Generation of a Functional Non-Shedding Collagen XVII Mouse Model: Relevance of Collagen XVII Shedding in Wound Healing  Joanna Jacków, Andreas Schlosser, Raija Sormunen, Alexander Nyström, Cassian Sitaru, Kaisa Tasanen, Leena Bruckner-Tuderman, Claus-Werner Franzke  Journal of Investigative Dermatology  Volume 136, Issue 2, Pages 516-525 (February 2016) DOI: 10.1016/j.jid.2015.10.060 Copyright © 2015 The Authors Terms and Conditions

Figure 1 In vitro generation of a murine non-shedding collagen XVII mutant. (a) Detection of the cleavage sites in murine collagen XVII: His-tagged C-terminally truncated collagen XVII ectodomain (Ecto 42 kDa) was purified by Ni-NTA affinity chromatography. (b) Immunoblot of Ecto 42 kDa. The corresponding Coomassie brilliant blue-stained band (c) was cut out from the gel, digested with either elastase or thermolysin, and subjected to multiple liquid chromatography tandem mass spectrometry analysis. (d) Schematic representation of the N-termini identified after MS analysis within the NC14A linker domain of murine collagen XVII. (e) Identification of non-sheddable collagen XVII: Five collagen XVII mutants with deletions within the NC14A domain were generated. (f) The expression and shedding of all collagen XVII deletion mutants were detected by immunoblot analysis using the antibody EC-7. Journal of Investigative Dermatology 2016 136, 516-525DOI: (10.1016/j.jid.2015.10.060) Copyright © 2015 The Authors Terms and Conditions

Figure 2 Generation and verification of collagen XVII non-shedding knock-in mice. (a) Constitutive knock-in Col17a1ΔNS/ΔNS allele was generated by introducing a 123 nucleotide deletion into exon 18. After flippase removal of the puromycine resistance cassette, one F3 site remained. (b) Genotyping was performed by PCR analysis spanning the 123 nucleotide deletion (primer set A, red) or the F3 region (primer set B, blue) in the genome of Col17a1ΔNS/ΔNS. (c, d) Immunoblot of Col17a1+/+ and Col17a1ΔNS/ΔNS skin lysates (c) or primary keratinocytes (d) with antibody EC-7 revealed complete loss of the ectodomain (Ecto) in the skin/keratinocytes of Col17a1ΔNS/ΔNS mice, whereas membrane-bound collagen XVII was detected in both genotypes (C XVII). Total ERK or β-actin was used as loading control. Representative blots from three different experiments. Journal of Investigative Dermatology 2016 136, 516-525DOI: (10.1016/j.jid.2015.10.060) Copyright © 2015 The Authors Terms and Conditions

Figure 3 Prevention of collagen XVII shedding did not alter skin architecture. (a) Newborn Col17a1ΔNS/ΔNS pups look similar to control littermates. (b) Hematoxylin and eosin staining of back skin revealed normal epidermal architecture in Col17a1ΔNS/ΔNS mice. (c) Confocal microscopy with EC-7 antibody demonstrated unchanged epidermal localization of Δ41 collagen XVII. (d) Immunofluorescence distribution and protein expression of α6β4 integrin and laminin-332 in Col17a1ΔNS/ΔNS skin. Scale bars = 20 μm. Right panel: Representative blots of Western blot quantification. Total ERK was loading control. (e) Immunoelectron microscopy with antibody EC-7 detected unchanged collagen XVII distribution (black arrows) in Col17a1ΔNS/ΔNS mice. (f) Transmission electron microscopy disclosed increased BM thickness in Col17a1ΔNS/ΔNS skin 1 and 8 weeks after birth. Scale bars = 500 nm. Values represent mean ± SEM, **P < 0.01 (n = 3 per genotype). Journal of Investigative Dermatology 2016 136, 516-525DOI: (10.1016/j.jid.2015.10.060) Copyright © 2015 The Authors Terms and Conditions

Figure 4 Normal epidermal differentiation in the skin of Col17a1ΔNS/ΔNS mice. (a) Immunofluorescence staining of P6 back skin for differentiation markers keratin 1 and loricrin. Scale bars = 20 μm. (b) Immunoblot analysis of P18 back skin with antibodies to transglutaminase 1 (TGM1), involucrin, loricrin, and keratins 1 and 5 revealed no differences in expression levels. Total β-tubulin was used as loading control. Representative blots from four independent experiments. (c) Immunohistochemical staining of P6 Col17a1ΔNS/ΔNS back skin for Ki-67 (red) revealed normal proliferation. The graph shows the percentage of Ki-67 positive cells per total cell number. Data ± SEM, n = 3. Scale bars = 200 μm. (d) Similar epidermal thickness of P18 back skin of Col17a1ΔNS/ΔNS mice and Col17a1+/+ littermates. Values represent mean ± SEM, n = 5 mice pairs. Journal of Investigative Dermatology 2016 136, 516-525DOI: (10.1016/j.jid.2015.10.060) Copyright © 2015 The Authors Terms and Conditions

Figure 5 Increased expression and shedding of collagen XVII in acute skin wounds. (a) Confocal immunofluorescence staining of normal human skin with selective collagen XVII antibodies showed that membrane-bound collagen XVII is distributed basolaterally (mAb NC16a-3; C XVII-membrane), whereas the ectodomain (HK139; C XVII-ectodomain) is restricted to the BM. (b) Increased collagen XVII immunoreactivity (white arrows) during early re-epithelialization of murine skin wounds. Immunofluorescence staining revealed extensive staining throughout the whole wound epithelium at day 3, whereas it was restricted to the epithelial tongues at day 6 postwounding. Red arrows indicate wound margins. (c) Immunofluorescence staining of acute human skin wounds with selective collagen XVII antibodies (as mentioned above) revealed elevated ectodomain release at the wound edges (white arrows). Scale bars = 100 μm. Journal of Investigative Dermatology 2016 136, 516-525DOI: (10.1016/j.jid.2015.10.060) Copyright © 2015 The Authors Terms and Conditions

Figure 6 Lack of collagen XVII shedding enhances re-epithelialization during cutaneous wound healing. (a) Closure of back skin wounds of Col17a1ΔNS/ΔNS and Col17a1+/+ mice were followed for 13 days. Representative photos at days 0–6 after wounding. Wound closure was quantified as percentage of the initial area at day 0. Results represent mean ± SEM, *P < 0.05, **P < 0.01 (n = 7). (b) Hematoxylin and eosin staining of wounds at day 6 postwounding demonstrated increased re-epithelialization in Col17a1ΔNS/ΔNS mice. Black arrows and dotted lines indicate wound margins and diameter, respectively. Red arrows and dotted lines indicate invading epithelial tips and not re-epithelialized wounds, respectively. Scale bar = 250 μm. (c) Re-epithelialization as percentage of wound covered by new epidermis at day 3 and 6 postwounding. Values represent mean ± SEM, *P < 0.05, ***P < 0.001 (n = 4). Journal of Investigative Dermatology 2016 136, 516-525DOI: (10.1016/j.jid.2015.10.060) Copyright © 2015 The Authors Terms and Conditions