Targeted histone H4 acetylation via phosphoinositide 3-kinase- and p70s6-kinase-dependent pathways inhibits iNOS induction in mesa
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《美国生理学杂志》
Departments of 1Internal Medicine and of 2Integrative Biology, Pharmacology
Physiology, The University of Texas Medical School at Houston
the 3Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, Houston, Texas
ABSTRACT
The inducible nitric oxide synthase (iNOS) gene plays an important role in the response to and propagation of injury in glomerular mesangial cells. Although several cis and trans regulatory factors have been characterized, epigenetic regulation of the iNOS gene has not been considered extensively. In this report, we explored the role of histone acetylation in interleukin (IL)-1-mediated iNOS induction in cultured murine mesangial cells. Treatment of cells with the histone deacetylase inhibitor trichostatin A (TSA, 200 nM) resulted in a time-dependent, selective increase in histone H4 acetylation. TSA treatment of cells stably transfected with an iNOS promoter-luciferase construct inhibited IL-1 induction of endogenous nitric oxide and iNOS protein production and iNOS promoter-luciferase activity. Chromatin immunoprecipitation assays revealed that, under basal conditions, acetylated histone H4 associated with the region –978 to –710 of the iNOS promoter, a region rich in gene control elements and that IL-1 significantly increased this binding, which was further accentuated by cotreatment with TSA. Blockade of the phosphoinositide 3-kinase pathway with LY-294002 or the p70s6-kinase pathway with rapamycin in the presence of TSA and IL-1 inhibited 389Thr phosphorylation of p70s6 kinase, promoted binding of acetylated histone H4 to the iNOS promoter, and further suppressed iNOS protein expression and iNOS promoter activity. Thus TSA diminishes IL-1-induced iNOS transcription through phosphoinositide 3-kinase- and p70s6 kinase-dependent pathways that increase site-specific histone H4 acetylation at the –978 to –710 region of the iNOS promoter. This novel epigenetic control mechanism extends the network of regulatory controls governing NO production in mesangial cells.
epigenetic; nitric oxide; transcription
NITRIC OXIDE (NO) is an endogenous cell signaling molecule involved in the regulation of key physiological and pathophysiological processes in the kidney and many other tissues. NO is produced by one of three NO synthases (NOS): neuronal NOS, endothelial NOS, and inducible NOS (iNOS). The iNOS gene is quiescent in most tissues until it is transcriptionally activated by diverse stimuli to produce large amounts of NO (20). Excessive NO production has been linked to several forms of glomerular injury (7, 16), and glomerular mesangial cells have been shown to generate cytokines, chemokines, and high-output NO when activated by immunological or inflammatory stimuli (14). The sustained flux of large amounts of NO produced by iNOS can result in cytotoxicity to both the host and the target cell. Accordingly, both positive and negative modulators have evolved to control tightly iNOS expression and to prevent untoward effects of excessive NO production.
iNOS transcription in mesangial cells is regulated in a complex manner by several constitutive and inducible transcription factors, including CREB (14), C/EBP (14), and NF-B (3). In contrast, transforming growth factor- (23), interleukin (IL)-13 (28), and STAT3 (37) suppress iNOS transcription. Epigenetic controls on iNOS transcription are also operative, and we have shown that hyperacetylation (37) and DNA methylation (36) limit iNOS activation in these cells. Although much is known about the cis and trans regulatory factors controlling activation of iNOS transcription by cytokines and bacterial lipopolysaccharide (LPS), relatively little is known about how iNOS transcription might be constrained and how local changes in chromatin structure might participate in this process.
At least two distinct mechanisms are thought to alter the repressive nature of bulk chromatin to regulate gene expression. One mechanism involves ATP-dependent nucleosome remodeling by which chromatin remodeling complexes transfer and slide histone cores, making DNA accessible to transcription factors and DNA binding factors that promote transcription (18). The second mechanism involves factors that directly modify core histone tails through acetylation, phosphorylation, methylation, or ubiquitination (10). Acetylation-related factors include histone acetyltransferases (HATs) and histone deacetylases (HDACs), which acetylate and deacetylate, respectively, lysine residues at the NH2-terminal tails of the core histones (5). The dynamic equilibrium of HATs and HDACs determines the net level of acetylation and, in turn, regulates transcription. Trichostatin A (TSA), an HDAC inhibitor, can promote core histone acetylation and affect the expression of 2% of cellular genes (30). We previously demonstrated that TSA diminished augmented cytokine induction of iNOS transcriptional activity in mesangial cells in part via an interaction of the HDAC2 isoform with NF-B p65 that apparently increased NF-B transactivation potential without altering its DNA-binding activity (36).
Multiple cytokines activate phosphatidylinositol 3-kinase (PI3K) and the downstream p70s6-kinase pathway in a variety of systems (19, 21, 22, 26). Cytokines, including IL-1, have also been shown in other systems to activate acetylation of histones H3 and H4 to modulate proinflammatory genes through different kinase pathways. Moreover, rapamycin, an immunosuppressant that binds and inhibits the function of mammalian target of rapamycin (mTOR) and downstream activation of p70s6 kinase, the major ribosomal kinase, has been shown to effect site-specific acetylation of histone H4 (25). Accordingly, we hypothesized that the PI3K and p70s6-kinase pathways, through regulation of targeted histone acetylation and local chromatin structure, participate in the control of iNOS gene transcription in mesangial cells. Using TSA as a tool to modulate histone acetylation, we now show that TSA inhibition of iNOS gene transcription is associated with increased binding of acetylated histone H4 in a targeted manner to the iNOS promoter by a mechanism that is dependent on the PI3K and p70s6 kinase pathways. This novel effect of histone H4 acetylation to restrict rather than augment gene expression may represent an important cell-specific mechanism to downregulate the inflammatory response and limit NO production.
EXPERIMENTAL METHODS
Cell culture and reagents. Wild-type mouse mesangial cells (ATCC CRL-1927) were maintained in Ham's F-12 plus DMEM supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5% FBS. Our previously characterized (35) mouse mesangial cell lines that stably express the proximal 1.6-kb murine iNOS promoter fused to the firefly luciferase coding region in pcDNA3.1-Zeo(+) [designated pcDNA3.1-Zeo(+)/iNOS-luc] were maintained in the same medium supplemented with 400 μg/ml Zeocin (Invitrogen). Cells transfected with the empty pcDNA3.1-Zeo(+) vector served as a control for other vector components, including the CMV promoter 5' to the multicloning site retained in both constructs. Vehicle, murine recombinant IL-1 (10 ng/ml; R&D Systems, Minneapolis, MN), TSA (200 nM; Sigma, St. Louis, MO), LY-294002 (50 μM; Calbiochem, San Diego, CA), rapamycin (20 ng/ml; Calbiochem), or insulin (1 μM; Sigma) was added to the cells as indicated in the text and legends for Figs. 1–6. Oligonucleotides were custom synthesized by SigmaGenosys (The Woodlands, TX). Lipofectamine 2000 reagent and TRIzol reagent were from Invitrogen (Carlsbad, CA). The Dual-Luciferase Reporter Assay System, the luciferase vectors pGL3-Basic and pRL-SV40, and Moloney murine leukemia virus (M-MLV) RT were from Promega. The bicinchoninic acid protein estimation kit was from Pierce Chemical. LY-294002 and rapamycin were from Calbiochem. Antibodies against iNOS, histone H4, and acetylated histone H4 or H3 were from Upstate Biotechnology (Charlottesville, VA). Anti-p70s6 kinase antibody and anti-phospho-p70s6 kinase antibody were from Cell Signaling (Beverly, MA). Anti-cyclooxygenase (COX)-2 antibody was from Cayman (Ann Arbor, MI).
Nitrite assays. Mesangial cells, wild type or transfected, were seeded in 24-well plates and treated with IL-1 for 24 h and the reagents indicated in the text and legends for Figs. 1–6. The medium was then collected, and the nitrite concentration was determined with the Griess Reagent System (Promega) according to the manufacturer's protocol.
Acid extraction of histone proteins. Confluent cells on 150-mm-diameter dishes were harvested in lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 1.5 mM phenylmethylsulfonyl fluoride, and 0.2 M HCl) for 30 min on ice. After centrifugation at 11,000 g for 10 min, the supernatants were dialyzed against 0.1 M acetic acid two times for 1 h each. The protein was quantified and stored at –80°C for further analysis.
Western blotting. Cytoplasmic and nuclear extracts from mouse mesangial cells were prepared using the Nuclear/Cytosol Fractionation Kit (BioVision) according to the manufacturer's manual. Samples (20 μg) of nuclear, cytoplasmic, whole cell, or histone protein extracts were resolved by SDS-PAGE, and the proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Hybond ECL; Amersham). The blots were probed with antibodies against iNOS, COX-2, acetylated histone H3, acetylated histone H4, histone H4, p70s6 kinase, phospho-p70s6 kinase, or -tubulin (all antibodies at 0.2 μg/ml) overnight at 4°C. The blots were washed extensively with a solution containing 50 mM Tris, pH 8.0, 138 mM NaCl, 2.7 mM KCl, and 0.05% Tween 20. The antigen-antibody complexes were detected by the enhanced chemiluminescence protocol using horseradish peroxidase-conjugated donkey anti-rabbit IgG as secondary antibody.
Chromatin immunoprecipitation assay and quantitative real-time PCR. Chromatin immunoprecipitation (ChIP) analysis was performed as described in our recent report (35), except that immunoprecipitation was performed with anti-acetylated histone H4 or rabbit nonimmune IgG, and the PCR was conducted with primers framing the murine iNOS 5'-flanking region –978 to –710 [5'-TGCTAGGGGGATTTTCCCTCTCTC-3' (nucleotides –978 to –955) and 5'-AGCCTTTAATCCCGGGATTCAGG-3' (nucleotides –732 to –710)]. The specificity of these primer sets for their targets was confirmed by agarose gel electrophoresis. PCR for the input was performed with genomic DNA. The input fraction corresponded to 1% of the chromatin solution before immunoprecipitation. The PCR products were analyzed on a 2% agarose gel and quantified by real-time PCR using the DyNAmo SYBR green qPCR kit (Finnzymes) and an MJ Research DNA Engine Opticon 2 System (South San Francisco, CA), as described in our previous work (35). Each quantitative PCR assay was repeated three times, and the results were averaged.
RT-PCR. RT-PCR for semiquantitative analysis of mRNA levels of the transfected Zeocin resistance gene encoded by the pcDNA3.1-Zeo(+) or pcDNA3.1-Zeo(+)/iNOS-luc plasmids and for endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH; as a housekeeping gene control) was performed as previously described (35) on cDNA synthesized using M-MLV RT from total RNA isolated with Tri-Zol reagent from transfected mesangial cells. Primers were designed to amplify specifically nucleotides +1 to +375 of the Zeocin resistance gene (sense: 5'-ATGGCCAAGTTGACCAGTGCC-3'; antisense: 5'-TCAGTCCTGCTCCTCGGCCAC-3') or nucleotides +238 to +757 of GAPDH (sense: 5'-CCACTAACATCAAATGGGGTGAGG-3'; antisense: 5'-TACTTGGCAGGTTTCTCCAGGC-3'). PCR conditions were an initial denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 45 s, 52°C for 45 s, 72°C for 2 min, and a final extension step of 72°C for 20 min. The PCR products were electrophoresed on 0.8% agarose gels and imaged with a Kodak ImageStation 1000 (Packard).
Data analysis. Quantitative data are presented as means ± SE and were analyzed by ANOVA. Significance was assigned at P < 0.05.
RESULTS
TSA suppresses IL-1 induction of endogenous NO production, iNOS protein, and the activity of a stably incorporated iNOS promoter. To extend our previous studies, which used transient transfections of iNOS promoter-luciferase constructs, we exploited the HDAC inhibitor TSA and mesangial cell lines that had been stably transfected with pcDNA3.1-Zeo(±)iNOS-luc, a mammalian expression vector containing the murine iNOS promoter fused to the firefly luciferase gene (35), to examine further the effects of acetylation status on endogenous NO production and iNOS protein and the activity iNOS promoter-luciferase DNA stably integrated in the genome wrapped by histones. TSA-treated mesangial cells harboring the iNOS promoter-luciferase DNA exhibited 45% lower IL-1-stimulated nitrite levels (Fig. 1A) and iNOS protein levels (Fig. 1B) compared with vehicle-treated controls, indicating that hyperacetylation inhibits cytokine induction of iNOS expression. In contrast, TSA significantly augmented protein expression of COX-2, another cytokine-inducible gene in mesangial cells (Fig. 1B), indicating that acetylation status has different effects depending on the specific gene in murine mesangial cells.
As seen in Fig. 1C, mesangial cell lines stably expressing the iNOS promoter-luciferase construct exhibited the expected induction of iNOS promoter-luciferase activity in response to IL-1 in the absence of TSA. In keeping with the nitrite and iNOS protein responses, mesangial cell lines treated with TSA exhibited a degree of induction of iNOS promoter activity in response to IL-1 that was 30% lower than controls (Fig. 1C). In the aggregate, these data in stably transfected cells are in keeping with our previous report in transiently transfected mesangial cells (36) and suggest that hyperacetylation decreases and hypoacetylation increases IL-1 induction of stably integrated iNOS promoter activity in mesangial cells and that the TSA inhibition of iNOS induction could be related to a direct effect on the acetylation of histones.
Because pcDNA3.1-Zeo(+)/iNOS-luc retains the CMV promoter of the parental pcDNA3.1-Zeo(+) plasmid 5' to the iNOS promoter fragment-luciferase segment, we analyzed by semiquantitative RT-PCR mRNA levels for the Zeocin resistance gene, which is also downstream of the CMV promoter in both constructs, as an internal control in the cells transfected with the parent pcDNA3.1-Zeo(+) plasmid or with pcDNA3.1-Zeo(+)iNOS-luc. As seen in Fig. 1D, these mRNA levels were comparable in the cells transfected with either plasmid. Moreover, the iNOS promoter activity results presented here with the pcDNA3.1-Zeo(+)-derived construct were virtually identical to those we previously reported using the same iNOS promoter segment cloned into the pGL3-Basic vector, which lacks the CMV promoter, in transient transfection assays in mesangial cells (36), and they precisely mirror the responses of the endogenous iNOS gene (Fig. 1B). Thus, in the aggregate, these results indicate that the differences in luciferase activity in cells transfected with pcDNA3.1-Zeo(+)/iNOS-luc we observed here were the result of differences in iNOS promoter activity and were not significantly affected by the CMV promoter retained in the plasmid (Fig. 1D).
TSA promotes acetylation of histone H4 and increases chromatin-associated acetylated histone H4 binding at –978/–710 of the iNOS promoter. We previously demonstrated that acetylation status limits iNOS expression in part through an effect of HDAC2 interacting with the transcription factor NF-B p65 (36). To determine whether histones are acetylated and involved in the regulation of the iNOS gene, Western blots of acid extracts of histone proteins from mesangial cells treated with TSA for various lengths of time (0–24 h) were blotted with antibodies directed against histones H3 and H4 and acetylated histone H3 and H4. TSA induced histone H4 acetylation in a time-dependent manner over the first 4 h of treatment, without affecting total histone H4 levels (Fig. 2A). Histone H3 levels and histone H3 acetylation were unaffected (data not shown). Given these results, we sought to determine whether TSA alters the acetylation state of histone H4 associated with the iNOS promoter in mesangial cells using ChIP assays of nucleosomes from vehicle- and TSA-treated cells. As shown in Fig. 2B, TSA treatment resulted in a large increase in chromatin-associated acetylated histone H4 at the iNOS promoter region –978/–710 beginning at 5 min and peaking at 1–4 h of treatment. This region of the iNOS promoter is of particular interest because it includes B and -interferon-activated sites (GAS) elements known to be important in iNOS transactivation gene (15, 34, 36). Treatment with IL-1 alone (Fig. 2C, lane 2) or TSA alone (Fig. 2C, lane 3) resulted in a small increase in association of acetylated histone H4 at the –978/–710 region of the iNOS promoter, whereas cells treated with TSA and IL-1 exhibited a marked increase in the binding of acetylated histone H4 compared with the cells treated with IL-1 alone (Fig. 2C, lane 4 compared with lane 2).
Inhibitors of PI3K and p70s6 kinase increase acetylation of histone H4 and chromatin-associated acetylated histone H4 at –978/–710 of the iNOS promoter and inhibit IL-1-induction of the iNOS gene. Although the PI3K and p70s6 kinase pathways have been shown to be involved in histone modification (33, 34, 49, 50) and the regulation of the iNOS gene (21, 26) in some cell types, the coupled effects of these pathways on histone modification and iNOS gene transcription have not been established. Accordingly, we sought to determine whether these pathways are involved in histone H4 acetylation at the iNOS promoter in mesangial cells. As shown in Fig. 3, IL-1 induced phosphorylation of p70s6 kinase at 389Thr, whereas TSA significantly limited IL-1-induced phosphorylation of p70s6 kinase. Pretreatment with the PI3K inhibitor LY-294002 or rapamycin combined with TSA further decreased IL-1-induced phosphorylation of p70s6 kinase. To determine whether these pathways directly affected histone H4 binding at the iNOS promoter, ChIP assays coupled with quantitative PCR were performed. TSA + IL-1-treated cells pretreated with LY-294002 or rapamycin exhibited increased acetylated histone H4 binding at –978/–710 of the iNOS promoter compared with TSA + IL-1-treated cells pretreated with vehicle (Fig. 4). These data indicate that PI3K and p70s6 kinase regulate site-specific histone H4 acetylation in a targeted manner at the iNOS promoter to augment cytokine induction of the iNOS gene in mesangial cells.
In contrast to the effect of TSA to increase the abundance of acetylated histone H4 in mesangial cells (Fig. 2A), neither LY-294002 nor rapamycin by themselves promoted acetylation of histone H4 on Western blots of acid extracts of histone proteins extracted from the cells (Fig. 5). Moreover, insulin, a known mTOR activator (33), had no significant effect on the abundance of acetylated histone H4 in vehicle-treated mesangial cells and did not antagonize the effect of TSA to promote histone H4 acetylation (Fig. 5). These results indicate that PI3K and p70s6 kinase pathways do not directly cause histone H4 acetylation under basal conditions.
Pretreatment with LY-294002 or rapamycin also significantly limited IL-1-induced iNOS protein expression in mesangial cells, whereas addition of TSA together with either drug completely abolished IL-1-induced iNOS protein expression (Fig. 6A), indicating a synergistic effect. A similar effect of LY-294002 or rapamycin was seen when iNOS promoter-luciferase activity was examined (Fig. 6B). In contrast, LY-294002 or rapamycin in the absence, but not the presence, of TSA significantly limited COX-2 protein expression, again indicating gene-specific effects (Fig. 6A).
DISCUSSION
High-output NO production is potentially cytotoxic to the host and innocent bystander cells, so controls to limit or terminate iNOS gene expression are important cell survival mechanisms. Pathways involved in the repression of iNOS gene activation in cytokine-induced mesangial cells are still incompletely defined, and their characterization could potentially identify novel anti-inflammatory processes. Modulation of chromatin structure plays an important role in transcriptional regulation, yet little is known about the influence of chromatin on iNOS transcription. In the present study, TSA promoted acetylated histone H4 binding to a specific region of the iNOS promoter harboring key enhancer elements and suppressed production of endogenous NO and iNOS protein and iNOS promoter activity in mesangial cells. IL-1-mediated induction of iNOS transcription in these cells was associated with selective acetylation of histone H4 and chromatin remodeling, and blockade of PI3K- or p70s6-kinase pathways increased site-specific histone H4 acetylation at the iNOS promoter to limit further iNOS gene expression. In the aggregate, the data identify PI3-kinase- and p70s6 kinase-dependent pathways that increase site-specific histone H4 acetylation at the iNOS promoter as another mechanism, in addition to its effects on the HDAC2-NF-B p65 interaction, by which TSA diminishes IL-1-induced iNOS transcription.
Inputs received at promoter DNA in the form of multiple transcription factor-binding events can mediate the recruitment of chromatin modifiers, such as HATs, HDACs, histone kinases, and methyltransferases. These resultant epigenetic changes and posttranslational modifications of histones generate specific patterns of acetylation, phosphorylation, methylation, or ubiquitination, generating a "histone code" (29) that extends the information content of the DNA code. Histone acetylation represents a central switch that allows interconversion between permissive and restricted transcriptional states. Targeted acetylation at promoter nucleosomes through recruitment or dismissal of HAT and HDAC activities negotiates the acetylation status of chromatin. Many transcription factors regulate transcription by physically recruiting HATs and HDACs to promoters. The region –978/–710 of the murine iNOS promoter analyzed in this report contains a B enhancer element positioned at –971 to –962 that is critical for cytokine inducibility of the iNOS gene (34). Similarly, we and others have demonstrated the functional importance of two GAS at –942 to –934 (15) and at –879 to –871 (37) in activation of the iNOS gene in response to cytokines and LPS. Histone acetylation has been shown to regulate NF-B-dependent gene accessibility in some promoter contexts, but direct acetylation of NF-B subunits p65 and p50 also regulates different NF-B functions, including transcriptional activation, DNA-binding affinity, and IB assembly (9, 17, 24). Finally, acetyltransferases and deacetylases interact directly with several proteins involved in the NF-B signaling pathway, including NF-B itself, IB, IKK, and IKK. These interactions probably allow acetylation of NF-B itself, of other transcription factors, and of histones associated with NF-B-regulated genes (1, 4, 9, 17).
The large differences detected by ChIP assay in the extent of chromatin-associated, acetylated histone H4 at the iNOS promoter in mesangial cells treated with IL-1 alone vs. IL-1 + TSA suggests differences in chromatin structure in the two settings. Stimulus-specific patterns of histone acetylation likely result in different conformational changes of chromatin structure that are associated with distinct patterns of transcription factor association with the iNOS promoter and probably different kinetics of iNOS expression. One can speculate that a specific pattern of modified histone tails is required to recruit the basal transcription machinery and that TSA can distort this pattern and thus reduce the cytokine-induced transcriptional response. Further experiments will be needed to determine whether acetylated histone H4 binds to the iNOS promoter to condense chromatin DNA, preventing the accessibility of transcription factor(s), or whether the TSA-induced remodeling of the iNOS promoter is triggered by acetylation of a transacting factor(s), which might lead to site-specific binding to the iNOS promoter and thereby direct the translational nucleosome positioning to repress the iNOS promoter. We also cannot exclude the involvement of other histone modifications, such as methylation or phosphorylation, in IL-1-induced iNOS gene expression.
Much of what is known about TOR signaling in gene transcription and chromatin modifications has come from studies in yeast. In this organism, TOR is involved in regulating ribosomal protein gene expression via a nutrient supply-dependent and rapamycin-sensitive mechanism that, in response to ample nutrient supply, involves targeted recruitment of the Esa1-histone acetylase complex to these promoters, resulting in histone H4 acetylation and changes in chromatin structure that activate transcription. In response to nutrient limitation or rapamycin, the Esa1-histone acetylase complex is released from ribosomal protein gene promoters, and deacetylation mediated by the Rpd3-Sin3 HDAC complex resident at ribosomal protein gene promoters occurs, with reversion of chromatin to the repressed state (25). In mammalian cells, it has been proposed that the ability of the TSC2 tumor suppressor gene to regulate vascular endothelial growth factor involves regulation of HDACs (6). Thus there appears to be precedent for involvement of mTOR in histone acetylation.
In the current study, the fact that rapamycin, LY-294002, and insulin alone did not alter histone H4 acetylation under basal conditions leads to the conclusion that the actions of the PI3K and p70s6 kinase/mTOR pathways on histone H4 acetylation are indirect. The fact that the effects of TSA together with rapamycin or LY-294002 on histone H4 acetylation at the –978/–710 region of the iNOS promoter and IL-1-mediated induction of iNOS promoter activity were at least partially additive suggests that either the inhibitor combination gives more effective blockade of a single HDAC target or that separate perhaps partially overlapping TSA-sensitive and TSA-insensitive mechanisms are involved in regulating histone H4 acetylation. Thus the PI3K and p70s6 kinase/mTOR pathways could either augment or facilitate recruitment of TSA-insensitive HDAC activity, such as that of SIRT1, an NAD-dependent and TSA-insensitive deacetylase (11), and/or inhibit involvement of proteins with intrinsic HAT activity, such as p300 (9). For example, PI3K inhibitors have been shown to limit the expression of SIRT1 in a human melanoma cell line (31) and to alter p300 HAT activity (8). Further studies will be required to test the mechanistic details of our model in the complex context of IL-1-mediated changes in transcription factor, coregulatory protein, and local chromatin environment at the iNOS promoter.
The role of IL-1 in the activation of the PI3-kinase and p70s6 kinase and on NO production has been studied in different cells. In agreement with our data, LY-294002 and rapamycin blocked iNOS induction in murine macrophages in response to LPS or cytokines, suggesting that PI3K and p70s6-kinase are essential signals for the expression of iNOS and production of NO (21, 26). A similar inductive effect of LY-294002 on iNOS expression was observed in vascular smooth muscle cells (19). In contrast, others (13, 22) found that LY-294002 induced LPS- or IL-1-stimulated iNOS induction in C6 glioma cells. The reasons for the cell-specific effects of LY-294002 and rapamycin on iNOS expression are unclear, but the effects on targeted histone acetylation reported here likely represents at least one mechanism for this divergence. Thus, although it is generally believed that transcriptional activity correlates with histone acetylation, as it was for COX-2 in this study, recent reports indicate that several genes in addition to iNOS are inhibited by histone acetylation (2, 12, 32). As in our study with mesangial cells, butyrate, another HDAC inhibitor, suppressed iNOS transcription in DLD-1 colonic epithelial cells (27). These results suggest that cell type and promoter contexts are important for this mechanism.
The ability of acetylated histone H4 to influence the activity of the iNOS gene in mesangial cells lends further complexity to the intricate regulation of this gene. Although our findings demonstrated that the PI3K/p70s6 pathways regulate these processes, our results cannot be explained by simple inhibition of the kinase pathway activation, because there is an opposite effect for COX-2 gene activation. Further elucidation of the mechanisms by which TSA suppresses iNOS expression and cross talk between the PI3K/p70s6 pathways and histone acetylation in mesangial cells may provide novel clues for controlling excessive NO production during renal diseases.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health Grants RO1-DK-50745 and P50 GM-38529, a Department of Defense "T5" grant, and endowment funds from The James T. and Nancy Willerson Chair (to B. C. Kone).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Physiology, The University of Texas Medical School at Houston
the 3Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, Houston, Texas
ABSTRACT
The inducible nitric oxide synthase (iNOS) gene plays an important role in the response to and propagation of injury in glomerular mesangial cells. Although several cis and trans regulatory factors have been characterized, epigenetic regulation of the iNOS gene has not been considered extensively. In this report, we explored the role of histone acetylation in interleukin (IL)-1-mediated iNOS induction in cultured murine mesangial cells. Treatment of cells with the histone deacetylase inhibitor trichostatin A (TSA, 200 nM) resulted in a time-dependent, selective increase in histone H4 acetylation. TSA treatment of cells stably transfected with an iNOS promoter-luciferase construct inhibited IL-1 induction of endogenous nitric oxide and iNOS protein production and iNOS promoter-luciferase activity. Chromatin immunoprecipitation assays revealed that, under basal conditions, acetylated histone H4 associated with the region –978 to –710 of the iNOS promoter, a region rich in gene control elements and that IL-1 significantly increased this binding, which was further accentuated by cotreatment with TSA. Blockade of the phosphoinositide 3-kinase pathway with LY-294002 or the p70s6-kinase pathway with rapamycin in the presence of TSA and IL-1 inhibited 389Thr phosphorylation of p70s6 kinase, promoted binding of acetylated histone H4 to the iNOS promoter, and further suppressed iNOS protein expression and iNOS promoter activity. Thus TSA diminishes IL-1-induced iNOS transcription through phosphoinositide 3-kinase- and p70s6 kinase-dependent pathways that increase site-specific histone H4 acetylation at the –978 to –710 region of the iNOS promoter. This novel epigenetic control mechanism extends the network of regulatory controls governing NO production in mesangial cells.
epigenetic; nitric oxide; transcription
NITRIC OXIDE (NO) is an endogenous cell signaling molecule involved in the regulation of key physiological and pathophysiological processes in the kidney and many other tissues. NO is produced by one of three NO synthases (NOS): neuronal NOS, endothelial NOS, and inducible NOS (iNOS). The iNOS gene is quiescent in most tissues until it is transcriptionally activated by diverse stimuli to produce large amounts of NO (20). Excessive NO production has been linked to several forms of glomerular injury (7, 16), and glomerular mesangial cells have been shown to generate cytokines, chemokines, and high-output NO when activated by immunological or inflammatory stimuli (14). The sustained flux of large amounts of NO produced by iNOS can result in cytotoxicity to both the host and the target cell. Accordingly, both positive and negative modulators have evolved to control tightly iNOS expression and to prevent untoward effects of excessive NO production.
iNOS transcription in mesangial cells is regulated in a complex manner by several constitutive and inducible transcription factors, including CREB (14), C/EBP (14), and NF-B (3). In contrast, transforming growth factor- (23), interleukin (IL)-13 (28), and STAT3 (37) suppress iNOS transcription. Epigenetic controls on iNOS transcription are also operative, and we have shown that hyperacetylation (37) and DNA methylation (36) limit iNOS activation in these cells. Although much is known about the cis and trans regulatory factors controlling activation of iNOS transcription by cytokines and bacterial lipopolysaccharide (LPS), relatively little is known about how iNOS transcription might be constrained and how local changes in chromatin structure might participate in this process.
At least two distinct mechanisms are thought to alter the repressive nature of bulk chromatin to regulate gene expression. One mechanism involves ATP-dependent nucleosome remodeling by which chromatin remodeling complexes transfer and slide histone cores, making DNA accessible to transcription factors and DNA binding factors that promote transcription (18). The second mechanism involves factors that directly modify core histone tails through acetylation, phosphorylation, methylation, or ubiquitination (10). Acetylation-related factors include histone acetyltransferases (HATs) and histone deacetylases (HDACs), which acetylate and deacetylate, respectively, lysine residues at the NH2-terminal tails of the core histones (5). The dynamic equilibrium of HATs and HDACs determines the net level of acetylation and, in turn, regulates transcription. Trichostatin A (TSA), an HDAC inhibitor, can promote core histone acetylation and affect the expression of 2% of cellular genes (30). We previously demonstrated that TSA diminished augmented cytokine induction of iNOS transcriptional activity in mesangial cells in part via an interaction of the HDAC2 isoform with NF-B p65 that apparently increased NF-B transactivation potential without altering its DNA-binding activity (36).
Multiple cytokines activate phosphatidylinositol 3-kinase (PI3K) and the downstream p70s6-kinase pathway in a variety of systems (19, 21, 22, 26). Cytokines, including IL-1, have also been shown in other systems to activate acetylation of histones H3 and H4 to modulate proinflammatory genes through different kinase pathways. Moreover, rapamycin, an immunosuppressant that binds and inhibits the function of mammalian target of rapamycin (mTOR) and downstream activation of p70s6 kinase, the major ribosomal kinase, has been shown to effect site-specific acetylation of histone H4 (25). Accordingly, we hypothesized that the PI3K and p70s6-kinase pathways, through regulation of targeted histone acetylation and local chromatin structure, participate in the control of iNOS gene transcription in mesangial cells. Using TSA as a tool to modulate histone acetylation, we now show that TSA inhibition of iNOS gene transcription is associated with increased binding of acetylated histone H4 in a targeted manner to the iNOS promoter by a mechanism that is dependent on the PI3K and p70s6 kinase pathways. This novel effect of histone H4 acetylation to restrict rather than augment gene expression may represent an important cell-specific mechanism to downregulate the inflammatory response and limit NO production.
EXPERIMENTAL METHODS
Cell culture and reagents. Wild-type mouse mesangial cells (ATCC CRL-1927) were maintained in Ham's F-12 plus DMEM supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5% FBS. Our previously characterized (35) mouse mesangial cell lines that stably express the proximal 1.6-kb murine iNOS promoter fused to the firefly luciferase coding region in pcDNA3.1-Zeo(+) [designated pcDNA3.1-Zeo(+)/iNOS-luc] were maintained in the same medium supplemented with 400 μg/ml Zeocin (Invitrogen). Cells transfected with the empty pcDNA3.1-Zeo(+) vector served as a control for other vector components, including the CMV promoter 5' to the multicloning site retained in both constructs. Vehicle, murine recombinant IL-1 (10 ng/ml; R&D Systems, Minneapolis, MN), TSA (200 nM; Sigma, St. Louis, MO), LY-294002 (50 μM; Calbiochem, San Diego, CA), rapamycin (20 ng/ml; Calbiochem), or insulin (1 μM; Sigma) was added to the cells as indicated in the text and legends for Figs. 1–6. Oligonucleotides were custom synthesized by SigmaGenosys (The Woodlands, TX). Lipofectamine 2000 reagent and TRIzol reagent were from Invitrogen (Carlsbad, CA). The Dual-Luciferase Reporter Assay System, the luciferase vectors pGL3-Basic and pRL-SV40, and Moloney murine leukemia virus (M-MLV) RT were from Promega. The bicinchoninic acid protein estimation kit was from Pierce Chemical. LY-294002 and rapamycin were from Calbiochem. Antibodies against iNOS, histone H4, and acetylated histone H4 or H3 were from Upstate Biotechnology (Charlottesville, VA). Anti-p70s6 kinase antibody and anti-phospho-p70s6 kinase antibody were from Cell Signaling (Beverly, MA). Anti-cyclooxygenase (COX)-2 antibody was from Cayman (Ann Arbor, MI).
Nitrite assays. Mesangial cells, wild type or transfected, were seeded in 24-well plates and treated with IL-1 for 24 h and the reagents indicated in the text and legends for Figs. 1–6. The medium was then collected, and the nitrite concentration was determined with the Griess Reagent System (Promega) according to the manufacturer's protocol.
Acid extraction of histone proteins. Confluent cells on 150-mm-diameter dishes were harvested in lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 1.5 mM phenylmethylsulfonyl fluoride, and 0.2 M HCl) for 30 min on ice. After centrifugation at 11,000 g for 10 min, the supernatants were dialyzed against 0.1 M acetic acid two times for 1 h each. The protein was quantified and stored at –80°C for further analysis.
Western blotting. Cytoplasmic and nuclear extracts from mouse mesangial cells were prepared using the Nuclear/Cytosol Fractionation Kit (BioVision) according to the manufacturer's manual. Samples (20 μg) of nuclear, cytoplasmic, whole cell, or histone protein extracts were resolved by SDS-PAGE, and the proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Hybond ECL; Amersham). The blots were probed with antibodies against iNOS, COX-2, acetylated histone H3, acetylated histone H4, histone H4, p70s6 kinase, phospho-p70s6 kinase, or -tubulin (all antibodies at 0.2 μg/ml) overnight at 4°C. The blots were washed extensively with a solution containing 50 mM Tris, pH 8.0, 138 mM NaCl, 2.7 mM KCl, and 0.05% Tween 20. The antigen-antibody complexes were detected by the enhanced chemiluminescence protocol using horseradish peroxidase-conjugated donkey anti-rabbit IgG as secondary antibody.
Chromatin immunoprecipitation assay and quantitative real-time PCR. Chromatin immunoprecipitation (ChIP) analysis was performed as described in our recent report (35), except that immunoprecipitation was performed with anti-acetylated histone H4 or rabbit nonimmune IgG, and the PCR was conducted with primers framing the murine iNOS 5'-flanking region –978 to –710 [5'-TGCTAGGGGGATTTTCCCTCTCTC-3' (nucleotides –978 to –955) and 5'-AGCCTTTAATCCCGGGATTCAGG-3' (nucleotides –732 to –710)]. The specificity of these primer sets for their targets was confirmed by agarose gel electrophoresis. PCR for the input was performed with genomic DNA. The input fraction corresponded to 1% of the chromatin solution before immunoprecipitation. The PCR products were analyzed on a 2% agarose gel and quantified by real-time PCR using the DyNAmo SYBR green qPCR kit (Finnzymes) and an MJ Research DNA Engine Opticon 2 System (South San Francisco, CA), as described in our previous work (35). Each quantitative PCR assay was repeated three times, and the results were averaged.
RT-PCR. RT-PCR for semiquantitative analysis of mRNA levels of the transfected Zeocin resistance gene encoded by the pcDNA3.1-Zeo(+) or pcDNA3.1-Zeo(+)/iNOS-luc plasmids and for endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH; as a housekeeping gene control) was performed as previously described (35) on cDNA synthesized using M-MLV RT from total RNA isolated with Tri-Zol reagent from transfected mesangial cells. Primers were designed to amplify specifically nucleotides +1 to +375 of the Zeocin resistance gene (sense: 5'-ATGGCCAAGTTGACCAGTGCC-3'; antisense: 5'-TCAGTCCTGCTCCTCGGCCAC-3') or nucleotides +238 to +757 of GAPDH (sense: 5'-CCACTAACATCAAATGGGGTGAGG-3'; antisense: 5'-TACTTGGCAGGTTTCTCCAGGC-3'). PCR conditions were an initial denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 45 s, 52°C for 45 s, 72°C for 2 min, and a final extension step of 72°C for 20 min. The PCR products were electrophoresed on 0.8% agarose gels and imaged with a Kodak ImageStation 1000 (Packard).
Data analysis. Quantitative data are presented as means ± SE and were analyzed by ANOVA. Significance was assigned at P < 0.05.
RESULTS
TSA suppresses IL-1 induction of endogenous NO production, iNOS protein, and the activity of a stably incorporated iNOS promoter. To extend our previous studies, which used transient transfections of iNOS promoter-luciferase constructs, we exploited the HDAC inhibitor TSA and mesangial cell lines that had been stably transfected with pcDNA3.1-Zeo(±)iNOS-luc, a mammalian expression vector containing the murine iNOS promoter fused to the firefly luciferase gene (35), to examine further the effects of acetylation status on endogenous NO production and iNOS protein and the activity iNOS promoter-luciferase DNA stably integrated in the genome wrapped by histones. TSA-treated mesangial cells harboring the iNOS promoter-luciferase DNA exhibited 45% lower IL-1-stimulated nitrite levels (Fig. 1A) and iNOS protein levels (Fig. 1B) compared with vehicle-treated controls, indicating that hyperacetylation inhibits cytokine induction of iNOS expression. In contrast, TSA significantly augmented protein expression of COX-2, another cytokine-inducible gene in mesangial cells (Fig. 1B), indicating that acetylation status has different effects depending on the specific gene in murine mesangial cells.
As seen in Fig. 1C, mesangial cell lines stably expressing the iNOS promoter-luciferase construct exhibited the expected induction of iNOS promoter-luciferase activity in response to IL-1 in the absence of TSA. In keeping with the nitrite and iNOS protein responses, mesangial cell lines treated with TSA exhibited a degree of induction of iNOS promoter activity in response to IL-1 that was 30% lower than controls (Fig. 1C). In the aggregate, these data in stably transfected cells are in keeping with our previous report in transiently transfected mesangial cells (36) and suggest that hyperacetylation decreases and hypoacetylation increases IL-1 induction of stably integrated iNOS promoter activity in mesangial cells and that the TSA inhibition of iNOS induction could be related to a direct effect on the acetylation of histones.
Because pcDNA3.1-Zeo(+)/iNOS-luc retains the CMV promoter of the parental pcDNA3.1-Zeo(+) plasmid 5' to the iNOS promoter fragment-luciferase segment, we analyzed by semiquantitative RT-PCR mRNA levels for the Zeocin resistance gene, which is also downstream of the CMV promoter in both constructs, as an internal control in the cells transfected with the parent pcDNA3.1-Zeo(+) plasmid or with pcDNA3.1-Zeo(+)iNOS-luc. As seen in Fig. 1D, these mRNA levels were comparable in the cells transfected with either plasmid. Moreover, the iNOS promoter activity results presented here with the pcDNA3.1-Zeo(+)-derived construct were virtually identical to those we previously reported using the same iNOS promoter segment cloned into the pGL3-Basic vector, which lacks the CMV promoter, in transient transfection assays in mesangial cells (36), and they precisely mirror the responses of the endogenous iNOS gene (Fig. 1B). Thus, in the aggregate, these results indicate that the differences in luciferase activity in cells transfected with pcDNA3.1-Zeo(+)/iNOS-luc we observed here were the result of differences in iNOS promoter activity and were not significantly affected by the CMV promoter retained in the plasmid (Fig. 1D).
TSA promotes acetylation of histone H4 and increases chromatin-associated acetylated histone H4 binding at –978/–710 of the iNOS promoter. We previously demonstrated that acetylation status limits iNOS expression in part through an effect of HDAC2 interacting with the transcription factor NF-B p65 (36). To determine whether histones are acetylated and involved in the regulation of the iNOS gene, Western blots of acid extracts of histone proteins from mesangial cells treated with TSA for various lengths of time (0–24 h) were blotted with antibodies directed against histones H3 and H4 and acetylated histone H3 and H4. TSA induced histone H4 acetylation in a time-dependent manner over the first 4 h of treatment, without affecting total histone H4 levels (Fig. 2A). Histone H3 levels and histone H3 acetylation were unaffected (data not shown). Given these results, we sought to determine whether TSA alters the acetylation state of histone H4 associated with the iNOS promoter in mesangial cells using ChIP assays of nucleosomes from vehicle- and TSA-treated cells. As shown in Fig. 2B, TSA treatment resulted in a large increase in chromatin-associated acetylated histone H4 at the iNOS promoter region –978/–710 beginning at 5 min and peaking at 1–4 h of treatment. This region of the iNOS promoter is of particular interest because it includes B and -interferon-activated sites (GAS) elements known to be important in iNOS transactivation gene (15, 34, 36). Treatment with IL-1 alone (Fig. 2C, lane 2) or TSA alone (Fig. 2C, lane 3) resulted in a small increase in association of acetylated histone H4 at the –978/–710 region of the iNOS promoter, whereas cells treated with TSA and IL-1 exhibited a marked increase in the binding of acetylated histone H4 compared with the cells treated with IL-1 alone (Fig. 2C, lane 4 compared with lane 2).
Inhibitors of PI3K and p70s6 kinase increase acetylation of histone H4 and chromatin-associated acetylated histone H4 at –978/–710 of the iNOS promoter and inhibit IL-1-induction of the iNOS gene. Although the PI3K and p70s6 kinase pathways have been shown to be involved in histone modification (33, 34, 49, 50) and the regulation of the iNOS gene (21, 26) in some cell types, the coupled effects of these pathways on histone modification and iNOS gene transcription have not been established. Accordingly, we sought to determine whether these pathways are involved in histone H4 acetylation at the iNOS promoter in mesangial cells. As shown in Fig. 3, IL-1 induced phosphorylation of p70s6 kinase at 389Thr, whereas TSA significantly limited IL-1-induced phosphorylation of p70s6 kinase. Pretreatment with the PI3K inhibitor LY-294002 or rapamycin combined with TSA further decreased IL-1-induced phosphorylation of p70s6 kinase. To determine whether these pathways directly affected histone H4 binding at the iNOS promoter, ChIP assays coupled with quantitative PCR were performed. TSA + IL-1-treated cells pretreated with LY-294002 or rapamycin exhibited increased acetylated histone H4 binding at –978/–710 of the iNOS promoter compared with TSA + IL-1-treated cells pretreated with vehicle (Fig. 4). These data indicate that PI3K and p70s6 kinase regulate site-specific histone H4 acetylation in a targeted manner at the iNOS promoter to augment cytokine induction of the iNOS gene in mesangial cells.
In contrast to the effect of TSA to increase the abundance of acetylated histone H4 in mesangial cells (Fig. 2A), neither LY-294002 nor rapamycin by themselves promoted acetylation of histone H4 on Western blots of acid extracts of histone proteins extracted from the cells (Fig. 5). Moreover, insulin, a known mTOR activator (33), had no significant effect on the abundance of acetylated histone H4 in vehicle-treated mesangial cells and did not antagonize the effect of TSA to promote histone H4 acetylation (Fig. 5). These results indicate that PI3K and p70s6 kinase pathways do not directly cause histone H4 acetylation under basal conditions.
Pretreatment with LY-294002 or rapamycin also significantly limited IL-1-induced iNOS protein expression in mesangial cells, whereas addition of TSA together with either drug completely abolished IL-1-induced iNOS protein expression (Fig. 6A), indicating a synergistic effect. A similar effect of LY-294002 or rapamycin was seen when iNOS promoter-luciferase activity was examined (Fig. 6B). In contrast, LY-294002 or rapamycin in the absence, but not the presence, of TSA significantly limited COX-2 protein expression, again indicating gene-specific effects (Fig. 6A).
DISCUSSION
High-output NO production is potentially cytotoxic to the host and innocent bystander cells, so controls to limit or terminate iNOS gene expression are important cell survival mechanisms. Pathways involved in the repression of iNOS gene activation in cytokine-induced mesangial cells are still incompletely defined, and their characterization could potentially identify novel anti-inflammatory processes. Modulation of chromatin structure plays an important role in transcriptional regulation, yet little is known about the influence of chromatin on iNOS transcription. In the present study, TSA promoted acetylated histone H4 binding to a specific region of the iNOS promoter harboring key enhancer elements and suppressed production of endogenous NO and iNOS protein and iNOS promoter activity in mesangial cells. IL-1-mediated induction of iNOS transcription in these cells was associated with selective acetylation of histone H4 and chromatin remodeling, and blockade of PI3K- or p70s6-kinase pathways increased site-specific histone H4 acetylation at the iNOS promoter to limit further iNOS gene expression. In the aggregate, the data identify PI3-kinase- and p70s6 kinase-dependent pathways that increase site-specific histone H4 acetylation at the iNOS promoter as another mechanism, in addition to its effects on the HDAC2-NF-B p65 interaction, by which TSA diminishes IL-1-induced iNOS transcription.
Inputs received at promoter DNA in the form of multiple transcription factor-binding events can mediate the recruitment of chromatin modifiers, such as HATs, HDACs, histone kinases, and methyltransferases. These resultant epigenetic changes and posttranslational modifications of histones generate specific patterns of acetylation, phosphorylation, methylation, or ubiquitination, generating a "histone code" (29) that extends the information content of the DNA code. Histone acetylation represents a central switch that allows interconversion between permissive and restricted transcriptional states. Targeted acetylation at promoter nucleosomes through recruitment or dismissal of HAT and HDAC activities negotiates the acetylation status of chromatin. Many transcription factors regulate transcription by physically recruiting HATs and HDACs to promoters. The region –978/–710 of the murine iNOS promoter analyzed in this report contains a B enhancer element positioned at –971 to –962 that is critical for cytokine inducibility of the iNOS gene (34). Similarly, we and others have demonstrated the functional importance of two GAS at –942 to –934 (15) and at –879 to –871 (37) in activation of the iNOS gene in response to cytokines and LPS. Histone acetylation has been shown to regulate NF-B-dependent gene accessibility in some promoter contexts, but direct acetylation of NF-B subunits p65 and p50 also regulates different NF-B functions, including transcriptional activation, DNA-binding affinity, and IB assembly (9, 17, 24). Finally, acetyltransferases and deacetylases interact directly with several proteins involved in the NF-B signaling pathway, including NF-B itself, IB, IKK, and IKK. These interactions probably allow acetylation of NF-B itself, of other transcription factors, and of histones associated with NF-B-regulated genes (1, 4, 9, 17).
The large differences detected by ChIP assay in the extent of chromatin-associated, acetylated histone H4 at the iNOS promoter in mesangial cells treated with IL-1 alone vs. IL-1 + TSA suggests differences in chromatin structure in the two settings. Stimulus-specific patterns of histone acetylation likely result in different conformational changes of chromatin structure that are associated with distinct patterns of transcription factor association with the iNOS promoter and probably different kinetics of iNOS expression. One can speculate that a specific pattern of modified histone tails is required to recruit the basal transcription machinery and that TSA can distort this pattern and thus reduce the cytokine-induced transcriptional response. Further experiments will be needed to determine whether acetylated histone H4 binds to the iNOS promoter to condense chromatin DNA, preventing the accessibility of transcription factor(s), or whether the TSA-induced remodeling of the iNOS promoter is triggered by acetylation of a transacting factor(s), which might lead to site-specific binding to the iNOS promoter and thereby direct the translational nucleosome positioning to repress the iNOS promoter. We also cannot exclude the involvement of other histone modifications, such as methylation or phosphorylation, in IL-1-induced iNOS gene expression.
Much of what is known about TOR signaling in gene transcription and chromatin modifications has come from studies in yeast. In this organism, TOR is involved in regulating ribosomal protein gene expression via a nutrient supply-dependent and rapamycin-sensitive mechanism that, in response to ample nutrient supply, involves targeted recruitment of the Esa1-histone acetylase complex to these promoters, resulting in histone H4 acetylation and changes in chromatin structure that activate transcription. In response to nutrient limitation or rapamycin, the Esa1-histone acetylase complex is released from ribosomal protein gene promoters, and deacetylation mediated by the Rpd3-Sin3 HDAC complex resident at ribosomal protein gene promoters occurs, with reversion of chromatin to the repressed state (25). In mammalian cells, it has been proposed that the ability of the TSC2 tumor suppressor gene to regulate vascular endothelial growth factor involves regulation of HDACs (6). Thus there appears to be precedent for involvement of mTOR in histone acetylation.
In the current study, the fact that rapamycin, LY-294002, and insulin alone did not alter histone H4 acetylation under basal conditions leads to the conclusion that the actions of the PI3K and p70s6 kinase/mTOR pathways on histone H4 acetylation are indirect. The fact that the effects of TSA together with rapamycin or LY-294002 on histone H4 acetylation at the –978/–710 region of the iNOS promoter and IL-1-mediated induction of iNOS promoter activity were at least partially additive suggests that either the inhibitor combination gives more effective blockade of a single HDAC target or that separate perhaps partially overlapping TSA-sensitive and TSA-insensitive mechanisms are involved in regulating histone H4 acetylation. Thus the PI3K and p70s6 kinase/mTOR pathways could either augment or facilitate recruitment of TSA-insensitive HDAC activity, such as that of SIRT1, an NAD-dependent and TSA-insensitive deacetylase (11), and/or inhibit involvement of proteins with intrinsic HAT activity, such as p300 (9). For example, PI3K inhibitors have been shown to limit the expression of SIRT1 in a human melanoma cell line (31) and to alter p300 HAT activity (8). Further studies will be required to test the mechanistic details of our model in the complex context of IL-1-mediated changes in transcription factor, coregulatory protein, and local chromatin environment at the iNOS promoter.
The role of IL-1 in the activation of the PI3-kinase and p70s6 kinase and on NO production has been studied in different cells. In agreement with our data, LY-294002 and rapamycin blocked iNOS induction in murine macrophages in response to LPS or cytokines, suggesting that PI3K and p70s6-kinase are essential signals for the expression of iNOS and production of NO (21, 26). A similar inductive effect of LY-294002 on iNOS expression was observed in vascular smooth muscle cells (19). In contrast, others (13, 22) found that LY-294002 induced LPS- or IL-1-stimulated iNOS induction in C6 glioma cells. The reasons for the cell-specific effects of LY-294002 and rapamycin on iNOS expression are unclear, but the effects on targeted histone acetylation reported here likely represents at least one mechanism for this divergence. Thus, although it is generally believed that transcriptional activity correlates with histone acetylation, as it was for COX-2 in this study, recent reports indicate that several genes in addition to iNOS are inhibited by histone acetylation (2, 12, 32). As in our study with mesangial cells, butyrate, another HDAC inhibitor, suppressed iNOS transcription in DLD-1 colonic epithelial cells (27). These results suggest that cell type and promoter contexts are important for this mechanism.
The ability of acetylated histone H4 to influence the activity of the iNOS gene in mesangial cells lends further complexity to the intricate regulation of this gene. Although our findings demonstrated that the PI3K/p70s6 pathways regulate these processes, our results cannot be explained by simple inhibition of the kinase pathway activation, because there is an opposite effect for COX-2 gene activation. Further elucidation of the mechanisms by which TSA suppresses iNOS expression and cross talk between the PI3K/p70s6 pathways and histone acetylation in mesangial cells may provide novel clues for controlling excessive NO production during renal diseases.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health Grants RO1-DK-50745 and P50 GM-38529, a Department of Defense "T5" grant, and endowment funds from The James T. and Nancy Willerson Chair (to B. C. Kone).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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