Thyroid Hormones Enhance Mitochondrial Function in Human Epidermis Silvia Vidali, Jérémy Chéret, Melanie Giesen, Swantje Haeger, Majid Alam, Rachel E.B. Watson, Abigail K. Langton, Matthias Klinger, Jana Knuever, Wolfgang Funk, Barbara Kofler, Ralf Paus  Journal of Investigative Dermatology  Volume 136, Issue 10, Pages 2003-2012 (October 2016) DOI: 10.1016/j.jid.2016.05.118 Copyright © 2016 The Authors Terms and Conditions

Figure 1 MTCO1, TFAM, and PGC1α IR AND complex I, II, and IV activity. Full-thickness human skin organ culture biopsy samples were treated with (a, e, i, m, q) vehicle, (b, f, j, n, r) T3 (100 pmol/L), or (c, g, k, o, s) T4 (100 nmol/L) for 24 hours. (a–d) MTCO1 IHC, (e–h) TFAM IF, (i–l) PGC1α IF, (m–p) complex I and (q–t) complex II and IV activity studies were performed. (d) MTCO1 IR, (h) TFAM IR, (l) PGC1α IR, (p) complex I, and (t) complex II and IV activity were measured with ImageJ software in precise and standardized epidermal areas. Scale bar = 100 μm. n = 8–9 samples (three subjects analyzed). Mean ± standard error of the mean (one-way analysis of variance), ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. CI, complex I; CII/IV, complex II and IV; Ep, epidermis; IF, immunofluorescence; IHC, immunohistochemistry; IR, immunoreactivity; MTCO1, mitochondrially encoded cytochrome c oxidase I; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; T3, triiodothyronine; T4, thyroxin; TFAM, mitochondrial transcription factor A. Journal of Investigative Dermatology 2016 136, 2003-2012DOI: (10.1016/j.jid.2016.05.118) Copyright © 2016 The Authors Terms and Conditions

Figure 2 MMP-1 IR and activity. Full-thickness human skin organ culture treated with (a, e, i, m) vehicle, (b, f, j, n) T3 (100 pmol/L), or (c, g, k, o) T4 (100 nmol/L) for (a–c, i–k) 24 hours or (e–g, m–o) 6 days, during which time the organ-cultured human skin remains vital (Lu et al., 2007). (a–h) MMP-1 IF was performed to detect pro- and active MMP-1. (i–p) In situ zymography (ISZ) was performed to measure MMP-1 activity. Scale bar = 100 μm. (d, h) MMP-1 IR and (l, p) MMP-1 activity were measured in precise and standardized epidermal and/or dermal areas. n = 6–12 samples (three subjects for MMP-1 IR, 24 hours; four subjects for MMP-1 ISZ, 24 hours; two subjects for MMP-1 IR and ISZ, 6 days). Mean ± standard error of the mean (one-way analysis of variance), ∗∗P < 0.01. It is normally believed that MMP activity resides in the dermis; however, there is increasing evidence that MMPs (and LOX/LOXLs) have additional activity in the epidermis (Ågren et al., 2015; Fisher et al., 2009). It is possible that MMPs are released epidermally and then diffuse to the papillary dermis to act on collagens and elastic fiber components. AUC, area under the curve; Der, dermis; Ep, epidermis; h, hours; hrs, hours; IF, immunofluorescence; IR, immunoreactivity; ISZ, in situ zymography; MMP, matrix metalloproteinase; T3, triiodothyronine; T4, thyroxin. Journal of Investigative Dermatology 2016 136, 2003-2012DOI: (10.1016/j.jid.2016.05.118) Copyright © 2016 The Authors Terms and Conditions

Figure 3 MMP-2 and -9 activity and collagen-III IR. Full-thickness human skin biopsy samples treated with (a, e, i, m) vehicle, (b, f, j, n) T3 (100 pmol/L), or (c, g, k, o) T4 (100 nmol/L) for (a–c, i–k) 24 hours or (e–g, m–o) 6 days. (a–h) In situ zymography assay was performed to detect the activity of MMP-2 and -9. (i–p) Collagen-III IF was performed. Scale bar = 100 μm. (d, h) MMP-2 and -9 activity and (l, p) collagen-III IR measured in precise and standardized epidermal and/or dermal areas. n = 6–12 samples (four subjects for the 24-hour treatment, two subjects for the 6-day treatment). Mean ± standard error of the mean (one-way analysis of variance), ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. AUC, area under the curve; Coll, collagen; Ep, epidermis; h, hours; hrs, hours; IF, immunofluorescence; IR, immunoreactivity; ISZ, in situ zymography; MMP, matrix metalloproteinase; T3, triiodothyronine; T4, thyroxin. Journal of Investigative Dermatology 2016 136, 2003-2012DOI: (10.1016/j.jid.2016.05.118) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Sirt1 IR and mTORC1/2 IR. Full-thickness human skin organ culture treated with (a, e, i) vehicle, (b, f, j) T3 (100 pmol/L), or (c, g, k) T4 (100 nmol/L) for (a–c, i–l) 24 hours or (e–g) 6 days. (a–h) Sirt-1 IHC. Scale bar = 100 μm. (d, h) Sirt1 IR was measured in precise and standardized epidermal area. n = 6–9 samples (three subjects for the 24-hour incubation; two subjects for the 6-day incubation). Mean ± standard error of the mean (one-way analysis of variance). (i–l) mTORC1/2 immunofluorescence. Scale bar = 100 μm. (l) mTORC1/2 IR was measured in precise and standardized epidermal area. n = 9–11 samples (four subjects). Mean ± standard error of the mean (one-way analysis of variance), ∗P < 0.05. Ep, epidermis; hrs, hours; IHC, immunohistochemistry; IR, immunoreactivity; mTORC1/2, mammalian target of rapamycin complex 1/2; sirt1, sirtuin 1; T3, triiodothyronine; T4, thyroxin. Journal of Investigative Dermatology 2016 136, 2003-2012DOI: (10.1016/j.jid.2016.05.118) Copyright © 2016 The Authors Terms and Conditions