Suberoylanilide Hydroxamic Acid Induces Akt-mediated Phosphorylation of p300, Which Promotes Acetylation and Transcriptional Activation of RelA/p65
2006; Elsevier BV; Volume: 281; Issue: 42 Linguagem: Inglês
10.1016/s0021-9258(19)84048-6
ISSN1083-351X
AutoresYuan Liu, Chadrick E. Denlinger, Brian K. Rundall, Philip W. Smith, David R. Jones,
Tópico(s)Ubiquitin and proteasome pathways
ResumoWe have previously demonstrated that the transcription factor NF-κB is activated by histone deacetylase inhibitors in a PI3K/Akt-dependent manner. The molecular mechanisms governing this process have not been well described. By virtue of their inhibitory action, it is unclear whether the addition of histone deacetylase inhibitors simply preserves the acetylation status of RelA/p65 or whether they actively stimulate signaling cascades that result in increased acetylation and transcription of NF-κB. Here we provide evidence that suberoylanilide hydroxamic acid stimulates NF-κB transcription through a signaling cascade that involves activation of both the serine/threonine kinase Akt and the p300 acetyltransferase. Using newly developed phosphospecific antibodies to p300 (pSer1834), and site-directed mutant proteins, we find that suberoylanilide hydroxamic acid stimulates Akt activity, which is required to phosphorylate p300 at Ser1834. Akt-mediated phosphorylation of p300 dramatically increases its acetyltransferase activity as measured by an increased acetylation of RelA/p65 at Lys310, a modification that is required for full NF-κB transcription. Importantly, coordinate activation of Akt/p300 pathway by suberoylanilide hydroxamic acid occurs at the chromatin level, resulting in recruitment of activated Akt (pSer473), p300 (pSer1834), acetylated RelA/p65 (Lys310), and RNA polymerase II to the NF-κB-dependent cIAP-2 and Bfl-1/A1 promoters. These studies provide evidence that histone deacetylase inhibitors, such as suberoylanilide hydroxamic acid, not only inhibit deacetylase activity but also stimulate active NF-κB transcription and cell survival through signaling pathways involving Akt and increased p300 acetyltransferase activity. We have previously demonstrated that the transcription factor NF-κB is activated by histone deacetylase inhibitors in a PI3K/Akt-dependent manner. The molecular mechanisms governing this process have not been well described. By virtue of their inhibitory action, it is unclear whether the addition of histone deacetylase inhibitors simply preserves the acetylation status of RelA/p65 or whether they actively stimulate signaling cascades that result in increased acetylation and transcription of NF-κB. Here we provide evidence that suberoylanilide hydroxamic acid stimulates NF-κB transcription through a signaling cascade that involves activation of both the serine/threonine kinase Akt and the p300 acetyltransferase. Using newly developed phosphospecific antibodies to p300 (pSer1834), and site-directed mutant proteins, we find that suberoylanilide hydroxamic acid stimulates Akt activity, which is required to phosphorylate p300 at Ser1834. Akt-mediated phosphorylation of p300 dramatically increases its acetyltransferase activity as measured by an increased acetylation of RelA/p65 at Lys310, a modification that is required for full NF-κB transcription. Importantly, coordinate activation of Akt/p300 pathway by suberoylanilide hydroxamic acid occurs at the chromatin level, resulting in recruitment of activated Akt (pSer473), p300 (pSer1834), acetylated RelA/p65 (Lys310), and RNA polymerase II to the NF-κB-dependent cIAP-2 and Bfl-1/A1 promoters. These studies provide evidence that histone deacetylase inhibitors, such as suberoylanilide hydroxamic acid, not only inhibit deacetylase activity but also stimulate active NF-κB transcription and cell survival through signaling pathways involving Akt and increased p300 acetyltransferase activity. Recent advances in the understanding of chromatin modifications in cancer have lead to the development of several different histone deacetylase inhibitors (HDI). 3The abbreviations used are: HDI, histone deacetylase inhibitor; SAHA, suberoylanilide hydroxamic acid; NSCLC, non-small cell lung cancer; HAT, histone acetyltransferases; HDAC, histone deacetylases; ChIP, chromatin immunoprecipitation; WT, wild type; DN-Akt, dominant-negative mutant of Akt; M-Akt, myristoylated Akt; MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; RT, reverse transcriptase; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAF, p300/CBP-associated factor. 3The abbreviations used are: HDI, histone deacetylase inhibitor; SAHA, suberoylanilide hydroxamic acid; NSCLC, non-small cell lung cancer; HAT, histone acetyltransferases; HDAC, histone deacetylases; ChIP, chromatin immunoprecipitation; WT, wild type; DN-Akt, dominant-negative mutant of Akt; M-Akt, myristoylated Akt; MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; RT, reverse transcriptase; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAF, p300/CBP-associated factor. Suberoylanilide hydroxamic acid (SAHA) is a HDI currently in clinical trials (1Kelly W.K. Richon V.M. O'Connor O. Curley T. MacGregor-Curtelli B. Tong W. Klang M. Schwartz L. Richardson S. Rosa E. Drobnjak M. Cordon-Cordo C. Chiao J.H. Rifkind R. Marks P.A. Scher H. Clin. Cancer Res. 2003; 9: 3578-3588PubMed Google Scholar, 2Kelly W.K. O'Connor O.A. Krug L.M. Chiao J.H. Heaney M. Curley T. MacGregore-Cortelli B. Tong W. Secrist J.P. Schwartz L. Richardson S. Chu E. Olgac S. Marks P.A. Scher H. Richon V.M. J. Clin. Oncol. 2005; 23: 3923-3931Crossref PubMed Scopus (811) Google Scholar). SAHA has been shown to induce cell cycle arrest through up-regulation of p21WAF1, as well as induce apoptosis through mitochondrial-mediated processes involving caspase activation and processing of Bcl-2 family members (3Henderson C. Mizzau M. Paroni G. Maestro R. Schneider C. Brancolini C. J. Biol. Chem. 2003; 278: 12579-12589Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 4Richon V.M. Sandhoff T.W. Rifkind R.A. Marks P.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10014-10019Crossref PubMed Scopus (1015) Google Scholar, 5Richon V.M. Emiliani S. Verdin E. Webb Y. Breslow R. Rifkind R.A. Marks P.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3003-3007Crossref PubMed Scopus (839) Google Scholar). In addition, p53 may be required for the enhanced cytotoxicity following treatment with SAHA, although it is likely tumor- and cell line-dependent (3Henderson C. Mizzau M. Paroni G. Maestro R. Schneider C. Brancolini C. J. Biol. Chem. 2003; 278: 12579-12589Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 6Huang L. Sowa Y. Sakai T. Pardee A.B. Oncogene. 2000; 19: 5712-5719Crossref PubMed Scopus (198) Google Scholar, 7Xu W.S. Perez G. Ngo L. Gui C.Y. Marks P.A. Cancer Res. 2005; 65: 7832-7839Crossref PubMed Scopus (115) Google Scholar). We, and others (8Denlinger C.E. Rundall B.K. Jones D.R. Semin. Thorac. Cardiovasc. Surg. 2004; 16: 28-39Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 9Mayo M.W. Denlinger C.E. Broad R.M. Yeung F. Reilly E.T. Shi Y. Jones D.R. J. Biol. Chem. 2003; 278: 18980-18989Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 10Dai Y. Rahmani M. Dent P. Grant S. Mol. Cell. Biol. 2005; 25: 5429-5444Crossref PubMed Scopus (215) Google Scholar) have shown that isolated HDI treatment fails to induce cell death in non-small cell lung cancer (NSCLC) and human leukemia cells secondary to up-regulation of NF-κB-dependent transcription. This process can occur in an Akt-dependent (9Mayo M.W. Denlinger C.E. Broad R.M. Yeung F. Reilly E.T. Shi Y. Jones D.R. J. Biol. Chem. 2003; 278: 18980-18989Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) or independent manner (11Rahmani M. Yu C. Reese E. Ahmed W. Hirsch K. Dent P. Grant S. Oncogene. 2003; 22: 6231-6242Crossref PubMed Scopus (93) Google Scholar). Mechanisms underlying HDI-induced NF-κB activation have been shown to involve increased nuclear translocation of RelA/p65 and enhanced acetylation of RelA/p65 (10Dai Y. Rahmani M. Dent P. Grant S. Mol. Cell. Biol. 2005; 25: 5429-5444Crossref PubMed Scopus (215) Google Scholar, 12Chen L. Fischle W. Verdin E. Greene W.C. Science. 2001; 293: 1653-1657Crossref PubMed Scopus (1047) Google Scholar). Greene and co-workers (13Chen L.F. Greene W.C. J. Mol. Med. 2003; 81: 549-557Crossref PubMed Scopus (243) Google Scholar, 14Chen L.F. Mu Y. Greene W.C. EMBO J. 2002; 21: 6539-6548Crossref PubMed Scopus (635) Google Scholar, 15Yeung F. Hoberg J.E. Ramsey C.S. Keller M.D. Jones D.R. Frye R.A. Mayo M.W. EMBO J. 2004; 23: 2369-2380Crossref PubMed Scopus (2186) Google Scholar) and others have shown that distinct biological activities of NF-κB are regulated by interactions between histone acetyltransferases (HAT) and deacetylases (HDAC) that modulate the acetylation and subsequent activation of NF-κB. The coactivators p300 and/or p/CAF (p300/CBP-associated factor) and their HAT domains have been shown to be necessary for phorbol 12-myristate 13-acetate and cytokine-mediated acetylation of RelA/p65 (16Kiernan R. Bres V. Ng R.W. Coudart M.P. El Messaoudi S. Sardet C. Jin D.Y. Emiliani S. Benkirane M. J. Biol. Chem. 2003; 278: 2758-2766Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 17Hoberg J.E. Popko A.E. Ramsey C.S. Mayo M.W. Mol. Cell. Biol. 2006; 26: 457-471Crossref PubMed Scopus (159) Google Scholar). HDACs that physically interact with NF-κB and are responsible for basal repression of NF-κB activation have been shown to be HDAC-1 (18Ashburner B.P. Westerheide S.D. Baldwin Jr., A.S. Mol. Cell. Biol. 2001; 21: 7065-7077Crossref PubMed Scopus (625) Google Scholar) as well as HDAC-2 (15Yeung F. Hoberg J.E. Ramsey C.S. Keller M.D. Jones D.R. Frye R.A. Mayo M.W. EMBO J. 2004; 23: 2369-2380Crossref PubMed Scopus (2186) Google Scholar) and HDAC-3 (12Chen L. Fischle W. Verdin E. Greene W.C. Science. 2001; 293: 1653-1657Crossref PubMed Scopus (1047) Google Scholar, 16Kiernan R. Bres V. Ng R.W. Coudart M.P. El Messaoudi S. Sardet C. Jin D.Y. Emiliani S. Benkirane M. J. Biol. Chem. 2003; 278: 2758-2766Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). Interestingly, acetylation of specific lysine (K) residues on RelA/p65 govern specific functions of NF-κB (12Chen L. Fischle W. Verdin E. Greene W.C. Science. 2001; 293: 1653-1657Crossref PubMed Scopus (1047) Google Scholar, 13Chen L.F. Greene W.C. J. Mol. Med. 2003; 81: 549-557Crossref PubMed Scopus (243) Google Scholar). For example, acetylation of Lys221 regulates DNA binding, nuclear export of NF-κB, and IκBα assembly, whereas acetylation of Lys310, and to a lesser extent Lys221, are more involved in transcriptional regulation of NF-κB (14Chen L.F. Mu Y. Greene W.C. EMBO J. 2002; 21: 6539-6548Crossref PubMed Scopus (635) Google Scholar). Similarly, Kiernan et al. (16Kiernan R. Bres V. Ng R.W. Coudart M.P. El Messaoudi S. Sardet C. Jin D.Y. Emiliani S. Benkirane M. J. Biol. Chem. 2003; 278: 2758-2766Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar) found that acetylation of Lys122 and Lys123 promotes post-induction removal of RelA/p65 from DNA and facilitates its nuclear exportation. HDI-induced acetylation of RelA/p65 is well established (7Xu W.S. Perez G. Ngo L. Gui C.Y. Marks P.A. Cancer Res. 2005; 65: 7832-7839Crossref PubMed Scopus (115) Google Scholar, 19Chen L.F. Williams S.A. Mu Y. Nakano H. Duerr J.M. Buckbinder L. Greene W.C. Mol. Cell. Biol. 2005; 25: 7966-7975Crossref PubMed Scopus (362) Google Scholar), but it is unknown which lysine residue(s) on RelA/p65 are involved in this process. Whereas HDIs can activate NF-κB-dependent transcription, it is unclear what molecular mechanisms actually govern this process. Previous work from our group has suggested that this may be a PI3K/Akt-dependent process (9Mayo M.W. Denlinger C.E. Broad R.M. Yeung F. Reilly E.T. Shi Y. Jones D.R. J. Biol. Chem. 2003; 278: 18980-18989Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). We hypothesize that HDIs activate NF-κB by de-repressing RelA/p65 acetylation through their intrinsic deacetylase inhibitory function as well more directly activating NF-κB through specific signal transduction pathways involving the serine/threonine kinase Akt. In this report we demonstrate evidence that SAHA enhances NF-κB-dependent transcriptional activity through two separate mechanisms both of which result in enhanced acetylation of RelA/p65 on Lys310. One mechanism involves SAHA inhibition of HDAC-1-mediated repression which preserves the Lys310 acetyl-mark on RelA/p65 and promotes NF-κB transcription. Using newly developed phosphospecific antibodies we have also identified a novel second mechanism through which SAHA activates NF-κB that involves signal transduction pathways requiring Akt-mediated phosphorylation of p300 on serine 1834 which, in turn, directly increases the acetyltransferase activity of p300 and the acetylation of RelA/p65. Furthermore, inhibition of the PI3K/Akt pathway markedly decreases SAHA enhanced recruitment of endogenous Akt (pSer473), acetyl-RelA/p65 (Lys310), and p300 (pSer1834) to the chromatin of NF-κB-dependent promoters cIAP-2 and Bfl-1/A1. Thus, SAHA activates NF-κB-dependent transcription via two distinct and likely complementary mechanisms. The discovery of this transcriptional activation involving phosphorylation and acetylation events surrounding HDI therapies provides clues for future drug development and combination treatment strategies. Cell Culture Reagents, Antibodies, and Plasmids—Four tumor-igenic NSCLC lines (NCI-H157 (p53 mutant), NCI-H358 (p53-null), NCI-H460 (p53 wild-type(WT)), NCI-A549 (p53 WT)) and 293T cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured in RPMI 1640 or Dulbecco's modified Eagle's Medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and 1% penicillin/streptomycin (Invitrogen). The 3x-κB luciferase reporter construct (3x-κB Luc), the Gal4-luciferase construct (Gal4-Luc), the Gal4-p65 fusion protein which has the yeast Gal4 DNA binding domain fused to full-length p65-(1–551) were generated as previously described (9Mayo M.W. Denlinger C.E. Broad R.M. Yeung F. Reilly E.T. Shi Y. Jones D.R. J. Biol. Chem. 2003; 278: 18980-18989Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 20Mayo M.W. Norris J.L. Baldwin A.S. Methods Enzymol. 2001; 333: 73-87Crossref PubMed Scopus (61) Google Scholar). The plasmid encoding FLAG-tagged p65WT was provided by Dr. Denis C. Guttridge, (Columbus, Ohio). The pCMV2-FLAG tagged p65K310R mutant construct was made using the quick-change mutagenesis kit (Stratagene, La Jolla, CA). The pCI-p300 and pCI-p300ΔHAT plasmids were kindly provided by Dr. Joan Boyes (London, England) and pCI-FLAG-tagged p/CAF, pcDNA-FLAG-tagged HDAC-1, pcDNA-HDAC-2, and pcDNA-Myc-tagged HDAC3 and dominant-negative mutant of Akt (DN-Akt), and myristoylated (M-Akt) expression plasmids were provided by Marty W. Mayo (Charlottesville, VA). The p300S1834A expression construct was kindly provided by Dr. Terry G. Unterman (Chicago, IL). The plasmid encoding HA-tagged p300 was purchased from Upstate Biotechnology (Lake Placid, NY). The antibodies used were: p300, HDAC-1, HDAC-2, and HDAC-3 from Upstate Biotechnology (Lake Placid, NY); Akt, phospho-Akt (Ser473), and pan-acetyl-lysine (polyclonal) from Cell Signaling (Beverly, MA); anti-FLAG M2 and anti-β-tubulin antibodies from Sigma-Aldrich; RNA Pol II, RelA/p65, cIAP2, Bfl-1/A1, and normal rabbit IgG from Santa Cruz Biotechnologies. Antibodies against acetyl-p65 (Lys310) were kindly provided by Dr. Marty W. Mayo (Charlottesville, VA) (17Hoberg J.E. Popko A.E. Ramsey C.S. Mayo M.W. Mol. Cell. Biol. 2006; 26: 457-471Crossref PubMed Scopus (159) Google Scholar). The phospho-p300 (Ser1834) antibody was developed in conjunction with Upstate Biotechnology (Lake Placid, NY). SAHA was provided by Merck, Inc. (Whitehouse Station, NJ). LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, LY, PI3K/Akt inhibitor, IC50, 1.4 μm), PD98059 (2′-amino-3′-methoxyflavone, MEK/MAP kinase inhibitor, IC50, 2 μm), SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole, p38 MAP kinase inhibitor, IC50, 34 nm), DMNB (4,5-dimethoxy-2-nitrobenzaldehyde DNA-dependent protein kinase inhibitor, IC50, 15 μm), and SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one1,9-pyrazoloanthrone, JNK inhibitor, IC50, 40 nm) were purchased from Calbiochem (La Jolla, CA). The siRNA Akt1 and negative control were obtained from Dharmacon (Lafayette, CO). Quantitative Reverse Transcriptase Polymerase Chain Reaction (Quantitative RT-PCR)—Human NSCLC cell lines and 293T cells at 80% confluence were left untreated, or treated with SAHA (5 μm) for 2 h. Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instruction. cDNAs were synthesized using the Advantage RT for PCR enzyme kit (Clontech) and both cIAP-2 and Bfl-1/A1 gene expression were determined by Quantitative-PCR with an iCycler IQ (Bio-Rad). Human cIAP-2 primers: 5′-GCTGTGATGGTGGACTCAGG-3′ and 5′-CATCCGTCAAGTTCAGCCA-3′; Bfl-1/A1 primers: 5′-TCATATTTTGTTGCGGAGTTCA-3′ and 5′-TTTGAACCTAAATCTGGCTGGA-3′. Quantitative PCR reaction conditions were identical to the standard PCR reaction except that each 25 μl of reaction included 1 μl of 1:3000 dilution of SYBR Green I dye (Molecular Probes, Eugene, OR) to indicate amplified DNA. The human HPRT gene was amplified at the same time as a reference gene, the primers: 5′-TTGGAAAGGGTGTTTATTCCTCA-3′; and 5′-TCCAGCAGGTCAGCAAAGAA-3′. Threshold cycle (TC) was defined as the cycle number at which fluorescence intensity exceeds a fixed threshold. The expression of cIAP-2 or Bfl-1/A1 genes (ΔTC) was normalized to endogenous HPRT (TCR). ΔTC was calculated by subtracting the TC value of the reference (TCR) from the TC value of the sample (TCS)(ΔTC = TCS – TCR). The relative expression of treated cells (ΔTCT) to the corresponding values obtained for untreated cells (ΔTCU) was determined using the formula: 2ΔTCU/2ΔTCT. Luciferase Reporter Gene Assays—Cells were plated at 40–60% confluence 24 h before transfection. The next day cells were transiently transfected with plasmids and/or reporter genes using Polyfect reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions. Luciferase reporter assays were performed as described (9Mayo M.W. Denlinger C.E. Broad R.M. Yeung F. Reilly E.T. Shi Y. Jones D.R. J. Biol. Chem. 2003; 278: 18980-18989Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). All transfections were normalized with CMV-β-galactosidase activity. Luminescence was normalized to protein concentrations and all transfection data are the mean ± S.D. of three independent experiments performed in triplicate analysis. For RNA interference, reporters and plasmids and/or siRNA (100 nm) were co-transfected into 293T cells with oligofectamine (Invitrogen) according to the manufacturer's protocol. RelA/p65 Acetylation Assays—Acetylation assays were performed as described (14Chen L.F. Mu Y. Greene W.C. EMBO J. 2002; 21: 6539-6548Crossref PubMed Scopus (635) Google Scholar). 293T cells were co-transfected with expression plasmids encoding FLAG-tagged p65 and HA-tagged p300. In select experiments, expression vectors encoding p300ΔHAT, FLAG-p/CAF or the p300S1834A mutant were used instead of p300, or with an expression vector encoding FLAG-tagged p65K310R mutant replacing FLAG-tagged p65WT. Alternatively, cells were also co-transfected with expression vectors encoding FLAG-tagged HDAC-1, HDAC-2, or Myc-tagged HDAC-3, M-Akt or DN-Akt. 24 h following transfection, cells were left untreated, or treated with SAHA (5 μm), LY (25 μm) or both for 2 h. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with a panacetyl antibody. Co-immunoprecipitation Assays, Phosphorylation Assays, and Western Blotting—293T cells were transfected with expression vectors encoding FLAG-tagged p65 and 24-h post-transfection, cells were left untreated or treated with SAHA (5 μm) for 2 h. In select experiments, cells at 80% confluence were treated as noted above. Primary antibodies: anti-RelA/p65 (20 μg/1000 μg protein) or anti-p300 (20 μg/1000 μg protein) were mixed with precleared lysates for 1.5 h at 4 °C before the addition of 20 μl of protein agarose A/G (Santa Cruz Biotechnology), and reactions were tumbled overnight at 4 °C. The agarose beads were then extensively washed, followed by immunoblot analysis. For phosphorylation assays, NSCLC cells and 293T cells at 80% confluence were left untreated, or treated with SAHA (5 μm), LY (25 μm) or SAHA (5 μm) plus LY (25 μm) for 2 h followed by immunoblot with phospho-p300 Ser1834 polyclonal antibody or phospho-Akt Ser473 polyclonal antibody. Standard Western blot was performed. Proteins were separated on SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad). Primary antibodies were used at 1:1000 dilution and secondary antibodies (Promega, Madison, WI) were used at 1:5000 dilution in blocking solution. SuperSignal West Pico chemiluminescent substrate kit (Pierce) was used to detect protein signal. ChIP Assays—293T cells at 80% confluence were left untreated, or treated with SAHA (5 μm), LY (25 μm) or SAHA (5 μm) plus LY (25 μm) for 2 h. ChIP assays were performed as previously described (21Kuo M.H. Allis C.D. Methods. 1999; 19: 425-433Crossref PubMed Scopus (486) Google Scholar). DNA immunoprecipitated by 4 μlof antibody (anti-p65, anti-acetyl-p65 Lys310, anti-p300, antiphospho-p300 Ser1834, anti-Akt, anti-Akt Ser473, anti-RNA Pol II or normal rabbit IgG) was purified. The regions of human cIAP-2 and Bfl-1/A1 promoters containing κB binding sites were targeted for amplification. The sequences of primers for promoters used were: cIAP-2 forward primer 5′-CACGAGCAATGAAGCAAATG-3′; reverse primer 5′-GTGCACTGGTGCTTTCCTTT-3′; Bfl-1/A1 forward primers 5′-CCCGAGTAGCT-GGGATTACA-3′; reverse primers 5′-CCTAGCACTTTGGGAGGACA-3′. A non-NF-κB-regulated gene GAPDH promoter was amplified as control (15Yeung F. Hoberg J.E. Ramsey C.S. Keller M.D. Jones D.R. Frye R.A. Mayo M.W. EMBO J. 2004; 23: 2369-2380Crossref PubMed Scopus (2186) Google Scholar). Human HPRT gene was amplified as an internal control to correct for differences in DNA loading. Quantitative PCR was performed as above. PCR data were analyzed as previously described (22Chakrabarti S.K. James J.C. Mirmira R.G. J. Biol. Chem. 2002; 277: 13286-13293Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Cell Viability and Apoptosis Assays—A549 and H157 NSCLC cells were transfected with expression vectors encoding the p65 WT or p65 K310R mutant and the β-galactosidase reporter. Seventy-two hours later, cells were treated for 2 h with escalating doses of SAHA (0, 1, 2, 5, 10 μm). Cell viability was determined based on quantitation of ATP present using Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega) according to the manufacturer's directions. Apoptosis was quantified with caspase-3 activity assays as previously described (Calbiochem, San Diego CA) (23Denlinger C.E. Rundall B.K. Jones D.R. J. Thorac. Cardiovasc. Surg. 2005; 130: 1422-1429Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). β-Galactosidase activities were analyzed as control for transfection efficiency. Statistical Analysis—Results of all experiments represent the mean ± S.D. of three separate experiments performed in triplicate, unless otherwise noted. Statistical differences between treatment groups were determined by a two-tailed, unpaired Student's t test when appropriate. p values < 0.05 were considered significant. SAHA Activates NF-κB-dependent Transcription through p300-mediated Acetylation of RelA/p65 on Lysine 310—To determine if SAHA would increase the expression of the endogenous NF-κB-dependent anti-apoptotic genes, cIAP-2 and Bfl-1/A1 (24Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2573) Google Scholar, 25Zong W.X. Edelstein L.C. Chen C. Bash J. Gelinas C. Genes Dev. 1999; 13: 382-387Crossref PubMed Scopus (643) Google Scholar), quantitative RT-PCR and Western blot analyses were performed. SAHA significantly increased both cIAP-2 and Bfl-1/A1 transcripts and protein levels in NSCLC and 293T cells (Fig. 1A). Because acetylation of RelA/p65 enhances the transcriptional activity of NF-κB (12Chen L. Fischle W. Verdin E. Greene W.C. Science. 2001; 293: 1653-1657Crossref PubMed Scopus (1047) Google Scholar), we next determined if SAHA would potentiate p300-mediated acetylation of RelA/p65. SAHA dramatically increased p300-mediated acetylation of RelA/p65 (Fig. 1B) without affecting total RelA/p65 levels. Moreover, despite equivalent expression of p300WT and p300 with a deleted HAT domain (p300ΔHAT), the HAT domain of p300 was required for basal and SAHA-induced acetylation of RelA/p65. Several groups have established that the coactivator p/CAF, like p300, can enhance the acetylation and subsequent transcriptional activity of NF-κB (16Kiernan R. Bres V. Ng R.W. Coudart M.P. El Messaoudi S. Sardet C. Jin D.Y. Emiliani S. Benkirane M. J. Biol. Chem. 2003; 278: 2758-2766Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 26Sheppard K.A. Rose D.W. Haque Z.K. Kurokawa R. McInerney E. Westin S. Thanos D. Rosenfeld M.G. Glass C.K. Collins T. Mol. Cell. Biol. 1999; 19: 6367-6378Crossref PubMed Google Scholar). As shown in Fig. 1C, only p300, and not p/CAF, lead to the accumulation of acetylated RelA/p65 in the presence of SAHA. Acetylation of Lys310 on RelA/p65 has been shown by others to be primarily responsible for transcriptional activation of RelA/p65 (14Chen L.F. Mu Y. Greene W.C. EMBO J. 2002; 21: 6539-6548Crossref PubMed Scopus (635) Google Scholar, 15Yeung F. Hoberg J.E. Ramsey C.S. Keller M.D. Jones D.R. Frye R.A. Mayo M.W. EMBO J. 2004; 23: 2369-2380Crossref PubMed Scopus (2186) Google Scholar). Acetylation assays were repeated with FLAG-p65WT or FLAG-p65K310R, a mutant protein that maintains a positive charge at Lys310 but is incapable of acetylation at this residue. Accordingly, we found that only p65WT, but not the p65K310R mutant protein was acetylated in the presence of p300 and SAHA (Fig. 1D). The absence of any demonstrable acetylation with the p65K310R mutant protein suggests that SAHA preferentially promotes acetylation of this lysine residue in vivo. To determine the transcriptional relevance of this acetylation event 3x-κB luciferase assays were performed in the NSCLC and 293T cells following ectopic expression of either the p65WT or p65K310R expression constructs. Inability to acetylate Lys310 dramatically decreased SAHA-induced NF-κB transcriptional activity compared with cells-overexpressing p65WT in multiple NSCLC cell lines. Collectively, these data suggest that SAHA preferentially promotes acetylation of RelA/p65 on Lys310 in a p300-dependent manner, which results in enhanced transcriptional activity of NF-κB. SAHA Activates NF-κB Transcription through De-repression of HDAC-1 on RelA/p65—HDAC-1, -2, and -3 have been shown to bind to RelA/p65 (13Chen L.F. Greene W.C. J. Mol. Med. 2003; 81: 549-557Crossref PubMed Scopus (243) Google Scholar, 16Kiernan R. Bres V. Ng R.W. Coudart M.P. El Messaoudi S. Sardet C. Jin D.Y. Emiliani S. Benkirane M. J. Biol. Chem. 2003; 278: 2758-2766Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 18Ashburner B.P. Westerheide S.D. Baldwin Jr., A.S. Mol. Cell. Biol. 2001; 21: 7065-7077Crossref PubMed Scopus (625) Google Scholar). Whereas SAHA is known to inhibit both class I and class II deacetylases (4Richon V.M. Sandhoff T.W. Rifkind R.A. Marks P.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10014-10019Crossref PubMed Scopus (1015) Google Scholar), it is likely that specific HDACs are involved in modulating NF-κB transcriptional activity. To address this question in our model system, co-immunoprecipitation assays were performed which demonstrate that HDAC-1, but not HDAC-2 or -3, associates with RelA/p65 (Fig. 2A). Interestingly, the addition of SAHA had no influence on p65-HDAC-1 interactions, suggesting that acetylation is not required for this interaction. To determine if HDAC-1 or HDAC-2, or -3 could repress either basal or SAHA-mediated acetylation of RelA/p65 acetylation assays were repeated. As shown in Fig. 2B, overexpression of both HDAC-1 and -2 suppressed both basal and inducible acetylation of RelA/p65 relative to controls. Additionally, HDAC-1 appears to provide superior deacetylation of RelA/p65, relative to HDAC-2, although this effect was partially reversed by SAHA. Interestingly, and in contrast to findings of others (12Chen L. Fischle W. Verdin E. Greene W.C. Science. 2001; 293: 1653-1657Crossref PubMed Scopus (1047) Google Scholar), HDAC-3 was unable to significantly affect either basal or SAHA-induced acetylation of RelA/p65. Control experiments confirmed expression of p65, p300, HDAC-1, -2, and -3. We next determined whether overexpression of specific HDACs would functionally inhibit the transactivation potential of RelA/p65 in the presence of SAHA. As shown in Fig. 2C, HDAC-1 dramatically suppressed basal and SAHA-induced transactivation potential of RelA/p65 in both NSCLC cell lines. Whereas HDAC-2 and -3 also suppressed the transactivation potential of RelA/p65, this was not significant and their effects were less than that observed with HDAC-1. Potential explanations for the observed decrease in transcriptional activity of NF-κB in the presence of only a modest deacetylation of RelA/p65 following SAHA exposure include that these deacetylases may be preferentially deacetylating other important coactivators or assembly proteins necessary for NF-κB transcription. Collectively, these data (Figs. 1 and 2) suggest that one mechanism through which SAHA activates RelA/p65 transcription involv
Referência(s)