Histone Deacetylases Augment Cytokine Induction of the iNOS Gene

Cell Culture and Reagents

Mouse mesangial cells (American Type Culture Collection [ATCC, Manassas, VA] CRL-1927) were maintained in Ham’s F12 plus DMEM supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5% fetal bovine serum (FBS). RAW 264.7 macrophage cells (ATCC) were cultured in DMEM supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FBS. Vehicle, IL-1β (10 ng/ml), or LPS (1 μg/ml) + IFN-γ (100 U/ml) was added to the cells as indicated in the text and figure legends. Mouse recombinant IL-1β and IFN-γ were from R & D Systems (Minneapolis, MN) and BioSource (Camarillo, CA), respectively. Polyclonal antibodies recognizing NF-κB p65 and p50 were from Santa Cruz Biotechnologies (Santa Cruz, CA). Oligonucleotides were custom synthesized by Genosys (The Woodlands, TX). Lipofectamine 2000 reagent was from Invitrogen (Carlsbad, CA). The Dual-Luciferase Reporter Assay System and the luciferase vectors pGL3-Basic and pRL-SV40 were from Promega (Madison, WI). The BCA protein estimation kit was from Pierce Chemical (Indianapolis, IN). Glutathione-Sepharose 4B beads, pGEX-5X-3 vector, and ECL reagents were from Amersham Pharmacia Biotech (Piscataway, NJ).

Plasmids

The NF-κB reporter construct, p36B(−)(NF-κB)₃-luc, which contains three tandem copies of the κB binding element (GGGGACTCTCCC) upstream of the SV40 early promoter sequence and fused to the coding sequence forthe luciferase gene (23), was provided by Dr. Bharat Aggarwal (University of Texas MD Anderson Cancer Center, Houston, TX). piNOS-luc, which contains the murine iNOS promoter/enhancer and a portion of exon 1 (nucleotides −1486 to +145) in pGL3-Basic, has been previously characterized (11). The FLAG-tagged HDAC2 expression plasmid pME18S-FLAG-HDAC2 (24) was from Dr. Edward Seto (University of South Florida, Tampa, FL). FLAG-tagged mammalian expression plasmids pBJ-HDAC1, pBJ-HDAC4, pBJ-HDAC5, and pBJ-HDAC6 (25) were from Dr. Stuart L. Schreiber (Harvard University, Cambridge, MA). To generate fusion with GST, cDNA encoding murine NF-κB p65 was amplified by RT-PCR from mesangial cell RNA and subcloned into pGEX-5X-3 at the EcoRI and NotI sites to maintain the appropriate reading frame and verified by DNA sequence analysis.

Nitrite Assays

Mesangial cells and RAW 264.7 cells were seeded in 24-well plates for 24 h, transiently transfected in some experiments, and then stimulated with IL-1β or LPS + IFN-γ for 24 h. The medium was then collected and the nitrite concentration determined with the Griess Reagent System (Promega) according to the manufacturer’s protocol.

Transient Transfections

Mesangial cells or RAW 264.7 cells were seeded in 24-well plates and grown to 90 to 95% confluency in complete medium without antibiotics and transfected the next day using the LipoFectamine 2000 reagent according to the manufacturer’s protocol and a total of 1 μg/well of plasmid DNAs. The amount of transfected DNA was kept constant by addition of appropriate amounts of the parental empty expression vector. Transfection efficiencies were normalized by cotransfection with 20 ng/well of the Renilla luciferase expression plasmid pRL-SV40. For trans-repression experiments, 0.5 μg of piNOS-luc, p36B(−)(NF-κB)₃-luc, or promoterless expression vector was cotransfected with 0.5 μg of pME18S-FLAG-HDAC2, pBJ-HDAC1, pBJ-HDAC4, pBJ-HDAC5, or pBJ-HDAC6 or insertless expression vector along with 0.02 μg pRL-SV40. Twenty-four hours after transfection, complete medium was added with vehicle, IL-1β, or LPS + IFN-γ. Twenty-four hours later, cell lysates were prepared and firefly and Renilla luciferase activities in 100 μl of lysate samples were measured as described previously in our laboratory (11). In some experiments, as indicated in the text and figure legends, trichostatin A (TSA; 200 nM) was added after transfection for 24 h before cell lysates were prepared. This concentration of TSA was chosen on the basis of its effectiveness in inhibiting HDAC activity in numerous published studies (20,21,24).

Preparation of Nuclear Extracts

Nuclear extracts were prepared from time-paired vehicle-, IL-1β-, or LPS + IFN-γ–treated mesangial cells as detailed in our earlier work (11,26).

Electrophoretic Mobility Shift Assays (EMSA)

NF-κB element double-stranded oligonucleotides from the murine iNOS promoter for use as probes and for competition studies (sense strand shown; consensus binding element italicized; “u” and “d” represent “upstream” and “downstream”) were: NF-κBu - 978 5′-TGCTAGGGGGATTTTCCCTCTCTC-3′ -955; NF-κBd -92 5′-CCAACTGGGGACTCTCCCTTTGGG-3′ -69. The probes were end-labeled with [γ³²P]ATP (3000 Ci/mmol) using T4 polynucleotide kinase. Binding reactions were performed in 20 μl of solution for 30 min at room temperature by incubating 10 μg of nuclear extract protein with duplex DNA probe (∼2 × 10⁵ cpm) in reaction buffer (13 mM HEPES, pH 7.9, 65 mM NaCl, 0.14 mM EDTA, 1 mM MgCl₂, 1mM dithiothreitol, 8% glycerol, and 50 μg/ml poly[dl-dC]). For supershift assays, antibodies (2 μg) specific for NF-κB p65 or p50 were added to the binding reaction and incubated on ice for 10 min before the addition of labeled probe. Aliquots of the reactions were resolved on 5% native polyacrylamide gels in 0.5× Tris borate-EDTA buffer. The gels were dried and exposed to x-ray film with an enhancing screen at −70°C to detect the DNA-protein and DNA-protein-antibody complexes. Experiments were replicated a minimum of three times, as indicated in the figure legends.

Immunoprecipitation and Western Blot Analyses

Stimulated mesangial cells were harvested and lysed in nuclear extract buffer (20 mM Tris-HCl, pH 8.0, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% Glycerol, 0.5 mM PMSF). These lysates were then precleared by incubating with 20 μl/ml protein A/G agarose beads (Santa Cruz) for 1 h at 4°C. After brief centrifugation, the supernatant was added to anti-HDAC2 antibody (0.2 μg/ml) or control IgG in nuclear extract buffer overnight at 4°C, followed by the addition of 20 μl of protein A/G agarose beads and incubation for 1 h at 4°C. Immunoprecipitates were washed four times in nuclear extract buffer, resuspended in sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min, and analyzed on 8% SDS-PAGE gels. The proteins were electrophorectically transferred to PVDF membranes (Hybond ECL, Amersham) and probed with an anti–NF-κB p65 antibody (0.2 μg/ml) overnight at 4°C as indicated in the Results section and figure legends. 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 ECL protocol using horseradish peroxidase–conjugated donkey anti-rabbit IgG as secondary antibody.

In Vitro Translation and GST Pull-Down Assay

A cDNA encoding HDAC2 with an upstream T7 promoter sequence was generated by PCR using the HDAC2 expression plasmid pME18S-FLAG-HDAC2 as a template. Using this cDNA, HDAC2 was transcribed and translated from the presence of [³⁵S]methionine using the TNT T7 Quick for PCR DNA kit (Promega) according to the manufacturer’s protocol. A GST-fusion protein constructed to contain full-length NF-κB p65 was purified from sonicates of isopropyl β-d-thiogalactoside–induced DH5α bacterial cells according to the manufacturer’s instructions (Amersham Pharmacia Biotech) and incubated with 50 μl of glutathione-Sepharose 4B beads for 1 h at 4°C. After centrifugation, the pellets was collected and resuspended in lysis buffer (PBS containing protease inhibitor cocktail). For in vitro binding reaction, 20 μl of purified GST or GST-NF-κB p65 was incubated in protein binding buffer (20 mM Tris, pH 8.0, 150 mM KCl, 1 mM EDTA, 4 mM MgCl₂, 0.2% NP-40, 10% glycerol) with 10 μl of [³⁵S]methionine-labeled full-length HDAC2 translation product at 4°C overnight. The samples were then washed four times in binding buffer, boiled in SDS sample buffer, and analyzed by SDS-PAGE gel and autography.

Statistical Analyses

Quantitative data are presented as mean ± SEM and were analyzed by ANOVA. Significance was assigned at P < 0.05.

TSA Inhibits Induction of Endogenous NO Production and iNOS Promoter Activity in Response to IL-1β or LPS + IFN-γ

To determine whether HDAC activity and the state of cellular acetylation contributes to the regulation of iNOS-generated NO production in mesangial cells and RAW 264.7 macrophages, nitrite production induced by treatment with IL-1β or LPS + IFN-γ was measured in the presence of vehicle or TSA, a potent and specific HDAC inhibitor. iNOS is the only NOS isoform appreciably expressed in these cell types and induced by these stimuli (reference 27 and unpublished observations). IL-1β stimulated mesangial cells that had been treated with TSA exhibited a 79% lower nitrite levels compared with IL-1β–stimulated controls, whereas LPS + IFN-γ–treated mesangial cells exposed to TSA exhibited nearly 72% lower levels of nitrite production compared with LPS + IFN-γ–treated controls (Figure 1A). Similarly, in RAW 264.7 cells, TSA inhibited LPS + IFN-γ–stimulated nitrite production by ∼50% (Figure 1B).Thus, TSA exerts prominent effects on cytokine induction of NO production by the endogenous iNOS gene.

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Figure 1. Trichostatin A (TSA) inhibits induction of endogenous nitric oxide (NO) production. (A) Mesangial cells were treated with vehicle or TSA (200 nM) together with interleukin–1β (IL-1β) or LPS + interferon-γ (IFN-γ) for 24 h, and the nitrite concentration in the culture supernatant was measured. (B) RAW 264.7 cells were similarly treated with vehicle or TSA in the presence and absence of LPS + IFN-γ for 24 h, after which nitrite determinations on the culture supernatants were made (n = 5). * P < 0.05 versus corresponding “−TSA” samples.

To determine whether the inhibitory effect of TSA included constraints on iNOS transcription, vehicle or TSA was added to mesangial cells and RAW 264.7 cells that had been transfected with piNOS-luc, which contains the murine iNOS promoter fused to the firefly luciferase gene. The transfected cells were then treated with medium, IL-1β, or LPS + IFN-γ, and the induction of iNOS reporter gene activity was measured. As seen in Figure 2A, mesangial cells treated with IL-1β or LPS + IFN-γ in the absence of TSA exhibited the expected induction of iNOS promoter-luciferase activity. In contrast, mesangial cells treated with 200 nM TSA exhibited a ∼35% reduction in the fold-induction of iNOS promoter activity in response to IL-1β or LPS + IFN-γ. Similarly, in RAW 264.7 cells, TSA inhibited by ∼40% iNOS promoter activity stimulated by LPS + IFN-γ (Figure 2B). These data suggest that hyperacetylation decreases cytokine induction of iNOS promoter activity in mesangial cells and macrophages.

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Figure 2. TSA inhibits induction of the murine inducible nitric oxide synthase (iNOS) promoter. Mesangial cells (A) or RAW 264.7 cells (B) were transiently transfected with piNOS-luc, containing the wild-type iNOS promoter, as well as with a Renilla luciferase expression plasmid for normalization of transfection efficiencies. After transfection, the cells were treated with vehicle or TSA (200 nM) together with vehicle, IL-1β, or LPS + IFN-γ for 24 h, and the luciferase activity in cell lysates was measured (n = 5) * P < 0.05 versus corresponding “−TSA” samples.

Overexpression of HDAC Isoforms in Mesangial Cells Augments Induction of the iNOS Promoter in Response to IL-1β or LPS + IFN-γ

To address more directly the effects of acetylation status on iNOS promoter induction, HDAC isoforms were individually overexpressed in transactivation assays of the iNOS promoter-reporter gene construct. Transfection of mesangial cells with HDAC1, -2, -4, -5, or -6 together with piNOS-luc did not significantly alter iNOS promoter activity under basal conditions, but resulted in differing degrees of superinduction of iNOS promoter activity in response to IL-1β or LPS + IFN-γ (Figure 3A). HDAC1 had no discernible effect on iNOS promoter activation, whereas HDAC2 promoted a ∼35% superinduction and HDAC6 a ∼30% superinduction of promoter activity in response to IL-1β and LPS + IFN-γ. HDAC4 overexpression resulted in negligible effects in response to IL-1β, but it exerted a ∼70% increase in iNOS promoter activity over controls in response to LPS + IFN-γ. HDAC5 was similarly more potent in superinducing the iNOS promoter in response to LPS + IFN-γ compared with IL-1β. In RAW 264.7 cells, overexpression of HDAC1, -5, and -6, but not HDAC2 or HDAC4, significantly increased iNOS promoter activity (Figure 3B). Thus there are clear, cell type-specific differences between mesangial cells and RAW 264.7 cells in HDAC isoform action on iNOS gene transcription.

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Figure 3. Overexpression of histone deacetylase (HDAC) isoforms augments induction of the murine iNOS promoter. (A) Mesangial cells were transiently cotransfected with the HDAC isoform expression plasmids encoding HDAC1, -2, -4, -5, or -6 and piNOS-luc, containing the wild-type iNOS promoter, as well as with a Renilla luciferase expression plasmid. After transfection, the cells were treated with vehicle, IL-1β, or LPS + IFN-γ for 24 h, and the luciferase activity in cell lysates was measured (n = 5). *P < 0.05 versus vehicle-treated cells not transfected with an HDAC (“none”); #P < 0.05 versus corresponding cytokine-treated cells not transfected with an HDAC (“none”). (B) RAW 264.7 cells were similarly cotransfected, but the cells were treated with LPS + IFN-γ for 24 h, and the luciferase activity in cell lysates was measured (n = 5). *P < 0.05 versus empty vector.

Of these HDAC isoforms, we elected to focus on HDAC2 because of its reported indirect influence on NF-κB, a known stimulator of iNOS gene transcription. Transfection of increasing amounts of HDAC2 into mesangial cells resulted in a threshold effect with increased IL-1β or LPS + IFN-γ–stimulated iNOS promoter activity evident beginning at 0.5 μg of transfected DNA (Figure 4).

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Figure 4. Dose-response of HDAC2 overexpression on iNOS promoter activity. Mesangial cells were cotransfected with increasing amounts of HDAC2 expression plasmid and a fixed amount of the iNOS promoter construct piNOS-luc as well as with a Renilla luciferase expression plasmid. After transfection, the cells were treated with vehicle, IL-1β, or LPS + IFN-γ for 24 h, and the luciferase activity in cell lysates was measured (n = 3).

TSA Inhibits and HDAC2 Overexpression Augments Induction of a NF-κB–Responsive Promoter in Response to IL-1β

Cytokine induction of the murine iNOS promoter is known to be partially signaled by NF-κB. This transcription factor is known to interact with HDAC1, -2, and -3 (21,28,29) and to be influenced functionally by changes in acetylation (21). We accordingly hypothesized that effects on NF-κB or its interaction with cognate binding elements might mediate the reciprocal effects of TSA treatment and HDAC overexpression to modulate iNOS promoter activity. Specifically, augmented activation of NF-κB or its coregulatory proteins might lead to more prominent iNOS gene activation. The effects of TSA treatment and of HDAC2 overexpression on the activity of an NF-κB promoter were accordingly examined in transfection experiments in mesangial cells. As shown in Figure 5A, TSA caused a 60% decrease in IL-1β–stimulated NF-κB promoter activity compared with vehicle-treated controls. In accordance with these findings, overexpression of HDAC2 augmented induction of the NF-κB promoter by nearly 40% in response to IL-1β (Figure 5B). These results indicate that acetylation alters the functional activity of NF-κB.

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Figure 5. TSA inhibits and HDAC2 promotes induction of an nuclear factor–κB (NF-κB) element reporter construct. (A) Mesangial cells were transiently transfected with p36B(−)(NF-κB)₃-luc, which contains three tandem copies of the κB binding element, as well as with a Renilla luciferase expression plasmid. After transfection, the cells were treated with vehicle or TSA (200 nM) with IL-1β for 24 h, and the luciferase activity in cell lysates was measured. The IL-1β–stimulated values are shown (n = 5). *P < 0.05 versus “−TSA.” (B) Mesangial cells were cotransfected with p36B(−)(NF-κB)₃-luc, empty vector, or an HDAC2 expression plasmid and the Renilla luciferase vector. After transfection, the cells were treated with or without IL-1β for 24 h, and the luciferase activity in cell lysates was measured. The IL-1β-stimulated values are shown (n = 6).*P < 0.05 versus “vector.”

Gel shift studies demonstrated two κB-dependent DNA-protein complexes under basal conditions in nuclear extracts harvested from mesangial cells (Figure 6). The higher molecular weight complex was markedly upregulated after IL-1β treatment. Supershift/antibody competition assays showed that anti-NF-κB p65 greatly diminished the intensity of this higher molecular weight complex and produced a supershift of the complex, whereas anti-p50 outcompeted the lower molecular weight complex (Figure 6). These data are consistent with the basal expression of NF-κB p50/p50 homodimers and the inducible expression of NF-κB p65/p65 homodimers. Comparable results were found when either the NF-κBu or NF-κBd probes were used. TSA treatment of thecells did not significantly affect NF-κB DNA binding activity in nuclear extracts prepared from stimulated mesangial cells (Figure 6). In the aggregate, these results suggested that acetylation status influences the transactivation potential of NF-κB, rather than its DNA binding properties, in its interaction with NF-κB responsive promoters.

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Figure 6. TSA treatment does not alter NF-κB DNA binding activity in IL-1β- or LPS + IFN-γ–treated mesangial cells. Mesangial cells were pretreated with TSA (200 nM) for 1 h before stimulation with IL-1β for 15 min. Nuclear extracts were prepared, and electrophoretic mobility shift assays (EMSA) and supershift/antibody competition assays with an NF-κB consensus binding site probe and antibodies to NF-κB p65 and p50 were performed (n = 3).

HDAC2 and NF-κB p65 Interact In Vitro and in Mesangial Cells In Vivo

To determine if HDAC2 and p65 interact in vivo in mesangial cells, cells were treated with vehicle or IL-1β and nuclear extracts were prepared for coimmunoprecipitation assays. Co-immunoprecipitations were performed with an anti-HDAC2 antibody, and proteins present in the immunoprecipitates were revealed by immunoblotting with anti-NF-κB p65 antibodies. As seen in Figure 7A, HDAC2 and NF-κB p65 coimmunoprecipitated from cells treated with IL-1β but not untreated cells. The lack of co-immunoprecipitation in the untreated cells (lane 1) serves as a negative control. Treatment of the cells with TSA did not appreciably alter the abundance of the co-immunoprecipitated proteins from the IL-1β–treated cells (Figure 7A). Importantly, the two endogenous proteins co-immunoprecipitated, and overexpression was not required. In agreement with these findings, radiolabeled HDAC2 transcribed and translated in vitro was specifically retained by a GST-NF-κB p65 fusion protein but not by GST alone (Figure 7B).

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Figure 7. HDAC2 directly interacts with NF-κB p65. (A) Coimmunoprecipitation of HDAC2 and NF-κB p65 from IL-1β–treated mesangial cells treated with and without TSA. Nuclear extracts were prepared and immunoprecipitated (IP) with polyclonal antibodies directed against HDAC2 or nonimmune IgG, separated by SDS-PAGE, and immunoblotted (IB) with polyclonal antibodies directed against NF-κB p65. Data are representative of three independent experiments. (B) GST pull-down experiment in which ³⁵S-labeled, in vitro-translated HDAC2 was incubated with GST as a negative control or GST-NF-κB p65 fusion protein. After extensive washes, proteins retained by GST or GST-NF-κB p65 were eluted and analyzed by SDS-PAGE and autoradiography (n = 3).