Intracellular HMGB1 transactivates the human IL1B gene promoter through association with an Ets transcription factor PU.1.
High mobility group box 1 protein (HMGB1), originally described as a non-histone, DNA binding protein, was recently identified as a late mediator of inflammation via its extracellular release from activated macrophages/monocytes. In the present study, we report that intracellular HMGB1 synergizes with a macrophage/monocyte-specific E26 transformation-specific sequence (Ets) transcription factor PU.1 to transactivate the promoter of the IL1B gene coding a 31-kDa proIL-1beta protein. The -131 to +12 IL1B promoter, which possesses a PU.1 binding motif essential for its transactivation, was induced when HMGB1 expression vector was transfected into murine RAW264.7 macrophage cells. Our glutathione S-transferase-pulldown and coimmunoprecipitation assays demonstrated direct physical interaction of HMGB1 with PU.1. Deletion of the PU.1 winged helix-turn-helix DNA-binding domain inhibited the association of the two proteins. In electrophoretic mobility shift assay using recombinant PU.1 protein, a ternary complex of PU.1, HMGB1 and PU.1-binding element within the IL1B promoter was generated. The importance of PU.1 was further supported by our observation that induction of the IL1B promoter was obtained only after PU.1 expression in PU.1-deficient murine EL4 thymoma cells. Thus, our data raise the possibility of a novel mechanism which sustains and amplifies inflammatory reactions through physical interaction of PU.1 with intracellular HMGB1 in macrophages/monocytes.
Mouri
Fumihiko
F
The first Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan.
Tsukada
Junichi
J
Mizobe
Takamitsu
T
Higashi
Takehiro
T
Yoshida
Yasuhiro
Y
Minami
Yasuhiro
Y
Izumi
Hiroto
H
Kominato
Yoshihiko
Y
Kohno
Kimitoshi
K
Tanaka
Yoshiya
Y
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D024243:HMGB1 Protein / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D053583:Interleukin-1beta / Q000235:genetics*; D008264:Macrophages; D051379:Mice; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D000099232:Proto-Oncogene Protein Spi-1
D024243:HMGB1 Protein; D053583:Interleukin-1beta; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1