title: BMP-2 induces ATF4 phosphorylation in chondrocytes through a COX-2/PGE2 dependent signaling pathway.
abstract: OBJECTIVE
  Bone morphogenic protein (BMP)-2 is approved for fracture non-union and spine fusion. We aimed to further dissect its downstream signaling events in chondrocytes with the ultimate goal to develop novel therapeutics that can mimic BMP-2 effect but have less complications.
  
  
  METHODS
  BMP-2 effect on cyclooxygenase (COX)-2 expression was examined using Real time quantitative PCR (RT-PCR) and Western blot analysis. Genetic approach was used to identify the signaling pathway mediating the BMP-2 effect. Similarly, the pathway transducing the PGE2 effect on ATF4 was investigated. Immunoprecipitation (IP) was performed to assess the complex formation after PGE2 binding.
  
  
  RESULTS
  BMP-2 increased COX-2 expression in primary mouse costosternal chondrocytes (PMCSC). The results from the C9 Tet-off system demonstrated that endogenous BMP-2 also upregulated COX-2 expression. Genetic approaches using PMCSC from ALK2(fx/fx), ALK3(fx/fx), ALK6(-/-), and Smad1(fx/fx) mice established that BMP-2 regulated COX-2 through activation of ALK3-Smad1 signaling. PGE-2 EIA showed that BMP-2 increased PGE2 production in PMCSC. ATF4 is a transcription factor that regulates bone formation. While PGE2 did not have significant effect on ATF4 expression, it induced ATF4 phosphorylation. In addition to stimulating COX-2 expression, BMP-2 also induced phosphorylation of ATF4. Using COX-2 deficient chondrocytes, we demonstrated that the BMP-2 effect on ATF4 was COX-2-dependent. Tibial fracture samples from COX-2(-/-) mice showed reduced phospho-ATF4 immunoreactivity compared to wild type (WT) ones. PGE2 mediated ATF4 phosphorylation involved signaling primarily through the EP2 and EP4 receptors and PGE2 induced an EP4-ERK1/2-RSK2 complex formation.
  
  
  CONCLUSIONS
  BMP-2 regulates COX-2 expression through ALK3-Smad1 signaling, and PGE2 induces ATF4 phosphorylation via EP4-ERK1/2-RSK2 axis.
authors.affiliation: Department of Biochemistry, Rush University Medical Center, 1735 W. Harrison St, Chicago, IL 60612, USA; Department of Orthopaedics, Rush University Medical Center, 1611 W. Harrison St, Chicago, IL 60612, USA. Electronic address: tianfang_li@rush.edu.
authors.forename: T-F
authors.initials: TF
authors.lastname: Li
authors.affiliation: Department of Orthopaedics, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan; Center for Musculoskeletal Research, Department of Orthopaedics, University of Rochester, 601 Elmwood Ave., NY 14642, USA.
authors.forename: K
authors.initials: K
authors.lastname: Yukata
authors.affiliation: Department of Orthopaedics, The First Affiliated Hospital, Nanjing Medical University, 300 Guangzhou Rd., Nanjing, Jiangsu 210029, China.
authors.forename: G
authors.initials: G
authors.lastname: Yin
authors.affiliation: Center for Musculoskeletal Research, Department of Orthopaedics, University of Rochester, 601 Elmwood Ave., NY 14642, USA.
authors.forename: T
authors.initials: T
authors.lastname: Sheu
authors.affiliation: Department of Biomedical Genetics, Center for Oral Biology, and James P. Wilmot Cancer Center, University of Rochester, 601 Elmwood Ave., Rochester, NY 14642, USA.
authors.forename: T
authors.initials: T
authors.lastname: Maruyama
authors.affiliation: Center for Musculoskeletal Research, Department of Orthopaedics, University of Rochester, 601 Elmwood Ave., NY 14642, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Jonason
authors.affiliation: Department of Biomedical Genetics, Center for Oral Biology, and James P. Wilmot Cancer Center, University of Rochester, 601 Elmwood Ave., Rochester, NY 14642, USA.
authors.forename: W
authors.initials: W
authors.lastname: Hsu
authors.affiliation: Center for Musculoskeletal Research, Department of Orthopaedics, University of Rochester, 601 Elmwood Ave., NY 14642, USA.
authors.forename: X
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry, Rush University Medical Center, 1735 W. Harrison St, Chicago, IL 60612, USA.
authors.forename: G
authors.initials: G
authors.lastname: Xiao
authors.affiliation: Department of Medicine, Institute of Clinical Medicine, University of Helsinki, PO Box 700 (Haartmaninkatu 8, Biomedicum 1), 00029 HUS, Finland.
authors.forename: Y T
authors.initials: YT
authors.lastname: Konttinen
authors.affiliation: Department of Biochemistry, Rush University Medical Center, 1735 W. Harrison St, Chicago, IL 60612, USA.
authors.forename: D
authors.initials: D
authors.lastname: Chen
authors.affiliation: Center for Musculoskeletal Research, Department of Orthopaedics, University of Rochester, 601 Elmwood Ave., NY 14642, USA. Electronic address: Regis_OKeefe@urmc.rochester.edu.
authors.forename: R J
authors.initials: RJ
authors.lastname: O'Keefe
mesh_terms: D051701:Activating Transcription Factor 4 / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D010766:Phosphorylation; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction
chemical_list: D055396:Bone Morphogenetic Protein 2; D051701:Activating Transcription Factor 4; D051546:Cyclooxygenase 2; D015232:Dinoprostone
keywords: ATF4; BMP-2; COX-2; Chondrocytes