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• W1989469359 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) E2, and interleukin (IL)-6 have been reported in OA cartilage in vivo, and in shear-activated chondrocytes in vitro. Although PGE2 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 PGE2 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-κB 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-κB 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. 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) E2, and interleukin (IL)-6 have been reported in OA cartilage in vivo, and in shear-activated chondrocytes in vitro. Although PGE2 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 PGE2 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-κB 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-κB 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. Excessive chronic or repetitive mechanical loading of articular cartilage producing hydrostatic stress, tensile strain, and fluid flow (1Carter D.R. Beaupré G.S. Wong M. Smith R.L. Andriacchi T.P. Schurman D.J. Clin. Orthop. Relat. Res. 2004; suppl. 427: S69-S77Crossref Scopus (267) Google Scholar) leads to irreversible cartilage erosion and osteoarthritic (OA) 2The abbreviations used are: OAosteoarthritisCOX-2cyclooxygenase-2PGE2prostaglandin E2EPE prostanoidIL-6interleukin-6PKAprotein kinase API3-Kphosphatidylinositol 3-kinaseGPCRG-protein-coupled receptorGiinhibitory GGsstimulatory GRASFsrheumatoid arthritis activated synovial fibroblastsGAPDHglyceraldehyde-3-phosphate dehydrogenaseAbantibody. disease (2Buckwalter J.A. Martin J.A. Brown T.D. Biorheology. 2006; 43: 603-609PubMed Google Scholar). Numerous in vitro studies support the concept that low fluid shear (<10 dyn/cm2) is chondroprotective (3Yokota H. Goldring M.B. Sun H.B. J. Biol. Chem. 2003; 278: 47275-47280Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), whereas high shear stress (>10 dyn/cm2) elicits the release of pro-inflammatory cytokines such as interleukin-6 (IL-6) (4Mohtai M. Gupta M.K. Donlon B. Ellison B. Cooke J. Gibbons G. Schurman D.J. Smith R.L. J. Ortho. Res. 1996; 14: 67-73Crossref PubMed Scopus (102) Google Scholar), and mediates matrix degradation (3Yokota H. Goldring M.B. Sun H.B. J. Biol. Chem. 2003; 278: 47275-47280Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) and chondrocyte cell death (5Healy Z.R. Lee N.H. Gao X. Goldring M.B. Talalay P. Kensler T.W. Konstantopoulos K. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14010-14015Crossref PubMed Scopus (110) Google Scholar, 6Zhu F. Wang P. Kontrogianni-Konstantopoulos A. Konstantopoulos K. Cell Death Differ. 2010; (in press)Google Scholar). Although OA is classified as a non-inflammatory joint disease, prostaglandins and cytokines are believed to play a role in the pathogenesis and progression of the disease. Prior work has shown that OA cartilage spontaneously releases prostaglandin (PG)E2 at 50-fold higher levels than in normal cartilage (7Amin A.R. Attur M. Patel R.N. Thakker G.D. Marshall P.J. Rediske J. Stuchin S.A. Patel I.R. Abramson S.B. J. Clin. Invest. 1997; 99: 1231-1237Crossref PubMed Scopus (369) Google Scholar). The superinduction of PGE2 is mediated by cyclooxygenase-2 (COX-2) protein, whose levels are markedly up-regulated in OA-affected cartilage (7Amin A.R. Attur M. Patel R.N. Thakker G.D. Marshall P.J. Rediske J. Stuchin S.A. Patel I.R. Abramson S.B. J. Clin. Invest. 1997; 99: 1231-1237Crossref PubMed Scopus (369) Google Scholar). The clinical correlates of OA are joint pain, dysfunction, and restricted motion. IL-6 has been implicated in pain signaling (8Brenn D. Richter F. Schaible H.G. Arthritis Rheum. 2007; 56: 351-359Crossref PubMed Scopus (189) Google Scholar). Moreover, IL-6 mRNA expression occurs in human chondrocytes from OA but not normal cartilage (4Mohtai M. Gupta M.K. Donlon B. Ellison B. Cooke J. Gibbons G. Schurman D.J. Smith R.L. J. Ortho. Res. 1996; 14: 67-73Crossref PubMed Scopus (102) Google Scholar). Catabolic and pro-inflammatory mediators such as PGE2 and IL-6 alter matrix homeostasis and participate in the destruction of articular cartilage, thereby contributing to OA. osteoarthritis cyclooxygenase-2 prostaglandin E2 E prostanoid interleukin-6 protein kinase A phosphatidylinositol 3-kinase G-protein-coupled receptor inhibitory G stimulatory G rheumatoid arthritis activated synovial fibroblasts glyceraldehyde-3-phosphate dehydrogenase antibody. Accumulating evidence suggests that IL-6 synthesis is positively regulated by either endogenous COX-2-derived or exogenously added PGE2 in many different cells including macrophages (9Chen B.C. Liao C.C. Hsu M.J. Liao Y.T. Lin C.C. Sheu J.R. Lin C.H. J. Immunol. 2006; 177: 681-693Crossref PubMed Scopus (74) Google Scholar), osteoblasts (10Takaoka Y. Niwa S. Nagai H. J. Biochem. 1999; 126: 553-558Crossref PubMed Scopus (29) Google Scholar), synovial (11Inoue H. Takamori M. Shimoyama Y. Ishibashi H. Yamamoto S. Koshihara Y. Br. J. Pharmacol. 2002; 136: 287-295Crossref PubMed Scopus (115) Google Scholar), and gingival (12Noguchi K. Shitashige M. Endo H. Kondo H. Ishikawa I. J. Periodontal Res. 2002; 37: 29-36Crossref PubMed Scopus (35) Google Scholar) fibroblasts. PGE2 exerts its biological functions via binding to four distinct transmembrane G-protein-coupled receptors (GPCRs) termed E prostanoid (EP) 1, EP2, EP3, and EP4. Following PGE2 binding, the EP receptors activate distinct intracellular signaling pathways, which may account for the pleiotropic effects of this prostaglandin. EP1 couples to Gq protein and raises intracellular calcium (13Sugimoto Y. Narumiya S. J. Biol. Chem. 2007; 282: 11613-11617Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar). EP2 and EP4 elevate intracellular cAMP levels by activating adenylate cyclase via stimulatory G (Gs) proteins (13Sugimoto Y. Narumiya S. J. Biol. Chem. 2007; 282: 11613-11617Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar). The major signaling pathway of the EP3 receptor is the inhibition of adenylate cyclase, and thus reduction of intracellular cAMP, via inhibitory G (Gi) proteins (13Sugimoto Y. Narumiya S. J. Biol. Chem. 2007; 282: 11613-11617Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar). Human chondrocytes primed with exogenous PGE2 synthesize IL-6 via an unknown signaling pathway (14Li X. Ellman M. Muddasani P. Wang J.H. Cs-Szabo G. van Wijnen A.J. Im H.J. Arthritis Rheum. 2009; 60: 513-523Crossref PubMed Scopus (128) Google Scholar). Although high fluid shear (16 dyn/cm2) has been reported to induce IL-6 mRNA and protein expression in human chondrocytes (4Mohtai M. Gupta M.K. Donlon B. Ellison B. Cooke J. Gibbons G. Schurman D.J. Smith R.L. J. Ortho. Res. 1996; 14: 67-73Crossref PubMed Scopus (102) Google Scholar), the underlying mechanism of this process has yet to be elucidated. Because OA is often a consequence of excessive mechanical forces (2Buckwalter J.A. Martin J.A. Brown T.D. Biorheology. 2006; 43: 603-609PubMed Google Scholar) and given that application of high fluid shear to chondrocytes recapitulates the earmarks of OA (3Yokota H. Goldring M.B. Sun H.B. J. Biol. Chem. 2003; 278: 47275-47280Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 5Healy Z.R. Lee N.H. Gao X. Goldring M.B. Talalay P. Kensler T.W. Konstantopoulos K. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14010-14015Crossref PubMed Scopus (110) Google Scholar, 6Zhu F. Wang P. Kontrogianni-Konstantopoulos A. Konstantopoulos K. Cell Death Differ. 2010; (in press)Google Scholar), we here delineate the signaling pathway of IL-6 induction in shear-activated human T/C-28a2 chondrocytes. Specifically, we demonstrate that COX-2-derived PGE2 signals via EP2 and EP3 receptors to regulate intracellular cAMP levels in chondrocytes, which in turn stimulate protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. PKA and PI3-K/Akt transactivate the NF-κB p65 subunit via phosphorylation at Ser-276 and Ser-536, respectively, which in turn binds to the IL-6 promoter and mediates IL-6 mRNA synthesis in shear-stimulated chondrocytes. The COX-2 selective inhibitor NS398, the EP and DP receptor antagonist AH6809, the selective agonist for EP3 receptor sulprostone, PGE2, forskolin, the PKA inhibitor H89, and the NF-κB inhibitor 6-amino-4-(4-phenoxyphenylethylamino) quinazoline (QNZ) were obtained from Enzo Life Sciences International Inc (Plymouth Meeting, PA). The EP3 and p65 cDNA plasmids were supplied from Origene Technologies (Rockville, MD) and subcloned to the pCMV6-XL vector. The IL-6 promoter reporter constructs pIL6-luc651 (-651/+1) and pIL6-luc651 ΔNF-κB (NF-κB site mutation) were gifts from Dr. Eickelberg (15Eickelberg O. Pansky A. Mussmann R. Bihl M. Tamm M. Hildebrand P. Perruchoud A.P. Roth M. J. Biol. Chem. 1999; 274: 12933-12938Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The PI3-K inhibitors, LY294002 and wortmannin, were from Sigma-Aldrich. p65 siRNA and antibodies specific for β-actin, COX-2, Akt, p-Akt (Ser-473), NF-κB p65, p-p65 (Ser-276), p-p65 (Ser-536), CREB, and p-CREB (Ser-133) were purchased from Cell Signaling Technology, Inc. (Danvers, MA). CREB1 and ATF4 siRNAs as well as monoclonal antibodies specific for p65 and IL-6 were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). The cAMP enzyme immunoassay kit and antibodies specific for EP2 and EP3 receptors were from Cayman Chemical. All reagents for qRT-PCR and SDS-PAGE experiments were purchased from Bio-Rad Laboratories. Reagents for electrophoretic mobility shift assays (EMSA) were obtained from Pierce Chemical Company. The Dual-Luciferase Reporter Assay kit was purchased from Promega (Madison, WI). All other reagents were from Invitrogen (Carlsbad, CA), unless otherwise specified. Human primary articular chondrocytes (Cell Applications) or T/C-28a2 chondrocytic cells were grown (37 °C in 5% CO2) on glass slides in F12/Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (5Healy Z.R. Lee N.H. Gao X. Goldring M.B. Talalay P. Kensler T.W. Konstantopoulos K. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14010-14015Crossref PubMed Scopus (110) Google Scholar, 16Abulencia J.P. Gaspard R. Healy Z.R. Gaarde W.A. Quackenbush J. Konstantopoulos K. J. Biol. Chem. 2003; 278: 28388-28394Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 17Healy Z.R. Zhu F. Stull J.D. Konstantopoulos K. Am. J. Physiol. Cell Physiol. 2008; 294: C1146-C1157Crossref PubMed Scopus (34) Google Scholar, 18Wang P. Zhu F. Konstantopoulos K. Am. J. Physiol. Cell Physiol. 2010; 298: C1445-C1456Crossref PubMed Scopus (89) Google Scholar). Before shear exposure, cells were incubated for 18 h in serum-free medium supplemented with 1% Nutridoma-SP (Roche), a low serum replacement that maintains chondrocyte phenotype and establishes quiescence in the monolayer (19Goldring M.B. Birkhead J.R. Suen L.F. Yamin R. Mizuno S. Glowacki J. Arbiser J.L. Apperley J.F. J. Clin. Invest. 1994; 94: 2307-2316Crossref PubMed Scopus (385) Google Scholar, 20Goldring M.B. Methods Mol. Med. 2004; 100: 37-52PubMed Google Scholar). Cells were then subjected to a shear stress level of 20 dyn/cm2 for prescribed periods of time in medium containing 1% Nutridoma-SP, using a streamer gold flow device (Flexcell International, Hillsborough, NC). In select experiments, the pharmacological agents were added to the medium at the indicated concentrations just before the onset of shear exposure. It is well established that transmission of the shear stress signal throughout the cell involves a complex interplay between cytoskeletal and biochemical constituents, and results in changes in structure, metabolism, and gene expression (21Papadaki M. Eskin S.G. Biotechnol. Prog. 1997; 13: 209-221Crossref PubMed Scopus (129) Google Scholar). For ectopic expression of EP3 receptor or p65, T/C-28a2 chondrocytes were transfected with 1.6 μg/slide of plasmid containing the EP3 or p65 cDNA by using Lipofectamine 2000. In control experiments, cells were transfected with 1.6 μg/slide of the empty vector pCMV6-XL (OriGene Technologies). In select experiments, T/C-28a2 cells were transfected with 1.6 μg/slide of the IL-6 promoter reporter construct pIL-6-luc651 or pIL6-luc651 ΔNF-κB. In RNA interference assays, T/C-28a2 cells were transfected with 100 nm siRNA oligonucleotide sequence specific for CREB1, ATF4, or p65. In control experiments, cells were transfected with 100 nm control siRNA. For EP2 shRNA experiments, T/C-28a2 chondrocytes were transfected with 1.6 μg/slide of plasmid containing the EP2 shRNA or scramble shRNA control. The following oligonucleotide sequences were used to specifically target the EP2 mRNA (underlined, sense and antisense sequences; boldface italicized, loop with linker): top strand, 5′-GATCCCCGATCCAGCTGCCTATTGATTTCAAGAGAATCAATAGGCAGCTGGATCTTTTTC-3′; bottom strand, 5′-TCGAGAAAAAGATCCAGCTGCCTATTGATTCTCTTGAAATCAATAGGCAGCTGGATCGGG-3′. Top and bottom strands were annealed, subcloned into the XhoI/BglII sites of pSuper, and the resulting construct sequence was verified. 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 (5Healy Z.R. Lee N.H. Gao X. Goldring M.B. Talalay P. Kensler T.W. Konstantopoulos K. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14010-14015Crossref PubMed Scopus (110) Google Scholar, 17Healy Z.R. Zhu F. Stull J.D. Konstantopoulos K. Am. J. Physiol. Cell Physiol. 2008; 294: C1146-C1157Crossref PubMed Scopus (34) Google Scholar). qRT-PCR assays were performed on the iCycler iQ detection system (Bio-Rad) using total RNA, the iScript one-step RT-PCR kit with SYBR green (Bio-Rad) and primers. The GenBankTM accession numbers and forward (F-) and reverse (R-) primers are as follows: Cox-2 (NM_000963), F-TGAGCATCTACGGTTTGCTG, R-AACTGCTCATCACCCCATTC; EP1 (NM_000955), F-TGGGCCTCTGGTTGTGCTTA, R-TTCGGCCTCCACCTTCTTTG; EP2 (NM_000956), F-CGTGCACCTACTTCGCTTTC, R-GAGGTCCCATTTTTCCTTTC; EP3 (NM_198712), F-TTCTGCACCCGCCTCAACCA, R-AGGAGAGCCCGAAAACAGTCAT; EP4 (NM_000958), F-TCGCGCAAGGAGCAGAAGGAGACG, R-GGACGGTGGCGAGAATGAGGAAGG; IL-6 (NM_000600), F-ATGAACTCCTTCTCCACAAGCGC, R-GAAGAGCCCTCAGGCTGGACT; CREB1 (NM_134442), F-CCAGGTATCTATGCCAGCAG, R-TCTGTGTTCCGGAGAAAAGTC; ATF4 (NM_182810), F-CATTCCTCGATTCCAGCAAAGCAC, R-TTCTCCAACATCCAATCTGTCCCG; p65 (NM_001145138), F-CTGCAGTTTGATGATGAAGA, R-TAGGCGAGTTATAGCCTCAG; GAPDH (NM_002046), F-CCACCCATGGCAAATTCCATGGCA, R-TCTAGACGGCAGGTCAGGTCCACC. GAPDH was used as internal control. Reaction mixtures were incubated at 50 °C for 15 min followed by 95 °C for 5 min, and then 35 PCR cycles were performed with the following temperature profile: 95 °C 15 s, 58 °C 30 s, 68 °C 1 min, 77 °C 20 s. Data were collected at the (77 °C 20 s) step to remove possible fluorescent contribution from dimer primers (22Zhu F. Massana R. Not F. Marie D. Vaulot D. FEMS Microbiol. Ecol. 2005; 52: 79-92Crossref PubMed Scopus (446) Google Scholar). Gene expression values were normalized to GAPDH. T/C-28a2 cells, from sheared and matched static control specimens, were lysed in radioimmune precipitation assay buffer (25 mm Tris·HCl, pH 7.6, 150 mm NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) containing a mixture of proteinase inhibitors (Pierce). The protein content of the cell lysates was determined using bicinchoninic acid (BCA) protein assay reagent (Pierce). Total cell lysates (4 μg) were subjected to SDS-PAGE, transferred to a membrane, and probed with a panel of specific antibodies. Each membrane was only probed using one antibody. β-Actin was used as loading control. All Western hybridizations were performed at least in triplicate using a different cell preparation each time. Cytosolic and nuclear extracts were isolated using the NE-PER nuclear and cytoplasmic extraction kit (Pierce) following the manufacturer's instructions as previously described (17Healy Z.R. Zhu F. Stull J.D. Konstantopoulos K. Am. J. Physiol. Cell Physiol. 2008; 294: C1146-C1157Crossref PubMed Scopus (34) Google Scholar). A 5′-biotinylated oligonucleotide probe (5′-GGGATTTTCC-3′) was synthesized containing the NF-κB cis-element present on the IL-6 promoter. EMSAs were performed with a commercially available nonradioisotopic EMSA kit (LightShift Chemiluminescence EMSA kit; Pierce). Briefly, nuclear extracts (1–2 μg) were incubated in 10× binding buffer (supplemented with 50 ng of poly(dI-dC), 2.5% glycerol, 0.05% Nonidet P-40, 5 mm MgCl2, and 0.25 mg of bovine serum albumin), containing 20 fmol of biotinylated, double-stranded probe for NF-κB for 30 min on ice. For competition binding, a 200-fold excess of unlabeled (cold) probe was incubated with nuclear extracts before the inclusion of the biotinylated one. For supershift assays, the nuclear extracts were preincubated for 30 min on ice with an anti-p65 antibody. The biotinylated oligonucleotide probe specific for NF-κB was then added to the reaction mixture and incubated for another 30 min on ice. To exclude the possibility of nonspecific binding, a 5′-biotinylated random probe (5′-TCATTTTGTC-3′) designed using a random sequence generator was used in shift and supershift assays (supplemental Fig. S1). The protein-DNA complexes were resolved on a native 6% polyacrylamide retardation gel in 0.5× Tris borate-EDTA running buffer at 10 mA for 1 h, transferred to a nylon membrane (Pierce), visualized using the LightShift Chemiluminescence kit (Pierce) and exposed to Kodak x-ray film (Pierce). cAMP levels were determined by cyclic AMP enzyme immunoassay kit following the manufacturer’s instructions (Cayman Chemical). The protein concentration of total cell lysate was used as loading control, and the results were expressed as pmol of cAMP/μg of total protein. IL-6 expression has been detected in chondrocytes from OA but not normal cartilage (4Mohtai M. Gupta M.K. Donlon B. Ellison B. Cooke J. Gibbons G. Schurman D.J. Smith R.L. J. Ortho. Res. 1996; 14: 67-73Crossref PubMed Scopus (102) Google Scholar). Prior work has shown that high fluid shear stress (16 dyn/cm2) induces IL-6 production in human articular chondrocytes in vitro (4Mohtai M. Gupta M.K. Donlon B. Ellison B. Cooke J. Gibbons G. Schurman D.J. Smith R.L. J. Ortho. Res. 1996; 14: 67-73Crossref PubMed Scopus (102) Google Scholar). However, the signaling pathway of IL-6 induction in human chondrocytes in response to elevated levels of fluid shear remains unknown. In light of accumulating evidence suggesting that OA is often a consequence of abnormal mechanical forces (23Martin J.A. Buckwalter J.A. Biorheology. 2006; 43: 517-521PubMed Google Scholar), we here aimed to delineate the mechanism by which shear stress induces IL-6 expression in human chondrocytes. The human T/C-28a2 chondrocyte cell line was chosen as a model system, because T/C-28a2 cells have been shown to behave much like primary human chondrocytes when cultured under appropriate conditions (19Goldring M.B. Birkhead J.R. Suen L.F. Yamin R. Mizuno S. Glowacki J. Arbiser J.L. Apperley J.F. J. Clin. Invest. 1994; 94: 2307-2316Crossref PubMed Scopus (385) Google Scholar, 20Goldring M.B. Methods Mol. Med. 2004; 100: 37-52PubMed Google Scholar). In view of previous observations showing a positive correlation between endogenous COX-2-mediated PGE2 production and IL-6 synthesis (24Anderson G.D. Hauser S.D. McGarity K.L. Bremer M.E. Isakson P.C. Gregory S.A. J. Clin. Invest. 1996; 97: 2672-2679Crossref PubMed Scopus (533) Google Scholar, 25Hinson R.M. Williams J.A. Shacter E. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 4885-4890Crossref PubMed Scopus (259) Google Scholar), we first evaluated the effects of shear stress on COX-2 expression and formation of cAMP, because PGE2 is a well-known activator of the cAMP signaling pathway. In accord with prior data (17Healy Z.R. Zhu F. Stull J.D. Konstantopoulos K. Am. J. Physiol. Cell Physiol. 2008; 294: C1146-C1157Crossref PubMed Scopus (34) Google Scholar), high shear stress (20 dyn/cm2) rapidly induces COX-2 mRNA (Fig. 1A) and protein (Fig. 1B) expression in human T/C-28a2 chondrocytes and cAMP production (Fig. 1C). IL-6 mRNA and protein up-regulation follows later, after 30–60 min of cell stimulation with fluid shear (Fig. 1, D and E). Treatment of T/C-28a2 chondrocytes with the selective COX-2 inhibitor NS398 (30 μm) 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. To validate previously published observations suggesting that T/C-28a2 cells represent an appropriate model for studying chondrocyte function in vitro (19Goldring M.B. Birkhead J.R. Suen L.F. Yamin R. Mizuno S. Glowacki J. Arbiser J.L. Apperley J.F. J. Clin. Invest. 1994; 94: 2307-2316Crossref PubMed Scopus (385) Google Scholar, 20Goldring M.B. Methods Mol. Med. 2004; 100: 37-52PubMed Google Scholar), we examined the responses of human primary articular chondrocytes to shear stress (20 dyn/cm2). Our data revealing significant similarities between primary articular chondrocytes and T/C-28a2 cells in the induction of IL-6 mRNA synthesis in response to shear stress (Fig. 1D) reinforce the aforementioned notion. The effects of PGE2 are mediated via four different transmembrane GPCRs, namely EP1–4, which are involved in the activation of phospholipase C (EP1) and activation (EP2, EP4) or inhibition (EP3) of adenyl cyclase (13Sugimoto Y. Narumiya S. J. Biol. Chem. 2007; 282: 11613-11617Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar). In agreement with previously published data from primary human chondrocytes (26Aoyama T. Liang B. Okamoto T. Matsusaki T. Nishijo K. Ishibe T. Yasura K. Nagayama S. Nakayama T. Nakamura T. Toguchida J. J. Bone Miner Res. 2005; 20: 377-389Crossref PubMed Scopus (47) Google Scholar), T/C-28a2 cells express EP2, EP3 and very low levels of EP4, but lack EP1, receptors (supplemental Fig. S2A). As a next step, we assessed the influence of shear stress on EP receptor expression. Application of high fluid shear up-regulates EP2 (Fig. 2, A and C) while it down-regulates EP3 (Fig. 2, B and D) receptor expression at both the mRNA and protein levels in T/C-28a2 chondrocytes. No significant changes are noted in the mRNA expression levels of EP1 and EP4 receptors in sheared relative to static control T/C-28a2 chondrocytes (supplemental Fig. S2B). The divergent effects of shear stress on EP2 and EP3 receptor expression are in line with the enhanced cAMP accumulation observed in shear-activated chondrocytes (Fig. 1C). Thus, we next investigated the effects of an EP2 receptor antagonist, AH6809 (9Chen B.C. Liao C.C. Hsu M.J. Liao Y.T. Lin C.C. Sheu J.R. Lin C.H. J. Immunol. 2006; 177: 681-693Crossref PubMed Scopus (74) Google Scholar, 27Attur M. Al-Mussawir H.E. Patel J. Kitay A. Dave M. Palmer G. Pillinger M.H. Abramson S.B. J. Immunol. 2008; 181: 5082-5088Crossref PubMed Scopus (155) Google Scholar), and an EP3 receptor agonist, sulprostone (14Li X. Ellman M. Muddasani P. Wang J.H. Cs-Szabo G. van Wijnen A.J. Im H.J. Arthritis Rheum. 2009; 60: 513-523Crossref PubMed Scopus (128) Google Scholar), on shear-induced cAMP formation and IL-6 synthesis. Incubation of T/C-28a2 chondrocytes with AH6809 (3 μm) does not alter the mRNA, and protein expression levels of EP2 receptor in shear-stimulated cells (Fig. 2, A and C). However, this pharmacological intervention dramatically attenuates shear-induced cAMP production (Fig. 2G) and IL-6 mRNA expression (Fig. 2E) in T/C-28a2 chondrocytes. Similarly, sulprostone (1 μm), although it does not impair the EP3 receptor transcript and protein levels (Fig. 2, B and D), inhibits the accumulation of cAMP (Fig. 2G) and IL-6 synthesis (Fig. 2F) in sheared chondrocytes. To verify the involvement of EP2 receptor in the signaling pathway of IL-6 induction in shear-stimulated chondrocytes, and given the rather non-selective nature of AH6809 (28Chan C.L. Jones R.L. Lau H.Y. Br. J. Pharmacol. 2000; 129: 589-597Crossref PubMed Scopus (54) Google Scholar), experiments were carried out by transfecting T/C-28a2 cells with an EP2 shRNA-containing plasmid. This genetic intervention effectively knocks down EP2 protein (Fig. 3A) and mRNA (Fig. 3C) expression in sheared T/C-28a2 chondrocytes relative to cells transfected with a scramble shRNA control. EP2 receptor knockdown also inhibits the shear-induced accumulation of intracellular cAMP (Fig. 3E) and IL-6 mRNA synthesis (Fig. 3F). Similarly, to confirm the role of EP3 receptor in this signaling pathway, and in view of its shear-induced down-regulation, T/C-28a2 chondrocytes were transfected with a plasmid containing the cDNA of EP3 receptor. The efficacy of EP3 receptor overexpression is demonstrated at both the translational (Fig. 3B) and transcriptional (Fig. 3D) levels relative to cells transfected with an empty vector. Moreover, this genetic intervention results in a drastic reduction of cAMP formation (Fig. 3E) and IL-6 synthesis (Fig. 3F) in sheared T/C-28a2 chondrocytes. Taken together, our data reveal that shear stress up-regulates and down-regulates EP2 and EP3 receptor expression, respectively, thereby elevating intracellular cAMP levels, which in turn play a key role in the induction of IL-6. We next aimed to delineate the signaling mechanism of IL-6 up-regulation in sheared chondrocytes. In view of our observations showing that shear-induced cAMP formation precedes IL-6 mRNA expression in human chondrocytes (Fig. 1), we examined the potential contribution of downstream effectors of cAMP activity to this signaling cascade, which include PI3-K and PKA (29Ciullo I. Diez-Roux G. Di Domenico M. Migliaccio A. Avvedimento E.V. Oncogene. 2001; 20: 1186-1192Crossref PubMed Scopus (88) Google Scholar, 30Fujino H. West K.A. Regan J.W. J. Biol. Chem. 2002; 277: 2614-2619Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Exposure of T/C-28a2 chondrocytes to high shear stress increases the phosphorylation levels of Akt at Ser-473 and CREB at Ser-133 without affecting total Akt and CREB levels (Fig. 4). The shear-induced Akt and CREB phosphorylation are significantly suppressed by treating T/C-28a2 cells with the selective COX-2 inhibitor NS398 (Fig. 4A), the EP2 receptor antagonist AH6809 or the EP3 receptor agonist sulprostone (Fig. 4B). Along these lines, selective EP2 receptor knockdown or EP3 receptor overexpression (Fig. 4C) reverses shear-induced Akt and CREB phosphorylation to near background levels in T/C-28a2 chondrocytes. To evaluate the involvement of PI3-K in the regulation of shear-induced IL-6 mRNA synthesis in human chondrocytes, T/C-28a2 cells were treated with the selective PI3-K inhibitors, LY294002 (30 μm) or wortmannin (10 μm). These pharmacological inhibitors abrogate the phosphorylation of Akt at Ser-473 without affecting total Akt levels (Fig. 5D), and markedly attenuate shear-induced IL-6 mRNA synthesis (Fig. 5, A and B). Furthermore, t" @default.
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