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/EBPβ-dependent pathway

Abstract

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.

Shear-induced Interleukin-6 Synthesis in Chondrocytes

“…Through the use of RNAi, we demonstrated the functional role of the NF-B p65 subunit in shear-induced IL-6 mRNA synthesis in human T/C-28a2 chondrocytes. In agreement with our previously published results (17), p65 knockdown did not affect the extent of shearinduced COX-2 expression in chondrocytes (data not shown), thereby suggesting that COX-2 is upstream of NF-B and eliminating the possibility of a feedback mechanism from NF-B to COX-2. The key role of NF-B in shear-induced IL-6 transcriptional activity is further demonstrated by the use of wild-type and NF-B mutated IL-6 promoterreporter constructs.…”

“…In view of previous observations showing a positive correlation between endogenous COX-2-mediated PGE 2 production and IL-6 synthesis (24, 25), we first evaluated the effects of shear stress on COX-2 expression and formation of cAMP, because PGE 2 is a well-known activator of the cAMP signaling pathway. In accord with prior data (17), high shear stress (20 dyn/cm 2 ) rapidly induces COX-2 mRNA (Fig. 1A) and protein (Fig.…”

“…Transfected cells were allowed to recover for at least 12 h in growth medium and then incubated overnight in medium containing 1% Nutridoma-SP before their exposure to shear or static conditions. In promoter activity experiments, luciferase activities were measured by using the Dual-Luciferase Reporter Assay kit (Promega), as previously described (5,17).…”

“…Preparation of Cytosolic and Nuclear Extracts-Cytosolic and nuclear extracts were isolated using the NE-PER nuclear and cytoplasmic extraction kit (Pierce) following the manufacturer's instructions as previously described (17).…”

An integrated microfluidic device for characterizing chondrocyte metabolism in response to distinct levels of fluid flow stimulus

“…It seemed that there was a threshold level between 3.1 and 7.5 dyne/ cm 2 . Previous study also found that COX-2 expression could be detected above the threshold level of 5 dyne/cm 2 in chondrocytes, which correlated well with our current results (Healy et al 2008). Therefore, our finding suggested that moderate level of stimulation could also result in detrimental cytokine release in chondrocytes.…”