Fos and Jun Proteins Are Specifically Expressed During Differentiation of Human Keratinocytes
2005; Elsevier BV; Volume: 124; Issue: 1 Linguagem: Inglês
10.1111/j.0022-202x.2004.23558.x
ISSN1523-1747
AutoresDenis Mehic, Latifa Bakiri, Minoo Ghannadan, Erwin F. Wagner, Erwin Tschachler,
Tópico(s)Biotin and Related Studies
ResumoActivator protein 1 (AP-1) proteins play key roles in the regulation of cell proliferation and differentiation. In this study we investigated the expression of Fos and Jun proteins in different models of terminal differentiation of human keratinocytes and in skin from psoriasis patients. All Jun and Fos proteins, with the exception of FosB, were efficiently expressed in keratinocytes in monolayer cultures. In contrast, in normal epidermis as well as in organotypic epidermal cultures, the expression pattern of AP-1 proteins was dependent on the differentiation stage. Fos proteins were readily detected in nuclei of keratinocytes of basal and suprabasal layers. JunB and JunD were expressed in all layers of normal epidermis. Interestingly, expression of c-Jun started suprabasally, then disappeared and became detectable again in distinct cells of the outermost granular layer directly at the transition zone to the stratum corneum. In psoriatic epidermis, c-Jun expression was prominent in both hyperproliferating basal and suprabasal keratinocytes, whereas c-Fos expression was unchanged. These data indicate that AP-1 proteins are expressed in a highly specific manner during terminal differentiation of keratinocytes and that the enhanced expression of c-Jun in basal and suprabasal keratinocytes might contribute to the pathogenesis of psoriasis. Activator protein 1 (AP-1) proteins play key roles in the regulation of cell proliferation and differentiation. In this study we investigated the expression of Fos and Jun proteins in different models of terminal differentiation of human keratinocytes and in skin from psoriasis patients. All Jun and Fos proteins, with the exception of FosB, were efficiently expressed in keratinocytes in monolayer cultures. In contrast, in normal epidermis as well as in organotypic epidermal cultures, the expression pattern of AP-1 proteins was dependent on the differentiation stage. Fos proteins were readily detected in nuclei of keratinocytes of basal and suprabasal layers. JunB and JunD were expressed in all layers of normal epidermis. Interestingly, expression of c-Jun started suprabasally, then disappeared and became detectable again in distinct cells of the outermost granular layer directly at the transition zone to the stratum corneum. In psoriatic epidermis, c-Jun expression was prominent in both hyperproliferating basal and suprabasal keratinocytes, whereas c-Fos expression was unchanged. These data indicate that AP-1 proteins are expressed in a highly specific manner during terminal differentiation of keratinocytes and that the enhanced expression of c-Jun in basal and suprabasal keratinocytes might contribute to the pathogenesis of psoriasis. activator protein 1 fetal calf serum human dermal fibroblasts normal human epidermal keratinocytes phosphate-buffered saline skin equivalents Terminal differentiation of epidermal keratinocytes begins when cells migrate out of the basal epidermal layer, stop to proliferate and move outwards to the spinous and granular layers. Keratinocytes thereby undergo defined changes finally giving rise to the mature horny layer of the epidermis composed of tightly connected corneocytes devoid of nuclei that function to protect the body from dehydration and injury (Fuchs and Byrne, 1994Fuchs E. Byrne C. The epidermis: Rising to the surface.Curr Opin Genet Dev. 1994; 4: 725-736https://doi.org/10.1016/0959-437X(94)90140-XCrossref PubMed Scopus (218) Google Scholar). The Activator protein 1 (AP-1) transcription factor consists of homo- or heterodimers of members of the Fos (c-Fos, FosB, Fra-1, and Fra-2) and Jun (c-Jun, JunB, and JunD) proteins (Shaulian and Karin, 2001Shaulian E. Karin M. AP-1 in cell proliferation and survival.Oncogene. 2001; 20: 2390-2400https://doi.org/10.1038/sj.onc.1204383Crossref PubMed Scopus (1282) Google Scholar). Loss- and gain-of-function experiments in mice have demonstrated the central role of AP-1 in many biological processes including cell proliferation, differentiation, oncogenic transformation, and apoptosis (Jochum et al., 2001Jochum W. Passegue E. Wagner E.F. AP-1 in mouse development and tumorigenesis.Oncogene. 2001; 20: 2401-2412https://doi.org/10.1038/sj.onc.1204389Crossref PubMed Scopus (572) Google Scholar). AP-1 proteins play important roles during terminal differentiation of epidermal keratinocytes and therefore the epidermis represents a highly informative and easily accessible in vivo system to study AP-1 functions. It is well known that AP-1 regulates many genes encoding critical players of skin homeostasis (Angel et al., 2001Angel P. Szabowski A. Schorpp-Kistner M. Function and regulation of AP-1 subunits in skin physiology and pathology.Oncogene. 2001; 20: 2413-2423https://doi.org/10.1038/sj.onc.1204380Crossref PubMed Scopus (312) Google Scholar). These include transglutaminase type I (Liew and Yamanishi, 1992Liew F.M. Yamanishi K. Regulation of transglutaminase 1 gene expression by 12-O-tetradecanoylphorbol-13-acetate, dexamethasone, and retinoic acid in cultured human keratinocytes.Exp Cell Res. 1992; 202: 310-315https://doi.org/10.1016/0014-4827(92)90080-RCrossref PubMed Scopus (32) Google Scholar), different cytokeratin gene family members, such as K1 (Lu et al., 1994Lu B. Rothnagel J.A. Longley M.A. Tsai S.Y. Roop D.R. Differentiation-specific expression of human keratin 1 is mediated by a composite AP-1/steroid hormone element.J Biol Chem. 1994; 269: 7443-7449Abstract Full Text PDF PubMed Google Scholar), K5 (Casatorres et al., 1994Casatorres J. Navarro J.M. Blessing M. Jorcano J.L. Analysis of the control of expression and tissue specificity of the keratin 5 gene, characteristic of basal keratinocytes. Fundamental role of an AP-1 element.J Biol Chem. 1994; 269: 20489-20496Abstract Full Text PDF PubMed Google Scholar), K14, and K17 (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 Exp. 1997; 6: 361-370PubMed Google Scholar) as well as structural epidermal proteins like involucrin (Welter et al., 1995Welter J.F. Crish J.F. Agarwal C. Eckert R.L. Fos-related antigen (Fra-1), junB, and junD activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity.J Biol Chem. 1995; 270: 12614-12622Crossref PubMed Scopus (172) Google Scholar), loricrin (DiSepio et al., 1995DiSepio D. Jones A. Longley M.A. Bundman D. Rothnagel J.A. Roop D.R. The proximal promoter of the mouse loricrin gene contains a functional AP-1 element and directs keratinocyte-specific but not differentiation-specific expression.J Biol Chem. 1995; 270: 10792-10799https://doi.org/10.1074/jbc.270.18.10792Crossref PubMed Scopus (94) Google Scholar), and profilaggrin (Jang et al., 1996Jang S.I. Steinert P.M. Markova N.G. Activator protein 1 activity is involved in the regulation of the cell type-specific expression from the proximal promoter of the human profilaggrin gene.J Biol Chem. 1996; 271: 24105-24114https://doi.org/10.1074/jbc.271.39.24105Crossref PubMed Scopus (85) Google Scholar). Although in mice inactivation of some AP-1 proteins did not reveal an overt skin phenotype (Angel et al., 2001Angel P. Szabowski A. Schorpp-Kistner M. Function and regulation of AP-1 subunits in skin physiology and pathology.Oncogene. 2001; 20: 2413-2423https://doi.org/10.1038/sj.onc.1204380Crossref PubMed Scopus (312) Google Scholar), it is difficult to speculate about AP-1 functions in mouse skin as loss of JunB and Fra-1 results in embryonic lethality (Eferl and Wagner, 2003Eferl R. Wagner E.F. AP-1: A double-edged sword in tumorigenesis.Nat Rev Cancer. 2003; 3: 859-868https://doi.org/10.1038/nrc1209Crossref PubMed Scopus (1492) Google Scholar). Interestingly, the conditional inactivation of c-Jun in the mouse epidermis has demonstrated that c-Jun plays an essential role in regulating eyelid closure, keratinocyte proliferation, and skin tumor development through anti-epidermal growth factor receptor signaling (Li et al., 2003Li G. Gustafson-Brown C. Hanks S.K. et al.c-Jun is essential for organization of the epidermal leading edge.Dev Cell. 2003; 4: 865-877https://doi.org/10.1016/S1534-5807(03)00159-XAbstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar; Zenz et al., 2003Zenz R. Scheuch H. Martin P. et al.c-Jun regulates eyelid closure and skin tumor development through EGFR signaling.Dev Cell. 2003; 4: 879-889https://doi.org/10.1016/S1534-5807(03)00161-8Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Expression of individual AP-1 proteins has also been critically implicated in cytokine-mediated mesenchymal–epithelial interactions in the skin (Szabowski et al., 2000Szabowski A. Maas-Szabowski N. Andrecht S. Kolbus A. Schorpp-Kistner M. Fusenig N.E. Angel P. c-Jun and JunB antagonistically control cytokine-regulated mesenchymal–epidermal interaction in skin.Cell. 2000; 103: 745-755Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). Disturbances in tissue homeostasis and an aberrant, altered expression of cytokines are likely to be implicated in human pathological diseases such as psoriasis. Psoriasis is a chronic and inflammatory skin disease, which affects around 3% of the population and is characterized by epidermal abnormalities including keratinocyte hyperproliferation and altered differentiation leading to parakeratosis and desquamation (Stern, 1997Stern R.S. Psoriasis.Lancet. 1997; 350: 349-353https://doi.org/10.1016/S0140-6736(97)05257-4Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In one study decreased transcription of c-fos and c-jun has been described in psoriasis (Basset-Seguin et al., 1991Basset-Seguin N. Escot C. Moles J.P. Blanchard J.M. Kerai C. Guilhou J.J. C-fos and c-jun proto-oncogene expression is decreased in psoriasis: An in situ quantitative analysis.J Invest Dermatol. 1991; 97: 672-678Abstract Full Text PDF PubMed Google Scholar). In contrast, targeted v-fos expression to mouse keratinocytes efficiently induced epidermal hyperplasia (Greenhalgh et al., 1993Greenhalgh D.A. Rothnagel J.A. Wang X.J. et al.Hyperplasia, hyperkeratosis and benign tumor production in transgenic mice by a targeted v-fos oncogene suggest a role for fos in epidermal differentiation and neoplasia.Oncogene. 1993; 8: 2145-2157PubMed Google Scholar) indicating that the functions of AP-1 in hyperproliferative skin disorders are still not well understood. Individual AP-1 proteins are differentially expressed in epidermal layers, although findings on AP-1 expression in the epidermis from different investigators are somehow discordant. In normal human skin, expression of c-Fos and c-Jun has been reported to be predominantly confined to basal and lower spinous cells (Basset-Seguin et al., 1990Basset-Seguin N. Escot C. Blanchard J.M. Kerai C. Verrier B. Mion H. Guilhou J.J. High levels of c-fos proto-oncogene expression in normal human adult skin.J Invest Dermatol. 1990; 94: 418-422Abstract Full Text PDF PubMed Google Scholar), whereas in another study c-Fos has been localized to all epidermal layers and c-Jun to granular layers of both authentic and reconstructed skin (Briata et al., 1993Briata P. D'Anna F. Franzi A.T. Gherzi R. AP-1 activity during normal human keratinocyte differentiation: Evidence for a cytosolic modulator of AP-1/DNA binding.Exp Cell Res. 1993; 204: 136-146Crossref PubMed Scopus (44) Google Scholar). In another study, analysis of all AP-1 proteins localized FosB and Fra-2 in lower, c-Jun and c-Fos in upper, Fra-1 in suprabasal and JunB as well as JunD in all layers of human neonatal epidermis (Welter and Eckert, 1995Welter J.F. Eckert R.L. Differential expression of the fos and jun family members c-fos, fosB, Fra-1, Fra-2, c-jun, junB and junD during human epidermal keratinocyte differentiation.Oncogene. 1995; 11: 2681-2687PubMed Google Scholar). We assumed that unspecific antibodies against AP-1 proteins are responsible for some of the contradicting results on AP-1 expression in different models of epidermal differentiation. Using in vitro translated AP-1 proteins and cells deficient in different AP-1 proteins, we first examined the reactivity of different antibodies with individual AP-1 proteins. These antibodies were subsequently used to analyze AP-1 expression in different models of terminal differentiation of human keratinocytes such as monolayer cultures, in vitro reconstructed epidermis as well as normal and psoriatic epidermis. We present novel findings on the specific expression of AP-1 proteins during terminal differentiation of human keratinocytes and also demonstrate high expression of c-Jun in psoriatic epidermis. Previous reports on AP-1 expression in human epidermis showed that primary antibodies developed in rabbits produced a high background staining (Welter and Eckert, 1995Welter J.F. Eckert R.L. Differential expression of the fos and jun family members c-fos, fosB, Fra-1, Fra-2, c-jun, junB and junD during human epidermal keratinocyte differentiation.Oncogene. 1995; 11: 2681-2687PubMed Google Scholar). Therefore, we investigated the specificity of commercially available antibodies against AP-1 proteins (Table S1). Download .pdf (.01 MB) Help with pdf files Table S1Supplementary Table 1 To test antibody cross-reactivity between different subunits, in vitro translated AP-1 proteins were analyzed by western blotting. Among the Fos proteins, the rabbit anti-Fra-1 antibody cross-reacted with c-Fos, whereas the other anti-Fos antibodies were detected only the specific subunits (Fig S1A). Similarly, among Jun proteins rabbit anti-JunD cross-reacted with c-Jun, whereas both mouse monoclonal antibodies anti-c-Jun and JunB detected only the corresponding Jun proteins (Fig S1B). To evaluate whether the respective antibodies also detect the different endogenous AP-1 proteins, serum-starved quiescent human dermal fibroblasts (HDF) were stimulated 3 h with serum and AP-1 reactivity was analyzed by immunostaining. Whereas in resting fibroblasts only Fra-2 and JunD were detected, all Fos and all Jun proteins were induced and accumulated in the nucleus upon serum stimulation (Fig S1C, D). No nuclear staining and a low cytoplasmic background staining were observed when serum-stimulated fibroblasts were stained with corresponding isotypes or normal rabbit IgG (Fig S1C, D). Thus, the antibodies used in these experiments detect all human AP-1 proteins by immunostaining and are, with the exception of Fra-1 and JunD AP-1 subunit, specific. Download .pdf (.27 MB) Help with pdf files Figure S1Supplementary Figure 1 Since keratinocytes in monolayer cultures exhibit a differentiated phenotype after prolonged confluence (Eckert et al., 1997Eckert R.L. Crish J.F. Robinson N.A. The epidermal keratinocyte as a model for the study of gene regulation and cell differentiation.Physiol Rev. 1997; 77: 397-424Crossref PubMed Scopus (331) Google Scholar; Chaturvedi et al., 1999Chaturvedi V. Qin J.Z. Denning M.F. Choubey D. Diaz M.O. Nickoloff B.J. Apoptosis in proliferating, senescent, and immortalized keratinocytes.J Biol Chem. 1999; 274: 23358-23367Crossref PubMed Scopus (187) Google Scholar), we studied differentiation-associated AP-1 expression at different time points by immunostaining and Western blot analysis. c-Fos localized to the nucleus of pre-confluent, as well as in 1 and 2 d post-confluent cells, showed a weak cytoplasmic localization at day 4 and virtually disappeared at day 6 after confluence had been reached (Figure 1a). Using western blot analysis, c-Fos was detected in nuclear extracts of pre-confluent as well as 1 and 2 d post-confluent normal human epidermal keratinocytes (NHEK) (Figure 1b). The disappearance of c-Fos in later differentiation stages suggests that it is dispensable for the expression of terminal differentiation-associated proteins and the maintenance of differentiation. FosB, which has been shown previously to be unable to bind to the promoter of several epidermal genes, could not be detected by immunostaining at any time point and western blot analysis only detected a very weak signal in proliferating NHEK (Figure 1a, b). This is in agreement with earlier findings demonstrating only low FosB RNA levels presenting NHEK (Gandarillas and Watt, 1995Gandarillas A. Watt F.M. Changes in expression of members of the fos and jun families and myc network during terminal differentiation of human keratinocytes.Oncogene. 1995; 11: 1403-1407PubMed Google Scholar; Rossi et al., 1998Rossi A. Jang S.I. Ceci R. Steinert P.M. Markova N.G. Effect of AP1 transcription factors on the regulation of transcription in normal human epidermal keratinocytes.J Invest Dermatol. 1998; 110: 34-40https://doi.org/10.1046/j.1523-1747.1998.00071.xCrossref PubMed Scopus (90) Google Scholar). Although only weakly expressed in pre-confluent cultures, both Fra proteins were induced in 1 or 2 d post-confluent cells and were found in nuclear location also at later stages of differentiation (Figure 1a). Both Fra proteins were also detectable in nuclear extracts of post-confluent keratinocytes (Figure 1b). In contrast to Fos proteins, all Jun proteins were detected in pre-confluent cells as well as at all time points after confluence had been reached (Figure 1a). Western blot analysis confirmed that Jun proteins were present at all time points in nuclear extracts of NHEK with higher expression levels in post-confluent than pre-confluent cells (Figure 1b). Together these data demonstrate that whereas Jun proteins are continuously expressed at all stages of keratinocyte differentiation, Fos proteins show a differential expression pattern in monolayer cultures. Therefore we assume that in late stages of keratinocyte differentiation, when c-Fos is absent, Fra-1 and/or Fra-2 may replace it as a partner for Jun. Keratinocytes co-cultured with fibroblasts from SE mimic the natural architecture of the epidermis in a simplified form and represent a powerful model to study keratinocyte differentiation (Fusenig et al., 1994Fusenig N.E. Leigh I. Watt B. Lane F. Epithelial–mesenchymal interactions regulate keratinocyte growth and differentiation in vitro.The Keratinocyte Handbook. Cambridge University Press, 1994: 71-94Google Scholar). The expression of different AP-1 components was next analyzed during terminal differentiation of keratinocytes in such organotypic cultures. SE were followed up to 8 d after initiating differentiation by lifting the epidermis to the air–liquid interface (Figure 2a). Under these conditions differentiation markers like filaggrin or caspase-14 were expressed as previously described (Eckhart et al., 2000Eckhart L. Declercq W. Ban J. et al.Terminal differentiation of human keratinocytes and stratum corneum formation is associated with caspase-14 activation.J Invest Dermatol. 2000; 115: 1148-1151https://doi.org/10.1046/j.1523-1747.2000.00205.xCrossref PubMed Scopus (171) Google Scholar; Rendl et al., 2002Rendl M. Ban J. Mrass P. et al.Caspase-14 expression by epidermal keratinocytes is regulated by retinoids in a differentiation-associated manner.J Invest Dermatol. 2002; 119: 1150-1155https://doi.org/10.1046/j.1523-1747.2002.19532.xCrossref PubMed Scopus (93) Google Scholar). Although in an earlier study c-Fos was detected in organotypic cultures only after prolonged culture (Basset-Seguin et al., 1994Basset-Seguin N. Demoly P. Moles J.P. et al.Comparative analysis of cellular and tissular expression of c-fos in human keratinocytes: Evidence of its role in cell differentiation.Oncogene. 1994; 9: 765-771PubMed Google Scholar), in our study c-Fos was present in the cytoplasm of basal cells already at day 2 after initiating differentiation. Its expression increased and extended to suprabasal cells as differentiation progressed resulting in nuclear localization from day 4 onwards. In the uppermost layers of nucleated cells corresponding to the granular layer of the epidermis, c-Fos expression was detected only in few cells (Figure 2b). As observed in monolayer cultures of keratinocytes, we did not detect FosB at any differentiation stage (Figure 2b). Both Fra-1 and Fra-2 showed faint cytoplasmic staining in SE up to day 6 when expression was also found in the nucleus (Figure 2b). These data demonstrate that Fra proteins are available as partners for the AP-1 complex in late stage of keratinocyte differentiation confirming their potential roles in regulating keratinocyte gene expression (Welter et al., 1995Welter J.F. Crish J.F. Agarwal C. Eckert R.L. Fos-related antigen (Fra-1), junB, and junD activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity.J Biol Chem. 1995; 270: 12614-12622Crossref PubMed Scopus (172) Google Scholar; Medvedev et al., 1999Medvedev A. Saunders N.A. Matsuura H. Chistokhina A. Jetten A.M. Regulation of the transglutaminase I gene. Identification of DNA elements involved in its transcriptional control in tracheobronchial epithelial cells.J Biol Chem. 1999; 274: 3887-3896https://doi.org/10.1074/jbc.274.6.3887Crossref PubMed Scopus (39) Google Scholar; Hanley et al., 2001Hanley K. Wood L. Ng D.C. et al.Cholesterol sulfate stimulates involucrin transcription in keratinocytes by increasing Fra-1, Fra-2, and Jun D.J Lipid Res. 2001; 42: 390-398Abstract Full Text Full Text PDF PubMed Google Scholar). The time course of their translocation to the nucleus indicates that Fra proteins may play a role in the homeostasis of already differentiated epidermis rather than in the actual differentiation process. Jun proteins were expressed from days 2 to 8 of SE differentiation. At all time points analyzed c-Jun expression showed a unique expression pattern. Staining was restricted to the basal and first layer of suprabasal cells as well as to cells of the granular layer, but absent in most cells in between. Moreover, the expression in the granular layer was more strongly than in the basal layers (Figure 2c). JunB was expressed at all time points of investigation and localized to the nucleus. The distribution, however, developed from a uniform to a scattered pattern in all epidermal layers from days 6 to 8 (Figure 2c). JunD predominantly accumulated in the cytoplasm at day 2, but was found in the nuclei of basal cells at day 4. The distinct distribution of JunD first became clearly detectable in suprabasal cells at day 8 (Figure 2c). Thus, during terminal differentiation of SE cultures, where the authentic epidermal architecture is reflected in a more genuine manner than in keratinocytes in monolayer cultures, the different AP-1 proteins showed differentiation-stage-associated expression (summarized in Figure 5). Although c-Jun expression in the uppermost layers has already been reported (Briata et al., 1993Briata P. D'Anna F. Franzi A.T. Gherzi R. AP-1 activity during normal human keratinocyte differentiation: Evidence for a cytosolic modulator of AP-1/DNA binding.Exp Cell Res. 1993; 204: 136-146Crossref PubMed Scopus (44) Google Scholar; Welter and Eckert, 1995Welter J.F. Eckert R.L. Differential expression of the fos and jun family members c-fos, fosB, Fra-1, Fra-2, c-jun, junB and junD during human epidermal keratinocyte differentiation.Oncogene. 1995; 11: 2681-2687PubMed Google Scholar), we show for the first time that c-Jun expression differed at different stages of keratinocyte differentiation, i.e. c-Jun was prominently expressed in basal cells of SE, not detectable in upper suprabasal cells and reoccurred in cells of the granular layer. We next assessed the expression of AP-1 proteins in human epidermis. Only marginal background staining was observed when isotype controls or normal rabbit IgG were used as primary antibodies (Figure 3a). When using the specific antibodies, a highly differential expression of AP-1 proteins was found in the human epidermis. c-Fos was expressed in the nuclei of all epidermal layers with slightly decreased intensity in the granular keratinocytes (Figure 3b). Our results are in agreement with two earlier reports on c-Fos expression (Basset-Seguin et al., 1990Basset-Seguin N. Escot C. Blanchard J.M. Kerai C. Verrier B. Mion H. Guilhou J.J. High levels of c-fos proto-oncogene expression in normal human adult skin.J Invest Dermatol. 1990; 94: 418-422Abstract Full Text PDF PubMed Google Scholar; Briata et al., 1993Briata P. D'Anna F. Franzi A.T. Gherzi R. AP-1 activity during normal human keratinocyte differentiation: Evidence for a cytosolic modulator of AP-1/DNA binding.Exp Cell Res. 1993; 204: 136-146Crossref PubMed Scopus (44) Google Scholar), but in contrast to another report (Welter and Eckert, 1995Welter J.F. Eckert R.L. Differential expression of the fos and jun family members c-fos, fosB, Fra-1, Fra-2, c-jun, junB and junD during human epidermal keratinocyte differentiation.Oncogene. 1995; 11: 2681-2687PubMed Google Scholar). These differences might be because of the use of different antibodies. Like in NHEK and SE, FosB was not detectable in human epidermis (Figure 3b). Interestingly, Fra-1 was detected predominantly in the nuclei of spinous and to a lower extent in basal layers, whereas Fra-2 expression was mainly confined to the nuclei of basal and spinous cells (Figure 3b). These findings are in agreement with published observations (Welter and Eckert, 1995Welter J.F. Eckert R.L. Differential expression of the fos and jun family members c-fos, fosB, Fra-1, Fra-2, c-jun, junB and junD during human epidermal keratinocyte differentiation.Oncogene. 1995; 11: 2681-2687PubMed Google Scholar) with the notable difference that we found Fra proteins predominantly in the nuclei. These data indicate that Fra proteins are available as partners for the AP-1 complex confirming their potential roles in regulating epidermal gene expression in vivo. This is further supported by studies on the role of these family proteins in the activation of promoters of several differentiation-associated keratinocyte genes (Welter et al., 1995Welter J.F. Crish J.F. Agarwal C. Eckert R.L. Fos-related antigen (Fra-1), junB, and junD activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity.J Biol Chem. 1995; 270: 12614-12622Crossref PubMed Scopus (172) Google Scholar; Medvedev et al., 1999Medvedev A. Saunders N.A. Matsuura H. Chistokhina A. Jetten A.M. Regulation of the transglutaminase I gene. Identification of DNA elements involved in its transcriptional control in tracheobronchial epithelial cells.J Biol Chem. 1999; 274: 3887-3896https://doi.org/10.1074/jbc.274.6.3887Crossref PubMed Scopus (39) Google Scholar; Hanley et al., 2001Hanley K. Wood L. Ng D.C. et al.Cholesterol sulfate stimulates involucrin transcription in keratinocytes by increasing Fra-1, Fra-2, and Jun D.J Lipid Res. 2001; 42: 390-398Abstract Full Text Full Text PDF PubMed Google Scholar). Among Jun proteins, c-Jun showed an interesting expression pattern. Although virtually absent from basal cells, c-Jun-stained cells of the first suprabasal and lower spinous layers was not detected in cells of the upper spinous layer, but intense staining reappeared in cells of the granular layer (Figure 3c). By contrast anti-JunB and JunD antibodies stained all epidermal layers. The intensity of the JunB signal increased in the upper spinous layers and in the granular layer, whereas JunD was expressed at equal levels in all epidermal layers (Figure 3c). Although c-Jun expression in cells of the granular layer has been reported previously (Briata et al., 1993Briata P. D'Anna F. Franzi A.T. Gherzi R. AP-1 activity during normal human keratinocyte differentiation: Evidence for a cytosolic modulator of AP-1/DNA binding.Exp Cell Res. 1993; 204: 136-146Crossref PubMed Scopus (44) Google Scholar; Welter and Eckert, 1995Welter J.F. Eckert R.L. Differential expression of the fos and jun family members c-fos, fosB, Fra-1, Fra-2, c-jun, junB and junD during human epidermal keratinocyte differentiation.Oncogene. 1995; 11: 2681-2687PubMed Google Scholar), we show for the first time that c-Jun is regularly present in suprabasal and infrequently also in basal cells. As also depicted and quantified in Figure 4c, c-Jun expression in the outermost nucleated cells of the granular layer of normal epidermis strongly exceeded that in the other layers. Together with the findings that also JunB was stronger expressed in these cells, our data indicate that Jun proteins might play a central role in the last steps of keratinocyte terminal differentiation. Furthermore, as described above c-Fos expression was reduced in the outermost cells of the granular layer; our data also suggest that in this location Jun homodimers might preferentially form. The data on the expression of AP-1 family members in authentic epidermis are summarized in Figure 5. Keratinocyte hyperproliferation and altered differentiation are hallmarks of psoriasis. We assumed an involvement of AP-1 in these steps and postulated that AP-1 expression could be altered in psoriasis. Since heterogeneous cytokine expression and inflammation in psoriatic skin induced a strong background staining with rabbit polyclonal antibodies raised against several AP-1 proteins, the analysis of AP-1 expression in psoriasis was limited to c-Fos and c-Jun. In contrast to the previously re
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