metallothionein and Keloid

The keloid fibroblast (KF) is known to have higher proliferative capacity than normal dermal fibroblast (NF). Metallothionein (MT), a metal-binding protein, has been reported to promote cell proliferation. In this study, we evaluated the expression of MT isoforms at the mRNA level in fetal bovine serum (FBS)-stimulated proliferating KF. Although the morphological appearance of NF and KF was similar when viewed under light, confocal and transmission electron microscopy, there was surprisingly a generally lower expression of MT isoforms in KF when compared with NF and also reduced MT staining in dermal fibroblasts of keloids as opposed to normal skin. Primary cultures of KF grown in 5% FBS or 10% FBS compared to without FBS demonstrated significantly higher proliferative activity and more abundant deposition of collagen. Contrary to expectation, MT-1A, -1F, -1G, -1X and -2A isoforms were significantly down-regulated in proliferating KF. Moreover, stimulating KF with TGF β1, which is known to promote collagen synthesis and keloid formation, increased expression of Collagen 1A and 3A genes accompanied by reduction in MT-2A gene expression. Furthermore, down-regulation of the MT-2A gene in proliferating KF by siRNA-mediated silencing enhanced cell proliferation with concomitant up-regulation of the NF-κB gene and 10 of 13 other NF-κB pathway-related genes analysed but no alteration of the Collagen 1 and Collagen 3 gene expression. It would appear that down-regulation of MT isoforms in proliferating KF, in particular MT-2A, enhances keloidogenesis with the possible involvement of the NF-κB signalling pathway.

Topics: Cell Proliferation; Cells, Cultured; Collagen; Culture Media, Serum-Free; Down-Regulation; Fibroblasts; Gene Expression; Humans; In Vitro Techniques; Keloid; Keratinocytes; Metallothionein; NF-kappa B; Protein Isoforms; RNA, Small Interfering; Serum; Signal Transduction; Skin; Transforming Growth Factor beta1; Up-Regulation

Keloids are proliferative growths of dermal collagen, usually resulting from excessive tissue response during wound healing. There is evidence that keratinocytes may promote keloidogenesis via epithelial-mesenchymal interactions. Metallothioneins (MTs) are known to be involved in the fundamental cellular processes of growth and apoptosis. In this study, we evaluated the expression of MT isoforms in normal and keloid keratinocytes. The expression patterns of ten functional MT isoforms were assessed using real-time RT-PCR in primary cultures of normal and keloid keratinocytes. The MT-2A isoform was the most abundant MT isoform in both normal and keloid keratinocytes while the MT-1B isoform was absent. There was a significant increase in the mRNA expression of four MT isoforms, viz. MT-1A, 1E, 1F and 2A in keloid keratinocytes as compared to normal keratinocytes. Up-regulation of MT-1A, 1E, 1F and 2A isoforms may play a part in the development of keloids by paracrine signaling.

Topics: Cells, Cultured; Humans; Keloid; Keratinocytes; Metallothionein; Microscopy, Electron, Transmission; Protein Isoforms; RNA, Messenger; Up-Regulation