The Aldo-Keto Reductase AKR1B10 Is Up-Regulated in Keloid Epidermis, Implicating Retinoic Acid Pathway Dysregulation in the Pathogenesis of Keloid Disease  Natalie Jumper, Tom Hodgkinson, Guyan Arscott, Yaron Har-Shai, Ralf Paus, Ardeshir Bayat  Journal of Investigative Dermatology  Volume 136, Issue 7, Pages 1500-1512 (July 2016) DOI: 10.1016/j.jid.2016.03.022 Copyright © 2016 The Authors Terms and Conditions

Figure 1 AKR1B10 overexpression in keloid versus normal skin. (a) Classical retinoic acid biosynthesis pathway. (b) Keloid biopsy site diagram including intralesional (center), perilesional (margin), and extralesional (adjacent normal-appearing skin) sites. (c) AKR1B10 quantitative real-time PCR validation of microarray data. Bars represent quantitative real-time PCR of epidermal sites within keloid compared with normal skin. The line graph shows the corresponding microarray data. (d) AKR1B10 immunohistochemistry expression in keloid (n = 14) and normal skin tissue (n = 12). Keloid shows strong red suprabasal epidermal staining for AKR1B10 (black arrows). No expression was observed in normal skin or negative control. Micrographs i–iii, scale bar = 100 μm; iv–vi, scale bar = 50 μm. Data are represented as mean ± standard error of the mean from at least three independent experiments. ∗P < 0.01, ∗∗P < 0.005 using Student’s t test. ADH, alcohol dehydrogenase; AKR1B10, aldo-keto reductase family 1 member B10; ALDH, aldehyde dehydrogenase; GE, gene expression; NAD, nicotinamide adenine dinucleotide (+ is oxidised form); NADH, hydroxylamine reductase; PPAR, peroxisome proliferator–activated receptor-α; RA, retinoic acid; RAR, retinoic acid receptor; (R)DH, retinol dehydrogenase; (R)ALDH, retinaldehyde dehydrogenase; RXR, retinoid X receptor; SDR, short-chain dehydrogenase. Journal of Investigative Dermatology 2016 136, 1500-1512DOI: (10.1016/j.jid.2016.03.022) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Keloid tissue retinoic acid pathway dysregulation. (a) Immunofluorescence showing increased epidermal staining intensity of AKR1B10 (green) and CRABP2 (red) for keloid (n = 5) (scale bar = 200 μm) compared with normal skin tissue (n = 6) (scale bar = 100 μm). (b) Magnified images of merged AKR1B10 and CRABP2 for both keloid and normal skin tissue. Strong yellow staining in keloid epidermis, indicated by the black arrows, shows co-localization of AKR1B10 and CRABP2, particularly in the suprabasal layers. Normal skin epidermis, by contrast, remains red-orange. Scale bar = 50 μm. (c) Detailed diagram of the classical retinoic acid biosynthesis pathway. Green represents up-regulation and red represents down-regulation of molecules identified from microarray data and confirmed on quantitative real-time PCR, which is available in Supplementary Table S1. ADH, alcohol dehydrogenase; AKR1B10, aldo-keto reductase family 1 member B10; ALDH, aldehyde dehydrogenase; atRA, all-trans-retinoic acid; CRABP, cellular retinoic acid binding protein; CRBP1, cellular retinol binding protein 1; CYP26, cytochrome P450, family 26; FABP5, fatty acid binding protein 5; LRAT, lecithin:retinol acetyltransferase; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced NADP; PPAR, peroxisome proliferator–activated receptor; RA, retinoic acid; (R)ALDH, retinaldehyde dehydrogenase; RAR, retinoic acid receptor; RARE, retinoic acid response element; RDH, retinol dehydrogenase; RXR, retinoid X receptor; SDR, short-chain dehydrogenase; STRA6, stimulated by retinoic acid 6; TTR-RBP, transthyretin-retinol binding protein. Journal of Investigative Dermatology 2016 136, 1500-1512DOI: (10.1016/j.jid.2016.03.022) Copyright © 2016 The Authors Terms and Conditions

Figure 3 AKR1B10 overexpression in normal skin keratinocytes. (a) AKR1B10 expression quantified by Western blot test (n = 10). (b) Quantitative real-time PCR (n = 10). (c) Immunocytochemistry (n = 8). Arrows identify transfected keratinocytes. Scale bar = 50 μm. (d, e) Quantitative real-time PCR for retinoic acid pathway molecule expression and downstream signaling after AKR1B10 overexpression. (f) Keratin 14 and 6b quantitative real-time PCR in transfected versus nontransfected keratinocytes. (g) AKR1B10-transfected versus nontransfected relative luciferase activity, treated with 10 μmol/L retinol or 1 μmol/L all-trans-retinoic acid (n = 10). Data from at least three independent means ± standard error of the mean. ∗P < 0.05, ∗∗P < 0.005, Student’s t test. AKR, aldo-keto reductase; AKR1B10, aldo-keto reductase 1, member B10; ALDH1a1, aldehyde dehydrogenase 1, member A1; atRA, all-trans-retinoic acid; CRABP, cellular retinoic acid binding protein; CYP26B1, cytochrome P450, family 26, subfamily B1; K, keratin; LRAT, lecithin:retinol acetyltransferase; N, not transfected with AKR1B10; RARE, retinoic acid response element; T, transfected with AKR1B10; TGF-β, transforming growth factor-beta. Journal of Investigative Dermatology 2016 136, 1500-1512DOI: (10.1016/j.jid.2016.03.022) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Effect of conditioned media on keloid fibroblasts and normal skin fibroblasts. (a) Fold change difference (quantitative real-time PCR) in the expression of profibrotic genes by keloid fibroblasts (n = 5) and normal skin fibroblasts (n = 5) treated with AKR1B10-transfected or nontransfected medium (n = 6). (b) Quantitative real-time PCR and (c) in-cell Western blot test for collagen I and III expression for fibroblasts treated with AKR1B10-transfected or nontransfected medium (n = 3). In-cell Western blot test fluorescence images accompany the graphs. Each bar on all graphs (a–c) represents fold change of fibroblasts treated with transfected versus nontransfected medium and associated significance with that fold change. Data are mean ± standard error of the mean from at least three independent experiments. ∗P < 0.05 using Student’s t test. AKR1B10, aldo-keto reductase family 1, member B10; Col, collagen; CTGF, connective tissue growth factor; FGF, fibroblast growth factor; KF, keloid fibroblasts; NSF, normal skin fibroblasts; NT, treated with non-transfected keratinocyte medium; T, treated with transfected keratinocyte medium; TGFB1, transforming growth factor-β1; TGFB2, transforming growth factor-β2. Journal of Investigative Dermatology 2016 136, 1500-1512DOI: (10.1016/j.jid.2016.03.022) Copyright © 2016 The Authors Terms and Conditions