Histone Deacetylase Inhibition Down-Regulates Cyclin D1 Transcription by Inhibiting Nuclear Factor-KB/p65 DNA Binding

Histone deacetylase (HDAC) inhibitors are emerging as a promising new class of cancer therapeutic agents. HDAC inhibitors relieve the deacetylation of histone proteins. However, little is known about the nonhistone targets of HDAC inhibitors and their roles in gene regulation. In this study, we addressed the molecular basis of the down-regulation of the nuclear factor-KB (NF-KB)–responsive gene cyclin D1 by the HDAC inhibitor trichostatin A in mouse JB6 cells. Cyclin D1 plays a critical role in cell proliferation and tumor progression. Trichostatin A inhibits cyclin D1 expression in a NF-KB-dependent manner in JB6 cells. Electrophoretic mobility shift assay studies showed that trichostatin A treatment prevents p65 dimer binding to NF-KB sites on DNA. Moreover, a chromatin immunoprecipitation assay shows that trichostatin A treatment inhibits endogenous cyclin D1 gene transcription by preventing p65 binding to the cyclin D1 promoter. However, acetylation of p65 is not affected by trichostatin A treatment. Instead, trichostatin A enhances p52 acetylation and increases p52 protein level by enhancing p100 processing. This is the first report that trichostatin A, a HDAC inhibitor, activates p100 processing and relieves the repression of p52 acetylation. The enhanced acetylation of p52 in the nuclei may operate to cause nuclear retention of p65 by increasing the p52/p65 interaction and preventing IKBA-p65 binding. The enhanced p52 acetylation coincides with decreased p65 DNA binding, suggesting a potential role of p52 acetylation in NF-KB regulation. Together, the results provide the first demonstration that HDAC inhibitor trichostatin A inhibits cyclin D1 gene transcription through targeting transcription factor NF-KB/p65 DNA binding. NF-KB is therefore identified as a transcription factor target of trichostatin A treatment. (Mol Cancer Res 2005;3(2):100–9) Introduction Histone deacetylase (HDAC) inhibitors suppress cell proliferation and induce tumor cell growth arrest and apoptosis (1, 2). They selectively affect transcription of a small portion of the genes, including cell cycle regulators, such as p21, and exert potent antitumor effects both in vivo and in vitro (3-5). However, with the exception of the hyperacetylation of histone proteins, little is known of the molecular basis of HDAC inhibitor target gene regulation and of the nonhistone acetylation targets (6). The transcription factor nuclear factor-nB (NF-nB) consists of two groups of NF-nB members. The first group consisting of Rel proteins [RelA (also known as p65), RelB, and c-Rel] is synthesized in the mature form. The second group consisting of NF-nB1 (p105) and NF-nB2 (p100) is processed to produce the mature p50 and p52 proteins, respectively (7). These two groups dimerize to form heterodimers or homodimers that bind to a common sequence motif known as the nB site. Among these dimers, RelA/p65-containing complexes are responsible for most of the transcriptional activity of NF-nB in many models. Two major NF-nB regulation pathways have been proposed. In resting cells, NF-nB dimers are held captive in the cytoplasm by specific inhibitory InB proteins (InBa, InBh, InBq, p105, and p100). Activation of NF-nB is triggered by stimuli, such as tumor necrosis factor-a (TNF-a), which activates the InB kinase (IKK) complex (8, 9). Activated IKK then phosphorylates NF-nB-bound InB, and this targets InB for ubiquitin-dependent degradation, allowing the liberated NF-nB dimers to translocate to the nucleus to activate NF-nBresponsive genes (10-12). Proteolytic processing of p105 and p100 is another important pathway of NF-nB regulation. The processing of p105 is largely a constitutive event (13, 14). In contrast to the high abundance of p50 in most cell types, the processing of p100 yields p52 in low abundance (15). The production of p52 is tightly controlled and highly selective and plays an important role in NF-nB activation (16, 17). Certain cytokines selectively activate the catalytic subunit (IKKa) and, along with another protein kinase called NF-nB-inducible kinase, trigger this processing-dependent pathway. Together, IKKa and NF-nB-inducible kinase induce the phosphorylationdependent proteolytic removal of the InB-like COOH-terminal domain of p100. This allows dimers containing p52 to translocate to the nucleus (18, 19). Post-translational modifications of NF-nB proteins are yet another level of NF-nB regulation. Like p65 phosphorylation Received 4/19/04; revised 12/7/04; accepted 1/10/05. 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. Requests for reprints: Jing Hu, Gene Regulation Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute-Frederick, Building 567, Room 188, Frederick, MD 21702. Phone: 301-846-6216; Fax: 301846-6907. E-mail: [email protected] Copyright D 2005 American Association for Cancer Research. Mol Cancer Res 2005;3(2). February 2005 100 on April 13, 2017. © 2005 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from (20), p65 acetylation is also important for NF-nB activation. Whether acetylation of p65 activates or suppresses NF-jB target gene transcription seems to depend on the biological context of the cell and the discrete acetylation sites of the NF-nB subunits (21, 22). Acetylation of p65 at Lys, Lys, and Lys activates NF-nB by inhibiting InBa binding to p65 and preventing the nuclear export of the NF-nB complex. Acetylation of p65 at Lys and Lys suppresses NF-nBdependent gene transcription by reducing its binding to nBcontaining DNA, facilitating its removal by InBa and subsequent export to the cytoplasm (23). In addition to acetylation of p65, acetylation of NF-nB p50 can be regulated (24). Acetylation of p50 increases its DNA binding and further enhances NF-nB transcriptional activity. Enhanced p50 acetylation correlates with increased p50 binding to cyclooxygenase-2 promoter and transcriptional activation (25). NF-nB activation induces a variety of genes that are involved in cell proliferation and cell survival, including cyclin D1 (7, 26). The HDAC inhibitors interrupt cell cycle progression in G1 and G2-M phase, resulting in growth arrest through induction of p21 and suppression of cyclin D1 expression (27-29). The HDAC inhibitor-induced p21 expression is mediated by Sp1 (28), whereas the molecular mechanism of the suppression of cyclin D1 is largely unknown. In this report, we show that down-regulation of cyclin D1 mRNA and protein by HDAC inhibitor trichostatin A in mouse JB6 cells is due to the lack of p65 binding to the cyclin D1 promoter. Although acetylation of p65 is unaffected by trichostatin A, trichostatin A activates p100 processing and selectively enhances acetylation of p52. Hyperacetylation of p52 coincides with diminished p65 DNA binding, suggesting a potential role of p52 acetylation in the regulation of NF-nB and NF-nB-controlled gene expression. Results Trichostatin A Treatment Suppresses Cyclin D1 Transcriptional Activation HDAC inhibitors stimulate the expression of growthinhibitory genes, such as p21 and p27 , and suppress the proliferation of cancer cells (4). Expression of cyclin D1, a positive cell cycle regulator that plays a critical role in tumor development (30, 31), is inhibited by HDAC inhibitors (27). In this study, we tested whether and how trichostatin A suppresses cyclin D1 expression. As shown in Fig. 1A, trichostatin A exposure suppressed cyclin D1 protein expression. Positive controls TNF-a and 12-O-tetradecanoylphorbol-13-acetate (TPA) are shown to enhance cyclin D1 expression. Coexposure of trichostatin A and TNF-a down-regulated cyclin D1 protein expression, indicating that trichostatin A overrides the inducing effect of TNF-a on cyclin D1 gene expression. We next examined whether the down-regulation of cyclin D1 occurs at a pretranslational level. Northern blotting results (Fig. 1B) show that cyclin D1 mRNA expression is suppressed by trichostatin A treatment in JB6 cells. Again, trichostatin A also abolished TNF-a-induced cyclin D1 mRNA expression. Assay of transfected cyclin D1 promoter reporter activity suggests that trichostatin A completely inhibits TNF-a-induced transcription from the cyclin D1 promoter (Fig. 1C). In contrast to the dramatic trichostatin A suppression of endogenous basal cyclin D1 mRNA expression (Fig. 1B), basal transcription from the transfected cyclin D1 promoter showed little or no inhibition by trichostatin A (Fig. 1C). This may suggest that, whereas trichostatin A inhibition of endogenous TNF-a-induced expression is transcriptional, trichostatin A inhibition of endogenous basal expression (Fig. 1B) is not transcriptional. Alternatively, the cyclin D1 promoter may resemble that of metallothionein (32) in containing separate elements for regulating induced and basal transcription. The partial cyclin D1 promoter sequence ( 66) used in the transfection experiment may contain elements needed to regulate induced transcription but may lack one or more elements needed to regulate basal transcription. Another possibility is that a transfected promoter is not functioning in a chromatin context and consequently does not recapitulate all of the regulation (in this case, basal regulation) that is seen with endogenous genes (33). In summary, trichostatin A inhibits TNF-a-induced and basal expression of endogenous cyclin D1. Observations with a transfected cyclin D1 promoter suggest that the regulation of TNF-a-induced but not basal cyclin D1 expression is