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Regulation of MyD88 Aggregation and the MyD88-dependent Signaling Pathway by Sequestosome 1 and Histone Deacetylase 6*

  • Takeshi Into
    Correspondence
    To whom correspondence should be addressed: Dept. of Oral Microbiology, Asahi University School of Dentistry, 1851-1 Hozumi, Mizuho, Gifu 501-0296, Japan. Tel./Fax: 81-58-329-1422;
    Affiliations
    From the Department of Oral Microbiology, Asahi University School of Dentistry, 1851-1 Hozumi, Mizuho, Gifu 501-0296,
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  • Megumi Inomata
    Affiliations
    From the Department of Oral Microbiology, Asahi University School of Dentistry, 1851-1 Hozumi, Mizuho, Gifu 501-0296,
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  • Shumpei Niida
    Affiliations
    the Laboratory of Genomics and Proteomics, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8522, and
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  • Yukitaka Murakami
    Affiliations
    From the Department of Oral Microbiology, Asahi University School of Dentistry, 1851-1 Hozumi, Mizuho, Gifu 501-0296,
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  • Ken-ichiro Shibata
    Affiliations
    the Laboratory of Oral Molecular Microbiology, Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, Sapporo 060-8586, Japan
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  • Author Footnotes
    * This work was supported by the Science Research Promotion Fund (2010) (to T. I.) from the Promotion and Mutual Aid Corporation for Private Schools of Japan and a grant from the Miyata Science Research Foundation in Asahi University (to T. I.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.
Open AccessPublished:September 13, 2010DOI:https://doi.org/10.1074/jbc.M110.126904
      MyD88 is an essential adaptor molecule for Toll-like receptors (TLRs) and interleukin (IL)-1 receptor. MyD88 is thought to be present as condensed forms or aggregated structures in the cytoplasm, although the reason has not yet been clear. Here, we show that endogenous MyD88 is present as small speckle-like condensed structures, formation of which depends on MyD88 dimerization. In addition, formation of large aggregated structures is related to cytoplasmic accumulation of sequestosome 1 (SQSTM1; also known as p62) and histone deacetylase 6 (HDAC6), which are involved in accumulation of polyubiquitinated proteins. A gene knockdown study revealed that SQSTM1 and HDAC6 were required for MyD88 aggregation and exhibited a suppressive effect on TLR ligand-induced expression of IL-6 and NOS2 in RAW264.7 cells. SQSTM1 and HDAC6 were partially involved in suppression of several TLR4-mediated signaling events, including activation of p38 and JNK, but they hardly affected degradation of IκBα (inhibitor of nuclear factor κB). Biochemical induction of MyD88 oligomerization induced recruitment of SQSTM1 and HDAC6 to the MyD88-TRAF6 signaling complex. Repression of SQSTM1 and HDAC6 enhanced formation of the MyD88-TRAF6 complex and conversely decreased interaction of the ubiquitin-specific negative regulator CYLD with the complex. Furthermore, ubiquitin-binding regions on SQSTM1 and HDAC6 were essential for MyD88 aggregation but were not required for interaction with the MyD88 complex. Thus, our study reveals not only that SQSTM1 and HDAC6 are important determinants of aggregated localization of MyD88 but also that MyD88 activates a machinery of polyubiquitinated protein accumulation that has a modulatory effect on MyD88-dependent signal transduction.

      Introduction

      MyD88 was originally identified as an inducible protein during terminal differentiation of M1 myeloleukemic cells upon interleukin (IL)-6 stimulation (
      • Lord K.A.
      • Abdollahi A.
      • Hoffman-Liebermann B.
      • Liebermann D.A.
      ). The essential function of MyD88 was later revealed to be a universal adaptor molecule for type 1 IL-1 receptor (IL-1R)
      The abbreviations used are: IL-1R
      IL-1 receptor
      EGFP
      enhanced green fluorescent protein
      ODN
      oligodeoxynucleotide
      Pam3CSK4
      the synthetic bacterial lipopeptide N-palmitoyl-(S)-dipalmitoylglyceryl-CSKKKK
      qRT-PCR
      quantitative reverse transcription-coupled PCR
      TLR
      Toll-like receptor
      UBA
      ubiquitin-associated
      BUZ
      bound to ubiquitin zinc finger.
      and Toll-like receptors (TLRs) (
      • Muzio M.
      • Ni J.
      • Feng P.
      • Dixit V.M.
      ,
      • Wesche H.
      • Henzel W.J.
      • Shillinglaw W.
      • Li S.
      • Cao Z.
      ,
      • Medzhitov R.
      • Preston-Hurlburt P.
      • Kopp E.
      • Stadlen A.
      • Chen C.
      • Ghosh S.
      • Janeway Jr., C.A.
      ). MyD88 is composed of three distinct regions, an N-terminal death domain, an intermediary domain, and a Toll/IL-1 homology domain at the C terminus (
      • Hardiman G.
      • Jenkins N.A.
      • Copeland N.G.
      • Gilbert D.J.
      • Garcia D.K.
      • Naylor S.L.
      • Kastelein R.A.
      • Bazan J.F.
      ). After receptor ligation, MyD88 interacts with the Toll/IL-1 homology domain of IL-1R/TLRs and then activates the signaling pathway through dimerization and utilizing death domain-containing IL-1R-associated kinases (
      • Muzio M.
      • Ni J.
      • Feng P.
      • Dixit V.M.
      ,
      • Akira S.
      • Uematsu S.
      • Takeuchi O.
      ,
      • O'Neill L.A.
      • Bowie A.G.
      ). This pathway is further activated through the ubiquitin E3 ligase TRAF6 (tumor necrosis factor (TNF) receptor-associated factor 6) that works together with a ubiquitin-conjugating enzyme complex consisting of UBC13 and UEV1A to catalyze Lys63-linked polyubiquitination, which then activates the TAK1 kinase. TAK1 activates IκB kinases and cascades of mitogen-activated protein kinases (MAPKs), ultimately leading to early phase activation of nuclear factor (NF)-κB and AP-1 and the transcription of genes encoding various proinflammatory mediators, such as TNF, NOS2 (nitric-oxide synthase 2), and IL-6 (
      • Akira S.
      • Uematsu S.
      • Takeuchi O.
      ,
      • O'Neill L.A.
      • Bowie A.G.
      ).
      In contrast to many findings in the signaling function of MyD88, the cellular distribution of MyD88 has still been unclear. Most studies on the subcellular localization have shown that MyD88 is present as condensed forms, such as discrete foci, fibrillar aggregates, and inclusion bodies, in the cytoplasm (
      • Kawai T.
      • Sato S.
      • Ishii K.J.
      • Coban C.
      • Hemmi H.
      • Yamamoto M.
      • Terai K.
      • Matsuda M.
      • Inoue J.
      • Uematsu S.
      • Takeuchi O.
      • Akira S.
      ,
      • Kagan J.C.
      • Medzhitov R.
      ,
      • Nishiya T.
      • Kajita E.
      • Horinouchi T.
      • Nishimoto A.
      • Miwa S.
      ,
      • Jaunin F.
      • Burns K.
      • Tschopp J.
      • Martin T.E.
      • Fakan S.
      ). Nishiya et al. (
      • Nishiya T.
      • Kajita E.
      • Horinouchi T.
      • Nishimoto A.
      • Miwa S.
      ) found that aggregated structures of MyD88 have irregular morphologies and do not reside in known particular organelles. They also showed that the entire molecule except the Toll/IL-1 homology domain was required for forming the structures. However, because overexpressed MyD88 automatically induces death domain-dependent activation of downstream signaling pathways (
      • Medzhitov R.
      • Preston-Hurlburt P.
      • Kopp E.
      • Stadlen A.
      • Chen C.
      • Ghosh S.
      • Janeway Jr., C.A.
      ,
      • Nishiya T.
      • Kajita E.
      • Horinouchi T.
      • Nishimoto A.
      • Miwa S.
      ,
      • Aliprantis A.O.
      • Yang R.B.
      • Weiss D.S.
      • Godowski P.
      • Zychlinsky A.
      ,
      • Häcker H.
      • Redecke V.
      • Blagoev B.
      • Kratchmarova I.
      • Hsu L.C.
      • Wang G.G.
      • Kamps M.P.
      • Raz E.
      • Wagner H.
      • Häcker G.
      • Mann M.
      • Karin M.
      ), it is still not known whether such condensed distribution is an artificial observation ascribed to overexpression.
      Here we show that endogenous MyD88 is present as a dimerized form to exhibit small speckle-like structures. In addition, the aggregated distribution is associated with the mechanism for protein accumulation by sequestosome 1 (SQSTM1; also known as p62) and histone deacetylase 6 (HDAC6). Both SQSTM1 and HDAC6 are important molecules for accumulation of Lys63-linked polyubiquitinated proteins to form protein aggregates, which ultimately leads to lysosomal degradation by autophagy (
      • Ding W.X.
      • Yin X.M.
      ). Our results indicate that oligomerized MyD88 recruits SQSTM1 and HDAC6 to the signaling complex, which may be an important step for formation of aggregated structures. In addition, SQSTM1 and HDAC6 are involved in down-regulation of formation of the MyD88-TRAF6 complex and have a suppressive effect on the signal transduction. Thus, our results may provide novel information to elucidate the enigmatic subcellular localization and complex functioning of MyD88.

      DISCUSSION

      Our results suggest that MyD88 is physiologically present as a dimerized form and the formation of aggregated structures of MyD88 depends on its potential to activate machineries of protein accumulation through SQSTM1 and HDAC6. MyD88 can activate downstream signaling through the ubiquitin E3 ligase TRAF6, which is currently known to induce Lys63-linked polyubiquitination of TRAF6 itself and target proteins (
      • Deng L.
      • Wang C.
      • Spencer E.
      • Yang L.
      • Braun A.
      • You J.
      • Slaughter C.
      • Pickart C.
      • Chen Z.J.
      ). In accordance with this, overexpressed TRAF6 forms aggregated structures in the cytoplasm (
      • Wooten M.W.
      • Geetha T.
      • Babu J.R.
      • Seibenhener M.L.
      • Peng J.
      • Cox N.
      • Diaz-Meco M.T.
      • Moscat J.
      ,
      • Sanz L.
      • Diaz-Meco M.T.
      • Nakano H.
      • Moscat J.
      ). SQSTM1 and HDAC6 have been found to be important molecules for selective recognition of Lys63-linked polyubiquitination and their accumulation in the cytoplasm. Recent findings have provided important evidence that Lys63-linked polyubiquitination is a signaling event for the selective removal of misfolded proteins by autophagy (
      • Ding W.X.
      • Yin X.M.
      ,
      • Kirkin V.
      • McEwan D.G.
      • Novak I.
      • Dikic I.
      ). SQSTM1 mediates accumulation of polyubiquitinated proteins for formation of sequestosomes (inclusion bodies), whereas HDAC6 mediates formation of aggresomes (
      • Ding W.X.
      • Yin X.M.
      ,
      • Kirkin V.
      • McEwan D.G.
      • Novak I.
      • Dikic I.
      ). Although the biological importance of the formation of sequestosomes and aggresomes for cell signal transduction has not generally been understood, the present study revealed that SQSTM1 and HDAC6 suppress the formation of the MyD88-TRAF6 complex through regulation of CYLD recruitment and limit activation of several signaling events, especially activation of p38 and JNK. However, these molecules did not have a distinct regulatory effect on NF-κB signaling. Therefore, it is possible that the suppressive effects are more preferentially exerted through inhibition of MAPKKKs downstream of TRAF6, such as MEKK3, Tpl-2, and ASK1, than through inhibition of TAK1 that mediates NF-κB signaling. The functions of SQSTM1 and HDAC6 are summarized in Fig. 9. Such functions of these molecules may be useful to protect cells from strong extracellular stimuli or cytotoxic signaling because the MyD88-dependent signaling pathway is cytotoxic under certain cellular circumstances (
      • Aliprantis A.O.
      • Yang R.B.
      • Weiss D.S.
      • Godowski P.
      • Zychlinsky A.
      ,
      • Lehnardt S.
      • Wennekamp J.
      • Freyer D.
      • Liedtke C.
      • Krueger C.
      • Nitsch R.
      • Bechmann I.
      • Weber J.R.
      • Henneke P.
      ,
      • Peck-Palmer O.M.
      • Unsinger J.
      • Chang K.C.
      • Davis C.G.
      • McDunn J.E.
      • Hotchkiss R.S.
      ).
      Figure thumbnail gr9
      FIGURE 9Schematic of MyD88-induced protein accumulation pathways through SQSTM1 and HDAC6. See “Discussion” for details. Ub, ubiquitin.
      Our results indicate that the ability of TLRs to form MyD88 condensed structures is not so high. Although TLRs are thought to activate signaling pathways through MyD88 recruitment, it is possible that TLRs only transiently interact with MyD88, which may allow recycling of MyD88 to avoid aggregation (through SQSTM1 and HDAC6) and to induce further signal transduction. Alternatively, it is possible that MyD88 is rapidly degraded after TLR signal transduction, in which a specific ubiquitin E3 ligase may rapidly ubiquitinate MyD88. Recently, a novel ubiquitin E3 ligase, Nrdp1, was revealed to directly bind MyD88 for Lys48-linked polyubiquitination, then allowing its degradation (
      • Wang C.
      • Chen T.
      • Zhang J.
      • Yang M.
      • Li N.
      • Xu X.
      • Cao X.
      ). Lys48-linked ubiquitination is known to target substrates for proteasomal degradation rather than for autophagic degradation (
      • Sun S.C.
      ).
      It is increasingly becoming evident that the MyD88-dependent signaling pathway is intricately regulated by protein ubiquitination and deubiquitination. TRAF6 advances the signaling processes itself through autoubiquitination. A20 serves as a ubiquitin-editing enzyme through removing Lys63-linked polyubiquitin chains from TRAF6 and then ligating Lys48-linked polyubiquitin chains, leading to restriction of TLR responses (
      • Boone D.L.
      • Turer E.E.
      • Lee E.G.
      • Ahmad R.C.
      • Wheeler M.T.
      • Tsui C.
      • Hurley P.
      • Chien M.
      • Chai S.
      • Hitotsumatsu O.
      • McNally E.
      • Pickart C.
      • Ma A.
      ,
      • Wertz I.E.
      • O'Rourke K.M.
      • Zhou H.
      • Eby M.
      • Aravind L.
      • Seshagiri S.
      • Wu P.
      • Wiesmann C.
      • Baker R.
      • Boone D.L.
      • Ma A.
      • Koonin E.V.
      • Dixit V.M.
      ). TRIM30α, a ubiquitin E3 ligase, limits TLR signaling by targeting TBL2 and TBL3, causing their degradation (
      • Shi M.
      • Deng W.
      • Bi E.
      • Mao K.
      • Ji Y.
      • Lin G.
      • Wu X.
      • Tao Z.
      • Li Z.
      • Cai X.
      • Sun S.
      • Xiang C.
      • Sun B.
      ). CYLD, a member of the ubiquitin-specific protease family, negatively regulates TLR-induced inflammatory responses by deubiquitination of TRAF6 (
      • Yoshida H.
      • Jono H.
      • Kai H.
      • Li J.D.
      ,
      • Trompouki E.
      • Hatzivassiliou E.
      • Tsichritzis T.
      • Farmer H.
      • Ashworth A.
      • Mosialos G.
      ). In this study, we found that the Lys63-linked polyubiquitin-binding molecules SQSTM1 and HDAC6 separately serve as modulators for the MyD88-dependent signaling pathway through binding with the MyD88 signaling complex that contains TRAF6 and CYLD. Our result shown in Fig. 6C suggests that SQSTM1 is more preferentially recruited to the MyD88 complex than HDAC6, probably through its higher affinity, because SQSTM1 repression enhanced HDAC6 recruitment to the complex. We found that HDAC6 regulates the early phase recruitment of TRAF6 and CYLD to the MyD88 signaling complex. It has recently been suggested that HDAC6 is rapidly translocated to the actin-enriched membrane ruffles after growth factor stimulation and subsequently associates with endocytic vesicles (macropinosomes) in an Hsp90-dependent manner (
      • Gao Y.S.
      • Hubbert C.C.
      • Lu J.
      • Lee Y.S.
      • Lee J.Y.
      • Yao T.P.
      ). For HDAC6 regulation of formation of the MyD88 signaling complex, such a mechanism may be deeply associated with the case of MyD88-dependent TLR signal transduction. Indeed, initiation of the MyD88 signaling has been shown to be induced in the site of membrane ruffles and endocytic vesicles in an actin-dependent manner (
      • Kagan J.C.
      • Medzhitov R.
      ). SQSTM1 seemed to function as a modulator of TRAF6 dissociation from the MyD88 signaling complex (Fig. 7C). Recently, SQSTM1 has been shown to be involved not only in binding with TRAF6 and CYLD but also in increased polyubiquitination and destabilization of IRF8 (
      • Wooten M.W.
      • Geetha T.
      • Babu J.R.
      • Seibenhener M.L.
      • Peng J.
      • Cox N.
      • Diaz-Meco M.T.
      • Moscat J.
      ,
      • Kim J.Y.
      • Ozato K.
      ), which ultimately results in attenuation of inflammatory responses. Several results of our study may support such conclusions.
      Protein aggregates are widely found in diseases in both the brain and the liver. These include Lewy bodies in Parkinson disease, neurofibrillary tangles in Alzheimer disease, and huntingtin aggregates. In the liver, Mallory bodies in steatohepatitis, hyaline bodies in hepatocellular carcinoma, and α1-antitrypsin aggregates are known. Importantly, all of these aggregates are attributed to accumulation of polyubiquitinated proteins, and polyubiquitinated protein-sequestering molecules, including SQSTM1 and HDAC6, have been found in the aggregates (
      • Komatsu M.
      • Waguri S.
      • Koike M.
      • Sou Y.S.
      • Ueno T.
      • Hara T.
      • Mizushima N.
      • Iwata J.
      • Ezaki J.
      • Murata S.
      • Hamazaki J.
      • Nishito Y.
      • Iemura S.
      • Natsume T.
      • Yanagawa T.
      • Uwayama J.
      • Warabi E.
      • Yoshida H.
      • Ishii T.
      • Kobayashi A.
      • Yamamoto M.
      • Yue Z.
      • Uchiyama Y.
      • Kominami E.
      • Tanaka K.
      ,
      • Kawaguchi Y.
      • Kovacs J.J.
      • McLaurin A.
      • Vance J.M.
      • Ito A.
      • Yao T.P.
      ,
      • Olzmann J.A.
      • Li L.
      • Chudaev M.V.
      • Chen J.
      • Perez F.A.
      • Palmiter R.D.
      • Chin L.S.
      ,
      • Kirkin V.
      • McEwan D.G.
      • Novak I.
      • Dikic I.
      ,
      • Bjørkøy G.
      • Lamark T.
      • Brech A.
      • Outzen H.
      • Perander M.
      • Overvatn A.
      • Stenmark H.
      • Johansen T.
      ,
      • Kirkin V.
      • Lamark T.
      • Sou Y.S.
      • Bjørkøy G.
      • Nunn J.L.
      • Bruun J.A.
      • Shvets E.
      • McEwan D.G.
      • Clausen T.H.
      • Wild P.
      • Bilusic I.
      • Theurillat J.P.
      • Øvervatn A.
      • Ishii T.
      • Elazar Z.
      • Komatsu M.
      • Dikic I.
      • Johansen T.
      ). Given that MyD88 has a capability to form aggregates through SQSTM1 and HDAC6, dysregulation of MyD88 expression may lead to development of diseases associated with polyubiquitinated protein aggregates.

      Acknowledgments

      We thank Margaret K. Offermann (Emory University School of Medicine), Michael A. Farrar (University of Minnesota), and Hans Häcker (Department of Infectious Diseases, St. Jude Children's Research Hospital) for providing DNA constructs used in this study.

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