Abstract 283: Redox-mediated Regulation of Histone deacetylase4, a Class II HDAC, by Thioredoxin1 through Interaction with DnaJb5, a Heat Shock Protein 40
Thioredoxin1 (Trx1) reduces redox-sensitive proteins and regulates cell growth and death. We previously reported that cardiac hypertrophy induced by pressure-overload is suppressed in mice with cardiac specific overexpression of Trx1 (Tg-Trx1). To elucidate the mechanisms by which Trx1 suppresses cardiac hypertrophy, we performed DNA microarray analysis using Tg-Trx1 mouse hearts. We identified DnaJb5, a heat shock protein 40, as a gene significantly upregulated by Trx1. Immunostaining and immunoblot analyses indicated that Trx1 and DnaJb5 are co-localized in the nucleus of myocytes. Pull-down and immunoprecipitation assays showed that DnaJb5 interacts with TBP-2, a Trx1-binding protein. DnaJb5 did not disturb the interaction between Trx1 and TBP-2, and enhanced the Trx1 reducing activity. Both Trx1 and DnaJb5 attenuated phenylephrine (PE)-induced activation of NFAT and myocyte hypertrophy in vitro. Using transgenic mice harboring an NFAT luciferase reporter, we confirmed that Trx1 suppresses both NFAT activation and cardiac hypertrophy induced by PE in vivo. We also found that DnaJb5 binds directly to histone deacetylase 4 (HDAC4), a class II HDAC. An HDAC4 mutant lacking the minimal region responsible for the interaction with DnaJb5 (residues 628 – 881) was localized in the cytosol, in contrast to the nuclear localization of the wild-type HDAC4, suggesting the importance of the interaction for the nuclear localization of HDAC4. Overexpression of Trx1 suppressed PE-induced nuclear export of HDAC4 in myocytes. Using mass spectroscopy, we found that HDAC4 forms a disulfide bond between Cys-667 and -669, which was reduced by Trx1. The HDAC4 Cys667/669Ser mutant was localized in the cytosol, and its nuclear export was suppressed by leptomycin B, an inhibitor of exportin, suggesting that the redox modification induces nuclear export regardless of phosphorylation. Consistently, the Cys667/669Ser substitution abolished the suppressive effect of HDAC4 on NFAT activity and cardiac hypertrophy. Collectively, these results show that Trx1 upregulates DnaJb5, which recruits HDAC4 into the complex formed by Trx1-TBP-2-DnaJb5, thereby reducing HDAC4, retaining its nuclear localization, and suppressing NFAT activity and cardiac hypertrophy.