title: Inhibition of BMP2-induced bone formation by the p65 subunit of NF-κB via an interaction with Smad4.
abstract: Bone morphogenic proteins (BMPs) stimulate bone formation in vivo and osteoblast differentiation in vitro via a Smad signaling pathway. Recent findings revealed that the activation of nuclear factor-κB (NF-κB) inhibits BMP-induced osteoblast differentiation. Here, we show that NF-κB inhibits BMP signaling by directly targeting the Smad pathway. A selective inhibitor of the classic NF-κB pathway, BAY11-770682, enhanced BMP2-induced ectopic bone formation in vivo. In mouse embryonic fibroblasts (MEFs) prepared from mice deficient in p65, the main subunit of NF-κB, BMP2, induced osteoblastic differentiation via the Smad complex to a greater extent than that in wild-type MEFs. In p65(-/-) MEFs, the BMP2-activated Smad complex bound much more stably to the target element than that in wild-type MEFs without affecting the phosphorylation levels of Smad1/5/8. Overexpression of p65 inhibited BMP2 activity by decreasing the DNA binding of the Smad complex. The C-terminal region, including the TA2 domain, of p65 was essential for inhibiting the BMP-Smad pathway. The C-terminal TA2 domain of p65 associated with the MH1 domain of Smad4 but not Smad1. Taken together, our results suggest that p65 inhibits BMP signaling by blocking the DNA binding of the Smad complex via an interaction with Smad4. Our study also suggests that targeting the association between p65 and Smad4 may help to promote bone regeneration in the treatment of bone diseases.
authors.affiliation: Department of Health Improvement (S.H.-T., G.S., C.N., S.K., H.T., E.J.) and Department of Oral Function (S.H.-T., T.M., C.K.), Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580, Japan; Department of Physiological Science and Molecular Biology (H.F.), Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan; Division of Pathophysiology (T.K., S.O., M.S.), Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka-shi, Saitama 350-1241, Japan; Section of Pharmacology (K.N., K.A., K.O.), Department of Bio-Matrix, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan; Technology and Development Team for BioSignal Program (T.D.), Subteam for BioSignal Integration, RIKEN BioResource Center, Tsukuba-shi, Ibaraki 305-0074, Japan; Laboratory of Molecular and Cellular Biochemistry (M.H.), Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; and Center for Oral Biological Research (C.K., E.J.), Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580, Japan.
authors.forename: Shizu
authors.initials: S
authors.lastname: Hirata-Tsuchiya
authors.forename: Hidefumi
authors.initials: H
authors.lastname: Fukushima
authors.forename: Takenobu
authors.initials: T
authors.lastname: Katagiri
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ohte
authors.forename: Masashi
authors.initials: M
authors.lastname: Shin
authors.forename: Kenichi
authors.initials: K
authors.lastname: Nagano
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Aoki
authors.forename: Takahiko
authors.initials: T
authors.lastname: Morotomi
authors.forename: Goro
authors.initials: G
authors.lastname: Sugiyama
authors.forename: Chihiro
authors.initials: C
authors.lastname: Nakatomi
authors.forename: Shoichiro
authors.initials: S
authors.lastname: Kokabu
authors.forename: Takahiro
authors.initials: T
authors.lastname: Doi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takeuchi
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ohya
authors.forename: Masamichi
authors.initials: M
authors.lastname: Terashita
authors.forename: Masato
authors.initials: M
authors.lastname: Hirata
authors.forename: Chiaki
authors.initials: C
authors.lastname: Kitamura
authors.forename: Eijiro
authors.initials: E
authors.lastname: Jimi
mesh_terms: D000818:Animals; D001846:Bone Development; D001847:Bone Diseases / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism*; D002454:Cell Differentiation / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism; D010012:Osteogenesis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D051901:Smad4 Protein / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000378:metabolism*
chemical_list: C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D011994:Recombinant Proteins; C498144:Rela protein, mouse; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D051996:Transcription Factor RelA