Effect of AP1 Transcription Factors on the Regulation of Transcription in Normal Human Epidermal Keratinocytes Antonello Rossi, Shyh-Ing Jang, Roberta.

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Effect of AP1 Transcription Factors on the Regulation of Transcription in Normal Human Epidermal Keratinocytes Antonello Rossi, Shyh-Ing Jang, Roberta Ceci, Peter M. Steinert, Nedialka G. Markova Journal of Investigative Dermatology Volume 110, Issue 1, Pages 34-40 (January 1998) DOI: 10.1046/j.1523-1747.1998.00071.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Transient expression of the CAT-reporter gene under the control of K5 and TGM1 5'-upstream sequences. Fragments spanning the designated lengths of the K5 and TGM1 5'-flanking regions were subcloned into the promoterless pCAT-Basic reporter vector carrying the chloramphenicol acetyl transferase gene CAT. The positions and the sequences of the wild-type and the mutant AP1 motifs are shown. NHEK cells were transfected and the activity of CAT was determined. The values for CAT were normalized by protein content and β-gal activity of the cotransfected vector pTKβ. They are the average of at least three independent experiments, each with duplicate samples. Journal of Investigative Dermatology 1998 110, 34-40DOI: (10.1046/j.1523-1747.1998.00071.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Bandshift analysis. The sequences of the wild-type (P) and the mutant variants of the AP1 motifs used in the binding reactions are shown above the bandshift profiles for K5 (a), TGM1 (b), involucrin (c), and loricrin (d). Five micrograms of NHEK nuclear extract was combined with 2–4 × 104 cpm of the 5'-end-labeled double-stranded oligonucleotide under the conditions described inMaterials and Methods. The DNA-protein complexes were resolved on 6% polyacrylamide gels in 0.5 × TBE buffer. Lanes 1, binding profile of the wild-type oligonucleotides P with NHEK extract;lanes 2, competition of the binding with a 100-fold molar excess of unlabeled P; lanes 3, mutant AP1 oligonucleotides; lanes 4, consensus AP1 oligonucleotide bearing the sequence TGAGTCA. Journal of Investigative Dermatology 1998 110, 34-40DOI: (10.1046/j.1523-1747.1998.00071.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Supershift analysis. Constructs were of involucrin AP1–5 (a) and loricrin AP1 (b) DNA/protein complexes with anti-human IgG (lanes 1) and with antibodies specific for c-jun (lanes 2), junB (lanes 3), junD (lanes 4), c-fos (lanes 5), fra1 (lanes 6), and fra2 (lanes 7). The arrowheads on the right point to positions of the supershifted complexes. Journal of Investigative Dermatology 1998 110, 34-40DOI: (10.1046/j.1523-1747.1998.00071.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effect of AP1 factors on the activity of the K5-pCAT constructs. (a) K5-pCAT wild-type and mutant constructs; (b) 1.0mg of the corresponding K5-pCAT constructs were cotransfected into NHEK cells together with 0.5mg of plasmid DNA expressing c-fos, c-jun, TAM, junB, or junD cDNA or with 0.5mg of expression vector DNA only. The relative values of the transient CAT activity are presented as a fold change over the activity of K5-pCAT construct –750/+32 (shown as Control), cotransfected with the RSV expression vector for c-fos and c-jun, with the CMV expression vector for TAM and jun B and with the SV40 expression vector for jun D, respectively. (c) Effect of AP1 proteins in F9 cells under the conditions described in (b). Journal of Investigative Dermatology 1998 110, 34-40DOI: (10.1046/j.1523-1747.1998.00071.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effect of AP1 factors on the activity of the TGM1-pCAT constructs. (a) Constructs used. Transfected into NHEK cells (b) or F9 cells (c). The relative values of the transient CAT activity are presented as a fold change over the activity of TGM1-pCAT construct –483/+9 (shown as Control), cotransfected with the RSV expression vector for c-fos and c-jun, with the CMV expression vector for TAM and junB, and with the SV40 expression vector for junD, respectively. Journal of Investigative Dermatology 1998 110, 34-40DOI: (10.1046/j.1523-1747.1998.00071.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of AP1 factors on the activity of the involucrin-pCAT constructs. (a) Constructs used. Transfected into NHEK cells (b) or F9 cells (c). The relative values of the transient CAT activity are presented as a fold change over the activity of involucrin-pCAT construct –2473/+9 (shown as Control), cotransfected with the RSV expression vector for c-fos and c-jun, with the CMV expression vector for TAM and junB, and with the SV40 expression vector for junD, respectively. Journal of Investigative Dermatology 1998 110, 34-40DOI: (10.1046/j.1523-1747.1998.00071.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Effect of AP1 factors on the activity of the loricrin-pCAT constructs. (a) Constructs used. Transfected into NHEK cells (b) or F9 cells (c). The relative values of the transient CAT activity are presented as a fold change over the activity of loricrin-pCAT construct –1329/+9 (shown as Control), cotransfected with the RSV expression vector for c-fos and c-jun, with the CMV expression vector for TAM and junB, and with the SV40 expression vector for junD, respectively. Journal of Investigative Dermatology 1998 110, 34-40DOI: (10.1046/j.1523-1747.1998.00071.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions