Induction of Transcription Factor AP-2 by Inflammatory Cytokines in Human Keratinocytes  Noritaka Oyama, Keiji Iwatsuki, Yoshimi Homma, Fumio Kaneko  Journal of Investigative Dermatology  Volume 113, Issue 4, Pages 600-606 (October 1999) DOI: 10.1046/j.1523-1747.1999.00734.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Time-dependent induction of AP-2 by IL-6 in normal human keratinocytes. (A) Subconfluent cultures of normal human keratinocytes were treated with 10 ng IL-6 per ml for indicated periods. Crude nuclear extracts were prepared as described in Materials and Methods, and aliquot (≈25 μg nuclear protein per lane) was subjected to SDS–PAGE (10% acrylamide). Samples were immunoblotted with anti-AP-2 rabbit polyclonal antibody and peroxidase-conjugated secondary antibody. Specific bands were visualized with a chemiluminescence detection system. A representative of three independent experiments is shown. Molecular weight markers are indicated on the left. The arrow represents the position of AP-2 protein. (B) Subconfluent cultures of normal human keratinocytes were treated with 10 ng IL-6 per ml for the indicated periods. RNA isolated from the cells was reverse-transcribed, and then followed by semiquantitative reverse transcriptase–PCR as described in Materials and Methods. An aliquot (5 μl of reaction mixture per lane) was subjected to gel electrophoresis (2% agarose) and PCR products were detected by staining with ethidium bromide. A representative of three independent experiments is shown. Molecular weight markers are indicated on the left. Arrows represent the positions of AP-2 and β-actin products. The treatment of vehicle control (phosphate-buffered saline) was without any effects in the expression of AP-2 protein and mRNA (data not shown). Journal of Investigative Dermatology 1999 113, 600-606DOI: (10.1046/j.1523-1747.1999.00734.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Kinetic profiles of AP-2 expression by IL-6 in normal human keratinocytes. (A) Subconfluent cultures of normal human keratinocytes were treated with 10 ng IL-6 per ml for indicated periods in the presence or absence of either 0.2 μg actinomycin D per ml or 30 μg cycloheximide per ml. RNA isolated from the cells was reverse-transcribed, and then followed by semiquantitative reverse transcriptase–PCR as described in Materials and Methods. An aliquot (5 μl of reaction mixture per lane) was subjected to gel electrophoresis (2% agarose) and detected by staining with ethidium bromide. Molecular weight markers are indicated on the left. Arrows represent the positions of AP-2 and β-actin products. (B) Specific signals for AP-2 and β-actin mRNA were analyzed by densitometry and assigned for arbitrary units. The expression of AP-2 mRNA was normalized with β-actin. Each value represents the relative intensity of AP-2 mRNA (mean ± SEM of three independent experiments). Cell viabilities after incubation with actinomycin D and cycloheximide were more than 95%, as determined by dye-exclusion test using Trypan blue. Journal of Investigative Dermatology 1999 113, 600-606DOI: (10.1046/j.1523-1747.1999.00734.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effects of neutralizing antibodies on IL-6-dependent AP-2 induction in normal human keratinocytes. Subconfluent cultures of normal human keratinocytes were treated with 10 ng IL-6 per ml for 4 h in the presence or absence of the indicated neutralizing antibodies or a mixture of all neutralizing antibodies except anti-IL-6 antibody (denoted as “mix”), and crude nuclear extracts were isolated as described in Materials and Methods. An aliquot (≈25 μg nuclear protein per lane) was subjected to SDS–PAGE (10% acrylamide), then samples were immunoblotted with anti-AP-2 rabbit polyclonal antibody and peroxidase-conjugated secondary antibody. Specific bands were visualized with a chemiluminescence detection system. The arrow represents the position of AP-2 protein. Molecular weight markers are indicated on the left. Journal of Investigative Dermatology 1999 113, 600-606DOI: (10.1046/j.1523-1747.1999.00734.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effects of cytokines on AP-2 expression in normal human keratinocytes. (A) Subconfluent cultures of normal human keratinocytes were treated with 10 ng per ml of indicated cytokines for 4 h. Crude nuclear extracts were prepared as described in Materials and Methods, and an aliquot (≈25 μg nuclear protein/lane) was subjected to SDS–PAGE (10% acrylamide). Samples were immunoblotted with anti-AP-2 rabbit polyclonal antibody and peroxidase-conjugated secondary antibody. Specific bands were visualized with a chemiluminescence detection system. The arrow represents the position of AP-2 protein. (B) (Lower) RNA isolated from the cells was reverse-transcribed, then followed by semiquantitative reverse transcriptase–PCR, as described in Materials and Methods. An aliquot (5 μl of reaction mixture per lane) was subjected to gel electrophoresis (2% agarose) and detected by staining with ethidium bromide. The arrow represents the position of AP-2 product. Molecular weight markers are indicated on the left. (Upper) Specific signals for AP-2 and β-actin mRNA were analyzed by densitometry and assigned for arbitrary units. The expression of AP-2 mRNA was normalized with β-actin. Each value represents the relative intensity of AP-2 mRNA (mean ± SEM of three independent experiments). Journal of Investigative Dermatology 1999 113, 600-606DOI: (10.1046/j.1523-1747.1999.00734.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 AP-2 expression induced by IL-6 in normal human keratinocytes, PLK, and HSC-5 cells. Three types of keratinocytes cultured in serum- or growth factor-free medium for 24 h, were independently treated with the indicated amounts (0–10 ng per ml) of IL-6 for 4 h. Crude nuclear extracts from these cells were prepared as described in Materials and Methods, and an aliquot (≈25 μg nuclear protein per lane) was subjected to SDS–PAGE (10% acrylamide). Samples were then immunoblotted with anti-AP-2 rabbit polyclonal antibody and peroxidase-conjugated secondary antibody. Specific bands were visualized with a chemiluminescence detection system. Representatives of three different donors in each group are shown. The arrow represents the position of AP-2 protein. Molecular weight markers are indicated on the left. Journal of Investigative Dermatology 1999 113, 600-606DOI: (10.1046/j.1523-1747.1999.00734.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Immunohistochemical detection of AP-2. Each skin section was incubated with anti-AP-2 rabbit polyclonal antibody, and then with biotin-labeled peroxidase. Positive signal was developed with diaminobenzidine substrate and counter-stained by methyl green. As a negative control, the sections treated with normal rabbit serum did not present any signals (data not shown). Representative results are shown. (A, B) Normal skin with low and high magnifications, respectively; (C, D) psoriatic lesional skin with low and high magnifications, respectively; (E, F) SCC lesional skin. In (F) the border areas between proliferated (left side), and well-differentiated lesions (right side) are shown. The latter contains abrupt keratinization. Scale bars: 120 μm (A, C, F); 150 μm (B, D, E). Journal of Investigative Dermatology 1999 113, 600-606DOI: (10.1046/j.1523-1747.1999.00734.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Gel mobility shift assay for AP-2 protein. Nuclear extracts from normal human keratinocytes or HSC-5 were incubated with radio-labeled oligonucleotide probe. The resulting samples were separated on nondenaturing 4% polyacrylamide gel, and then the specific bands were detected by autoradiography. Arrows represent the positions of the free probe and AP-2-DNA complex. Each lane contains the sample as follows: lane 1, radio-labeled AP-2 probe only; lane 2, nuclear extracts from IL-6-treated normal human keratinocytes were incubated with radio-labeled AP-2 probe; lane 3, nuclear extracts from nonstimulated normal human keratinocytes were incubated with radio-labeled AP-2 probe; lane 4, nuclear extracts from IL-6-treated normal human keratinocytes were incubated with radio-labeled AP-2 probe and an excess amount (≈100-fold molar) of cold AP-2 oligonucleotide; lane 5, nuclear extracts from IL-6-treated normal human keratinocytes were incubated with radio-labeled AP-2 probe and an excess amount (≈100-fold molar) of cold AP-1 oligonucleotide; lane 6, nuclear extracts from nonstimulated HSC-5 cells were incubated with radio-labeled AP-2 probe. Journal of Investigative Dermatology 1999 113, 600-606DOI: (10.1046/j.1523-1747.1999.00734.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions