Keratinocytes Express the CD146 (Muc18/S-Endo) Antigen in Tissue Culture and During Inflammatory Skin Diseases1 Wolfgang Weninger, Michael Rendl, Michael.

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Keratinocytes Express the CD146 (Muc18/S-Endo) Antigen in Tissue Culture and During Inflammatory Skin Diseases1 Wolfgang Weninger, Michael Rendl, Michael Mildner, Christoph Mayer, Jozef Ban, Alexandra Geusau, Gottfried Bayer, Adrian Tanew, Otto Majdic, Erwin Tschachler Journal of Investigative Dermatology Volume 115, Issue 2, Pages 219-224 (August 2000) DOI: 10.1046/j.1523-1747.2000.00039.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Cultured KC express the CD146 antigen at cell to cell contact sites. (a) Confocal laser microscopy of CD146 expression by KC cultured in KBM for 24 h. The staining reveals expression on the cell surface mainly at the cell-cell contact sites. (b) Isotype-matched control. Scale bar: 25 μm. Journal of Investigative Dermatology 2000 115, 219-224DOI: (10.1046/j.1523-1747.2000.00039.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Upregulation of CD146 on KC expression by growth factor withdrawal. KC and SkMel28 melanoma cells were analyzed for CD146 expression by flow cytometry. (a) KC cultured for 48 h in KGM (thin line) showed weak CD146 expression (mean channel of fluorescence (MCF): 192; isotype: dotted line, MCF: 150) which was increased when KC were cultured in KBM (bold line) without growth factors (MCF: 239; isotype: dashed line, MCF: 145). (b) A similar regulation was seen when SkMel28 cells were cultured for 48 h in medium without growth supplements (bold line, MCF: 531; isotype: dashed line, MCF: 133) compared to fully supplemented medium (thin line, MCF: 455; isotype: dotted line, MCF: 114). (c) Culture of KC 48 h in KBM plus PMA (thin line, MCF: 185; isotype: dotted line, MCF: 151) reduced CD146 expression compared to KBM alone (bold line, same as in part a). Journal of Investigative Dermatology 2000 115, 219-224DOI: (10.1046/j.1523-1747.2000.00039.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Western blot analysis of CD146 expression in cultured KC and KC-derived cell lines. (a) When western blot analysis was performed with an anti-CD146 MoAb, a distinct band of ≈113 kDa band was detected in lysates of SkMel28 melanoma cells (lane 1) and in lysates of three different preparations of human foreskin KC cultured in KBM (lanes 4–6). No reactivity was found in A431 (lane 2) and HaCaT cell-lysates (lane 3). (b) CD146 expression was increased in KC cultured without growth factors (KBM, lane 3) as compared with culture in KGM (lane 2). Addition of PMA decreased CD146 levels of KC cultured both in KGM (lane 4) and KBM (lane 5). Equal loading was confirmed by Ponceau-S staining of the blot prior immunostaining (lower panel). Journal of Investigative Dermatology 2000 115, 219-224DOI: (10.1046/j.1523-1747.2000.00039.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Detection of CD146 mRNA transcripts in cultured KC by reverse transcriptase–PCR. Upper panel: total RNA was extracted from cultured cells and cDNA was synthesized. PCR amplification products were subjected to agarose gel electrophoresis. Cells analyzed were: A431 cells (lane 1), HaCaT cells (lane 2), two different preparations of foreskin KC (lanes 3 and 4), SkMel28 (lane 5), and RHTP49 melanoma cells (lane 6). Lane 7, H2O control. Lower panel: Southern hybridization of the agarose gel shown in the upper panel using a CD146-specific [α-32P]-labeled oligonucleotide probe. Journal of Investigative Dermatology 2000 115, 219-224DOI: (10.1046/j.1523-1747.2000.00039.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Expression of CD146 in normal human skin and in psoriasis. Immunohistochemical staining was performed on cryostat sections using an anti-CD146 monoclonal antibody. (a) Normal adult skin. No staining of interfollicular epidermal KC is observed in normal adult skin. Vascular smooth muscle cells (arrows) served as an internal positive control. (b) Distinct CD146 expression is detectable on cells in the outer root sheath of the hair follicle in the portion below the area of the sebaceous gland (arrows). (c, d) In psoriasis suprabasal KC show distinct membrane staining for CD146. Scale bar: (a, c) 50 μm; (b, d) 25 μm. Journal of Investigative Dermatology 2000 115, 219-224DOI: (10.1046/j.1523-1747.2000.00039.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of CD146 in Bowen's disease, basal cell carcinomas, and squamous cell carcinomas. Immunohistochemical staining for CD146 was performed on cyryostat section of (a) Bowen's disease (arrows mark the border between normal epidermis and the CD146 negative lesion) and (b) basal cell carcinoma. Singly positive cells are visible within the tumor nodules (arrowheads). (c) A squamous cell carcinoma. For (d) serial section to c was stained with a pan-cytokeratin antibody. Scale bar: 25 μm. Journal of Investigative Dermatology 2000 115, 219-224DOI: (10.1046/j.1523-1747.2000.00039.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions