Shear-induced interleukin-6 synthesis in chondrocytes: roles of E prostanoid (EP) 2 and EP3 in cAMP/protein kinase A- and PI3-K/Akt-dependent NF-kappaB activation.
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.
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Pu
P
Wang
Fei
F
Zhu
Norman H
NH
Lee
Konstantinos
K
Konstantopoulos
D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D015232:Dinoprostone / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism*; D018078:Receptors, Prostaglandin E / Q000378:metabolism*; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058308:Receptors, Prostaglandin E, EP3 Subtype; D015398:Signal Transduction; D013314:Stress, Mechanical
D015850:Interleukin-6; D016328:NF-kappa B; C545644:PTGER2 protein, human; C545647:PTGER3 protein, human; D018078:Receptors, Prostaglandin E; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058308:Receptors, Prostaglandin E, EP3 Subtype; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D015232:Dinoprostone