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Originally published In Press as doi:10.1074/jbc.M604478200 on August 21, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31359-31368, October 20, 2006
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Suberoylanilide Hydroxamic Acid Induces Akt-mediated Phosphorylation of p300, Which Promotes Acetylation and Transcriptional Activation of RelA/p65*

Yuan Liu1, Chadrick E. Denlinger1, Brian K. Rundall, Philip W. Smith, and David R. Jones2

From the Department of Surgery, University of Virginia School of Medicine, Charlottesville, Virginia 22908

Received for publication, May 10, 2006 , and in revised form, July 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously demonstrated that the transcription factor NF-{kappa}B 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-{kappa}B. Here we provide evidence that suberoylanilide hydroxamic acid stimulates NF-{kappa}B 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-{kappa}B 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-{kappa}B-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-{kappa}B transcription and cell survival through signaling pathways involving Akt and increased p300 acetyltransferase activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent advances in the understanding of chromatin modifications in cancer have lead to the development of several different histone deacetylase inhibitors (HDI).3 Suberoylanilide hydroxamic acid (SAHA) is a HDI currently in clinical trials (1, 2). 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 (3–5). In addition, p53 may be required for the enhanced cytotoxicity following treatment with SAHA, although it is likely tumor- and cell line-dependent (3, 6, 7).

We, and others (8–10) 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-{kappa}B-dependent transcription. This process can occur in an Akt-dependent (9) or independent manner (11). Mechanisms underlying HDI-induced NF-{kappa}B activation have been shown to involve increased nuclear translocation of RelA/p65 and enhanced acetylation of RelA/p65 (10, 12).

Greene and co-workers (13–15) and others have shown that distinct biological activities of NF-{kappa}B are regulated by interactions between histone acetyltransferases (HAT) and deacetylases (HDAC) that modulate the acetylation and subsequent activation of NF-{kappa}B. 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 (16, 17). HDACs that physically interact with NF-{kappa}B and are responsible for basal repression of NF-{kappa}B activation have been shown to be HDAC-1 (18) as well as HDAC-2 (15) and HDAC-3 (12, 16). Interestingly, acetylation of specific lysine (K) residues on RelA/p65 govern specific functions of NF-{kappa}B (12, 13). For example, acetylation of Lys221 regulates DNA binding, nuclear export of NF-{kappa}B, and I{kappa}B{alpha} assembly, whereas acetylation of Lys310, and to a lesser extent Lys221, are more involved in transcriptional regulation of NF-{kappa}B (14). Similarly, Kiernan et al. (16) 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 (7, 19), but it is unknown which lysine residue(s) on RelA/p65 are involved in this process.

Whereas HDIs can activate NF-{kappa}B-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 (9). We hypothesize that HDIs activate NF-{kappa}B by de-repressing RelA/p65 acetylation through their intrinsic deacetylase inhibitory function as well more directly activating NF-{kappa}B through specific signal transduction pathways involving the serine/threonine kinase Akt. In this report we demonstrate evidence that SAHA enhances NF-{kappa}B-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-{kappa}B transcription. Using newly developed phosphospecific antibodies we have also identified a novel second mechanism through which SAHA activates NF-{kappa}B 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-{kappa}B-dependent promoters cIAP-2 and Bfl-1/A1. Thus, SAHA activates NF-{kappa}B-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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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-{kappa}B luciferase reporter construct (3x-{kappa}B 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 (9, 20). 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-p300{Delta}HAT 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%20Biotechnology">Upstate Biotechnology (Lake Placid, NY). The antibodies used were: p300, HDAC-1, HDAC-2, and HDAC-3 from Upstate%20Biotechnology">Upstate Biotechnology (Lake Placid, NY); Akt, phospho-Akt (Ser473), and pan-acetyl-lysine (polyclonal) from Cell Signaling (Beverly, MA); anti-FLAG M2 and anti-beta-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) (17). The phospho-p300 (Ser1834) antibody was developed in conjunction with Upstate%20Biotechnology">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 ({Delta}TC) was normalized to endogenous HPRT (TCR). {Delta}TC was calculated by subtracting the TC value of the reference (TCR) from the TC value of the sample (TCS)({Delta}TC = TCS – TCR). The relative expression of treated cells ({Delta}TCT) to the corresponding values obtained for untreated cells ({Delta}TCU) was determined using the formula: 2{Delta}TCU/2{Delta}TCT.

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 (9). All transfections were normalized with CMV-beta-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 (14). 293T cells were co-transfected with expression plasmids encoding FLAG-tagged p65 and HA-tagged p300. In select experiments, expression vectors encoding p300{Delta}HAT, 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 (21). 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 {kappa}B 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-{kappa}B-regulated gene GAPDH promoter was amplified as control (15). 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 (22).

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 beta-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) (23). beta-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
SAHA Activates NF-{kappa}B-dependent Transcription through p300-mediated Acetylation of RelA/p65 on Lysine 310—To determine if SAHA would increase the expression of the endogenous NF-{kappa}B-dependent anti-apoptotic genes, cIAP-2 and Bfl-1/A1 (24, 25), 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-{kappa}B (12), 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 (p300{Delta}HAT), 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-{kappa}B (16, 26). 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 (14, 15). 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-{kappa}B 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-{kappa}B 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-{kappa}B.


Figure 1
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  		FIGURE 1.
SAHA activates NF-{kappa}B-dependent transcription through p300-mediated acetylation of RelA/p65 on lysine 310. A, NSCLC cells (H358 and H460), and 293T cells were left untreated (No add), or treated with SAHA (5 µM) for 2 h. Quantitative RT-PCR and Western blot were used to analyze mRNA and protein expression for the NF-{kappa}B-dependent genes, cIAP-2 and Bfl-1/A1. Gene expression was calculated as the ratio of mRNA expression in SAHA-treated cells to untreated cells. beta-Tubulin was used as a loading control. B, 293T cells were transfected with expression plasmids encoding FLAG-tagged p65 and either p300WT or p300{Delta}HAT. The transfected cells were not treated or treated with SAHA (5 µM) for 2 h. Acetylated RelA/p65 was determined as described under "Experimental Procedures." Equal protein expression of p300 was determined by Western blotting. C, 293T cells were transfected with expression plasmids FLAG-tagged p65. Cells were also co-transfected with HA-tagged p300 or FLAG-tagged p/CAF. Acetylated RelA/p65 was determined as above. Western blot analysis confirmed expression of HA-tagged p300 and FLAG-tagged p/CAF. D, 293T cells were transfected with expression plasmids encoding HA-tagged p300 and either FLAG-tagged p65WT or the FLAG-p65K310R mutant. Cells were left alone or treated with SAHA (5 µM) for 2 h and acetylation assays for RelA/p65 performed. E, NSCLC cells and 293T cells were transiently co-transfected with 3x-{kappa}B-luciferase reporter and expression vector encoding p65WT or p65K310R. Cells were left untreated (No add), or treated with SAHA (5 µM) for 12 h. Equivalent expression of p65WT and p65K310R proteins was confirmed by Western blot. (*) p < 0.01 and ({ddagger}) p < 0.05 compared with p65WT plus SAHA.

 
SAHA Activates NF-{kappa}B Transcription through De-repression of HDAC-1 on RelA/p65—HDAC-1, -2, and -3 have been shown to bind to RelA/p65 (13, 16, 18). Whereas SAHA is known to inhibit both class I and class II deacetylases (4), it is likely that specific HDACs are involved in modulating NF-{kappa}B 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.


Figure 2
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  		FIGURE 2.
HDAC-1 preferentially binds with RelA/p65 and attenuates SAHA-induced RelA/p65 acetylation and transcription. A, IP Westerns: 293T cells were transfected with expression plasmid encoding FLAG-tagged p65. Cells were left alone or treated with SAHA (5 µM) for 2 h. Cell lysates were immunoprecipitated with {alpha}-p65, and immunoblotted for the presence of the respective endogenous HDAC. Western blot for input protein showed that relatively equal amounts of FLAG-tagged RelA/p65, or endogenous HDAC proteins were both expressed and immunoprecipitated. B, 293T cells were co-transfected with expression vectors encoding FLAG-tagged p65, HA-tagged p300 and either FLAG-tagged HDAC-1, HDAC-2, Myc-tagged HDAC-3, or empty vector as control. The transfected cells were left alone or treated with SAHA (5 µM) for 2 h and acetylation assays performed. Equivalent expression of FLAG-tagged HDAC-1, HDAC-2, and Myc-tagged HDAC-3 was confirmed by Western blotting. C, NSCLC cells were transiently co-transfected with Gal4 luciferase reporter and expression plasmid encoding the fusion protein Gal4-p65. Cells were also co-transfected with expression plasmid encoding HDAC-1, -2, -3 or an empty vector as control. Cells were subsequently left untreated (No add), or treated with SAHA (5 µM) for 12 h, and luciferase activity was determined. (*) p < 0.01 and ({ddagger}) p < 0.05 compared with empty vector plus SAHA.

 
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 (12), 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.


Figure 3
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  		FIGURE 3.
Akt is required for SAHA-induced acetylation of RelA/p65. A, NSCLC cells were transiently transfected with 3x-{kappa}B-luciferase reporter. Cells were left untreated (No add), or treated with SAHA (5 µM), as well as pharmacologic inhibitors of kinases known to be involved in NF-{kappa}B activation at doses 2x the manufacturer's described IC50. (*) p < 0.01 compared with SAHA without an inhibitor. B, 293T cells were co-transfected with FLAG-tagged p65 and HA-tagged p300. Cells were also co-transfected with expression vector encoding DN-Akt or empty vector control. Cells were then left alone or treated with SAHA (5 µM) for 2 h. Acetylation assays were repeated. HA-p300 and nuclear concentrations of FLAG-p65 were determined by Western blotting in parallel experiments. C, endogenous acetylation of RelA/p65 was determined in the 293T and the H358 and H460 NSCLC cells following treatment with SAHA (5 µM) and the PI3K/Akt pharmacologic inhibitor LY (25 µM) for 2 h. Endogenous RelA/p65 protein levels are shown as a control. D, 293T cells were left alone or treated with SAHA (5 µM), LY (25 µM), or SAHA (5 µM) plus LY (25 µM). Whole cell lysates were immunoprecipitated with {alpha}-p300 then immunoblotted for endogenous RelA/p65. Western blot of input protein showed relatively equal amounts of endogenous p300 and RelA/p65 proteins were both expressed and immunoprecipitated.

 
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-{kappa}B 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-{kappa}B transcription. Collectively, these data (Figs. 1 and 2) suggest that one mechanism through which SAHA activates RelA/p65 transcription involves de-repression of HDAC-1 deacetylase activity preserving the pro-transcription acetyl mark on Lys310.

Akt Is Required for p300-dependent Acetylation of RelA/p65—We have previously demonstrated that histone deacetylase inhibitors activate NF-{kappa}B through a PI3K/Akt mechanism (9). Alternatively, other kinases have been shown to regulate NF-{kappa}B transcriptional activity. To examine this possibility NSCLC cells were transfected with a 3x-{kappa}B reporter gene and subsequently treated with or without SAHA in the presence of pharmacologic inhibitors of the following kinases: LY (PI3K/Akt), PD98059 (MEK/MAP kinase), SB203580 (p38), DNMB (DNA-PK), or SP600125 (JNK). As shown in Fig. 3A, only inhibition of the PI3K/Akt pathway with LY resulted in a significant decrease in SAHA-mediated activation of NF-{kappa}B. Because LY has been shown to also block the DNA-PK pathway (27), it was important to observe no reduction in NF-{kappa}B activation following pharmacologic inhibition of DNA-PK.

Because we have established that the transactivation potential of NF-{kappa}B is dependent on acetylation at Lys310 on RelA/p65 (Fig. 1, D and E), and that Akt is required for HDI-mediated NF-{kappa}B-dependent transcription (9), we wanted to know whether Akt was required for acetylation of RelA/p65. As shown in Fig. 3B, in the absence of Akt activity, RelA/p65 acetylation is largely abolished both basally and following exposure to SAHA. This occurs without a significant diminution of nuclear p65 levels suggesting that Akt is not regulating acetylation of RelA/p65 through enhanced nuclear translocation or decreased nuclear exportation. Realizing that transient transfection assays have inherent limitations, we examined if SAHA could increase endogenous acetylation of RelA/p65 in our model system. As shown in Fig. 3C, SAHA does increase endogenous RelA/p65 acetylation relative to controls, and, importantly, this effect was blocked following the addition of LY.

A potential mechanism through which inhibition of the PI3K/Akt pathway could be affecting RelA/p65 acetylation and subsequent transcriptional activation would be through facilitating a physical uncoupling of p300 and RelA/p65 such that the ability of the p300 HAT domain to acetylate RelA/p65 would be lost. As shown in Fig. 3D, RelA/p65 does physically interact with p300 in the presence of SAHA, but this interaction is minimally affected following treatment with LY. Thus, while there is some decrease in p300 and p65 binding following inhibition of the PI3K/Akt pathway this does not account for the complete loss of RelA/p65 acetylation as demonstrated in Fig. 3C.

Collectively these experiments establish that Akt is required for p300-dependent, SAHA-mediated acetylation of RelA/p65 acetylation but that inhibition of the PI3K/Akt pathway does not significantly affect RelA/p65-p300 interactions.

SAHA-induced Akt Phosphorylation Promotes Phosphorylation of p300 (Ser1834), Which Is Required for Acetylation of RelA/p65—Akt phosphorylates p300 on serine 1834 as demonstrated in an in vitro kinase assay using our newly developed phosphospecific p300 (Ser1834) antibody (Fig. 4A). Moreover, as shown in Fig. 4B, exposure of 293T and NSCLC cells to SAHA results in a robust phosphorylation of p300 (Ser1834). Additionally, there is evidence of increased activation of Akt by SAHA as measured by increased amounts of phospho-Akt (Ser473). Furthermore, the addition of LY dramatically suppressed phosphorylation at both the p300 (Ser1834) and Akt (Ser473) sites. Confirmation of the importance of Akt in regulating the transcriptional activity of p300 was demonstrated as siRNA to Akt1 completely blocked SAHA-induced activation of p300 transcriptional activity (Fig. 4C).


Figure 4
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  		FIGURE 4.
SAHA phosphorylates Akt (Ser473) and p300 (Ser1834), which control RelA/p65 acetylation and transcription. A, cold immunokinase assays were performed to prove the specificity of newly developed phospho-p300 Ser1834 antibody by incubating WT GST-p300 or mutant GST-p300 (S1834A) proteins (5 µg/reaction) with recombinant Akt (+) or without kinase (–). Western blot analysis was used to detect Akt-induced phosphorylated p300 using {alpha}-p300 (Ser1834) (1:2000) developed in collaboration with Upstate%20Biotechnology">Upstate Biotechnology. Coomassie Blue-stained gel (lower panel) displays equal levels of GST-p300 proteins per lane. B, 293T and NSCLC cells were not treated or treated with SAHA (5 µM), LY (25 µM), or SAHA (5 µM) plus LY (25 µM) for 2 h. Endogenous phospho-p300 (Ser1834), phospho-Akt (Ser473), p300, and Akt were probed by using immunoblot. beta-Tubulin was detected as a loading control. C, 293T cells were transiently transfected with expression vectors encoding the fusion protein Gal4-p300 and the Gal4-Luciferase reporter. Cells were also co-transfected with siRNA Akt1 or siRNA control. Cells were left untreated (No add), or treated with SAHA (5 µM) for 12 h. Luciferase activities were determined. (*) p < 0.01 compared with siRNA control plus SAHA. D, 293T cells were co-transfected with 3x-{kappa}B-luciferase reporter and expression vector encoding FLAG-tagged p65. These cells were also co-transfected with expression plasmid encoding full-length p300WT or p300S1834A mutant constructs, or an empty vector control. In addition, a plasmid encoding M-Akt was co-transfected as indicated. Cells were then left untreated (No add), or treated with SAHA (5 µM) for 12 h. Luciferase activities were determined. ({ddagger}) p < 0.05 compared with p300WT with (lane 4) and without M-Akt (lane 5). E, acetylation assays were repeated in 293T cells transfected with expression constructs as noted in the figure. Protein expression of the transfected constructs was confirmed by Western blot. F, similar acetylation assay is performed except 293T cells were left untreated or treated with SAHA (5 µM) for 2 h.

 
To confirm the requirement of Akt-mediated phosphorylation of p300 (Ser1834) on NF-{kappa}B transcriptional activity, 3x-{kappa}B luciferase assays were performed in the presence or absence of SAHA and ectopic expression of either p300WT or p300S1834A and M-Akt. Importantly, there was a significant decrease in NF-{kappa}B transcriptional activity in cells expressing the p300S1834A construct compared with those overexpressing p300WT (Fig. 4D, compare lane 3 to lane 5). Akt further stimulated p300WT, but not p300S1834A, mediated NF-{kappa}B transcriptional activity (Fig. 4D, compare lane 4 to lane 6). The observation that cells overexpressing both p300S1834A and M-Akt are not completely rescued from SAHA-induced activation of NF-{kappa}B, suggests that Akt is activating NF-{kappa}B independent of phosphorylation of p300. Taken together these results establish a direct link between SAHA induced Akt-mediated phosphorylation of p300 (Ser1834) to acetylation of RelA/p65 (Lys310) and transcriptional activation of NF-{kappa}B.

To further investigate the importance of p300 (Ser1834) on acetylation of RelA/p65, acetylation assays were repeated. As expected, there was complete inhibition of acetylation of RelA/p65 in cells overexpressing the mutant p300S1834A construct. Moreover, this absence of acetylation on RelA/65 could not be overcome even with overexpression of M-Akt (Fig. 4E). Finally, identical results on the acetylation of RelA/p65 were seen in cells treated with SAHA as shown in Fig. 4F.

While it is possible that Akt may phosphorylate other serine residues on p300, phosphorylation of Ser1834 is required for p300 acetyltransferase activity and acetylation of RelA/p65 following treatment with SAHA. Collectively, these results provide compelling evidence of a novel transcriptional mechanism-linking phosphorylation and acetylation events through which SAHA induces NF-{kappa}B activation.

Recruitment of SAHA-induced, Phospho-Akt (Ser473), Phospho-p300 (Ser1834), and Acetyl-RelA/p65 (Lys310) to NF-{kappa}B-regulated Promoters Is Akt-dependent—To determine whether Akt kinase activity was required for recruitment of phospho-Akt (Ser473), phospho-p300 (Ser1834), and acetyl-RelA/p65 (Lys310) to NF-{kappa}B-dependent promoters, ChIP assays were performed across both the cIAP-2 and Bfl-1/A1 promoters (Fig. 5, A and B) in the presence or absence of SAHA/LY. Using our phospho- and acetyl-specific antibodies one can observe a SAHA-enhanced recruitment of endogenous phospho-Akt (Ser473), phospho-p300 (Ser1834), and acetyl-RelA/p65 (Lys310) to chromatin. Importantly, the addition of the PI3K/Akt inhibitor LY markedly diminished recruitment of these same proteins to the promoter regions even in the presence of SAHA. The dramatic decrease in RNA polymerase II at both promoters following LY treatment suggests a loss of the resulting enhancesome formation that occurs following SAHA exposure. Fig. 5A represents the raw data and illustrates the total levels of proteins across the promoters. The identical data are shown in Fig. 5B and is normalized to each antibody to reflect changes in binding for that specific protein depending on experimental conditions. Confirmation of the correct size of the mRNA product is confirmed using semi-quantitative PCR (Fig. 5C).


Figure 5
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  		FIGURE 5.
Inhibition of PI3K/Akt pathway attenuates SAHA-induced recruitment of acetyl-p65 Lys310, phospho-p300 (Ser1834), phospho-Akt (Ser473), and RNA polymerase II formation on both cIAP2 and Bfl-1/A1 promoters. A, 293T cells were left untreated (No add), or treated with SAHA (5µM), LY (25µM), or SAHA (5 µM) plus LY (25 µM) for 2 h. ChIP analysis was performed on two NF-{kappa}B-regulated genes, cIAP2 and Bfl-1/A1; GAPDH served as a non-NF-{kappa}B-regulated gene control. Quantitative PCR was performed and the raw data are presented as percent of input DNA. B, exact same data from experiment 5A is presented as promoter fold expression by normalizing (to 1) the amplified promoter regions including the {kappa}B binding site(s) with no treatment for each antibody. C, products of PCR were resolved on 1.5% agarose gels.

 
Interestingly, examination of our GAPDH control gene (Fig. 5A) suggests that Akt is not recruited to this promoter despite the presence of p300. This suggests that RelA/p65, not p300, may in fact actively recruit Akt to NF-{kappa}B-regulated promoters in response to SAHA treatment.

Inability to Acetylate RelA/p65 on Lys310 Promotes SAHA-induced Apoptosis—To examine the biologic relevance of acetylation of RelA/p65 on Lys310, cell viability and caspase-3 assays were performed in NSCLC cells. As shown in Fig. 6, the inability to acetylate RelA/p65 on Lys310 results in a significant increase in cell death (Fig. 6A) and apoptosis (Fig. 6B) with escalating doses of SAHA. Overexpression of the RelA/p65 K310R mutant in our NSCLC model system results in dramatically less cell viability prior to drug exposure compared with cells-overexpressing RelA/p65 WT, suggesting that the loss of this acetyl mark is important in regulating even basal tumor cell survival.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Whereas HDIs alter only 2% of the expression of transcribed genes (28), there is increasing evidence that HDIs may play an important role in the treatment of both hematologic and non-hematologic malignancies (29). Unfortunately, both preclinical data from our group and early phase clinical trials utilizing isolated HDI therapy have demonstrated little tumoricidal activity, regardless of the specific HDI examined (2, 9, 30–32). We have previously shown that one explanation for this clinical observation may be that HDIs, despite inhibiting all tumor cell deacetylase activity, activate the anti-apoptotic transcription factor NF-{kappa}B (8, 9, 33).

Regulation of NF-{kappa}B-dependent transcription has been thought to more commonly occur through proteasome-mediated degradation of its cytosolic inhibitor protein I{kappa}B, increased nuclear translocation of p65/p50 heterodimers, and increased DNA binding of -{kappa}B subunits (34). It has only recently been shown that acetylation of RelA/p65 and p50 at distinct lysine residues can also regulate its transcriptional activity (14, 16, 35). Our study extends the previous work on acetylation of NF-{kappa}B subunits by confirming that Lys310 on RelA/p65 is the primary lysine residue acetylated following SAHA treatment. This observation is supported by findings of Greene and co-workers (14) who demonstrated that acetylation of Lys310 following overexpression of RelA/p65 increased NF-{kappa}B transcriptional activity. Additionally, Yeung et al. (15) found that SIRT1, a class III HDAC, completely silences NF-{kappa}B transcription by deacetylating RelA/p65 at Lys310. Whereas SAHA is likely to promote acetylation of other lysine residues on RelA/p65, the complete loss of acetylation in cells overexpressing the RelA/p65K310R mutant, coupled with the dramatic decreases in NF-{kappa}B transcriptional activity strongly suggest that Lys310 is the predominate lysine residue involved in HDI-mediated acetylation and activation of NF-{kappa}B.

Several coactivator proteins with HAT domains, including CBP, p/CAF, SRC-1, -2, -3, and p300 are known to associate with and facilitate the transcriptional activity of NF-{kappa}B (13, 16, 26, 36). We found that the HAT domain of p300, and not p/CAF, was required for SAHA-induced acetylation of RelA/p65 at lysine 310. As suggested by several groups, it is likely that requirements for coactivator activation are stimulus and transcription factor dependent (35, 37, 38). For instance, Furia et al. (35) found that only p50, not RelA/p65, was acetylated following treatment with the HIV viral protein Tat. Furthermore, both p/CAF and p300 have been shown to have separate and distinct functions with other transcription factors including both the Sp1/KLF and the hepatocyte nuclear factor-1{alpha} families (37, 38).


Figure 6
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  		FIGURE 6.
RelA/p65 Lys310 mutant sensitizes NSCLC cells to SAHA-induced apoptosis. A549 and H157 NSCLC cells were transiently transfected with the RelA/p65 WT or K310R mutant vectors. Cells were subsequently treated with a dose escalation of SAHA (0, 1, 2, 5, 10 µM) for an additional 2 h. Cell viability (A) and caspase-3 activity (B) were determined as markers for apoptosis. (*) p < 0.05 and (**) p < 0.01 compared with RelA/p65 K310R group; (#) p < 0.05 compared with SAHA 0 µM groups.

 
Data presented in this article demonstrate two mechanisms through which SAHA can activate NF-{kappa}B transcription (Fig. 7). First, SAHA activates NF-{kappa}B through de-repression of HDAC-1-mediated inhibition of RelA/p65. Thus, SAHA is able to preserve or "restore" the acetyl mark on RelA/p65(Lys310) which promotes NF-{kappa}B transcription. Given that SAHA inhibits histone deacetylase activity this result would be expected although our observation of relative HDAC-1 specificity in our model system is interesting. In agreement with our findings, Ashburner et al. (18) demonstrated that HDAC-1 but not HDAC-2 was found to directly interact with RelA/p65. Alternatively, other groups have shown that only HDAC-3, not HDAC-1 or -2 deactylates RelA/p65, most likely through mechanisms involving the corepressors NCoR/SMRT (12, 17). All of these studies were performed in different cell lines and under different treatment conditions, suggesting that HDAC specificity for RelA/p65 deacetylation may depend on these and other factors.

The second mechanism through which SAHA activates NF-{kappa}B-dependent transcription involves specific Akt-mediated phosphorylation events that transcriptionally upregulate RelA/p65 through its enhanced acetylation. We initially observed that SAHA promotes activation of Akt through enhanced phosphorylation of serine 473 which has not been previously reported. Based on this observation, and our previous findings (9), we examined the co-activator p300 for the well characterized Akt consensus motif (RXRXXp(S/T)) (39) and identified only one potential Akt consensus site in the C-terminal region (1829–1834). A recent study by Huang et al. (40) emphasized the importance of this Akt consensus site following treatment with TNF in activating the HAT domain and transcriptional activity of p300. Furthermore, it has been previously demonstrated that Akt co-localizes with p300 (40, 41). By utilizing site-directed mutagenesis of p300 (Ser1834) and our newly developed phosphospecific Ser1834 antibody we were able to demonstrate that Akt-mediated phosphorylation of this specific serine is required for acetylation and activation of RelA/p65 following treatment with SAHA.


Figure 7
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  		FIGURE 7.
SAHA enhances RelA/p65 acetylation and transcriptional activation through two distinct pathways. 1, SAHA induces phosphorylation of Akt at serine 473 (which is completely blocked by LY). Activated Akt phosphorylates the coactivator p300 at serine 1834. This phosphorylation event is required for p300-mediated acetylation of the RelA/p65 subunit of NF-{kappa}Bat lysine 310. Acetyl-Lys310-RelA/p65 transcriptionally up-regulates NF-{kappa}B-regulated anti-apoptotic gene products. 2, SAHA attenuates histone deacetylase activity, most notably HDAC-1, resulting in enhanced NF-{kappa}B activation through de-repression of p300-mediated acetylation of RelA/p65.

 
The importance of this specific signal transduction pathway in activating HDI-induced NF-{kappa}B-dependent transcription is highlighted in the ChIP analysis (Fig. 5). SAHA dramatically increased binding of endogenous phospho-Akt (Ser473) and p300 (Ser1834), as well as acetyl RelA/p65 (Lys310) to both the cIAP-2 and Bfl-1/A1 promoters, while the addition of the PI3K/Akt inhibitor LY294002 completely ameliorated this recruitment. This is powerful evidence that SAHA-induced phosphorylation and activation of Akt regulates the anti-apoptotic biology observed in NSCLC following treatment with HDIs and that this occurs at the chromatin level (8, 9, 33). This finding may have important clinical applicability as the majority of NSCLC, as well as many other cancers, overexpress Akt (42), and as such, are primed for SAHA-induced activation of anti-apoptotic NF-{kappa}B pathways. Furthermore, we have recently demonstrated the importance of Akt in regulating tumor response to HDI therapy in both in vivo and xenograft NSCLC models (9, 23).

Both of the mechanisms described in this article through which SAHA increases NF-{kappa}B transcription result in increased acetylation of RelA/p65 on Lys310. While others have shown that acetylation of Lys310 on RelA/p65 is a marker of increased NF-{kappa}B transcription (14, 15), we have identified the biologic relevance of this specific acetylation event as it relates to apoptosis. As shown in Fig. 6, the inability to activate RelA/p65 through acetylation on Lys310 markedly enhances apoptotic tumor cell death following treatment with SAHA, suggesting that this lysine residue may be a putative target for drug development.

Whereas our findings focused on Akt-dependent mechanisms that modulate SAHA-induced activation of NF-{kappa}B, it is possible that other signal transduction pathways may be involved. It has recently been shown that TNF stimulates RelA/p65 acetylation on Lys310 through IKK-mediated phosphorylation of serines 276 and/or 529 on RelA/p65, and enhances assembly of phospho-RelA/p65 with p300 (19). While we did not explore the ability of SAHA to activate IKK-mediated phosphorylation of RelA/p65, this may be another mechanism of enhanced RelA/p65 acetylation. Other putative mechanisms of p300 activation include the recruitment of other coactivators such as p/CAF or SRC-1 to the CH3 domain of p300 that would enhance acetylation and transcription following p300 activation by Akt (40). Finally, HDI can activate the HAT domain of p300 independent of a phosphorylation event by promoting autoacetylation of key lysine residues on p300, such as Lys1499, as demonstrated by Thompson et al. (43).

In summary, this article establishes dual mechanisms through which SAHA activates the anti-apoptotic transcription factor NF-{kappa}B. One mechanism, explored to various degrees by other investigators (13, 16, 18), involves inhibition of the transcriptional repression provided by HDACs, specifically HDAC-1 in our system. In addition, we have identified a previously unreported signal transduction pathway through which SAHA promotes Akt activation via phosphorylation of serine 473, which in turn activates p300 through phosphorylation of serine 1834. This specific phosphorylation event is required to acetylate RelA/p65 on lysine 310, which results in recruitment of endogenous acetylated RelA/p65 to NF-{kappa}B-dependent promoters. These specific signaling events need to be considered as future oncopharmacologic therapies for HDI-resistant malignancies are designed.


   FOOTNOTES
 
* This research was supported in part by Grants K08 CA83920 (to D. R. J) and F32 CA101497 (to C. E. D.) from the NCI, National Institutes of Health and the American Association for Cancer Research (to D. R. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this article. Back

2 To whom correspondence should be addressed: Dept. of Surgery, Box 800679, University of Virginia, Charlottesville, VA 22908-0679. Tel.: 434-243-6443; Fax: 434-982-1026; E-mail: djones{at}virginia.edu.

3 The 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. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Denis C. Guttridge, Ph.D (The Ohio State University), Dr. Rong Li, Ph.D (University of Virginia), Dr. Marty W. Mayo, Ph.D (University of Virginia), and Sally J. Parsons, Ph.D. (University of Virginia) for helpful review and critique of the manuscript.



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 DISCUSSION
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