S100A12 Induced in the Epidermis by Reduced Hydration Activates Dermal Fibroblasts and Causes Dermal Fibrosis  Jingling Zhao, Aimei Zhong, Emily E. Friedrich, Shengxian Jia, Ping Xie, Robert D. Galiano, Thomas A. Mustoe, Seok Jong Hong  Journal of Investigative Dermatology  Volume 137, Issue 3, Pages 650-659 (March 2017) DOI: 10.1016/j.jid.2016.10.040 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Reduction of hydration increased the expression of S100A12 in keratinocytes. (a) Increased expression of S100A12 was found in human hypertrophic and keloid scar. S100A12 expression in human normal skin, hypertrophic, and keloid scars. Skin sections were stained with S100A12-specific antibodies and visualized with fluorescence-labeled secondary antibodies (red). Nuclei were counterstained with DAPI (blue). The dermal-epidermal junction of the skin is denoted by white lines. Scale bar = 100 μm. (b) Schematic drawing of monolayer and stratified culture of HaCaT. (c) S100A12 protein expression. Whole-cell extract from HaCaT cells that were stratified for 10–14 days (S) and that were cultured in a monolayer (M) until 90% confluency was used for analysis. (d) Schematic drawing of control and reduced-hydration conditions used for the stratified HaCaT culture. (e) S100A12 expression analysis. Stratified HaCaT cells were cultured for 16 hours in reduced-hydration (RH) and control (C) conditions. (c, e) Protein from HaCaT cells (20 μg) was loaded in 12% polyacrylamide gel, and expression of S100A12 protein was detected with anti-S100A12 antibody by Western blot analysis. β-actin was detected as a loading control. The signal in X-ray film was quantified by densitometry using the ImageJ program (National Institutes of Health, Bethesda, MD). Expression of S100A12 was normalized to that of β-actin. n = 4, ∗∗P < 0.01. (f) S100A12 immunostaining. Stratified HaCaT culture sections were stained with S100A12 antibody. Scale bar = 200 μm. Journal of Investigative Dermatology 2017 137, 650-659DOI: (10.1016/j.jid.2016.10.040) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Differentiation of keratinocytes were affected by S100A12 knockdown. Knockdown of S100A12 in HaCaT. S100A12 gene was knocked down by lentivirus-mediated RNA interference in HaCaT. Both wild-type and S100A12-knockdown HaCaT cells were stratified. (a, b) Expression of S100A12 was (a) detected by Western blot analysis and (b) quantified using the ImageJ program (National Institutes of Health) as described in Figure 1. n = 3, ∗∗P < 0.01. (c) Hematoxylin and eosin staining. Scale bar = 200 μm. (d–e) Immunofluorescence staining. (d) Expression of S100A12 (red) was detected with anti-S100A12 antibody. (e) Expression of cytokeratins 14 (red) and 10 (green) was detected with anti-serum to these proteins. Nuclei were stained with DAPI. Scale bar = 200 μm. K/D, knockdown; WT, wild type. Journal of Investigative Dermatology 2017 137, 650-659DOI: (10.1016/j.jid.2016.10.040) Copyright © 2016 The Authors Terms and Conditions

Figure 3 S100A12 from epidermal keratinocytes is critical for the activation of dermal fibroblasts. (a) Quantification of secreted S100A12 in the culture medium. The amount of secreted S100A12 protein in the culture medium of monolayer and stratified HaCaT cells was quantified by ELISA. n = 4, ∗∗P < 0.01. (b) Schematic drawing of keratinocyte-fibroblast co-culture model in control and reduced-hydration conditions. (c–e) Stratified S100A12 knockdown or wild-type HaCaT cells in a cell culture insert were co-cultured with human foreskin fibroblasts plated in the lower chamber. After 16 hours of culture in the reduced-hydration and control conditions, expressions of (c) α-SMA, (d) pro-collagen l, and (e) F-actin in fibroblasts were analyzed by immunofluorescence staining. Scale bar = 100 μm. (f–h) Quantification of the fluorescence intensity using the ImageJ program (National Institutes of Health). Signals from fibroblasts co-cultured with wild-type HaCaT in the control condition were set as 1. n = 4, ∗P < 0.05, ∗∗P < 0.01. α-SMA, α-smooth muscle actin; K/D, knockdown; WT, wild type. Journal of Investigative Dermatology 2017 137, 650-659DOI: (10.1016/j.jid.2016.10.040) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Secreted S100A12 protein from keratinocytes activates dermal fibroblasts. Stratified S100A12 knockdown or wild-type HaCaT cells were cultured in the reduced-hydration and control conditions for 16 hours and conditioned medium was collected. (a) The amount of S100A12 was measured by ELISA. (b–g) Conditioned medium from keratinocytes was incubated with human foreskin fibroblasts and cultured for 16 hours. Immunofluorescence staining for (b) α-SMA, (c) pro-collagen l, and (d) F-actin in fibroblasts. Scale bar = 100 μm. (e–g) Quantification of the fluorescence intensity using the ImageJ program (National Institutes of Health). Signals from fibroblasts treated with conditioned medium of wild-type HaCaT cultured in the control condition were set as 1. n = 4, ∗P < 0.05, ∗∗P < 0.01. α-SMA, α-smooth muscle actin; K/D, knockdown; WT, wild type. Journal of Investigative Dermatology 2017 137, 650-659DOI: (10.1016/j.jid.2016.10.040) Copyright © 2016 The Authors Terms and Conditions

Figure 5 S100A12 activates dermal fibroblasts through RAGE and TLR4. Purified recombinant human S100A12 protein was added at 1 μg/ml into culture medium of foreskin fibroblasts in the presence or absence of RAGE- or TLR4-specific inhibitors, FPS-ZM1 (500 nmol/L) or TAK-242 (400 nmol/L), respectively. (a) After 16 hours, fibroblasts were analyzed for the expression of α-SMA, pro-collagen l, and F-actin by immunofluorescence. Scale bar = 100 μm. (b–d) Quantification of the fluorescence intensity using the ImageJ program (National Institutes of Health). Signals from untreated control were set as 1. n = 4, ∗P < 0.05, ∗∗P < 0.01. TLR4, toll-like receptor 4; α-SMA, α-smooth muscle actin; Pro-Col-I, pro-collagen-1. Journal of Investigative Dermatology 2017 137, 650-659DOI: (10.1016/j.jid.2016.10.040) Copyright © 2016 The Authors Terms and Conditions

Figure 6 Scar hypertrophy is increased by exogenous S100A12 treatment in the rabbit ear model. Full-thickness excisional wounds of 7-mm diameter were created on the surface of the rabbit ears at day 0. Recombinant rabbit S100A12 protein (10 μg) was delivered into the surrounding tissue of wounds at postoperative days (PODs) 15, 19, and 23. Histological analysis was performed on wounds harvested on POD 28. (a) Hematoxylin and eosin staining at POD 28. Representative pictures of scar tissues injected with saline and S100A12 are shown. Scale bars = 500 μm. (b–c) The scar elevation index (SEI) measurement. The SEI is a ratio of area of newly formed dermis (total scar area)/estimated area of nonscarred dermis (unwounded dermis). An SEI of 1 indicates that the wound healed with no scar hypertrophy, and an SEI greater than 1 represents a raised hypertrophic scar. (b) Schematic diagram for SEI. (c) Measuring SEI in control and S100A12-treated groups. n = 12, ∗∗P < 0.01. Journal of Investigative Dermatology 2017 137, 650-659DOI: (10.1016/j.jid.2016.10.040) Copyright © 2016 The Authors Terms and Conditions