Regulation of chondrocyte metabolism by transcription factor C/EBP families

2011 Fiscal Year Final Research Report

• Project/Area Number: 21591930
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Orthopaedic surgery
• Research Institution: The University of Tokyo
• Principal Investigator: MIURA Toshiki 東京大学, 医学部附属病院, 講師 (20376479)
• Co-Investigator(Kenkyū-buntansha): KAWAGUCHI Hiroshi 東京大学, 医学部附属病院, 准教授 (40282660) ; CHIKUDA Hirotaka 東京大学, 医学部附属病院, 助教 (30345219) ; ITOH Ideya 東京大学, 医学部附属病院, 助教 (30436464)
• Co-Investigator(Renkei-kenkyūsha): OGATA Naoshi 東京大学, 医学部附属病院, 講師 (10361495)
• Project Period (FY): 2009 – 2011
• Keywords: 骨 / 軟骨代謝学

Research Abstract

The endochondral ossification is a crucial process in normal skeletal growth and osteoarthritis(OA) development. Aiming at elucidation of the molecular mechanism underlying this process and identification of therapeutic targets for OA, we examined the signal network around CCAAT/enhancer-binding protein-β(C/EBP-β), a regulator of chondrocyte functions. Computational predictions and a C/EBP motif-reporter assay identified RUNX2 as the most potent transcriptional partner of C/EBP-βin chondrocytes.
Both C/EBP-βand RUNX2 were co-expressed in the terminal differentiation stage of chondrocytes in several cell culture systems, in the mouse limb cartilage, and in the OA joint cartilage of mice and humans. To determine the involvement of C/EBP-βand RUNX2 in the endochondral ossification process, we generated their compound knockout mice : Cebpb^<-/->; Runx2^<+/-> and Cebpb^<+/->; Runx2^<+/->, because Runx2^<-/-> mice died just after birth. The Cebpb^<-/->; Runx2^<+/-> mice showed more severe dwa rfism than the Cebpb^<-/-> and Runx2^<+/-> littermates with impaired cartilage degradation but unaffected chondrocyte differentation. Although Cebpb^<+/->; Runx2^<+/-> mice showed normal skeletal growth, they exhibited greater suppression of knee OA development in surgical and age-related OA models than their Cebpb^<+/-> and Runx2^<+/-> littermates. Histological analyses in the limb cartilage and the OA joint cartilage showed that the compound knockout caused a considerable decrease of matrix metalloproteinase-13(MMP-13) among various factors related to endochondral ossification. In cultured chondrocytes, C/EBP-βand RUNX2 cooperatively enhanced endogenous expression and promoter activity of MMP13 shown by real-time RT-PCR and luciferase assay. The specific and direct binding of C/EBP-βand RUNX2 as a protein complex to a C/EBP-binding motif and an OSE2 motif in the MMP13 promoter was shown by EMSA and ChIP-reIP assay. Lastly, to identify the upstream mechanism, we performed a screening of transcription factors using a human CEBPB promoter fragment among putative factors regulating chondrocyte differentiation, and identified hypoxia-inducible factor-2α(HIF-2α) as the most potent and functional inducer of C/EBP-βexpression in chondrocytes through specific binding to a hypoxia-responsive element. In conclusion, C/EBP-βand RUNX2, with MMP-13 as the target and HIF-2αas the inducer, cooperatively control cartilage degradation. This molecular network in chondrocytes may represent a therapeutic target for OA.

Research Products

• [Journal Article] Lack of a chondroprotective effect of cyclooxygenase 2 inhibition in a surgically induced model of osteoarthritis in mice (2012)
  • Author(s): Fukai A, Kamekura S, Chikazu D, Nakagawa T, Hirata M, Saito T, Hosaka Y, Ikeda T, Nakamura K, Chung UI, and Kawaguchi H
  • Journal Title: Arthritis Rheum-US Volume: 64 Pages: 198-203
  • Peer Reviewed
• [Journal Article] C/EBPβand RUNX2 cooperate to degrade cartilage with MMP-13 as the target and HIF-2αas the inducer in chondrocytes (2012)
  • Author(s): Hirata M, Kugimiya F, Fukai A, Saito T, Yano F, Ikeda T, Mabuchi A, Sapkota BR, Akune T, Nishida N, Yoshimura N, Nakagawa T, Tokunaga K, Nakamura K, Chung UI, and Kawaguchi H
  • Journal Title: Hum Mol Genet Volume: 21 Pages: 1111-1123
  • Peer Reviewed
• [Journal Article] The Gαq signal in osteoblasts is inhibitory to the osteoanabolic action of PTH (2011)
  • Author(s): Ogata N, Shinoda Y, Wettschureck N, Offerman S, Takeda S, Nakamura K, Segre GV, Chung UI, and Kawaguchi H
  • Journal Title: J Biol Chem Volume: 286 Pages: 13733-13740
  • Peer Reviewed
• [Journal Article] Akt1 in murine chondrocytes controls cartilage calcification during endochondral ossification under physiologic and pathologic conditions (2010)
  • Author(s): Fukai A, Kawamura N, Saito T, Oshima Y, Ikeda T, Kugimiya F, Higashikawa H, Yano F, Ogata N, Nakamura K, Chung UI, and Kawaguchi H
  • Journal Title: Arthritis Rheum Volume: 62 Pages: 826-836
  • Peer Reviewed
• [Journal Article] Mechanisms underlying catabolic and anabolic functions of parathyroid hormone on bone by combination of culture systems of mouse cells (2010)
  • Author(s): Shinoda Y, Kawaguchi H, Higashikawa A, Hirata M, Miura T, Saito T, Nakamura K, Chung UI, and Ogata N
  • Journal Title: J Cell Biochem Volume: 109 Pages: 755-763
  • Peer Reviewed
• [Journal Article] Transcriptional regulation of endochondral ossification by HIF-2αduring skeletal growth and osteoarthritis development (2010)
  • Author(s): Saito T, Fukai A, Mabuchi A, Ikeda T, Yano F, Ohba S, Nishida N, Akune T, Yoshimura N, Nakagawa T, Nakamura K, Tokunaga K, Chung UI, and Kawaguchi H
  • Journal Title: Nat Med Volume: 16 Pages: 678-686
  • Peer Reviewed
• [Journal Article] Identification of the core element responsive to runt-related transcription factor 2 in the promoter of human type x collagen gene (2009)
  • Author(s): Higashikawa A, Saito T, Ikeda T, Kamekura S, Kawamura N, Kan A, Oshima Y, Ohba S, Ogata N, Takeshita K, Nakamura K, Chung UI, and Kawaguchi H
  • Journal Title: Arthritis Rheum Volume: 60 Pages: 166-178
  • Peer Reviewed
• [Journal Article] C/EBPβpromotes transition from proliferation to hypertrophic differentiation of chondrocytes through transactivation of p57^<Kip2> (2009)
  • Author(s): Hirata M, Kugimiya F, Fukai A, Ohba S, Kawamura N, Ogasawara T, Kawasaki Y, Saito T, Yano F, Ikeda T, Nakamura K, Chung UI, and Kawaguchi H
  • Journal Title: PLoS ONE Volume: 4 Pages: e4543
  • Peer Reviewed
• [Journal Article] Transcriptional induction of SOX9 by NF_<-κ> B family member RelA in chondrogenic cells (2009)
  • Author(s): Ushita M, Saito T, Ikeda T, Yano F, Higashikawa A, Ogata N, Chung UI, Nakamura K, and Kawaguchi H
  • Journal Title: Osteoarthritis Cartilage Volume: 17 Pages: 1065-1075
  • Peer Reviewed
• [Journal Article] Screening of chondrogenic factors by a real-time fluorescence monitoring cell line ATDC5-C2ER : Identification of sorting nexin 19 as a novel factor (2009)
  • Author(s): Kan A, Ikeda T, Saito T, Yano F, Fukai A, Ogata N, Nakamura K, Chung UI, and Kawaguchi H
  • Journal Title: Arthritis Rheum Volume: 60 Pages: 3314-3323
  • Peer Reviewed
• [Presentation] C/EBP-βand RUNX2 cooperatively control cartilage degradation with MMP-13 as the target and HIF-αas the inducer in chondrocytes (2011)
  • Author(s): Hirata M, Kugimiya F, Fukai A, Saito T, Mabuchi A, Sapkota BJ, Akune T, Yano F, Ikeda T, Nishida N, Yoshimura N, Nakagawa T, Nakamura K, Tokunaga K, Chung UI, Kawaguchi H
  • Organizer: 33th annual meeting of the American Society for Bone and Mineral Research(ASBMR)
  • Place of Presentation: San Diego, California, USA
  • Year and Date: 20110916-20
• [Presentation] Endochondral ossification signals as potential therapeutic targets for osteoarthritis(Invited Lecture) (2011)
  • Author(s): Kawaguchi H
  • Organizer: 2011 Gordon Research Conference
  • Place of Presentation: Ventura, CA, USA
  • Year and Date: 20110306-11
• [Presentation] Transcriptional induction of ADAMTS5 by NF_<-κ>Βfamily member RelA/p65 in chondrocytes during osteoarthritis development (2011)
  • Author(s): Kobayashi H, Hirata M, Itoh S, Saito T, Nakamura K, Chung UI, and Kawaguchi H
  • Organizer: 2011 Gordon Research Conference : Cartilage Biology & Pathology
  • Place of Presentation: Ventura, CA, USA
  • Year and Date: 20110306-11
• [Presentation] C/EBP-βand RUNX2 in chondrocytes control cartilage degradation (2011)
  • Author(s): Hirata M, Kugimiya F, Fukai A, Saito T, Mabuchi A, Sapkota BR, Akune T, Yano F, Ikeda T, Nishida N, Yoshimura N, Nakagawa T, Nakamura K, Tokunaga K, Chung UI, and Kawaguchi H
  • Organizer: 2011 Gordon Research Conference : Cartilage Biology & Pathology
  • Place of Presentation: Ventura, CA, USA
  • Year and Date: 20110306-11
• [Presentation] Overview of molecular backgrounds of osteoarthritis (2011)
  • Author(s): Kawaguchi H
  • Organizer: The 3rd International Cartilage and Osteoarthritis Symposium(ICOA 2011)(Keynote Lecture)
  • Place of Presentation: Suwon, Korea
  • Year and Date: 2011-07-05
• [Presentation] A local application of recombinant human fibroblast growth factor-2 for tibial shaft ractures : A randomized, double-blind, placebo-controlled trial (2010)
  • Author(s): Kawaguchi H, Oka H, Jingushi S, Izumi T, Fukunaga M, Sato K, Matsushita T, and Nakamura K
  • Organizer: 32th annual meeting of the American Society for Bone and Mineral Research(ASBMR)
  • Place of Presentation: Toronto, Canada
  • Year and Date: 20101015-19
• [Presentation] A Transcription factor p63 controls extensive steps of endochondral ossification through distinct functions of the isoforms(Young Investigator Award) (2010)
  • Author(s): Taniguchi Y, Saito T, Ikeda T, Chung UI, Nakamura H, Kawaguchi H
  • Organizer: 32th annual meeting of the American Society for Bone and Mineral Research(ASBMR)
  • Place of Presentation: Toronto, Canada
  • Year and Date: 20101015-19
• [Presentation] Endochondral ossification signal : A potential therapeutic target for osteoarthritis(invited lecture) (2010)
  • Author(s): Kawaguchi H
  • Organizer: 2010 World Congress on Osteoarthritis(OARSI)
  • Place of Presentation: Brussels, Belgium
  • Year and Date: 20100923-26
• [Presentation] HIF2A/NF-kappa B signal in chondrocytes controls extensive steps of osteoarthritis development in mice and humans(Young Investigator Award) (2010)
  • Author(s): Fukai A, Saito T, Mabuchi A, Ikeda T, Yano F, Ohba S, Nishida N, Akune T, Yoshimura N, Nakagawa T, Nakamura K, Tokunaga K, Chung UI, Kawaguchi H
  • Organizer: 2010 World Congress on Osteoarthritis(OARSI)
  • Place of Presentation: Brussels, Belgium
  • Year and Date: 20100923-26
• [Presentation] Molecular network on the C/EBP-beta axis including Runx2, MMP13, and HIF2A controls osteoarthritis development(Young Investigator Award). (2010)
  • Author(s): Hirata M, Kugimiya F, Fukai A, Saito T, Yano F, Ikeda T, Nakamura K, Chung UI, Kawaguchi H
  • Organizer: 2010 World Congress on Osteoarthritis(OARSI)
  • Place of Presentation: Brussels, Belgium
  • Year and Date: 20100923-26
• [Presentation] A randomized, double-blind, placebo-controlled trial of local application of recombinant human fibroblast growth factor-2 for tibial shaft fractures (2010)
  • Author(s): Kawaguchi H, Matsushita T, Oka H, Jingushi S, Izumi T, Fukunaga M, Sato K, Nakamura K
  • Organizer: 56th Annual Meeting of the Orthopaedic Research Society(ORS)
  • Place of Presentation: New Orleans, Louisiana, USA
  • Year and Date: 20100306-09
• [Presentation] Akt1 in chondrocytes controls cartilage calcification during skeletal growth and osteophyte formation in osteoarthritis (2010)
  • Author(s): Fukai A, Kawamura N, Saito T, Oshima Y, Ikeda T, Yano F, Ogata N, Nakamura K, Chung UI, Kawaguchi H
  • Organizer: 56th Annual Meeting of the Orthopaedic Research Society(ORS)
  • Place of Presentation: New Orleans, Louisiana, USA
  • Year and Date: 20100306-09
• [Presentation] Molecular backgrounds of cartilage degradation during osteoarthritis progression(Invited Lecture) (2010)
  • Author(s): Kawaguchi H
  • Organizer: 2010 Anti-aging and Well-being International Symposium
  • Place of Presentation: Daegu, Korea
  • Year and Date: 2010-02-19
• [Presentation] Therapeutic target molecules for cartilage degradation in osteoarthritis progression(Invited Lecture) (2010)
  • Author(s): Kawaguchi H
  • Organizer: System Biology and Drug Discovery
  • Place of Presentation: Seoul, Korea
  • Year and Date: 2010-02-09
• [Presentation] Comprehensive control of endochondral ossification by HIF-2αduring skeletal growth and osteoarthritis progression (2009)
  • Author(s): Saito T, Fukai A, Ikeda T, Yano F, Hirata M, Kan A, Nakamura K, Chung UI, and Kawaguchi H
  • Organizer: 31th annual meeting of the American Society for Bone and Mineral Research(ASBMR)
  • Place of Presentation: Denver, Colorado, USA
  • Year and Date: 20090911-15
• [Presentation] Distinct transcriptional control of chondrocyte hypertrophy and cartilage degeneration by C/EBP-beta and Runx2 during endochondral ossification (2009)
  • Author(s): Hirata M, Kugimiya F, Fukai A, Saito T, Kan A, Higashikawa A, Yano F, Ikeda T, Nakamura K, Chung UI, and Kawaguchi H
  • Organizer: 2009 World Congress on Osteoarthritis(OARSI)
  • Place of Presentation: Montreal, Quebec, Canada
  • Year and Date: 20090910-13
• [Presentation] Akt1 in chondrocytes controls cartilage calcification during osteophyte formation in osteoarthritis (2009)
  • Author(s): Fukai A, Kawamura N, Saito T, Ikeda T, Ogata N, Nakamura K, Chung UI, and Kawaguchi H
  • Organizer: 2009 World Congress on Osteoarthritis(OARSI)
  • Place of Presentation: Montreal, Quebec, Canada
  • Year and Date: 20090910-13
• [Presentation] Cyclic GMP-dependent protein kinase II promotes chondrocyte hypertrophy and skeletal growth (2009)
  • Author(s): Kawaguchi H, Chikuda H, Kawasaki Y, Hofmann F
  • Organizer: 4th International Conference on cGMP
  • Place of Presentation: Rosensburg, Germany
  • Year and Date: 20090619-21
• [Presentation] Molecular backgrounds of cartilage degeneration during osteoarthritis progression(Special Lecture : OARSI-KORS Co-sponsored Symposium) (2009)
  • Author(s): Kawaguchi H
  • Organizer: The 35th Annual Meeting of the Korean Orthopaedic Research Society
  • Place of Presentation: Seoul, Korea
  • Year and Date: 2009-10-14
• [Presentation] Transcriptional regulation of cartilage degeneration during osteoarthritis progression (2009)
  • Author(s): Kawaguchi H
  • Organizer: The 2nd International Cartilage and Osteoarthritis Symposium(Plenary Lecture)
  • Place of Presentation: Seoul, Korea
  • Year and Date: 2009-10-10
• [Remarks]
  • URL: http://www.u-tokyo-ortho.jp/