1 TNFα REPRESSES BMP SIGNALING BY INTERFERING WITH THE DNA BINDING OF SMADS THROUGH THE ACTIVATION OF NF-κ B Masato Yamazaki 1,2 , Hidefumi Fukushima 1 , Masashi Shin 1 , Takenobu Katagiri 3 , Takahiro Doi 4 , Tetsu Takahashi 2 and Eijiro Jimi 1,5 From the 1 Department of Biosciences and 2 Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu; 3 Division of Pathophysiology and Translational Research, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama; 4 Technology and Development Team for the BioSignal Program, Subteam for BioSignal Integration, RIKEN BioResource Center, Koyadai, Tsukuba; 5 Center for Oral Biological Research, Kyushu Dental College, Kitakyushu, Fukuoka,Japan Running title: TNFα inhibits BMP-induced osteoblast differentiation through NF-κB activation Address all correspondence to: Dr Eijiro Jimi, Division of Molecular Signaling and Biochemistry, Department of Biosciences, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580, Japan. Phone: +81-93-285-3047; Fax: +81-93-582-6000; Email: ejimi@kyu-dent.ac.jp Bone morphogenetic proteins (BMPs) induce not only bone formation in vivo but also osteoblast differentiation of mesenchymal cells in vitro. Tumor necrosis factor α (TNFα ) inhibits both osteoblast differentiation and bone formation induced by BMPs. However, the molecular mechanisms of these inhibitions remain unknown. In this study, we found that TNFα inhibited the alkaline phosphatase activity and markedly reduced BMP2- and Smad-induced reporter activity in MC3T3-E1 cells. TNFα had no effect on the phosphorylation of Smad1, Smad5 and Smad8 or on the nuclear translocation of the Smad1-Smad4 complex. In p65-deficient mouse embryonic fibroblasts, overexpression of p65, a subunit of NF-κ B, inhibited BMP2- and Smad-induced reporter activity in a dose-dependent manner. Furthermore, this p65-mediated inhibition of BMP2- and Smad-responsive promoter activity was restored after inhibition of NF-κ B by the overexpression of the dominant negative Iκ Bα . Although TNFα failed to affect receptor-dependent formation of the Smad1-Smad4 complex, p65 associated with the complex. Chromatin immunoprecipitation and electrophoresis mobility shift assays revealed that TNFα suppressed the DNA binding of Smad proteins to the target gene. Importantly, the specific NF-κ B inhibitor, BAY11-7082, abolished these phenomena. These results suggest that TNFα inhibits BMP signaling by interfering with the DNA binding of Smads through the activation of NF-κ B. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β superfamily (TGF-β) that were originally identified by their ability to induce ectopic bone formation when implanted into muscle tissue (1, 2). BMP signaling is transduced by two types of transmembrane serine-threonine kinase receptor, type I and type II (3, 4). After type II receptors phosphorylate type I receptors in a ligand-dependent fashion, activated type I receptors phosphorylate downstream molecules in the cytoplasm. After BMP type I receptors phosphorylate Smad1, Smad5 and Smad8 (Smad1,5,8), the three Smads form heteromeric complexes with Smad4 and other transcription factors. These complexes translocate into the http://www.jbc.org/cgi/doi/10.1074/jbc.M109.070540 The latest version is at JBC Papers in Press. Published on October 23, 2009 as Manuscript M109.070540 Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc. by guest, on February 19, 2013 www.jbc.org Downloaded from