3′) within many promoters (Vinson et al., 1989Vinson C.R. Sigler P.B. McKnight S.L. Scissors-Grip model for DNA recognition by a family of leucie zipper proteins.Science. 1989; 246: 911-916Crossref PubMed Scopus (717) Google Scholar; Johnson, 1993Johnson P.F. Identification of C/EBP basic region resodues involved in DNA sequence recognition and half-site spacing preference.Mol Cell Biol. 1993; 13: 6919-6930Crossref PubMed Scopus (101) Google Scholar). The C/EBP family contains at least seven members that include C/EBPα, C/EBPβ (also termed NF-IL6, NF-M, IL-6DBP, CRP2, or LAP), C/EBPδ (NF-IL6β or CRP3), C/EBPγ (Ig/EBP-1), CRP1, d/CEBP, and CHOP10 (GADD153) (for review, see Wedel and Loms Ziegler-Heitbrock, 1995Wedel A. Loms Ziegler-Heitbrock H.W. The C/EBP family of transcription factors.Immunobiol. 1995; 193: 171-185Crossref PubMed Scopus (195) Google Scholar). C/EBP proteins are members of the bZIP family of DNA-binding proteins and consist of three structural components: a C-terminal leucine zipper, a basic DNA-binding region (Landschulz et al., 1988Landschulz W.H. Johnson P.F. McKnight S.L. The leucine zipper: A hypothetical structure common to a new class of DNA binding proteins.Sci. 1988; 240: 1759-1764Crossref PubMed Scopus (2465) Google Scholar; Vinson et al., 1989Vinson C.R. Sigler P.B. McKnight S.L. Scissors-Grip model for DNA recognition by a family of leucie zipper proteins.Science. 1989; 246: 911-916Crossref PubMed Scopus (717) Google Scholar), and an N-terminal transactivating region (Friedman and McKnight, 1990Friedman A.D. McKnight S.L. Identification of two polypeptide segments of CCAAT/enhancer-binding protein required fot transcriptional activation of the serum albumin gene.Genes Dev. 1990; 4: 1416-1426Crossref PubMed Scopus (123) Google Scholar). C/EBP proteins share amino acid sequence similarities within their C-terminal basic region/leucine zipper domain. The leucine zipper domain is responsible for dimerization, whereas the basic region is responsible for binding to specific DNA sequences (Vinson et al., 1989Vinson C.R. Sigler P.B. McKnight S.L. Scissors-Grip model for DNA recognition by a family of leucie zipper proteins.Science. 1989; 246: 911-916Crossref PubMed Scopus (717) Google Scholar). Both homo- and heterodimers of C/EBP isoforms can form and bind to C/EBP sites within the promoters/enhancers of certain genes (Friedman et al., 1989Friedman A.D. Landschulz W.H. McKnight S.L. CCAAT/enhancer binding protein activates the promoter of the serum albumin gene in cultured hepatoma cells.Genes Dev. 1989; 3: 1314-1322Crossref PubMed Scopus (360) Google Scholar; Descombes et al., 1990Descombes P. Chojkier M. Lichisteiner S. Falvey E. Schibler U. LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein.Genes Dev. 1990; 4: 1541-1551Crossref PubMed Scopus (410) Google Scholar; Cao et al., 1991Cao Z. Umek R.M. Mcnight S.L. Regulated espression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells.Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1297) Google Scholar; Williams et al., 1991Williams S.C. Cantwell C.A. Johnson P.F. A family of C/EBP-related proteins capable of forming covalently linked leucine zipper dimers in vitro.Genes Dev. 1991; 5: 1553-1567Crossref PubMed Scopus (434) Google Scholar). The expression of C/EBP isoforms is most prominent in adipocytes, hepatocytes, intestinal tissues, lung (Birkenmeier et al., 1989Birkenmeier E.H. Gwynn B. Howard S. Jerry J. Gordon J.I. Landschulz W.H. McKinght S.L. Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein.Genes Dev. 1989; 3: 1146-1156Crossref PubMed Scopus (457) Google Scholar; Cao et al., 1991Cao Z. Umek R.M. Mcnight S.L. Regulated espression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells.Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1297) Google Scholar), monocytes/macrophage (Natsuka et al., 1992Natsuka S. Akira S. Nishio Y. Hashimoto S. Sugita T. Isshiki H. Kishimoto T. Macrophage differentiation specific expression of NF-IL6, a transcription factor for IL-6.Blood. 1992; 79: 460-466Crossref PubMed Google Scholar), and ovarian follicles (Piontkewitz et al., 1993Piontkewitz Y. Enerback S. Hedin L. Expression and hormonal regulation of the CCAAT/enhancer binding protein-α during differentiation of rat ovarian follicles.Endocrinol. 1993; 133: 2327-2333Crossref PubMed Scopus (37) Google Scholar). C/EBPα is produced in tissues capable of gluconeogenesis and lipogenesis, especially liver and fat (Birkenmeier et al., 1989Birkenmeier E.H. Gwynn B. Howard S. Jerry J. Gordon J.I. Landschulz W.H. McKinght S.L. Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein.Genes Dev. 1989; 3: 1146-1156Crossref PubMed Scopus (457) Google Scholar), and it is considered to play a direct role in regulating transcription of some of the enzymes involved in controlling these metabolic processes (McKnight et al., 1989McKnight S.L. Lane M.D. Gluecksohn-Waelsch S. Is CCAAT/enhancer-binding protein a central regulator of energy metabolism?.Genes Dev. 1989; 3: 2021-2024Crossref PubMed Scopus (190) Google Scholar). Many lines of evidence indicate that C/EBPα plays a key role in the differentiation of preadipocytes into adipocytes as it appears to function both by inhibiting the clonal expansion that precedes adipocyte terminal differentiation and by activating the coordinate expression of a group of adipocyte genes whose promoters possess C/EBP-binding sites (Christy et al., 1989Christy R.J. Yang V.W. Ntambi J.M. et al.Differentiation-induced gene expression in 3T3-L1 preadipocytes: CCAAT/enhancer binding protein interacts with and activates the promoters of two adipocyte-specific genes.Genes Dev. 1989; 3: 1323-1335Crossref PubMed Scopus (453) Google Scholar; Cao et al., 1991Cao Z. Umek R.M. Mcnight S.L. Regulated espression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells.Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1297) Google Scholar; Umek et al., 1991Umek R.M. Friedman A.D. McKnight S.L. CCAAT-enhancer binding protein: A component of a differentiation switch.Science. 1991; 251: 288-292Crossref PubMed Scopus (561) Google Scholar; Freytag et al., 1994Freytag S.O. Paielli D.L. Gilbert J.D. Ectopic expression of the CCAAT/enhancer-binding protein a promotes the adipogenic program in a variety of mouse fibroblastic cells.Genes Dev. 1994; 8: 1654-1663Crossref PubMed Scopus (377) Google Scholar). Recent studies indicate that C/EBPα inhibits cell proliferation of fibrosarcoma cells through the upregulation of the cyclin-dependent kinase inhibitor, p21 (Timchenko et al., 1996Timchenko N.A. Wilde M. Nakanishi M. Smith J.R. Darlington G.J. CCAAT/enhancer-binding protein α (C/EBPα) inhibits cell proliferation through the p21 (WAF-1/CIP-1/SDI-1) protein.Genes Dev. 1996; 10: 804-815Crossref PubMed Scopus (341) Google Scholar). C/EBPβ plays a role at the early stages of preadipocyte differentiation and leads to the expression of C/EBPα, after which C/EBPβ is downregulated as the adipocytes acquire the complete fat-specific phenotype (Cao et al., 1991Cao Z. Umek R.M. Mcnight S.L. Regulated espression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells.Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1297) Google Scholar; Yeh et al., 1995Yeh W.-C. Cao Z. Classon M. McKnight S.L. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins.Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (784) Google Scholar). C/EBPβ is more widely expressed than C/EBPα, e.g., C/EBPβ expression increases during differentiation of macrophage, myeloid, and plasma cells (Cooper et al., 1992Cooper C. Johnson D. Roman C. Avitahl N. Tucker P. Calame K. The C/EBP family of transcriptional activators is functionally important for Ig VH promoter activity in vivo and in vitro.J Immunol. 1992; 149: 3225-3231PubMed Google Scholar; Natsuka et al., 1992Natsuka S. Akira S. Nishio Y. Hashimoto S. Sugita T. Isshiki H. Kishimoto T. Macrophage differentiation specific expression of NF-IL6, a transcription factor for IL-6.Blood. 1992; 79: 460-466Crossref PubMed Google Scholar; Scott et al., 1992Scott L.M. Civin C.I. Roth P. Friedman A.D. A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells.Blood. 1992; 80: 1725-1735Crossref PubMed Google Scholar), suggesting its involvement in the differentiation of these lineages. Moreover, there is evidence that C/EBPβ is involved in the regulation of the expression of several cytokines and C/EBPβ binding motifs are found in the regulatory regions of IL-1β, IL-6, IL-8, tumor necrosis factor-α, and granulocyte-colony stimulating factor (G-CSF) (Akira et al., 1990Akira S. Issiki H. Sugita T. et al.A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family.EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1176) Google Scholar; Mukaida et al., 1990Mukaida N. Mahe Y. Matsushima K. Cooperative interaction of NF-κB- and C/EBP-like factor binding elements in activating the interleukin-8 gene by proinflammatory cytokines.J Biol Chem. 1990; 265: 21128-21133Abstract Full Text PDF PubMed Google Scholar; Drouet et al., 1991Drouet C. Shakhov A.N. Jongeneel C.V. Enhancers and transcription factors controlling the inducibility of the tumor necrosis factor-α promoter in primary macrophages.J Immunol. 1991; 147: 1694-1700PubMed Google Scholar; Natsuka et al., 1992Natsuka S. Akira S. Nishio Y. Hashimoto S. Sugita T. Isshiki H. Kishimoto T. Macrophage differentiation specific expression of NF-IL6, a transcription factor for IL-6.Blood. 1992; 79: 460-466Crossref PubMed Google Scholar; Zhang and Rom, 1993Zhang Y. Rom W.N. Regulation of the interleukin-1β (IL-1 β) gene by mycobacterial components and lipopolysaccharide is mediated by two nuclear factor-IL6 motifs.Mol Cell Biol. 1993; 13: 3831-3837Crossref PubMed Google Scholar). Like C/EBPβ, C/EBPδ also appears to play a role at the early stages of preadipocyte differentiation (Cao et al., 1991Cao Z. Umek R.M. Mcnight S.L. Regulated espression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells.Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1297) Google Scholar; Yeh et al., 1995Yeh W.-C. Cao Z. Classon M. McKnight S.L. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins.Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (784) Google Scholar). Recent studies have demonstrated that C/EBPδ is important for regulating the acute phase expression of the human C3 (the third component of complement) gene in the presence of IL-1 (Juan et al., 1993Juan T.S.-C. Wilson D.R. Wilde M.D. Darlington G.J. Participation of the transcription factor C/EBPδ in the acute-phase regulation of the human gene for complement component C3.Proc Natl Acad Sci USA. 1993; 90: 2584-2588Crossref PubMed Scopus (117) Google Scholar), suggesting a role of C/EBPδ in the regulation of several IL-1 target genes. As C/EBP proteins play a fundamental role in the differentiation of preadipocytes to adipocytes and in the regulation of the expression of many different genes encoding cytokines in several cell types, we reasoned that C/EBP may be expressed in epidermis and they may play an important role in differentiation and/or cytokine expression. Our results demonstrate that C/EBPα, C/EBPβ, and C/EBPδ mRNA are highly expressed in mouse epidermis and in primary keratinocytes. Furthermore, C/EBPα and C/EBPβ proteins are preferentially expressed in specific suprabasal keratinocytes in epidermis and their expression is upregulated during calcium-induced differentiation of primary keratinocytes. Antibodies for C/EBP isoforms were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Keratin 1 polyclonal antibody was purchased from Berkeley Antibody Company (Richmond, CA). Biotinylated secondary goat anti-rabbit IgG was purchased from Boehringer Mannheim (Indianapolis, IN). Peroxidase-conjugated streptavidin and 5,5′-diaminobenzidine were purchased from BioGenex (San Ramon, CA). Protease inhibitors, sodium orthovanadate, and DNAse were purchased from Sigma (St. Louis, MO). [α-32P]dCTP was purchased from DuPont-New England Nuclear Research Products (Boston, MA). Fetal bovine serum and trypsin were purchased from GIBCO BRL (Gaithersburg, MD). EMEM (Ca++ free) was purchased from BioWhittaker (Walkersville, MD). Epidermal growth factor was purchased from United States Biochemical (Cleveland, OH). Female CD-1 mice, 6–7 wk old, or pregnant CD-1 mice were purchased from Charles River Laboratories (Raleigh, NC). The mice were kept in our facility for 1 wk prior to use and were fed rodent chow (Agway Food, Granville Milling, Creedmore, NC) and water ad libitum. The mice were kept on corn cob bedding and placed on a 12 h light/dark cycle until they were used. The hair of the dorsal skin of 6–7 wk old mice was clipped with electric clippers. Epidermis was removed from the dermis (Goodell et al., 1996Goodell A.L. Oh H.-S. Meyer S.A. Smart R.C. Epidermal protein kinase C-β2 is highly sensitive to downregulation and is exculsively expressed in langerhans cells: Down regulation is associated with attenuated contact hypersensitivity.J Invest Dermatol. 1996; 107: 354-359Crossref Scopus (18) Google Scholar) and total RNA was isolated from the epidermis/other tissues or from primary mouse keratinocytes using the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction as described previously (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62247) Google Scholar). Thirty micrograms of each RNA sample was subjected to electrophoresis on a 1% agarose-formaldehyde gel, transferred to a Zeta-probeGT nylon membrane (Bio-Rad, Hercules, CA), and prehybridized at 65°C for 10 min in a solution containing 0.25 M sodium phosphate buffer (pH 7.2) and 7% sodium dodesyl sulfate. Hybridization was conducted overnight at 65°C with 25 ng of 32P-labeled C/EBPα rat cDNA isolated from the pMSV-C/EBPα plasmid, a gift from Dr. S. L. McKnight (University of Texas Southwestern Medical Center, Dallas, TX). After hybridization, the membrane was washed and exposed to Kodak X-OMAT AR film in a cassette with intensifying screens at –80°C. The same membrane was stripped and reprobed with radiolabeled cornifin-α/SPRR1 cDNA, a gift from Dr. A.M. Jetten (National Institute of Environmental Health Sciences, Research Triangle Park, NC), 7S RNA cDNA, a gift from Dr. A. Balmain (The Beatson Institute for Cancer Research, Glasgow, U.K.), or polymerase chain reaction cloned mouse genes corresponding to C/EBPβ and C/EBPδ. Mouse genomic DNA was utilized to polymerase chain reaction clone the intronless C/EBPβ and C/EBPδ genes using the following forward and reverse primer for C/EBPβ, 5′-TTCTACTACGAGCCCGACTGCC-3′ and 5′-CAGCTTGTCCACCGTCTTCTTG-3′, respectively, and the following forward and reverse primers for C/EBPδ, 5′-CCAGATTTTCATTTCGCTCCAG-3′ and 5′-TCGCAGGTCCCAAAGAAACTAG-3′, respectively. The identity of C/EBPβ and C/EBPδ polymerase chain reaction products was confirmed by the size of the amplified product and restriction enzyme analysis. 7S RNA cDNA was used to confirm that equal amounts of RNA were loaded (Balmain et al., 1982Balmain A. Krumlauf R. Vass J.K. Birnie G.D. Cloning and characterization of the abundant cytoplasmic 7S RNA from mouse cells.Nucl Acids Res. 1982; 10: 4259-4277Crossref PubMed Scopus (147) Google Scholar). All cDNA were radiolabeled by random priming using a random priming kit (GIBCO BRL) and [α-32P]dCTP (3000 Ci per mmol, 10 μCi per μl) and purified from unincorporated [α-32P]dCTP using Push Columns (Stratagene, La Jolla, CA). The preparation of epidermal cells from mouse skin was performed as described by Goodell et al., 1996Goodell A.L. Oh H.-S. Meyer S.A. Smart R.C. Epidermal protein kinase C-β2 is highly sensitive to downregulation and is exculsively expressed in langerhans cells: Down regulation is associated with attenuated contact hypersensitivity.J Invest Dermatol. 1996; 107: 354-359Crossref Scopus (18) Google Scholar. The epidermal cell nuclei were isolated as described by Chapin et al., 1994Chapin R.B. Brady P.S. Barke R.A. Brady L.J. Hepatic CCAAT/enhancer binding protein (C/EBP-α and C/EBP-β) expression changes with riboflavin deficiency, diet restriction and starvation in rats.J Nutr. 1994; 124: 2365-2375Google Scholar. The nuclei were resuspended in RIPA buffer [phosphate buffered saline (PBS) containing 1% Nonidet P-40, 0.5% sodium deoxycholic acid, and 0.1% sodium dodecyl sulfate] with protease inhibitors (100 μg aprotinin per ml and 0.1 mM phenylmethylsulfonyl fluoride) and tyrosine phosphatase inhibitor (1 mM sodium orthovanadate). The resuspension was sonicated, incubated on ice for 1 h, and subjected to centrifugation for 20 min at 4°C, 11,000 × g. In some experiments, epidermal cells or primary keratinocytes were placed in the above buffer, sonicated, incubated on ice for 1 h, and subjected to centrifugation for 20 min at 4°C, 11,000 × g. The supernatant was stored at –80°C prior to analysis and the protein concentration was determined using the Lowry method (Lowry et al., 1951Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein determination with folin phenol reagent.J Biol Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Epidermal homogenate, C/EBPα and C/EBPβ standard proteins, and molecular weight markers (GIBCO BRL) were boiled in sodium dodecyl sulfate sample loading buffer for 4 min and subjected to reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis through Tris-Glycine gels. The C/EBPα and C/EBPβ standard proteins were gifts from Drs. Peter Johnson and Esta Sterneck (National Cancer Institute, Bethesda, MD). The separated proteins were electrophorectically transferred to an Immobilon P membrane (Millipore, Bedford, MA). Following incubation in blocking buffer (PBS with 1% bovine serum albumin, 5% milk, and 0.1% Tween) for 1 h at room temperature, the membranes were probed overnight at 4°C with rabbit polyclonal IgG raised against C/EBPα (1:5000), C/EBPβ (1:2500), keratin 1 AF109 (1:500), or cornifin-α/SPRR1 (1:5000), a gift from Dr. A.M. Jetten (National Institute of Environmental Health Sciences, Research Triangle Park, NC). The membranes were then probed with a horseradish peroxidase-linked donkey anti-rabbit Ig (Amersham, Arlington Heights, IL) for 1 h at room temperature. Detection was made with a luminol system (ECL system, Amersham), and the resulting light was then recorded on to Kodak BioMax MR film. Upon completion of autoradiography, membranes were stained with amido black to confirm equal protein content in all the lanes. Skin samples (n = 5), 7,12-dimethylbenz[a]anthracene-initiated/12-O-tetradecanoylphorbol-13-acetate-promoted squamous papillomas (n = 8), and squamous cell carcinomas (n = 4) were frozen in OCT compound (Miles, Elkhart, IN). Frozen sections (5 μm) on Super Frost Plus slides (Fisher, Pittsburgh, PA) were air dried for 30 min at room temperature and fixed in ice-cold acetone for 10 min on ice followed by rinsing in three changes of PBS. The endogenous peroxidase activity was quenched by incubation in 0.1% H2O2 in PBS for 10 min at room temperature. After rinsing in three changes of PBS, the nonspecific binding of antibodies was blocked by incubating the sections in 1% normal goat serum in PBS for 30 min at room temperature. The excessive blocking solution was drained and the sections were then incubated with the primary polyclonal antibodies against C/EBPα (1:25,000) and C/EBPβ (1:10,000) in 1% bovine serum albumin in PBS at 4°C overnight. After washing with PBS, the samples were incubated with a biotinylated goat-anti-rabbit IgG for 30 min at room temperature followed by a 30 min incubation with peroxidase conjugated streptavidin. The avidin/biotin-peroxidase complexes were visualized by incubation with 5,5′-diaminobenzidine (following the manufacturer’s protocol). The sections were counterstained lightly with Harris-modified hematoxylin, dehydrated, and permounted with cover glasses. No C/EBP isoform staining was observed when the primary antibody was omitted and the control rabbit serum was applied. Primary keratinocytes were isolated from newborn CD-1 mice (1–2 d old) by trypsin floatation (Hennings et al., 1980Hennings H. Michael D. Cheng C. Steinert P. Holbrook K. Yuspa S.H. Calcium regulation of growth and differentiation of mouse epidermal cells in culture.Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1448) Google Scholar). Isolated epidermal cells were plated at 6 × 106 cells per 60 mm plate in Ca++ free EMEM supplemented with 10% fetal bovine serum and 4 ng epidermal growth factor per ml for 4 h to enhance keratinocyte attachment. Cells were then gently washed with Mg++ and Ca++ free PBS twice to remove any remaining calcium and then cultured in Ca++ free EMEM supplemented with 8% chelex-treated fetal bovine serum and 4 ng epidermal growth factor per ml. Calcium chloride was then added to produce a final concentration of 0.05 mM Ca++. After 3 d in culture, calcium chloride was added to produce a final concentration 0.12 mM Ca++ or 1.5 mM Ca++ (Yuspa et al., 1989Yuspa S.H. Kilkenny A.E. Steinert P.M. Roop D.R. Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro.J Cell Biol. 1989; 109: 1207-1217Crossref PubMed Scopus (488) Google Scholar). At various times, keratinocytes were harvested for the isolation of protein and RNA for western and northern analysis, respectively. In order to determine whether C/EBPα, C/EBPβ, and C/EBPδ mRNA are expressed in mouse epidermis, total RNA was isolated from the epidermis and northern blot analysis was conducted. For comparison purposes, northern analysis was also conducted on total RNA isolated from several other organs or tissues known to express specific isoforms of C/EBP (Birkenmeier et al., 1989Birkenmeier E.H. Gwynn B. Howard S. Jerry J. Gordon J.I. Landschulz W.H. McKinght S.L. Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein.Genes Dev. 1989; 3: 1146-1156Crossref PubMed Scopus (457) Google Scholar; Cao et al., 1991Cao Z. Umek R.M. Mcnigh DA - 1998/6// PY - 1998/6// DO - 10.1046/j.1523-1747.1998.00199.x VL - 110 IS - 6 SP - 939-945 SN - 0022-202X KW - cytokines KW - skin KW - transcription factor ER - TY - PCOMM TI - On the effect of estrogen receptor agonists and antagonists on the mouse hair follicle cycle AU - Smart, RC AU - Oh, HS AB - The experiments we reported in our letter were described as executed, and the mouse strains investigated were chosen for the reasons stated. The experiments were planned and conducted completely independent of one another but, unfortunately, the same mistake was made in both cases – the concentrations used were not as reported in the original report. Although we take responsibility for this oversight, we also recognize that the dosage listing is not entirely conventional to the field. Before starting the repeat experiments we consulted several independent researchers outside our respective laboratories; in all cases the understood dosage was interpreted exactly as we had. When we repeated the work using the twice weekly protocol and the concentrations used originally by Oh and Smart (Proc Natl Acad Sci 93:12525), β-estradiol did indeed inhibit the normal progression of spontaneous anagen in pigmented mice (C57B16). From additional and subsequent studies that we have since executed, we have learned several important features about the role of estrogen receptor-mediated signaling in murine hair growth control that we did not formerly appreciate. We would hope to share these data in a future report. We are indebted to Drs. Oh and Smart for calling our attention to this interesting phenomenon and regret the confusion our mistake might have caused. Note from the Editor: Due to an editorial office error, Drs. Smart and Oh were not given a chance to reply to the original letter about their paper by Drs. Stenn, Paus, and Filippi (J Invest Dermatol, 110:95 1998). We apologize to all the authors for this mistake. DA - 1998/7// PY - 1998/7// DO - 10.1046/j.1523-1747.1998.00257.x SP - 175-175 ER -