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Gauthier Y, Cario-Andre M, Lepreux S et al. (2003) Clinical Implications Melanocyte detachment after skin friction in ● In vitiligo, all the skin is affected. non lesional skin of patients with generalized vitiligo. Br J Dermatol 148:95–101 ● fi Modi cation of pigment distributions in the skin may be associated with Haass NK, Herlyn M (2005) Normal human melanocyte other alterations, such as defects in the epidermal barrier. homeostasis as a paradigm for understanding mela- noma. J Invest Dermatol Symp Proc 10:153–63 ● Stabilization of the disease, by targeting nonlesional skin, is an additional challenge for improving the benefits of treatment. Harris JE, Harris TH, Weninger W et al. (2012) A mouse model of vitiligo with focused epider- ● Full-body phototherapy may stabilize the disease by inducing the differentia- mal depigmentation requires IFN-γ for auto- + tion of melanocytes, with consequent improvements in cell–cell adhesion. reactive CD8 T-cell accumulation in the skin. JInvestDermatol132:1869–76 ● Counteracting extracellular and intracellular oxidative stress may con- Jin Y, Birlea SA, Fain PR et al. (2012) Genome-wide tribute to reducing melanocyte detachment and disease progression. association analyses identify 13 new susceptibility loci for generalized vitiligo. Nat Genet 44:676–80 Kovacs D, Abdel-Raouf H, Al-Khayyat M et al. (2015) Vitiligo: characterization of melano- Cario-André M, Pain C, Gauthier Y et al. (2007) The Recently, immunohistochemical examina- cytes in repigmented skin after punch grafting. melanocytorrhagic hypothesis of vitiligo tested tion of E-cadherin in tissue sam- J Eur Acad Dermatol Venereol 29:581–90 on pigmented, stressed, reconstructed epider- ples collected from patients with vitiligo, mis. Pigment Cell Res 20:385–93 Richmond JM, Frisoli ML, Harris JE (2013) Innate after punch grafting, revealed that melano- immune mechanisms in vitiligo: danger Dell’Anna ML, Picardo M (2006) A review and a from within. Curr Opin Immunol 25:676–82 cytes from normally pigmented donor new hypothesis for non-immunological patho- sites may migrate toward lesional skin genic mechanisms in vitiligo. Pigment Cell Res Schallreuter KU, Salem MA, Gibbons NC et al. (2012) 19:406–11 Blunted epidermal L-tryptophan metabolism in and repopulate the depigmented areas vitiligo affects immune response and ROS because of decreased E-cadherin expres- Denat L, Kadekaro AL, Marrot L et al. (2014) scavenging by Fenton chemistry, part 2: Epider- sion (Kovacs et al., 2015). The mecha- Melanocytes as instigators and victims of mal H2O2/ONOO( − ) − mediated stress in viti- oxidative stress. JInvestDermatol134:1512–8 nisms that underly the activation of ligo hampers indoleamine 2,3-dioxygenase and Eleftheriadou V, Thomas K, van Geel N et al. (2015) aryl hydrocarbon receptor-mediated immune melanocytes after punch grafting have not Developing core outcome set for vitiligo response signaling. FASEB J 26:2471–85 been fully explained. Nevertheless, the clinical trials: international e-Delphi consen- Wagner RY, Luciani F, Cario-Andre M et al. (2015) process might be regulated by epidermal sus. Pigment Cell Melanoma Res Altered E-cadherin levels and distribution in and dermal cells that should be able to Ezzedine K, Eleftheriadou V, Whitton M et al. melanocytes precedes manifestations of vitiligo. – manage cell adhesion actively. (2015) Vitiligo. Lancet J Invest Dermatol 135:1810 20 + Therapeutic options for vitiligo are still Ezzedine K, Lim HW, Suzuki T et al. (2012) Revised Wu J, Zhou M, Wan Y et al. (2013) CD8 T cells classification/nomenclature of vitiligo and related from vitiligo perilesional margins induce auto- limited. Although some mechanisms under- issues: the Vitiligo Global Issues Consensus Con- logous melanocyte apoptosis. Mol Med Rep 7: lying the interplay between oxidative stress ference. Pigment Cell Melanoma Res 25:E1–13 237–41 and immunity have been postulated (Richmond et al., 2013), understanding See related letter to the editor on pg 1921 mechanisms that cause oxidative stress could provide valuable information to identify new therapeutic targets. The chal- lenge will be to maintain cells metabo- EDA Fibronectin in Keloids Create a lically active as requirement for sustaining the energy demand and coping with oxida- Vicious Cycle of Fibrotic Tumor Formation tive stress. Moreover, the analysis has to be 1 1 2 extended to pigmented skin to both identify Rhiannon M. Kelsh , Paula J. McKeown-Longo and Richard A.F. Clark early events in “silent” vitiligo melanocytes During the early phase of wound healing, first plasma fibronectin (FN) and then in situ and prevent the spread of the disease. FN are deposited at the site of injury. In situ FN––FN made by tissue cells at the injury –– CONFLICT OF INTEREST site oftencontainsanextradomainA(EDA)insert. Multiple wound-related signal The authors state no conflict of interest. transduction pathways control the deposition of EDA FN, and the EDA insert can in turn trigger pathways that induce inflammation, increased extracellular matrix REFERENCES molecule deposition including FN and collagen, and activation of fibroblasts. Together Ardigo M, Malizewsky I, Dell’Anna ML et al. (2007) these pathways can create a vicious cycle that leads to fibrosis or keloid formation. Preliminary evaluation of vitiligo using in vivo Journal of Investigative Dermatology (2015) 135, 1714–1718. doi:10.1038/jid.2015.155 reflectance confocal microscopy. J Eur Acad Dermatol Venereol 21:1344–50 Bellei B, Pitisci A, Ottaviani M et al. (2013) Vitiligo: 1Center for Cell Biology & Cancer Research, Albany Medical College, Albany, New York, USA and a possible model of degenerative diseases. 2Departments of Dermatology and Biomedical Engineering, Stony Brook University, Stony Brook, New York, PLoS One 8:e59782 USA Bertolotti A, Boniface K, Vergier B et al. (2014) Correspondence: Paula J. McKeown-Longo, Center for Cell Biology & Cancer Research, Mail Code 165, Type I interferon signature in the initiation of Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208, USA or Richard A.F. Clark, the immune response in vitiligo. Pigment Cell Departments of Dermatology and Biomedical Engineering, Stony Brook University, HSC T-16, 060, Stony Melanoma Res 27:398–407 Brook, New York 11794-8165, USA. E-mail: [email protected] or [email protected]

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FN is an extracellular matrix (ECM)

EDA glycoprotein critical for embryogenesis, EDB morphogenesis, and wound repair (Clark, fi 2014). Although plasma bronectin (FN), NH CH which is synthesized by the liver, v circulates in blood as non-complexed SS molecules in a 1:10 ratio with fibrinogen SS (Greiling and Clark, 1997), little FN ECM NH CH exists in fully developed tissue (Yamada

and Clark, 1996). However, tissue injury EDB or inflammation leads to rapid plasma FN EDA deposition in complex with fibrin followed by so-called cell FN deposition by tissue cells a few days later (Yamada Alternatively spliced Fn I Fn II Fn III Variable region and Clark, 1996). Plasma FN exists as Fn III v 250 kDa glycoprotein consisting of two fi fi chains, one with and one without a Figure 1. Depiction of the modular structure of bronectin. Plasma bronectin consists of two chains, made up of repeating, modular structures: type I (blue), type II (green), and type III (yellow). One chain variable domain (v). Although cell FN contains the variable region (purple) and the other does not. Alternative splicing of the gene results in the comes from the same gene as plasma FN, synthesis of fibronectin that contains the extra domains A (EDA) and B (EDB; pink). it contains additional spliced variants with extra domains A and/or B (EDA and/or EDB; Figure 1). Both plasma and circumstances of tissue remodeling, such mTOR signaling pathway facilitates cell FN are polymerized into fibrils and as during wound repair, tissue injury, phosphorylation and activation of the deposited into the ECM of most tissues. inflammation, and fibrosis (ffrench- splicing factor SF2/ASF (also known Most of the secondary structure of FN is Constant et al., 1989; Peters et al., as SRSF1), resulting in the increased organized into individually folded 1989; Satoi et al., 2000; Singh et al., expression of EDA FN. It was proposed domains based on amino acid homology 2004; Muro et al., 2008). The mecha- that the modulation of AKT activity was (Petersen et al., 1983). These domains, nisms that trigger upregulation of the EDA a direct result of TGF-β-induced termed Type I, II, and III, also represent FN isoform are both cell type specificand downregulation of PTEN (White et al., biologically sites that participate in the context dependent, making it critical to 2010). These studies suggest that TGF-β formation of matrix fibrils and provide understand the regulation of this process family members can control EDA levels binding sites for cells, other matrix mole- in response to extracellular signals. The by altering activity and/or expression cules, and growth factors (Schwarzbauer molecular mechanism controlling alter- levels of splicing factors. and DeSimone, 2011; Zhu and Clark, native mRNA splicing of the EDA exon In addition to TGF-β, growth factors, 2014). As FN is extremely sensitive to depends on spliceosome assembly and such as HGF and EGF, as well as stress proteolysis, FN must be deposited RNA secondary structure, as well as the signals, have been implicated in regulat- continuously from blood or from in situ serine/arginine (SR)-rich family of proteins ing increased levels of EDA FN (Inoue tissue cell production to sustain its pre- (Lavigueur et al., 1993; Mermoud et al., et al., 1999). Interestingly, it is known sence (Deno et al., 1983). With healing of 1994 Cramer et al., 1997; Buratti and that HGF works through the PI3K a wound or resolution of inflammation, Baralle, 2004). Growth factors, stress cascade to increase levels of EDA FN both plasma FN and cell FN disappear signals, ECM proteins, cytokines, and (Magnuson et al., 1991; Seebacher et al., rapidly from the tissue (Welch et al., 1990). stiffness have all been connected 1988; Blaustein et al., 2004), further Here we focus on FN containing EDA upstream of these SR proteins, ultimately implicating this signaling pathway in (EDA FN) that has been found to persist in leading to increased EDA FN expression regulating alternative splicing of FN. sites of skin fibrosis (Bhattacharyya et al., (Naro and Sette, 2013). Recent evidence suggests that cell–ECM 2014) and in keloids, as reported in this Over two decades ago, transforming interactions will alter levels of EDA FN. issue (Andrews et al., 2015). growth factor-β (TGF-β) was first identi- Srebrow et al. (2002) demonstrated that fied as a modulator of the EDA isoform a laminin-rich basement membrane can EDA FN inductive pathways levels in fibroblasts, resulting in an modulate the alternative splicing of EDA. During embryonic development, the EDA increased ratio of EDA to total FN Treatment of fibroblasts with type III and domain of FN is highly expressed. How- (Balza et al., 1988; Borsi et al., 1990; V collagens promoted the splicing and ever, its expression is transient as it reviewed in Leask and Abraham increased assembly of EDA FN (Zoppi disappears with the increasing age of an (2004)). Differential cell type–specific et al., 2012). Furthermore, the EDA organism (Vartio et al., 1987; Pagani signaling axes downstream of TGF-β domain itself has recently been et al., 1991). In fact, the EDA domain of have also been identified in the implicated in promoting FN synthesis FN is largely absent from normal, adult regulation of EDA FN splicing. In and fibrillogenesis by dermal fibroblasts tissue (Oyama et al., 1989). It becomes mouse embryonic fibroblasts, it was (Shinde et al., 2015). Further studies upregulated, however, under specific shown that activation of the PI3K/AKT/ designed to understand the molecular

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TGF-β Keratinocyte EMT Fibroblast TGF-β ECM Epithelial to mesenchymal ECM Cytokines molecules Cytokines transition stiffness

α α4 β7 9 β1 α4 β1 TLR4 TLR4 TLR4 TLR4

9 10 EDA V

EDA+Fn

Keloid fibroblasts

Figure 2. Feed-forward fibrotic loop contributes to Keloid formation. The influence of the pro-fibrotic cytokine, TGF-β, on both epithelial and fibroblast cell types can result in increased synthesis and deposition of EDA FN. Integrin receptors and TLR-4 on the surface of both cell types mediate intracellular signal transduction pathways in response to EDA FN resulting in cytokine production, epithelial–mesenchymal transition (EMT), fibroblast activation, and synthesis of matrix proteins. These cellular outcomes in response to EDA FN all contribute to the fibro-inflammatory environment driving keloid formation. EDA, extra domain A; FN, fibronectin; TGF-β, transforming growth factor-β; TLR-4, toll-like receptor 4.

mechanisms regulating EDA FN splicing EDA has also recently received atten- Other receptors, in addition to TLR-4, should provide important insights into tion due to its contribution to patholo- have been identified for the EDA novel strategies for the treatment of gical inflammation. The major receptor domain of FN: integrins α4β1, α9β1, fibrosis and excessive tissue remodeling believed to mediate EDA's role in and α4β7 (Liao et al., 2002; Shinde seen in hypertrophy and keloids. inflammation is the innate immune et al., 2008; Kohan et al., 2010). The receptor, toll-like receptor 4 (TLR-4). role of these receptors in EDA-mediated EDA FN triggered pathways EDA FN has been shown to activate fibrosis and inflammation is still some- EDA FN and TGF-β, coupled with inflammatory pathways in dermal fibro- what uncertain. EDA FN was shown to α β mechanical stress, are considered the blasts, mast cells, cytotoxic T Cells, and work through 4 1 to promote a con- fl leading in uences in the differentiation monocytes via TLR-4 (Okamura et al., tractile phenotype in human dermal fi fi fi of broblasts into myo broblasts, a 2002; Gondokaryono et al., 2007; broblasts (Shinde et al., 2015). EDA fi β critical cell type in brotic disease Lasarte et al., 2007; McFadden et al., has also been shown to mediate TGF- - fi (Desmouliere et al., 1993; Vyalov et al., 2011; Kelsh et al., 2014). dependent myo broblast differentiation 1993; Serini et al.,1998).Animalmodels and collagen synthesis via the α4β7- of scleroderma, atherosclerosis, cardiac Understanding the mole- dependent activation of the MAPK/ allograft rejection, and adverse airway ERK1/2 pathway. Integrin α9β1 has remodeling have demonstrated that EDA cular pathways involving also been shown to regulate PI3K/AKT FN promotes the fibrotic phenotypes that the expression of EDA and ERK1/2 activation in response to drive pathological progression (Tan et al., fibronectin and its ability EDA FN, ultimately leading to 2004; Arslan et al., 2011; Kohan et al., epithelial–mesenchymal transition in 2011; Booth et al., 2012; Bhattacharyya to enhance stimulation lung cancer cells (Sun et al., 2014). et al., 2014). In addition, EDA FN has of those pathways––i.e., The role of each of these receptors in been used clinically as an indicator of the progression of fibrotic disease fibrotic disease and tissue remodeling in a positive feedback remains an important question. humans (van der Straaten et al., 2004; loop––it is hoped that Wound repair of full-thickness skin Peters et al., 2011). Recently, EDA FN new, more effective injury ideally ends after a few months was shown to increase in skin lesions with a flat, non-tender scar that is ~ 70% and serum from patients with treatment of keloids can as strong as normal skin (Gurtner et al., scleroderma (Bhattacharyya et al., 2014). break this vicious cycle. 2011). Hypertrophic scars and keloids,

1716 Journal of Investigative Dermatology (2015), Volume 135 COMMENTARY

however, sometimes intervene. These insufficient to induce skin fibrosis novel mechanism for maintaining and ampli- two abnormal responses to wound (Campaner et al., 2006). Moreover, in fying fibrosis in scleroderma. Am J Pathol 182: – repair have fundamentally different at least one scenario, TLR-4 was found 192 205 clinical and histological appearances to activate the beta-catenin signaling. Bhattacharyya S, Tamaki Z, Wang W et al. (2014) FibronectinEDA promotes chronic cutaneous and distinct clinical outcomes. Hypertro- Hence, TLR-4 stimulation by EDA FN fibrosis through Toll-like receptor signaling. phic scars do not invade, but rather might augment TGF-β responses both Sci Transl Med 6:232ra50 generate tensile stress, on the surroun- directly and indirectly creating a vicious Blaustein M, Pelisch F, Coso OA et al. (2004) ding tissue, whereas keloids do the cycle of keloidal fibrotic tumor forma- Mammary epithelial-mesenchymal interaction fi opposite (Bolognia et al.,2012).Given regulates bronectin alternative splicing via tion. The activation of innate immune phosphatidylinositol 3-kinase. JBiolChem these facts it is not surprising that hyper- pathway signaling downstream of TLR- 279:21029–37 fi trophic scar broblasts, but not keloid -4, culminating in the production of Bolognia JL, Jorizzo JL, Schaffer JV (2012) Derma- fibroblasts, express α-smooth muscle cytokines and other pro-inflammatory tology 3rd (edn). Elsevier fi actin, indicative of a myo broblast mediators, suggests that EDA FN has the Booth AJ, Wood SC, Cornett AM et al. (2012) phenotype that can generate substantial ability to act as a damage-associated Recipient-derived EDA fibronectin pro- fi tensile forces on surrounding ECM motes cardiac allograft brosis. JPathol226: molecular pattern (DAMP). Endogenous 609–18 (Ehrlich et al., 1994). In contrast, keloid DAMPs that arise from the ECM, fi Borsi L, Castellani P, Risso AM et al. (1990) broblasts demonstrate a greater such as the EDA FN, are often gene- Transforming growth factor-beta regulates the propensity to proliferate in serum-free rated during tissue remodeling and splicing pattern of fibronectin messenger RNA medium (Russell et al.,1988),resistcell fibrosis. Subsequently, these DAMPs precursor. FEBS Lett 261:175–8 apoptosis (Chodon et al., 2000), and can trigger inflammation and further Buratti E, Baralle FE (2004) Influence of RNA have increased sensitivity to TGF-β, fi secondary structure on the pre-mRNA splicing contribute to the brotic phenotype in process. Mol Cell Biol 24:10505–4 which induces synthesis and deposition a vicious, feed-forward cycle (Huebener of ECM molecules including FN and Campaner AB, Ferreira LM, Gradnani A et al. and Schwabe, 2013). Targeting the (2006) Upregulation of TGF-beta1 expression collagen (Babu et al., 1992; Bettinger pathways regulating the expression of may be necessary but is not sufficient for β β et al., 1996). In fact, TGF- 1and 2, the EDA FN as well as its receptors may be excessive scarring. J Invest Dermatol 126: β 1168–76 TGF- isoforms related to increased key in controlling EDA FN–mediated fibroblast ECM production and tissue fl fi Chodon T, Sugihara T, Igawa HH et al. (2000) in ammation and brosis. fi fibrosis, are increased in keloid Keloid-derived broblasts are refractory to Fas- fi mediated apoptosis and neutralization of broblasts compared with normal CONFLICT OF INTEREST autocrine transforming growth factor-beta1 fibroblasts and are thought to be largely The authors state no conflict of interest. can abrogate this resistance. Am J Pathol 157: fi ’ 1661–9 responsible for broblasts aggressive ACKNOWLEDGMENTS fibroproliferative nature in keloids (Xia This was supported by NIH CA058626 to PM-L. Clark RAF (2014) Wound repair: basic biology to et al.,2004).Furthermore,TGF-β1and tissue engineering. In: Lanza RP, Langer RC, β Vacanti JP (eds). Principles of Tissue Engineering, 2 also increase the expression of EDA REFERENCES 4th edn. Elsevier: Amsterdam, Boston FN (Viedt et al., 1995) and thus may be Akhmetshina A, Palumbo K, Dees C et al. (2012) Colwell AS, Phan TT, Kong W et al. (2005) responsible for the increased EDA FN Activation of canonical Wnt signalling is Hypertrophic scar fibroblasts have increased observed in keloids (Andrews et al., required for TGF-beta-mediated fibrosis. Nat connective tissue growth factor expression 2015). Interestingly, EDA FN stimulates Commun 3:735 after transforming growth factor-beta stimula- tion. 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