Nitric Oxide Inhibits Macrophage-Colony Stimulating Factor Gene Transcription in Vascular Endothelial Cells

Macrophage-colony stimulating factor (M-CSF) contributes to atherogenesis by regulating macrophage-derived foam cells in atherosclerotic lesions. Here we report that nitric oxide (NO) inhibits the expression of M-CSF in human vascular endothelial cells independent of guanylyl cyclase activation. The induction of M-CSF mRNA expression by either oxidized low density lipoprotein (ox-LDL) or tumor necrosis factor-α (TNFα) was attenuated by NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP), and 3-morpholinosydnonimine, but not by cGMP analogues, glutathione, or nitrite. Inhibition of endogenous NO production by N-monomethyl-L-arginine (L-NMA) also increased M-CSF expression in control and TNFα-stimulated cells. Nuclear run-on assays and transfection studies using M-CSF promoter constructs linked to chloramphenicol acetyltransferase reporter gene indicated that NO repressed M-CSF gene transcription through nuclear factor-κB (NF-κB). Electrophoretic mobility shift assays demonstrated that activation of NF-κB by L-NMA, ox-LDL, and TNFα was attenuated by GSNO and SNP, but not by glutathione or cGMP analogues. Since the induction of M-CSF expression depends upon NF-κB activation, the ability of NO to inhibit NF-κB activation and M-CSF expression may contribute to some of NO's antiatherogenic properties. Macrophage-colony stimulating factor (M-CSF) contributes to atherogenesis by regulating macrophage-derived foam cells in atherosclerotic lesions. Here we report that nitric oxide (NO) inhibits the expression of M-CSF in human vascular endothelial cells independent of guanylyl cyclase activation. The induction of M-CSF mRNA expression by either oxidized low density lipoprotein (ox-LDL) or tumor necrosis factor-α (TNFα) was attenuated by NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP), and 3-morpholinosydnonimine, but not by cGMP analogues, glutathione, or nitrite. Inhibition of endogenous NO production by N-monomethyl-L-arginine (L-NMA) also increased M-CSF expression in control and TNFα-stimulated cells. Nuclear run-on assays and transfection studies using M-CSF promoter constructs linked to chloramphenicol acetyltransferase reporter gene indicated that NO repressed M-CSF gene transcription through nuclear factor-κB (NF-κB). Electrophoretic mobility shift assays demonstrated that activation of NF-κB by L-NMA, ox-LDL, and TNFα was attenuated by GSNO and SNP, but not by glutathione or cGMP analogues. Since the induction of M-CSF expression depends upon NF-κB activation, the ability of NO to inhibit NF-κB activation and M-CSF expression may contribute to some of NO's antiatherogenic properties. The 1The abbreviations used are: M-CSFmacrophage colony stimulating factorNOnitric oxideGSNOS-nitrosoglutathioneTNFαtumor necrosis factor αox-LDLoxidized low density lipoproteinRSVRous sarcoma virusTBARSthiobarbituric acid reactive substancesCATchloramphenicol acetyltransferaseSNPsodium nitroprussideL-NMAN-monomethyl-L-arginine. 1The abbreviations used are: M-CSFmacrophage colony stimulating factorNOnitric oxideGSNOS-nitrosoglutathioneTNFαtumor necrosis factor αox-LDLoxidized low density lipoproteinRSVRous sarcoma virusTBARSthiobarbituric acid reactive substancesCATchloramphenicol acetyltransferaseSNPsodium nitroprussideL-NMAN-monomethyl-L-arginine. activation of mononuclear phagocytes in the vessel wall is an important event in atherogenesis(1Libby P. Clinton S.K. Curr. Opin. Lipidol. 1993; 4: 355-363Crossref Scopus (124) Google Scholar). Macrophage-colony stimulating factor (M-CSF)1 regulates macrophage growth (2Stanley E.R. Chen D.M. Lin H.S. Nature. 1978; 274: 168-170Crossref PubMed Scopus (151) Google Scholar) and differentiation (3Munn D.H. Armstrong E. Cancer Res. 1993; 53: 2603-2613PubMed Google Scholar) and may contribute to the development of macrophage-derived foam cells in atherosclerotic lesions (4). Expression of M-CSF in vascular endothelial cells is induced by minimally modified low density lipoprotein (LDL) (5Rajavashisth T.B. Andalibi A. Territo M.C. Berliner J.A. Navab M. Fogelman A.M. Lusis A.J. Nature. 1990; 344: 254-257Crossref PubMed Scopus (608) Google Scholar) and various cytokines such as interleukin-1 and TNFα(6Clinton S.K. Underwood R. Hayes L. Sherman M.L. Kufe D.W. Libby P. Am. J. Pathol. 1992; 140: 301-316PubMed Google Scholar). Atherosclerotic lesions contain both oxidized lipids (7Ylä-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. J. Clin. Invest. 1989; 84: 1086-1095Crossref PubMed Google Scholar) and inflammatory cytokines (8Tipping P.G. Hancock W.W. Am. J. Pathol. 1993; 142: 1721-1728PubMed Google Scholar) which may induce the local expression of M-CSF. Indeed, human and rabbit atherosclerotic lesions contain increased levels of M-CSF compared to normal arterial tissues(5Rajavashisth T.B. Andalibi A. Territo M.C. Berliner J.A. Navab M. Fogelman A.M. Lusis A.J. Nature. 1990; 344: 254-257Crossref PubMed Scopus (608) Google Scholar, 6Clinton S.K. Underwood R. Hayes L. Sherman M.L. Kufe D.W. Libby P. Am. J. Pathol. 1992; 140: 301-316PubMed Google Scholar, 7Ylä-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. J. Clin. Invest. 1989; 84: 1086-1095Crossref PubMed Google Scholar). Consequently, factors which regulate the expression of M-CSF may modulate atherogenesis. macrophage colony stimulating factor nitric oxide S-nitrosoglutathione tumor necrosis factor α oxidized low density lipoprotein Rous sarcoma virus thiobarbituric acid reactive substances chloramphenicol acetyltransferase sodium nitroprusside N-monomethyl-L-arginine. macrophage colony stimulating factor nitric oxide S-nitrosoglutathione tumor necrosis factor α oxidized low density lipoprotein Rous sarcoma virus thiobarbituric acid reactive substances chloramphenicol acetyltransferase sodium nitroprusside N-monomethyl-L-arginine. Nitric oxide exerts many antiatherogenic actions via stimulation of guanylyl cyclase activity(9Garg U.C. Hassid A. J. Clin. Invest. 1989; 83: 1774-1777Crossref PubMed Scopus (1991) Google Scholar, 10Radomski M.W. Palmer R.M. Moncada S. Br. J. Pharmacol. 1987; 92: 639-646Crossref PubMed Scopus (1006) Google Scholar, 11Bath P.M. Hassall D.G. Gladwin A.M. Palmer R.M. Martin J.F. Arterioscler. Thromb. 1991; 11: 254-260Crossref PubMed Scopus (300) Google Scholar). Abnormal endothelial-derived nitric oxide activity contributes to impaired vascular responses in atherosclerotic vessels of humans and animals(12Bossaler C. Habib G.B. Yamamoto H. Williams C. Wells S. Henry P.D. J. Clin. Invest. 1987; 79: 174-179Crossref Scopus (430) Google Scholar, 13Tanner F.C. Noll G. Boulanger C.M. Lüscher T.F. Circulation. 1991; 83: 2012-2020Crossref PubMed Scopus (303) Google Scholar). Inhibition of endogenous NO production by Nω-nitro-L-arginine methyl ester promotes vasoconstriction and endothelial-leukocyte adhesion, processes which are mitigated, to some extent, by addition of cGMP analogues(14Kubes P. Suzuki M. Granger D.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4651-4655Crossref PubMed Scopus (2808) Google Scholar, 15Kurose I. Kubes P. Wolf R. Anderson D.C. Paulson J. Miyasaka M. Granger D.N. Circ. Res. 1993; 73: 164-171Crossref PubMed Scopus (337) Google Scholar). Furthermore, enriching the diets of cholesterol-fed rabbits with L-arginine, the precursor of NO, improves endothelial-dependent relaxation, reduces leukocyte attachment to the endothelial surface, and limits the extent of atherosclerotic lesions(16Cooke J.P. Singer A.H. Tsao P. Zera P. Rowan R.A. Billingham M.E. J. Clin. Invest. 1992; 90: 1168-1172Crossref PubMed Scopus (635) Google Scholar). Although many effects of nitric oxide are attributed to its stimulation of guanylyl cyclase, little is known regarding other cellular pathway(s) mediated by nitric oxide. The findings of our recent study indicate that the regulation of endothelial vascular cell adhesion molecule-1 expression by NO is not mediated by cGMP, but rather is associated with the inhibition of nuclear binding protein, NF-κB(17De Caterina R. Libby P. Peng H.-B. Thannickal V.J. Rajavashisth T.B. Gimbrone M.A. Shin W.S. Liao J.K. J. Clin. Invest. 1995; 96: 60-68Crossref PubMed Scopus (1573) Google Scholar). The induction of various inflammatory cytokines important in atherogenesis requires activation of NF-κB(18Libermann T.A. Baltimore D. Mol. Cell. Biol. 1990; 10: 2327-2334Crossref PubMed Google Scholar, 19Kunsch R. Lang R.K. Rosen C.A. Shannon M.F. J. Immunol. 1994; 153: 153-164PubMed Google Scholar, 20Neish A.S. Williams A.J. Palmer H.J. Whitley M.Z. Collins T. J. Exp. Med. 1992; 176: 1583-1593Crossref PubMed Scopus (384) Google Scholar). NF-κB was originally described as a heterodimeric cytosolic protein in B-cells which, upon activation, translocated into the nucleus where it binds to specific decameric sequences in the IgG κ light chain enhancer(21Sen R. Baltimore D. Cell. 1986; 46: 705-716Abstract Full Text PDF PubMed Scopus (1924) Google Scholar). 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Since cellular adhesion molecules and proinflammatory cytokines participate in atherogenesis and share common κB binding motifs in their transcriptional promoters, we hypothesized that NO may regulate their gene expression through NF-κB. This study, therefore, tested whether NO could regulate the expression of an important proatherogenic molecule, M-CSF, through NF-κB. All standard culture reagents were obtained from JRH Bioscience (Lenexa, KS). Glutathione, nitrite, sodium nitroprusside, dimethyl sulfoxide, dithiothreitol, L-arginine, heparin sulfate, cupric sulfate (CuSO4), polymyxin B, butylated hydroxytoluene, thiobarbituric acid, and 1,1,3,3-tetramethoxypropane, phenylmethylsulfonyl fluoride, and cGMP analogues, 8-bromo-cGMP and dibutyryl cGMP, were purchased from Sigma. GSNO was synthesized from glutathione and nitrite as described previously(26Kowaluk E.A. Fung H.-L. J. Pharmacol. Exp. Ther. 1990; 255: 1256-1264PubMed Google Scholar). Purified human low density lipoprotein (LDL, Lot No. 730793) and N-monomethyl-L-arginine (L-NMA) were obtained from Calbiochem. The Limulus amebocyte lysate kinetic assay was performed by BioWhittaker (Walkersville, MD). Recombinant human TNFα was purchased from Endogen, Inc. (Boston, MA). [α-32P]CTP (3000 Ci/mmol), [γ-32P]ATP (3000 Ci/mmol), 32Pi (1000 Ci/mmol), and [3H]chloramphenicol (37 Ci/mmol) were supplied by DuPont NEN. The oligonucleotide corresponding to the two tandem κB sequences in the M-CSF promoter was synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX). Rabbit polyclonal antisera to NF-κB subunits, p65 and p50, were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Nylon transfer membranes were purchased from Schleicher and Schuell. The expression vectors containing the RSV promoter linked to NF-κB subunits, p65 and p50, were kindly provided by G. Nabel (University of Michigan). The human M-CSF promoter constructs linked to the chloramphenicol acetyltransferase (CAT) reporter gene were generously provided by D. Kufe (Dana Farber Cancer Institute, Boston, MA). Human saphenous vein and bovine aortic endothelial cells were cultured and characterized as described previously(27Liao J.K. Shin W.S. Lee W.Y. Clark S.L. J. Biol. Chem. 1995; 270: 319-324Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar). Only endothelial cells of less than three passages were used. Cells were pretreated with NO donors for 30 min prior to addition of LDL or TNFα. Cellular viability was determined by morphology and trypan blue exclusion. Native LDL (density 1.02-1.06 g/ml) from a single donor was isolated using a sequential ultracentrifugation method in the presence of butylated hydroxytoluene and polymyxin B as described previously(28Liao J.K. J. Biol. Chem. 1994; 269: 12987-12992Abstract Full Text PDF PubMed Google Scholar). Its identity was confirmed by SDS-polyacrylamide gel electrophoresis. Cholesterol, triglyceride, and protein content were determined as described previously(27Liao J.K. Shin W.S. Lee W.Y. Clark S.L. J. Biol. Chem. 1995; 270: 319-324Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar). Oxidized LDL (80% lipid, 20% protein) was prepared by exposing samples of native LDL to CuSO4 (5 μM) at 37°C for 2 to 24 h. Both native and oxidized LDL were dialyzed with three changes of sterile buffer (150 mM NaCl, 0.01% EDTA, and 100 μg/ml polymyxin B, pH 7.4) before filtering through a 0.2-μm membrane. The degree of LDL oxidation was estimated by measuring the amounts of thiobarbituric acid reactive substances (TBARS) produced using a colorimetric assay standardized with malondialdehyde(29Yagi K. Biochem. Med. 1976; 15: 212-216Crossref PubMed Scopus (2044) Google Scholar). The TBARS value is expressed as nanomoles of malondialdehyde per mg of LDL protein. was using and by ultracentrifugation amounts of were by gel by and for 2 prior to of a human M-CSF was performed using with [α-32P]CTP and a of The membranes and were at in a buffer containing mM sodium buffer and μg/ml and in at before at for h. All were with as an control MD). endothelial cells (5 were with 32Pi for prior to the addition of 8-bromo-cGMP at the indicated and for an h. The study was by the addition of sodium acid and sodium Cells were and by a from cellular were determined by the method of J. Biol. Chem. Full Text PDF PubMed Google Scholar). were in buffer containing pH and and at for The and known were by SDS-polyacrylamide gel The were with and acid and by a gel before at for h. from endothelial cells were and in transcription with was performed as J.K. Shin W.S. Lee W.Y. Clark S.L. J. Biol. Chem. 1995; 270: 319-324Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar). containing M-CSF, and were membranes using a and the membranes were to at for in a buffer containing mM sodium buffer and μg/ml The membranes were with for at before for at bovine rather than human endothelial cells were used of their by the method M-CSF promoter and linked to the chloramphenicol acetyltransferase (CAT) gene were H. S. M. Kufe D. 1991; PubMed Google Scholar). Cells were with the indicated promoter constructs or cells were with or TNFα studies with and GSNO was and were and GSNO was h. an control for transfection was in cellular were prepared using buffer μg/ml μg/ml mM phenylmethylsulfonyl fluoride, mM EDTA, mM 100 mM NaCl, mM pH 7.4) and activity was determined by the cellular with [3H]chloramphenicol and for at 37°C as described PubMed Scopus Google Scholar). The activity was as the of to M-CSF promoter activity was expressed as the of activity to the activity of was performed three in Nuclear were prepared as R.M. Res. 11: PubMed Scopus Google Scholar). The NF-κB oligonucleotide corresponding to the two tandem κB in the M-CSF promoter was with [γ-32P]ATP and and by Nuclear were to NF-κB oligonucleotide in buffer containing 2 of of bovine mM mM NaCl, mM dithiothreitol, mM EDTA, and were gel at for in low buffer at the indicated was to the nuclear for min before addition of In some GSNO or NF-κB oligonucleotide was to the nuclear min prior to addition of from and in transcription assay were by the of W. of Scholar). All are expressed as compared to and and were to the of changes in activity and was for of less than were effects of oxidized or cellular and cellular and viability were for LDL L-NMA, and 8-bromo-cGMP levels of The native LDL was of protein and The TBARS value was which increased to and and 24 of with endothelial cells in LDL samples which have oxidized by to CuSO4 (5 μM) TBARS from 2 to 24 h. that TNFα induced the mRNA expression of M-CSF in a with induction 2 and TNFα stimulation oxidized LDL TBARS also induced the mRNA expression of M-CSF, compared to that of TNFα induction was and at 24 The induction of M-CSF upon the degree of LDL oxidation as by the presence of thiobarbituric acid reactive substances (TBARS) to oxidized LDL to M-CSF expression than native LDL or oxidized LDL of the effects of native TBARS and oxidized LDL M-CSF mRNA expression at 24 with to The of TNFα M-CSF mRNA expression at is shown for for was by to were performed The induction of M-CSF mRNA expression by oxidized LDL TBARS or TNFα was attenuated by S-nitrosoglutathione in a other NO donors, sodium nitroprusside and 3-morpholinosydnonimine, but not glutathione or nitrite, M-CSF mRNA expression induced by oxidized LDL TBARS that this was to NO Inhibition of endogenous NO production by a in M-CSF mRNA expression compared to levels of to TNFα-stimulated cells M-CSF mRNA expression by compared to that of TNFα stimulation GSNO was in both and TNFα-stimulated M-CSF mRNA expression by and of control and TNFα-stimulated endothelial cells with the cGMP 8-bromo-cGMP M-CSF mRNA expression minimally that the activation of endothelial guanylyl cyclase not this of The cGMP protein activity in a as demonstrated by in protein in endothelial cells In the presence of GSNO not the of M-CSF mRNA induced by TNFα The of M-CSF mRNA in the presence and of GSNO was not In transcription studies a transcriptional activity of the M-CSF gene standard culture with oxidized LDL TBARS or TNFα increased M-CSF gene transcription and to gene NO transcriptional activity of the M-CSF gene induced by oxidized LDL or but not gene studies using amounts of that our was and The density of M-CSF was standardized to the density of its corresponding The of was determined by the of to the the effects of NO M-CSF gene we bovine aortic endothelial cells using two M-CSF promoter and linked to the chloramphenicol acetyltransferase (CAT) reporter H. S. M. Kufe D. 1991; PubMed Google Scholar). of the M-CSF promoter binding sequences for and and promoter two tandem κB the promoter κB with TNFα or oxidized LDL TBARS increased promoter activity by and of with the expression in a induction in promoter activity compared to a induction with a of and and induction with with GSNO was in promoter activity induced by TNFα oxidized LDL and with p65 or in with p50 promoter activity was than that of with with produced promoter with TNFα or oxidized LDL produced a induction of the containing promoter GSNO not promoter activity induced by ox-LDL, or with was not to GSNO not and or the promoter Electrophoretic mobility shift assays demonstrated and activation of NF-κB by TNFα 30 min In the degree of NF-κB activation by native LDL TBARS was compared to TNFα and the TBARS value was to endothelial cell of native of NF-κB by oxidized LDL TBARS in a that of TNFα 30 GSNO and sodium nitroprusside attenuated the activation of NF-κB by both oxidized LDL and TNFα of 8-bromo-cGMP not activation of NF-κB that NO's was not to guanylyl cyclase activation. GSNO was to cells rather than to nuclear that NO not with NF-κB binding to of NF-κB was also endogenous NO production was by produced NF-κB activation to a extent than TNFα. studies indicate that with NO activity by not Although the of NF-κB activation by was less compared to that by NF-κB activation was by with GSNO The indicated was specific for NF-κB in the presence of to p50 and p65, this was and have shown that both endogenous and nitric oxide (NO) can the expression of a proatherogenic stimulating factor (M-CSF) induced by two TNFα and oxidized Since M-CSF may contribute to the development of macrophage-derived foam M.E. S. Witztum J.L. Steinberg D. Am. J. Pathol. 1992; 140: Google Scholar, 6Clinton S.K. Underwood R. Hayes L. Sherman M.L. Kufe D.W. Libby P. Am. J. Pathol. 1992; 140: 301-316PubMed Google inhibition of M-CSF expression by NO may by which NO can atherogenesis. NO's M-CSF mRNA expression was not mediated by stimulation of guanylyl cyclase cGMP not inhibit M-CSF This is in to other antiatherogenic effects of NO which are mediated by cGMP such as vascular C. Habib G.B. Yamamoto H. Williams C. Wells S. Henry P.D. J. Clin. Invest. 1987; 79: 174-179Crossref Scopus (430) Google Scholar) and inhibition of M.W. Palmer R.M. Moncada S. Br. J. Pharmacol. 1987; 92: 639-646Crossref PubMed Scopus (1006) Google Scholar). our findings a by which NO can modulate the expression of an important M-CSF. studies and nuclear run-on assays indicated that the regulation of M-CSF expression at the of M-CSF gene of the M-CSF promoter that two tandem κB binding from the were for transcriptional induction by TNFα and oxidized with studies the of nuclear binding protein NF-κB in transcriptionally the M-CSF H. S. M. Kufe D. 1991; PubMed Google Scholar). the the κB promoter activity in to TNFα and oxidized LDL that other binding could also participate and in the M-CSF Electrophoretic mobility shift assays demonstrated that the transcriptional of the M-CSF gene by NO was to the inhibition of NF-κB activation. NO not inhibit the binding of NF-κB to its the addition of NO to nuclear of TNFα-stimulated cells not the activation of NF-κB. cellular in the cell which NO's NF-κB activation. with p65 in a in M-CSF promoter activity compared to that with the of p65 and Since the p50 can bind κB but is a H.-B. Nabel K. A.S. Collins T. Nabel Mol. Cell. Biol. 1993; PubMed Google with p65 and p50 to a the p65 with the and the p50 for κB in less promoter activity compared to that of the p65 Furthermore, the ability of NO to M-CSF promoter activity in cells with either p65 or in with p50 that NO's effects are mediated through NF-κB transcriptional activation of the M-CSF gene in to H. S. M. 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A. 1990; PubMed Scopus Google Scholar). in the local of an inflammatory atherosclerotic NO can at its and the of endothelial cells to and vascular cells less to findings a antiatherogenic of NO which is independent of its guanylyl Although we report the effects of NO NF-κB activation and M-CSF effects may to other inflammatory cytokines and adhesion molecules which contain κB in their transcriptional that NO is an important of both and G. Nabel for and expression vectors and H. and D. Kufe for M-CSF promoter