Elucidation of the signaling network of COX-2 induction in sheared chondrocytes: COX-2 is induced via a Rac/MEKK1/MKK7/JNK2/c-Jun-C/EBPbeta-dependent pathway.
Shear stress is a pathophysiologically relevant mechanical signal in cartilage biology and tissue engineering. Cyclooxygenase-2 (COX-2) is a pivotal proinflammatory enzyme, which is induced by mechanical loading-derived shear stress in chondrocytes. In the present study, we investigated the transcriptional machinery and signaling pathway regulating shear-induced COX-2 expression in human chondrocytic cells. Deletion and mutation analyses of the human cox-2 promoter reveal that the CCAAT/enhancer-binding protein (C/EBP) and activator protein-1 (AP-1) predominantly contribute to the shear-induced cox-2 promoter activity. Supershift assays disclose that C/EBPbeta, but not C/EBPalpha or C/EBPdelta, binds to the C/EBP site, whereas c-Jun binds to AP-1. Individual gene knockdown experiments demonstrate the direct regulation of C/EBPbeta expression by c-Jun, and the critical roles of both c-Jun and C/EBPbeta in shear-induced COX-2 synthesis. Our studies also indicate that Rac and, to a lesser extent, Cdc42 transactivate MEKK1, which is, in turn, responsible for activation of mitogen-activated protein kinase kinase 7 (MKK7). MKK7 regulates c-Jun NH(2)-terminal kinase 2 activation, which, in turn, triggers the phosphorylation of c-Jun that controls shear-mediated COX-2 upregulation in chondrocytes. Reconstructing the signaling network regulating shear-induced COX-2 expression and inflammation may provide insights to optimize conditions for culturing artificial cartilage in bioreactors and for developing therapeutic interventions for arthritic disorders.
Healy
Zachary R
ZR
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA.
Zhu
Fei
F
Stull
Joshua D
JD
Konstantopoulos
Konstantinos
K
D001665:Binding Sites; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002467:Cell Nucleus / Q000201:enzymology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000201:enzymology*; D051546:Cyclooxygenase 2 / Q000096:biosynthesis* / Q000235:genetics; D004252:DNA Mutational Analysis; D004790:Enzyme Induction; D006801:Humans; D048688:MAP Kinase Kinase 7 / Q000378:metabolism*; D048728:MAP Kinase Kinase Kinase 1 / Q000378:metabolism*; D010957:Plasmids; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D015398:Signal Transduction
D022782:CCAAT-Enhancer-Binding Protein-beta; D016755:Proto-Oncogene Proteins c-jun; D051546:Cyclooxygenase 2; D051057:Proto-Oncogene Proteins c-akt; D048728:MAP Kinase Kinase Kinase 1; D048688:MAP Kinase Kinase 7; C482569:MAP2K7 protein, human