Smad3 signalling plays an important role in keloid pathogenesis via epithelial–mesenchymal interactions

Abstract

Smad signalling plays important roles in developmental and cancer biology as well as in fibropathogenesis. Its role in keloid biology is not known. Epithelial-mesenchymal interactions, originally described in normal skin, have recently been established to play a significant role in keloid pathogenesis, and demonstrate the important influence of keratinocyte paracrine factor signalling on fibroblast behaviour. The present study investigated the role of downstream Smad cascade induction in this interaction. Normal fibroblasts (NF) and keloid fibroblasts (KF) were co-cultured in serum-free medium with normal keratinocytes (NK) or keloid keratinocytes (KK) for 5 days, after which fibroblast cell lysates were subjected to western blot and immunoprecipitation analysis to quantify the levels of Smad and Smad2/3/4 binding complex. In another set of experiments, wild-type (wt), Smad2-null (Smad2-/-) and Smad3-null (Smad3-/-) mouse embryonic fibroblasts (MEF) were assayed for cell proliferation and collagen production after serum-free co-culture with KK or exposure to conditioned media collected from serum-free KK/KF co-culture. Compared to normal skin, keloids expressed high basal levels of TGFbetaR1 and TGFbetaR2, Smad2, 3 and 4 and phospho-Smad2. Upregulation of TGFbetaR1 and TGFbetaR2, Smad3 and p-Smad2 was observed in KF co-cultured with KK, together with enhanced Smad3 phosphorylation and Smad2/3/4 binding complex production. When MEF-wt, MEF-Smad2-/- or MEF-Smad3-/- were co-cultured with KK or exposed to KK/KF co-culture conditioned media, enhanced proliferation and collagen production were seen in MEF-wt and MEF-Smad2-/- but not in MEF-Smad3-/- cells. The activation of Smad signalling, importantly that of Smad3, appears to be one facet of the complex epithelial-mesenchymal interactions in keloid pathogenesis, resulting in active KF proliferation and collagen-ECM production in co-culture with KK. This finding suggests the suppression of Smad signalling as a novel approach in keloid therapy.

Adenoviral Overexpression and Small Interfering RNA Suppression Demonstrate That Plasminogen Activator Inhibitor-1 Produces Elevated Collagen Accumulation in Normal and Keloid Fibroblasts

“…47 Such choices for analysis will also minimize cell selection but may allow inclusion of a small proportion vascular cells or epithelial cells that inform epithelial-mesenchymal communication to support the fibroblast keloid phenotype. 18 The molecular mechanisms that drive inherent collagen accumulation and dictate other aspects of the keloid phenotype remain elusive, but several possibilities have been suggested by both gene arrays and directed studies. Among these are two that interact with the PAI-1-uPA system.…”

“…[5][6][7][8][9][10][11][12] Modifications in growth factor-regulated cell growth and apoptosis, intracellular signal transduction, and epidermis-dermis interactions have been reported. [13][14][15][16][17][18][19][20][21] Changes in the level of genes involved in the above phenomena were also detected in gene profiling studies. 22,23 Despite these findings, knowledge-based specific approaches for prevention and efficacious treatment of keloids are still absent, and the lack of proper in vitro and animal models that recapitulate the pathophysiology of keloids further hampers progress.…”

Altered expression of hyaluronan synthase and hyaluronidase mRNA may affect hyaluronic acid distribution in keloid disease compared with normal skin

“…While expression of mRNA and proteins often correspond, the level of correlation can vary (9). Wound healing is a complex process, with many growth factors and cytokines implicated in the regulation of both HA synthesis and degradation and the remodelling of ECM (10-12), several of which are differentially regulated and implicated in KD (13,14). A number of ECM molecules are involved in tissue remodelling, the majority of these such as collagen I, fibronectin and other glycosaminoglycans are increased in KD (1); however, the reasons for dysregulated HAS and HYAL expression, their role in lower HA concentrations in KD tissue and any subsequent effect in vivo remain elusive.…”

Transforming growth factor-β and fibrosis

“…In addition, increased expression of TβRI and TBRⅡ, and increased phosphorylation of Smad3 in keloid fibroblasts, have also been reported [34] , supporting the hypothesis that TGF-β/Smad signaling plays a central role in keloid pathogenesis. Further more, the activation of Smad signaling, importantly that of Smad3, appears to be one facet of the complex epithelial-mesenchymal interactions in keloid pathogenesis, resulting in active keratinocyte proliferation and collagen production by fibroblasts [35] . Skin tissue fibrosis may also be a sequel of both radiotherapy or accidental exposure to gamma irradiation [36] .…”