Caspase-mediated Specific Cleavage of Human Histone Deacetylase 4

Histone deacetylase 4 (HDAC4) is a class II HDAC implicated in controlling gene expression important for diverse cellular functions, but little is known about how its expression and stability are regulated. We report here that this deacetylase is unusually unstable, with a half-life of less than 8 h. Consistent with the instability of HDAC4 protein, its mRNA was also highly unstable (with a half-life of less than 4 h). The degradation of HDAC4 could be accelerated by exposure of cells to ultraviolet irradiation. HDAC4 degradation was not dependent on proteasome or CRM1-mediated export activity but instead was caspase-dependent and was detectable in diverse human cancer lines. Of two potential caspase consensus motifs in HDAC4, both lying within a region containing proline-, glutamic acid-, serine-, and threonine-rich (PEST) sequences, we identified, by site-directed mutagenesis, Asp-289 as the prime cleavage site. Notably, this residue is not conserved among other class IIa members, HDAC5, -7, and -9. Finally, the induced expression of caspase-cleavable HDAC4 led to markedly increased apoptosis. These results therefore unexpectedly link the regulation of HDAC4 protein stability to caspases, enzymes that are important for controlling cell death and differentiation. Histone deacetylase 4 (HDAC4) is a class II HDAC implicated in controlling gene expression important for diverse cellular functions, but little is known about how its expression and stability are regulated. We report here that this deacetylase is unusually unstable, with a half-life of less than 8 h. Consistent with the instability of HDAC4 protein, its mRNA was also highly unstable (with a half-life of less than 4 h). The degradation of HDAC4 could be accelerated by exposure of cells to ultraviolet irradiation. HDAC4 degradation was not dependent on proteasome or CRM1-mediated export activity but instead was caspase-dependent and was detectable in diverse human cancer lines. Of two potential caspase consensus motifs in HDAC4, both lying within a region containing proline-, glutamic acid-, serine-, and threonine-rich (PEST) sequences, we identified, by site-directed mutagenesis, Asp-289 as the prime cleavage site. Notably, this residue is not conserved among other class IIa members, HDAC5, -7, and -9. Finally, the induced expression of caspase-cleavable HDAC4 led to markedly increased apoptosis. These results therefore unexpectedly link the regulation of HDAC4 protein stability to caspases, enzymes that are important for controlling cell death and differentiation. Histone deacetylases (HDACs) 1The abbreviations used are: HDAC, histone deacetylase; PEST, proline-, glutamic acid-, serine-, and threonine-rich; LMB, leptomycin B; GFP, green fluorescent protein. have been increasingly implicated in mediating diverse fundamental cellular activities. Based on sequence homology with their yeast orthologs, mammalian HDACs have been divided into three classes. Class I HDACs include HDAC1, -2, -3, -7, -8, and -11, whereas class II HDACs contain HDAC4, -5, -6, -7, -9, and -10 (for recent reviews, see Refs. 1Khochbin S. Verdel A. Lemercier C. Seigneurin-Berny D. Curr. Opin. Genet. Dev. 2001; 11: 162-166Crossref PubMed Scopus (330) Google Scholar, 2Grozinger C.M. Schreiber S.L. Chem. Biol. 2002; 9: 3-16Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 3Verdin E. Dequiedt F. Kasler H.G. Trends. Genet. 2003; 19: 286-293Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 4Yang X.J. Seto E. Curr. Opin. Genet. Dev. 2003; 13: 143-153Crossref PubMed Scopus (186) Google Scholar). Among class II, HDAC4, -5, -7, and -9 form a subclass known as class IIa, whereas HDAC6 and -10 constitute class IIb. A third class of mammalian HDACs includes the Sir2-like proteins Sirt1–7 (5Imai S. Johnson F.B. Marciniak R.A. McVey M. Park P.U. Guarente L. Cold Spring Harbor Symp. Quant. Biol. 2000; 65: 297-302Crossref PubMed Scopus (119) Google Scholar, 6North B.J. Verdin E. Genome Biol. 2004; 5: 224Crossref PubMed Scopus (439) Google Scholar). The most well characterized function of HDACs is the deacetylation of core histones, which in turn leads to the compaction of nucleosomes to repress gene transcription. HDACs have also been implicated in the deacetylation of nonhistone targets. For example, HDAC6 regulates the deacetylation of tubulin, which may in turn promote cell motility (7North B.J. Marshall B.L. Borra M.T. Denu J.M. Verdin E. Mol. Cell. 2003; 2: 437-444Abstract Full Text Full Text PDF Scopus (1237) Google Scholar, 8Hubbert C. Guardiola A. Shao R. Kawaguchi Y. Ito A. Nixon A. Yoshida M. Wang X.F. Yao T.P. Nature. 2002; 417: 455-458Crossref PubMed Scopus (1789) Google Scholar, 9Matsuyama A. Shimazu T. Sumida Y. Saito A. Yoshimatsu Y. Seigneurin-Berny D. Osada H. Komatsu Y. Nishino N. Khochbin S. Horinouchi S. Yoshida M. EMBO J. 2002; 21: 6820-6831Crossref PubMed Scopus (574) Google Scholar). Despite the increasing repertoire of cellular activities that have been found to involve HDACs, relatively little is known regarding mechanisms regulating their expression. For example, HDAC1 binding to the CCAAT/enhancer-binding protein α-promoter increased upon treatment of cells with a proteasome inhibitor, but the protein levels were not directly assessed (10Wiper-Bergeron N. Wu D. Pope L. Schild-Poulter C. Hache R.J. EMBO J. 2003; 22: 2135-2145Crossref PubMed Scopus (119) Google Scholar). HDAC5 and HDAC6 are ubiquitinated, but it is unclear how their stability is regulated (11Hook S.S. Orian A. Cowley S.M. Eisenman R.N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13425-13430Crossref PubMed Scopus (171) Google Scholar). Interestingly, HDAC1 and HDAC4 undergo sumoylation, a post-translational modification that is reminiscent of ubiquitination but does not appear to regulate protein degradation (12Colombo R. Boggio R. Seiser C. Draetta G.F. Chiocca S. EMBO Rep. 2002; 11: 1062-1068Crossref Scopus (79) Google Scholar, 13Kirsh O. Seeler J.S. Pichler A. Gast A. Muller S. Miska E. Mathieu M. Harel-Bellan A. Kouzarides T. Melchior F. Dejean A. EMBO J. 2002; 21: 2682-2691Crossref PubMed Scopus (265) Google Scholar). To assess how the levels of HDAC4 and other HDACs might be controlled, we measured their protein stability following the inhibition of de novo synthesis. HDAC4 was found to be exceptionally unstable, with a half-life of less than 8 h, far less stable than HDAC1, -2, -3, and -6. Analysis of truncation mutants of HDAC4 revealed that the instability was mediated by a proline-, glutamic acid-, serine-, and threonine-rich (PEST)-containing region that also contains two potential caspase cleavage sites. Surprisingly, the degradation of HDAC4 was neither proteasome-dependent nor inhibited by blocking active nuclear export but was blocked by the inhibition of caspase activity. Ultraviolet irradiation greatly accelerated the caspase-dependent degradation of HDAC4 protein. In addition, Asp-289 appeared to be essential for caspase cleavage and is not conserved among HDAC5, -7, and -9. The induced expression of caspase-cleavable HDAC4 led to increased apoptosis of HeLa cells when compared with induced expression of HDAC4 mutated at the caspase cleavage site. Together, these novel findings link the differential regulation of class IIa HDAC proteins to components of the intracellular apoptotic machinery. Cell Culture, Reagents, and Treatments—All cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA), and grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 15% fetal bovine serum at 37 °C in 5% CO2. Actinomycin, cycloheximide, lactacystin, ALLN, and MG-132 were from Sigma, leptomycin-B was from Bio-Source, and the cell-permeable caspase inhibitors DEVD-CHO (inhibitor of caspase-3) and YVAD-CHO (inhibitor of caspase-1) were from either Calbiochem or Bio-Source (Camarillo, CA), and both were used at 20 μm final concentration. All inhibitors were prepared as concentrated stock solutions in Me2SO. Caffeine, hydroxyurea, okadaic acid, and aphidicolin were all obtained from Sigma and used at the following final concentrations: caffeine, 2 mm; hydroxyurea, 2 mm; okadaic acid, 100 μm; and aphidicolin, 2 μg/ml. UV was performed with a GS UV Linker (Bio-Rad), whereas γ irradiation (ionizing irradiation) was delivered using a high dose rate cesium irradiator (12.84 grays/min). Mock-treated control cells were handled in similar manners except that no drugs were used. Analytic Methods—Cell lysates were prepared via scraping on ice and pelleting at 4 °C followed by resuspension in Laemmli buffer and sonication. For immunoblotting, samples (10 μg/lane) were boiled for 5 min and separated via SDS-PAGE and then transferred to nitrocellulose membranes. After transfer, the membranes were blocked with 5% nonfat milk in phosphate-buffered saline and then probed with the indicated primary antibodies followed by the appropriate secondary antibodies conjugated with horseradish peroxidase. Anti-HDAC4 polyclonal antibodies were purified as described previously (14Kao G.D. McKenna W.G. Guenther M.G. Muschel R.J. Lazar M.A. Yen T.J. J. Cell Biol. 2003; 160: 1017-1027Crossref PubMed Scopus (160) Google Scholar). Specifically, these antibodies were generated against either the SalI-HindIII fragment of HDAC4 (when translated, encompasses 601 amino acids in the N-terminal portion of the protein) or the larger SalI-XhoI fragment of HDAC4 (when translated, spans 784 amino acids starting from the N-terminal portion of the protein). Anti-HDAC2 antibodies were from Biomol, anti-HDAC1 was from Santa Cruz Biotechnology, and anti-HDAC3 and anti-HDAC6 were from Cell Signaling. Washes were performed with phosphate-buffered saline with 0.1% Tween. Finally, after probing with primary and secondary antibodies, the membranes were exposed to film after enhanced chemiluminescence (ECL) (Amersham Biosciences). Densitometry of immunoblots was performed on images obtained under nonsaturated conditions and quantitated with NIH Image 1.54 software. For pulse labeling experiments, 10 μCi/ml [35S]methionine (Amersham Biosciences cell labeling grade) was added to the cellular medium for 0.5–4 h. Cells were lysed as above, and cytoplasmic extracts were run by SDS-PAGE. For autoradiography, gels were then dried and exposed to x-ray film. Cell viability and death were assessed by direct visualization of cell morphology, trypan blue exclusion, Hoescht 33342 vital staining, and flow-assisted cytometric analysis of cells with sub-G1 DNA content. These methods showed good general agreement. Viable cells were defined as those excluding trypan blue, with uncondensed chromatin or mitotic chromosomes as visualized by vital staining and with G1 or greater DNA content. Reverse-transcriptase (RT)-PCR—Endogenous mRNA was isolated using TRIzol reagent (Invitrogen) as per the manufacturer's instructions, and assessed via RT-PCR. The Titan One Tube RT-PCR system was used with the following HDAC4, for all were performed under similar with results under a of and regarding the used are upon HDAC4 mutated in potential caspase cleavage were from that have been described previously Muschel R.J. McKenna W.G. J. Biol. PubMed Scopus Google Scholar, S. T. M. M. H. Y. Nature. PubMed Scopus Google Scholar). The were generated with the site-directed For the HDAC4 the used was and For the HDAC4 the used was and DNA was performed to the described green fluorescent protein were from Cells were with proteins using reagent of HDAC4 amino acids of human HDAC4 or mutated at Asp-289 was into the mammalian expression to HDAC4 or HDAC4 of the were by DNA Cells with these were in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum and in the of 100 and and then induced with as described by the of HDAC4 found previously that HDAC4 protein levels could be by (14Kao G.D. McKenna W.G. Guenther M.G. Muschel R.J. Lazar M.A. Yen T.J. J. Cell Biol. 2003; 160: 1017-1027Crossref PubMed Scopus (160) Google Scholar). In we that HDAC4 mRNA was Cells with to de novo were at and assessed for HDAC4, or mRNA A and HDAC4 mRNA levels was within and was by 8 h. HDAC4 mRNA therefore appeared to have a half-life of 4 or In under the mRNA levels were at but detectable at h, whereas mRNA was the of HDAC4 instability of HDAC4 mRNA that HDAC4 protein might be also be To assess protein cells with to de novo protein were at following treatment and assessed for protein In with the half-life of the HDAC4 protein levels were within and were by after the of A and levels of HDAC1, -2, -3, and To the that the instability of HDAC4 was to HeLa we to assess its levels in cancer and cancer cells after or HDAC4 protein levels in these cell lines treatment and but after exposure to in all and 5 with and These results that are cell in HDAC4 expression levels and its rate of instability is a general of this protein. HDAC4 or but on the instability of HDAC4 were with both antibodies not as well as polyclonal antibodies that we generated against the N-terminal We that the antibodies when compared with the antibodies against a larger portion of the protein, of to To the of antibodies, we generated a of antibodies against the N-terminal and which for when we used these antibodies to immunoblots from we in to the HDAC4 protein, a of Interestingly, as HDAC4 degradation in the of cycloheximide, the of the that this a these antibodies, we probed cells that were with or with lactacystin, a cell-permeable and of The cells were at for The degradation of HDAC4 not in the or of the proteasome with the MG-132 known as known as I or and all no on the degradation not We HDAC4 degradation might be by CRM1-mediated We cells with cycloheximide, either or with leptomycin a of CRM1-mediated nuclear In cell lysates that were after we found that HDAC4 degradation not appear to be in the or of and HDAC4 degradation by after treatment and was by both in the and in the of In to the of on HDAC4 the degradation of the of the mutated in the of LMB, was detectable after the of and Ultraviolet HDAC4 The degradation of HDAC4 after exposure to that cells might contain active mechanisms that are when de novo is a that might be induced by or as ultraviolet irradiation. We cells with ultraviolet irradiation or and cells at both led to the degradation of HDAC4, the rate of degradation was after UV irradiation. The degradation of HDAC4 was by 4 after whereas at this after cycloheximide, of HDAC4 HDAC4 accelerated degradation after UV in a as of UV led to levels of HDAC4 degradation A was at the dose but at a Interestingly, this showed that with the of the of HDAC4 as that this may degradation that after high UV We to conditions to these led to HDAC4 is to and DNA In HeLa irradiation does not by Muschel R.J. McKenna W.G. J. Biol. PubMed Scopus Google Scholar). and aphidicolin, the inhibition of in of intracellular and DNA both DNA and under to the death of cells S. T. M. M. H. Y. Nature. PubMed Scopus Google Scholar, Google Scholar). is a and and apoptosis in a of cell to caspase J. D. 2002; PubMed Scopus Google Scholar, H. Wang J. U. C. Cell 2001; PubMed Scopus Google Scholar). 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