Heat shock protein 90 inhibitor decreases collagen synthesis of keloid fibroblasts and attenuates the extracellular matrix on the keloid spheroid model

Abstract

The 90-kDa heat-shock protein (Hsp90) is very abundant cytosolicproteins representing more than 1% of totalproteins, andthis is induced in response to a widevariety of physiological and environmental stress. Interestingly,inhibition ofHsp90 by 17-allylamino-demethoxy-geldanamycin (17AAG), which is a geldanamycin analog thatspecifically inhibits the ATPase activity of Hsp90, compromises TGFβ-mediated transcriptionalresponses byenhancing TGF- β receptor I and II degradation, thus preventingSmad2/3 activation.Keloid scars are pathologicproliferations of the dermal skin layer resulting from excessive collagen deposition.Also, TGF-β is one of themost well-studied growth factors and seems to play the main role in the pathophysiology of keloids. Thus, weexamined whether Hsp90 might regulate TGF-β signaling in pathogenesis and treatment of keloids andinvestigated the expression of Hsp90 in keloid tissue and normal tissue by immunohistochemistry (IHC).Hsp 90 protein expression was examined byimmunohistochemistry in keloid tissues. HDFs and KFs weretreated with a various amount (5, 10, 20 μM) of 17AAG and mRNA levels of collagen type I and III wereassessed by real time RT-PCR. The effect of 17AAG onprotein expression of kinase B (Akt)and Smad 2/3complex was evaluated by Western blot assay.Additionally, the expression levels of major extracellular matrix(ECM)were investigated by Masson-Trichrome stain and immunohistochemistry in keloid spheroids treatedwith a various amount of 17AAG.Overexpression of Hsp90 in human keloid tissuesamplescompared to the adjascent normal tissue. Also,Hsp90 inhibitor such as 17AAG decreased the mRNA expression of type I collagen and Smad-2/3 complexin KFs.Masson’s trichrome staining of keloid sections revealed that collagen deposition was decreasedin spheroidstreated with 17AAG. In addition, dense and coarse collagen bundles were replaced by thin and shallow collagen2bundles. Immunohistochemical analysis using keloid spheroid showed that expressions of ECM proteinssuch ascollagen I, III, fibronectin, and elastin were markedly decreased in keloid spheroidstreated with 17AAG.These results suggest that the antifibrotic effect ofHsp90 inhibitor such as 17AAG may have therapeuticeffects on keloids.