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• W2043968008 abstract "Our previous works demonstrated that ligands of macrophage scavenger receptor (MSR) induce protein kinases (PKs) including protein-tyrosine kinase (PTK) and up-regulate urokinase-type plasminogen activator expression (Hsu, H. Y., Hajjar, D. P., Khan, K. M., and Falcone, D. J. (1998)J. Biol. Chem. 273, 1240–1246). To continue to investigate MSR ligand-mediated signal transductions, we focus on ligands, oxidized low density lipoprotein (OxLDL), and fucoidan induction of the cytokines tumor necrosis factor-α (TNF) and interleukin 1β (IL-1). In brief, in murine macrophages J774A.1, OxLDL and fucoidan up-regulate TNF production; additionally, fucoidan but not OxLDL induces IL-1 secretion, prointerleukin 1 (proIL-1, precursor of IL-1) protein, and proIL-1 message. Simultaneously, fucoidan stimulates activity of interleukin 1-converting enzyme. We further investigate the molecular mechanism by which ligand binding-induced PK-mediated mitogen-activated protein kinase (MAPK) in regulation of expression of proIL-1 and IL-1. Specifically, fucoidan stimulates activity of p21-activated kinase (PAK) and of the MAPKs extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. Combined with PK inhibitors and genetic mutants of Rac1 and JNK in PK activity assays, Western blotting analyses, and IL-1 enzyme-linked immunosorbent assay, the role of individual PKs in the regulation of proIL-1/IL-1 was extensively dissected. Moreover, tyrosine phosphorylation of pp60Src as well as association between pp60Src and Hsp90 play important roles in fucoidan-induced proIL-1 expression. We are the first to establish two fucoidan-mediated signaling pathways: PTK(Src)/Rac1/PAK/JNK and PTK(Src)/Rac1/PAK/p38, but not PTK/phospholipase C-γ1/PKC/MEK1/ERK, playing critical roles in proIL-1/IL-1 regulation. Our current results indicate and suggest a model for MSR ligands differentially modulating specific PK signal transduction pathways, which regulate atherogenesis-related inflammatory cytokines TNF and IL-1. Our previous works demonstrated that ligands of macrophage scavenger receptor (MSR) induce protein kinases (PKs) including protein-tyrosine kinase (PTK) and up-regulate urokinase-type plasminogen activator expression (Hsu, H. Y., Hajjar, D. P., Khan, K. M., and Falcone, D. J. (1998)J. Biol. Chem. 273, 1240–1246). To continue to investigate MSR ligand-mediated signal transductions, we focus on ligands, oxidized low density lipoprotein (OxLDL), and fucoidan induction of the cytokines tumor necrosis factor-α (TNF) and interleukin 1β (IL-1). In brief, in murine macrophages J774A.1, OxLDL and fucoidan up-regulate TNF production; additionally, fucoidan but not OxLDL induces IL-1 secretion, prointerleukin 1 (proIL-1, precursor of IL-1) protein, and proIL-1 message. Simultaneously, fucoidan stimulates activity of interleukin 1-converting enzyme. We further investigate the molecular mechanism by which ligand binding-induced PK-mediated mitogen-activated protein kinase (MAPK) in regulation of expression of proIL-1 and IL-1. Specifically, fucoidan stimulates activity of p21-activated kinase (PAK) and of the MAPKs extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. Combined with PK inhibitors and genetic mutants of Rac1 and JNK in PK activity assays, Western blotting analyses, and IL-1 enzyme-linked immunosorbent assay, the role of individual PKs in the regulation of proIL-1/IL-1 was extensively dissected. Moreover, tyrosine phosphorylation of pp60Src as well as association between pp60Src and Hsp90 play important roles in fucoidan-induced proIL-1 expression. We are the first to establish two fucoidan-mediated signaling pathways: PTK(Src)/Rac1/PAK/JNK and PTK(Src)/Rac1/PAK/p38, but not PTK/phospholipase C-γ1/PKC/MEK1/ERK, playing critical roles in proIL-1/IL-1 regulation. Our current results indicate and suggest a model for MSR ligands differentially modulating specific PK signal transduction pathways, which regulate atherogenesis-related inflammatory cytokines TNF and IL-1. macrophage scavenger receptor type A low density lipoprotein oxidized LDL interleukin 1β prointerleukin 1β tumor necrosis factor-α protein-tyrosine kinase phosphatidylinositol 3-kinase protein kinase C herbimycin A geldanamycin heat-shock protein 90 non-receptor protein-tyrosine kinase p21-activated kinase mitogen-activated protein kinase extracellular signal-regulated kinase c-Jun NH2-terminal protein kinase p38 mitogen-activated protein kinase interleukin 1-converting enzyme reverse transcription-polymerase chain reaction horseradish peroxidase glyceraldehyde phosphate dehydrogenase dominant negative JNK construct dominant negative Rac1 construct constitutive activated Rac1 construct enzyme-linked immunosorbent assay mitogen-activated protein kinase/extracellular signal-regulated kinase kinase SB203580 Overexpression of macrophage scavenger receptors (MSR)1 on activated macrophages and macrophage-derived foam cells in atherosclerotic lesions has been reported (2Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9931) Google Scholar, 3Sakaguchi H. Takeya M. Suzuki H. Hakamata H. Kodama T. Horiuchi S. Gordon S. van der Laan L.J. Kraal G. Ishibashi S. Kitamura N. Takahashi K. Lab. Invest. 1998; 78: 423-434PubMed Google Scholar, 4Goldstein J.L. Ho Y.K. Basu S.K. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 333-337Crossref PubMed Scopus (1925) Google Scholar, 5Naito M. Suzuki H. Mori T. Matsumoto A. Kodama T. Takahashi K. Am. J. Pathol. 1992; 141: 591-599PubMed Google Scholar). Scavenger receptors mediate the high affinity binding and internalization of modified lipoproteins including OxLDL (6Parthasarathy S. Fong L.G. Otero D. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 537-540Crossref PubMed Scopus (140) Google Scholar, 7Sparrow C.P. Parthasarathy S. Steinberg D. J. Biol. Chem. 1989; 264: 2599-2604Abstract Full Text PDF PubMed Google Scholar, 8Ottnad E. Parthasarathy S. Sambrano G.R. Ramprasad M.P. Quehenberger O. Kondratenko N. Green S. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1391-1395Crossref PubMed Scopus (139) Google Scholar), implying an important role in lipid-laden foam cell formation during atherosclerosis development and progression. In addition to OxLDL, a diverse group of polyanionic compounds has been listed as ligands for MSR (9Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1057) Google Scholar, 10Brown M.S. Goldstein J.L. Annu. Rev. Biochem. 1983; 52: 223-261Crossref PubMed Google Scholar). The broad nature of ligand specificity in MSR may explain receptor-multifaceted functions of macrophages such as adhesion (11Fraser I. Hughes D. Gordon S. Nature. 1993; 364: 343-346Crossref PubMed Scopus (307) Google Scholar, 12El Khoury J. Thomas C.A. Loike J.D. Hickman S.E. Cao L. Silverstein S.C. J. Biol. Chem. 1994; 269: 10197-10200Abstract Full Text PDF PubMed Google Scholar), clearance of pathologic substances (13Resnick D. Freedman N.J. Xu S. Krieger M. J. Biol. Chem. 1993; 268: 3538-3545Abstract Full Text PDF PubMed Google Scholar, 14Hampton R.Y. Golenbock D.T. Penman M. Krieger M. Raetz C.R. Nature. 1991; 352: 342-344Crossref PubMed Scopus (440) Google Scholar), host defenses (9Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1057) Google Scholar, 15Suzuki H. Kurihara Y. Takeya M. Kamada N. Kataoka M. Jishage K. Ueda O. Sakaguchi H. Higashi T. Suzuki T. Takashima Y. Kawabe Y. Cynshi O. Wada Y. Honda M. Kurihara H. Aburatani H. Doi T. Matsumoto A. Azuma S. Noda T. Toyoda Y. Itakura H. Yazaki Y. Kodama T. Nature. 1997; 386: 292-296Crossref PubMed Scopus (999) Google Scholar), and cytokine production (this paper and Refs. 9Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1057) Google Scholar and 16Palkama T. Immunology. 1991; 74: 432-438PubMed Google Scholar). However, the molecular mechanisms for MSR on macrophages possessing the capacity of cytokine induction remain unclear and need further investigation.The activated macrophages not only transform into foam cells via uptake of OxLDL, but also aberrantly produce a battery of inflammatory cytokines that play pivotal roles in the process of atherogenesis. It has been demonstrated that interleukin-1β (IL-1) and tumor necrosis factor-α (TNF) (2Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9931) Google Scholar, 17Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19074) Google Scholar, 18Schreyer S.A. Peschon J.J. LeBoeuf R.C. J. Biol. Chem. 1996; 271: 26174-26178Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 19Tipping P.G. Hancock W.W. Am. J. Pathol. 1993; 142: 1721-1728PubMed Google Scholar) are the major inflammatory cytokines (2Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9931) Google Scholar,18Schreyer S.A. Peschon J.J. LeBoeuf R.C. J. Biol. Chem. 1996; 271: 26174-26178Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 19Tipping P.G. Hancock W.W. Am. J. Pathol. 1993; 142: 1721-1728PubMed Google Scholar) altering the function of macrophages in ways that appear to promote atherosclerosis (20Hamilton T.A. Major J.A. Chisolm G.M. J. Clin. Invest. 1995; 95: 2020-2027Crossref PubMed Scopus (54) Google Scholar). Recent studies show that cytokines secreted from activated macrophages can be initiated or potentiated by one of the MSR ligands, OxLDL (21Jovinge S. Ares M.P. Kallin B. Nilsson J. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1573-1579Crossref PubMed Scopus (197) Google Scholar), thus dysregulating expression of MSR and likely promoting the development of localized inflammatory reactions. Indeed, the macrophage-derived inflammatory cytokines IL-1 and TNF cause adjacent endothelial cellular expression of procoagulant activity, changing the endothelial cell surface from antithrombotic to thrombotic.On the other hand, fucoidan, the principal polysaccharide sulfate ester occurring in the various species of Phaeophyceae (brown seaweed), although it is a non-lipoprotein, is recognized as an MSR ligand and effectively competes with OxLDL in MSR ligand binding studies (9Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1057) Google Scholar, 10Brown M.S. Goldstein J.L. Annu. Rev. Biochem. 1983; 52: 223-261Crossref PubMed Google Scholar). Up-regulation of MSR expression in human THP-1 monocytic macrophages upon phorbol 12-myristate 13-acetate priming has been reported (16Palkama T. Immunology. 1991; 74: 432-438PubMed Google Scholar, 22Hsu H.Y. Nicholson A.C. Hajjar D.P. J. Biol. Chem. 1996; 271: 7767-7773Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 23Moulton K.S. Wu H. Barnett J. Parthasarathy S. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8102-8106Crossref PubMed Scopus (62) Google Scholar). Binding of fucoidan to MSR stimulates urokinase-type plasminogen activator secretion in human THP-1 macrophages (1Hsu H.Y. Hajjar D.P. Khan K.M. Falcone D.J. J. Biol. Chem. 1998; 273: 1240-1246Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), in activated peritoneal murine macrophages, and in murine macrophage cell lines (J774A.1 and RAW264.7) (24Falcone D.J. McCaffrey T.A. Vergilio J.A. J. Biol. Chem. 1991; 266: 22726-22732Abstract Full Text PDF PubMed Google Scholar, 25Falcone D.J. Ferenc M.J. J. Cell. Physiol. 1988; 135: 387-396Crossref PubMed Scopus (33) Google Scholar). The expression of uPA via MSR ligand-mediated signaling molecules include protein-tyrosine kinases (PTK), phospholipase C-γ1, phosphatidylinositol 3-kinase (PI 3-kinase), and protein kinase C (PKC), indicating a molecular model for regulation of urokinase-type plasminogen activator expression by the specific ligand of MSR (1Hsu H.Y. Hajjar D.P. Khan K.M. Falcone D.J. J. Biol. Chem. 1998; 273: 1240-1246Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Furthermore, herbimycin A (HB) inhibits fucoidan-induced tyrosine phosphorylation of phospholipase C-γ1 (1Hsu H.Y. Hajjar D.P. Khan K.M. Falcone D.J. J. Biol. Chem. 1998; 273: 1240-1246Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), suggesting that an unidentified PTK is involved in fucoidan-induced signaling. In this paper, we demonstrate again that geldanamycin (GA) or HB inhibits fucoidan-induced phosphorylation of various protein tyrosines and expression of IL-1. It is known that GA or HB inhibits the activation of certain tyrosine kinases; however, their cellular target is heat-shock protein 90 (Hsp90) (26Pratt W.B. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 297-326Crossref PubMed Google Scholar), and its expression is induced by cell stress. Obviously, the MSR-induced cytokines IL-1 and TNF could lead to cellular stress including inflammation. Both GA and HB, benzoquinone ansamycin antibiotics, inhibit various signal transduction proteins including non-receptor protein-tyrosine kinase (NRPTK) pp60Src. Pharmacologically, HB or GA binds in a specific manner to Hsp90 and inhibits pp60Src·Hsp90 heterocomplex formation (26Pratt W.B. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 297-326Crossref PubMed Google Scholar, 27Xu Y. Singer M.A. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 109-114Crossref PubMed Scopus (164) Google Scholar, 28Whitesell L. Mimnaugh E.G. De Costa B. Myers C.E. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8324-8328Crossref PubMed Scopus (1317) Google Scholar). Although ligands of MSR such as fucoidan and polyinosinic acid induce IL-1 production in human THP-1 macrophages (16Palkama T. Immunology. 1991; 74: 432-438PubMed Google Scholar), they are unable to examine systematically the MSR ligand-regulated signaling cascade for IL-1 production in macrophages. Hence, we propose and investigate pp60Src and Hsp90, which may play certain roles in fucoidan-mediated effects such as the association between pp60Src and Hsp90 in forming a complex in the regulation of fucoidan-induced prointerleukin 1β (proIL-1) protein expression.In studies reported here, we observe tha both OxLDL and fucoidan up-regulate TNF production; additionally, fucoidan but not OxLDL induces proIL-1 protein and IL-1 secretion in murine macrophages J774A.1. We demonstrate for the first time that fucoidan differentially stimulates the signaling machinery including NRPTK, pp60Src, p21-activated kinase (PAK), and mitogen-activated protein kinases (MAPKs; i.e. ERK, JNK, and p38). Recent studies indicate that IL-1 secretion is post-transcriptionally regulated via IL-1 converting enzyme (ICE, caspase 1) (29Mallat Z. Ohan J. Leseche G. Tedgui A. Circulation. 1997; 96: 424-428Crossref PubMed Scopus (90) Google Scholar, 30Geng Y.J. Libby P. Am. J. Pathol. 1995; 147: 251-266PubMed Google Scholar, 31Cerretti D.P. Kozlosky C.J. Mosley B. Nelson N. Van Ness K. Greenstreet T.A. March C.J. Kronheim S.R. Druck T. Cannizzaro L.A. Science. 1992; 256: 97-100Crossref PubMed Scopus (991) Google Scholar, 32Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2185) Google Scholar). In here, we find that fucoidan-mediated specific signals regulate IL-1 secretion, and simultaneously, fucoidan induces ICE activity during alternation of proIL-1/IL-1 expression. Moreover, combining with pharmacological inhibitors and genetic mutants in protein kinase assays, we investigate the molecular mechanism for MSR ligand-mediated signal transduction pathways in the regulation of proIL-1/IL-1 expression. Specifically, we further dissect and confirm the role of several key signaling molecules in induction of proIL-1, and we establish two signaling cascades,i.e. the PTK(Src)/Rac1/PAK/JNK pathway and the PTK(Src)/Rac1/PAK/p38 pathway, in the regulation of proIL-1/IL-1 expression.EXPERIMENTAL PROCEDURESCell CulturesMurine macrophage J774A.1 cells were obtained from ATCC (Rockville, MD), propagated in RPMI 1640 medium supplemented with 10% heated-inactivated fetal bovine serum and 2 mml-glutamine (Life Technologies, Inc.), and cultured in a 37 °C, 5% CO2 incubator. Using the Histopaque®-1077 method, human monocyte-derived macrophages were obtained from the Taiwan Blood Center (Taipei, Taiwan) from the blood of healthy persons.MaterialsHistopaque®-1077, fucoidan, sodium orthovanadate, phenylmethylsulfonyl fluoride, bovine serum albumin (fraction V), GA, and curcumin were purchased from Sigma. LipofectAMINE PLUS® reagent was purchased from Life Technologies, Inc. An Immobilon® polyvinylidene difluoride membrane was purchased from Millipore Inc. (Bedford, MA). non-radioactive Western blot chemiluminescence Reagent, Renaissance®, and 10 Ci/mmol [γ-32P]ATP were purchased from PerkinElmer Life Sciences. REZOl®C&T was from PROtech Technology Co. (Taipei, Taiwan). A GeneAmp® RNA PCR kit for RT-PCR amplification was purchased from PerkinElmer Life Sciences. OxLDL was supplied by Dr. Ming-Shi Shiao (Veteran General Hospital, Taipei, Taiwan).Growth Factors and AntibodiesAnti-IL-1β, 3ZD monoclonal antibody (a gift from the National Institutes of Health, Bethesda, MD), anti-PAK, rabbit polyclonal IgG, anti-rabbit IgG-HRP, anti-mouse IgG-HRP and protein A/G plus agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine clone 4G10 (mouse monoclonal IgG2bκ) was purchased from Upstate Biotechnology, Inc. Monoclonal anti-Hsp90 and monoclonal anti-Src (Oncogene Inc.) were obtained from Calbiochem-Novabiochem Corp. (La Jolla, CA). The CaspACE® Assay System, Fluorometric was purchased fromPromega Co.Kinase Assay KitsThe p44/42 MAP kinase assay Kit, SAPK/JNK assay kit, and p38 MAP kinase assay kit were purchased from Cell Signaling Technology (Beverly, MA).Protein Kinase InhibitorsPD98059 was from Cell Signaling Technology. PP1 was from Biomol (Plymouth meeting, PA). Calphostin C, PP2, and HB were from Calbiochem-Novabiochem Corp. Wortmannin, LY294002, and SB203580 were from Sigma.OligonucleotidesPrimers for TNF, proIL-1/IL-1, and glyceraldehyde phosphate dehydrogenase (GAPDH) were synthesized from local MD Bio. Inc. (Taipei, Taiwan). The dominant negative JNK construct (DN-JNK) was a gift from Dr. Michael Karin (UCSD, San Diego, CA) (33Li Y.S. Shyy J.Y. Li S. Lee J. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (206) Google Scholar). The dominant negative Rac1 construct (DN-Rac1) and constitutive activated Rac1 construct (CA-Rac1) were gifts from Dr. S. Bagrodia (Cornell University, Ithaca, NY) (34Bagrodia S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar).Isolation of LDL for Preparation of OxLDL, RNA Isolation, RT and PCR Amplification for Detecting the Expression of TNF or ProIL-1/IL-1, Protein Assay (Determined by Bio-Rad Protein Assay), Western Blotting Analysis, and Enzyme-linked Immunosorbent Assay for Measurement of TNF and IL-1All detail methods and procedures have been presented previously (22Hsu H.Y. Nicholson A.C. Hajjar D.P. J. Biol. Chem. 1996; 271: 7767-7773Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 35Hsu H.Y. Twu Y.C. J. Biol. Chem. 2000; 275: 41035-41048Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar).Analysis of ICE (Caspase 1) ActivityTo analyze the ICE activity in J774A.1 cells we basically followed the protocols from the CaspACE® assay system, fluorometric. Briefly, the cells were incubated with 25 µg/ml fucoidan or 5 µg/ml OxLDL. At the indicated time, the cells were washed twice with ice-cold phosphate-buffered saline (without Ca+2, Mg+2), harvested in 300 µl of cell suspension buffer (25 mm pH 7.5 HEPES, 5 mmMgCl2, 5 mm EDTA, 5 mmdithiothreitol, 2 mm phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, from Promega Co.). The suspended cells were stored in 1.5-ml centrifugation tubes, frozen and thawed three times, and then centrifuged at 12,000 × gat 4 °C for 15 min; the pellets were discarded. The protein concentration of supernatant in cell extract was determined, and the rest of the supernatant was saved at −80 °C for future use. For assaying ICE activity, 32 µl of ICE-like enzyme assay buffer, 2 µl of dimethyl sulfoxide, and 10 µl of 100 mm dithiothreitol were added to 75 µg of protein of cell extract from tested samples at various times, and then adjusted with water to 98 µl. For each tested sample there was a negative control (the same as above tested sample with the addition of 2 µl of 2.5 mm ICE inhibitor (Ac-YVAD-CHO)) and a blank control (32 µl of ICE-like enzyme assay buffer, 10 µl of 100 mm dithiothreitol, 2 µl of dimethyl sulfoxide, and 54 µl of water). All tested samples were incubated at 30 °C for 30 min. After the incubation, 2 µl of 2.5 mm ICE substrate (Ac-YVAD-AMC) was added to each sample, followed by an additional 60-min incubation at 30 °C; then the intensity of the fluorescence of the tested sample was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Calculation of the relative fluorescence units and the caspase activity for each sample, as well as construction of standard curve and AMC calibration curves were as described in the Technical Bulletin fromPromega Co.Assay Activity of ERK, JNK, p38, and PAK in Fucoidan-treated J774A.1 Cells with or without PK InhibitorsMethods for assaying these PKs, including cell lysate preparation, immunoprecipitation of PK, in vitro PK reaction, analysis of PK activity, and quantification of PK activity were as described previously (35Hsu H.Y. Twu Y.C. J. Biol. Chem. 2000; 275: 41035-41048Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar).Transfection of DN-JNK, DN-Rac1, and CA-Rac1 into J774A.1 Cells and Assay of JNK and p38 Activity as well as Assay of ProIL-1 Protein Expression in the Transfected Cells upon Fucoidan StimulationMethods for transfection and MAPK activity assays were as described (35Hsu H.Y. Twu Y.C. J. Biol. Chem. 2000; 275: 41035-41048Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Briefly, the conditions for growing J774A.1 cells to be transfected were identical to those of regular J774A.1 cells, except the cells were subpassaged the day before transfection and replaced with fresh medium. The transient transfection of DN-JNK, DN-Rac1, CA-Rac1, or control (i.e. the empty expression vector) at 10 µg of DNA/100-mm plate into cells was conducted by using LipofectAMINE PLUS® reagent (Life Technologies, Inc.) as described previously (35Hsu H.Y. Twu Y.C. J. Biol. Chem. 2000; 275: 41035-41048Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The efficiency of both transfection and expression was monitored by the hemagglutinin tag expression. DN-Rac1-transfected cells were stimulated with fucoidan for 120 min and 240 min, respectively, then used to assay the activity of JNK and p38 as described above. DN-JNK-transfected cells and DN-Rac1-transfected cells were harvested after 24, 48, and 72 h (each sample was treated with fucoidan for 8 h), as well as CA-Rac1-stably transfected cells were harvested after fucoidan treatment for 4, 12, or 24 h, respectively. ProIL-1 protein expression in cells was detected by Western blotting analysis.Statistical AnalysisStatistical differences between the experimental groups were examined by analysis of variance, and statistical significance was determined at p < 0.05. The experiments were conducted three times or as indicated, and all data are expressed as the mean ± S.E.DISCUSSIONIn atherosclerotic lesions, activated macrophages via overexpressed scavenger receptors aberrantly taking up OxLDL are also the main source of secretion of inflammatory cytokines TNF (2Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9931) Google Scholar, 17Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19074) Google Scholar, 18Schreyer S.A. Peschon J.J. LeBoeuf R.C. J. Biol. Chem. 1996; 271: 26174-26178Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 19Tipping P.G. Hancock W.W. Am. J. Pathol. 1993; 142: 1721-1728PubMed Google Scholar) and IL-1 (17Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19074) Google Scholar, 19Tipping P.G. Hancock W.W. Am. J. Pathol. 1993; 142: 1721-1728PubMed Google Scholar). Yet it is unclear whether there is a relationship between receptor ligand binding and stimulation of cytokine expression. Fucoidan, a polyanionic polysaccharide, has been used as an effective competitor for OxLDL or modified LDL in studies of receptor binding (9Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1057) Google Scholar,10Brown M.S. Goldstein J.L. Annu. Rev. Biochem. 1983; 52: 223-261Crossref PubMed Google Scholar). Moreover, fucoidan stimulates production of proteases (1Hsu H.Y. Hajjar D.P. Khan K.M. Falcone D.J. J. Biol. Chem. 1998; 273: 1240-1246Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 9Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1057) Google Scholar, 24Falcone D.J. McCaffrey T.A. Vergilio J.A. J. Biol. Chem. 1991; 266: 22726-22732Abstract Full Text PDF PubMed Google Scholar) and cytokines in macrophages (9, 16, and this paper); however, there is no molecular mechanism to date for fucoidan-mediated reactions. Using ELISA, we demonstrate here that 24-h fucoidan induces about 50-fold more TNF production than OxLDL does in J774A.1 cells. Based on the time course of TNF production (Fig. 1, A versus B), there are different patterns and mechanisms for TNF induction by the two ligands, although TNF preexists in untreated cells. In addition, only the relatively lower concentration of OxLDL (5 µg/ml) induced TNF because there was no induction of TNF at a higher concentration of OxLDL (50 µg/ml) (data not shown), comparable to the previous demonstration (21Jovinge S. Ares M.P. Kallin B. Nilsson J. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1573-1579Crossref PubMed Scopus (197) Google Scholar). Using RT-PCR for TNF mRNA, there was no apparent difference of TNF message among cells treated with fucoidan and OxLDL and untreated cells. The fact that there was increased TNF production but no alteration of TNF message under fucoidan stimulation indicates that fucoidan regulation of TNF expression is likely at a post-transcriptional level.Neither IL-1 nor proIL-1 preexists in quiescent J774A.1 cells. Upon fucoidan stimulation, IL-1 can be detected by ELISA after 6–8 h; IL-1 secretion is consistent with the sequential times for synthesis of proIL-1 mRNA and proIL-1 protein under stimulation for 2 and 4 h, respectively. The ICE activity of fucoidan-treated cells peaks around 6 h and simultaneously IL-1 secretion increases, which reflects that active ICE hydrolyzes proIL-1 into IL-1 as in various cells (31Cerretti D.P. Kozlosky C.J. Mosley B. Nelson N. Van Ness K. Greenstreet T.A. March C.J. Kronheim S.R. Druck T. Cannizzaro L.A. Science. 1992; 256: 97-100Crossref PubMed Scopus (991) Google Scholar, 32Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2185) Google Scholar), although the reason for continuous increasing IL-1 needs further study. Our results indicate that there are complicated mechanisms, likely at transcriptional, post-transcriptional, and post-translational levels for fucoidan induction of proIL-1 protein and IL-1 secretion in macrophages. Surprisingly, there is no detectable expression of proIL-1/IL-1 in the cell incubated with OxLDL. One of explanations for the results is that binding of different ligands to the conserved lysine clusters of the collagen-like domain in MSR (43Kodama T. Freeman M. Rohrer L. Zabrecky J. Matsudaira P. Krieger M. Nature. 1990; 343: 531-535Crossref PubMed Scopus (836) Google Scholar, 44Acton S. Resnick D. Freeman M. Ekkel Y. Ashkenas J. Krieger M. J. Biol. Chem. 1993; 268: 3530-3537Abstract Full Text PDF PubMed Google Scholar, 45Doi T. Higashino K. Kurihara Y. Wada Y. Miyazaki T. Nakamura H. Uesugi S. Imanishi T. Kawabe Y. Itakura H. J. Biol. Chem. 1993; 268: 2126-2133Abstract Full Text PDF PubMed Google Scholar) may trigger on different signal transductions (44Acton S. Resnick D. Freeman M. Ekkel Y. Ashkenas J. Krieger M. J. Biol. Chem. 1993; 268: 3530-3537Abstract Full Text PDF PubMed Google Scholar,45Doi T. Higashino K. Kurihara Y. Wada Y. Miyazaki T. Nakamura H. Uesugi S. Imanishi T. Kawabe Y. Itakura H. J. Biol. Chem. 1993; 268: 2126-2133Abstract Full Text PDF PubMed Google Scholar). 2H.-Y. Hsu, S.-L. Chiu, M.-H. Wen, K.-Y. Chen, and K.-F. Hua, unpublished data. For the first time we demonstrate that different MSR ligands transduce diverse signaling and further lead to different biochemical reactions such as cytokine production.Ligation of MSR induces signal transduction pathways in macrophages (1Hsu H.Y. Hajjar D.P. Khan K.M. Falcone D.J. J. Biol. Chem. 1998; 273: 1240-1246Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar,16Palkama T. Immunology. 1991; 74: 432-438PubMed Google Scholar, 46Fong L.G. Le D. J. Biol. Chem. 1999; 274: 36808-36816Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 47Fong L.G. J. Lipid Res. 1996; 37: 574-587Abstract Full Text PDF PubMed Google Scholar); for example, binding of fucoidan to MSR in human monocytic macrophage THP-1 cells induced PKC signaling-mediated IL-1 secretion (16Palkama T. Immunology. 1991; 74: 432-438PubMed Google Scholar) and also stimulated protein tyrosine phosphorylation including phospholipase C-γ1, PI 3-kinase, and PKC activity leading to up-regulated urokinase-type plasminogen activator expression (1Hsu H.Y. Hajjar D.P. Khan K.M. Falcone D.J. J. Biol. Chem. 1998; 273" @default.
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