Effect of heat shock protein 90 on TGF-β induced collagen synthesis of human dermal fibroblast

Abstract

Heat shock proteins (Hsps) interact with non-native proteins under various stressful conditions, prevent abnormal protein folding, and aid in proper protein folding. Recently, it was shown that various Hsps are involved in the wound healing process. Hsp 90 is one of several stress proteins, and its expression and activity are upregulated under stressful conditions. It has been shown that Hsp 90 is involved in the fibrosis pathway through stabilization of TGF-β receptors and Src kinases, which are important in the intracellular fibrosis process. The aim of present study was to investigate the role of Hsp 90 in TGF-β induced collagen synthesis of human dermal fibroblasts. Human dermal fibroblast cells were cultured, and the effects of TGF-β, 17-N-allylamino-17 demethoxygeldanamycin (17AAG), and transfection of Hsp 90 plasmids on cell viability were evaluated. Real-time PCR, western blot, and immunofluorescence assay were performed to evaluate the influence of Hsp 90 level on TGF-β induced collagen synthesis. Western blot analysis was also used to investigate the Smad 2/3 and Akt pathways, and to identify the signal pathways involved in collagen synthesis. Finally, the expression of Hsp 90 and collagen were compared in keloid and control tissue by immunohistochemical analysis. The expression of collagen was significantly increased after treatment with TGF-β, both at the RNA and protein levels. Pretreatment with 17AAG inhibited TGF-β induced collagen synthesis. Overexpression of Hsp 90 increased TGF-β induced collagen synthesis. Pretreatment with 17AAG and overexpression of Hsp 90 affected the Smad pathway through modulation of Smad 2/3 phosphorylation. However, involvement of the Akt pathway was not obvious. Using human skin tissue, we found that expression of Hsp 90 was increased in keloid compared with control tissue. We found that inhibition or overexpression of Hsp 90 in human dermal fibroblast influenced TGF-β induced collagen synthesis through modulation of Smad 2/3 phosphorylation. Furthermore, the expression of Hsp 90 was increased in keloid tissue compared with control tissue, supporting the results of our in vitro experiments showing that Hsp 90 is involved in the TGF-β induced collagen synthesis.