Synergistic Collagenase Expression and Cartilage Collagenolysis Are Phosphatidylinositol 3-Kinase/Akt Signaling-dependent
2008; Elsevier BV; Volume: 283; Issue: 21 Linguagem: Inglês
10.1074/jbc.m710136200
ISSN1083-351X
AutoresGary J. Litherland, Craig Dixon, Rachel Lakey, Timothy Robson, Debra Jones, David A. Young, Tim E. Cawston, Andrew D. Rowan,
Tópico(s)NF-κB Signaling Pathways
ResumoThe phosphatidylinositol 3-kinase (PI3K) signaling pathway has emerged as a major regulator of cellular functions and has been implicated in several pathologies involving remodeling of extracellular matrix (ECM). The end stage of inflammatory joint diseases is characterized by excessive ECM catabolism, and in this study we assess the role of PI3K signaling in the induction of collagenolytic matrix metalloproteinases (MMPs) in human chondrocytes. We used the most potent cytokine stimulus reported to promote cartilage ECM catabolism, namely interleukin-1 (IL-1) in combination with oncostatin M (OSM). Both OSM and IL-6 (in the presence of its soluble receptor), but not IL-1 nor leukemia inhibitory factor, induced Akt phosphorylation in human chondrocytes. Inhibition of PI3K signaling using LY294002 blocked IL-1+OSM-mediated Akt phosphorylation, induction of MMP-1 and MMP-13, and cartilage collagenolysis. To further explore the role of downstream substrates within the PI3K pathway, complementary use of small molecule inhibitors and specific small interfering RNAs demonstrated that the PI3K subunit p110α and Akt1 were required for MMP-1 mRNA induction. MMP-13 induction was also reduced by loss of function of these molecules and by a lack of p110δ, 3-phosphoinositide-dependent kinase-1 or Akt3. We therefore propose that the activities of specific elements of the PI3K signaling pathway, including Akt, are necessary for the synergistic induction of MMP-1 and MMP-13 and the cartilage breakdown stimulated by IL-1+OSM. Our data provide new insight into the mechanism of synergy between IL-1 and OSM and highlight new therapeutic targets for inflammatory joint diseases that aim to repress the expression of collagenases. The phosphatidylinositol 3-kinase (PI3K) signaling pathway has emerged as a major regulator of cellular functions and has been implicated in several pathologies involving remodeling of extracellular matrix (ECM). The end stage of inflammatory joint diseases is characterized by excessive ECM catabolism, and in this study we assess the role of PI3K signaling in the induction of collagenolytic matrix metalloproteinases (MMPs) in human chondrocytes. We used the most potent cytokine stimulus reported to promote cartilage ECM catabolism, namely interleukin-1 (IL-1) in combination with oncostatin M (OSM). Both OSM and IL-6 (in the presence of its soluble receptor), but not IL-1 nor leukemia inhibitory factor, induced Akt phosphorylation in human chondrocytes. Inhibition of PI3K signaling using LY294002 blocked IL-1+OSM-mediated Akt phosphorylation, induction of MMP-1 and MMP-13, and cartilage collagenolysis. To further explore the role of downstream substrates within the PI3K pathway, complementary use of small molecule inhibitors and specific small interfering RNAs demonstrated that the PI3K subunit p110α and Akt1 were required for MMP-1 mRNA induction. MMP-13 induction was also reduced by loss of function of these molecules and by a lack of p110δ, 3-phosphoinositide-dependent kinase-1 or Akt3. We therefore propose that the activities of specific elements of the PI3K signaling pathway, including Akt, are necessary for the synergistic induction of MMP-1 and MMP-13 and the cartilage breakdown stimulated by IL-1+OSM. Our data provide new insight into the mechanism of synergy between IL-1 and OSM and highlight new therapeutic targets for inflammatory joint diseases that aim to repress the expression of collagenases. Phosphatidylinositol 3-kinase (PI3K) 3The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; ECM, extracellular matrix; MAPK, mitogen-activated protein kinase; RA, rheumatoid arthritis; OA, osteoarthritis; IL, interleukin; TNFα, tumor necrosis factor α; OSM, oncostatin M; MMP, matrix metalloproteinase; STAT, signal transducers and activators of transcription; AP-1, activator protein-1; LIF, leukemia inhibitory factor; TIMP, tissue inhibitor of metalloproteinases; sIL-6R, soluble IL-6 receptor; LDH, lactate dehydrogenase; PDK1, phosphoinositide-dependent kinase-1; RT, reverse transcription; ANOVA, analysis of variance. 3The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; ECM, extracellular matrix; MAPK, mitogen-activated protein kinase; RA, rheumatoid arthritis; OA, osteoarthritis; IL, interleukin; TNFα, tumor necrosis factor α; OSM, oncostatin M; MMP, matrix metalloproteinase; STAT, signal transducers and activators of transcription; AP-1, activator protein-1; LIF, leukemia inhibitory factor; TIMP, tissue inhibitor of metalloproteinases; sIL-6R, soluble IL-6 receptor; LDH, lactate dehydrogenase; PDK1, phosphoinositide-dependent kinase-1; RT, reverse transcription; ANOVA, analysis of variance. comprises a group of lipid kinases that catalyze the phosphorylation of the 3-position of the inositol moiety of phosphoinositides at the cell membrane (1Hawkins P.T. Anderson K.E. Davidson K. Stephens L.R. Biochem. Soc. Trans. 2006; 34: 647-662Crossref PubMed Scopus (453) Google Scholar). Activation of class I PI3K signaling plays a vital role in the response of many cytokine and growth factor receptors to control cellular processes such as growth, survival, proliferation, differentiation, and migration (1Hawkins P.T. Anderson K.E. Davidson K. Stephens L.R. Biochem. Soc. Trans. 2006; 34: 647-662Crossref PubMed Scopus (453) Google Scholar). Activity of the downstream serine/threonine kinase, Akt (protein kinase B), is of key importance for these events. This PI3K subgroup is further sub-divided: class IA comprises heterodimers consisting of a p110 catalytic subunit (α, β, or δ isoforms), closely associated with a p50, p55, or p85 regulatory subunit, which is responsible for recruitment to tyrosine kinase-associated receptors activated by cytokines and growth factors. In class IB enzymes, usually activated by G-protein-coupled receptors, the catalytic subunit is p110γ, and the regulatory subunit is p84 or p101 (1Hawkins P.T. Anderson K.E. Davidson K. Stephens L.R. Biochem. Soc. Trans. 2006; 34: 647-662Crossref PubMed Scopus (453) Google Scholar). Akt is a pivotal kinase in PI3K signaling, and mechanisms exist to limit Akt activation. Positive or negative regulation of PI3K/Akt signaling enables functional differences between receptors which, for example, determine the outcome of T cell activation (2Parry R.V. Riley J.L. Ward S.G. Trends Immunol. 2007; 28: 161-168Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). There are three mammalian Akt family members involved in a plethora of cellular signaling events. These isoforms have central roles in a variety of human cancers, with effects on tumor initiation and progression as well as metastasis. They also contribute to tumorigenesis at multiple levels such as regulation of cell motility and migration, and cellular interactions with the extracellular matrix (ECM) (3Stambolic V. Woodgett J.R. Trends Cell Biol. 2006; 16: 461-466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Cross-talk occurs between Akt and other signaling pathways such as the mitogen-activated protein kinases (MAPKs), which are themselves activated by pro-inflammatory stimuli (4Yart A. Chap H. Raynal P. Biochim. Biophys. Acta. 2002; 1582: 107-111Crossref PubMed Scopus (58) Google Scholar). Thus, PI3K pathway activation can influence other signaling pathways; combined with the ability of Akt to regulate the subcellular localization of downstream substrates (and hence their functionality, Ref. 5Kumar R. Hung M.C. Cancer Res. 2005; 65: 2511-2515Crossref PubMed Scopus (51) Google Scholar), this pathway is an important modulator of gene expression in inflammatory milieu where multiple cytokines are present.Inflammation is a major characteristic of joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA). Despite having different aetiologies, inflammatory mediators released by infiltrating immune cells as well as resident joint cells induce alterations in gene expression that can lead to ECM degradation (6Sakkas L.I. Platsoucas C.D. Arthritis Rheum. 2007; 56: 409-424Crossref PubMed Scopus (116) Google Scholar, 7Scrivo R. Di Franco M. Spadaro A. Valesini G. Ann. N. Y. Acad. Sci. 2007; 1108: 312-322Crossref PubMed Scopus (51) Google Scholar). Cytokines such as interleukin(IL-)1, IL-17, and tumor necrosis factor α (TNFα) are the key mediators thought to be involved in promoting inflammatory responses in such destructive joint diseases (8Goldring M.B. Goldring S.R. J. Cell. 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Arthritis Rheum. 2006; 54: 540-550Crossref PubMed Scopus (77) Google Scholar), and this has been proposed to be via the interplay of signal transduction pathways whereby signal transducers and activators of transcription (STAT), c-Fos, and alterations in activator protein-1 (AP-1) composition are important (20Catterall J.B. Carrere S. Koshy P.J. Degnan B.A. Shingleton W.D. Brinckerhoff C.E. Rutter J. Cawston T.E. Rowan A.D. Arthritis Rheum. 2001; 44: 2296-2310Crossref PubMed Scopus (74) Google Scholar). MMP gene expression is certainly reliant on transcription factors such as erythroblastosis 26 and nuclear factor-κB as well as AP-1 (21Vincenti M.P. Brinckerhoff C.E. Arthritis Res. 2002; 4: 157-164Crossref PubMed Scopus (584) Google Scholar), although synergistic MMP gene expression is likely to involve other signaling pathways that impinge on one or more of these factors. Thus, targeting specific pathways such as those activated by OSM may have the potential to suppress collagenolytic MMP gene expression during inflammation.Class I PI3K activity, particularly of PI3Kγ and PI3Kδ isoforms, represents a well established target for the treatment of inflammatory joint disease, based largely on the importance of these activities in T cell receptor-induced T cell activation and/or migration (22Rommel C. Camps M. Ji H. Nat. Rev. Immunol. 2007; 7: 191-201Crossref PubMed Scopus (358) Google Scholar, 23Alcazar I. Marques M. Kumar A. Hirsch E. Wymann M. Carrera A.C. Barber D.F. J. Exp. Med. 2007; 204: 2977-2987Crossref PubMed Scopus (86) Google Scholar). Moreover, PI3Kγ blockade ameliorates joint damage in murine models (24Camps M. Ruckle T. Ji H. Ardissone V. Rintelen F. Shaw J. Ferrandi C. Chabert C. Gillieron C. Francon B. Martin T. Gretener D. Perrin D. Leroy D. Vitte P.A. Hirsch E. Wymann M.P. Cirillo R. Schwarz M.K. Rommel C. Nat. 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Berenbaum F. Osteoarthritis Cartilage. 2007; 15: 138-146Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Chondrocyte synthesis of proteoglycan (40Starkman B.G. Cravero J.D. Delcarlo M. Loeser R.F. Biochem. J. 2005; 389: 723-729Crossref PubMed Scopus (141) Google Scholar) and tissue inhibitor of metalloproteinases (TIMP)-3 expression (41Qureshi H.Y. Ahmad R. Sylvester J. Zafarullah M. Cell Signal. 2007; 19: 1643-1651Crossref PubMed Scopus (63) Google Scholar) require PI3K/Akt activity, and this pathway has recently been implicated in OSM-induced expression of aggrecanase-1 and MMP-13 (42El Mabrouk M. Sylvester J. Zafarullah M. Biochim. Biophys. Acta. 2007; 1773: 309-320Crossref PubMed Scopus (41) Google Scholar).We propose that elements of the PI3K pathway are important determinants for the synergistic induction of collagenolytic MMPs in cartilage. In this study, we demonstrate activation of Akt by OSM (but not IL-1), and that MMP-1 and MMP-13 gene expression are dependent on distinct sets of signaling molecules within the PI3K pathway. These data therefore further support the differential regulation of these important collagenases (43Mengshol J.A. Vincenti M.P. Coon C.I. Barchowsky A. Brinckerhoff C.E. Arthritis Rheum. 2000; 43: 801-811Crossref PubMed Scopus (559) Google Scholar), confirm that OSM-activated PI3K signaling is an important contributor to MMP synergy, and that specific ablation of PI3K signaling events could function to preserve cartilage homeostasis during inflammation.EXPERIMENTAL PROCEDURESMaterials—All chemicals, including AS-604850, were obtained from Sigma unless otherwise stated and were of the highest purity available. All cytokines used were recombinant human. IL-1α was a generous gift from Dr. Keith Ray (Glaxo-SmithKline, Stevenage, UK). OSM and LIF were kindly donated by Prof. John Heath (Department of Biochemistry, University of Birmingham, Edgbaston, UK), while IL-6 and its soluble receptor (sIL-6R) were purchased from R&D Systems Ltd. (Abingdon, UK). LY294002, and Akt inhibitors IV and VIII were from Merck Chemicals (Nottingham, UK), compound 15e and TGX-221 were from Axxora (Nottingham, UK). All kinase inhibitors and siRNA reagents were screened for toxicity using the XTT assay of metabolic activity (Roche Applied Sciences), and used at concentrations that did not affect cell viability over a 24-h period.Chondrocytes—Human chondrocytes were obtained by the enzymatic digestion of macroscopically normal articular cartilage from OA patients undergoing joint replacement surgery as described (44Cleaver C.S. Rowan A.D. Cawston T.E. Ann. Rheum. Dis. 2001; 60: 150-157Crossref PubMed Scopus (35) Google Scholar). All subjects gave informed consent, and the study was approved by the Newcastle and North Tyneside Joint Ethics Committee. Bovine cartilage was dissected from nasal septi obtained from a local abattoir as described (45Shingleton W.D. Ellis A.J. Rowan A.D. Cawston T.E. J. Cell. Biochem. 2000; 79: 519-531Crossref PubMed Scopus (46) Google Scholar). Chondrocytes were only used as primary cultures, or for siRNA transfection, after a single passage.Cartilage Degradation Assay—Bovine nasal septum cartilage was dissected into ∼2 × 2 × 2-mm discs, plated into 24-well tissue culture plates (3 discs per well, n = 4) in serum-free medium and incubated for 14 days in the presence of IL-1 ± OSM (±LY294002 including a 24-h preincubation), changing medium after 7 days as previously described (45Shingleton W.D. Ellis A.J. Rowan A.D. Cawston T.E. J. Cell. Biochem. 2000; 79: 519-531Crossref PubMed Scopus (46) Google Scholar). The cartilage remaining at day 14 was digested with papain (45Shingleton W.D. Ellis A.J. Rowan A.D. Cawston T.E. J. Cell. Biochem. 2000; 79: 519-531Crossref PubMed Scopus (46) Google Scholar), and all samples were stored at –20 °C until assay.Viability of cartilage explants was assessed by screening for the production of lactate dehydrogenase (LDH) using the Cytotox 96 assay (Promega, Southampton, UK). No increase in LDH levels with any of the treatments including inhibitors has been found (data not shown).Proteoglycan, Collagen, and Collagenolytic Activity Assays—For proteoglycan release, a measurement of sulfated glycosaminoglycans was used as described previously (47Cawston T.E. Curry V.A. Summers C.A. Clark I.M. Riley G.P. Life P.F. Spaull J.R. Goldring M.B. Koshy P.J. Rowan A.D. Shingleton W.D. Arthritis Rheum. 1998; 41: 1760-1771Crossref PubMed Scopus (171) Google Scholar). Hydroxyproline measurements (46Bergman I. Loxley R. Anal. Chem. 1963; 35: 1961-1965Crossref Scopus (1290) Google Scholar) were used as an estimate of cartilage collagen, and the cumulative release calculated and expressed as a percentage of the total for each well (47Cawston T.E. Curry V.A. Summers C.A. Clark I.M. Riley G.P. Life P.F. Spaull J.R. Goldring M.B. Koshy P.J. Rowan A.D. Shingleton W.D. Arthritis Rheum. 1998; 41: 1760-1771Crossref PubMed Scopus (171) Google Scholar). Collagenolytic activity present in the culture medium from cartilage explants was determined using a diffuse fibril assay with 3H-acetylated collagen (48Koshy P.J. Rowan A.D. Life P.F. Cawston T.E. Anal. Biochem. 1999; 275: 202-207Crossref PubMed Scopus (32) Google Scholar). One unit of collagenase activity degrades 1 μgof collagen per min at 37 °C.Immunoblotting—Human articular chondrocytes were grown to 80–90% confluence, serum-starved overnight, and then stimulated with cytokines for 20 min. Cells were lysed with ice-cold buffer (50 mm Tris-Cl, pH 7.5, 1.2 m glycerol, 1 mm EGTA, 1 mm EDTA, 1 mm Na3VO4, 10 mm 2-glycerophosphate, 50 mm NaF, 5 mm sodium pyrophosphate, 1% (v/v) Triton X-100, 1 μm microcystin-LR; 0.1% (v/v) 2-mercaptoethanol, protease inhibitor mixture (Roche Applied Sciences)), particulate matter removed by centrifugation at 13,000 × g, 5 min at 4 °C, and lysates stored at –80 °C until use. Lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and subsequently probed using the following antibodies: phospho-Akt (Ser-473), phospho-Akt (Thr-308), Akt, 3-phosphoinositide-dependent kinase-1 (PDK1), and phospho-PDK1 (Ser-241) from Cell Signaling Technology (Danvers, MA); PI3K p110 from Santa Cruz Biotechnology (Autogen Bioclear, Wiltshire, UK); glyceraldehyde-3′-phosphate dehydrogenase (GAPDH) from Millipore (Watford, UK).Immunoassays—Primary human articular chondrocytes were stimulated with IL-1 ± OSM (±LY294002 preincubated with the cells for 1 h) for 48 h under serum-free conditions (10Rowan A.D. Koshy P.J. Shingleton W.D. Degnan B.A. Heath J.K. Vernallis A.B. Spaull J.R. Life P.F. Hudson K. Cawston T.E. Arthritis Rheum. 2001; 44: 1620-1632Crossref PubMed Scopus (119) Google Scholar). The medium was removed and assayed by specific immunoassay for MMP-1 (49Clark I.M. Powell L.K. Wright J.K. Cawston T.E. Hazleman B.L. Matrix. 1992; 12: 475-480Crossref PubMed Scopus (38) Google Scholar), MMP-13 (50Morgan T.G. Rowan A.D. Dickinson S.C. Jones D. Hollander A.P. Deehan D. Cawston T.E. Ann. Rheum. Dis. 2006; 65: 184-190Crossref PubMed Scopus (20) Google Scholar), and TIMP-1 (51Clark I.M. Powell L.K. Wright J.K. Cawston T.E. Matrix. 1991; 11: 76-85Crossref PubMed Scopus (46) Google Scholar). The MMP assays detected both the pro- and active forms.siRNA-mediated Gene Silencing—Primary human chondrocytes were prepared and cultured as above. For siRNA transfection, cells were trypsinized and re-seeded at ∼50% confluence. Maintenance of the chondrocytic phenotype was confirmed by assessment of SOX9 and Col2A1 expression by real-time PCR using Taqman® gene expression assays (Applied Biosystems, Foster City, CA). Dharmacon siGENOME™ SMARTpools® (Thermo Fisher Scientific, Lafayette, CO.) of 4 specific siRNA duplexes (total of 100 nm siRNA) were used to transfect chondrocytes using the Dharmafect™ 1 lipid reagent (Thermo Fisher). siRNA pools were used to target p110α (NM_006218, cat: 003018); p110β (NM_006219, cat: 003019); p110δ (NM_005026, cat: 006775); PDK1 (NM_002613, cat: 003017); Akt1 (NM_005163, cat: 003000); Akt2 (NM_001626, cat: 003001); Akt3 (NM_005465, cat: 003002). After a 28-h transfection, cells were washed in serum-free medium for 20 h prior to the addition of cytokines for a further 24 h. Total RNA was isolated and reverse-transcribed using the Cells-to-Signal™ kit (Ambion, Applied Biosystems, Warrington, UK) as directed. Expression of MMP-1 and MMP-13 mRNA was measured by real-time reverse transcription (RT)-PCR (see below). Depletion of gene-specific mRNA levels was calculated by comparison of expression levels with cells transfected with 100 nm siCONTROL (non-targeting siRNA 2, cat. 001210-02; Dharmacon).Real-time PCR of Relative mRNA Levels—RNA was stabilized in cell lysates in a 96-well format and cDNA synthesized using the Cells-to-Signal™ kit as directed. For TaqMan or SYBR Green PCR, mRNA levels for each gene were obtained from standard curves and corrected using 18 S ribosomal RNA levels. Cycling conditions (7900HT system, Applied Biosystems, Foster City, CA) for SYBR Green PCR (using Takara SYBR ExTaq premix; Lonza Biologics, Cambridge, UK) were 95 °C 10 s, then 40 cycles of 95 °C 5 s, then 60 °C 30 s, followed by a standard dissociation curve analysis. Cycling conditions for Taqman PCR (Jumpstart Taq Readymix; Sigma) were 2 min at 50 °C, 10 min at 95 °C, then 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. Primers and probe sequences for Taqman PCR were: MMP-1: For, 5′-AAGATGAAAGGTGGACCAAAATT-3′ and Rev, 5′-CCAAGAGAATGGCCGAGTTC-3′, Probe: 5′-FAM-CAGAGAGTACAACTTACATCGTGTTGCGGCTCTAMRA-3′; MMP-13: For, 5′-AAATTATGGAGGAGATGCCCATT-3′ and Rev, 5′-TCCTTGGAGTGGTCAAGACCTAA-3′, Probe: 5′-FAM-CTACAACTTGTTTCTTGTTGCTGCGCATGATAMRA-3′; 18 S ribosomal RNA: For, 5′-CGAATGGCTCATTAAATCAGTTATGG-3′ and Rev 5′-TATTAGCTCTAGAATTACCACAGTTATCC-3′, Probe: 5′-FAM-TCCTTTGGTCGCTCGCTCCTCTCCC-TAMRA-3′. Some Taqman assays used Universal Probe Library probes (Roche Applied Sciences) as directed: Akt2: For, 5′-ACGTGGATTCTCCAGACGA-3′ and Rev, 5′-GCTGCTTGAGGCTGTTGG-3′, Probe: 52; Akt3: For, 5′-TGGATTTACCTTATCCCCTCAA-3′ and Rev 5′-TGGCTTTGGTCGTTCTGTTT-3′, Probe: 59; PDK1: For, 5′-GTCTTATCCCCAGAGAGCAAAC-3′ and Rev, 5′-AGCAGCTCTGGAGAAACGTACT-3′, Probe: 81; p110β: For, 5′-CCCTTCTGAACTGGCTTAAAGA-3′ and Rev, 5′-GGACAGTGTAAATTCCTCAATGG-3′, Probe: 85. For SYBR Green PCR, primer sequences were: Akt1: For, 5′-CTGTCATCGAACGCACCTT-3′ and Rev, 5′-GTCTGGATGGCGGTTGTC-3′; p110α: For, 5′-CACGAGATCCTCTCTCTGAAATC-3′ and Rev, 5′-GGTAGAATTTCGGGGATAGTTACA-3′; p110γ: For, 5′-CCTCAACCATGAAGGAAACC-3′ and Rev, 5′-GCACCACGGGCTGTTTATAG-3′; p110δ: For, 5′-TTGCTGGTCTTTCTTGGACTATT-3′ and Rev, 5′-TTCTCCTCCTTGGTCCAGAAT-3′.Statistical Analyses—Statistical differences between sample groups were assessed using ANOVA with a post-hoc Bonferroni's multiple comparison test, where ***, p < 0.001; **, p < 0.01; *, p < 0.05. For clarity, only selected comparisons are presented in some figures.RESULTSOSM Stimulates Akt Phosphorylation in Human Chondrocytes—Stimulation of primary human articular chondrocytes with OSM and IL-6 (+sIL-6R), but not LIF, led to the phosphorylation of Akt at both Thr-308 and Ser-473 (Fig. 1A) indicating Akt activation. LIF treatment did induce STAT3 phosphorylation in these chondrocytes (data not shown). IL-1 stimulation did not result in Akt activation, and inclusion of LY294002 (5 μm) prevented OSM-mediated Akt phosphorylation (Fig. 1B). LY294002 similarly blocked IL-6-mediated activation of Akt (data not shown).PI3K Inhibition Blocks IL-1+OSM-mediated Cartilage Degradation—We examined the effect of inhibiting the PI3K/Akt pathway in an established model of cartilage degradation. The IL-1+OSM combination (0.02 ng/ml and 10 ng/ml, respectively) was used at pathologically relevant concentrations, and induced a reproducible and synergistic release of collagen from bovine cartilage as shown previously (47Cawston T.E. Curry V.A. Summers C.A. Clark I.M. Riley G.P. Life P.F. Spaull J.R. Goldring M.B. Koshy P.J. Rowan A.D. Shingleton W.D. Arthritis Rheum. 1998; 41: 1760-1771Crossref PubMed Scopus (171) Google Scholar), while inclusion of LY294002 throughout the 14-day culture resulted in a concentration-dependent inhibition of this release (Fig. 2). There was no increase in LDH release from inhibitor-treated cartilage (not shown) indicating that the observed effects were not due to toxicity of the inhibitor. A concentration-dependent reduction in the levels of active collagenolytic activity was also seen in the presence of LY294002 (Fig. 2). Although collagenase activity was significantly reduced, no inhibition of collagenolysis was observed at low inhibitor levels, consistent with previous findings when collagenase levels exceed 1 unit; such remaining collagenase activity is sufficient to catalyze maximum collagen release (52Shingleton W.D. Jones D. Xu X. Cawston T.E. Rowan A.D. Rheumatology (Oxford). 2006; 45: 958-965Crossref PubMed Scopus (8) Google Scholar). The most marked reduction in activity correlated with the inhibition of collagenolysis (10 μm LY294002) (Fig. 2). LY294002 did not affect IL-1+OSM-mediated proteoglycan release (Fig. 2, inset).FIGURE 2PI3K pathway inhibition blocks IL-1+OSM-induced collagenolysis. Cartilage explants were incubated with IL-1 (1 ng/ml) ± OSM (10 ng/ml) for 14 days, with fresh medium and cytokines at day 7. LY294002 was preincubated with cartilage for 24 h and throughout the stimulation period; DMSO (Dm) was included as a solvent control for the inhibitor. Cumulative collagen release by day 14 (black bars) was expressed as a percentage of the total for each treatment, and active collagenolytic activity in the day 14 culture supernatants (gray bars) was determined by bioassay (mean ± S.D., n = 4; ***, p < 0.001, LY294002-treated compared with IL-1+OSM; **, p < 0.01, ANOVA). Da
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