Targeting of Sp1 transcription factor: a novel therapeutic approach for keloids, an in vitro analysis.

Keloid scars are fibroproliferative disorders characterized by the accumulation of extracellular matrix (ECM) components resulting in a fibrotic condition. Several ECM promoters are regulated by Sp1. Thus, our aim was to investigate the role of Sp1 in keloid pathogenesis and investigate the antiproliferative and antifibrotic effects of Wp631 and mitoxantrone, potent inhibitors of Sp1-activated transcription. An elevated level of Sp1 was observed in tissue extracts obtained from keloid tissue. Serum stimulation elevated Sp1 levels in keloid fibroblasts (KF). Under coculture conditions Sp1 seemed to be downregulated. Wp631 and mitoxanthrone in serum growth factors resulted in a reduced expression of ECM components in KF. Both Wp631 and mitoxanthrone were also able to inhibit the proliferation of normal and keloid keratinocytes and fibroblasts significantly. As Wp631 seems to be potent in downregulating the ECM components in KF and also inhibiting the proliferation of these cells it could be explored as a possible therapeutic agent in the treatment of keloids.

mTOR as a potential therapeutic target for treatment of keloids and excessive scars.

Keloid is a dermal fibroproliferative disorder characterized by excessive deposition of extracellular matrix (ECM) components such as collagen, glycoproteins and fibronectin. The mammalian target of rapamycin (mTOR) is a serine/theronine kinase which plays an important role in the regulation of metabolic processes and translation rates. Published reports have shown mTOR as regulator of collagen expression and its inhibition induces a decrease in ECM deposition. Our aim was to investigate the role of mTOR in keloid pathogenesis and investigate the effect of rapamycin on proliferating cell nuclear antigen (PCNA), cyclin D1, collagen, fibronectin and alpha-smooth muscle actin (alpha-SMA) expression in normal fibroblasts (NF) and keloid fibroblasts (KF). Tissue extracts obtained from keloid scar demonstrated elevated expression of mTOR, p70KDa S6 kinase (p70S6K) and their activated forms, suggesting an activated state in keloid scars. Serum stimulation highlighted the heightened responsiveness of KF to mitogens and the importance of mTOR and p70S6K during early phase of wound healing. Application of rapamycin to monoculture NF and KF, dose- and time-dependently downregulates the expression of cytoplasmic PCNA, cyclin D1, fibronectin, collagen and alpha-SMA, demonstrating the anti-proliferative effect and therapeutic potential of rapamycin in the treatment of keloid scars. The inhibitory effect of rapamycin was found to be reversible following recovery in the expression of proteins following the removal of rapamycin from the culture media. These results demonstrate the important role of mTOR in the regulation of cell cycle and the expression of ECM proteins: fibronectin, collagen and alpha-SMA.

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

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.