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BMP-2 induces ATF4 phosphorylation in chondrocytes through a COX-2/PGE2 dependent signaling pathway

  • Li, T.-F.
  • Yukata, K.
  • Yin, G.
  • Sheu, T.
  • Maruyama, T.
  • Jonason, J.H.
  • Hsu, W.
  • Zhang, X.
  • Xiao, G.
  • Konttinen, Y.T.
  • Chen, D.
  • O'Keefe, R.J.1, 2, 3, 2, 3, 4, 5, 3, 6, 3, 7, 8, 9, 10, 11, 6, 12, 13, 14
  • 1 Department of Biochemistry
  • 2 Rush University Medical Center
  • 3 Department of Orthopaedics
  • 4 University of Tokushima
  • 5 Center for Musculoskeletal Research
  • 6 University of Rochester
  • 7 The First Affiliated Hospital
  • 8 Nanjing Medical University
  • 9 Department of Biomedical Genetics
  • 10 Center for Oral Biology
  • 11 and James P. Wilmot Cancer Center
  • 12 Department of Medicine
  • 13 Institute of Clinical Medicine
  • 14 University of Helsinki
Published Article
Osteoarthritis and Cartilage
Publication Date
Jan 01, 2014
Accepted Date
Dec 20, 2013
DOI: 10.1016/j.joca.2013.12.020


ObjectiveBone 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. MethodsBMP-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. ResultsBMP-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 ALK2fx/fx, ALK3fx/fx, ALK6−/−, and Smad1fx/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. ConclusionsBMP-2 regulates COX-2 expression through ALK3–Smad1 signaling, and PGE2 induces ATF4 phosphorylation via EP4-ERK1/2-RSK2 axis.

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