Title: Gemfibrozil, a Lipid-lowering Drug, Inhibits the Induction of Nitric-oxide Synthase in Human Astrocytes
Abstract: Gemfibrozil, a lipid-lowering drug, inhibited cytokine-induced production of NO and the expression of inducible nitric-oxide synthase (iNOS) in human U373MG astroglial cells and primary astrocytes. Similar to gemfibrozil, clofibrate, another fibrate drug, also inhibited the expression of iNOS. Inhibition of human iNOS promoter-driven luciferase activity by gemfibrozil in cytokine-stimulated U373MG astroglial cells suggests that this compound inhibits the transcription of iNOS. Since gemfibrozil is known to activate peroxisome proliferator-activated receptor-α (PPAR-α), we investigated the role of PPAR-α in gemfibrozil-mediated inhibition of iNOS. Gemfibrozil induced peroxisome proliferator-responsive element (PPRE)-dependent luciferase activity, which was inhibited by the expression of ΔhPPAR-α, the dominant-negative mutant of human PPAR-α. However, ΔhPPAR-α was unable to abrogate gemfibrozil-mediated inhibition of iNOS suggesting that gemfibrozil inhibits iNOS independent of PPAR-α. The human iNOS promoter contains consensus sequences for the binding of transcription factors, including interferon-γ (IFN-γ) regulatory factor-1 (IRF-1) binding to interferon-stimulated responsive element (ISRE), signal transducer and activator of transcription (STAT) binding to γ-activation site (GAS), nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and CCAAT/enhancer-binding protein β (C/EBPβ); therefore, we investigated the effect of gemfibrozil on the activation of these transcription factors. The combination of interleukin (IL)-1β and IFN-γ induced the activation of NF-κB, AP-1, C/EBPβ, and GAS but not that of ISRE, suggesting that IRF-1 may not be involved in cytokine-induced expression of iNOS in human astrocytes. Interestingly, gemfibrozil strongly inhibited the activation of NF-κB, AP-1, and C/EBPβ but not that of GAS in cytokine-stimulated astroglial cells. These results suggest that gemfibrozil inhibits the induction of iNOS probably by inhibiting the activation of NF-κB, AP-1, and C/EBPβ and that gemfibrozil, a prescribed drug for humans, may further find its therapeutic use in neuroinflammatory diseases. Gemfibrozil, a lipid-lowering drug, inhibited cytokine-induced production of NO and the expression of inducible nitric-oxide synthase (iNOS) in human U373MG astroglial cells and primary astrocytes. Similar to gemfibrozil, clofibrate, another fibrate drug, also inhibited the expression of iNOS. Inhibition of human iNOS promoter-driven luciferase activity by gemfibrozil in cytokine-stimulated U373MG astroglial cells suggests that this compound inhibits the transcription of iNOS. Since gemfibrozil is known to activate peroxisome proliferator-activated receptor-α (PPAR-α), we investigated the role of PPAR-α in gemfibrozil-mediated inhibition of iNOS. Gemfibrozil induced peroxisome proliferator-responsive element (PPRE)-dependent luciferase activity, which was inhibited by the expression of ΔhPPAR-α, the dominant-negative mutant of human PPAR-α. However, ΔhPPAR-α was unable to abrogate gemfibrozil-mediated inhibition of iNOS suggesting that gemfibrozil inhibits iNOS independent of PPAR-α. The human iNOS promoter contains consensus sequences for the binding of transcription factors, including interferon-γ (IFN-γ) regulatory factor-1 (IRF-1) binding to interferon-stimulated responsive element (ISRE), signal transducer and activator of transcription (STAT) binding to γ-activation site (GAS), nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and CCAAT/enhancer-binding protein β (C/EBPβ); therefore, we investigated the effect of gemfibrozil on the activation of these transcription factors. The combination of interleukin (IL)-1β and IFN-γ induced the activation of NF-κB, AP-1, C/EBPβ, and GAS but not that of ISRE, suggesting that IRF-1 may not be involved in cytokine-induced expression of iNOS in human astrocytes. Interestingly, gemfibrozil strongly inhibited the activation of NF-κB, AP-1, and C/EBPβ but not that of GAS in cytokine-stimulated astroglial cells. These results suggest that gemfibrozil inhibits the induction of iNOS probably by inhibiting the activation of NF-κB, AP-1, and C/EBPβ and that gemfibrozil, a prescribed drug for humans, may further find its therapeutic use in neuroinflammatory diseases. It is now increasingly clear that glial cells (astrocytes and microglia) in the central nervous system (CNS) 1The abbreviations used for: CNS, central nervous system; iNOS, inducible nitric-oxide synthase; IL, interleukin; TNF, tumor necrosis factor; INF, interferon; IRF-1, IFN-γ regulatory factor-1; PPAR, peroxisome proliferator-activated receptor; STAT, signal transducer and activator of transcription; NF-κB, nuclear factor-κB; AP-1, activator protein-1; C/EBPβ, CCAAT/enhancer-binding protein β; GAS, γ-activation site; DMEM, Dulbecco's modified Eagle's medium; l-NMA, l-NG-Monomethylarginine; d-NMA, d-NG-monomethylarginine; PPRE, peroxisome proliferator-responsive element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JAK, Janus kinase. induce the expression of inducible nitric-oxide synthase (iNOS) and the production of NO in response to proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) (1Feinstein D.L. Galea E. Roberts S. Berquist H. Wang H. Reis D.J. J. Neurochem. 1994; 62: 315-321Crossref PubMed Scopus (150) Google Scholar, 2Pahan K. Sheikh F.G. Namboodiri A.M.S. Singh I. J. Clin. Invest. 1997; 100: 2671-2679Crossref PubMed Scopus (513) Google Scholar, 3Pahan K. Sheikh F.G. Liu X. Hilger S. McKinney M. Petro T.M. J. Biol. Chem. 2001; 276: 7899-7905Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 4Jana M. Liu X. Koka S. Ghosh S. Petro T.M. Pahan K. J. Biol. Chem. 2001; 276: 44527-44533Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Although the NO produced by iNOS has bactericidal and tumoricidal properties, it also plays an important role in pathophysiologies of inflammatory neurological diseases including demyelinating disorders (e.g. multiple sclerosis, experimental allergic encephalopathy), neurodegenerative disorder like Alzheimer's disease, and in ischemic and traumatic brain injuries associated with the activation of glial cells and the production of proinflammatory cytokines (5Mitrovic B. Ignarro L.J. Montestruque S. Smoll A. Merril J.E. Neuroscience. 1994; 61: 575-585Crossref PubMed Scopus (247) Google Scholar, 6Merrill J.E. Ignarro L.J. Sherman M.P. Melinek J. Lane T.E. J. Immunol. 1993; 151: 2132-2141PubMed Google Scholar, 7Koprowski H. Zheng Y.M. Heber-Katz E. Fraser N. Rorke L. Fu Z.F. Hanlon C. Dietzshold B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3024-3027Crossref PubMed Scopus (472) Google Scholar, 8Akama K.T. Albanese C. Pestell R.G. Van Eldik L.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5795-5800Crossref PubMed Scopus (320) Google Scholar). NO derived from activated glial cells is assumed to contribute to oligodendrocyte degeneration in demyelinating diseases and neuronal death during ischemia and trauma (5Mitrovic B. Ignarro L.J. Montestruque S. Smoll A. Merril J.E. Neuroscience. 1994; 61: 575-585Crossref PubMed Scopus (247) Google Scholar, 6Merrill J.E. Ignarro L.J. Sherman M.P. Melinek J. Lane T.E. J. Immunol. 1993; 151: 2132-2141PubMed Google Scholar). Therefore, characterization of intracellular pathways required to transduce the signal from the cell surface to the nucleus for the induction of iNOS is an active area of investigation, since compounds capable of antagonizing signaling steps for the induction of iNOS may have therapeutic effect in NO-mediated pathophysiological conditions. Peroxisome proliferator-activated receptors (PPARs), members of the nuclear hormone receptor superfamily, have been implicated in a variety of human diseases (9Lemberger T. Desvergne B. Wahli W. Annu. Rev. Cell Dev. Biol. 1996; 12: 335-363Crossref PubMed Scopus (640) Google Scholar). Three isotypes have been described to date, PPAR-α, PPAR-β, and PPAR-γ (9Lemberger T. Desvergne B. Wahli W. Annu. Rev. Cell Dev. Biol. 1996; 12: 335-363Crossref PubMed Scopus (640) Google Scholar). Activation of PPAR-α mainly leads to the induction of a variety of genes such as those coding for the enzymes for β- and ω-oxidation of fatty acids (10Dreyer C. Krey G. Keller H. Givel F. Helftenbein G. Wahli W. Cell. 1992; 68: 879-887Abstract Full Text PDF PubMed Scopus (1214) Google Scholar). Gemfibrozil, an activator of PPAR-α, has been often prescribed in patients to lower the level of triglycerides (11Hsu H.C. Lee Y.T. Yeh H.T. Chen M.F. J. Lab. Clin. Med. 2001; 137: 414-421Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 12Bloomfield R.H. Davenport J. Babikian V. Brass L.M. Collins D. Wexler L. Wagner S. Papademetriou V. Rutan G. Robins S.J. Circulation. 2001; 103: 2828-2833Crossref PubMed Google Scholar). This drug decreases the risk of coronary heart disease by increasing the level of high density lipoprotein cholesterol and decreasing the level of low density lipoprotein cholesterol (11Hsu H.C. Lee Y.T. Yeh H.T. Chen M.F. J. Lab. Clin. Med. 2001; 137: 414-421Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 12Bloomfield R.H. Davenport J. Babikian V. Brass L.M. Collins D. Wexler L. Wagner S. Papademetriou V. Rutan G. Robins S.J. Circulation. 2001; 103: 2828-2833Crossref PubMed Google Scholar). Activation of PPAR-α is also capable of modifying the stress response by activation of heat shock factor 1 (HSF-1) and induction of HSP70 (13Amici C. Sistonen L. Santoro M.G. Morimoto R.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6227-6231Crossref PubMed Scopus (154) Google Scholar, 14Elia G. Amici C. Rossi A. Santoro M.G. Cancer Res. 1996; 56: 210-217PubMed Google Scholar). Recently it has been shown that activation of HSP70 inhibits the expression of iNOS in astrocytes (15Feinstein D.L. Galea E. Aquino D.A. Li G.C. Xu H. Reis D.J. J. Biol. Chem. 1996; 271: 17724-17732Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), suggesting that the expression of iNOS may also be regulated by activators of PPAR-α. Therefore, we investigated the effect of gemfibrozil on the expression of iNOS in cytokine-stimulated human U373MG astroglial cells and primary astrocytes. In the current work, we present evidence that gemfibrozil markedly inhibited the expression of iNOS and the production of NO in human astrocytes independent of PPAR-α. In addition, reporter gene assays reveal that gemfibrozil specifically inhibited cytokine-induced activation of NF-κB, AP-1, and C/EBPβ but not that of GAS. These results raise the possibility that gemfibrozil, a common lipid-lowering drug, may be of therapeutic value in human neuroinflammatory diseases. Fetal bovine serum and DMEM/F-12 were from Invitrogen. Human recombinant IFN-γ, IL-1β, and TNF-α were purchased from R & D Systems.l-NG-Monomethylarginine (l-NMA) andd-NG-monomethylarginine (d-NMA) were obtained from Biomol. Gemfibrozil and clofibrate were obtained from Sigma. Antibodies against human macrophage iNOS were obtained fromCalbiochem. 125I-Labeled protein A and [α-32P]dCTP (3000 Ci/mmol) were purchased from PerkinElmer Life Sciences. Peroxisome proliferator-responsive element (PPRE)-dependent reporter construct (tk-PPREx3-Luc) and dominant-negative mutant of CCAAT/enhancer-binding protein β (ΔC/EBPβ) were kindly provided by Dr. Ronald M. Evans of The Salk Institute and Dr. Steve Smale of the University of California at Los Angeles, respectively. Human CNS tissue was obtained from the Human Embryology Laboratory, University of Washington, Seattle. The CNS tissue from each specimen was processed separately and independently, as were subsequent cell cultures; there was no pooling of CNS tissue from distinct specimens. All the experimental protocols were reviewed and approved by the Institutional Review Board (IRB number 224-01-FB) of the University of Nebraska Medical Center. These cells were grown in a serum-free, defined medium (B16) enriched with 5 ng of basic fibroblast growth factor per ml for optimal growth of astrocytes and for the suppression of fibroblast growth (16McCarthy M. Wood C. Fedoseyeva L. Whittemore S. J. Neurovirol. 1995; 1: 275-285Crossref PubMed Scopus (32) Google Scholar). By immunofluorescence assay, these cultures homogeneously expressed glial fibrillary acidic protein (GFAP). Cells were trypsinized, subcultured, and stimulated with cytokines in serum-free DMEM/F-12 medium to induce the production of NO. The human U373MG astrocytoma cell line, purchased from the American Type Culture Collection (ATCC), was also maintained and stimulated under similar conditions. Synthesis of NO was determined by assay of culture supernatant for nitrite, a stable reaction product of NO with molecular oxygen, using Griess reagent (1Feinstein D.L. Galea E. Roberts S. Berquist H. Wang H. Reis D.J. J. Neurochem. 1994; 62: 315-321Crossref PubMed Scopus (150) Google Scholar, 2Pahan K. Sheikh F.G. Namboodiri A.M.S. Singh I. J. Clin. Invest. 1997; 100: 2671-2679Crossref PubMed Scopus (513) Google Scholar, 3Pahan K. Sheikh F.G. Liu X. Hilger S. McKinney M. Petro T.M. J. Biol. Chem. 2001; 276: 7899-7905Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 4Jana M. Liu X. Koka S. Ghosh S. Petro T.M. Pahan K. J. Biol. Chem. 2001; 276: 44527-44533Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Briefly, 400 μl of culture supernatant was allowed to react with 200 μl of Griess reagent and incubated at room temperature for 15 min. The optical density of the assay samples was measured spectrophotometrically at 570 nm. Fresh culture medium served as the blank in all experiments. Nitrite concentrations were calculated from a standard curve derived from the reaction of NaNO2 in the assay. Protein was measured by the procedure of Bradford (17Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Immunoblot analysis for iNOS was carried out as described earlier (2Pahan K. Sheikh F.G. Namboodiri A.M.S. Singh I. J. Clin. Invest. 1997; 100: 2671-2679Crossref PubMed Scopus (513) Google Scholar, 3Pahan K. Sheikh F.G. Liu X. Hilger S. McKinney M. Petro T.M. J. Biol. Chem. 2001; 276: 7899-7905Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 4Jana M. Liu X. Koka S. Ghosh S. Petro T.M. Pahan K. J. Biol. Chem. 2001; 276: 44527-44533Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Briefly, cells were detached by scraping, washed with Hanks' buffer, and homogenized in 50 mm Tris-HCl (pH 7.4) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml antipain, 5 μg/ml pepstatin A, and 5 μg/ml leupeptin). After electrophoresis the proteins were transferred onto a nitrocellulose membrane, and the iNOS band was visualized by immunoblotting with antibodies against human iNOS and125I-labeled protein A (2Pahan K. Sheikh F.G. Namboodiri A.M.S. Singh I. J. Clin. Invest. 1997; 100: 2671-2679Crossref PubMed Scopus (513) Google Scholar, 3Pahan K. Sheikh F.G. Liu X. Hilger S. McKinney M. Petro T.M. J. Biol. Chem. 2001; 276: 7899-7905Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 4Jana M. Liu X. Koka S. Ghosh S. Petro T.M. Pahan K. J. Biol. Chem. 2001; 276: 44527-44533Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Cells were taken out of the culture dishes directly by adding Ultraspec-II RNA reagent (Biotecx Laboratories, Inc.), and total RNA was isolated using Ultraspec-II RNA reagent (Biotecx Laboratories Inc.) according to the manufacturer's protocol. For Northern blot analyses, 20 μg of total RNA was electrophoresed on 1.2% denaturing formaldehyde-agarose gels, electrotransferred to Hybond-Nylon Membrane (AmershamBiosciences) and hybridized at 68 °C with32P-labeled cDNA probe using Express Hyb hybridization solution (Clontech) as described by the manufacturer. The cDNA probe was made by polymerase chain reaction amplification using two primers (forward primer, 5′-CTC CTT CAA AGA GGC AAA AAT A-3′; reverse primer, 5′-CAC TTC CTC CAG GAT GTT GT-3′) (2Pahan K. Sheikh F.G. Namboodiri A.M.S. Singh I. J. Clin. Invest. 1997; 100: 2671-2679Crossref PubMed Scopus (513) Google Scholar, 3Pahan K. Sheikh F.G. Liu X. Hilger S. McKinney M. Petro T.M. J. Biol. Chem. 2001; 276: 7899-7905Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 4Jana M. Liu X. Koka S. Ghosh S. Petro T.M. Pahan K. J. Biol. Chem. 2001; 276: 44527-44533Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,18Pahan K. Namboodiri A.M.S. Sheikh F.G. Smith B.T. Singh I. J. Biol. Chem. 1997; 272: 7786-7791Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). After hybridization filters were washed two or three times in solution I (2× SSC, 0.05% SDS) for 1 h at room temperature followed by solution II (0.1× SSC, 0.1% SDS) at 50 °C for another hour. The membranes were then dried and exposed to x-ray films (Eastman Kodak Co.). The same amount of RNA was hybridized with probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Construction of phiNOS(7.2)Luc, the 7.2-kb human iNOS promoter-luciferase construct, has been described previously (19Taylor B.S. de Vera M.E. Ganster R.W. Wang Q. Shapiro R.A. Morris S.M. Billiar T.R. Geller D.A. J. Biol. Chem. 1998; 273: 15148-15156Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Cells plated at 50–60% confluence in six-well plates were cotransfected with 1 μg of phiNOS(7.2)Luc and 50 ng of pRL-TK (a plasmid encoding Renilla luciferase, used as transfection efficiency control; Promega) by LipofectAMINE Plus (Invitrogen) following manufacturer's protocol (3Pahan K. Sheikh F.G. Liu X. Hilger S. McKinney M. Petro T.M. J. Biol. Chem. 2001; 276: 7899-7905Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 4Jana M. Liu X. Koka S. Ghosh S. Petro T.M. Pahan K. J. Biol. Chem. 2001; 276: 44527-44533Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Twenty-four h after transfection, cells were treated with different stimuli for 12 h. Firefly and Renilla luciferase activities were obtained by analyzing total cell extract according to standard instructions provided in the Dual Luciferase Kit (Promega) in a TD-20/20 Luminometer (Turner Designs). Relative luciferase activity of cell extracts was typically represented as the ratio of firefly luciferase value/Renilla luciferase value × 10−3. Cells plated at 50–60% confluence in six-well plates were cotransfected with 1 μg of either pNF-κB-Luc (NF-κB-dependent reporter construct), pAP-1-Luc (AP-1-dependent reporter construct), pC/EBPβ-Luc (C/EBPβ-dependent reporter construct), pGAS-Luc (GAS-dependent reporter construct), or pISRE-Luc (ISRE-dependent reporter construct) and 50 ng of pRL-TK using LipofectAMINE Plus. Construction of pC/EBPβ-Luc has been described earlier (4Jana M. Liu X. Koka S. Ghosh S. Petro T.M. Pahan K. J. Biol. Chem. 2001; 276: 44527-44533Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). This C/EBPβ-sensitive promoter contains four consensus C/EBPβ-binding sites. Other reporter constructs (pNF-κB-Luc, pAP-1-Luc, pGAS-Luc, and pISRE-Luc) were obtained from Stratagene. After 24 h of transfection, cells were treated with different stimuli for 6 h. Firefly and Renilla luciferase activities were obtained as described above. Statistical comparisons were made using one-way analysis of variance followed by Student's t test. Cells were cultured in serum-free media in the presence of IL-1β and IFN-γ. It is evident from Table I that IL-1β and IFN-γ alone were poor inducers of NO production. However, marked induction of NO production was observed by the combination of IL-1β and IFN-γ. This combination of cytokines was used to induce the production of NO in subsequent studies. The inhibition of cytokine-induced production of NO by arginase (an enzyme that degrades the substrate,l-arginine, of NOS) and l-NMA (a competitive inhibitor of NOS) but not by d-NMA (a negative control ofl-NMA) suggests that the combination of IL-1β and IFN-γ induces the production of NO in U373MG astroglial cells through NOS-mediated arginine metabolism (Table I). Next we examined the effect of gemfibrozil, an activator of PPAR-α (20Kliewer S.A. Forman B.M. Blumberg B. Ong E.S. Borgmeyer U. Mangelsdorf D.J. Umesono K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7355-7359Crossref PubMed Scopus (1281) Google Scholar), on the cytokine-induced nitrite production in U373MG glial cells. Gemfibrozil itself was neither stimulatory nor much inhibitory to NO production in control cells. However, gemfibrozil, when added 2 h before the addition of cytokines, markedly inhibited cytokine-induced production of NO (TableI).Table IInduction of NO production in human U373MG astroglial cellsTreatmentsNitrite productionμg/mg protein/24 hControl6.2 ± 0.8IL-1β only18.6 ± 1.9IFN-γ only15.9 ± 0.9IL-1β + IFN-γ196.3 ± 26.6IL-1β + IFN-γ + arginase21.2 ± 1.8ap < 0.001 versusIL-1β + IFN-γ.IL-1β + IFN-γ +l-NMA19.1 ± 2.1ap < 0.001 versusIL-1β + IFN-γ.IL-1β + IFN-γ +d-NMA194.7 ± 21.3Gemfibrozil only5.9 ± 1.1IL-1β + IFN-γ + gemfibrozil20.8 ± 2.8ap < 0.001 versusIL-1β + IFN-γ.U373MG glial cells preincubated in serum-free DMEM/F-12 for 1 h with arginase, l-NMA or d-NMA received the combination of IL-1β and IFN-γ. After 24 h of incubation, nitrite concentrations in the supernatants were measured as described under “Materials and Methods.” Data are expressed as the mean ± S.D. of three different experiments. The concentrations of different compounds were as follows: IL-1β, 10 ng/ml; IFN-γ, 10 units/ml; arginase, 100 units/ml; l-NMA, 0.1 mm;d-NMA, 0.1 mm; gemfibrozil, 200 μm.a p < 0.001 versusIL-1β + IFN-γ. Open table in a new tab U373MG glial cells preincubated in serum-free DMEM/F-12 for 1 h with arginase, l-NMA or d-NMA received the combination of IL-1β and IFN-γ. After 24 h of incubation, nitrite concentrations in the supernatants were measured as described under “Materials and Methods.” Data are expressed as the mean ± S.D. of three different experiments. The concentrations of different compounds were as follows: IL-1β, 10 ng/ml; IFN-γ, 10 units/ml; arginase, 100 units/ml; l-NMA, 0.1 mm;d-NMA, 0.1 mm; gemfibrozil, 200 μm. To determine whether inhibition of cytokine-induced NO production by gemfibrozil was simply due to delayed induction, we measured NO concentrations in cytokine-stimulated cultures maintained up to 48 h. When cells were stimulated in the absence of gemfibrozil, NO was detected in culture supernatants after 8 h and increased progressively thereafter for 48 h, the duration of the experiment (Fig. 1). However, when 200 μm gemfibrozil was added 2 h before the addition of the combination of IL-1β and IFN-γ, production of NO was significantly inhibited (Fig. 1). In our studies, maximal suppression of NO production was observed when gemfibrozil was added 2 h before the addition of cytokines (data not shown). When gemfibrozil was added after the addition of cytokines, the extent of inhibition progressively decreased (data not shown). It is evident from Fig.2 A that gemfibrozil dose-dependently inhibited cytokine-induced production of NO. Although at 50 μm concentration, gemfibrozil was not a potent inhibitor of cytokine-induced NO production, it inhibited the induction of NO production by more than 80% at 200 μmconcentration. To understand the mechanism of inhibition, we examined the effect of gemfibrozil on protein and mRNA level of iNOS in cytokine-stimulated cells. Consistent with the effect of gemfibrozil on cytokine-induced production of NO, gemfibrozil dose-dependently inhibited cytokine-induced expression of iNOS protein (Fig. 2 B) and mRNA (Fig.2 C).Figure 2Gemfibrozil dose-dependently inhibits the expression of iNOS in cytokine-stimulated human U373MG astroglial cells. Cells preincubated with different concentrations of gemfibrozil for 2 h in serum-free DMEM/F-12 received the combination of IL-1β (10 ng/ml) and IFN-γ (10 units/ml).A, after 24 h of stimulation, the concentration of nitrite was measured in the supernatants. Data are mean ± S.D. of three different experiments. a,p < 0.001 versuscontrol; b, p < 0.005 versusIL-1β+IFN-γ; c, p < 0.001versus IL-1β+IFN-γ. B, cell homogenates were immunoblotted with antibodies against mouse macrophage iNOS as described under “Materials and Methods.” C, after 6 h of stimulation, total RNA was isolated, and Northern blot analysis for iNOS mRNA was carried out as described under “Materials and Methods.”View Large Image Figure ViewerDownload (PPT) Next we investigated the possibility whether gemfibrozil inhibited cytokine-induced expression of iNOS mRNA by decreasing the stability of iNOS mRNA. Human U373MG astroglial cells were stimulated with the combination of IL-1β and IFN-γ under serum-free condition. After 6 h of stimulation, cells were treated with actinomycin D (an inhibitor of RNA synthesis) in the presence or absence of 200 μm of gemfibrozil. At different h of treatment with actinomycin D, the level of iNOS mRNA was analyzed by Northern blot. It is apparent from figure 3, A and B, that the relative rate of degradation of iNOS mRNA (iNOS/GAPDH) in the presence or absence of gemfibrozil at different time periods remained almost same suggesting that gemfibrozil-mediated inhibition of iNOS mRNA is not due to any alteration of the stability of iNOS mRNA. To investigate whether other fibrate drugs are also capable of inhibiting cytokine-induced production of NO and expression of iNOS in astrocytes, we examined the effect of clofibrate. Clofibrate is also a hypolipidemic drug that activates PPAR-α and induces proliferation of peroxisomes in rats and mice (9Lemberger T. Desvergne B. Wahli W. Annu. Rev. Cell Dev. Biol. 1996; 12: 335-363Crossref PubMed Scopus (640) Google Scholar, 20Kliewer S.A. Forman B.M. Blumberg B. Ong E.S. Borgmeyer U. Mangelsdorf D.J. Umesono K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7355-7359Crossref PubMed Scopus (1281) Google Scholar, 21Illingworth D.R. Bacon S. Arteriosclerosis. 1989; 9: I121-I134PubMed Google Scholar). Similar to gemfibrozil, clofibrate itself was neither stimulatory nor much inhibitory to NO production; however, it dose-dependently inhibited the production of NO (Fig.4 A) and the expression of iNOS protein (Fig. 4 B) in cytokine-stimulated U373MG astroglial cells. These studies suggest that fibrate drugs, in general, are inhibitory to cytokine-induced expression of iNOS in human astroglial cells. Human primary astrocytes have been shown to induce the expression of iNOS in the presence of different proinflammatory cytokines (22Pahan K. Liu X. McKinney M. Wood C. Sheikh F.G. Raymond J.R. J. Neurochem. 2000; 74: 2288-2295Crossref PubMed Scopus (85) Google Scholar, 23Pahan K. Liu X. Wood C. Raymond J.R. FEBS Lett. 2000; 472: 203-207Crossref PubMed Scopus (32) Google Scholar, 24Zhao M.L. Liu J.S.H. He D.K. Dickson D.W. Lee S.C. Brain Res. 1998; 813: 402-405Crossref PubMed Scopus (66) Google Scholar). Since gemfibrozil potently inhibited the expression of iNOS in human U373MG astroglial cells, we examined the effect of gemfibrozil on cytokine-induced expression of iNOS in human primary astrocytes. Different cytokines alone were poor inducers of NO production (Table II). However, the combination of IL-1β and IFN-γ markedly induced the production of NO. The addition of TNF-α to the combination of IL-1β and IFN-γ did not further increase the production of NO (Table II). Although gemfibrozil itself had no effect on NO production in control cells, preincubation of human primary astrocytes with 200 μm of gemfibrozil for 2 h markedly inhibited cytokine-induced production of NO (Table II).Table IIGemfibrozil inhibits the induction of NO production in human primary astrocytesTreatmentsNitrite productionμg/mg protein/24 hControl5.3 ± 0.7IL-1β24.5 ± 2.5IFN-γ6.1 ± 0.7TNF-α5.8 ± 0.8Gemfibrozil5.1 ± 0.6IL-1β + IFN-γ215.3 ± 22.3IL-1β + IFN-γ + TNF-α219.2 ± 28.5IL-1β + IFN-γ + gemfibrozil24.8 ± 2.8ap < 0.001 versusIL-1β + IFN-γ.IL-1β + IFN-γ + TNF-α + gemfibrozil25.7 ± 3.1bp < 0.001 versus IL-1β + IFN-γ + TNF-α.Human primary astrocytes preincubated in serum-free DMEM/F-12 for 2 h with 200 μm gemfibrozil, received IL-1β, IFN-γ, and TNF-α alone or in different combinations. After 24 h of incubation, nitrite concentrations in the supernatants. Data are expressed as the mean ± S.D. of three different experiments. The concentrations of different cytokines were as follows: IL-1β, 10 ng/ml; IFN-γ, 10 units/ml; TNF-α, 10 ng/ml.a p < 0.001 versusIL-1β + IFN-γ.b p < 0.001 versus IL-1β + IFN-γ + TNF-α. Open table in a new tab Human primary astrocytes preincubated in serum-free DMEM/F-12 for 2 h with 200 μm gemfibrozil, received IL-1β, IFN-γ, and TNF-α alone or in different combinations. After 24 h of incubation, nitrite concentrations in the supernatants. Data are expressed as the mean ± S.D. of three different experiments. The concentrations of different cytokines were as follows: IL-1β, 10 ng/ml; IFN-γ, 10 units/ml; TNF-α, 10 ng/ml. To understand the effect of gemfibrozil on the transcription of iNOS gene, U373MG glial cells were transfected with phiNOS(7.2)Luc, a construct containing the human iNOS promoter fused to the luciferase gene (19Taylor B.S. de Vera M.E. Ganster R.W. Wang Q. Shapiro R.A. Morris S.M. Billiar T.R. Geller D.A. J. Biol. Chem. 1998; 273: 15148-15156Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar), and activation of this promoter was measured after stimulating the cells with cytokines in the presence or absence of gemfibrozil. The combination of IL-1β and IFN-γ induced iNOS