Overexpression of Serpin Squamous Cell Carcinoma Antigens in Psoriatic Skin Atsushi Takeda, Dousei Higuchi, Tadahito Takahashi, Masashi Ogo, Peter Baciu,

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Overexpression of Serpin Squamous Cell Carcinoma Antigens in Psoriatic Skin Atsushi Takeda, Dousei Higuchi, Tadahito Takahashi, Masashi Ogo, Peter Baciu, Paul F. Goetinck, Toshihiko Hibino Journal of Investigative Dermatology Volume 118, Issue 1, Pages 147-154 (January 2002) DOI: 10.1046/j.0022-202x.2001.01610.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 SCCA gene locus and cDNA structure. (A) Structure of the SCCA gene. The exons are indicated by solid boxes with the respective numbers above them; introns are indicated by thin lines. The translation start codon (atg) and translation stop codon (tag) are shown. The scale is shown at the top of the figure. (B) Structure of SCCA1 and SCCA2 cDNA. The shadowed boxes show the open reading frame regions. The open boxes at each 3′ and 5′ region show noncoding regions. The scale marker indicates 0.2 kb. Arrowheads below the cDNA show the position and direction of primers used in PCR. Journal of Investigative Dermatology 2002 118, 147-154DOI: (10.1046/j.0022-202x.2001.01610.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunohistochemical staining of SCCA in psoriatic skin tissues. (A) Normal skin. Serum SCCA concentration is < 5 ng per ml. SCCA staining was negative. (B)-(I) Psoriatic skin. The expression of SCCA in psoriasis was classified into two patterns: (i) diffuse staining in the cytoplasm and nuclei of epidermal cells (early psoriatic lesion, B) and (ii) condensed staining in the nuclei and plasma membrane/intercellular space (active psoriatic lesion, D-I). (B) Early psoriatic lesion (serum SCCA concentration 9.4 ng per ml). Upper spinous and granular cells are weakly positive. Arrowheads indicate SCCA-positive cells in the dermis. (C) High magnification of (B). Many nuclei are also positive for SCCA. (D)–(F) Active psoriatic skin (serum SCCA concentration 21.8 ng per ml). (D) Boundary lesion. Note that SCCA protein is found only in the psoriatic lesion. (E) High magnification of the center part, characterized by elongated rete ridges. (F) Micropustules of Kogoj. Strong SCCA staining is obvious in this area. Note that parakeratotic cells were SCCA negative. (G)–(I) Very active psoriatic skin (serum SCCA concentration 38.2 ng per ml). Most of the cells except basal cells are positive for SCCA. (H) High magnification of (G), elongated rete ridges. Most of the suprabasal cells are stained. (I) Higher magnification of (G), upper layer. SCCA-positive nuclei and condensed membrane staining are frequently seen. Scale bars: (A) 100 µm; (D) 200 µm; (B, E, G) 50 µm; (F, H) 20 µm; (I) 10 µm. Journal of Investigative Dermatology 2002 118, 147-154DOI: (10.1046/j.0022-202x.2001.01610.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Electron micrographs of psoriatic skin sections treated with the antibody against SCCA. (A) Cornified cell layer. (B) Upper part of the spinous cell layer. (C) Higher magnification of (B). Gold particles were mainly localized along the periphery of the cell-cell contact area. (D) Higher magnification of (B). SCCA-positive particles are clearly shown in the nucleus designated N. (E) Filtrated-cell-rich region near the dermo-epidermal junction marked with dashes (ep, epidermis; de, dermis). Many immunoreactive particles were observed in the cytoplasmic granules of the filtrated cells (arrowheads) and in the surrounding degranulated cells (arrows). (F) Higher magnification of (E). Many immunoreactive gold particles were observed in the cytoplasmic granules of filtrated cells. Numbers on the scale bars indicate μm. Journal of Investigative Dermatology 2002 118, 147-154DOI: (10.1046/j.0022-202x.2001.01610.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Electron micrographs of psoriatic skin sections treated with the antibody against SCCA. (A) Cornified cell layer. (B) Upper part of the spinous cell layer. (C) Higher magnification of (B). Gold particles were mainly localized along the periphery of the cell-cell contact area. (D) Higher magnification of (B). SCCA-positive particles are clearly shown in the nucleus designated N. (E) Filtrated-cell-rich region near the dermo-epidermal junction marked with dashes (ep, epidermis; de, dermis). Many immunoreactive particles were observed in the cytoplasmic granules of the filtrated cells (arrowheads) and in the surrounding degranulated cells (arrows). (F) Higher magnification of (E). Many immunoreactive gold particles were observed in the cytoplasmic granules of filtrated cells. Numbers on the scale bars indicate μm. Journal of Investigative Dermatology 2002 118, 147-154DOI: (10.1046/j.0022-202x.2001.01610.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 In situ hybridization of SCCA mRNAs in psoriatic epidermis. (A) In situ hybridization using the antisense SCCA1 cRNA probe. Positive signals were localized in the suprabasal spinous layer, but not in the supracorneal and basal layers. (B) In situ hybridization with a sense SCCA1 cRNA probe. No positive signal was observed. Scale bars: 100 μm. Journal of Investigative Dermatology 2002 118, 147-154DOI: (10.1046/j.0022-202x.2001.01610.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Distinctive amplification for SCCA1 and SCCA2. Specificity of cDNA amplification was tested for SCCA1 (lanes 1 and 2) and SCCA2 (lanes 3 and 4) by PCR. PCR was performed using P1/P10 (lanes 1 and 3) and P5/P11 (lanes 2 and 4) primer pairs, respectively. Each template concentration (pGEMT-SCCA1 or pGEMT-SCCA2) was 2 ng. The right lane shows molecular weight markers (ϕX174RT DNA/HaeIII fragments). Journal of Investigative Dermatology 2002 118, 147-154DOI: (10.1046/j.0022-202x.2001.01610.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 RT-PCR analysis of SCCA mRNA expression. Expression of SCCA1 and SCCA2 mRNA in psoriatic skin as well as normal tissues was analyzed by RT-PCR. Lanes 1, 2, normal skin tissues; lanes 3–6, psoriatic skin tissues from four patients. Journal of Investigative Dermatology 2002 118, 147-154DOI: (10.1046/j.0022-202x.2001.01610.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Semiquantitation of SCCA1 and SCCA2 mRNA expression in psoriatic skin tissues. The intensity of the DNA band was analyzed by densitometry. Bar heights and error bars depict means ± SEM. Individual values are shown as solid dots. (a) Comparison of SCCA1 mRNA expression between normal tissues (N, n = 8) and psoriatic tissues (Pso, n = 9) (asterisk indicates p < 0.01). (b) SCCA2 mRNA expression. Data are expressed as nanograms of DNA per 10 μl of reaction mixture. Journal of Investigative Dermatology 2002 118, 147-154DOI: (10.1046/j.0022-202x.2001.01610.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions