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Immunomodulatory β-Glucan from Lentinus edodes Activates Mitogen-activated Protein Kinases and Nuclear Factor-κB in Murine RAW 264.7 Macrophages*

  • Xiaojuan Xu
    Correspondence
    To whom correspondence should be addressed: Dept. of Chemistry, Wuhan University, Wuchang Luojiashan, Wuhan 430072, China. Tel.: 86-27-6876-0313; Fax: 86-27-6875-4067;
    Affiliations
    Department of Chemistry, Wuhan University, Wuhan 430072, China

    Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan
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  • Chen Pan
    Affiliations
    Department of Chemistry, Wuhan University, Wuhan 430072, China
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  • Lina Zhang
    Affiliations
    Department of Chemistry, Wuhan University, Wuhan 430072, China
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  • Hitoshi Ashida
    Affiliations
    Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan
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  • Author Footnotes
    * This work was supported by Japan Society for the Promotion of Science Postdoctoral Fellowship 20.08431 (to X. X.), National Natural Science Foundation Grant 20874078, and Youth Technology Chenguang Project of Wuhan 200950431193.
Open AccessPublished:July 20, 2011DOI:https://doi.org/10.1074/jbc.M111.246470
      Lentinan, a cell wall β-glucan from the fruiting bodies of Lentinus edodes, is well known to be a biological defense modifier, but the signal transduction pathway(s) induced by Lentinan have not been elucidated. In this study, we extracted Lentinan (LNT-S) by ultrasonication from Lentinus edodes and report that, in murine RAW 264.7 macrophages, LNT-S glucan activated NF-κB p65 and triggered its nuclear translocation as determined by Western blotting. Moreover, LNT-S enhanced NF-κB-luciferase activity in the Dual-Luciferase gene system assay. Its upstream signaling molecules, MAPKs such as ERK1/2 and JNK1/2, were shown to be activated by assessing the level of phosphorylation in a time- and concentration-dependent manner, but its downstream proinflammatory enzyme, inducible NOS, was not observed. The data evaluated using a TNF-α ELISA kit and Griess reagent further demonstrated that no proinflammatory mediators such as TNF-α and NO were produced by LNT-S stimulation in RAW 264.7 cells. In contrast, LPS significantly induced inducible NOS expression and increased NO and TNF-α production, which are associated with activation of the NF-κB p65/p50 heterodimer complex. It is possible that LNT-S did not activate NF-κB p65/p50, and the activation of NF-κB p65 was not sufficient to stimulate cytokine production. These data demonstrate that LNT-S glucan carries out its immunomodulating activity by activating MAPK signaling pathways without secretion of TNF-α and NO.

      Introduction

      β-Glucans are known to possess significant biological and physiological activities, including antitumor activity (
      • Ross G.D.
      • Vetvicka V.
      • Yan J.
      • Xia Y.
      • Vetvicková J.
      ,
      • Vetvicka V.
      • Yvin J.C.
      ), antiviral activity (
      • Mayell M.
      ,
      • Chen J.
      • Seviour R.
      ), antibacterial activity (
      • Markova N.
      • Kussovski V.
      • Drandarska I.
      • Nikolaeva S.
      • Georgieva N.
      • Radoucheva T.
      ,
      • Markova N.
      • Michailova L.
      • Kussovski V.
      • Jourdanova M.
      • Radoucheva T.
      ,
      • Kournikakis B.
      • Mandeville R.
      • Brousseau P.
      • Ostroff G.
      ), antifungal activity (
      • Herre J.
      • Willment J.A.
      • Gordon S.
      • Brown G.D.
      ), and immunomodulating activity (
      • Wasser S.P.
      ,
      • Cleary J.A.
      • Kelly G.E.
      • Husband A.J.
      ,
      • Vetvicka V.
      • Vetvickova J.
      • Frank J.
      • Yvin J.C.
      ,
      • Shimojoh M.
      • Kojima T.
      • Nakajima K.
      • Hatta K.
      • Katoh A.
      • Kurita K.
      ). In addition, β-glucans are effective in wound healing (
      • Mayell M.
      ) and in lowering blood cholesterol (
      • Nicolosi R.
      • Bell S.J.
      • Bistrian B.R.
      • Greenberg I.
      • Forse R.A.
      • Blackburn G.L.
      ) and glucose (
      • Lo H.C.
      • Tsai F.A.
      • Wasser S.P.
      • Yang J.G.
      • Huang B.M.
      ) concentrations. Therefore, β-glucan is a promising product in biochemical and medical applications.
      Among the numerous β-glucans derived from different sources such as plants, animals, and microbials, Lentinan from Lentinus edodes is especially remarkable for its anticancer and immunomodulating activities (
      • Fang N.
      • Li Q.
      • Yu S.
      • Zhang J.
      • He L.
      • Ronis M.J.
      • Badger T.M.
      ,
      • Kupfahl C.
      • Geginat G.
      • Hof H.
      ,
      • Zheng R.
      • Jie S.
      • Hanchuan D.
      • Moucheng W.
      ) and is a β-(1,3)-glucan with one β-(1,6)-glucose branch every five glucose residues. Lentinan is currently used clinically as an antitumor agent (
      • Suga T.
      • Shiio T.
      • Maeda Y.Y.
      • Chihara G.
      ,
      • Okamura K.
      • Suzuki M.
      • Chihara T.
      • Fujiwara A.
      • Fukuda T.
      • Goto S.
      • Ichinohe K.
      • Jimi S.
      • Kasamatsu T.
      • Kawai N.
      ). It has been shown to stimulate natural killer cell activity (
      • Taguchi T.
      • Kaneko Y.
      ,
      • Nanba H.
      • Kuroda H.
      ,
      • Péter G.
      • Károly V.
      • Imre B.
      • János F.
      • Kaneko Y.
      ), macrophage/monocyte functions (secreting IL-1 and superoxide anion), phagocytosis, and cytotoxicity (
      • Fruehauf J.P.
      • Bonnard G.D.
      • Herberman R.B.
      ,
      • Akiyama T.
      • Kashima S.
      • Hayami T.
      • Izawa M.
      • Mitsugi K.
      • Hamuro J.
      ,
      • Abel G.
      • Szöllösi J.
      • Chihara G.
      • Fachet J.
      ,
      • Herlyn D.
      • Kaneko Y.
      • Powe J.
      • Aoki T.
      • Koprowski H.
      ,
      • Nanba H.
      • Mori K.
      • Toyomasu T.
      • Kuroda H.
      ,
      • Ladányi A.
      • Tímár J.
      • Lapis K.
      ). Lentinan was found to elevate the cytotoxic activity and TNF secretion of macrophages in vitro and in vivo (
      • Kupfahl C.
      • Geginat G.
      • Hof H.
      ,
      • Kerékgyártó C.
      • Virág L.
      • Tankó L.
      • Chihara G.
      • Fachet J.
      ). Pretreatment of bone marrow macrophages with Lentinan results in increased production of NO in vitro (
      • Kupfahl C.
      • Geginat G.
      • Hof H.
      ). However, Masihi et al. (
      • Masihi K.N.
      • Madaj K.
      • Hintelmann H.
      • Gast G.
      • Kaneko Y.
      ) demonstrated that pretreatment of Lentinan before LPS administration induces a striking inhibition of up to 89% of circulating TNF-α in bacillus Calmette-Guérin-primed mice. It is possible that the different immunomodulatory reaction was highly dependent on the genotype of the host. To our knowledge, most of the research has focused on secretion of proinflammatory cytokines or mediators such as TNF-α, IL-12, IFN-γ, and NO and on inflammatory mRNA expression from Lentinan-stimulated macrophages or monocytes (
      • Kupfahl C.
      • Geginat G.
      • Hof H.
      ,
      • Kerékgyártó C.
      • Virág L.
      • Tankó L.
      • Chihara G.
      • Fachet J.
      ,
      • Masihi K.N.
      • Madaj K.
      • Hintelmann H.
      • Gast G.
      • Kaneko Y.
      ,
      • Liu F.
      • Ooi V.E.
      • Fung M.C.
      ), and there have been few studies involved in the signal transduction for macrophage activation by Lentinan. In contrast, the signal transduction of β-glucans, in particular water-insoluble zymosan or yeast glucan from Saccharomyces cerevisiae as a model of immunomodulatory glucans, has been partly elucidated (
      • Gross O.
      • Gewies A.
      • Finger K.
      • Schäfer M.
      • Sparwasser T.
      • Peschel C.
      • Förster I.
      • Ruland J.
      ,
      • Li B.
      • Allendorf D.J.
      • Hansen R.
      • Marroquin J.
      • Ding C.
      • Cramer D.E.
      • Yan J.
      ). It has been reported that, after activation of receptor dectin-1 by zymosan, spleen tyrosine kinase (Syk) is recruited and induces the assembly of a scaffold consisting of the Card9 (caspase recruitment domain 9) protein and the adaptor proteins Bcl10 and Malt1; the Card9-Bcl10-Malt1 scaffold then couples dectin-1 to the canonical NF-κB pathway by activation of the IκB kinase complex, leading to nuclear translocation of NF-κB subunit p65 (
      • Gross O.
      • Gewies A.
      • Finger K.
      • Schäfer M.
      • Sparwasser T.
      • Peschel C.
      • Förster I.
      • Ruland J.
      ). Yan and co-workers have demonstrated that yeast glucan particles amplify phagocyte killing of iC3b-opsonized tumor cells via the complement receptor 3-Syk-PI3K pathway (
      • Li B.
      • Allendorf D.J.
      • Hansen R.
      • Marroquin J.
      • Ding C.
      • Cramer D.E.
      • Yan J.
      ) and activate murine resident macrophages to secrete proinflammatory cytokines, including TNF-α, MCP-1 (monocyte chemotactic protein-1), and IL-6, through MyD88- and Syk-dependent pathways (
      • Li B.
      • Cramer D.
      • Wagner S.
      • Hansen R.
      • King C.
      • Kakar S.
      • Ding C.
      • Yan J.
      ). Therefore, in this study, we explored activation of RAW 264.7 macrophages induced by Lentinan to clarify the signal transduction pathways used by Lentinan.

      DISCUSSION

      In this work, β-glucan from L. edodes (LNT-S) was shown to stimulate murine RAW 264.7 macrophage cells by activation of MAPKs ERK1/2 and JNK1/2 and transcription factor NF-κB (Fig. 1). Moreover, activation occurred in a time- and concentration-dependent manner. Western blotting indicated that the nuclear titer of NF-κB p65 increased in response to LPS and LNT-S treatment of the RAW 264.7 cells (Fig. 1B), which was probably due to its translocation from the cytoplasm. The increase in NF-κB-luciferase activity further demonstrated that the nuclear transcription factor was really activated by LNT-S. LPS is known to induce MAPK-dependent phosphorylation, thereby activating multiple transcription factors to translocate to the nucleus, facilitating DNA-binding activity, and leading to up-regulation of iNOS expression (
      • Kim J.W.
      • Kim C.
      ,
      • Tebo J.M.
      • Chaoqun W.
      • Ohmori Y.
      • Hamilton T.A.
      ). In particular, NF-κB is a major activator for TNF-α production in macrophages and a central target for activators or inhibitors of iNOS expression (
      • Ghosh S.
      • May M.J.
      • Kopp E.B.
      ,
      • Xie Q.W.
      • Kashiwabara Y.
      • Nathan C.
      ). For example, zymosan-induced TNF-α production in RAW 264.7 macrophage cells is associated essentially with activation of NF-κB, similar to LPS (
      • Young S.H.
      • Ye J.
      • Frazer D.G.
      • Shi X.
      • Castranova V.
      ). However, as shown in Figs. 1A and 3, LNT-S showed a major difference compared with LPS and zymosan; LNT-S could not induce iNOS expression or production of NO and TNF-α regardless of its activation of MAPKs (ERK1/2 and JNK1/2) and NF-κB. This phenomenon is very similar to that of water-soluble PGG-glucan, a β-(1,3)-glucan with β-(1,6)-branches that activates NF-κB p65 without inducing inflammatory cytokines such as TNF-α and IL-6 (
      • Adams D.S.
      • Pero S.C.
      • Petro J.B.
      • Nathans R.
      • Mackin W.M.
      • Wakshull E.
      ). This was attributed to the fact that PGG-glucan increased the titer of only p65, but not other NF-κB subunits such as p50, p52, p68, and p75, and that p65 formed a heterodimer complex (called NF-κB-like transcription factor) with another unknown unit.
      It is well known that the activated form of NF-κB is a heterodimer, which usually consists of two proteins, a p65 (also called RelA) subunit and a p50 subunit. Other subunits, including C-Rel, RelB, v-Rel, and p52, may also be part of activated NF-κB (
      • Adams D.S.
      • Pero S.C.
      • Petro J.B.
      • Nathans R.
      • Mackin W.M.
      • Wakshull E.
      ,
      • Gringhuis S.I.
      • den Dunnen J.
      • Litjens M.
      • van der Vlist M.
      • Wevers B.
      • Bruijns S.C.
      • Geijtenbeek T.B.
      ). Of these, the classic p65/p50 heterodimer complex has been demonstrated to be involved in the transcriptional regulation of the proinflammatory cytokines IL-1β and TNF-α (
      • Baldwin Jr., A.S.
      ,
      • Sweet M.J.
      • Hume D.A.
      ,
      • Barnes P.J.
      • Karin M.
      ) and the proinflammatory mediator NO. On the basis of the fact discussed above and the study of PGG-glucan, we can attribute this to the following: LNT-S increased the titer of p65, which could not form a heterodimer with p52 but maybe a complex called NF-κB-like transcription factor with another unknown unit and was not sufficient to stimulate inflammatory cytokine mRNA transcription and cytokine production. Whether this is true or not will be determined in future work.
      As we know, the well studied zymosan particle activates β-glucan receptor dectin-1, followed by recruiting Syk, leading to activation of NF-κB and proinflammatory cytokine production, whereas water-soluble PGG-glucan activates NF-κB without production of proinflammatory cytokines. LNT-S is more similar to PGG-glucan. Water solubility likely plays an important role in the regulation of immunomodulating activity. This work is one of the first reports demonstrating that Lentinan activates ERK1/2, JNK1/2, and NF-κB p65 without production of proinflammatory cytokines. Study of the upstream signaling pathway will be continued in our future work.
      In summary, our results suggest that the β-glucan extracted from L. edodes by ultrasonication directly stimulated macrophages by inducing phosphorylation of MAPKs ERK1/2 and JNK1/2, but not p38. Subunit p65 of nuclear transcription factor NF-κB was shown to translocate from the cytoplasm to the nucleus, and NF-κB-luciferase activity was also enhanced compared with the blank (PBS) and the positive control (LPS stimulation). However, TNF-α and NO were not produced in the supernatant of RAW 264.7 cells, which may be explained by the lack of formation of the active NF-κB p65/p50 heterodimer complex.

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