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Originally published online as doi:10.1189/jlb.1206739 on May 16, 2007

Published online before print May 16, 2007
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(Journal of Leukocyte Biology. 2007;82:436-447.)
© 2007 by Society for Leukocyte Biology

Simvastatin inhibits IFN-{gamma}-induced CD40 gene expression by suppressing STAT-1{alpha}

Sun Jung Lee, Hongwei Qin and Etty N. Benveniste1

Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA

1 Correspondence: Department of Cell Biology, 1918 University Blvd., MCLM 395A, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA. E-mail: tika{at}uab.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD40, a member of the TNF receptor superfamily, is critical for productive immune responses. Macrophages constitutively express CD40 at low levels, which are enhanced by IFN-{gamma}. IFN-{gamma}-induced CD40 expression involves activation of STAT-1{alpha} as well as NF-{kappa}B activation through an autocrine response to IFN-{gamma}-induced TNF-{alpha} production. Statins are 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, which exert anti-inflammatory effects independent of their cholesterol-lowering actions. Herein, we describe that simvastatin (SS) inhibits IFN-{gamma}-induced CD40 expression via the suppression of STAT-1{alpha} expression. This results in diminished STAT-1{alpha} recruitment to the CD40 promoter upon IFN-{gamma} treatment, in addition to reduced RNA Polymerase II recruitment and diminished levels of H3 and H4 histone acetylation. SS-mediated inhibition of STAT-1{alpha} occurs through suppression of constitutive STAT-1{alpha} mRNA and protein expression. The inhibitory effect of SS on CD40 and STAT-1{alpha} is dependent on HMG-CoA reductase activity, as the addition of mevalonate reverses the inhibitory effect. In addition, CD40 and/or STAT-1{alpha} expression is inhibited by GGTI-298 or Clostridium difficile Toxin A, a specific inhibitor of Rho family protein prenylation, indicating the involvement of small GTP-binding proteins in this process. Collectively, these data indicate that SS inhibits IFN-{gamma}-induced CD40 expression by suppression of STAT-1{alpha}, and altering transcriptional events at the CD40 promoter.

Key Words: statins • JAK-STAT pathway • macrophage


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IFN-{gamma} is a cytokine involved in many aspects of the immune response [1 ], and the major biological responses to IFN-{gamma} are mediated through the JAK-STAT pathway [2 ]. Seven mammalian STATs (STAT-1, -2, -3, -4, -5A, -5B, and -6) have been identified thus far, and STAT-1 is the major transcription factor in the IFN-{gamma} signal transduction pathway [2 ]. Binding of IFN-{gamma} to its receptor induces a series of events, which ultimately results in tyrosine phosphorylation of STAT-1, followed by homodimerization, nuclear translocation, and binding to IFN-{gamma}-activated sequence (GAS) elements in the promoter regions of IFN-{gamma}-inducible genes. Serine phosphorylation of STAT-1 is also important for optimal transcriptional activity [3 ].

The co-stimulatory molecule CD40 is involved in critical immunologic functions, including antigen presentation to T cells [4 ]. Aberrant CD40 expression is implicated in participating in human diseases, particularly autoimmune and inflammatory diseases [5 6 7 ]. Blocking the interaction between CD40 and its ligand CD154 is beneficial in animal models of autoimmune and neurodegenerative diseases [6 , 8 9 10 11 ]. IFN-{gamma} treatment leads to the induction of CD40 expression on macrophages and microglia, the endogenous macrophage of the brain [12 ]. Necessary for IFN-{gamma}-induced CD40 expression is STAT-1{alpha} activation, as well as TNF-{alpha} secretion and subsequent autocrine induction of NF-{kappa}B activation [13 , 14 ]. Although TNF-{alpha} signaling is necessary for optimal CD40 induction, TNF-{alpha} treatment alone has a modest effect on CD40 expression [14 ]. IFN-{gamma}-activated STAT-1{alpha} and NF-{kappa}B, along with constitutively expressed PU.1/Spi-B, bind to GAS, {kappa}B, etsA, and etsB elements, respectively, within the CD40 promoter, leading to CD40 gene transcription [12 , 14 ].

Statins competitively inhibit 3-hydroxy-3-methyglutaryl (HMG)-CoA reductase, which catalyzes the conversion of HMG-CoA to L-mevalonate (L-MVLT), a key intermediate in cholesterol biosynthesis [15 ]. Because of their cholesterol-lowering effects, statins are used widely and clinically for the treatment of cardiovascular disease [16 ]. Statins also have immunomodulatory and anti-inflammatory effects [17 18 19 20 ], although the mechanisms of action are not yet defined completely. Inhibition of HMG-CoA reductase reduces the synthesis of all MVLT pathway products. Indeed, MVLT is a precursor, not only for the synthesis of cholesterol but also for the synthesis of isoprenoids, including geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP). Isoprenylation is a functionally important post-translational modification of GTP-binding proteins such as Rho and Ras, which are important for protein trafficking, signaling, cytoskeletal structure, cell motility, and membrane transport [21 , 22 ]. By reducing the cellular pool of isoprenoids, statins cause the accumulation of inactive forms of Rho and Ras within the cytoplasm [23 ]. Thus, it is postulated that statins have significant cholesterol-independent effects, which result from inhibition of the isoprenoid pathway.

Several studies have shown that statins inhibit immunological functions of IFN-{gamma} such as expression of Class II MHC on APCs, thereby inhibiting T cell activation [24 25 26 ]. In addition, it has been shown previously that statins inhibit IFN-{gamma}-inducible expression of CD40 on primary macrophages and microglia [26 , 27 ]. Other effects of statins on primary macrophages and/or microglia include inhibition of IFN-β-induced expression of CCL5 [28 ], changes in microglial morphology [29 ], inhibition of macrophage migration and MMP-9 secretion [30 ], and inhibition of LPS-induced IL-6 secretion [31 ] (also see review article by Greenwood et al. [19 ]). In this study, we investigated how statins, especially simvastatin (SS), influence IFN-{gamma}-induced CD40 gene expression in macrophages and microglia. SS is a potent inhibitor of IFN-{gamma}-induced CD40 gene expression, and this effect is dependent on HMG-CoA reductase activity, as it was reversed by the addition of MVLT, supporting the previous report by Youssef et al. [26 ]. Chromatin immunoprecipitation (ChIP) assays demonstrate that SS inhibits IFN-{gamma}-induced acetylation of histones H3 and H4, and the recruitment of STAT-1{alpha} and RNA Polymerase II (Pol II) on the endogenous CD40 promoter. SS-mediated inhibition of CD40 expression results from SS suppression of constitutive STAT-1{alpha} expression. Collectively, these results implicate STAT-1 as a target for SS, resulting in the inhibition of STAT-1{alpha} expression and function, and subsequent inhibition of IFN-{gamma}-inducible genes such as CD40. These findings also provide information about the molecular basis by which SS mediates some of its anti-inflammatory properties.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant proteins and reagents
Recombinant murine IFN-{gamma} was purchased from R&D Systems (Minneapolis, MN, USA), and PE-conjugated rat IgG anti-mouse CD40 antibody (Clone 3/23) was purchased from BD PharMingen (San Diego, CA, USA). Activated SS, Clostridium difficile Toxin A, FTI-277, and GGTI-298 were purchased from Calbiochem (San Diego, CA, USA). STAT-4 and STAT-5 antibodies were purchased from Zymed Laboratories (San Francisco, CA, USA), and phospho-STAT-1, phospho-JAK1, and STAT-6 antibodies were from Cell Signaling Technology (Beverly, MA, USA). JAK1, JAK2, STAT-1, AcH3, and AcH4 antibodies were purchased from Upstate Biotechnology (Lake Placid, NY, USA), and all other antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). L-MVLT, GGPP, FPP, cholesterol, and methyl-β-cyclodextrin (CDX) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Moloney murine leukemia virus (MMLV) RT was purchased from Promega (San Luis Obispo, CA, USA).

Cells
The murine macrophage cell line RAW264.7 was maintained in DMEM supplemented with 10% FBS. Primary microglia from C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, USA) were prepared as described previously [12 ].

Immunoblotting and immunoprecipitation
Whole cell lysates were prepared in lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM PMSF-20]. Twenty to 30 µg whole cell lysates were boiled in sample buffer, separated on 8% SDS-PAGE, and transferred to nitrocellulose membranes, which were then blocked for 1 h in 5% nonfat dry milk/TBST (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20, pH 7.6) and incubated at 4°C overnight in the indicated primary antibodies containing 5% nonfat dry milk. Blots were washed with TBST (three times, 10 min/wash) and incubated for 45 min at room temperature with secondary antibody (HRP-conjugated anti-rabbit or anti-mouse, 1/2500 dilution). The blots were washed three times in TBST, and ECL was used for detection of bound antibody, as described previously [32 ]. Densitometry was used to quantify immunoblots, and all results were normalized by the respective actin values. The basal level of the untreated sample was set as 1.0, and fold induction (or inhibition) upon treatment with IFN-{gamma} and/or SS was compared with that. Immunoprecipitation was performed as described previously [33 ]. Cleared lysates were incubated overnight with 3 µg anti-JAK1 antibody and an additional incubation for 3 h with 50 µl protein A/G bead slurry at 4°C. After washing, the samples were separated on SDS/PAGE and analyzed by immunoblotting with antiphospho-JAK1 antibody.

EMSA
EMSA was performed with 5–10 µg nuclear extract in a total volume of 15 µl binding buffer and 40,000 cpm 32P-labeled oligonucleotide containing a GAS-binding sequence and incubated on ice for 15 min, as described previously [12 ]. Bound and free DNA was then resolved by electrophoresis through a 6% polyacrylamide gel. For supershift analysis, 1 µl of the indicated antibody was added, or for competition analysis, a 50-fold molar excess of the indicated, unlabeled oligonucleotides was added to the nuclear extracts and incubated for 30 min, followed by an additional incubation for 15 min with the labeled probe. After electrophoresis, gels were dried and visualized by autoradiography.

RNA isolation, riboprobes, and RNase protection assay
Total cellular RNA was isolated as described previously [34 ]. Riboprobes for murine CD40, IFN regulator factor 1 (IRF-1), and GAPDH, prepared from in vitro transcription with T7 polymerase, are 654, 367, and 270 nt in length, respectively. Total RNA (20 µg) was hybridized with CD40, IRF-1, and GAPDH riboprobes at 42°C overnight in 20 µl 40 mM PIPES (pH 6.4), 80% deionized formamide, 400 mM NaOAc, and 1 mM EDTA. The hybridized mixture was then treated with RNase A/T1 (1/200 dilution in 200 µl of the RNase digestion buffer) at 37°C for 30 min and analyzed by 5% denaturing (8 M urea) PAGE, and the gels were exposed to phosphorImager cassettes. The protected fragments of the CD40, IRF-1, and GAPDH riboprobes are 524, 314, and 212 nt in length, respectively. Quantification of the protected RNA fragments was performed by scanning with the PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA). Values for CD40 and IRF-1 mRNA expression were normalized to GAPDH mRNA levels for each experimental condition. GAPDH mRNA was used as a housekeeping gene, as its levels are not affected by cytokine or statin treatment.

RT-PCR
STAT-1 mRNA expression was analyzed by RT-PCR. Briefly, total cellular RNA was isolated from cells, and 1 µg RNA was used to reverse-transcribe mRNA into cDNA using MMLV RT. The cDNA was subjected to PCR with primers specific for mouse STAT-1 and GAPDH. Primer sequences were as follows: STAT-1 (sense): 5'-GGAGGTGAACCTGACTTCCA-3'; STAT-1 (antisense): 5'-TCTGGTGCTTCCTTTGGTCT-3'; GAPDH (sense): 5'-AACTTTGGCATTGTGGAAGG-3'; and GAPDH (antisense): 5'-CCCTGTTGCTGTAGCCGTAT-3'. PCR was performed using Taq DNA polymerase. After initial denaturing for 2 min at 94°C, 29 cycles of amplification (94°C for 30 s, 59°C for 45 s, and 72°C for 30 s) were performed, followed by a 5-min extension at 72°C. The PCR products were analyzed in 1.5% agarose gels, and densitometry was used to quantify the PCR results. GAPDH mRNA served as an internal control for sample loading and mRNA integrity, and all results were normalized by the respective GAPDH values.

Quantitative analysis of CD40 protein expression by flow cytometry
Cells were plated at 5.0 x 105 cells/well and allowed to grow for 12–16 h in media supplemented with 10% FBS. The cells were pretreated with SS for 12 h, stimulated with IFN-{gamma} for 12 h, and then stained for CD40 protein expression as described previously [34 ]. The cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA, USA). Negative controls were stained with an isotype-matched control antibody. Ten thousand cells were analyzed for each sample.

ChIP assay
ChIP assays were performed as described previously [35 ]. Nuclei from formaldehyde-cross-linked cells were resuspended in Tris-EDTA buffer and sonicated. The soluble chromatin was adjusted into radioimmunoprecipitation assay buffer (0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 140 mM NaCl) and precleared. Immunoprecipitation was performed with 4 µg appropriate antibodies, and the immune complexes were absorbed with protein A beads (Upstate Biotechology) blocked with salmon sperm DNA. Immunoprecipitated DNA was amplified by a primer pair (5'-CTACAGCCTCTGGATGGAGC-3' and 5'-TGCAGAACCGAAAGCGTCTC-3') corresponding to a 250-bp fragment from the mouse CD40 promoter and subjected to PCR. The PCR products were resolved in 1.5% agarose gels in 1x Tris/acetate/EDTA electrophoresis buffer, and the gels were stained with ethidium bromide. Densitometry was used to quantify the PCR results, and all results were normalized by the respective input values.

Statistical analysis
Data are presented as mean ± SEM, and the Student’s t-test was used to determine statistical differences. P values of <0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SS inhibits IFN-{gamma}-induced CD40 gene expression
IFN-{gamma} is a potent inducer of CD40 expression, and the transcription factors STAT-1{alpha} and NF-{kappa}B p65 are involved in transcriptional activation of the CD40 gene [12 13 14 ]. It has been shown previously that IFN-{gamma}-induced CD40 protein expression on macrophages can be inhibited by atorvastatin, and that this effect is dependent on HMG-CoA reductase activity [26 ]. Furthermore, the study by Townsend et al. [27 ] showed that lovastatin suppressed IFN-{gamma}-induced CD40 expression on microglia by inhibition of JAK1/2 activation. We tested the ability of SS to inhibit IFN-{gamma}-induced CD40 gene expression in RAW264.7 murine macrophage cells. These cells were treated with increasing concentrations of SS (0.1–10 µM) for 12 h prior to IFN-{gamma} treatment, and then, surface CD40 protein expression was examined. IFN-{gamma}-induced CD40 protein expression was inhibited by SS in a dose-dependent manner, and maximal inhibition was seen at 10 µM (~60% inhibition; Fig. 1A ). Figure 1B shows a representative flow cytometry result using 10 µM SS. The inhibitory effect of SS was also observed at the level of CD40 mRNA expression (~50% inhibition; Fig. 1C ). IRF-1, another IFN-{gamma}-inducible, STAT-1-dependent gene, was tested, and IRF-1 mRNA expression was also inhibited (~50% inhibition) by SS treatment (Fig. 1D) . In addition, we determined that the t1/2 of IFN-{gamma}-induced CD40 mRNA is >8 h and that SS treatment does not affect CD40 mRNA stability (data not shown). Therefore, the inhibitory effect of SS on CD40 mRNA is not mediated at the post-transcriptional level. Furthermore, SS did not affect cell viability at these concentrations and times, as determined by MTT assays (data not shown). Two other statins, mevastatin and lovastatin, also inhibited IFN-{gamma}-induced CD40 gene expression in RAW264.7 cells (data not shown), indicating that this effect was not unique to SS. We also examined the effect of SS on primary murine microglia and determined that SS inhibited IFN-{gamma}-induced CD40 protein expression in these cells (Fig. 1E) . As well, SS partially inhibited CD40 expression in primary murine bone marrow-derived macrophages (data not shown).


Figure 1
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  		Figure 1. SS inhibits IFN-{gamma}-induced CD40 protein and mRNA expression. (A) RAW264.7 cells were pretreated with different concentrations of SS (0–10 µM) for 12 h, and then were stimulated by IFN-{gamma} (10 ng/ml) for 12 h. Surface expression of CD40 was assessed by flow cytometry. IFN-{gamma} induction of CD40 protein was set at 100%, and SS treatment was compared with that. Mean ± SEM of four experiments. *, P < 0.001, compared with IFN-{gamma} treatment. (B) A representative flow cytometry result with SS (10 µM) treatment. Cells were pretreated with SS (10 µM) for 12 h prior to IFN-{gamma} treatment for 6 h (C) or 3 h (D). Total RNA (20 µg) was analyzed for CD40, IRF-1, and GAPDH mRNA expression. Values were normalized to GAPDH mRNA. The basal level of the untreated sample was set as 1.0, and fold induction was compared with that; representative of three independent experiments. (E) Primary murine microglia were pretreated with SS (10 µM) for 12 h and stimulated with IFN-{gamma} for 24 h, and then flow cytometry was performed for CD40 protein expression; representative of two experiments.

 
SS inhibits CD40 expression in a HMG-CoA reductase-dependent manner
We next determined whether the inhibitory effect of SS on CD40 expression was via the HMG-CoA reductase pathway. The primary effect of HMG-CoA reductase inhibitors is to block the synthesis of cholesterol, but it also reduces the cellular pools of all the metabolic intermediates in the biosynthesis pathway. Cells were incubated with several metabolic intermediates (MVLT, GGPP, FPP, or cholesterol) in the presence of SS. As shown in Figure 2A , the addition of MVLT reversed SS-mediated inhibition of IFN-{gamma}-induced CD40 protein expression, demonstrating that its effect was a result of inhibition of HMG-CoA reductase. GGPP and FPP are required for the post-translational modification of GTP-binding proteins of the Rho and Ras family, respectively. Interestingly, GGPP reversed the inhibitory effect of SS, while FPP was without effect. In addition, cholesterol had no effect on the inhibitory influence of SS. A similar effect of the metabolic intermediates was observed on SS-mediated inhibition of CD40 mRNA expression; i.e., MVLT and GGPP reversed the inhibition (data not shown). To examine further if inhibition of GTP-binding protein activation was responsible for the inhibitory effect of SS, cells were treated with GGTI-298 and FTI-277, which are specific inhibitors for Ras superfamily protein prenylation [36 , 37 ]. Treatment with GGTI-298 (5–20 µM) inhibited IFN-{gamma}-induced CD40 mRNA expression (Fig. 2B , Lanes 7–9), mimicking the effect of SS (Lane 3). In contrast, FTI-277 was without effect at all concentrations tested (Fig. 2B , Lanes 4–6). Similar results were observed on IFN-{gamma}-induced CD40 protein expression; i.e., GGTI-298 inhibited expression, and FTI-277 did not (data not shown). These results indicate that the effect of SS is mediated by inhibition of HMG-CoA reductase, subsequently inhibiting isoprenylation of Ras superfamily proteins by decreasing the pools of GGPP. In addition, blockade of cholesterol biosynthesis by SS does not account for its inhibitory effect on CD40 expression.


Figure 2
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  		Figure 2. The inhibitory effect of SS is dependent on MVLT and GGPP, but not FPP or cholesterol. (A) RAW264.7 cells were pretreated with SS (10 µM) or SS plus metabolic intermediates (500 µM MVLT, 5 µM GGPP, 5 µM FPP, or 500 µM cholesterol) for 12 h and then were stimulated by IFN-{gamma} (10 ng/ml) for 12 h. Surface expression of CD40 was assessed by flow cytometry. IFN-{gamma} induction of CD40 protein was set at 100%, and SS treatment compared with that. Mean ± SEM of four experiments. *, P < 0.001, compared with SS plus IFN-{gamma}. N.S., Not significant. (B, C) Cells were pretreated with SS (10 µM), FTI-277 (5–20 µM), or GGTI-298 (5–20 µM; B) or Toxin A (1–2.5 nM; C) for 12 h prior to IFN-{gamma} treatment for 6 h. Total RNA (20 µg) was analyzed for CD40 and GAPDH mRNA expression. Values were normalized to GAPDH mRNA. The basal level of the untreated sample was set as 1.0, and fold induction was compared with that; representative of three independent experiments.

 
The Rho family of G-proteins, including Rho, Rac, and Cdc42, is isoprenylated primarily by GGPP. As the inhibition of GGTI-298 mimicked the inhibitory effect of SS on CD40 expression, we hypothesized that this inhibitory effect of SS was a consequence of inhibition of Rho family protein function. We investigated the role of Rho family GTPase function on IFN-{gamma}-induced CD40 expression by using a specific inhibitor of this class of GTPases. C. difficile Toxin A ADP ribosylates Rho family GTPases and inhibits Rho family proteins (Rho, Rac, and Cdc42) specifically and robustly [38 ]. Cells were pretreated with increasing concentrations (1–2.5 nM) of Toxin A for 12 h and then stimulated with IFN-{gamma}. Toxin A treatment reduced IFN-{gamma}-induced CD40 mRNA levels (Fig. 2C , compare Lane 2 with Lanes 4 and 5), comparable with the extent of SS-mediated inhibition (Lane 3). These results strongly suggest that the inhibitory effect of SS is derived from its ability to suppress Rho family GTPase function by limiting GGPP levels and subsequently inhibiting isoprenylation.

SS inhibits recruitment of STAT-1{alpha} and RNA Pol II to the CD40 promoter and affects histone acetylation
To investigate further whether the inhibitory effect of SS was mediated at the level of CD40 transcription, we examined the influence of SS on CD40 promoter activity. We were unable to detect SS inhibition of IFN-{gamma}-induced CD40 promoter activity in transient transfection assays performed in RAW264.7 cells (data not shown), suggesting that the CD40 reporter construct does not behave similarly to the endogenous CD40 gene. Given the strong inhibitory effect of SS on IFN-{gamma}-induced CD40 mRNA expression, and the fact that this is not a result of SS-induced destabilization of the CD40 message, this strongly suggests an effect at the transcriptional level. This prompted us to examine the effect of SS on events occurring at the endogenous CD40 promoter.

IFN-{gamma}-induced CD40 expression involves recruitment of STAT-1{alpha} and NF-{kappa}B to the CD40 promoter [14 ]. STAT-1{alpha} activation and recruitment occur rapidly after IFN-{gamma} treatment, and NF-{kappa}B is activated at a later time as a result of endogenous TNF-{alpha} production induced by IFN-{gamma} (2–6 h time-frame) [13 , 14 ]. We examined whether SS inhibits the association of IFN-{gamma}-activated STAT-1{alpha} with the endogenous CD40 promoter. Recruitment of STAT-1{alpha} to the CD40 promoter was observed 15–30 min after addition of IFN-{gamma}, and this recruitment was inhibited by SS treatment, especially at the 30-min time-point (Fig. 3A ). We have shown previously that association of STAT-1{alpha} with the CD40 promoter and transcription of the CD40 gene are correlated with acetylation of histones H3 and H4 [39 , 40 ]. Histone acetylation is generally associated with the opening of the chromatin structure and with activation of transcription [41 ]. Treatment with IFN-{gamma} led to increased acetylation of H3 and H4 at 15–30 min, and SS treatment inhibited these responses substantially (Fig. 3A) . Pol II recruitment is important for CD40 gene transcription [39 ]. The binding of Pol II to the CD40 promoter was observed 15–30 min after IFN-{gamma} addition, and this recruitment was also inhibited by SS treatment (Fig. 3B) . IFN-{gamma}-induced NF-{kappa}B recruitment to the CD40 promoter was not inhibited by SS treatment; in fact, there was a slight enhancement of NF-{kappa}B recruitment (Fig. 3C) . The inclusion of rabbit IgG or mouse IgG serves as a negative control, and PCR analysis of input indicates that the soluble chromatin samples obtained from each time-point had equal amounts of chromatin fragments containing the CD40 promoter (Fig. 3A 3B 3C) . Taken together, these data indicate that SS inhibits CD40 expression at the transcriptional level by inhibiting STAT-1{alpha} and Pol II recruitment, and diminishing histone acetylation of the CD40 promoter.


Figure 3
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  		Figure 3. SS inhibits recruitment of STAT-1{alpha} and RNA Pol II to the CD40 promoter and affects histone acetylation. RAW264.7 cells were pretreated with 10 µM SS for 16 h, and then stimulated with IFN-{gamma} for 0.25, 0.5, or 4 h. Cells were cross-linked with formaldehyde, and the soluble chromatin was subjected to immunoprecipitation with antibodies against STAT-1, histone acetylation [H3 and H4 (AcH3 and AcH4, respectively)], or rabbit IgG (A), RNA Pol II or mouse IgG (B), or NF-{kappa}B p65 or rabbit IgG (C), followed by PCR. Input chromatin was subjected to PCR to control for variations in immunoprecipitation-starting material. Polyclonal rabbit IgG or mouse IgG were used as negative immunoprecipitation controls for nonspecific binding. The basal level of the untreated sample (UT) was set as 1.0, and fold induction was compared with that; representative of three independent experiments.

 
SS inhibits STAT-1{alpha} activation and expression
It has been reported that statins inhibit the phosphorylation of STAT-1 in some cell types [27 , 42 ], but the results are controversial, as other groups have not observed an inhibitory effect [43 , 44 ]. To explore this effect in detail, cells were pretreated with SS for 16 h prior to IFN-{gamma} treatment, and then, protein lysates were subjected to immunoblotting to detect the phosphorylation status of STAT-1. Tyrosine701 phosphorylation of STAT-1 was detected after 30 min of IFN-{gamma} treatment (Fig. 4A , Lane 3) and was inhibited by pretreatment with 10 µM SS (Lane 4). Serine727 phosphorylation of STAT-1 was also inhibited by 10 µM SS (data not shown). It is more interesting that SS substantially decreased the levels of constitutive STAT-1{alpha} protein (compare Lanes 2 and 4), while actin protein levels were not affected. These results suggest that SS may inhibit IFN-{gamma}-induced activation of STAT-1{alpha} by decreasing levels of the STAT-1{alpha} protein. The same inhibitory effect of SS was observed in primary murine microglia, but not primary murine astrocytes (data not shown), suggesting cell-type specific effects.


Figure 4
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  		Figure 4. SS inhibits STAT-1{alpha} activation and expression. RAW264.7 cells were pretreated with SS (10 µM) for 16 h, and then stimulated by IFN-{gamma} (10 ng/ml) for 30 min. (A) Total cell lysates were analyzed by immunoblotting with phospho (P)-STAT-1{alpha} or phospho-JAK2 antibodies, stripped, and reprobed with total STAT-1{alpha}, JAK2, or actin antibodies; representative of four experiments. (B) Cell lysates (1.0 mg) were immunoprecipiated (IP) with anti-JAK1 antibody, and then analyzed by immunoblotting (IB) with antiphospho-JAK1 antibody or anti-JAK1 antibody; representative of three experiments. (C) EMSA experiments were performed using nuclear extracts (10 µg), prepared from untreated RAW264.7 cells or those treated with SS for 16 h, IFN-{gamma} for 30 min, or SS (16 h), followed by IFN-{gamma} for 30 min, and then assayed using a GAS sequence end-labeled with [32P]ATP. For competition and supershift, a 50-fold molar excess of indicated oligonucleotides or 1 µl of the indicated antibodies was added to the nuclear extracts for 30 min before the addition of labeled probe. Bound and free probes were separated on 6% nondenaturing polyacrylamide gels in 0.5x Tris/boric acid/EDTA buffer; representative of three independent experiments.

 
Binding of IFN-{gamma} to its receptor activates JAK1 and JAK2, which are responsible for subsequent STAT-1{alpha} tyrosine phosphorylation and activation [2 , 45 ]. To determine the effect of SS on activation of the JAK proteins, the expression and phosphorylation status of JAK2 and JAK1 was assessed. SS did not inhibit IFN-{gamma}-induced phosphorylation of JAK2 (Fig. 4A , Lanes 3 and 4) nor did it affect the levels of total JAK2 protein (Lanes 2 and 4). As JAK1 protein levels are low in RAW264.7 cells, we performed immunoprecipitation experiments to determine the effect of SS on expression and activation of JAK1. As shown in Figure 4B , neither the phosphorylation of JAK1 (Lanes 3 and 4) nor total JAK1 protein expression (Lanes 2 and 4) was affected by SS. These results demonstrate that the inhibitory effect of SS on phosphorylation of STAT-1{alpha} is not a result of inhibition of JAK1 or JAK2 tyrosine phosphorylation.

We next determined if the inhibition of STAT-1{alpha} expression by SS decreased binding of STAT-1{alpha} to GAS elements identified functionally as important for IFN-{gamma}-inducible gene transcription. Cells were treated with SS for 16 h prior to IFN-{gamma} treatment, nuclear extracts were prepared, and EMSA was performed using a GAS oligonucleotide as the probe. Figure 4C demonstrates that there is no binding to the GAS oligonucleotide using nuclear extracts from untreated or SS-treated cells (Lanes 1 and 2). Upon treatment with IFN-{gamma}, there is complex formation over the GAS probe (Lane 3), which is diminished upon SS treatment (Lane 4). The complex was competed by a 50-fold excess of the GAS oligonucleotide (Lane 7), but not by a NF-{kappa}B oligonucleotide (Lane 8), indicating the specificity of binding. The identity of the IFN-{gamma}-induced complex was confirmed by supershifting with anti-STAT-1{alpha} (Lane 5) but not with anti-STAT-3 antibody (Lane 6). These results indicate that SS inhibition of STAT-1{alpha} binding to GAS elements results from a reduction in STAT-1{alpha} protein levels by SS.

SS inhibits STAT-1{alpha} protein and mRNA expression in a dose- and time-dependent manner
To further study the inhibitory effect of SS on STAT-1{alpha} expression, cells were treated with increasing concentrations of SS for 16 h. We observed a dose-dependent inhibition of STAT-1{alpha} protein expression by SS, with optimal inhibition at the concentration of 10 µM (Fig. 5A ). SS did not affect expression of NF-{kappa}B p65 or actin. SS also inhibited STAT-1{alpha} protein expression in a time-dependent manner. Upon treatment with SS for 16–20 h, STAT-1{alpha} protein levels were decreased substantially (Fig. 5B) , again with no effect on NF-{kappa}B p65 or actin expression. A similar result was obtained for STAT-1{alpha} mRNA expression (Fig. 5C and 5D) ; SS functioned in a dose- and time-dependent manner to inhibit STAT-1 mRNA levels. In addition, SS partially inhibited STAT-1{alpha} mRNA expression in primary microglia (Fig. 5E) . These results demonstrate that SS inhibits constitutively expressed STAT-1{alpha} protein and mRNA, without a general reduction in protein synthesis.


Figure 5
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  		Figure 5. SS inhibits constitutively expressed STAT-1{alpha} protein and mRNA in a dose- and time-dependent manner. RAW264.7 cells were treated with increasing concentrations of SS (0–10 µM) for 16 h (A, C) or with SS (10 µM) for 0–20 h (B, D). Total cell lysates were analyzed by immunoblotting with STAT-1{alpha}, NF-{kappa}B p65, or actin antibodies (A, B), and total RNA was analyzed by RT-PCR with primers specific for STAT-1 and GAPDH (C, D). Values were normalized to the respective actin or GAPDH levels. The basal level of the untreated sample was set as 1.0, and fold induction was compared with that; representative of three independent experiments. Primary murine microglia were treated with 10 µM SS for 24 h, and then total RNA was analyzed by RT-PCR for STAT-1 and GAPDH (E).

 
SS inhibits STAT-1{alpha} in a HMG-CoA reductase-dependent manner
As shown in Figure 2 , the inhibitory effect of SS on CD40 expression was mediated via blockade of the HMG-CoA reductase pathway, and inhibition of Rho protein function by decreasing the intracellular pools of GGPP likely contributes to the inhibitory mechanism of SS. As such, we next examined the effect of metabolic intermediates on SS-mediated inhibition of STAT-1{alpha} expression. As shown in Figure 6A , the metabolic intermediates MVLT, GGPP, and cholesterol alone had a modest enhancing effect on STAT-1{alpha} expression (Lanes 3–5). The addition of MVLT or GGPP reversed the inhibitory effect of SS on STAT-1{alpha} protein expression (compare Lanes 2, 6, and 7), while cholesterol had no effect (Lane 8). In addition, MVLT and GGPP reversed SS-mediated inhibition of STAT-1 mRNA expression (Fig. 6B , compare Lanes 6, 7, and 8), whereas cholesterol was without effect (Lane 9). We did test the effect of FPP supplementation on STAT-1{alpha} expression; however, the results were inconsistent. In some experiments, a modest reversal of the inhibitory effect of SS was observed, and in other experiments, no effect was observed (data not shown). To examine further if the inhibitory effect of SS was independent of cholesterol levels, cells were treated with CDX, which depletes cholesterol [46 ]. Treatment with 5 mM CDX for various time periods (0.5–4 h) did not affect STAT-1{alpha} protein expression (Fig. 6C) . Given that GGPP reversed the inhibitory effect of SS (Fig. 6A and 6B) , we examined the effect of GGTI-298 on STAT-1 expression. GGTI-298 treatment mimicked the inhibitory effect of SS on STAT-1{alpha} protein and mRNA expression (Fig. 7A and 7B ). These results suggest that the inhibitory effect of SS is a result of inhibition of HMG-CoA reductase, subsequently reducing the intracellular pool of GGPP. Therefore, SS-induced suppression of GGPP-mediated protein isoprenylation is likely involved in the inhibitory effect on STAT-1{alpha} expression.


Figure 6
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  		Figure 6. The inhibitory effect of SS is dependent on MVLT and GGPP. (A, B) Cells were treated with SS (10 µM) or SS plus metabolic intermediates (500 µM MVLT, 5 µM GGPP, or 500 µM cholesterol) for 16 h. (A) Total cell lysates were analyzed by immunoblotting with STAT-1{alpha}, NF-{kappa}B p65, or actin antibodies. (B) Total RNA was analyzed by RT-PCR with primers specific for STAT-1 and GAPDH. Values were normalized to the respective actin or GAPDH levels. The basal level of the untreated sample was set as 1.0, and fold induction was compared with that. The solvent ethanol was included as a control (Lane 2); representative of three independent experiments. (C) Cells were treated with 5 mM CDX for 0–4 h. Total cell lysates were analyzed by immunoblotting with STAT-1{alpha} and actin antibodies; representative of three independent experiments.

 

Figure 7
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  		Figure 7. GGTI-298 inhibits STAT-1 expression, mimicking the effect of SS. (A, B) Cells were treated with SS (10 µM) or GGTI-298 (5 or 10 µM) for 16 h. (A) Total cell lysates were analyzed by immunoblotting with STAT-1{alpha} or actin antibodies. (B) Total RNA was analyzed by RT-PCR with primers specific for STAT-1 and GAPDH. Values were normalized to the respective actin or GAPDH levels. The basal level of the untreated sample was set as 1.0, and fold induction was compared with that. The solvent DMSO was included as a control (Lane 2); representative of three independent experiments.

 
SS has a modest effect on other STAT proteins
We tested whether the inhibitory effect of SS was specific for STAT-1{alpha}, or if SS had an influence on other STAT family members. At a concentration of 10 µM, SS decreased the expression of STAT-3, -5, and -6 (Fig. 8 , Lane 4) slightly, and STAT-2 and -4 levels were not affected. These results indicate that SS has a modest inhibitory effect on STAT-3, -5, and -6, but the predominant inhibitory effect is on STAT-1{alpha}.


Figure 8
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  		Figure 8. SS has a modest inhibitory effect on constitutively expressed STAT-3, -5, and -6 proteins. Cells were treated with increasing concentrations of SS (0–10 µM) for 16 h. Cell lysates were obtained and subjected to immunoblotting. Blots were probed with a STAT-1{alpha} antibody, stripped, and reprobed with STAT-2, -3, -4, -5, and -6 or actin antibodies; representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we identify STAT-1{alpha} as a biological target for SS. Our data demonstrate that SS inhibits IFN-{gamma}-induced CD40 gene expression through inhibition of recruitment of STAT-1{alpha} and RNA Pol II, as well as acetylation of histones on the endogenous CD40 promoter. The inhibitory effect of SS on STAT-1{alpha} results from suppression of constitutive STAT-1{alpha} mRNA and protein expression. SS-mediated suppression of STAT-1{alpha} is independent of cholesterol reduction, but dependent on GGPP-mediated isoprenylation of Rho family members. As CD40 expression has been implicated in participating in autoimmune diseases such as multiple sclerosis (MS) [6 ] and neurodegenerative diseases such as Alzheimer’s disease [47 , 48 ], inhibition of CD40 through inhibition of STAT-1{alpha} expression may help to explain the beneficial effect of statins in animal models of these diseases [26 , 49 , 50 ].

Although the inhibitory effect of statins on expression of IFN-{gamma}-induced CD40 has been studied in numerous cell types [26 , 27 , 51 52 53 ], the molecular basis for suppression was not established. Consistent with a previous study [26 ], we observed that SS inhibited IFN-{gamma}-induced CD40 expression in a HMG-CoA reductase-dependent manner (Fig. 2A) . More importantly, our results indicate that SS-mediated inhibition of IFN-{gamma}-induced CD40 gene expression is a result of suppression of STAT-1{alpha} expression. This ultimately results in the lack of STAT-1{alpha} recruitment to the endogenous CD40 promoter upon IFN-{gamma} treatment, in addition to reduced RNA Pol II recruitment and diminished levels of IFN-{gamma}-induced H3 and H4 histone acetylation (Fig. 3) . SS has also been shown to inhibit expression of other IFN-{gamma}-inducible genes (CIITA, Class II MHC, IRF-1, and ICAM-1), which require STAT-1{alpha} for their expression [19 , 54 ] (Fig. 1D) . We propose that SS-mediated inhibition of STAT-1 expression is responsible for suppression of numerous IFN-{gamma}-inducible, STAT-1-dependent genes such as CD40, CIITA, IRF-1, and ICAM-1.

The inhibitory effect of SS on STAT-1{alpha} was mediated through inhibition of STAT-1{alpha} protein and mRNA expression (Figs. 4A and 5) . In subsequent experiments, we observed that SS decreased the DNA-binding ability of STAT-1{alpha} to GAS elements, which are functionally important for IFN-{gamma}-inducible gene activity (Fig. 4C) . The inhibitory effect of SS was quite pronounced for STAT-1{alpha} expression, although SS had a modest inhibitory effect on STAT-3, -5, and -6 (Fig. 8) . Moreover, SS did not inhibit other constitutively expressed transcription factors such as NF-{kappa}B p65 (Fig. 5) nor did it influence expression or phosphorylation of JAK1 and JAK2 (Fig. 4A and 4B) , indicating that SS is not a global inhibitor of protein synthesis. In this regard, our results differ from those of Townsend et al. [27 ], who demonstrated a modest inhibitory effect of lovastatin on IFN-{gamma}-induced JAK1 and JAK2 phosphorylation in microglia. This may be a result of the use of different statins (SS vs. lovastatin).

Many of the anti-inflammatory effects of statins are related to inhibition of HMG-CoA reductase activity [17 , 23 ]. In this regard, we demonstrated that SS-mediated inhibition of CD40 and STAT-1{alpha} expression occurred in a HMG-CoA reductase-dependent manner, as inclusion of the downstream metabolite, MVLT, reversed the inhibitory effect of SS (Figs. 2A and 6A and 6B) . Statins mediate immunomodulatory effects on T cells and APCs, primarily through inhibition of synthesis of isoprenoid compounds in the MVLT pathway [26 , 37 ]. Recently, it has been reported that farnesylated Ras and geranylgeranylated RhoA proteins are the targets of atorvastatin in T cells, which facilitates Th2 cell differentiation [37 ]. We found that exogenous addition of GGPP reversed the inhibitory effect of SS on CD40 and STAT-1{alpha} expression (Figs. 2A and 6) , suggesting that depleting cells of GGPP-dependent biochemical processes mediates the inhibitory effect of SS. We determined further that GGTI-298, a geranylgeranyl transferase I inhibitor, and C. difficile Toxin A, a specific inhibitor for Rho family proteins, inhibit CD40 and/or STAT-1{alpha} expression (Figs. 2B and 2C and 7) . Therefore, Rho family proteins function as positive regulators of IFN-{gamma}-induced CD40 expression, and SS decreases CD40 expression by inhibiting the prenylation of Rho family proteins. In addition, these results suggest that GGPP-mediated protein isoprenylation is important for STAT-1{alpha} expression, and interrupting this by SS negatively affects STAT-1{alpha} gene expression and the subsequent ability to induce CD40. The molecular basis by which this occurs is unknown at present. It has been reported that ZBP-89, which is a Krüppel-type zinc-finger protein, binds to a GC-rich element in the STAT-1 promoter and is required for STAT-1 constitutive expression [55 ]. Whether SS affects the expression and/or activity of ZBP-89 is not known at this time. Future experiments are planned to address these possibilities.

The inhibitory effect of SS on CD40 expression was not absolute; rather, we observed ~60% inhibition. This suggests that some component of IFN-{gamma}-induced CD40 gene expression is refractory to SS treatment; we believe this to be the NF-{kappa}B component. We have reported previously that optimal induction of CD40 expression by IFN-{gamma} involves TNF-{alpha} production, which then activates NF-{kappa}B [14 ]. It is interesting that SS did not inhibit IFN-{gamma}-induced TNF-{alpha} expression, translocation of NF-{kappa}B into the nucleus, or IFN-{gamma}-induced serine phosphorylation of NF-{kappa}B p65 (data not shown). As shown in Figure 3C , IFN-{gamma}-induced recruitment of NF-{kappa}B to the endogenous CD40 promoter was not inhibited by SS treatment. These results were surprising initially, as the ability of statins to inhibit NF-{kappa}B activation, nuclear translocation, and DNA binding has been shown in numerous cell types [52 , 56 57 58 59 ]. However, in these studies, the statins were acting in a rapid time-frame (15–60 min after stimulation with LPS or IL-1β) to inhibit NF-{kappa}B activation. In our system, NF-{kappa}B activation is caused by IFN-{gamma}-mediated TNF-{alpha} production, which occurs with delayed kinetics (2–6 h). Furthermore, a recent paper has demonstrated that SS treatment increases TNF-{alpha} production in macrophages, possibly via SS-mediated inhibition of c-Fos and induction/phosphorylation of c-Jun, which activates the TNF-{alpha} promoter [60 ]. Indeed, our results also demonstrate that SS induces TNF-{alpha} expression modestly, as well as enhances that induced by IFN-{gamma} (data not shown). Several recent reports have documented that SS does not influence TNF-{alpha}-induced NF-{kappa}B activation in microglia [28 ] and actually enhances TNF-{alpha} secretion by microglia [29 ]. In addition, statins enhance the secretion of proinflammatory cytokines such as IL-6, IL-18, IL-1, IL-12, and TNF-{alpha} from monocytes and dendritic cells [53 , 61 ]. Thus, for IFN-{gamma}-induced CD40 expression, SS inhibits the STAT-1{alpha}-mediated component of CD40 induction, while slightly potentiating the delayed NF-{kappa}B component, which may explain the partial inhibitory effect of SS on CD40 gene expression (Fig. 9 ). Our data and those of others indicate that statins exert pro- and anti-inflammatory effects; thus, given the multifaceted effects of statins, it is critical to understand the molecular mechanisms underlying their effects on gene expression.


Figure 9
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  		Figure 9. Proposed model of SS-mediated inhibition of CD40 gene expression. (A) IFN-{gamma}-induced CD40 gene expression involves recruitment of activated STAT-1{alpha}, activated NF-{kappa}B, RNA Pol II, and the coactivators CREB-binding protein (CBP)/p300 to the CD40 promoter, as well as acetylation of histones H3 and H4 (shown as purple circles). (B) SS suppresses constitutive STAT-1{alpha} expression. This results in inhibition of STAT-1{alpha} activation, subsequently leading to a reduction of IFN-{gamma}-induced CD40 gene expression. This inhibition is associated with reduced recruitment of STAT-1{alpha} and RNA Pol II to the CD40 promoter, as well as inhibition of IFN-{gamma}-induced H3 and H4 acetylation. In contrast, SS does not inhibit IFN-{gamma}-induced TNF-{alpha} production and recruitment of activated NF-{kappa}B to the CD40 promoter. Green circles indicate phosphorylation.

 
Oral administration of SS (Zocor) reduced the number of new, gadolinium-enhancing lesions in relapsing-remitting MS patients by 44% in a small 6-month open-label trial [62 ]. As well, atorvastatin mediated modest, but clinically significant, anti-inflammatory effects in patients with rheumatoid arthritis [63 ]. Statins have also been shown to reduce the risk of Alzheimer’s disease in subjects younger than 80 years [64 ]. These are all diseases associated with aberrant macrophage activation and elevated expression of CD40, Class II MHC, and numerous cytokines and chemokines [5 , 7 , 47 ]. We suggest that the ability of statins to mediate beneficial effects may be a result of the blunting of CD40 and Class II MHC expression on macrophages, thereby lessening antigen presentation and subsequent T cell activation [23 24 25 26 ]. The inhibition of CD40 and Class II MHC is likely a result of inhibition of STAT-1{alpha} expression, as both of these genes are dependent on STAT-1{alpha} for expression [12 , 54 ]. Furthermore, inhibition of CD40 expression on macrophages/microglia by statins attenuates CD40-mediated induction of cytokines/chemokines such as IL-12, IL-1, IL-8, and MCP-1 [27 , 51 ], which are implicated in inflammatory responses. The present study documents the complex biological effect of SS on intracellular signaling cascades and transcription factor expression/function in macrophages and microglia, which results in a skewing toward predominantly anti-inflammatory effects, and may underly the beneficial effects of statins in autoimmune diseases.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health grants NS45290 and NS36765 to E. N. B. and a Pilot Research grant from the National Multiple Sclerosis Society (PP1129) to E. N. B. We acknowledge the support of the University of Alabama at Birmingham Flow Cytometry Core Facility (AM20614).

Received December 20, 2006; revised April 5, 2007; accepted April 26, 2007.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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