6-Formylindolo[3,2-b]Carbazole Accelerates Skin Wound Healing via Activation of ERK, but Not Aryl Hydrocarbon Receptor  Saori Morino-Koga, Hiroshi Uchi, Chikage Mitoma, Zhouwei Wu, Mari Kiyomatsu, Yoko Fuyuno, Konosuke Nagae, Mao Yasumatsu, Mary Ann Suico, Hirofumi Kai, Masutaka Furue  Journal of Investigative Dermatology  Volume 137, Issue 10, Pages 2217-2226 (October 2017) DOI: 10.1016/j.jid.2016.10.050 Copyright © 2017 The Authors Terms and Conditions

Figure 1 FICZ accelerates wound closure in vitro and in vivo. (a) HaCaT cells or (c) NHEKs were treated with FICZ or DMSO for 5 hours (n = 3). (b) HaCaT cells or (d) NHEKs were scratched and incubated with FICZ (100 nM) or DMSO (n = 6). (e, f) HaCaT cells were scratched and incubated with FICZ, tryptophan, or DMSO (n = 6). (g–i) Representative photographs and the time courses of the relative wound area after wound creation in (g) balb/c, (h) db/+, and (i) db/db mice are shown (n = 5). The white (FICZ) and black (control) arrows showed complete cure. All data are presented as mean ± SE (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). Scale bar = 4 mm. FICZ, 6-formylindolo[3,2-b]carbazole; NHEK, normal human epidermal keratinocyte; SE, standard error. Journal of Investigative Dermatology 2017 137, 2217-2226DOI: (10.1016/j.jid.2016.10.050) Copyright © 2017 The Authors Terms and Conditions

Figure 2 FICZ promotes keratinocyte migration, but not proliferation. (a–c) HaCaT cells were treated (a) without or (b) with 5 μg/ml mitomycin C for 2 hours. Cells were scratched and incubated with 100 nM FICZ or DMSO (n = 6). (c) The relative wound area at 10 hours is shown. (d, e) HaCaT cells were treated with FICZ for 24 hours and the reaction products of (d) BrdU incorporation assay and (e) MTT assay were quantified (n = 5). (f, g) HaCaT cells were scratched and incubated with 100 nM FICZ or DMSO in the (f) absence or (g) presence of 2 μM cytochalasin D (n = 6). All data are presented as mean ± SE (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). FICZ, 6-formylindolo[3,2-b]carbazole; MTT, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; SE, standard error. Journal of Investigative Dermatology 2017 137, 2217-2226DOI: (10.1016/j.jid.2016.10.050) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Effect of FICZ is dependent of AhR. (a, c) HaCaT cells were treated with (a) BaP, (c) BNF, or DMSO for 5 hours (n = 3). (b, d) Cells were scratched and incubated with (b) BaP, (d) BNF, or DMSO (n = 6). (e, g) C57BL/6 and (f, h) DBA/2 mice were applied with ointments containing FICZ or DMSO for 5 hours (n = 4). (i, j) Representative photographs and the time courses of the relative wound area after wound creation in (i) C57BL/6 and (j) DBA/2 mice are shown (n = 5). The white (FICZ) and black (control) arrows showed complete cure. All data are presented as mean ± SE (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). Scale bar = 4 mm. AhR, aryl hydrocarbon receptor; BaP, benzo[a]pyrene; BNF, β-naphthoflavone; FICZ, 6-formylindolo[3,2-b]carbazole; SE, standard error. Journal of Investigative Dermatology 2017 137, 2217-2226DOI: (10.1016/j.jid.2016.10.050) Copyright © 2017 The Authors Terms and Conditions

Figure 4 FICZ promotes keratinocytes migration via an AhR-independent pathway. (a) HaCaT cells were transfected with AhR or control siRNA (n = 3). (b) Control siRNA or (c) AhR siRNA-transfected cells were scratched and incubated with FICZ (100 nM) or DMSO (n = 6). (d) HaCaT cells were treated with FICZ and/or CH-223191 for 6 hours (n = 3). (e, f) HaCaT cells were scratched and incubated with FICZ or DMSO in the (e) absence or (f) presence of 10 μM CH-223191 (n = 6). All data are presented as mean ± SE (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). AhR, aryl hydrocarbon receptor; FICZ, 6-formylindolo[3,2-b]carbazole; SE, standard error; siRNA, small interfering RNA. Journal of Investigative Dermatology 2017 137, 2217-2226DOI: (10.1016/j.jid.2016.10.050) Copyright © 2017 The Authors Terms and Conditions

Figure 5 FICZ activates the MEK/ERK pathway in human keratinocytes. (a, b) HaCaT cells were scratched and treated with 100 nM FICZ or DMSO for 6 hours (n = 3). (c) HaCaT cells were scratched and treated with 100 nM FICZ in the presence or absence of 10 μM CH-223191, 10 μM U0126, or 50 μM PD98059 for 6 hours. (d–h) HaCaT cells were scratched and treated with 100 nM FICZ or DMSO for 6 hours (n = 3). All data are presented as mean ± SE (∗P < 0.05, ∗∗P < 0.01). Akt, acutely transforming retrovirus AKT8 in rodent T-cell lymphoma; ERK, extracellular signal-regulated kinase; FICZ, 6-formylindolo[3,2-b]carbazole; JNK, c-Jun N-terminal kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase; RAF, rapidly accelerated fibrosarcoma; SE, standard error. Journal of Investigative Dermatology 2017 137, 2217-2226DOI: (10.1016/j.jid.2016.10.050) Copyright © 2017 The Authors Terms and Conditions

Figure 6 FICZ induces cell migration via the upregulation of pERK, but not EGFR. HaCaT cells were scratched and incubated with 100 nM FICZ or DMSO in the (a, c, e) absence or (b) presence of 10 µM U0126, (d) 50 µM PD98059, or (f) 100 nM PD153035 for 10 hours (n = 6). All data are presented as mean ± SE (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). ERK, extracellular signal-regulated kinase; FICZ, 6-formylindolo[3,2-b]carbazole; SE, standard error. Journal of Investigative Dermatology 2017 137, 2217-2226DOI: (10.1016/j.jid.2016.10.050) Copyright © 2017 The Authors Terms and Conditions