Collagen VII Half-Life at the Dermal-Epidermal Junction Zone: Implications for Mechanisms and Therapy of Genodermatoses  Tobias Kühl, Markus Mezger, Ingrid Hausser, Lin T. Guey, Rupert Handgretinger, Leena Bruckner-Tuderman, Alexander Nyström  Journal of Investigative Dermatology  Volume 136, Issue 6, Pages 1116-1123 (June 2016) DOI: 10.1016/j.jid.2016.02.002 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Tamoxifen-induced knockout on at the genomic and transcriptional levels. (a) Tamoxifen-inducible Col7a1 knockout mice were injected with tamoxifen on 5 consecutive days to induce excision of exon 2. Tissue was analyzed for collagen VII decay at indicated time points. (b) Analysis of gene excision efficiency. Col7a1 exon 2-3 PCR products will form only if excision has not taken place. (c) PCR products from 22–28 amplification cycles were analyzed on agarose gels and densitometrically quantified. This showed an average excision efficiency of 88.2%. Samples at different time points after tamoxifen injection have been analyzed. Values represent mean ± standard error of the mean of pooled time points normalized to vehicle-treated Cre+ mice (n = 11). (d) Col7a1 mRNA expression normalized to GAPDH. Values represent mean ± standard error of the mean; ∗∗∗P < 0.001; n = 7. mRNA, messenger RNA; TEM, transmission electron microscopy. Journal of Investigative Dermatology 2016 136, 1116-1123DOI: (10.1016/j.jid.2016.02.002) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Determination of in vivo half-life in skin for collagen VII. (a) Representative immunofluorescence staining of skin from vehicle-treated and tamoxifen-treated mice 18 weeks after last injection. n ≥ 3 mice per time point; bar = 100 μm. In the lower panel, arrows indicate remaining collagen VII-positive areas. (b) Western blots of back skin samples 2–18 weeks after the last tamoxifen injection. Blots were probed with antibodies to collagen VII, Erk1/2, and Gapdh as loading controls (Nystrom et al., 2015). (c) Densitometric quantification of blots in b. Values represent mean ± standard error of the mean of collagen VII in tamoxifen-treated mice normalized to vehicle-treated controls. n ≥ 2 mice. ∗∗P < 0.01, ∗∗∗P < 0.001. (d) Based on assumption of one-phase decay, collagen VII half-life in skin is estimated to be 4.4 weeks. R2 > 0.7. Erk, extracellular signal-regulated kinase; ns, not significant. Journal of Investigative Dermatology 2016 136, 1116-1123DOI: (10.1016/j.jid.2016.02.002) Copyright © 2016 The Authors Terms and Conditions

Figure 3 In vivo half-life of collagen VII is tissue-independent. (a and e) Immunofluorescence staining of (a) tongue and (e) esophagus from vehicle-treated and tamoxifen-treated mice 18 weeks after injection. n ≥ 3 mice; bar = 100 μm. Arrows indicate remaining collagen VII-positive areas. (b and f) Western blots of (b) tongue and (f) esophagus at indicated times after knockout induction. Blots probed with antibodies to collagen VII and Erk1/2 (loading control). (c and g) Quantification of blots as in b and f; values represent mean ± standard error of the mean of collagen VII expression in tamoxifen-treated mice normalized to vehicle-treated mice. n ≥ 2 mice. (d and h) Based on assumption of one-phase decay, the half-life of collagen VII in tongue and esophagus is 4.6 and 3.2 weeks, respectively; R2 > 0.7. Erk, extracellular signal-regulated kinase. ∗P < 0.05. Journal of Investigative Dermatology 2016 136, 1116-1123DOI: (10.1016/j.jid.2016.02.002) Copyright © 2016 The Authors Terms and Conditions

Figure 4 In vivo half-life of anchoring fibrils in murine back skin. (a) Transmission electron microscopy images of back skin of vehicle-treated mice and tamoxifen-treated mice 6 and 18 weeks after the last tamoxifen injection. Anchoring fibrils (white arrows) are visible in vehicle-treated mice but gradually decrease after gene knockout. The bottom panel is a magnification of the top panel. Top panel bar = 200 nm; bottom panel bar = 100 nm. (b) Quantification of anchoring fibrils. Values represent mean ± standard error of the mean of anchoring fibrils/μm lamina densa. ∗∗P < 0.01, ∗∗∗P < 0.001. (c) Based on assumption of one-phase decay, the half-life of anchoring fibrils is 4.5 weeks. (d) Hematoxylin and eosin staining of skin as in a. Arrows indicate dermal-epidermal separation. Bar = 100 μm. n = 2 mice per time point. Multiple images from two technical replicates per mice were analyzed; R2 > 0.7. Journal of Investigative Dermatology 2016 136, 1116-1123DOI: (10.1016/j.jid.2016.02.002) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Therapeutic collagen VII displays similar half-life to endogenous collagen VII. (a) Skin from PBS-treated or MSC-treated collagen VII hypomorphic mice 8 and 12 weeks after injections, stained with antibodies to human collagen VII (green). The arrows indicate linear collagen VII staining at the DEJZ. Bar = 50 μm. (b) Quantification of the signal intensity of collagen VII staining 8 and 12 weeks after MSC injections. The intensity of staining after 8 weeks was set to 100%, and values are expressed as the percentage of this. Note that collagen VII is decreased to about one half during the 4 weeks between the 8- and 12-week time points, suggesting that the half-life of exogenous collagen VII at the DEJZ is about 4 weeks. DAPI, 4′,6-diamidino-2-phenylindole. DEJZ, dermal-epidermal junction zone; MSC, mesenchymal stromal cell; PBS, phosphate-buffered saline. Journal of Investigative Dermatology 2016 136, 1116-1123DOI: (10.1016/j.jid.2016.02.002) Copyright © 2016 The Authors Terms and Conditions