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W2092699086 abstract "Previously, we showed that macrophages (MØ) from old mice have significantly higher levels of lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) production than young mice, due to increased cyclooxygenase-2 (COX-2) mRNA levels. The aim of the current study was to determine the underlying mechanisms of age-associated increase in COX-2 gene expression. The results demonstrate that increased COX-2 mRNA expression in the old mice is due to a higher rate of transcription rather than increased stability of COX-2 mRNA. Furthermore, the results show that LPS-induced ceramide levels from the old mice are significantly higher than those of young mice, whereas there is no age-related difference in concentration of its down stream metabolite, sphingosine. The addition of ceramide in the presence or absence of LPS resulted in a significant increase in PGE2 production in a dose- and time-dependent manner. Inhibition of ceramide conversion to sphingosine had no effect on this ceramide-induced effect. The ceramide-induced up-regulation in PGE2 production was mediated through increase in COX activity and transcriptional up-regulation of COX-2 mRNA. Collectively, these data suggest that the age-associated increase in MØ COX-2 mRNA is due to transcriptional up-regulation. Furthermore, this increase in transcription is mediated by higher cellular ceramide concentration in old MØ compared with that of young MØ. Previously, we showed that macrophages (MØ) from old mice have significantly higher levels of lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) production than young mice, due to increased cyclooxygenase-2 (COX-2) mRNA levels. The aim of the current study was to determine the underlying mechanisms of age-associated increase in COX-2 gene expression. The results demonstrate that increased COX-2 mRNA expression in the old mice is due to a higher rate of transcription rather than increased stability of COX-2 mRNA. Furthermore, the results show that LPS-induced ceramide levels from the old mice are significantly higher than those of young mice, whereas there is no age-related difference in concentration of its down stream metabolite, sphingosine. The addition of ceramide in the presence or absence of LPS resulted in a significant increase in PGE2 production in a dose- and time-dependent manner. Inhibition of ceramide conversion to sphingosine had no effect on this ceramide-induced effect. The ceramide-induced up-regulation in PGE2 production was mediated through increase in COX activity and transcriptional up-regulation of COX-2 mRNA. Collectively, these data suggest that the age-associated increase in MØ COX-2 mRNA is due to transcriptional up-regulation. Furthermore, this increase in transcription is mediated by higher cellular ceramide concentration in old MØ compared with that of young MØ. macrophage(s) prostaglandin E2 lipopolysaccharide cyclooxygenase sphingomyelinase interleukin mitogen-activated protein kinase extracellular signal-regulated kinase c-Jun N-terminal kinase(s) heteronuclear RNA reverse transcriptase-PCR high performance liquid chromatography radioimmune assay d-erythro-2-(N-myristoylamino)-1- phenyl-1-propanol Accumulating evidence indicates that T cell-mediated immune responses decline with aging (1Bryl E. Gazda M. Foerster J. Witkowski J.M. Blood. 2001; 98: 1100-1107Crossref PubMed Scopus (31) Google Scholar, 2Ginaldi L. DeMartinis M. D'Ostilio A. Marini L. Loreto F. Modesti M. Quaglino D. Am. J. Hematol. 2001; 67: 63-72Crossref PubMed Scopus (48) Google Scholar, 3Hsu H.C. Zhou T. Shi J. Yang P.A. Liu D. Zhang H.G. Bluethmann H. Mountz J.D. Mech. Ageing Dev. 2001; 122: 305-326Crossref PubMed Scopus (16) Google Scholar, 4Pahlavani M.A. Vargas D.A. FEBS Lett. 2001; 491: 114-118Crossref PubMed Scopus (24) Google Scholar, 5Ginaldi L. DeMartinis M. Modesti M. Loreto F. Corsi M.P. Quaglino D. Gerontology. 2000; 46: 242-248Crossref PubMed Scopus (45) Google Scholar, 6Fagnoni F.F. Vescovini R. Passeri G. Bologna G. Pedrazzoni M. Lavaghetto G. Casti A. Franceschi C. Passeri M. Sansoni P. Blood. 2000; 95: 2860-2868Crossref PubMed Google Scholar). We, as well as others, have demonstrated that, in addition to intrinsic changes in T cells, increased macrophage (MØ)1-derived prostaglandin E2 (PGE2) production contributes to the age-associated decline in T cell function (7Beharka A.A. Dayong W. Han S.N. Meydani S.N. Mech. Ageing Dev. 1997; 93: 59-77Crossref PubMed Scopus (123) Google Scholar). We further showed that the age-related increase in lipopolysaccharide (LPS)-induced MØ PGE2 production is due to increased cyclooxygenase-2 (COX-2) mRNA and protein levels, leading to increased COX enzyme activity (8Hayek M.G. Mura C., Wu, D. Beharka A.A. Han S.N. Paulson K.E. Hwang D. Meydani S.N. J. Immunol. 1997; 159: 2445-2451PubMed Google Scholar). PGE2 is synthesized from the precursor arachidonic acid by the two cyclooxygenase isoenzymes: cyclooxygenase 1 (COX-1), which is a ubiquitously expressed enzyme, and COX-2, which has a low basal expression but is rapidly induced by inflammatory stimuli such as LPS.Ceramide, a sphingolipid second messenger, generated from hydrolysis of membrane sphingomyelin by sphingomyelinase (SMase) or by de novo synthesis (9Vesper H. Schmelz E.M. Nikolova-Karakashian M.N. Dillehay D.L. Lynch D.V. Merrill A.H.J. J. Nutri. 1999; 129: 1239-1250Crossref PubMed Scopus (355) Google Scholar) has been shown to be involved in multiple signaling pathways, resulting in proliferation (10Pahlavani M.A. Vargas D.A. Immunopharmacology. 2000; 49: 345-354Crossref PubMed Scopus (3) Google Scholar, 11Marchell N.L. Uchida Y. Brown B.E. Elias P.M. Holleran W.M. J. Invest. Dermatol. 1998; 110: 383-387Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), differentiation (12Schneider C. Delorme N. Buisson-Legendre N. Bellon G. Emonard H. Btaouri H.E. Hornebeck W. Haye B. Martiny L. Lipids. 2000; 35: 1259-1268Crossref PubMed Scopus (5) Google Scholar), and apoptosis (13Herget T. Esdar C. Oehrlein S.A. Heinrich M. Schutze S. Maelicke A. van Echten-Deckert G. J. Biol. Chem. 2000; 275: 30344-30354Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 14Willaime S. Vanhoutte P. Caboche J. Lemaigre-Dubreuil Y. Mariani J. Brugg B. Eur. J. Neurosci. 2001; 13: 2037-2046Crossref PubMed Google Scholar, 15Selzner M. Bielawska A. Morse M.A. Rudiger H.A. Sindram D. Hannun Y.A. Clavien P.A. Cancer Res. 2001; 61: 1233-1240PubMed Google Scholar, 16Sugiki H. Hozumi Y. Maeshima H. Katagata Y. Mitsuhashi Y. Kondo S. Br. J. Dermatol. 2000; 143: 1154-1163Crossref PubMed Scopus (26) Google Scholar). In murine MØ, cell-permeable analogues of ceramide (C2- or C6-ceramide) and SMase have been shown to mimic LPS action (17Joseph C.K. Wright S.D. Bornmann W.G. Randolph J.T. Kumar E.R. Bittman R. Liu J. Kolesnick R.N. J. Biol. Chem. 1994; 269: 17606-17610Abstract Full Text PDF PubMed Google Scholar). Furthermore, ceramide increases expression of LPS-inducible genes in MØ from LPS-responsive, but not LPS-hyporesponsive mice (18Barber S.A. Perera P.-Y. Vogl S.N. J. Immunol. 1995; 155: 2303-2305PubMed Google Scholar). In several studies, LPS injection (19Haimovitz-Friedman A. Cordon-Cardo C. Bayoumy S. Garzotto M. McLoughlin M. Gallily R. Edwards C.K.R. Schuchman E.H. Fuks Z. Kolesnick R. J. Exp. Med. 1997; 186: 1831-1841Crossref PubMed Scopus (379) Google Scholar, 20Zimmermann C. Ginis I. Furuya K. Klimanis D. Ruetzler C. Spatz M. Hallenbeck J.M. Brain Res. 2001; 895: 59-65Crossref PubMed Scopus (78) Google Scholar, 21Memon R.A. Holleran W.M. Uchida Y. Moser A.H. Grunfeld C. Feingold K.R. J. Lipid Res. 2001; 42: 452-459Abstract Full Text Full Text PDF PubMed Google Scholar) as well as treatment of cultured cells with LPS (22MacKichan M.L. DeFranco A.L. J. Biol. Chem. 1999; 274: 1767-1775Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) resulted in increased intracellular ceramide generation.Exogenously added membrane-permeable analogues of ceramide (C2- and C6-ceramide) as well as bacterial SMase augmented IL-1-induced PGE2 production and COX-2 expression in human dermal fibroblast (23Ballou L.R. Chao C.P. Holness M.A. Barker S.C. Raghow R. J. Biol. Chem. 1992; 267: 20044-20050Abstract Full Text PDF PubMed Google Scholar) and granulose cell cultures (24Santana P. Lllanes L. Hernandez I. Gonzalez-Robayna I. Tabraue C. Gonzalez-Reyes J. Quintana J. Estevez F. Ruiz de Galarreta C.M. Fanjul L.F. Endocrinology. 1996; 137: 2480-2489Crossref PubMed Scopus (55) Google Scholar). Subsequent work showed that this effect was specific to COX-2 and not COX-1 (24Santana P. Lllanes L. Hernandez I. Gonzalez-Robayna I. Tabraue C. Gonzalez-Reyes J. Quintana J. Estevez F. Ruiz de Galarreta C.M. Fanjul L.F. Endocrinology. 1996; 137: 2480-2489Crossref PubMed Scopus (55) Google Scholar, 25Hayakawa M. Jayadev S. Tsujimoto M. Hannun Y.A. Ito F. Biochem. Biophys. Res. Commun. 1996; 220: 681-686Crossref PubMed Scopus (79) Google Scholar). In addition to ceramide, its metabolite, sphingosine (23Ballou L.R. Chao C.P. Holness M.A. Barker S.C. Raghow R. J. Biol. Chem. 1992; 267: 20044-20050Abstract Full Text PDF PubMed Google Scholar), has been shown to increase IL-1-induced PGE2 production. The effect of sphingosine, however, might be due to its retroconversion to ceramide (23Ballou L.R. Chao C.P. Holness M.A. Barker S.C. Raghow R. J. Biol. Chem. 1992; 267: 20044-20050Abstract Full Text PDF PubMed Google Scholar). Venable et al. (26Venable M.E. Lee J.Y. Smyth M.J. Bielawska A. Obeid L.M. J. Biol. Chem. 1995; 270: 30701-30708Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar) showed that senescent WI38 human fibroblast cells had higher ceramide levels and activity of neutral SMase. Age-related increases in brain and liver ceramide (27Palestini P. Masserini M. Fiorilli A. Calappi E. Tettamanti G. J. Neurochem. 1993; 61: 955-960Crossref PubMed Scopus (31) Google Scholar, 28Petkova D.H. Momchilova-Pankova A.B. Markovska T.T. Koumanov K.S. Exp. Gerontol. 1988; 23: 19-24Crossref PubMed Scopus (22) Google Scholar, 29Lightle S.A. Oakley J.I. Nikolova-Karakashian M.N. Mech. Ageing Dev. 2000; 120: 111-125Crossref PubMed Scopus (98) Google Scholar) and neutral SMase levels have also been reported. Since several lines of evidence indicated that LPS and ceramide share a common pathway(s) leading to induction of COX-2, we hypothesized that ceramide mediates the age-associated increase in COX-2 expression and PGE2 production.Recent studies have indicated that mitogen-activated protein kinase (MAPK) intermediates such as extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinases (JNK), and p38 (30Subbaramaiah K. Chung W.J. Dannenberg A.J. J. Biol. Chem. 1998; 273: 32943-32949Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) are involved in LPS- as well as ceramide-induced COX-2 gene expression. Moreover, several transcription factors, which bind COX-2 gene-regulatory regions such as AP-1, and cyclic AMP-response element-binding protein have been shown to be regulated by these MAPKs (30Subbaramaiah K. Chung W.J. Dannenberg A.J. J. Biol. Chem. 1998; 273: 32943-32949Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Thus, we further hypothesized that ceramide mediates the age-associated increase in MØ COX-2 mRNA expression via MAPKs.DISCUSSIONPGE2 is a proinflammatory eicosanoid, which has been indicated in pathogenesis of cardiovascular diseases, cancer, and inflammation. An age-related increase in PGE2 production has been demonstrated in animal models (8Hayek M.G. Mura C., Wu, D. Beharka A.A. Han S.N. Paulson K.E. Hwang D. Meydani S.N. J. Immunol. 1997; 159: 2445-2451PubMed Google Scholar, 41Shimizu N. Yamaguchi M. Uesu K. Goseki T. Abiko Y. J. Gerontol. A Biol. Sci. Med. Sci. 2000; 55: B489-B495Crossref PubMed Scopus (32) Google Scholar, 42Millatt L.J. Siragy H.M. Hypertension. 2000; 35: 643-647Crossref PubMed Scopus (9) Google Scholar) and in humans (43Schwab R. Hausman P.B. Rinnooy-Kan E. Weksler M.E. Immunology. 1985; 55: 677-684PubMed Google Scholar,44Wu D. Meydani M. Leka L.S. Nightingale Z. Handelman G. Blumberg J.B. Meydani S.N. Am. J. Clin. Nutr. 1999; 70: 536-543Crossref PubMed Scopus (90) Google Scholar). Previously we showed that the increase in MØ PGE2production with aging contributes to a decline in T cell-mediated immune function. Furthermore, we demonstrated that the age-associated increase in MØ PGE2 production is due to increased COX-2 mRNA and protein levels, leading to an increase in COX enzyme activity. Age-related increases in COX-2 gene have implications for many age-associated diseases such as cardiovascular, inflammatory, and neoplastic diseases. Thus, determining causative factors for the up-regulation of COX-2 gene expression is imperative. The aim of our current study was to determine the underlying molecular mechanisms responsible for the age-associated increase in PGE2production.The results demonstrated that the age-related increase in LPS-induced MØ COX-2 expression is due to an increase in transcription of COX-2 message rather than an increase in stability of COX-2 mRNA. COX-2 transcription is regulated by several factors such as cytokines (IL-6, IL-1β, tumor necrosis factor-α, IL-10), corticosteroids, and sphingolipids. Comparison of cytokine production between young and old MØ showed that IL-6, IL-1β, tumor necrosis factor-α, and IL-10 cannot account for the age-related difference in COX-2 mRNA, since there were no age-associated differences in MØ production of these cytokines (data not shown). Furthermore, we previously showed that the addition of recombinant IL-6 to MØ from young mice or that of anti-IL-6 antibody to MØ from old mice did not influence their ability to produce PGE2 (45Beharka A.A. Meydani M., Wu, D. Leka L.S. Meydani A. Meydani S.N. J. Gerontol. A Biol. Sci. Med. Sci. 2001; 56: B81-B88Crossref PubMed Scopus (114) Google Scholar). We did not test the role of glucocorticoids in our system, since glucocorticoids inhibit rather than induce COX-2 gene expression (46Masferrer J.L. Reddy S.T. Zweifel B.S. Seibert K. Needleman P. Gilbert R.S. Herschman H.R. J. Pharmacol. Exper. Ther. 1994; 270: 1340-1344PubMed Google Scholar, 47Vlahos R. Stewart A.G. Br. J. Pharmacol. 1999; 126: 1315-1324Crossref PubMed Scopus (48) Google Scholar, 48Lasa M. Brook M. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2001; 21: 771-780Crossref PubMed Scopus (216) Google Scholar), and aging has been shown to be associated with increased glucocorticoid levels (49Oxenkrug G.F. McIntyre I.M. Gershon S. J. Pineal Res. 1984; 1: 181-185Crossref PubMed Scopus (63) Google Scholar, 50Harris N.R. Rumbaut R.E. Pathophysiology. 2001; 8: 1-10Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We have also ruled out the age-related differences of MØ responsiveness to LPS treatment as a possible contributing factor, since the stimulation of MØ with other stimuli, including phytohemagglutinin and calcium ionophore A-23186, also resulted in age-related differences in PGE2 production (51Hayek M.G. Meydani S.N. Meydani M. Blumberg J.B. J. Gerontol. 1994; 49: B197-B207Crossref PubMed Scopus (78) Google Scholar). Collectively, these data indicate that the age-related difference in PGE2 production might be mediated through the postreceptor events.Another factor that has been shown to increase COX-2 expression is ceramide. Our results suggest that ceramide mediates the age-related increase in COX-2 expression. First we showed that old MØ have significantly higher LPS-induced ceramide levels compared with young mice, whereas there is no age difference in ceramide downstream metabolite sphingosine. Second, using C2-ceramide, we demonstrated that increasing ceramide levels in the young mice significantly increases PGE2 production and COX activity. These effects of ceramide were not altered when ceramide conversion to sphingosine was blocked. Our results further showed that ceramide significantly enhances PGE2 production by increasing COX-2 mRNA (Fig. 7) and that this increase in COX-2 gene expression is coupled to increased transcription of the COX-2 gene (Fig. 8).Additive effects of LPS and ceramide in young MØ (Fig. 6, Aand B) strongly support our hypothesis that ceramide mediates the higher LPS-induced COX-2 expression in old MØ. From our previous study (8Hayek M.G. Mura C., Wu, D. Beharka A.A. Han S.N. Paulson K.E. Hwang D. Meydani S.N. J. Immunol. 1997; 159: 2445-2451PubMed Google Scholar), we showed that old mouse MØ have higher levels of LPS-stimulated COX-2 mRNA, protein, and PGE2production. In a time frame that is consistent with age-associated up-regulation of COX-2 mRNA, ceramide also accumulated at a significantly higher concentration in the old mouse MØ compared with the MØ of young mice (Table I). Based on these observations, we suggest that LPS-induced higher accumulation of intracellular ceramide results in increased COX-2 mRNA, COX-2 protein, and PGE2 production in old mice. Although LPS stimulation in MØ from SMase knockout mice would provide further support for our proposed mechanisms, unfortunately only knockout mice for one isoform of SMase (i.e. the acidic form) are available. These animals, however, develop Niemann-Pick disease and die by the age of 10 months and thus would not be suitable to address the role of ceramide in COX-2 up-regulation of aged mice (typically more than 20 months old). Furthermore, the neutral and not the acidic SMase has been indicated in the observed age-related increase of ceramide levels in other tissues.The underlying mechanism for higher ceramide levels in old MØ was not determined in this study. The age-associated increase in LPS-induced ceramide generation in the old animals may be due to an increase in the sphingomyelinase activity (28Petkova D.H. Momchilova-Pankova A.B. Markovska T.T. Koumanov K.S. Exp. Gerontol. 1988; 23: 19-24Crossref PubMed Scopus (22) Google Scholar, 29Lightle S.A. Oakley J.I. Nikolova-Karakashian M.N. Mech. Ageing Dev. 2000; 120: 111-125Crossref PubMed Scopus (98) Google Scholar, 52Kim S.S. Kang M.S. Choi Y.M. Suh Y.H. Kim D.K. Biochem. Biophys. Res. Commun. 1997; 237: 583-587Crossref PubMed Scopus (18) Google Scholar), due to increased sphingomyelin levels, or due to both. Increases in age-related sphingomyelin levels have been observed in liver (29Lightle S.A. Oakley J.I. Nikolova-Karakashian M.N. Mech. Ageing Dev. 2000; 120: 111-125Crossref PubMed Scopus (98) Google Scholar, 53Jenkins K.J. Kramer J.K. J. Dairy Sci. 1988; 71: 435-441Abstract Full Text PDF PubMed Scopus (13) Google Scholar), brain (54Aureli T., Di Cocco M.E. Capuani G. Ricciolini R. Manetti C. Miccheli A. Conti F. Neurochem. Res. 2000; 25: 395-399Crossref PubMed Scopus (28) Google Scholar, 55Delion S. Chalon S. Guilloteau D. Lejeune B. Besnard J.C. Durand G. J. Lipid Res. 1997; 38: 680-689Abstract Full Text PDF PubMed Google Scholar), and nerve cells (56Giusto N.M. Roque M.E. Ilincheta de Boschero M.G. Lipids. 1992; 27: 835-839Crossref PubMed Scopus (60) Google Scholar). Moreover, GSH has been shown to inhibit neutral SMase activity and ceramide formation (57Yoshimura S. Banno Y. Nakashima S. Hayashi K. Yamakawa H. Sawada M. Sakai N. Nozawa Y. J. Neurochem. 1999; 73: 675-683Crossref PubMed Scopus (105) Google Scholar, 58Liu B. Andrieu-Abadie N. Levade T. Zhang P. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1998; 273: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Since we and others have shown that GSH levels decline with age (59Furukawa T. Meydani S.N. Blumberg J.B. Mech. Ageing Dev. 1987; 38: 107-117Crossref PubMed Scopus (84) Google Scholar, 60Sasaki T. Senda M. Kim S. Kojima S. Kubodera A. Nucl. Med. Biol. 2001; 28: 25-31Crossref PubMed Scopus (43) Google Scholar), it is feasible that the age-related decreases in GSH levels lead to increased neutral SMase activity, which in turn would result in higher ceramide levels in MØ from old mice.To determine signaling molecules responsible for the ceramide-induced age-associated increases in COX-2 mRNA, we tested the involvement of MAPKs. Although MAPKs have been indicated in LPS- and ceramide-induced COX-2 expression (30Subbaramaiah K. Chung W.J. Dannenberg A.J. J. Biol. Chem. 1998; 273: 32943-32949Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 39Chen C.C. Sun Y.T. Chen J.J. Chang Y.J. Mol. Pharmacol. 2001; 59: 493-500Crossref PubMed Scopus (121) Google Scholar, 61Zhang Z. Sheng H. Shao J. Beauchamp R.D. DuBois R.N. Neoplasia. 2000; 2: 523-530Crossref PubMed Scopus (67) Google Scholar), our results showed that the increase in LPS-induced ceramide generation leading to its subsequent up-regulation of COX-2 is unlikely to be due to increased activation of ERK, JNK, or p38 activities, because we found no differences in these MAPKs activity between young and old MØ. Although we were not able to differentiate the ERK-1 effects from that of ERK-2 or to determine the contribution of nuclear and cytosolic MAPKs separately, currently there is no evidence that these various types of MAPKs are differentially affected by age. Furthermore, our preliminary results indicate that the ceramide effect might be mediated through NF-κB activation. 2D. Wu, M. Marko, K. Claycombe, K. E. Paulson, S. N. Meydani, unpublished data.In conclusion, we have shown that increased MØ COX-2 gene transcription rather than increased COX-2 mRNA stability is responsible for the age-related increase in the rate of MØ COX-2 gene expression. We further demonstrated that LPS-induced increases in ceramide, but not its down stream metabolite, sphingosine, mediate the age-associated up-regulation of COX-2 mRNA, leading to increased COX-2 protein, enzyme activity, and PGE2 production. Findings from this study may serve as useful information toward developing therapeutic interventions to inhibit or delay age-associated dysregulation of the immune and inflammatory responses. Accumulating evidence indicates that T cell-mediated immune responses decline with aging (1Bryl E. Gazda M. Foerster J. Witkowski J.M. Blood. 2001; 98: 1100-1107Crossref PubMed Scopus (31) Google Scholar, 2Ginaldi L. DeMartinis M. D'Ostilio A. Marini L. Loreto F. Modesti M. Quaglino D. Am. J. Hematol. 2001; 67: 63-72Crossref PubMed Scopus (48) Google Scholar, 3Hsu H.C. Zhou T. Shi J. Yang P.A. Liu D. Zhang H.G. Bluethmann H. Mountz J.D. Mech. Ageing Dev. 2001; 122: 305-326Crossref PubMed Scopus (16) Google Scholar, 4Pahlavani M.A. Vargas D.A. FEBS Lett. 2001; 491: 114-118Crossref PubMed Scopus (24) Google Scholar, 5Ginaldi L. DeMartinis M. Modesti M. Loreto F. Corsi M.P. Quaglino D. Gerontology. 2000; 46: 242-248Crossref PubMed Scopus (45) Google Scholar, 6Fagnoni F.F. Vescovini R. Passeri G. Bologna G. Pedrazzoni M. Lavaghetto G. Casti A. Franceschi C. Passeri M. Sansoni P. Blood. 2000; 95: 2860-2868Crossref PubMed Google Scholar). We, as well as others, have demonstrated that, in addition to intrinsic changes in T cells, increased macrophage (MØ)1-derived prostaglandin E2 (PGE2) production contributes to the age-associated decline in T cell function (7Beharka A.A. Dayong W. Han S.N. Meydani S.N. Mech. Ageing Dev. 1997; 93: 59-77Crossref PubMed Scopus (123) Google Scholar). We further showed that the age-related increase in lipopolysaccharide (LPS)-induced MØ PGE2 production is due to increased cyclooxygenase-2 (COX-2) mRNA and protein levels, leading to increased COX enzyme activity (8Hayek M.G. Mura C., Wu, D. Beharka A.A. Han S.N. Paulson K.E. Hwang D. Meydani S.N. J. Immunol. 1997; 159: 2445-2451PubMed Google Scholar). PGE2 is synthesized from the precursor arachidonic acid by the two cyclooxygenase isoenzymes: cyclooxygenase 1 (COX-1), which is a ubiquitously expressed enzyme, and COX-2, which has a low basal expression but is rapidly induced by inflammatory stimuli such as LPS. Ceramide, a sphingolipid second messenger, generated from hydrolysis of membrane sphingomyelin by sphingomyelinase (SMase) or by de novo synthesis (9Vesper H. Schmelz E.M. Nikolova-Karakashian M.N. Dillehay D.L. Lynch D.V. Merrill A.H.J. J. Nutri. 1999; 129: 1239-1250Crossref PubMed Scopus (355) Google Scholar) has been shown to be involved in multiple signaling pathways, resulting in proliferation (10Pahlavani M.A. Vargas D.A. Immunopharmacology. 2000; 49: 345-354Crossref PubMed Scopus (3) Google Scholar, 11Marchell N.L. Uchida Y. Brown B.E. Elias P.M. Holleran W.M. J. Invest. Dermatol. 1998; 110: 383-387Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), differentiation (12Schneider C. Delorme N. Buisson-Legendre N. Bellon G. Emonard H. Btaouri H.E. Hornebeck W. Haye B. Martiny L. Lipids. 2000; 35: 1259-1268Crossref PubMed Scopus (5) Google Scholar), and apoptosis (13Herget T. Esdar C. Oehrlein S.A. Heinrich M. Schutze S. Maelicke A. van Echten-Deckert G. J. Biol. Chem. 2000; 275: 30344-30354Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 14Willaime S. Vanhoutte P. Caboche J. Lemaigre-Dubreuil Y. Mariani J. Brugg B. Eur. J. Neurosci. 2001; 13: 2037-2046Crossref PubMed Google Scholar, 15Selzner M. Bielawska A. Morse M.A. Rudiger H.A. Sindram D. Hannun Y.A. Clavien P.A. Cancer Res. 2001; 61: 1233-1240PubMed Google Scholar, 16Sugiki H. Hozumi Y. Maeshima H. Katagata Y. Mitsuhashi Y. Kondo S. Br. J. Dermatol. 2000; 143: 1154-1163Crossref PubMed Scopus (26) Google Scholar). In murine MØ, cell-permeable analogues of ceramide (C2- or C6-ceramide) and SMase have been shown to mimic LPS action (17Joseph C.K. Wright S.D. Bornmann W.G. Randolph J.T. Kumar E.R. Bittman R. Liu J. Kolesnick R.N. J. Biol. Chem. 1994; 269: 17606-17610Abstract Full Text PDF PubMed Google Scholar). Furthermore, ceramide increases expression of LPS-inducible genes in MØ from LPS-responsive, but not LPS-hyporesponsive mice (18Barber S.A. Perera P.-Y. Vogl S.N. J. Immunol. 1995; 155: 2303-2305PubMed Google Scholar). In several studies, LPS injection (19Haimovitz-Friedman A. Cordon-Cardo C. Bayoumy S. Garzotto M. McLoughlin M. Gallily R. Edwards C.K.R. Schuchman E.H. Fuks Z. Kolesnick R. J. Exp. Med. 1997; 186: 1831-1841Crossref PubMed Scopus (379) Google Scholar, 20Zimmermann C. Ginis I. Furuya K. Klimanis D. Ruetzler C. Spatz M. Hallenbeck J.M. Brain Res. 2001; 895: 59-65Crossref PubMed Scopus (78) Google Scholar, 21Memon R.A. Holleran W.M. Uchida Y. Moser A.H. Grunfeld C. Feingold K.R. J. Lipid Res. 2001; 42: 452-459Abstract Full Text Full Text PDF PubMed Google Scholar) as well as treatment of cultured cells with LPS (22MacKichan M.L. DeFranco A.L. J. Biol. Chem. 1999; 274: 1767-1775Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) resulted in increased intracellular ceramide generation. Exogenously added membrane-permeable analogues of ceramide (C2- and C6-ceramide) as well as bacterial SMase augmented IL-1-induced PGE2 production and COX-2 expression in human dermal fibroblast (23Ballou L.R. Chao C.P. Holness M.A. Barker S.C. Raghow R. J. Biol. Chem. 1992; 267: 20044-20050Abstract Full Text PDF PubMed Google Scholar) and granulose cell cultures (24Santana P. Lllanes L. Hernandez I. Gonzalez-Robayna I. Tabraue C. Gonzalez-Reyes J. Quintana J. Estevez F. Ruiz de Galarreta C.M. Fanjul L.F. Endocrinology. 1996; 137: 2480-2489Crossref PubMed Scopus (55) Google Scholar). Subsequent work showed that this effect was specific to COX-2 and not COX-1 (24Santana P. Lllanes L. Hernandez I. Gonzalez-Robayna I. Tabraue C. Gonzalez-Reyes J. Quintana J. Estevez F. Ruiz de Galarreta C.M. Fanjul L.F. Endocrinology. 1996; 137: 2480-2489Crossref PubMed Scopus (55) Google Scholar, 25Hayakawa M. Jayadev S. Tsujimoto M. Hannun Y.A. Ito F. Biochem. Biophys. Res. Commun. 1996; 220: 681-686Crossref PubMed Scopus (79) Google Scholar). In addition to ceramide, its metabolite, sphingosine (23Ballou L.R. Chao C.P. Holness M.A. Barker S.C. Raghow R. J. Biol. Chem. 1992; 267: 20044-20050Abstract Full Text PDF PubMed Google Scholar), has been shown to increase IL-1-induced PGE2 production. The effect of sphingosine, however, might be due to its retroconversion to ceramide (23Ballou L.R. Chao C.P. Holness M.A. Barker S.C. Raghow R. J. Biol. Chem. 1992; 267: 20044-20050Abstract Full Text PDF PubMed Google Scholar). Venable et al. (26Venable M.E. Lee J.Y. Smyth M.J. Bielawska A. Obeid L.M. J. Biol. Chem. 1995; 270: 30701-30708Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar) showed that senescent WI38 human fibroblast cells had higher ceramide levels and activity of neutral SMase. Age-related increases in brain and liver ceramide (27Palestini P. Masserini M. Fiorilli A. Calappi E. Tettamanti G. J. Neurochem. 1993; 61: 955-960Crossref PubMed Scopus (31) Google Scholar, 28Petkova D.H. Momchilova-Pankova A.B. Markovska T.T. Koumanov K.S. Exp. Gerontol. 1988; 23: 19-24Crossref PubMed Scopus (22) Google Scholar, 29Lightle S.A. Oakley J.I. Nikolova-Karakashian M.N. Mech. Ageing Dev. 2000; 120: 111-125Crossref PubMed Scopus (98) Google Scholar) and neutral SMase levels have also been reported. Since several lines of evidence indicated that LPS and ceramide share a common pathway(s) leading to induction of COX-2, we hypothesized that ceramide mediates the age-associated increase in COX-2 expression and PGE2 production. Recent studies have indicated that mitogen-activated protein kinase (MAPK) intermediates such as extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinases (JNK), and p38 (30Subbaramaiah K. Chung W.J. Dannenberg A.J. J. Biol. Chem. 1998; 273: 32943-32949Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) are involved in LPS- as well as ceramide-induced COX-2 gene expression. Moreover, several transcription factors, which bind COX-2 gene-regulatory regions such as AP-1, and cyclic AMP-response element-binding protein have been shown to be regulated by these MAPKs (30Subbaramaiah K. Chung W.J. Dannenberg A.J. J. Biol. Chem. 1998; 273: 32943-32949Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Thus, we further hypothesized that ceramide mediates the age-associated increase in MØ COX-2 mRNA expression via MAPKs. DISCUSSIONPGE2 is a proinflammatory eicosanoid, which has been indicated in pathogenesis of cardiovascular diseases, cancer, and inflammation. An age-related increase in PGE2 production has been demonstrated in animal models (8Hayek M.G. Mura C., Wu, D. Beharka A.A. Han S.N. Paulson K.E. Hwang D. Meydani S.N. J. Immunol. 1997; 159: 2445-2451PubMed Google Scholar, 41Shimizu N. Yamaguchi M. Uesu K. Goseki T. Abiko Y. J. Gerontol. A Biol. Sci. Med. Sci. 2000; 55: B489-B495Crossref PubMed Scopus (32) Google Scholar, 42Millatt L.J. Siragy H.M. Hypertension. 2000; 35: 643-647Crossref PubMed Scopus (9) Google Scholar) and in humans (43Schwab R. Hausman P.B. Rinnooy-Kan E. Weksler M.E. Immunology. 1985; 55: 677-684PubMed Google Scholar,44Wu D. Meydani M. Leka L.S. Nightingale Z. Handelman G. Blumberg J.B. Meydani S.N. Am. J. Clin. Nutr. 1999; 70: 536-543Crossref PubMed Scopus (90) Google Scholar). Previously we showed that the increase in MØ PGE2production with aging contributes to a decline in T cell-mediated immune function. Furthermore, we demonstrated that the age-associated increase in MØ PGE2 production is due to increased COX-2 mRNA and protein levels, leading to an increase in COX enzyme activity. Age-related increases in COX-2 gene have implications for many age-associated diseases such as cardiovascular, inflammatory, and neoplastic diseases. Thus, determining causative factors for the up-regulation of COX-2 gene expression is imperative. The aim of our current study was to determine the underlying molecular mechanisms responsible for the age-associated increase in PGE2production.The results demonstrated that the age-related increase in LPS-induced MØ COX-2 expression is due to an increase in transcription of COX-2 message rather than an increase in stability of COX-2 mRNA. COX-2 transcription is regulated by several factors such as cytokines (IL-6, IL-1β, tumor necrosis factor-α, IL-10), corticosteroids, and sphingolipids. Comparison of cytokine production between young and old MØ showed that IL-6, IL-1β, tumor necrosis factor-α, and IL-10 cannot account for the age-related difference in COX-2 mRNA, since there were no age-associated differences in MØ production of these cytokines (data not shown). Furthermore, we previously showed that the addition of recombinant IL-6 to MØ from young mice or that of anti-IL-6 antibody to MØ from old mice did not influence their ability to produce PGE2 (45Beharka A.A. Meydani M., Wu, D. Leka L.S. Meydani A. Meydani S.N. J. Gerontol. A Biol. Sci. Med. Sci. 2001; 56: B81-B88Crossref PubMed Scopus (114) Google Scholar). We did not test the role of glucocorticoids in our system, since glucocorticoids inhibit rather than induce COX-2 gene expression (46Masferrer J.L. Reddy S.T. Zweifel B.S. Seibert K. Needleman P. Gilbert R.S. Herschman H.R. J. Pharmacol. Exper. Ther. 1994; 270: 1340-1344PubMed Google Scholar, 47Vlahos R. Stewart A.G. Br. J. Pharmacol. 1999; 126: 1315-1324Crossref PubMed Scopus (48) Google Scholar, 48Lasa M. Brook M. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2001; 21: 771-780Crossref PubMed Scopus (216) Google Scholar), and aging has been shown to be associated with increased glucocorticoid levels (49Oxenkrug G.F. McIntyre I.M. Gershon S. J. Pineal Res. 1984; 1: 181-185Crossref PubMed Scopus (63) Google Scholar, 50Harris N.R. Rumbaut R.E. Pathophysiology. 2001; 8: 1-10Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We have also ruled out the age-related differences of MØ responsiveness to LPS treatment as a possible contributing factor, since the stimulation of MØ with other stimuli, including phytohemagglutinin and calcium ionophore A-23186, also resulted in age-related differences in PGE2 production (51Hayek M.G. Meydani S.N. Meydani M. Blumberg J.B. J. Gerontol. 1994; 49: B197-B207Crossref PubMed Scopus (78) Google Scholar). Collectively, these data indicate that the age-related difference in PGE2 production might be mediated through the postreceptor events.Another factor that has been shown to increase COX-2 expression is ceramide. Our results suggest that ceramide mediates the age-related increase in COX-2 expression. First we showed that old MØ have significantly higher LPS-induced ceramide levels compared with young mice, whereas there is no age difference in ceramide downstream metabolite sphingosine. Second, using C2-ceramide, we demonstrated that increasing ceramide levels in the young mice significantly increases PGE2 production and COX activity. These effects of ceramide were not altered when ceramide conversion to sphingosine was blocked. Our results further showed that ceramide significantly enhances PGE2 production by increasing COX-2 mRNA (Fig. 7) and that this increase in COX-2 gene expression is coupled to increased transcription of the COX-2 gene (Fig. 8).Additive effects of LPS and ceramide in young MØ (Fig. 6, Aand B) strongly support our hypothesis that ceramide mediates the higher LPS-induced COX-2 expression in old MØ. From our previous study (8Hayek M.G. Mura C., Wu, D. Beharka A.A. Han S.N. Paulson K.E. Hwang D. Meydani S.N. J. Immunol. 1997; 159: 2445-2451PubMed Google Scholar), we showed that old mouse MØ have higher levels of LPS-stimulated COX-2 mRNA, protein, and PGE2production. In a time frame that is consistent with age-associated up-regulation of COX-2 mRNA, ceramide also accumulated at a significantly higher concentration in the old mouse MØ compared with the MØ of young mice (Table I). Based on these observations, we suggest that LPS-induced higher accumulation of intracellular ceramide results in increased COX-2 mRNA, COX-2 protein, and PGE2 production in old mice. Although LPS stimulation in MØ from SMase knockout mice would provide further support for our proposed mechanisms, unfortunately only knockout mice for one isoform of SMase (i.e. the acidic form) are available. These animals, however, develop Niemann-Pick disease and die by the age of 10 months and thus would not be suitable to address the role of ceramide in COX-2 up-regulation of aged mice (typically more than 20 months old). Furthermore, the neutral and not the acidic SMase has been indicated in the observed age-related increase of ceramide levels in other tissues.The underlying mechanism for higher ceramide levels in old MØ was not determined in this study. The age-associated increase in LPS-induced ceramide generation in the old animals may be due to an increase in the sphingomyelinase activity (28Petkova D.H. Momchilova-Pankova A.B. Markovska T.T. Koumanov K.S. Exp. Gerontol. 1988; 23: 19-24Crossref PubMed Scopus (22) Google Scholar, 29Lightle S.A. Oakley J.I. Nikolova-Karakashian M.N. Mech. Ageing Dev. 2000; 120: 111-125Crossref PubMed Scopus (98) Google Scholar, 52Kim S.S. Kang M.S. Choi Y.M. Suh Y.H. Kim D.K. Biochem. Biophys. Res. Commun. 1997; 237: 583-587Crossref PubMed Scopus (18) Google Scholar), due to increased sphingomyelin levels, or due to both. Increases in age-related sphingomyelin levels have been observed in liver (29Lightle S.A. Oakley J.I. Nikolova-Karakashian M.N. Mech. Ageing Dev. 2000; 120: 111-125Crossref PubMed Scopus (98) Google Scholar, 53Jenkins K.J. Kramer J.K. J. Dairy Sci. 1988; 71: 435-441Abstract Full Text PDF PubMed Scopus (13) Google Scholar), brain (54Aureli T., Di Cocco M.E. Capuani G. Ricciolini R. Manetti C. Miccheli A. Conti F. Neurochem. Res. 2000; 25: 395-399Crossref PubMed Scopus (28) Google Scholar, 55Delion S. Chalon S. Guilloteau D. Lejeune B. Besnard J.C. Durand G. J. Lipid Res. 1997; 38: 680-689Abstract Full Text PDF PubMed Google Scholar), and nerve cells (56Giusto N.M. Roque M.E. Ilincheta de Boschero M.G. Lipids. 1992; 27: 835-839Crossref PubMed Scopus (60) Google Scholar). Moreover, GSH has been shown to inhibit neutral SMase activity and ceramide formation (57Yoshimura S. Banno Y. Nakashima S. Hayashi K. Yamakawa H. Sawada M. Sakai N. Nozawa Y. J. Neurochem. 1999; 73: 675-683Crossref PubMed Scopus (105) Google Scholar, 58Liu B. Andrieu-Abadie N. Levade T. Zhang P. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1998; 273: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Since we and others have shown that GSH levels decline with age (59Furukawa T. Meydani S.N. Blumberg J.B. Mech. Ageing Dev. 1987; 38: 107-117Crossref PubMed Scopus (84) Google Scholar, 60Sasaki T. Senda M. Kim S. Kojima S. Kubodera A. Nucl. Med. Biol. 2001; 28: 25-31Crossref PubMed Scopus (43) Google Scholar), it is feasible that the age-related decreases in GSH levels lead to increased neutral SMase activity, which in turn would result in higher ceramide levels in MØ from old mice.To determine signaling molecules responsible for the ceramide-induced age-associated increases in COX-2 mRNA, we tested the involvement of MAPKs. Although MAPKs have been indicated in LPS- and ceramide-induced COX-2 expression (30Subbaramaiah K. Chung W.J. Dannenberg A.J. J. Biol. Chem. 1998; 273: 32943-32949Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 39Chen C.C. Sun Y.T. Chen J.J. Chang Y.J. Mol. Pharmacol. 2001; 59: 493-500Crossref PubMed Scopus (121) Google Scholar, 61Zhang Z. Sheng H. Shao J. Beauchamp R.D. DuBois R.N. Neoplasia. 2000; 2: 523-530Crossref PubMed Scopus (67) Google Scholar), our results showed that the increase in LPS-induced ceramide generation leading to its subsequent up-regulation of COX-2 is unlikely to be due to increased activation of ERK, JNK, or p38 activities, because we found no differences in these MAPKs activity between young and old MØ. Although we were not able to differentiate the ERK-1 effects from that of ERK-2 or to determine the contribution of nuclear and cytosolic MAPKs separately, currently there is no evidence that these various types of MAPKs are differentially affected by age. Furthermore, our preliminary results indicate that the ceramide effect might be mediated through NF-κB activation. 2D. Wu, M. Marko, K. Claycombe, K. E. Paulson, S. N. Meydani, unpublished data.In conclusion, we have shown that increased MØ COX-2 gene transcription rather than increased COX-2 mRNA stability is responsible for the age-related increase in the rate of MØ COX-2 gene expression. We further demonstrated that LPS-induced increases in ceramide, but not its down stream metabolite, sphingosine, mediate the age-associated up-regulation of COX-2 mRNA, leading to increased COX-2 protein, enzyme activity, and PGE2 production. Findings from this study may serve as useful information toward developing therapeutic interventions to inhibit or delay age-associated dysregulation of the immune and inflammatory responses. PGE2 is a proinflammatory eicosanoid, which has been indicated in pathogenesis of cardiovascular diseases, cancer, and inflammation. An age-related increase in PGE2 production has been demonstrated in animal models (8Hayek M.G. Mura C., Wu, D. Beharka A.A. Han S.N. Paulson K.E. Hwang D. Meydani S.N. J. Immunol. 1997; 159: 2445-2451PubMed Google Scholar, 41Shimizu N. Yamaguchi M. Uesu K. Goseki T. Abiko Y. J. Gerontol. A Biol. Sci. Med. Sci. 2000; 55: B489-B495Crossref PubMed Scopus (32) Google Scholar, 42Millatt L.J. Siragy H.M. Hypertension. 2000; 35: 643-647Crossref PubMed Scopus (9) Google Scholar) and in humans (43Schwab R. Hausman P.B. Rinnooy-Kan E. Weksler M.E. Immunology. 1985; 55: 677-684PubMed Google Scholar,44Wu D. Meydani M. Leka L.S. Nightingale Z. Handelman G. Blumberg J.B. Meydani S.N. Am. J. Clin. Nutr. 1999; 70: 536-543Crossref PubMed Scopus (90) Google Scholar). Previously we showed that the increase in MØ PGE2production with aging contributes to a decline in T cell-mediated immune function. Furthermore, we demonstrated that the age-associated increase in MØ PGE2 production is due to increased COX-2 mRNA and protein levels, leading to an increase in COX enzyme activity. Age-related increases in COX-2 gene have implications for many age-associated diseases such as cardiovascular, inflammatory, and neoplastic diseases. Thus, determining causative factors for the up-regulation of COX-2 gene expression is imperative. The aim of our current study was to determine the underlying molecular mechanisms responsible for the age-associated increase in PGE2production. The results demonstrated that the age-related increase in LPS-induced MØ COX-2 expression is due to an increase in transcription of COX-2 message rather than an increase in stability of COX-2 mRNA. COX-2 transcription is regulated by several factors such as cytokines (IL-6, IL-1β, tumor necrosis factor-α, IL-10), corticosteroids, and sphingolipids. Comparison of cytokine production between young and old MØ showed that IL-6, IL-1β, tumor necrosis factor-α, and IL-10 cannot account for the age-related difference in COX-2 mRNA, since there were no age-associated differences in MØ production of these cytokines (data not shown). Furthermore, we previously showed that the addition of recombinant IL-6 to MØ from young mice or that of anti-IL-6 antibody to MØ from old mice did not influence their ability to produce PGE2 (45Beharka A.A. Meydani M., Wu, D. Leka L.S. Meydani A. Meydani S.N. J. Gerontol. A Biol. Sci. Med. Sci. 2001; 56: B81-B88Crossref PubMed Scopus (114) Google Scholar). We did not test the role of glucocorticoids in our system, since glucocorticoids inhibit rather than induce COX-2 gene expression (46Masferrer J.L. Reddy S.T. Zweifel B.S. Seibert K. Needleman P. Gilbert R.S. Herschman H.R. J. Pharmacol. Exper. Ther. 1994; 270: 1340-1344PubMed Google Scholar, 47Vlahos R. Stewart A.G. Br. J. Pharmacol. 1999; 126: 1315-1324Crossref PubMed Scopus (48) Google Scholar, 48Lasa M. Brook M. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2001; 21: 771-780Crossref PubMed Scopus (216) Google Scholar), and aging has been shown to be associated with increased glucocorticoid levels (49Oxenkrug G.F. McIntyre I.M. Gershon S. J. Pineal Res. 1984; 1: 181-185Crossref PubMed Scopus (63) Google Scholar, 50Harris N.R. Rumbaut R.E. Pathophysiology. 2001; 8: 1-10Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We have also ruled out the age-related differences of MØ responsiveness to LPS treatment as a possible contributing factor, since the stimulation of MØ with other stimuli, including phytohemagglutinin and calcium ionophore A-23186, also resulted in age-related differences in PGE2 production (51Hayek M.G. Meydani S.N. Meydani M. Blumberg J.B. J. Gerontol. 1994; 49: B197-B207Crossref PubMed Scopus (78) Google Scholar). Collectively, these data indicate that the age-related difference in PGE2 production might be mediated through the postreceptor events. Another factor that has been shown to increase COX-2 expression is ceramide. Our results suggest that ceramide mediates the age-related increase in COX-2 expression. First we showed that old MØ have significantly higher LPS-induced ceramide levels compared with young mice, whereas there is no age difference in ceramide downstream metabolite sphingosine. Second, using C2-ceramide, we demonstrated that increasing ceramide levels in the young mice significantly increases PGE2 production and COX activity. These effects of ceramide were not altered when ceramide conversion to sphingosine was blocked. Our results further showed that ceramide significantly enhances PGE2 production by increasing COX-2 mRNA (Fig. 7) and that this increase in COX-2 gene expression is coupled to increased transcription of the COX-2 gene (Fig. 8). Additive effects of LPS and ceramide in young MØ (Fig. 6, Aand B) strongly support our hypothesis that ceramide mediates the higher LPS-induced COX-2 expression in old MØ. From our previous study (8Hayek M.G. Mura C., Wu, D. Beharka A.A. Han S.N. Paulson K.E. Hwang D. Meydani S.N. J. Immunol. 1997; 159: 2445-2451PubMed Google Scholar), we showed that old mouse MØ have higher levels of LPS-stimulated COX-2 mRNA, protein, and PGE2production. In a time frame that is consistent with age-associated up-regulation of COX-2 mRNA, ceramide also accumulated at a significantly higher concentration in the old mouse MØ compared with the MØ of young mice (Table I). Based on these observations, we suggest that LPS-induced higher accumulation of intracellular ceramide results in increased COX-2 mRNA, COX-2 protein, and PGE2 production in old mice. Although LPS stimulation in MØ from SMase knockout mice would provide further support for our proposed mechanisms, unfortunately only knockout mice for one isoform of SMase (i.e. the acidic form) are available. These animals, however, develop Niemann-Pick disease and die by the age of 10 months and thus would not be suitable to address the role of ceramide in COX-2 up-regulation of aged mice (typically more than 20 months old). Furthermore, the neutral and not the acidic SMase has been indicated in the observed age-related increase of ceramide levels in other tissues. The underlying mechanism for higher ceramide levels in old MØ was not determined in this study. The age-associated increase in LPS-induced ceramide generation in the old animals may be due to an increase in the sphingomyelinase activity (28Petkova D.H. Momchilova-Pankova A.B. Markovska T.T. Koumanov K.S. Exp. Gerontol. 1988; 23: 19-24Crossref PubMed Scopus (22) Google Scholar, 29Lightle S.A. Oakley J.I. Nikolova-Karakashian M.N. Mech. Ageing Dev. 2000; 120: 111-125Crossref PubMed Scopus (98) Google Scholar, 52Kim S.S. Kang M.S. Choi Y.M. Suh Y.H. Kim D.K. Biochem. Biophys. Res. Commun. 1997; 237: 583-587Crossref PubMed Scopus (18) Google Scholar), due to increased sphingomyelin levels, or due to both. Increases in age-related sphingomyelin levels have been observed in liver (29Lightle S.A. Oakley J.I. Nikolova-Karakashian M.N. Mech. Ageing Dev. 2000; 120: 111-125Crossref PubMed Scopus (98) Google Scholar, 53Jenkins K.J. Kramer J.K. J. Dairy Sci. 1988; 71: 435-441Abstract Full Text PDF PubMed Scopus (13) Google Scholar), brain (54Aureli T., Di Cocco M.E. Capuani G. Ricciolini R. Manetti C. Miccheli A. Conti F. Neurochem. Res. 2000; 25: 395-399Crossref PubMed Scopus (28) Google Scholar, 55Delion S. Chalon S. Guilloteau D. Lejeune B. Besnard J.C. Durand G. J. Lipid Res. 1997; 38: 680-689Abstract Full Text PDF PubMed Google Scholar), and nerve cells (56Giusto N.M. Roque M.E. Ilincheta de Boschero M.G. Lipids. 1992; 27: 835-839Crossref PubMed Scopus (60) Google Scholar). Moreover, GSH has been shown to inhibit neutral SMase activity and ceramide formation (57Yoshimura S. Banno Y. Nakashima S. Hayashi K. Yamakawa H. Sawada M. Sakai N. Nozawa Y. J. Neurochem. 1999; 73: 675-683Crossref PubMed Scopus (105) Google Scholar, 58Liu B. Andrieu-Abadie N. Levade T. Zhang P. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1998; 273: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Since we and others have shown that GSH levels decline with age (59Furukawa T. Meydani S.N. Blumberg J.B. Mech. Ageing Dev. 1987; 38: 107-117Crossref PubMed Scopus (84) Google Scholar, 60Sasaki T. Senda M. Kim S. Kojima S. Kubodera A. Nucl. Med. Biol. 2001; 28: 25-31Crossref PubMed Scopus (43) Google Scholar), it is feasible that the age-related decreases in GSH levels lead to increased neutral SMase activity, which in turn would result in higher ceramide levels in MØ from old mice. To determine signaling molecules responsible for the ceramide-induced age-associated increases in COX-2 mRNA, we tested the involvement of MAPKs. Although MAPKs have been indicated in LPS- and ceramide-induced COX-2 expression (30Subbaramaiah K. Chung W.J. Dannenberg A.J. J. Biol. Chem. 1998; 273: 32943-32949Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 39Chen C.C. Sun Y.T. Chen J.J. Chang Y.J. Mol. Pharmacol. 2001; 59: 493-500Crossref PubMed Scopus (121) Google Scholar, 61Zhang Z. Sheng H. Shao J. Beauchamp R.D. DuBois R.N. Neoplasia. 2000; 2: 523-530Crossref PubMed Scopus (67) Google Scholar), our results showed that the increase in LPS-induced ceramide generation leading to its subsequent up-regulation of COX-2 is unlikely to be due to increased activation of ERK, JNK, or p38 activities, because we found no differences in these MAPKs activity between young and old MØ. Although we were not able to differentiate the ERK-1 effects from that of ERK-2 or to determine the contribution of nuclear and cytosolic MAPKs separately, currently there is no evidence that these various types of MAPKs are differentially affected by age. Furthermore, our preliminary results indicate that the ceramide effect might be mediated through NF-κB activation. 2D. Wu, M. Marko, K. Claycombe, K. E. Paulson, S. N. Meydani, unpublished data. In conclusion, we have shown that increased MØ COX-2 gene transcription rather than increased COX-2 mRNA stability is responsible for the age-related increase in the rate of MØ COX-2 gene expression. We further demonstrated that LPS-induced increases in ceramide, but not its down stream metabolite, sphingosine, mediate the age-associated up-regulation of COX-2 mRNA, leading to increased COX-2 protein, enzyme activity, and PGE2 production. Findings from this study may serve as useful information toward developing therapeutic interventions to inhibit or delay age-associated dysregulation of the immune and inflammatory responses. We thank Dr. Daniel Hwang for the donation of COX-2 cDNA (see “Experimental Procedures”). We also thank Stephanie Marco for preparation of the manuscript." @default.
W2092699086 created "2016-06-24" @default.
W2092699086 creator A5030986047 @default.
W2092699086 creator A5033246045 @default.
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W2092699086 date "2002-08-01" @default.
W2092699086 modified "2023-09-30" @default.
W2092699086 title "Ceramide Mediates Age-associated Increase in Macrophage Cyclooxygenase-2 Expression" @default.
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