Collagen XVII Shedding Suppresses Re-Epithelialization by Directing Keratinocyte Migration and Dampening mTOR Signaling  Joanna Jacków, Stefanie Löffek, Alexander Nyström, Leena Bruckner-Tuderman, Claus-Werner Franzke  Journal of Investigative Dermatology  Volume 136, Issue 5, Pages 1031-1041 (May 2016) DOI: 10.1016/j.jid.2016.01.012 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Lack of collagen XVII shedding enhances re-epithelialization in mice. (a) H&E staining of paraffin sections of wound edges (red arrows) at day 3 in Col17a1ΔNS/ΔNS mice. Quantification of the length of the epithelial tongues at days 3 and 6 after wounding. Values represent mean ± SEM, *P < 0.05 (n = 4). (b) Proliferation rate in the epithelial tongues. Ki-67 stained skin at day 3 after wounding. Quantification of the Ki-67 positive cells at days 3 and 6 after wounding. Values represent mean ± SEM, **P < 0.01, ***P < 0.001 (n = 4). (c) Expression of the collagen XVII interacting proteins α6β4 integrin and laminin-332 at day 3 after wounding. Values represent mean of relative staining intensity ± SD, *P < 0.05 (n = 4). Scale bars = 200 μm (a), 100 μm (b), 50 μm (c). H&E, hematoxylin and eosin; SD, standard deviation; SEM, standard error of the mean. Journal of Investigative Dermatology 2016 136, 1031-1041DOI: (10.1016/j.jid.2016.01.012) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Col17a1ΔNS/ΔNS keratinocytes show hypermotility and increased proliferation. Primary Col17a1ΔNS/ΔNS versus Col17a1+/+ keratinocytes were assessed for (a) in vitro re-epithelialization for 16 hours and (b) single cell motility for 4 hours by time-lapse video microscopy with images in every 4 minutes. Directionality: (distance from the origin)/total distance; 40 cells per genotype were analyzed. Values represent mean ± SEM, *P < 0.05, **P < 0.01 (n = 4). (c) Stress fiber formation in Col17a1ΔNS/ΔNS cells demonstrated by β-actin staining. (d) Visualization and quantification of spread Col17a1+/+ and Col17a1ΔNS/ΔNS keratinocytes on uncoated culture plates within 3 hours after seeding. Values represent mean ± SEM, ***P < 0.001 (n = 3). (e) Cell proliferation was calculated as percentage of BrdU positive cells per total cell number. Values represent mean ± SEM, ***P < 0.001 (n = 4). Scale bars = 25 μm (a), 20 μm (c), 50 μm (d, e). SEM, standard error of the mean. Journal of Investigative Dermatology 2016 136, 1031-1041DOI: (10.1016/j.jid.2016.01.012) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Lack of collagen XVII shedding in keratinocytes alters localization of the endodomain, β4 integrin, and laminin-332. Migrating Col17a1+/+ and Col17a1ΔNS/ΔNS keratinocytes were stained 6 hours after seeding with antibodies to collagen XVII ectodomain (Ecto-CXVII), endodomain (Endo-CXVII), β4 integrin, laminin-332 (Lam332), and β-actin. The collagen XVII endodomain staining was completely lost in the periphery/protrusions of Col17a1ΔNS/ΔNS keratinocytes. This was accompanied by altered β4 integrin staining and scattered laminin-332 deposition. Scale bars = 20 μm. Journal of Investigative Dermatology 2016 136, 1031-1041DOI: (10.1016/j.jid.2016.01.012) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Adhesion-dependent activation of the PI3K/Akt/mTOR pathway is increased in Col17a1ΔNS/ΔNS keratinocytes. (a) WB analysis with subsequent densitometric quantification of different signaling pathway proteins in subconfluent primary Col17a1ΔNS/ΔNS and Col17a1+/+ keratinocytes 6 hours after seeding. Values represent mean ± SD, *P < 0.05 (n = 3). (b, c) Time-resolved analysis of the adhesion dependency of Akt signaling in Col17a1ΔNS/ΔNS and Col17a1+/+ keratinocytes plated on uncoated culture dishes (b) or dishes coated with 1 μg/ml laminin-332 (c). Representative blots from experiments with keratinocytes derived from four mice per genotype. (d) Addition of 20 ng/ml EGF significantly increased Akt phosphorylation in Col17a1ΔNS/ΔNS cells. (e) Integrin α6 blocking: WB analysis of EGF-induced Akt phosphorylation of Col17a1ΔNS/ΔNS keratinocytes using a GoH3 antibody. (f) Loss of collagen XVII shedding promotes α6β4-driven mTOR activation. Akt, acutely transforming retrovirus AKT8 in rodent T-cell lymphoma; mTOR, mechanistic target of rapamycin; PI3K, phosphoinositide 3-kinase; SD, standard deviation; WB, western blotting. Journal of Investigative Dermatology 2016 136, 1031-1041DOI: (10.1016/j.jid.2016.01.012) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Increased mTOR activation at wound edges drives wound closure in Col17a1ΔNS/ΔNS mice. (a) Immunofluorescence staining of skin wounds at day 3 after wounding demonstrates increased phosphorylation of S6 ribosomal protein (pS6) in Col17a1ΔNS/ΔNS mice. Mean of staining intensity ± SEM, *P < 0.05 (n = 4). (b, c) Daily administration of mTOR inhibitor rapamycin represses wound closure in Col17a1ΔNS/ΔNS mice. Representative H&E staining of reduced epidermal tongues and its quantification 5 days after wounding (b). Arrows indicate wound edges. Values represent mean ± SEM, ***P < 0.001 (n = 4 mice). Proliferation rate in the epidermal tongues was assessed as the ratio of Ki-67 positive cells to total keratinocytes at day 5 after wounding (c). Values represent mean ± SEM, **P < 0.01 (n = 4 mice). Scale bars = 200 μm (a), 100 μm (b), 50 μm (c). H&E, hematoxylin and eosin; mTOR, mechanistic target of rapamycin; SD, standard deviation; SEM, standard error of the mean. Journal of Investigative Dermatology 2016 136, 1031-1041DOI: (10.1016/j.jid.2016.01.012) Copyright © 2016 The Authors Terms and Conditions

Figure 6 mTOR signaling accelerates proliferation and migration velocity in Col17a1ΔNS/ΔNS keratinocytes. (a) In vitro re-epithelialization of Col17a1ΔNS/ΔNS keratinocytes treated with either vehicle or rapamycin (75 nM) was followed by photographic documentation in every 4 hours for 24 hours. Scale bars = 200 μm. (b) For assessment of cell proliferation, cells were pretreated with rapamycin for 24 hours, followed by BrdU incorporation for 2 hours. The graph shows the percentage of BrdU positive cells. Values represent mean ± SEM, ***P < 0.001. (c, d) Single cell motility of vehicle- and rapamycin-treated Col17a1+/+ or Col17a1ΔNS/ΔNS keratinocytes was investigated. Forty cells per genotype were analyzed. Values represent mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.01 (n = 3). (e) β-actin immunofluorescence staining of untreated or rapamycin-treated Col17a1ΔNS/ΔNS keratinocytes. Scale bar = 20 μm. mTOR, mechanistic target of rapamycin; SEM, standard error of the mean. Journal of Investigative Dermatology 2016 136, 1031-1041DOI: (10.1016/j.jid.2016.01.012) Copyright © 2016 The Authors Terms and Conditions