Heat Shock Protein 90 Inhibitor Induces Apoptosis and Attenuates Activation of Hepatic Stellate Cells

Abstract

Activated hepatic stellate cells (HSCs) are major participants in hepatic fibrosis; thus, the induction of HSC apoptosis has been proposed as an antifibrotic treatment strategy. Heat shock protein (Hsp) 90 is a molecular chaperone that stabilizes major signal transduction proteins, and its inhibitors have antitumor activity. In this study, the susceptibility of HSCs to an Hsp90 inhibitor was evaluated. LX-2 cells, an immortalized human HSC line, 17-(allylamino)-17-demethoxygeldanamycin (17AAG), an Hsp90 inhibitor, and monensin, an acidic sphingomyelinase inhibitor, were used in this study. Cellular apoptosis was quantified by 4`,6-diamidino-2-phenylindole dihydrochloride staining, and signaling cascades were explored using immunoblotting and immunoprecipitation techniques. Nuclear factor (NF) kappa B activities were evaluated by immunofluorescent microscopy and enzyme-linked immunosorbent assay. Collagen alpha 1 and alpha-smooth muscle actin expressions were determined by real-time reverse transcription-polymerase chain reaction and immunoblotting, respectively. It was found that 17AAG induced HSC apoptosis and that caspase 8 cleavage preceded the downstream activation of apoptotic signaling cascades. Furthermore, this caspase 8 activation was dependent on ceramide generation by acidic sphingomyelinase. In addition, 17AAG prevented NF kappa B nuclear translocation and activation, specifically by inducing complex formation between NF kappa B and the glucocorticoid receptor. In accordance, NF kappa B-dependent cellular FLICE-like inhibitory protein expression level was found to be reduced by 17AAG. Finally, 17AAG down-regulated collagen alpha 1 and alpha-smooth muscle actin expression levels in HSCs before inducing apoptosis. These results demonstrate that the Hsp90 inhibitor induces HSC apoptosis via a sphingomyelinase- and NF kappa B-dependent mechanism. Because this inhibitor also reduces HSC activation before apoptosis, Hsp90 inhibitor treatment might be therapeutically useful as an antifibrotic strategy in a variety of liver diseases.