Transcriptional Regulation of the Elafin Gene in Human Keratinocytes Arno Pol, Rolph Pfundt, Patrick Zeeuwen, Henri Molhuizen, Joost Schalkwijk  Journal of Investigative Dermatology  Volume 120, Issue 2, Pages 301-307 (February 2003) DOI: 10.1046/j.1523-1747.2003.12043.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Nucleotide sequence of the human elafin gene promoter region. Numbering is relative to the first base of the ATG translation codon (printed in bold). The borders of the fragments used for the luciferase transfection constructs are indicated with arrows (⇓). The putative “CCAAT” and “TATA” boxes are double underlined. The region of multiple transcription start sites is underlined with a dotted line, the preferential transcription start sites are printed in italics and bold. Consensus binding sequences for known transcription factors are indicated by name and underlined. Journal of Investigative Dermatology 2003 120, 301-307DOI: (10.1046/j.1523-1747.2003.12043.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Deletion mapping of the elafin promoter.(A) Schematic representation of the luciferase constructs containing deletion fragments of the elafin promoter. Nucleotide positions are given relative to the translation start codon and indicate the orientation of the deletion fragment. (B) Relative promoter activity of the various elafin promoter reporter constructs, defined as the relative luciferase activity. The bars represent the relative luciferase activity found in cell lysates of transient transfected primary human keratinocytes cultured in either KGM/-GF or KGM/FCS. Data are representative of three independent transfection experiments, and reported as mean ± SEM. Journal of Investigative Dermatology 2003 120, 301-307DOI: (10.1046/j.1523-1747.2003.12043.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Determination of the region where transcription of the elafin gene initiates. Relative distribution of transcription start sites in the elafin gene promoter determined by 5′-RACE. Nucleotide positions (relative to the translation start site) are indicated on the X-axis. The graph summarizes the results of 15 analyzed RACE clones. Journal of Investigative Dermatology 2003 120, 301-307DOI: (10.1046/j.1523-1747.2003.12043.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Elafin promoter activity in different cell types. Primary human keratinocytes, U138-MG glioblastoma cells, primary human skin fibroblasts, and epithelial A431 cells were cotransfected with either empty reporter construct pSLA4 and EF2-βGAL or with transfection clone pSPL1000 and EF2-βGAL. Twenty-four hours after transfection cell cultures received fresh medium containing either 10%FCS (glioblastoma, fibroblast, epithelial A431) or 5%FCS (keratinocyte). After a final incubation of 24 h, cells were harvested and assayed for luciferase enzyme activity. Values represent the promoter activity of pSPL1000 compared to that of pSLA4 (assigned a value of 1). Journal of Investigative Dermatology 2003 120, 301-307DOI: (10.1046/j.1523-1747.2003.12043.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effect of TNFα and RA on the mRNA expression of elafin and elafin-EGFP in cell cultures of stably transfected HaCaT cell lines. Both unstipulated and TNFα-stimulated (25 ng/ml) cultures of the 0.8 kb-and the 4 kb-elafinprom clone were treated with indicated amounts of RA for 72 h. Northern blot analysis was performed on total RNA isolated from these cultures. Samples were hybridized using a probe directed against elafin. Journal of Investigative Dermatology 2003 120, 301-307DOI: (10.1046/j.1523-1747.2003.12043.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 RA counteracts the TNFα-dependent induction of endogenous elafin but not the TNFα-dependent induction of the elafin-EGFP fusion protein.(A) Both unstimulated and TNFα-stimulated cultures of the 0.8 kb-elafinprom clone, were treated with indicated amounts of RA for 72 h. Levels of endogenous elafin were determined from cell culture medium using a sandwich-type ELISA specific for elafin. Values represent the mean ± SD (n=4). *p<0.005 versus control values of unstimulated samples, and *p<0.005 versus control values of TNFα stimulated samples, by one-way analysis of variance with Duncan's multiple comparison test. (B) Both unstimulated and TNFα-stimulated cultures of the 0.8 kb-elafinprom clone and untransfected HaCaT were treated with indicated amounts of RA for 72 h. A sandwich-type ELISA specific for elafin-EGFP was used to determine levels of fusion protein from cell culture medium. Assay background values were obtained from culture supernatant of either unstimulated or TNFα-stimulated untransfected HaCaT. Levels of elafin-EGFP from samples of the 0.8 kb-elafinprom clone were expressed relative to these background values. Values represent the mean ± SD (n=4). An effect of RA on levels of elafin-EGFP could not be demonstrated (one-way analysis of variance). Journal of Investigative Dermatology 2003 120, 301-307DOI: (10.1046/j.1523-1747.2003.12043.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions