Partial epithelial-mesenchymal transition in keloid scars: regulation of keloid keratinocyte gene expression by transforming growth factor-β1

Abstract

BackgroundKeloids are an extreme form of abnormal scarring that result from a pathological fibroproliferative wound healing process. The molecular mechanisms driving keloid pathology remain incompletely understood, hindering development of targeted, effective therapies. Recent studies in our laboratory demonstrated that keloid keratinocytes exhibit adhesion abnormalities and display a transcriptional signature reminiscent of cells undergoing epithelial-mesenchymal transition (EMT), suggesting a role for EMT in keloid pathology. In the current study, we further define the EMT-like phenotype of keloid scars and investigate regulation of EMT-related genes in keloid.MethodsPrimary keratinocytes from keloid scar and normal skin were cultured in the presence or absence of transforming growth factor beta 1 (TGF-β1) +/− inhibitors of TGF-β1 and downstream signaling pathways. Gene expression was measured using quantitative polymerase chain reaction. Migration was analyzed using an in vitro wound healing assay. Proteins in keloid scar and normal skin sections were localized by immunohistochemistry. Statistical analyses utilized SigmaPlot (SyStat Software, San Jose, CA) or SAS® (SAS Institute, Cary, NC).ResultsIn keloid and normal keratinocytes, TGF-β1 regulated expression of EMT-related genes, including hyaluronan synthase 2, vimentin, cadherin-11, wingless-type MMTV integration site family, member 5A, frizzled 7, ADAM metallopeptidase domain 19, and interleukin-6. Inhibition of canonical TGF-β1 signaling in keloid keratinocytes significantly inhibited expression of these genes, and TGF-β1 stimulation of normal keratinocytes increased their expression. The inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway or the p38 mitogen-activated protein kinase pathway attenuated TGF-β1-induced expression of subsets of these genes. Migration of keloid keratinocytes, previously shown to be increased compared with normal keratinocytes, was significantly reduced by inhibition of TGF-β1 or ERK1/2 signaling. Biomarkers of EMT, including reduced E-cadherin and increased active β-catenin, were observed in keloid epidermis in vivo. However, evidence of basement membrane breakdown in keloid scar was not observed.ConclusionsThe results suggest that keloid keratinocytes exist in an EMT-like metastable state, similar to activated keratinocytes in healing wounds. The EMT-like gene expression pattern of keloid keratinocytes is regulated by canonical and non-canonical TGF-β1 signaling pathways. Therefore, interventions targeting TGF-β1-regulated EMT-like gene expression in keloid keratinocytes may serve to suppress keloid scarring.Electronic supplementary materialThe online version of this article (doi:10.1186/s41038-016-0055-7) contains supplementary material, which is available to authorized users.

The effect of tranilast on fibroblast activation protein α (FAP-α) expression in normal and keloid fibroblasts in vitro

“…Recent studies have noted the roles of keratinocytes in the pathogenesis of keloids [29,30]. Hahn et al demonstrated that keloid keratinocytes exhibit adhesion abnormalities and display a transcriptional signature reminiscent of cells undergoing epithelial-mesenchymal transition (EMT).…”

Presence of galectin-1 in the epidermal and dermal thickening of keloid tissues

“…This is why most studies on keloids focus on dermis and few on epidermis [4,17]. However, recent studies describe the epidermis of keloids as thickened, primarily due to hyperproliferation [4,7,17] and altered terminal differentiation [18] of keratinocytes,; some of them, indicating that keratinocytes also participate in the regulation of fibroblasts activity and might contribute to keloid pathogenesis [4,7,19]. On the other hand, several studies consider genetic predisposition as an important factor in the keloid formation and progression [2,5,9,20] whilst others deem the role of local mechanical Hematoxylin staining (H&E) from a biopsy of keloid showing reduced rete ridges, epidermal thickening, hyperkeratosis and spongiosis.…”

“…frequently occur on the anterior portion of the chest, shoulders, upper back, arms, cheeks and earlobes [1,2,[5][6][7][8][9][10]. Histopathologically, keloids are defined as inflammatory disorders characterized by exhibiting a thickened dermis containing numerous fibroblasts, abnormal vascularization, increased inflammatory immune cells, abundant hyalinised collagen bundles (keloidal collagen) and extracellular matrix (ECM) components including collagen, elastin, fibronectin and proteoglycans such as syndecan and versican, mainly produced by activated fibroblasts [1][2][3][4][5][6][7][8][9][10][11][12][13][14].…”

“…Moreover, there is evidence suggesting that epigenetic modifications would be involved in the pathogenesis of keloids by regulation of fibroblasts activation, proliferation and apoptosis, as well as collagen and elastin synthesis [3,[20][21][22]. Nevertheless, the etiology and pathophysiology of keloids is not fully elucidated yet [3][4][5][6][7]18].…”

“…Histopathologically, keloids are defined as inflammatory disorders characterized by exhibiting a thickened dermis containing numerous fibroblasts, abnormal vascularization, increased inflammatory immune cells, abundant hyalinised collagen bundles (keloidal collagen) and extracellular matrix (ECM) components including collagen, elastin, fibronectin and proteoglycans such as syndecan and versican, mainly produced by activated fibroblasts [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. These histological alterations proper of the keloid tissue have been associated with the elevated production of growth factors such as TGF-β, FGF-2, PDGF, EGF, IGF and VEGF which regulate fibroblast proliferation, ECM synthesis and angiogenesis, and pro-inflammatory cytokines including IL-1α, IL-1β, IL-6 and TNFα which promote fibroblast activation and consequently an excessive deposition of ECM components [1,2,[5][6][7]9,10,15,16]. This is why most studies on keloids focus on dermis and few on epidermis [4,17].…”