PI3 Kinase δ Is a Key Regulator of Synoviocyte Function in Rheumatoid Arthritis
2012; Elsevier BV; Volume: 180; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2012.01.030
ISSN1525-2191
AutoresBeatrix Bartók, David L. Boyle, Yi Liu, Pingda Ren, Scott T. Ball, William D. Bugbee, Christian Rommel, Gary S. Firestein,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoClass I phosphoinositide 3 kinase (PI3K) δ is a promising therapeutic target for rheumatoid arthritis (RA) because of its contribution to leukocyte biology. However, its contribution in fibroblasts has not been studied as a mechanism that contributes to efficacy. We investigated the expression and function of PI3Kδ in synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that PI3Kδ is highly expressed in RA synovium, especially in the synovial lining. Using quantitative PCR and Western blot analysis, we found that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis (OA) synovium. PI3Kδ was also expressed in cultured FLS, along with PI3Kα and PI3Kβ, whereas PI3Kγ was not detectable. PI3Kδ mRNA expression was selectively induced by inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) but not by growth factors platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ). The use of inhibitors that block individual PI3K isoforms, including the novel selective PI3Kδ inhibitor INK007, showed that PI3Kδ is required for PDGF- and TNF-induced Akt activation. PI3Kδ inhibition also diminished PDGF-mediated synoviocyte growth and sensitized cells to H2O2-induced apoptosis. These data are the first documentation of increased PI3Kδ expression in both RA synovium and cultured synoviocytes. Furthermore, these are the first data demonstrating that PI3Kδ is a major regulator of PDGF-mediated fibroblast growth and survival via Akt. Thus, targeting PI3Kδ in RA could modulate synoviocyte function via anti-inflammatory and disease-altering mechanisms. Class I phosphoinositide 3 kinase (PI3K) δ is a promising therapeutic target for rheumatoid arthritis (RA) because of its contribution to leukocyte biology. However, its contribution in fibroblasts has not been studied as a mechanism that contributes to efficacy. We investigated the expression and function of PI3Kδ in synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that PI3Kδ is highly expressed in RA synovium, especially in the synovial lining. Using quantitative PCR and Western blot analysis, we found that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis (OA) synovium. PI3Kδ was also expressed in cultured FLS, along with PI3Kα and PI3Kβ, whereas PI3Kγ was not detectable. PI3Kδ mRNA expression was selectively induced by inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) but not by growth factors platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ). The use of inhibitors that block individual PI3K isoforms, including the novel selective PI3Kδ inhibitor INK007, showed that PI3Kδ is required for PDGF- and TNF-induced Akt activation. PI3Kδ inhibition also diminished PDGF-mediated synoviocyte growth and sensitized cells to H2O2-induced apoptosis. These data are the first documentation of increased PI3Kδ expression in both RA synovium and cultured synoviocytes. Furthermore, these are the first data demonstrating that PI3Kδ is a major regulator of PDGF-mediated fibroblast growth and survival via Akt. Thus, targeting PI3Kδ in RA could modulate synoviocyte function via anti-inflammatory and disease-altering mechanisms. Class I phosphoinositide 3 kinases (PI3K), such as PI3Kδ, are emerging as novel therapeutic targets to treat inflammatory diseases.1Harris S.J. Foster J.G. Ward S.G. PI3K isoforms as drug targets in inflammatory diseases: lessons from pharmacological and genetic strategies.Curr Opin Investig Drugs. 2009; 10: 1151-1162PubMed Google Scholar, 2Rommel C. Camps M. Ji H. PI3K delta and PI3K gamma: partners in crime in inflammation in rheumatoid arthritis and beyond?.Nat Rev Immunol. 2007; 7: 191-201Crossref PubMed Scopus (371) Google Scholar, 3Williams O. Houseman B.T. Kunkel E.J. Aizenstein B. Hoffman R. Knight Z.A. Shokat K.M. Discovery of dual inhibitors of the immune cell PI3Ks p110delta and p110gamma: a prototype for new anti-inflammatory drugs.Chem Biol. 2010; 17: 123-134Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar PI3Ks are members of a conserved family of lipid kinases that regulate cell functions such as proliferation, differentiation, cell survival, cell motility, and metabolism. They are heterodimers that include a catalytic domain and a regulatory domain and are subdivided into multiple classes. The three members of the class IA subgroup (PI3Kα, β and δ) are generally activated by receptor tyrosine kinases (RTKs). One class IB kinase member, namely PI3Kγ, is activated by G-protein–coupled receptors (GPCRs). All PI3Ks exert their biological activity via generation of secondary messenger phosphatidylinositol 3, 4, 5-triphosphate (PIP3) at the plasma membrane, which serves as a docking site for additional signaling molecules such as protein kinases.4Vanhaesebroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Synthesis and function of 3-phosphorylated inositol lipids.Annu Rev Biochem. 2001; 70: 535-602Crossref PubMed Scopus (1384) Google Scholar Interest in targeting PI3Kδ for autoimmune disease is due, in part, to an expression pattern that is restricted to bone marrow–derived cells and endothelial cells.5Vanhaesebroeck B. Welham M.J. Kotani K. Stein R. Warne P.H. Zvelebil M.J. Higashi K. Volinia S. Downward J. Waterfield M.D. P110delta, a novel phosphoinositide 3-kinase in leukocytes.Proc Natl Acad Sci USA. 1997; 94: 4330-4335Crossref PubMed Scopus (378) Google Scholar, 6Puri K.D. Doggett T.A. Douangpanya J. Hou Y. Tino W.T. Wilson T. Graf T. Clayton E. Turner M. Hayflick J.S. Diacovo T.G. Mechanisms and implications of phosphoinositide 3-kinase delta in promoting neutrophil trafficking into inflamed tissue.Blood. 2004; 103: 3448-3456Crossref PubMed Scopus (191) Google Scholar, 7Puri K.D. Doggett T.A. Huang C.Y. Douangpanya J. Hayflick J.S. Turner M. Penninger J. Diacovo T.G. The role of endothelial PI3Kgamma activity in neutrophil trafficking.Blood. 2005; 106: 150-157Crossref PubMed Scopus (142) Google Scholar PI3Kα and PI3Kβ are ubiquitously expressed, and the respective knockout mice are embryonic lethal. PI3Kδ kinase inactive mice have a normal lifespan but exhibit attenuated immune responses. Analysis of these mice revealed that PI3Kδ has distinct roles in innate and adaptive immunity. When PI3Kδ was inactivated in mice, defective mast cell function, B-cell development and antibody production, altered T cell differentiation was observed.8Okkenhaug K. Bilancio A. Farjot G. Priddle H. Sancho S. Peskett E. Pearce W. Meek S.E. Salpekar A. Waterfield M.D. Smith A.J. Vanhaesebroeck B. Impaired B and T cell antigen receptor signaling in p110delta PI 3-kinase mutant mice.Science. 2002; 297: 1031-1034PubMed Google Scholar, 9Clayton E. Bardi G. Bell S.E. Chantry D. Downes C.P. Gray A. Humphries L.A. Rawlings D. Reynolds H. Vigorito E. Turner M. A crucial role for the p110delta subunit of phosphatidylinositol 3-kinase in B cell development and activation.J Exp Med. 2002; 196: 753-763Crossref PubMed Scopus (391) Google Scholar, 10Ramadani F. Bolland D.J. Garcon F. Emery J.L. Vanhaesebroeck B. Corcoran A.E. Okkenhaug K. The PI3K isoforms p110alpha and p110delta are essential for pre-B cell receptor signaling and B cell development.Sci Signal. 2010; 3 (ra60)PubMed Google Scholar, 11Patton D.T. Garden O.A. Pearce W.P. Clough L.E. Monk C.R. Leung E. Rowan W.C. Sancho S. Walker L.S. Vanhaesebroeck B. Okkenhaug K. Cutting edge: the phosphoinositide 3-kinase p110 delta is critical for the function of CD4+CD25+Foxp3+ regulatory T cells.J Immunol. 2006; 177: 6598-6602PubMed Google Scholar, 12Jou S.T. Carpino N. Takahashi Y. Piekorz R. Chao J.R. Wang D. Ihle J.N. Essential, nonredundant role for the phosphoinositide 3-kinase p110delta in signaling by the B-cell receptor complex.Mol Cell Biol. 2002; 22: 8580-8591Crossref PubMed Scopus (327) Google Scholar In contrast, the PI3Kγ isoform, which is also mainly expressed in hematopoietic cells, plays a critical role in chemokine signaling and cell recruitment.13Hirsch E. Katanaev V.L. Garlanda C. Azzolino O. Pirola L. Silengo L. Sozzani S. Mantovani A. Altruda F. Wymann M.P. Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation.Science. 2000; 287: 1049-1053Crossref PubMed Scopus (1117) Google Scholar, 14Li Z. Jiang H. Xie W. Zhang Z. Smrcka A.V. Wu D. Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction.Science. 2000; 287: 1046-1049Crossref PubMed Scopus (762) Google Scholar, 15Jones G.E. Prigmore E. Calvez R. Hogan C. Dunn G.A. Hirsch E. Wymann M.P. Ridley A.J. Requirement for PI 3-kinase gamma in macrophage migration to MCP-1 and CSF-1.Exp Cell Res. 2003; 290: 120-131Crossref PubMed Scopus (96) Google Scholar, 16Ferrandi C. Ardissone V. Ferro P. Ruckle T. Zaratin P. Ammannati E. Hauben E. Rommel C. Cirillo R. Phosphoinositide 3-kinase gamma inhibition plays a crucial role in early steps of inflammation by blocking neutrophil recruitment.J Pharmacol Exp Ther. 2007; 322: 923-930Crossref PubMed Scopus (54) Google Scholar Given its role in inflammation, PI3Kδ is a potential therapeutic target for a variety of autoimmune and inflammatory diseases.1Harris S.J. Foster J.G. Ward S.G. PI3K isoforms as drug targets in inflammatory diseases: lessons from pharmacological and genetic strategies.Curr Opin Investig Drugs. 2009; 10: 1151-1162PubMed Google Scholar, 2Rommel C. Camps M. Ji H. PI3K delta and PI3K gamma: partners in crime in inflammation in rheumatoid arthritis and beyond?.Nat Rev Immunol. 2007; 7: 191-201Crossref PubMed Scopus (371) Google Scholar, 17Fung-Leung W.P. Phosphoinositide 3-kinase delta (PI3Kdelta) in leukocyte signaling and function.Cell Signal. 2011; 23: 603-608Crossref PubMed Scopus (87) Google Scholar Based on these concepts, we studied the expression and function of class I PI3Kisoforms in RA synovium and cultured fibroblast-like synoviocytes (FLS), a critical cell that mediates tissue destruction in RA.18Bartok B. Firestein G.S. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis.Immunol Rev. 2010; 233: 233-255Crossref PubMed Scopus (1384) Google Scholar PI3Kγ protein or mRNA was not detected in synoviocytes. However, we unexpectedly found high expression of PI3Kδ in the RA synovial intimal lining and in FLS, where it was induced by pro-inflammatory cytokines and contributed to cell survival and growth. This is the first study showing that this isoform controls functions critical to inflammation in fibroblasts. The data suggest that PI3Kδ could play a role in synovial lining hyperplasia as well as the activation and recruitment of inflammatory cells in RA. This study was approved by the Institutional Review Board of University of California San Diego School of Medicine, and informed consent was obtained from all participants. Synovial tissue was obtained from patients with OA and RA at the time of total joint replacement or synovectomy, as previously described.19Alvaro-Gracia J.M. Zvaifler N.J. Brown C.B. Kaushansky K. Firestein G.S. Cytokines in chronic inflammatory arthritis VI Analysis of the synovial cells involved in granulocyte-macrophage colony-stimulating factor production and gene expression in rheumatoid arthritis and its regulation by IL-1 and tumor necrosis factor-alpha.J Immunol. 1991; 146: 3365-3371PubMed Google Scholar The diagnosis of RA conformed to American College of Rheumatology 1987 revised criteria.20Arnett F.C. Edworthy S.M. Bloch D.A. McShane D.J. Fries J.F. Cooper N.S. Healey L.A. Kaplan S.R. Liang M.H. Luthra H.S. et al.The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis.Arthritis Rheum. 1988; 31: 315-324Crossref PubMed Scopus (18946) Google Scholar The samples were either processed for cell culture or snap frozen for immunohistochemistry. For preparation of FLS, the synovium was minced and incubated with 1 mg/mL of collagenase type VIII (Sigma Chemicals, St. Louis, MO) in serum-free RPMI 1640 (Gibco BRL, Grand Island, NY) for 1 hour at 37°C, filtered, extensively washed, and cultured in DMEM (Gibco BRL) supplemented with 10% fetal bovine serum (FBS; Gemini Bio Products, Calabasas, CA), penicillin, streptomycin, gentamicin, and glutamine in a humidified 5% CO2 atmosphere. Cells were allowed to adhere overnight; nonadherent cells were removed, and adherent FLS were split at 1:3 when 70% to 80% confluent. FLS were used from passage 3 through 9, during which time they were a homogeneous population of cells (<1% CD11b positive, <1% phagocytic, and <1% FcγRII and FcγRIII receptor positive). FLS were cultured and used at 80% confluence. Cells were synchronized in 0.1% FBS for 24 hours before the addition of the appropriate stimulus. Cytokines and platelet-derived growth factor BB (PDGF) were obtained from R&D Laboratories (Minneapolis, MN). PI3K inhibitors were provided by Intellikine (La Jolla, CA) with the exception of CAL-101 which was obtained from Selleck Chemicals (Houston, TX) and AS-252424, which was obtained from Caymen Chemical (Ann Arbor, MI). All of the compounds were dissolved in dimethyl sulfoxide (DMSO) at 10 mmol/L. Concentrations for A66 (PI3Kα), TGX-221 (PI3Kβ), AS-252424 (PI3Kγ), and CAL-101 (PI3Kδ) were chosen based on previous reports.21Sun M. Hillmann P. Hofmann B.T. Hart J.R. Vogt P.K. Cancer-derived mutations in the regulatory subunit p85alpha of phosphoinositide 3-kinase function through the catalytic subunit p110alpha.Proc Natl Acad Sci USA. 2010; 107: 15547-15552Crossref PubMed Scopus (129) Google Scholar, 22Condliffe A.M. Davidson K. Anderson K.E. Ellson C.D. Crabbe T. Okkenhaug K. Vanhaesebroeck B. Turner M. Webb L. Wymann M.P. Hirsch E. Ruckle T. Camps M. Rommel C. Jackson S.P. Chilvers E.R. Stephens L.R. Hawkins P.T. Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils.Blood. 2005; 106: 1432-1440Crossref PubMed Scopus (259) Google Scholar, 23Hayer S. Pundt N. Peters M.A. Wunrau C. Kuhnel I. Neugebauer K. Strietholt S. Zwerina J. Korb A. Penninger J. Joosten L.A. Gay S. Ruckle T. Schett G. Pap T. PI3Kgamma regulates cartilage damage in chronic inflammatory arthritis.FASEB J. 2009; 23: 4288-4298Crossref PubMed Scopus (58) Google Scholar, 24Herman S.E. Gordon A.L. Wagner A.J. Heerema N.A. Zhao W. Flynn J.M. Jones J. Andritsos L. Puri K.D. Lannutti B.J. Giese N.A. Zhang X. Wei L. Byrd J.C. Johnson A.J. Phosphatidylinositol 3-kinase-delta inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals.Blood. 2010; 116: 2078-2088Crossref PubMed Scopus (486) Google Scholar, 25Hoellenriegel J. Meadows S.A. Sivina M. Wierda W.G. Kantarjian H. Keating M.J. Giese N. O'Brien S. Yu A. Miller L.L. Lannutti B.J. Burger J.A. The phosphoinositide 3′-kinase delta inhibitor CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia.Blood. 2011; 118: 3603-3612Crossref PubMed Scopus (459) Google Scholar, 26Lannutti B.J. Meadows S.A. Herman S.E. Kashishian A. Steiner B. Johnson A.J. Byrd J.C. Tyner J.W. Loriaux M.M. Deininger M. Druker B.J. Puri K.D. Ulrich R.G. Giese N.A. CAL-101, a p110delta selective phosphatidylinositol-3-kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3K signaling and cellular viability.Blood. 2011; 117: 591-594Crossref PubMed Scopus (628) Google Scholar Rabbit monoclonal P-Akt (Ser473), P-Akt (Thr308), pan-Akt, P-GSK3β (Ser9), pan-GSK3β, PI3K p110α, p110β, p110γ, and secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA). Mouse monoclonal PI3K p110δ antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). INK007 is a potent PI3Kδ inhibitor discovered through structure-based design. INK007 was synthesized with the methods described in the patent (patent no. WO2009088986). Some compounds were synthesized as described in patents (patents WO2004016607, WO2007129161, and WO2009080705). Staining protocols were performed as previously described.27Elices M.J. Tsai V. Strahl D. Goel A.S. Tollefson V. Arrhenius T. Wayner E.A. Gaeta F.C. Fikes J.D. Firestein G.S. Expression and functional significance of alternatively spliced CS1 fibronectin in rheumatoid arthritis microvasculature.J Clin Invest. 1994; 93: 405-416Crossref PubMed Scopus (134) Google Scholar, 28Chabaud-Riou M. Firestein G.S. Expression and activation of mitogen-activated protein kinase kinases-3 and -6 in rheumatoid arthritis.Am J Pathol. 2004; 164: 177-184Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Cryosections (5 μm) of synovial tissue were cut, fixed in cold acetone for 10 minutes, and incubated with the appropriate Abs overnight at 4°C. Isotype-matched antibodies served as a negative control. Endogenous peroxidase was depleted with 0.3% hydrogen peroxide and sections and then stained with secondary antibodies (Vector Laboratories, Burlingame, CA). The signal was developed using diaminobenzidine and sections were counterstained with hematoxylin. Cells were plated in six-well plates, grown until 70% to 80% confluence, and subsequently serum starved (0.1% FBS/DMEM) for 24 hours for synchronization. Cells were washed with cold PBS, and protein was extracted using PhosphoSafe buffer (Novagene, Madison, WI) supplemented with Complete Proteinase Inhibitors (Roche Applied Science, Indianapolis, IN). RIPA buffer was used for protein extraction from synovial tissue. The protein concentrations of tissue and FLS were determined using the Micro BCA protein assay kit (Thermo Scientific, Rockford, IL). Samples containing 25 μg of protein from cultured FLS or 50 μg of protein from synovial tissue were resolved on Invitrogen (Carlsbad, CA) NuPage 4% to 12% precast gels and transferred to a PVDF membrane. The membranes were blocked with 5% dry milk, incubated with primary Ab at 4°C overnight, followed by horseradish peroxidase–conjugated secondary Ab for 1 hour. Membranes were developed with Immun-Star Western ECL substrate (Bio-Rad, Hercules, CA) and imaged using the VersaDoc imaging system (Bio-Rad) and QuantityOne software (Hercules, CA) for image capture and densitometry. All Western blot data were analyzed from a single membrane. RNA isolation and RT-PCR were performed as previously described.29Boyle D.L. Rosengren S. Bugbee W. Kavanaugh A. Firestein G.S. Quantitative biomarker analysis of synovial gene expression by real-time PCR.Arthritis Res Ther. 2003; 5: R352-R360Crossref PubMed Google Scholar Forward and reverse primers as well as fluorogenic TaqMan FAM/TAMRA-labeled hybridization probes were used (Assays on Demand, Applied Biosystems). To control for sample cellularity, human GAPDH forward and reverse primers and labeled probe were included in separate PCR reactions. The threshold cycle (Ct) was determined for each sample using GeneAmp software. Resulting threshold Ct data were normalized to standard curves constructed from cDNA from human PBMC, yielding cell equivalents. The ratio between the gene of interest and GAPDH cell equivalents (relative expression units [REU]) is reported. For the MTT assay, 3 × 103 FLS/well were plated into 96-well plates in 10% FBS/DMEM. After 24 hours, the medium was replaced with low-serum medium (0.1% FBS/DMEM) for 24 hours for synchronization. On day 0, medium was replaced with 1% FBS and cells were treated with PI3K inhibitor at the indicated concentration or with DMSO for 1 hour. PDGF or medium alone was added to the appropriate wells. The experiment was performed in quadruplicate wells. Cell growth was estimated on days 0, 2, 4, 6, and 7 after incubation with MTT for 4 hours and was read at 550 nm with a spectrophotometer. For cell survival and apoptosis assays, 3 × 103 FLS/well were plated onto 96-well plate in 10% FBS/DMEM, then after 24 hours was replaced with starving medium (0.1% FBS/DMEM) for 24 hours for synchronization. Cells were incubated with PI3K inhibitors for 1 hour, then followed by PDGF stimulation. The next day cells were treated with 0.1 mmol/L H2O2 for 6 hours and viability was determined using an MTT assay. Apoptosis was determined using a Cell Death Detection ELISAPLUS kit (Roche Applied Science, Mannheim, Germany). Statistical analyses were performed using the paired Student's t-test. A comparison was considered significant if P was less than 0.05. We initially examined PI3K protein expression in synovial tissues using immunohistochemistry. PI3Kδ is highly expressed in the rheumatoid synovium, with especially prominent staining in the synovial lining (representative example in Figure 1A). A similar pattern was observed for PI3Kα (not shown). We next examined relative expression of each PI3K isoform in RA and OA synovial tissues. Using quantitative PCR, we found that PI3Kδ mRNA expression is significantly greater in RA compared with OA (P < 0.05; n = 6 from each; Figure 1B). There was a trend for increased PI3Kγ mRNA in RA, although it did not reach statistical significance (P = 0.06). There were no significant differences for PI3Kα or β mRNA between OA and RA (Figure 1B). Increased PI3Kδ protein expression in RA synovium was confirmed with Western blot analysis, with 1.5-fold higher levels in RA compared with OA (P < 0.04) (Figure 1C). Because PI3Kδ protein is localized to intimal lining cells, we evaluated its expression and regulation in cultured fibroblast-like synoviocytes (FLS). As expected, mRNA for α and β isoforms was detected. Surprisingly, PI3Kδ gene expression was readily detected in all FLS lines, with no difference between RA and OA cells (n = 8 each). mRNA for PI3Kγ was not detected in any FLS line (n = 12 RA and OA) even though control cells were positive (Ramos human B cells, MCP-1 human monocytes, and PBMCs; data not shown) and a previous report noted PI3Kγ in FLS.23Hayer S. Pundt N. Peters M.A. Wunrau C. Kuhnel I. Neugebauer K. Strietholt S. Zwerina J. Korb A. Penninger J. Joosten L.A. Gay S. Ruckle T. Schett G. Pap T. PI3Kgamma regulates cartilage damage in chronic inflammatory arthritis.FASEB J. 2009; 23: 4288-4298Crossref PubMed Scopus (58) Google Scholar Protein expression for PI3Kδ was confirmed with Western blot analysis (n = 5) (Figure 2A) and immunohistochemistry (Figure 2B). The lack of PI3Kγ in synoviocytes was confirmed with Western blot analysis (n = 5) (Figure 2A) and remained undetected even after overnight stimulation with TNF (see Supplemental Figure S1 at http://ajp.amjpathol.org). PI3Kα and β protein was also detected (n = 5) (Figure 2A). To determine whether PI3Kδ expression is regulated by inflammatory mediators implicated in RA, we stimulated FLS with tumor necrosis factor (TNF), interleukin-1 (IL-1), platelet-derived growth factor (PDGF), or transforming growth factor β (TGFβ) for 4 to 48 hours and assayed PI3K mRNA using quantitative PCR. PI3Kδ mRNA increased four- to fivefold after TNF or IL-1 stimulation within 6 hours and peaked at 24 hours (Figure 3A for TNF data, n = 4, P < 0.04), whereas PDGF or TGFβ had no effect (data not shown). PI3Kδ induction by TNF or IL-1 was concentration dependent, with maximal effects seen at 50 ng/mL and 2 ng/mL, respectively (Figure 3B). PI3Kα and PI3Kβ mRNA levels were not affected by IL-1 or TNF (Figure 3C). Akt is a key signaling molecule in the PI3K pathway. Full activation requires phosphorylation at two sites: Thr308 and Ser473.30Fruman D.A. Bismuth G. Fine tuning the immune response with PI3K.Immunol Rev. 2009; 228: 253-272Crossref PubMed Scopus (180) Google Scholar To investigate a potential role of PI3Kδ and Akt in synoviocytes, cells were stimulated with cytokines (TNF, IL-1), lipopolysaccharide (LPS), or growth factors (PDGF, TGFβ). P-Akt (Thr308), and P-Akt (Ser473) was detected within 5 minutes after PDGF stimulation and remained elevated at least for 60 minutes. There was a transient and low level of P-Akt detected in response to TNF, IL-1, or lipopolysaccharide, whereas TGFβ had no effect (n = 3) (Figure 4A). GSK3β is another signaling molecule downstream of Akt that is constitutively active and is inactivated on phosphorylation. P-GSK3β was also detected within 1 minute, peaked at 15 minutes, and decreased by 60 minutes (see Supplemental Figure S2 at http://ajp.amjpathol.org). To evaluate the functional relevance of PI3Kδ induction, we pretreated FLS with TNF and determined whether the cytokine potentiates PDGF responses. As shown in Figure 4B, TNF synergized with low concentration of PDGF and increased Akt phosphorylation. Because PDGF is the most effective inducer of P-Akt, we concentrated our subsequent studies on determining whether PI3Kδ plays a role in PDGF-mediated Akt activation. These studies focused on a panel of inhibitors, including the PI3Kδ inhibitor INK007 (Figure 5A and Table 1). In addition, several other PI3K inhibitors were used at previously reported effective concentrations22Condliffe A.M. Davidson K. Anderson K.E. Ellson C.D. Crabbe T. Okkenhaug K. Vanhaesebroeck B. Turner M. Webb L. Wymann M.P. Hirsch E. Ruckle T. Camps M. Rommel C. Jackson S.P. Chilvers E.R. Stephens L.R. Hawkins P.T. Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils.Blood. 2005; 106: 1432-1440Crossref PubMed Scopus (259) Google Scholar, 23Hayer S. Pundt N. Peters M.A. Wunrau C. Kuhnel I. Neugebauer K. Strietholt S. Zwerina J. Korb A. Penninger J. Joosten L.A. Gay S. Ruckle T. Schett G. Pap T. PI3Kgamma regulates cartilage damage in chronic inflammatory arthritis.FASEB J. 2009; 23: 4288-4298Crossref PubMed Scopus (58) Google Scholar: panPI3K (GDC-0941), PI3Kα selective (A66), PI3Kβ selective (TGX-221), PI3Kγ (AS-252424), and broad-spectrum panPI3K/mTOR (LY249002). Cells were pretreated with the inhibitors followed by PDGF stimulation for 30 minutes. Western blot analysis showed modest or no effect of the PI3Kα or PI3Kβ inhibitors whereas the pan-PI3K inhibition completely prevented Akt activation (Figure 5A). Unexpectedly, INK007 (δ) decreased P-Akt (Thr308) and P-Akt (Ser473) in a concentration-dependent fashion with an EC50 of 500 nmol/L (Figure 5C and see Supplemental Figure S3 at http://ajp.amjpathol.org). Consistent with our observation that PI3Kγ was not detected in FLS, the PI3Kγ inhibitor had no effect on Akt activation. When FLS were treated with a combination of INK007 (PI3Kδ) and A66 (PI3Kα), PDGF-induced Akt phosphorylation was also completely prevented even though A66 alone had a limited effect (Figure 5, C and D). These findings suggest that PI3Kδ is a key isoform responsible for PDGF-mediated Akt activation in RA FLS, with PI3Kδ contributing to Akt phosphorylation (Figure 5B). PI3Kδ inhibition did not affect P-GSK3β downstream of Akt (see Supplemental Figure S4 at http://ajp.amjpathol.org) or mitogen-activated protein kinase phosphorylation (data not shown). To confirm that the effect of INK007 on P-Akt is due to delta inhibition, we used two other PI3Kδ inhibitors with different chemical structures and selectivity profiles: INK055 (PI3Kγ/δ) and CAL-101 (PI3Kδ).24Herman S.E. Gordon A.L. Wagner A.J. Heerema N.A. Zhao W. Flynn J.M. Jones J. Andritsos L. Puri K.D. Lannutti B.J. Giese N.A. Zhang X. Wei L. Byrd J.C. Johnson A.J. Phosphatidylinositol 3-kinase-delta inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals.Blood. 2010; 116: 2078-2088Crossref PubMed Scopus (486) Google Scholar, 25Hoellenriegel J. Meadows S.A. Sivina M. Wierda W.G. Kantarjian H. Keating M.J. Giese N. O'Brien S. Yu A. Miller L.L. Lannutti B.J. Burger J.A. The phosphoinositide 3′-kinase delta inhibitor CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia.Blood. 2011; 118: 3603-3612Crossref PubMed Scopus (459) Google Scholar, 26Lannutti B.J. Meadows S.A. Herman S.E. Kashishian A. Steiner B. Johnson A.J. Byrd J.C. Tyner J.W. Loriaux M.M. Deininger M. Druker B.J. Puri K.D. Ulrich R.G. Giese N.A. CAL-101, a p110delta selective phosphatidylinositol-3-kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3K signaling and cellular viability.Blood. 2011; 117: 591-594Crossref PubMed Scopus (628) Google Scholar As shown in Figure 5E, both compounds, like INK007, decreased P-Akt induction. Because TNF induces PI3Kδ expression and synergizes with PDGF–induced Akt activation, we tested the effect of PI3Kδ inhibitors INK007 and CAL-101 on TNF-induced P-Akt. P-Akt induction was completely suppressed, suggesting that PI3Kδ is responsible for Akt activation in response to TNF (Figure 5F). Similar results were obtained when cells were stimulated with IL-1β (data not shown).Table 1Profile of PI3K InhibitorsCompoundPI3KαPI3KβPI3KδPI3KγGDC-0941 (pan)3345118207A66 (α)70>200001805018810TGX-221 (β)500073500100AS252424 (γ)27317370277935INK007 (δ)10933470.37.4INK055 (γ/δ)>10000>1000053CAL-101 (δ)8205652.589IC50s (nM) for inhibition of the catalytic subunits of class I PI3Ks by various compounds based on isolated enzyme assays. Open table in a new tab IC50s (nM) for inhibition of the catalytic subunits of class I PI3Ks by various compounds based on isolated enzyme assays. Akt is a major regulator of cell proliferation and survival in multiple cell types. Therefore, we determined whether PI3Kδ inhibition interferes with cell growth in vitro. FLS were pretreated with INK007 (δ), INK055 (γ/δ), CAL-101 (δ) or the panPI3K inhibitor and then cultured in the presence of PDGF for 7 days. The PI3Kδ inhibitors decreased cell growth in a concentration dependent manner (Figure 6, A–D). The EC50 for the PI3Kδ inhibitor IN
Referência(s)