TY - JOUR
T1 - Elucidation of the signaling network of COX-2 induction in sheared chondrocytes
T2 - COX-2 is induced via a Rac/MEKK1/MKK7/JNK2/c-Jun-C/EBPβ- dependent pathway
AU - Healy, Zachary R.
AU - Zhu, Fei
AU - Stull, Joshua D.
AU - Konstantopoulos, Konstantinos
PY - 2008/5
Y1 - 2008/5
N2 - 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/EBPβ, but not C/EBPα or C/EBPδ, binds to the C/EBP site, whereas c-Jun binds to AP-1. Individual gene knockdown experiments demonstrate the direct regulation of C/EBPβ expression by c-Jun, and the critical roles of both c-Jun and C/EBPβ 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 NH2-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.
AB - 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/EBPβ, but not C/EBPα or C/EBPδ, binds to the C/EBP site, whereas c-Jun binds to AP-1. Individual gene knockdown experiments demonstrate the direct regulation of C/EBPβ expression by c-Jun, and the critical roles of both c-Jun and C/EBPβ 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 NH2-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.
KW - Cyclooxygenase
KW - Enhancer-binding protein
KW - Mechanobiology
KW - Mitogen-activated protein kinase kinase
KW - Shear stress
KW - Signal transduction
KW - c-Jun NH -terminal kinase
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U2 - 10.1152/ajpcell.00542.2007
DO - 10.1152/ajpcell.00542.2007
M3 - Article
C2 - 18367585
AN - SCOPUS:48249114442
SN - 0363-6143
VL - 294
SP - C1146-C1157
JO - American Journal of Physiology - Cell Physiology
JF - American Journal of Physiology - Cell Physiology
IS - 5
ER -