Activation of Proteinase-Activated Receptor-2 by Human Kallikrein-Related Peptidases Kristina Stefansson, Maria Brattsand, Dirk Roosterman, Cordula Kempkes,

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Activation of Proteinase-Activated Receptor-2 by Human Kallikrein-Related Peptidases Kristina Stefansson, Maria Brattsand, Dirk Roosterman, Cordula Kempkes, Georgeta Bocheva, Martin Steinhoff, Torbjörn Egelrud Journal of Investigative Dermatology Volume 128, Issue 1, Pages 18-25 (January 2008) DOI: 10.1038/sj.jid.5700965 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Preparations of recombinant human tissue KLK protein precursors and active enzymes. Coomassie brilliant blue stained SDS-PAGE. Journal of Investigative Dermatology 2008 128, 18-25DOI: (10.1038/sj.jid.5700965) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Ca2+ responses to trypsin and KLK in KNRK-PAR2 cells. KNRK-PAR2 cells were treated with (a) 1μM trypsin (T), (b) 10μM KLK5EK, (c) 10μM KLK7EK, (d) 10μM KLK8, and (e) 1μM KLK14. Trypsin or KLK was added at indicated time points (arrows). If no response could be detected, the integrity of the cells was verified by addition of 100nM trypsin (T). Journal of Investigative Dermatology 2008 128, 18-25DOI: (10.1038/sj.jid.5700965) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Dose response curves for Ca2+ mobilization in response to trypsin and KLK. (a) KNRK-PAR2 cells were treated with active trypsin (circles), KLK5EK (triangles), KLK7EK (crosses), KLK8 (diamonds) or KLK14 (squares). Means±SEM, n≥3 for trypsin, KLK5EK, and KLK14. For KLK7EK and KLK8, experiments were carried out in duplicate at 1μM, at lower concentrations single experiments were performed. *1: statistically significantly different from baseline (n=3, P=0.04); *2: statistically significantly different from baseline (n=4, P=0.003). (b) Human dermal microvascular endothelial cells (hDMEC) cells were treated with active trypsin (circles), KLK5EK (triangles), or KLK14 (squares) with concentrations as indicated in the figure. 100% Ca2+ corresponds to fluorescence ratio 340/380nm=2. Journal of Investigative Dermatology 2008 128, 18-25DOI: (10.1038/sj.jid.5700965) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Confocal photomicrographs of KNRK-PAR2 cells stimulated with trypsin or KLK. KNRK-PAR2 cells were stimulated with KLK5EK, KLK7EK, KLK8, KLK14, or trypsin, all at 1μM. Negative control shows cells incubated in DMEM/0.1% BSA only. Cells incubated with rEK looked like cells in negative control (data not shown). Cells were stained with anti-Flag antibody toward extracellular Flag epitope and anti-HA antibody toward intracellular HA11 epitope. Merged: superimposition of images in the same row. Yellow color in superimposed images indicates no degradation of extracellular parts of PAR2. Journal of Investigative Dermatology 2008 128, 18-25DOI: (10.1038/sj.jid.5700965) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Immunoreactivity of KLK14 and PAR2 in inflamed skin tissues. Immunoreactivity of KLK14 as well as PAR2 was found widely distributed in the upper squamous and granular layer in patients with inflammatory skin disorders. (a–d) Atopic dermatitis, (e–h) rosacea. Staining was performed with antisera specific for (a, b, e, f) KLK14 or (c, g) PAR2. (d, h) Negative controls. Inset size bars=50μM (b, f) or 100μM. Journal of Investigative Dermatology 2008 128, 18-25DOI: (10.1038/sj.jid.5700965) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions