Background: Keloid disease is a fibroproliferative disorder, with an ill-defined treatment that is characterized by excessive extracellular matrix deposition. Mechanical tension promotes deposition of extracellular matrix and overexpression of tension-related proteins, which is associated with keloid disease. The aim of this study was to investigate the effect of tension-related proteins on extracellular matrix steady-state synthesis in primary keloid fibroblasts.
Methods: Keloid fibroblasts (n = 10) and normal skin (n = 4) fibroblast cultures were established from passages 0 to 3. A panel of 21 tension-related genes from microarray data were assessed at mRNA (quantitative reverse-transcriptase polymerase chain reaction) and protein (in-cell Western blotting) levels. Three genes were significantly altered in keloid tissue and fibroblasts, and their functional role was assessed using siRNA knockdown.
Results: Hsp27, α2β1-integrin, and PAI-2 were significantly up-regulated (p < 0.05)in keloid tissue and fibroblasts compared with normal skin. Hsp27, α2β1-integrin, and PAI-2 expression was inhibited by RNA interference. Both the mRNA and protein levels of Hsp27, α2β1-integrin, and PAI-2 significantly decreased (p < 0.05) in keloid fibroblasts at 48 hours after transfection. After down-regulation of Hsp27, α2β1-integrin, and PAI-2, the expression of intracellular extracellular matrix was significantly reduced (p < 0.05). Water-soluble tetrazolium salt-1 assay showed that transfection of Hsp27, α2β1-integrin, and PAI-2 did not influence the viability/metabolic activity of keloid fibroblasts.
Conclusions: This study demonstrates overexpression of key tension-related proteins in keloid tissue and keloid fibroblasts. Knockdown of Hsp27, PAI-2, and α2β1-integrin by RNA interference attenuates the expression of mRNA and protein levels and certain other extracellular matrix molecules.