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) κB activities were evaluated by immunofluorescent microscopy and enzyme-linked immunosorbent assay. Collagen α1 and α-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 NFκB nuclear translocation and activation, specifically by inducing complex formation between NFκB and the glucocorticoid receptor. In accordance, NFκB-dependent cellular FLICE-like inhibitory protein expression level was found to be reduced by 17AAG. Finally, 17AAG down-regulated collagen α1 and α-smooth muscle actin expression levels in HSCs before inducing apoptosis. These results demonstrate that the Hsp90 inhibitor induces HSC apoptosis via a sphingomyelinase- and NFκB-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.
Footnotes
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This work was supported by the Korea Health 21 Research and Development Project[Grant 0412-CR01-0704-0001]; and the Korean Foundation of Liver Research [Grant 2008].
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.109.151860.
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ABBREVIATIONS: HSC, hepatic stellate cell; Hsp, heat shock protein; 17AAG, 17-(allylamino)-17-demethoxygeldanamycin; TGF, transforming growth factor; LPS, lipopolysaccharide; SNP, sodium nitroprusside; SIN, 3-morpholinosydnonimine; ELISA, enzyme-linked immunosorbent assay; NF, nuclear factor; cFLIP, cellular FLICE-like inhibitory protein; PCR, polymerase chain reaction; GR, glucocorticoid receptor; IKK, IκB kinase.
- Received February 3, 2009.
- Accepted March 26, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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