Modulation of metallothionein isoforms is associated with collagen deposition in proliferating keloid fibroblasts in vitro.
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.
Toh
Pearl P-C
PP
Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Li
Jasmine J
JJ
Yip
George W-C
GW
Lo
Soo-Ling
SL
Guo
Chun-Hua
CH
Phan
Toan-Thang
TT
Bay
Boon-Huat
BH
D049109:Cell Proliferation; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics / Q000378:metabolism*; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D015536:Down-Regulation / Q000235:genetics; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007627:Keloid / Q000473:pathology*; D015603:Keratinocytes / Q000378:metabolism / Q000473:pathology; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D044967:Serum / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics; D012867:Skin / Q000378:metabolism / Q000473:pathology; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D015854:Up-Regulation / Q000235:genetics
D016895:Culture Media, Serum-Free; C530099:MT1A protein, human; C541541:MT1E protein, human; C501902:MT1F protein, human; C499629:MT1G protein, human; C491053:MT2A protein, human; D016328:NF-kappa B; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1; C407523:metallothionein isoform 1; D003094:Collagen; D008668:Metallothionein