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• W2018274375 abstract "Involucrin is a marker of human keratinocyte differentiation. Previous studies show that the human involucrin gene promoter has two distinct regulatory regions – the proximal regulatory region (PRR) and the distal regulatory region (DRR). To study the role of these regions in vivo, we have constructed human involucrin promoter transgenic mice and monitored the impact of specific promoter mutations on involucrin gene expression. In this study, we monitor the impact of specific mutations on expression in a range of surface epithelia. We begin by confirming previous observations made in footpad epidermis by showing that the full-length involucrin promoter drives differentiation-appropriate expression in other surface epithelia, including epidermis, cervix, and esophagus. We further show that mutation of the activator protein AP1-5 site in the DRR completely eliminates transgene expression in all of these tissues. In contrast, mutation of the DRR Sp1 site reduces overall expression, but does not alter the differentiation dependence. Additional studies identify a DRR immediate suprabasal element (ISE). Deletion of the ISE results in a loss of transgene expression in the immediate suprabasal layers. Our studies also indicate that the PRR is important for appropriate transgene expression. Mutation of a PRR C/EBP (CCAAT enhancer binding protein) transcription factor binding site results in patchy/discontinuous expression. These studies suggest that AP1, Sp1, and C/EBP transcription factors are required for appropriate differentiation-dependent involucrin expression, and that the mechanism of regulation is similar in most surface epithelia. Involucrin is a marker of human keratinocyte differentiation. Previous studies show that the human involucrin gene promoter has two distinct regulatory regions – the proximal regulatory region (PRR) and the distal regulatory region (DRR). To study the role of these regions in vivo, we have constructed human involucrin promoter transgenic mice and monitored the impact of specific promoter mutations on involucrin gene expression. In this study, we monitor the impact of specific mutations on expression in a range of surface epithelia. We begin by confirming previous observations made in footpad epidermis by showing that the full-length involucrin promoter drives differentiation-appropriate expression in other surface epithelia, including epidermis, cervix, and esophagus. We further show that mutation of the activator protein AP1-5 site in the DRR completely eliminates transgene expression in all of these tissues. In contrast, mutation of the DRR Sp1 site reduces overall expression, but does not alter the differentiation dependence. Additional studies identify a DRR immediate suprabasal element (ISE). Deletion of the ISE results in a loss of transgene expression in the immediate suprabasal layers. Our studies also indicate that the PRR is important for appropriate transgene expression. Mutation of a PRR C/EBP (CCAAT enhancer binding protein) transcription factor binding site results in patchy/discontinuous expression. These studies suggest that AP1, Sp1, and C/EBP transcription factors are required for appropriate differentiation-dependent involucrin expression, and that the mechanism of regulation is similar in most surface epithelia. activator protein CCATT enhancer binding protein distal regulatory region human involucrin immediate suprabasal element proximal regulatory region The epidermis is a differentiating system in which basal proliferative cells undergo discrete rounds of proliferation. The daughter cells from this division undergo profound morphology changes as they proceed through the differentiation process (Alonso and Fuchs, 2003Alonso L. Fuchs E. Stem cells of the skin epithelium.Proc Natl Acad Sci USA. 2003; 100: 11830-11835Crossref PubMed Scopus (374) Google Scholar). The ultimate fate of these cells is to be lost from the body surface after performing their protective function. These morphological changes are driven by changes in gene expression. During this process, a specific set of differentiation-appropriate genes are turned on as part of the differentiation program. Several of these genes encode proteins that are used to assemble the cornified envelope. Involucrin is a cornified envelope precursor protein (Rice and Green, 1979Rice R.H. Green H. Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.Cell. 1979; 18: 681-694Abstract Full Text PDF PubMed Scopus (621) Google Scholar; Green, 1980Green H. The keratinocyte as differentiated cell type.Harvey Lect. 1980; 74: 101-139PubMed Google Scholar; Watt and Green, 1981Watt F.M. Green H. Involucrin synthesis is correlated with cell size in human epidermal culture.J Cell Biol. 1981; 90: 738-742Crossref PubMed Scopus (212) Google Scholar; Etoh et al., 1986Etoh Y. Simon M. Green H. Involucrin acts as a transglutaminase substrate at multiple sites.Biochem Biophys Res Commun. 1986; 136: 51-56Crossref PubMed Scopus (34) Google Scholar; Yaffe et al., 1992Yaffe M.B. Beegen H. Eckert R.L. Biophysical characterization of involucrin reveals a molecule ideally suited to function as an intermolecular cross-bridge of the keratinocyte cornified envelope.J Biol Chem. 1992; 267: 12233-12238Abstract Full Text PDF PubMed Google Scholar, Yaffe et al., 1993Yaffe M.B. Murthy S. Eckert R.L. Evidence that involucrin is a covalently linked constituent of highly purified cultured keratinocyte cornified envelopes.J Invest Dermatol. 1993; 100: 3-9Abstract Full Text PDF PubMed Google Scholar; Eckert et al., 1993Eckert R.L. Yaffe M.B. Crish J.F. Murthy S. Rorke E.A. Welter J.F. Involucrin – structure and role in envelope assembly.J Invest Dermatol. 1993; 100: 613-617Abstract Full Text PDF PubMed Google Scholar; Steinert and Marekov, 1997Steinert P.M. Marekov L.N. Direct evidence that involucrin is a major early isopeptide cross-linked component of the keratinocyte cornified cell envelope.J Biol Chem. 1997; 272: 2021-2030Crossref PubMed Scopus (193) Google Scholar). Involucrin is not expressed in basal cells, but expression ensues in the immediate suprabasal layer (spinous layer) and is maintained throughout the granular layer. As such, involucrin is considered an early marker of the differentiation process. A major goal of our effort is to understand the mechanisms that regulate differentiation-dependent gene expression of involucrin as a model for epidermal gene regulation. Previous studies identified two segments within the human involucrin (hINV) promoter that are required for optimal expression in cultured human epidermal keratinocytes (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; Banks et al., 1998Banks E.B. Crish J.F. Welter J.F. Eckert R.L. Characterization of human involucrin promoter distal regulatory region transcriptional activator elements – a role for Sp1 and AP1 binding sites.Biochem J. 1998; 331: 61-68Crossref PubMed Scopus (68) Google Scholar; Efimova et al., 1998Efimova T. LaCelle P. Welter J.F. Eckert R.L. Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.J Biol Chem. 1998; 273: 24387-24395Crossref PubMed Scopus (137) Google Scholar; Agarwal et al., 1999Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. CCAAT/enhancer-binding proteins. A role in regulation of human involucrin promoter response to phorbol ester.J Biol Chem. 1999; 274: 6190-6194Crossref PubMed Scopus (41) Google Scholar). These elements, called the distal regulatory region (DRR) and the proximal regulatory region (PRR), are required for optimal expression in cultured cells (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). The DRR contains an activator protein (AP1 site, AP1-5, and an adjacent Sp1 site that have been implicated as being required for expression in footpad epidermis (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; Banks et al., 1998Banks E.B. Crish J.F. Welter J.F. Eckert R.L. Characterization of human involucrin promoter distal regulatory region transcriptional activator elements – a role for Sp1 and AP1 binding sites.Biochem J. 1998; 331: 61-68Crossref PubMed Scopus (68) Google Scholar; Crish et al., 1998Crish J.F. Zaim T.M. Eckert R.L. The distal regulatory region of the human involucrin promoter is required for expression in epidermis.J Biol Chem. 1998; 273: 30460-30465Crossref PubMed Scopus (56) Google Scholar). The PRR contains CCAAT enhancer binding protein (C/EBP) and AP1 (AP1-1) sites that are required for optimal expression in cultured human epidermal keratinocytes (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; Efimova et al., 1998Efimova T. LaCelle P. Welter J.F. Eckert R.L. Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.J Biol Chem. 1998; 273: 24387-24395Crossref PubMed Scopus (137) Google Scholar; Agarwal et al., 1999Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. CCAAT/enhancer-binding proteins. A role in regulation of human involucrin promoter response to phorbol ester.J Biol Chem. 1999; 274: 6190-6194Crossref PubMed Scopus (41) Google Scholar). Transgenic studies indicate a role for the AP1-5 site in epidermis, as mutation of this site results in absence of expression (Crish et al., 1993Crish J.F. Howard J.M. Zaim T.M. Murthy S. Eckert R.L. Tissue-specific and differentiation-appropriate expression of the human involucrin gene in transgenic mice: an abnormal epidermal phenotype.Differentiation. 1993; 53: 191-200Crossref PubMed Scopus (76) Google Scholar, Crish et al., 1998Crish J.F. Zaim T.M. Eckert R.L. The distal regulatory region of the human involucrin promoter is required for expression in epidermis.J Biol Chem. 1998; 273: 30460-30465Crossref PubMed Scopus (56) Google Scholar, Crish et al., 2002Crish J.F. Bone F. Banks E.B. Eckert R.L. The human involucrin gene contains spatially distinct regulatory elements that regulate expression during early versus late epidermal differentiation.Oncogene. 2002; 21: 738-747Crossref PubMed Scopus (39) Google Scholar). In the present report, we describe transgenic mouse studies that further define the role of these elements. In particular, we monitor the effects of altering the DRR and PRR sites on expression in a range of surface epithelia. We confirm an important role for the DRR AP1-5 site and Sp1 sites. Our studies further indicate that a DNA sequence element located in the DRR segment, the immediate suprabasal element (ISE), is required for appropriate expression. In addition, the PRR C/EBP factor binding site is also required for appropriate expression. Finally, our studies suggest that hINV expression requires similar cis-acting DNA regulatory elements in a range of surface epithelia. This suggests that common mechanisms regulate involucrin expression in diverse epithelia. We began by analyzing the expression of the full-length hINV transgene, hINV H6B. Animals that harbor the hINV H6B transgene were created and expression was analyzed by immunoblot and immunohistology. The structure of the H6B transgene is shown in Figure 1a. As shown in Figure 1b, hINV immunoreactive material is detected in the footpad, epidermis, and esophagus of hINV H6B transgenic mice. Staining of sections with anti-hINV reveals an absence of hINV protein expression in the basal layer (arrow) of footpad, cervix, epidermis, and esophagus. However, strong expression is observed in the suprabasal layers in each tissue. As expected, replacing the rabbit anti-hINV antibody with a rabbit anti-IgG as primary antibody results in an absence of detectable staining, demonstrating specificity of the reagents. In addition, staining is not observed in the underlying connective tissue layer in any tissue, indicating that expression is epithelial-specific. Staining of non-transgenic mice reveals an absence of staining with the hINV-specific antibody (not shown) (Crish et al., 1998Crish J.F. Zaim T.M. Eckert R.L. The distal regulatory region of the human involucrin promoter is required for expression in epidermis.J Biol Chem. 1998; 273: 30460-30465Crossref PubMed Scopus (56) Google Scholar, Crish et al., 2002Crish J.F. Bone F. Banks E.B. Eckert R.L. The human involucrin gene contains spatially distinct regulatory elements that regulate expression during early versus late epidermal differentiation.Oncogene. 2002; 21: 738-747Crossref PubMed Scopus (39) Google Scholar). Figure 1c shows that mutation of the AP1-5 site in H6B(AP1-5mm) results in a complete loss of expression in all epithelia, as confirmed by immunoblot and immunohistology. These studies confirm our previous reported findings in footpad epidermis (Crish et al., 1993Crish J.F. Howard J.M. Zaim T.M. Murthy S. Eckert R.L. Tissue-specific and differentiation-appropriate expression of the human involucrin gene in transgenic mice: an abnormal epidermal phenotype.Differentiation. 1993; 53: 191-200Crossref PubMed Scopus (76) Google Scholar, Crish et al., 1998Crish J.F. Zaim T.M. Eckert R.L. The distal regulatory region of the human involucrin promoter is required for expression in epidermis.J Biol Chem. 1998; 273: 30460-30465Crossref PubMed Scopus (56) Google Scholar, Crish et al., 2002Crish J.F. Bone F. Banks E.B. Eckert R.L. The human involucrin gene contains spatially distinct regulatory elements that regulate expression during early versus late epidermal differentiation.Oncogene. 2002; 21: 738-747Crossref PubMed Scopus (39) Google Scholar) and show that the AP1-5 site has an important role in several surface epithelia. Previous cell culture studies implicate the AP1-5 and Sp1 sites within the DRR as being important for regulation (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; Banks et al., 1998Banks E.B. Crish J.F. Welter J.F. Eckert R.L. Characterization of human involucrin promoter distal regulatory region transcriptional activator elements – a role for Sp1 and AP1 binding sites.Biochem J. 1998; 331: 61-68Crossref PubMed Scopus (68) Google Scholar, Banks et al., 1999Banks E.B. Crish J.F. Eckert R.L. Transcription factor Sp1 activates involucrin promoter activity in non-epithelial cell types.Biochem J. 1999; 337: 507-512Crossref PubMed Google Scholar; Efimova et al., 1998Efimova T. LaCelle P. Welter J.F. Eckert R.L. Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.J Biol Chem. 1998; 273: 24387-24395Crossref PubMed Scopus (137) Google Scholar; Bikle et al., 2002Bikle D.D. Ng D. Oda Y. Hanley K. Feingold K. Xie Z. The vitamin D response element of the involucrin gene mediates its regulation by 1,25-dihydroxyvitamin D3.J Invest Dermatol. 2002; 119: 1109-1113Crossref PubMed Scopus (49) Google Scholar). To examine the role of these elements in greater detail, we cloned the DRR immediately upstream of the hINV basal promoter to create DRR-P3.4B (Figure 2a). We have previously shown that the basal promoter transgene, P3.4B, does not drive expression in mice (Crish et al., 1998Crish J.F. Zaim T.M. Eckert R.L. The distal regulatory region of the human involucrin promoter is required for expression in epidermis.J Biol Chem. 1998; 273: 30460-30465Crossref PubMed Scopus (56) Google Scholar, Crish et al., 2002Crish J.F. Bone F. Banks E.B. Eckert R.L. The human involucrin gene contains spatially distinct regulatory elements that regulate expression during early versus late epidermal differentiation.Oncogene. 2002; 21: 738-747Crossref PubMed Scopus (39) Google Scholar) (see Figure 3b). As shown in Figure 2b, linking the isolated DRR to the hINV minimal promoter drives normal differentiation-appropriate expression. Mutation of the AP1-5 site, in the context of the isolated DRR element in DRR-P3.4B(AP1-5mm), results in a complete loss of hINV expression in all tissues (Figure 2c). We also mutated the Sp1 site, located immediate downstream of the AP1-5 site (Banks et al., 1998Banks E.B. Crish J.F. Welter J.F. Eckert R.L. Characterization of human involucrin promoter distal regulatory region transcriptional activator elements – a role for Sp1 and AP1 binding sites.Biochem J. 1998; 331: 61-68Crossref PubMed Scopus (68) Google Scholar, Banks et al., 1999Banks E.B. Crish J.F. Eckert R.L. Transcription factor Sp1 activates involucrin promoter activity in non-epithelial cell types.Biochem J. 1999; 337: 507-512Crossref PubMed Google Scholar), to create DRR-P3.4B(Sp1m). As shown in Figure 2d, mutation of the Sp1 site results in a reduction in hINV expression, as evidenced by the decreased hINV level relative to the β-actin (see immunoblot). However, although transgene expression level is reduced, appropriate suprabasal expression is maintained (Figure 2d).Figure 3DRR truncation analysis – a role for AP1-5/Sp1. (a) The left panel shows schematics of the various hINV transgenes. H6B, the full-length transgene, is shown at the top, with the DRR and PRR segments identified. Each DRR-derived segment is linked in an identical configuration to hINV minimal promoter, P3.4B. DRR-P3.4B encodes the full-length (nucleotides −2,473/−1,953) DRR segment. The ends of the DRR-derived DNA segment present in each construct are indicated in nucleotides. The positions of the Sp1, C/EBP, and AP1-1 and AP1-5 sites are indicated. Other conventions are as in the legend to Figure 1. The right panel indicates the status of transgene expression in esophagus, cervix, epidermis, and footpad. +++ indicates optimal expression, + indicates reduced expression, − indicates absence of expression, normal indicates that the transgene is expressed in a differentiation-appropriate manner indistinguishable from expression of hINV H6B, and high suprabasal indicates that the transgene expression is confined to the high suprabasal layers. Similar results were observed in each of five independent founder-derived transgenic lines for each transgene. (b) Immunohistological staining of the footpad of hINV DRR truncation construct transgenic mice. Footpad sections were prepared, stained with anti-hINV and secondary antibody, and visualized as described in Materials and Methods. The arrow indicates the epidermal basal layer.View Large Image Figure ViewerDownload (PPT) Given the importance of the AP1-5 and Sp1 sites within the DRR, we examined additional DRR modifications. These constructs are shown in Figure 3a. Analysis of these transgenic mice reveals that constructs including nucleotides −2,473/−1,993 and −2,473/−2,043 produce expression patterns identical to those observed for the complete DRR (nucleotides −2,473/−1,953). In contrast, the DRR-P3.4B(−2,473/−2,097) transgene, which lacks the segment immediately downstream of the AP1-5/Sp1 sites, is expressed at low levels and expression is confined to the high suprabasal layers (ie absent in the immediate suprabasal layers) (Figure 3b). A transgene, DRR-P3.4B(−2,140/−2,088), encoding only the 52-nucleotide segment containing the AP1-5 and Sp1 sites, drives expression in a manner similar to that observed for DRR-P3.4B(−2,473/−2,097). Taken together, these results indicate that sequence elements within nucleotides −2,097/−2,043 are required, in conjunction with the AP1-5/Sp1 sites, for appropriate differentiation-dependent expression. In addition, a transgene containing the isolated downstream site(s), DRR-P3.4B(−2,097/−1,950), does not drive transcription in any tissue. The minimal promoter transgene, P3.4B, does not drive involucrin expression (Crish et al., 2000Crish J.F. Bone F. Balasubramanian S. Zaim T.M. Wagner T. Yun J. et al.Suprabasal expression of the human papillomavirus type 16 oncoproteins in mouse epidermis alters expression of cell cycle regulatory proteins.Carcinogenesis. 2000; 21: 1031-1037Crossref PubMed Scopus (29) Google Scholar). To compare the relative level of transgene expression, we immunoblotted extracts of epidermis from individual transgenic lines using anti-hINV (Figure 4). In this experiment, as in all other experiments in this paper, all lines tested were of the F1 or subsequent generation, and transgene expression was observed to be maintained in each generation (not shown). As expected, owing to differences in integration site in the murine host genome, transgene expression is variable among individual lines for each construct. However, several general trends are apparent. First, mutation of the AP1-5 site, in the context of the full-length promoter or in the context of the DRR, uniformly results in a complete loss of transgene expression – in six hINV H6B(AP1-5mm) lines and seven DRR-P3.4B(AP1-5mm) lines. Second, mutation of the Sp1 site results in a consistent partial reduction in transgene expression – four of five DRR-P3.4B(Sp1m) lines (Figure 4a). Third, trimming of the DRR results in a reduction in expression level – four DRR-P3.4B(−2,473/−2,097) and three DRR-P3.4B(−2,140/−2,088) lines (Figure 4b). Finally, no expression is observed in six DRR-P3.4B(−2,097/−1,950) and in six P3.4B lines. We next examined the function of the PRR segment. This region encodes C/EBP and AP1 (AP1-1) sites that are important for promoter activity in cultured keratinocytes (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; Agarwal et al., 1999Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. CCAAT/enhancer-binding proteins. A role in regulation of human involucrin promoter response to phorbol ester.J Biol Chem. 1999; 274: 6190-6194Crossref PubMed Scopus (41) Google Scholar; Eckert et al., 2004Eckert R.L. Crish J.F. Efimova T. Dashti S.R. Deucher A. Bone F. et al.Regulation of involucrin gene expression.J Invest Dermatol. 2004; 123: 13-22Crossref PubMed Scopus (109) Google Scholar). However, the in vivo role of this region has not been studied. We therefore mutated the AP1-1 site and/or the C/EBP site to produce the transgenes shown in Figure 5a. Mutation of the AP1-1 site results in no apparent change from the normal pattern of expression (Figure 5b). In contrast, mutation of the C/EBP site (Figure 5c) results in an atypical pattern of discontinuous expression. This phenotype is also observed in C/EBPm/AP1-1mm mice (not shown), indicating that the altered expression is due to the absence of a functional C/EBP site. This phenotype is observed in footpad and in the other tested epithelia (Figure 5c). The process and signals that convert a basal cell to a differentiated surface epithelial cell are not well understood. Nor are the molecular events that regulate gene expression during this process. We have attempted to understand this process by focusing on the mechanisms that regulate expression of a single gene – involucrin (Eckert et al., 1993Eckert R.L. Yaffe M.B. Crish J.F. Murthy S. Rorke E.A. Welter J.F. Involucrin – structure and role in envelope assembly.J Invest Dermatol. 1993; 100: 613-617Abstract Full Text PDF PubMed Google Scholar, Eckert et al., 2004Eckert R.L. Crish J.F. Efimova T. Dashti S.R. Deucher A. Bone F. et al.Regulation of involucrin gene expression.J Invest Dermatol. 2004; 123: 13-22Crossref PubMed Scopus (109) Google Scholar). Involucrin is selectively expressed in the differentiating suprabasal cells in surface epithelia (Rice and Green, 1979Rice R.H. Green H. Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.Cell. 1979; 18: 681-694Abstract Full Text PDF PubMed Scopus (621) Google Scholar; Banks-Schlegel and Green, 1981Banks-Schlegel S. Green H. Involucrin synthesis and tissue assembly by keratinocytes in natural and cultured human epithelia.J Cell Biol. 1981; 90: 732-737Crossref PubMed Scopus (272) Google Scholar). As cells complete the final stages in terminal differentiation, involucrin is incorporated via the action of transglutaminase to form part of the crosslinked protective surface (Rice and Green, 1979Rice R.H. Green H. Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.Cell. 1979; 18: 681-694Abstract Full Text PDF PubMed Scopus (621) Google Scholar; Yaffe et al., 1992Yaffe M.B. Beegen H. Eckert R.L. Biophysical characterization of involucrin reveals a molecule ideally suited to function as an intermolecular cross-bridge of the keratinocyte cornified envelope.J Biol Chem. 1992; 267: 12233-12238Abstract Full Text PDF PubMed Google Scholar, Yaffe et al., 1993Yaffe M.B. Murthy S. Eckert R.L. Evidence that involucrin is a covalently linked constituent of highly purified cultured keratinocyte cornified envelopes.J Invest Dermatol. 1993; 100: 3-9Abstract Full Text PDF PubMed Google Scholar; Eckert et al., 1993Eckert R.L. Yaffe M.B. Crish J.F. Murthy S. Rorke E.A. Welter J.F. Involucrin – structure and role in envelope assembly.J Invest Dermatol. 1993; 100: 613-617Abstract Full Text PDF PubMed Google Scholar; Nemes et al., 1999Nemes Z. Marekov L.N. Fesus L. Steinert P.M. A novel function for transglutaminase 1: attachment of long-chain omega-hydroxyceramides to involucrin by ester bond formation.Proc Natl Acad Sci USA. 1999; 96: 8402-8407Crossref PubMed Scopus (207) Google Scholar; Nemes and Steinert, 1999Nemes Z. Steinert P.M. Bricks and mortar of the epidermal barrier.Exp Mol Med. 1999; 31: 5-19Crossref PubMed Scopus (415) Google Scholar). Cell culture-based studies indicate that the AP1-5 site is required for optimal hINV promoter activity (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; Banks et al., 1998Banks E.B. Crish J.F. Welter J.F. Eckert R.L. Characterization of human involucrin promoter distal regulatory region transcriptional activator elements – a role for Sp1 and AP1 binding sites.Biochem J. 1998; 331: 61-68Crossref PubMed Scopus (68) Google Scholar; Bikle et al., 2002Bikle D.D. Ng D. Oda Y. Hanley K. Feingold K. Xie Z. The vitamin D response element of the involucrin gene mediates its regulation by 1,25-dihydroxyvitamin D3.J Invest Dermatol. 2002; 119: 1109-1113Crossref PubMed Scopus (49) Google Scholar). Mutation of this site, in the context of the full-length hINV promoter, results in a partial loss of promoter activity, whereas its mutation in the context of the isolated DRR results in a complete loss of promoter activity. The finding that simultaneous mutation of AP1-5 in the DRR and AP1-1 in the PRR results in a near-complete loss of activity suggested that both these sites have important functional roles (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). A role for the AP1-5 site has also been confirmed in vivo in mouse footpad, esophagus, and cervix, where mutation of this site results in a loss of expression (Crish et al., 2002Crish J.F. Bone F. Banks E.B. Eckert R.L. The human involucrin gene contains spatially distinct regulatory elements that regulate expression during early versus late epidermal differentiation.Oncogene. 2002; 21: 738-747Crossref PubMed Scopus (39) Google Scholar). Our present studies confirm these observations and extend these findings to the epidermis. These findings suggest that the AP1-5 site and AP1 transcription factors play a central role in maintaining hINV gene expression across a range of surface epithelial tissues. In contrast, and much to our surprise, mutation of the PRR AP1-1 site causes no change in hINV transgene expression. Thus, although AP1-1 appears to be functionally important in vitro, it does not appear to play a major role in vivo. It is possible that the AP1-1 site is not utilized owing to chromatin-mediated inaccessibility. Previous studies identified an Sp1 site, located downstream and adjacent to the AP1-5 site, as being important for optimal involucrin promoter activity in cultured keratinocytes (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; Banks et al., 1998Banks E.B. Crish J.F. Welter J.F. Eckert R.L. Characterization of human involucrin promoter distal regulatory region transcriptional activator elements – a role for Sp1 and AP1 binding sites.Biochem J. 1998; 331: 61-68Crossref PubMed Scopus (68) Google Scholar; Crish et al., 2000Crish J.F. Bone F. Balasubramanian S. Zaim T.M. Wagner T. Yun J. et al.Suprabasal expression of the human papillomavirus type 16 oncoproteins in mouse epidermis alters expression of cell cycle regulatory proteins.Carcinogenesis. 2000; 21: 1031-1037Crossref PubMed Scopus (29) Google Scholar). In this context, it has been suggested that Sp1 proteins may facilitate AP1 factor loading at the AP1-5 site – this is based on intracellular transcriptional regulation studies (Banks et al., 1998Banks E.B. Crish J.F. Welter J.F. Eckert R.L. Characterization of human involucrin promoter distal regulatory region transcriptional activator elements – a role for Sp1 and AP1 binding sites.Biochem J. 1998; 331: 61-68Crossref PubMed Scopus (68) Google Scholar). Indeed, our present in vivo studies confirm a role for the DRR Sp1 site. Its mutation results in a lower level of transgene expression. Moreover, like AP1-5, this site appears to be a universal mediator, as mutation of the Sp1 site reduces hINV expression in all tissues examined, including footpad, cervix, epidermis, and esophagus. This may be due to an impact of Sp1 interaction on AP1 factor binding to the adjacent AP1-5 site, a possibility that has been suggested (Banks et al., 1998Banks E.B. Crish J.F. Welter J.F. Eckert R.L. Characterization of human involucrin promoter distal regulatory region transcriptional activator elements – a role for Sp1 and AP1 binding sites.Biochem J. 1998; 331: 61-68Crossref PubMed Scopus (68) Google Scholar). However, it is interesting that although the Sp1 site mutation lowers overall expression, the expression remains differentiation-dependent. It is interesting that this Sp1 site is absolutely essential for involucrin expression in the corneal epithelium (Adhikary et al., 2004Adhikary G. Crish J.F. Gopalakrishna R. Bone F. Eckert R.L. Involucrin expression in the corneal epithelium – an essential role for Sp1 transcription factors.Invest Ophthalmol Vis Sci. 2004; 46: 3109-3120Crossref Scopus (20) Google Scholar, Adhikary et al., 2005Adhikary G. Crish J.F. Bone F. Gopalakrishnan R. Lass J. Eckert R.L. An involucrin promoter AP1 transcription factor binding site is required for expression of involucrin in the corneal epithelium in vivo.Invest Ophthalmol Vis Sci. 2005; 46: 1219-1227Crossref PubMed Scopus (17) Google Scholar). To evaluate the role of the DRR in greater detail, we tested the ability of DRR subsegments to drive gene expression. Truncation of the DRR to generate smaller and smaller segments defines multiple, spatially distinct elements as required for differentiation-appropriate gene expression. Removal of nucleotides to position −2,043 results in no change in expression as compared to the full-length DRR segment. However, removal of an additional 54 nucleotides to position −2,097 (Figure 3) results in a reduction in overall expression level and reduced expression in the immediate suprabasal layers (Figures 3 and 4). Expression is also reduced when the hINV AP1-5/Sp1 site-containing segment is tested in isolation. Moreover, expression is absent in the immediate suprabasal tissue but maintained in the high suprabasal layers. In these experiments, we chose to remove the intervening sequence when creating the DRR constructs. Although it is possible that this may change the regulatory properties of this element, we decided that this approach was preferable to inserting a large segment of spacer DNA, which may also alter regulation in an unknown manner. Promoter analysis studies in cultured keratinocytes indicate that the hINV promoter C/EBP and AP1-1 sites, located immediately upstream of the transcription start site, are required for promoter activity (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; Efimova et al., 1998Efimova T. LaCelle P. Welter J.F. Eckert R.L. Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.J Biol Chem. 1998; 273: 24387-24395Crossref PubMed Scopus (137) Google Scholar; Balasubramanian et al., 2000Balasubramanian S. Agarwal C. Efimova T. Dubyak G.R. Banks E. Welter J. et al.Thapsigargin suppresses phorbol ester-dependent human involucrin promoter activity by suppressing CCAAT-enhancer-binding protein alpha (C/EBPalpha) DNA binding.Biochem J. 2000; 350: 791-796Crossref PubMed Google Scholar, Balasubramanian et al., 2002Balasubramanian S. Efimova T. Eckert R.L. Green tea polyphenol stimulates a Ras, MEKK1, MEK3, and p38 cascade to increase activator protein 1 factor-dependent involucrin gene expression in normal human keratinocytes.J Biol Chem. 2002; 277: 1828-1836Crossref PubMed Scopus (128) Google Scholar; Efimova and Eckert, 2000Efimova T. Eckert R.L. Regulation of human involucrin promoter activity by novel protein kinase C isoforms.J Biol Chem. 2000; 275: 1601-1607Crossref PubMed Scopus (75) ; Balasubramanian and Eckert, 2004Balasubramanian S. Eckert R.L. Green tea polyphenol and curcumin inversely regulate human involucrin promoter activity via opposing effects on CCAAT/enhancer-binding protein function.J Biol Chem. 2004; 279: 24007-24014Crossref PubMed Scopus (66) Google Scholar). In the present study, we examined the effect of mutating these sites separately and together. Mutation of the AP1-1 site produces no apparent change in expression. The most remarkable change is observed following mutation of the C/EBP site. These animals display an unusual pattern of intense expression alternating with reduced or absent expression. This pattern is observed in all tested surface epithelia, but is particularly evident in the footpad. In vitro DNA binding assays, using extracts prepared from cultured keratinocytes, indicate that the C/EBP site binds C/EBPα (Agarwal et al., 1999Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. CCAAT/enhancer-binding proteins. A role in regulation of human involucrin promoter response to phorbol ester.J Biol Chem. 1999; 274: 6190-6194Crossref PubMed Scopus (41) Google Scholar) and that transcription is enhanced by co-transfection of the hINV promoter with C/EBPα (Agarwal et al., 1999Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. CCAAT/enhancer-binding proteins. A role in regulation of human involucrin promoter response to phorbol ester.J Biol Chem. 1999; 274: 6190-6194Crossref PubMed Scopus (41) Google Scholar; Balasubramanian et al., 2000Balasubramanian S. Agarwal C. Efimova T. Dubyak G.R. Banks E. Welter J. et al.Thapsigargin suppresses phorbol ester-dependent human involucrin promoter activity by suppressing CCAAT-enhancer-binding protein alpha (C/EBPalpha) DNA binding.Biochem J. 2000; 350: 791-796Crossref PubMed Google Scholar). Finally, the mitogen-activated protein kinase signaling cascade is known to increase C/EBPα level and binding to DNA (Efimova et al., 2002Efimova T. Deucher A. Kuroki T. Ohba M. Eckert R.L. Novel protein kinase C isoforms regulate human keratinocyte differentiation by activating a p38 delta mitogen-activated protein kinase cascade that targets CCAAT/enhancer-binding protein alpha.J Biol Chem. 2002; 277: 31753-31760Crossref PubMed Scopus (83) Google Scholar). These results suggest that interfering with C/EBP factor input has dramatic effects on hINV expression. Based on the available in vivo studies, we propose that the DRR consists of at least three key regulatory binding sites (Figure 6). The AP1-5 site is absolutely required for expression and appears to function as an on/off switch; the Sp1 site, located adjacent to the AP1-5 site, appears to function to maximize expression; and a third region, a putative ISE, appears to be required for expression in the immediate suprabasal layers (Figure 6). The PRR C/EBP site is also important, and is required for continuous expression in all tissues (Figure 6 and Table 1). Based on these findings, we propose that a transcriptional complex forms on the DRR that includes JunB, JunD, and Fra-1 at the AP1-5 site, Sp1 at the Sp1 site, and unknown differentiation-regulatory factors at the ISE (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; Banks et al., 1998Banks E.B. Crish J.F. Welter J.F. Eckert R.L. Characterization of human involucrin promoter distal regulatory region transcriptional activator elements – a role for Sp1 and AP1 binding sites.Biochem J. 1998; 331: 61-68Crossref PubMed Scopus (68) Google Scholar), and that a parallel complex forms over the PRR that includes C/EBP factors (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; Agarwal et al., 1999Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. CCAAT/enhancer-binding proteins. A role in regulation of human involucrin promoter response to phorbol ester.J Biol Chem. 1999; 274: 6190-6194Crossref PubMed Scopus (41) Google Scholar), and that ultimately, these factors may come into contact to fully activate differentiation-appropriate involucrin gene expression.Table 1Role of transcription factor binding sites in the hINV promoterTranscription factor binding siteLocationIn vivo functionAP1-5DRRMutation results in complete loss of expression in all tissuesSp1DRRRequired for optimal level of expression, mutation results in reduced expressionISEDRR (immediately 3′ of AP1-5/Sp1 sites)Required for expression in early differentiated layersC/EBPPRRMutation results in discontinuous expressionAP1-1PRREssential for promoter activity in cultured keratinocytes - in vivo mutation has minimal impact on expression Open table in a new tab The H6B transgene is a 6 kb HindIII/BamHI fragment containing the complete hINV gene that was derived by restricting Charon 4AλI-3 with HindIII/BamHI and subcloning the resulting 6 kb fragment into HindIII/BamHI-restricted pSP64 to yield pSP64λI-3 H6B (Eckert and Green, 1986Eckert R.L. Green H. Structure and evolution of the human involucrin gene.Cell. 1986; 46: 583-589Abstract Full Text PDF PubMed Scopus (307) Google Scholar). Each of the constructs was derived from this construct using previously described methods (Crish et al., 1993Crish J.F. Howard J.M. Zaim T.M. Murthy S. Eckert R.L. Tissue-specific and differentiation-appropriate expression of the human involucrin gene in transgenic mice: an abnormal epidermal phenotype.Differentiation. 1993; 53: 191-200Crossref PubMed Scopus (76) Google Scholar, Crish et al., 1998Crish J.F. Zaim T.M. Eckert R.L. The distal regulatory region of the human involucrin promoter is required for expression in epidermis.J Biol Chem. 1998; 273: 30460-30465Crossref PubMed Scopus (56) Google Scholar, Crish et al., 2002Crish J.F. Bone F. Banks E.B. Eckert R.L. The human involucrin gene contains spatially distinct regulatory elements that regulate expression during early versus late epidermal differentiation.Oncogene. 2002; 21: 738-747Crossref PubMed Scopus (39) Google Scholar). For microinjection, the transgenes were released from plasmid sequences by restriction with HindIII/BamHI. Mouse embryos, obtained from B6CBA × B6CBA mating, were injected with each individual transgene. The injected embryos were placed into surrogate mothers (Crish et al., 1993Crish J.F. Howard J.M. Zaim T.M. Murthy S. Eckert R.L. Tissue-specific and differentiation-appropriate expression of the human involucrin gene in transgenic mice: an abnormal epidermal phenotype.Differentiation. 1993; 53: 191-200Crossref PubMed Scopus (76) Google Scholar, Crish et al., 1998Crish J.F. Zaim T.M. Eckert R.L. The distal regulatory region of the human involucrin promoter is required for expression in epidermis.J Biol Chem. 1998; 273: 30460-30465Crossref PubMed Scopus (56) Google Scholar) and the resulting offspring were characterized for transgene presence by hINV tail DNA blotting. The hINV cDNA probe does not crossreact with the endogenous mouse involucrin gene; thus, the presence of hINV gene sequences can be directly detected (Crish et al., 1993Crish J.F. Howard J.M. Zaim T.M. Murthy S. Eckert R.L. Tissue-specific and differentiation-appropriate expression of the human involucrin gene in transgenic mice: an abnormal epidermal phenotype.Differentiation. 1993; 53: 191-200Crossref PubMed Scopus (76) Google Scholar). A minimum of three and a maximum of eight individual transgenic founders were characterized for each transgene tested, and all lines were bred to the F1 and subsequent generations prior to testing. All the experiments were approved by the Institutional Animal Care and Use Committee. It is important to note that all of the transgenes utilized in the present study retain the hINV coding sequence in a native configuration with respect to the hINV promoter segments. To assay for transgene expression, we monitored the presence of hINV protein by immunoblot and by immunohistology. Our antibodies readily distinguish the hINV protein produced from the transgene from the endogenous murine involucrin protein (LaCelle et al., 1998LaCelle P.T. Lambert A. Ekambaram M.C. Robinson N.A. Eckert R.L. In vitro cross-linking of recombinant human involucrin.Skin Pharmacol Appl Skin Physiol. 1998; 11: 214-226Crossref PubMed Scopus (28) Google Scholar). To detect hINV expression in mouse tissues, total protein extracts were prepared from epidermal samples following release of the epidermis with dispase treatment. The samples were then prepared in Laemmli sample buffer, and equivalent amounts of protein from each sample were electrophoresed on an acrylamide gel and transferred to nitrocellulose for antibody-dependent detection of hINV. The blot was incubated with a primary antibody prepared against recombinant hINV (LaCelle et al., 1998LaCelle P.T. Lambert A. Ekambaram M.C. Robinson N.A. Eckert R.L. In vitro cross-linking of recombinant human involucrin.Skin Pharmacol Appl Skin Physiol. 1998; 11: 214-226Crossref PubMed Scopus (28) Google Scholar) diluted 1:600 and visualized using a chemiluminescent detection system (Crish et al., 1993Crish J.F. Howard J.M. Zaim T.M. Murthy S. Eckert R.L. Tissue-specific and differentiation-appropriate expression of the human involucrin gene in transgenic mice: an abnormal epidermal phenotype.Differentiation. 1993; 53: 191-200Crossref PubMed Scopus (76) Google Scholar). The blot was then stripped and incubated for detection of β-actin as a loading normalization control. For immunohistology, sections were prepared as previously described (Crish et al., 1993Crish J.F. Howard J.M. Zaim T.M. Murthy S. Eckert R.L. Tissue-specific and differentiation-appropriate expression of the human involucrin gene in transgenic mice: an abnormal epidermal phenotype.Differentiation. 1993; 53: 191-200Crossref PubMed Scopus (76) Google Scholar, Crish et al., 1998Crish J.F. Zaim T.M. Eckert R.L. The distal regulatory region of the human involucrin promoter is required for expression in epidermis.J Biol Chem. 1998; 273: 30460-30465Crossref PubMed Scopus (56) Google Scholar) and then incubated with rabbit anti-hINV followed by a peroxidase-conjugated goat anti-rabbit IgG and visualization by peroxidase substrate deposition. This work utilized the facilities of the Skin Diseases Research Center of Northeast Ohio (NIH, AR39750) and the Case Western Reserve University Comprehensive Cancer Center (P30 CA43703), and was supported by a grant from the National Institutes of Health (R.L.E.). Drs Crish and Gopalakrishnan were primarily responsible for completion of this project and both made essential contributions." @default.
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• W2018274375 title "The Distal and Proximal Regulatory Regions of the Involucrin Gene Promoter have Distinct Functions and Are Required for In Vivo Involucrin Expression" @default.
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