Abstract
Mechanical overloading of cartilage producing hydrostatic stress, tensile strain, and fluid flow can adversely affect chondrocyte function and precipitate osteoarthritis (OA). Application of high fluid shear stress to chondrocytes recapitulates the earmarks of OA, as evidenced by the release of pro-inflammatory mediators, matrix degradation, and chondrocyte apoptosis. Elevated levels of cyclooxygenase-2 (COX-2), prostaglandin (PG) E(2), and interleukin (IL)-6 have been reported in OA cartilage in vivo, and in shear-activated chondrocytes in vitro. Although PGE(2) positively regulates IL-6 synthesis in chondrocytes, the underlying signaling pathway of shear-induced IL-6 expression remains unknown. Using the human T/C-28a2 chondrocyte cell line as a model system, we demonstrate that COX-2-derived PGE(2) signals via up-regulation of E prostanoid (EP) 2 and down-regulation of EP3 receptors to raise intracellular cAMP, and activate protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. PKA and PI3-K/Akt transactivate the NF-kappaB p65 subunit via phosphorylation at Ser-276 and Ser-536, respectively. Binding of p65 to the IL-6 promoter elicits IL-6 synthesis in sheared chondrocytes. Selective knockdown of EP2 or ectopic expression of EP3 blocks PKA- and PI3-K/Akt-dependent p65 activation and markedly diminishes shear-induced IL-6 expression. Similar inhibitory effects on IL-6 synthesis were observed by inhibiting PKA, PI3-K, or NF-kappaB using pharmacological and/or genetic interventions. Reconstructing the signaling network regulating shear-induced IL-6 expression in chondrocytes may provide insights for developing therapeutic strategies for arthritic disorders and for culturing artificial cartilage in bioreactors.
Highlights
Excessive chronic or repetitive mechanical loading of articular cartilage producing hydrostatic stress, tensile strain, and fluid flow [1] leads to irreversible cartilage erosion and osteoarthritic (OA)2 disease [2]
Shear Stress Induces IL-6 Expression in Human Chondrocytes via a COX-2-dependent Pathway— IL-6 expression has been detected in chondrocytes from OA but not membrane, and probed with a panel of specific antibodies
Preparation of Cytosolic and Nuclear Extracts—Cytosolic In light of accumulating evidence suggesting that OA is often and nuclear extracts were isolated using the NE-PER nuclear a consequence of abnormal mechanical forces [23], we here and cytoplasmic extraction kit (Pierce) following the manufac- aimed to delineate the mechanism by which shear stress turer’s instructions as previously described [17]
Summary
Excessive chronic or repetitive mechanical loading of articular cartilage producing hydrostatic stress, tensile strain, and fluid flow [1] leads to irreversible cartilage erosion and osteoarthritic (OA)2 disease [2]. With the selective COX-2 inhibitor NS398 (30 M) markedly suppresses both intracellular cAMP accumulation (Fig. 1C) and IL-6 mRNA expression (Fig. 1D), suggesting the potential involvement of cAMP in shear-induced IL-6 synthesis in human chondrocytes.
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