Keloid Pathogenesis: Potential Role of Cellular Fibronectin with the EDA Domain

JP Andrews et al. FN-EDA in Keloids

Table 2. FLG genotyping results for patients of white European ethnicity Cases, n (%) Comorbid atopic disease

22 (44.9)

Controls, n (%) Undetermined

FLG wild-type genotype

38 (77.6)

88 (88.0)

FLG heterozygote genotype

10 (20.4)

12 (12.0)

FLG homozygote or compound heterozygote Incomplete genotyping data Total χ2 analysis Odds ratio (95% CI)

0

0

1 (2.0)

0

49 (100.0)

100 (100.0)

P = 0.16 1.92 (0.77–4.85)

Abbreviations: CI, confidence interval; FLG, filaggrin. Cases with Fitzpatrick skin type of IV–VI were excluded from the genetic analysis because FLG loss-offunction mutations remain ill defined in these ethnic groups.

ACKNOWLEDGMENTS We are grateful to the patients who participated in this study. We acknowledge the help of Robert Dawe in recruitment of patients to the study and the Photobiology technicians for help in coordinating sample collection. SJB holds a Wellcome Trust Intermediate Fellowship (086398/Z/08/Z), and the Centre for Dermatology and Genetic Medicine, University of Dundee, is funded by a Wellcome Trust Strategic Award (098439/Z/12/Z) to WHIM.

Catriona P. Harkins1, Alex Waters1, Alastair Kerr1, Linda Campbell2, W.H. Irwin McLean2, Sara J. Brown3,4 and Sally H. Ibbotson1,4 1 Photobiology Unit, Department of Dermatology, University of Dundee, Ninewells Hospital and Medical School, Dundee, Scotland, UK; 2Dermatology and Genetic Medicine, Division of Molecular Medicine, College of Life Sciences and College of Medicine, Dentistry and Nursing, University of Dundee, Dundee, Scotland, UK and 3 Dermatology and Genetic Medicine, Division

of Cancer Research, College of Medicine, Dentistry and Nursing, Ninewells Hospital and Medical School, Dundee, Scotland, UK E-mail [email protected] 4 Joint senior authors. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

REFERENCES Barresi C, Stremnitzer C, Mlitz V (2011) Increased sensitivity of histidinemic mice to UVB radiation suggests a crucial role of endogenous urocanic acid in photoprotection. J Invest Dermatol 131:188–94 Baurecht H, Irvine AD, Novak N et al. (2007) Toward a major risk factor for atopic eczema: meta-analysis of filaggrin polymorphism data. J Allergy Clin Immunol 120:1406–12 Brown SJ, McLean WH (2012) One remarkable molecule: filaggrin. J Invest Dermatol 132: 751–62

Dawe RS, Crombie IK, Ferguson J (2000) The natural history of chronic actinic dermatitis. Arch Dermatol 136:1215–20 Dawe RS (2005) Chronic actinic dermatitis in the elderly - recognition and treatment. Drugs Aging 22:201–7 de Fine Olivarius F, Wulf HC, Crosby J (1996) The sunscreening effect of urocanic acid. Photodermatol Photoimmunol Photomed 12:95–9 Frain-Bell W, Lakshmipathi T, Rogers J et al. (1974) The syndrome of chronic photosensitivity and actinic recticuloid. Br J Dermatol 1974: 617–34 Hawk JLM (2004) Chronic actinic dermatitis. Photodermatol Photoimmunol Photomed 20: 312–4 Menage HD, Sattar NK, Haskard DO et al. (1996) A study of the kinetics and pattern of E-selectin, VCAM-1 and ICAM-1 expression in chronic actinc dermatitis. Br J Dermatol 134:262–8 Kerr A, Ibbotson S (2006) Chronic actinic dermatitis. Exp Rev Dermatol 1:451–61 Kezic S, O'Reagan GM, Yau N et al. (2011) Levels of filaggrin degradation products are influenced by both filaggrin genotype and atopic dermatitis severity. Allergy 66:934–40 Mildner M, Jin J, Eckhart L et al. (2010) Knockdown of filaggrin impairs diffusion barrier function and increases UV sensitivity in a human skin model. J Invest Dermatol 130:2286–94 Palmer CNA, Irvine AD, Terron-Kwiatkowski A et al. (2006) Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis. Nat Genet 38:441–6 Smith FJD, Irvine AD, Terron-Kwiatkowski A et al. (2006) Loss-of-function mutations in the gene encoding filaggrin cause icthyosis vulgaris. Nat Genet 337:337–42 Thyssen JP, Linneberg A, Ross-Hansen K et al. (2013) Filaggrin mutations are strongly associated with contact sensitization in individuals with dermatitis. Contact Dermatitis 68: 273–6

See related commentary on pg 1714

Keloid Pathogenesis: Potential Role of Cellular Fibronectin with the EDA Domain Journal of Investigative Dermatology (2015) 135, 1921–1924; doi:10.1038/jid.2015.50; published online 12 March 2015

TO THE EDITOR Fibronectins (FNs) are high molecular weight glycoproteins present in

extracellular connective tissue matrices (ECM) and extracellular fluids, including blood plasma. The human FN gene

Abbreviations: cFN, cellular fibronectin; ECM, extracellular matrix; EDA, extra domain A; FN, fibronectin; PBS, phosphate-buffered saline; TGF-β, transforming growth factor-β; TLR4, toll-like receptor 4; WT, wild type Accepted article preview online 16 Fenruary 2015; published online 12 March 2015

consists of 45 exons, and the primary mRNA transcripts are alternatively spliced to form up to 20 different mRNA variants (White et al., 2008). The FNs interact with other matrix macromolecules, such as collagens, glycosaminoglycans, and fibrin, as well as cell surface receptors, including www.jidonline.org 1921

JP Andrews et al. FN-EDA in Keloids

integrins α9β1, α5β1, and αvβ3, and tolllike receptor 4 (TLR4; Charo et al., 1990; Okamura et al., 2001). The precise role of individual isoforms of FN in ECM biology and pathology remains unclear. One of the alternatively spliced exons encodes the extra domain A (EDA), also known as extra type III repeat, that is regulated developmentally and is found exclusively in cellular FN (cFN) but not in plasma FN (Muro et al., 1999). The latter form is synthesized by hepatocytes and secreted into the circulation, whereas cFN is produced by a variety of cells, including fibroblasts and epithelial cells, and is deposited as fibrils in the ECM. The in vivo role of the EDA variant has been studied by constructing mouse strains either constitutively expressing (FN-EDA+/+) or excluding it (FN-EDA− / − ; Muro et al., 2003). No embryonic lethality or postnatal malformations were observed in the case of the homozygous mutant FN-EDA − / − mice, suggesting that EDA is not required for normal development. However, significant abnormalities were observed in adult FN-EDA− / − animals. Specifically, the EDA peptide segment 90

is not found in the skin of wild-type (WT) animals, but FN-EDA was shown to be essential for normal wound healing, particularly with respect to re-epithelization. Although wound healing in EDA+/+ mice was indistinguishable from EDAwt/wt mice, the wounds in FN-EDA− / − mice showed ulcerations in the epidermis resulting in delay of re-epithelialization. Furthermore, both FN-EDA+/+ and FN-EDA− / − mice had shortened life spans compared with WT animals. Compelling recent evidence has accumulated implicating a crucial role for FN-EDA not only in normal wound healing but also in fibroproliferative disorders characterized by increased production and deposition of ECM. Treatment of normal fibroblasts with transforming growth factor-β (TGF-β), a critical cytokine in the pathogenesis of a variety of fibroproliferative disorders, results in a substantial increase in FNEDA production (Balza et al., 1988; Chalmers, 2011). Interestingly, FN-EDA upregulation precedes that of collagen, and FN-EDA is required for the induction of the myofibroblastic phenotype by TGF-β. Furthermore, FN-EDA-null mice

failed to develop significant lung fibrosis after bleomycin administration (Muro et al., 2003). Recently, Varga and co-workers (Bhattacharyya et al., 2014) demonstrated increased FN-EDA in the skin and circulation of patients with scleroderma. In addition, exogenous FN-EDA potently stimulated collagen production and myofibroblast differentiation in vitro, which was mediated by TLR4. These observations stimulated our investigation of the role of FN-EDA in the development of keloids, an abnormal fibroproliferative response elicited by trauma to the skin of genetically susceptible individuals; the pathogenesis of this disorder remains obscure (Bran et al., 2009). Our results suggest that FN-EDA may have a critical role in the keloid disease process. Discarded tissue specimens were obtained anonymously from patients undergoing cosmetic surgical excision of keloids and from patients undergoing panniculectomy (for patient information, see Supplementary Table S1 online). The use of discarded tissue was approved by the Institutional Review Board of Thomas Jefferson University. The tissues

**

Fold gene expression

80 70 60 50 40 30 20 10 0 Keloid

Control

K1 K3–1 K3–2 C6

C7

C8

K1 K3–1 K3–2 C6

C7

C8

220 kD

42 kD Figure 1. Expression of FN-EDA in keloid tissues. Note 70-fold increase in mRNA levels in keloid tissue versus control (n = 5 for both groups; mean ± SE; P = 0.005851). (a) Western blot results for tissue protein isolates from three keloid samples and three control samples (b). A total of 20 μg protein was loaded into each well; the presence of fibronectin-EDA (220 kD) is demonstrated in keloid extracts versus near total absence in controls. Expression was normailzed in each sample to β-actin levels present in each protein isolate (42 kD). Immunofluorescence of frozen sections of keloid (c, left and d; scale bar = 30 μm) and control skin (c, right). Note the heavy decoration of the surfaces of the type III collagen fibers (shown in red) with FN-EDA antibody (shown in green) in keloids in contrast to a complete absence of FN-EDA in control skin (c, right). EDA, extra domain A; FN, fibronectin.

1922 Journal of Investigative Dermatology (2015), Volume 135

JP Andrews et al. FN-EDA in Keloids

were dissected, sterilized with betadine solution, subcutaneous adipose tissue was removed, and the samples were prepared for gene expression analysis. Total RNA was extracted from all specimens using the RNeasy Kit (Fibrous Tissue Kit, Qiagen, Alameda, CA) and reverse transcribed to cDNA. Primer pairs were designed to amplify FN-EDA gene sequences as well as the housekeeping gene, glyceraldehyde-3-phosphodehydrogenase, as a control (for primer sequence information, see Supplementary Table S2 online). Relative quantitative RT-PCR was performed to investigate the expression of total FN, as well as FN-EDA in both keloid and control specimens. As demonstrated previously (Kischer and Hendrix, 1983; Sible et al., 1994), the total FN expression was increased in keloids by 10.7-fold over controls. However, the results indicated a significant, up to 70-fold (P = 0.005851), increase in FNEDA mRNA in keloid tissues in comparison to controls (n = 5 both groups; Figure 1a). Thus, the relative increase in cFN-EDA in comparison to total FN was ~ 7-fold higher in keloids. To investigate whether the increased mRNA transcript levels result in similarly increased protein levels in keloid tissues, total protein was extracted from keloids and control tissue specimens via bead-mill homogenization in the presence of protein lysis buffer containing Radio-Immunoprecipitation Assay Buffer (RIPA) buffer, phenylmethylsulfonyl Fluoride (PMSF), and EDTA-free protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). 20 μg of total protein from each sample was subjected to SDS/PAGE (4–20% gel) in the presence of reducing agent. Protein was electrotransferred to polyvinylidene fluoride membrane, and nonspecific binding sites on the membrane were blocked by incubation in 5% milk for 1 hour at room temperature. The blot was incubated with anti-FN EDA antibody (IST-9) in 1:200 dilution overnight at 4 °C and anti-β actin primary antibody (ab8224, Abcam, Cambridge, MA) and then incubated with Licor 800cw infrared anti-mouse secondary antibody at 1:5,000 dilution for 1 hour at room temperature. After several washings, the

signal was determined with Odyssey infrared image scanner (LiCor Biotechnology, Lincoln, NE). Western blotting of the protein from keloids, as illustrated in Figure 1b, revealed a significantly enhanced level of FN-EDA of the molecular weight of ~ 220 kDa, corresponding to a FN monomer, whereas very little, if any, protein was found in extracts of control specimens. Localization of FN-EDA in keloid tissues was determined by immunofluorescence staining with the same antibody (IST-9 monoclonal hybridoma supernatant, undiluted) as used for western blotting, recognizing a 10-amino acid peptide sequence within the EDA segment, and an antibody to type III collagen (Rockland No. 600401-105-0.1), overnight at 4 °C followed by 3 washes in phosphate-buffered saline (PBS). Sections were incubated for 1 hour at room temperature with 1:200 dilutions in PBS of Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit secondary antibodies (Invitrogen, Grand Island, NY). After 3 washes in PBS, sections were then incubated at room temperature for 15 minutes with a 1:1,000 dilution of DAPI in PBS (Southern Biotech, Birmingham, AL). After washing 3X with PBS, sections were mounted in Fluoromount G (Southern Biotech) and visualized. The results indicated intense staining of FN-EDA in association with broad collagen fibers within the keloids, as visualized by anti-type III collagen antibody, whereas little, if any, staining was noted in control skin (Figure 1c and d). Thus, the keloidal lesions contain an abundance of FN-EDA. Although total FN expression has been shown to be higher in keloid fibroblasts compared with control dermal fibroblasts (Kischer and Hendrix, 1983; Sible et al., 1994), the high level of FN-EDA expression in keloid tissue has not been previously reported. Although the mechanisms of the high level of FN-EDA expression in keloid tissue and its virtual absence in normal tissue remain to be determined, its presence may explain in part the continuous excess matrix production. EDA acting through the TLR4 receptor may enhance the activation of fibroblasts, resulting in increased production of TGF-β and subsequent production

and accumulation of ECM molecules, particularly collagen, creating a positive feedback loop, thus establishing progressive fibrosis. This hypothesis suggests that blocking FN-EDA synthesis or its interactions with cell surface receptors may provide suitable strategies to interrupt the fibrotic reaction in keloids. CONFLICT OF INTEREST

The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank the clinical faculty and residents of the Department of Dermatology and Cutaneous Biology at Sidney Kimmel Medical College at Thomas Jefferson University for assistance in obtaining keloid samples. Patrick J Greaney, Division of Plastic Surgery at Thomas Jefferson University, provided control skin tissue. Carol Kelly assisted in manuscript preparation. The authors wish to thank the National Institutes of Health (T32-AR060715) and the Orion-Farmos Research Foundation for their support for research and medical education.

Jonathan P. Andrews1, Jaana Marttala1, Edward Macarak1,2, Joel Rosenbloom1,2 and Jouni Uitto1 1

Department of Dermatology and Cutaneous Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA and 2The Joan and Joel Rosenbloom Research Center for Fibrotic Diseases, Thomas Jefferson University, Philadelphia, Pennsylvania, USA E-mail: [email protected] SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

REFERENCES Balza E, Borsi L, Allemanni G et al. (1988) Transforming growth factor beta regulates the levels of different fibronectin isoforms in normal human cultured fibroblasts. FEBS Lett 228:42–4 Bhattacharyya S, Tamaki Z, Wang W et al. (2014) Fibronectin EDA promotes chronic cutaneous fibosis through Toll-like receptor signaling. Sci Transl Med 6:232ra50 Bran GM, Goessler UR, Hormann K et al. (2009) Keloids: current concepts of pathogenesis (review). Int J Mol Med 24:283–93 Chalmers RL (2011) The evidence for the role of transforming growth factor-beta in the formation of abnormal scarring. Int Wound J 8:218–23 Charo IF, Nannizzi L, Smith JW et al. (1990) The vitronectin receptor alpha v beta 3 binds fibronectin and acts in concert with alpha 5 beta 1 in promoting cellular attachment and spreading on fibronectin. J Cell Biol 111: 2795–800 Kischer CW, Hendrix MJ (1983) Fibronectin (FN) in hypertrophic scars and keloids. Cell Tissue Res 231:29–37

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JP Andrews et al. FN-EDA in Keloids

Muro AF, Caputi M, Pariyarath R et al. (1999) Regulation of fibronectin EDA exon alternative splicing: possible role of RNA secondary structure for enhancer display. Mol Cell Biol 19:2657–71 Muro AF, Chauhan AK, Gajovic S et al. (2003) Regulated splicing of the fibronectin EDA

exon is essential for proper skin wound healing and normal lifespan. J Cell Biol 162: 149–60 Okamura Y, Watari M, Jerud ES et al. (2001) The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem 276: 10229–33

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Sible JC, Eriksson E, Smith SP et al. (1994) Fibronectin gene expression differs in normal and abnormal human wound healing. Wound Repair Regen 2:3–19 White ES, Baralle FE, Muro AF (2008) New insights into form and function of fibronectin splice variants. J Pathol 216:1–14