Pyridinyl imidazole inhibitors, particularly SB203580, have been widely used to elucidate the roles of p38 mitogen-activated protein (MAP) kinase (p38/HOG/SAPKII) in a wide array of biological systems. Studies by this group and others have shown that SB203580 can have antiproliferative activity on cytokine-activated lymphocytes. However, we recently reported that the antiproliferative effects of SB203580 were unrelated to p38 MAP kinase activity. This present study now shows that SB203580 can inhibit the key cell cycle event of retinoblastoma protein phosphorylation in interleukin-2-stimulated T cells. Studies on the proximal regulator of this event, the phosphatidylinositol 3-kinase/protein kinase B (PKB)(Akt/Rac) kinase pathway, showed that SB203580 blocked the phosphorylation and activation of PKB by inhibiting the PKB kinase, phosphoinositide-dependent protein kinase 1. The concentrations of SB203580 required to block PKB phosphorylation (IC50 3–5 μm) are only approximately 10-fold higher than those required to inhibit p38 MAP kinase (IC50 0.3–0.5 μm). These data define a new activity for this drug and would suggest that extreme caution should be taken when interpreting data where SB203580 has been used at concentrations above 1–2 μm. Pyridinyl imidazole inhibitors, particularly SB203580, have been widely used to elucidate the roles of p38 mitogen-activated protein (MAP) kinase (p38/HOG/SAPKII) in a wide array of biological systems. Studies by this group and others have shown that SB203580 can have antiproliferative activity on cytokine-activated lymphocytes. However, we recently reported that the antiproliferative effects of SB203580 were unrelated to p38 MAP kinase activity. This present study now shows that SB203580 can inhibit the key cell cycle event of retinoblastoma protein phosphorylation in interleukin-2-stimulated T cells. Studies on the proximal regulator of this event, the phosphatidylinositol 3-kinase/protein kinase B (PKB)(Akt/Rac) kinase pathway, showed that SB203580 blocked the phosphorylation and activation of PKB by inhibiting the PKB kinase, phosphoinositide-dependent protein kinase 1. The concentrations of SB203580 required to block PKB phosphorylation (IC50 3–5 μm) are only approximately 10-fold higher than those required to inhibit p38 MAP kinase (IC50 0.3–0.5 μm). These data define a new activity for this drug and would suggest that extreme caution should be taken when interpreting data where SB203580 has been used at concentrations above 1–2 μm. interleukin mitogen-activated protein protein kinase B 3-phosphoinositide-dependent protein kinase retinoblastoma phosphatidylinositol c-Jun NH2-terminal kinase stress-activated protein kinase 4-morpholinepropanesulfonic acid dithiothreitol phosphatidylinositol 3,4,5-trisphosphate Interleukin-2 (IL-2)1 is a potent T cell growth factor that mediates its effects via a high affinity heterotrimeric receptor comprising α, β, and γc subunits. Several intracellular signaling pathways are known to be activated by IL-2, including the p42/44 mitogen-activated protein kinase (MAP kinase, also known as ERK2/1), the p38 and p54 MAP kinases (also called stress kinases, or HOG and JNK, respectively), the phosphatidyl inositol 3′ (PI) 3-kinase pathway and the Jak/STAT (signal transducer and activator of transcription) pathways. Our earlier studies using the MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor PD098059 (1.Crawley J.B. Willcocks J. Foxwell B.M. Eur. J. Immunol. 1996; 26: 2717-2723Crossref PubMed Scopus (42) Google Scholar) and those of others (2.Minami Y. Oishi I. Liu Z.J. Nakagawa S. Miyazaki T. Taniguchi T. J. Immunol. 1994; 152: 5680-5690PubMed Google Scholar, 3.Evans G.A. Goldsmith M.A. Johnston J.A. Xu W. Weiler S.R. Erwin R. Howard O.M.Z. Abraham R.T. O'Shea J.J. Greene W.C. Farrar W.L. J. Biol. Chem. 1995; 270: 28858-28863Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) have indicated that the p42/44 MAP kinase pathway is not required for IL-2-driven proliferation. In contrast, a pyridinyl imidazole inhibitor of p38 MAP kinase, SB203580, inhibited IL-2-driven T cell proliferation with an IC50 of 3–5 μm, suggesting a possible role for p38 MAP kinase in this process (4.Crawley J.B. Rawlinson L. Lali F.V. Page T.H. Saklatvala J. Foxwell B.M.J. J. Biol. Chem. 1997; 272: 15023-15027Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Recently, we have further investigated the role of p38 MAP kinase in proliferation by mapping the subdomains of the IL-2 receptor β chain involved in the activation of the kinase. As previously shown for p42/44 MAP kinase, activation of p38 and p54 MAP kinases required the acidic rich A region of the IL-2 receptor β chain (5.Hunt A.E. Lali F.V. Lord J.D. Nelson B.H. Miyazaki T. Tracey K.J. Foxwell B.M.J. J. Biol. Chem. 1999; 274: 7591-7597Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). However, the A region is not required for proliferation (2.Minami Y. Oishi I. Liu Z.J. Nakagawa S. Miyazaki T. Taniguchi T. J. Immunol. 1994; 152: 5680-5690PubMed Google Scholar, 5.Hunt A.E. Lali F.V. Lord J.D. Nelson B.H. Miyazaki T. Tracey K.J. Foxwell B.M.J. J. Biol. Chem. 1999; 274: 7591-7597Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), indicating that neither p38 MAP kinase nor p54 MAP kinase is essential for this function. Furthermore, CNI-1493 (6.Bianchi M. Bloom O. Raabe T. Cohen P.S. Chesney J. Sherry B. Schmidtmayerova H. Calandra T. Zhang X. Bukrinsky M. Ulrich P. Cerami A. Tracey K.J. J. Exp. Med. 1996; 183: 927-936Crossref PubMed Scopus (123) Google Scholar, 7.Cohen P.S. Nakshatri H. Dennis J. Caragine T. Bianchi M. Cerami A. Tracey K.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3967-3971Crossref PubMed Scopus (102) Google Scholar), an inhibitor of p38 and p54 MAP kinase activation by IL-2 was unable to inhibit proliferation (5.Hunt A.E. Lali F.V. Lord J.D. Nelson B.H. Miyazaki T. Tracey K.J. Foxwell B.M.J. J. Biol. Chem. 1999; 274: 7591-7597Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Surprisingly, SB203580 was still able to inhibit proliferation in the absence of IL-2 stimulated p38 MAP kinase activation. It has already been reported that SB203580 does inhibit p54 MAP kinase activity (8.Whitmarsh A.J. Yang S.H. Su M.S. Sharrocks A.D. Davis R.J. Mol. Cell. Biol. 1997; 17: 2360-2371Crossref PubMed Scopus (438) Google Scholar,9.Clerk A. Sugden P.H. FEBS Lett. 1998; 426: 93-96Crossref PubMed Scopus (207) Google Scholar), but the possibility that the anti-proliferative effects of SB203580 may be mediated by effects on p54 MAP kinase can also be discounted by the studies of Hunt et al. (5.Hunt A.E. Lali F.V. Lord J.D. Nelson B.H. Miyazaki T. Tracey K.J. Foxwell B.M.J. J. Biol. Chem. 1999; 274: 7591-7597Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The above data suggest the existence of a novel target of SB203580 that is critical for mitogenic signaling. To help understand the mechanism of action and possible targets of SB203580, the effect of this inhibitor on cell cycle-regulated proteins was examined. We found that the IL-2-induced hyperphosphorylation of Rb was greatly reduced in SB203580-treated cells, whereas the drug had no effect on cytokine-induced p27kip degradation or Myc expression. Subsequent studies investigated the effects of SB203580 on PI 3-kinase/protein kinase B (PKB), as these kinases have been implicated in the regulation of Rb phosphorylation (10.Brennan P. Babbage J.W. Burgering B.M. Groner B. Reif K. Cantrell D.A. Immunity. 1997; 7: 679-689Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). We observed that although PI 3-kinase activity was unaffected, the phosphorylation of PKB on Thr308 and Ser473 was inhibited, resulting in an inhibition of IL-2-induced kinase activity. The concentrations of SB203580 required were commensurate with antiproliferative effects of the drug and provide evidence that 3-phosphoinositide-dependent protein kinase 1 (PDK1; the Thr308 kinase) is inhibited by SB203580. It has recently been reported that PDK1 also acts as the Ser473 kinase (11.Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). These results could provide a mechanism for the antiproliferative effect of SB203580. Furthermore these data will have important implications for the interpretation of results from numerous published studies in which SB203580 has been used at concentrations in excess of 2 μm. IL-2 was a generous gift from Dr. P. Lomedico (Roche Molecular Biochemicals). Antiphospho-specific Ser473 and Thr308 PKB were from New England Biolabs (Hitchin, Herts, UK), whereas sheep anti-PKB and other reagents for in vitroPKB kinase assay were sold as a kit by Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-Rb was from Pharmingen (San Diego, CA), and the monoclonal anti-cyclin D3 and anti-p27kip1 were from Pierce. Rabbit anti-p70S6 kinase was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and was used for both immunoprecipitation and Western blotting. Rabbit anti-c-Myc antibody was from Santa Cruz (Santa Cruz, CA). SB203580 was purchased from Calbiochem-Novabiochem, and U5 anti-p85α monoclonal antibody was a generous gift from Professor D. Cantrell (Imperial Cancer Research Fund, London). Rabbit antisera to p38 MAP kinase was from Professor J. Saklatvala (Kennedy Institute of Rheumatology, London). Second-layer antibodies (horseradish peroxidase-conjugated) were purchased from DAKO (DAKO A/S Denmark). Recombinant PDK1 and recombinant PKB were kindly provided by Dr. L. Stephens (Babraham Institute, Cambridge, UK) and Dr. D. Alessi (Dundee, UK). CN1-1493 was from Dr. K. Tracey (Picower Institute of Medical Research New York). The IL-2-dependent murine T cell line, CT6, was grown and maintained as described previously (12.Willcocks J.L. Hales A. Page T.H. Foxwell B.M. Eur. J. Immunol. 1993; 23: 716-720Crossref PubMed Scopus (9) Google Scholar). These cells were rested by washing three times in RPMI and culturing overnight in RPMI, 5% fetal calf serum in the absence of growth factor, antibiotics, or β-mercaptoethanol supplements. 2–5 × 106 rested CT6 cells were resuspended in 2 ml of RPMI, 5% fetal calf serum and preincubated with inhibitors or vehicle control as indicated in figure legends. Cells were then stimulated with 20 ng/ml recombinant human IL-2 for 5 min at 37 °C and pelleted in a minifuge for 30 s, medium was aspirated, and the pellet was lysed in the appropriate buffer. BA/F3 cells stably expressing deletion mutants of IL-2 receptor β chain (a generous gift from Professor T. Taniguchi, Tokyo, Japan) were maintained in glutamine containing RPMI further supplemented with 5% fetal calf serum and 0.2 μg/ml G418 (Calbiochem-Novabiochem) as described previously (13.Hatakeyama M. Mori H. Doi T. Taniguchi T. Cell. 1989; 59: 837-845Abstract Full Text PDF PubMed Scopus (302) Google Scholar). Human peripheral blood mononuclear cells were prepared from buffy coat leukophoresis residues (North London Blood Transfusion Service, Colindale, London UK) and activated with 50 ng/ml OKT3 for 48 h. The cells were then washed extensively, rested overnight, and washed again before activating with IL-2; such cell preparations were >90% T cells (14.Page T.H. Lali F.V. Groome N. Foxwell B.M. J. Immunol. 1997; 158: 5727-5735PubMed Google Scholar). Cellular proliferation assays were performed by measurement of [3H]thymidine incorporation as described previously (12.Willcocks J.L. Hales A. Page T.H. Foxwell B.M. Eur. J. Immunol. 1993; 23: 716-720Crossref PubMed Scopus (9) Google Scholar). Cells were lysed in Buffer A (see below) for Western blotting and PKB kinase assays. Kinase assays were performed according to the manufacturer's instructions. Briefly, 4 μg of sheep anti-PKBα was immobilized on 25 μl of protein G-Sepharose overnight (or 1.5 h) and washed in Buffer A (50 mm Tris, pH 7.5, 1 mm EDTA, 1 mmEGTA, 0.5 mm Na3VO4, 0.1% β-mercaptoethanol, 1% Triton X-100, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 0.1 mmphenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, pepstatin, leupeptin, and 1 μm microcystin). The immobilized anti-PKB was then incubated with 0.5 ml of lysate (from 5 × 106 cells) for 1.5 h and washed three times in 0.5 ml of Buffer A supplemented with 0.5 m NaCl, two times in 0.5 ml of Buffer B (50 mm Tris-HCl, pH 7.5, 0.03% (w/v) Brij-35, 0.1 mm EGTA, and 0.1% β-mercaptoethanol), and twice with 100 μl of assay dilution buffer; 5× assay dilution buffer is 100 mm MOPS, pH 7.2, 125 mmβ-glycerophosphate, 25 mm EGTA, 5 mm sodium orthovanadate, 5 mm DTT. To the PKB enzyme immune complex was added 10 μl of assay dilution buffer, 40 μm protein kinase A inhibitor peptide, 100 μm PKB-specific substrate peptide, and 10 μCi of [γ-32P]ATP, all made up in assay dilution buffer. The reaction was incubated for 20 min at room temperature with shaking, then samples were pulse spun, and 40 μl of reaction volume were removed into another tube to which was added 20 μl of 40% trichloroacetic acid to stop the reaction. This was mixed and incubated for 5 min at room temperature, and 40 μl was transferred onto P81 phosphocellulose paper and allowed to bind for 30 s. The P81 pieces were washed three times in 0.75% phosphoric acid then in acetone at room temperature. γ-32P incorporation was then measured by scintillation counting. Cells were lysed in PI 3-kinase lysis buffer (40 mm Tris-HCl, pH 7.5, 200 mm NaCl, 1 mm EGTA supplemented with 1 mm DTT, 1 mm Na3VO4, and 10 μg/ml each of aprotinin, pepstatin, leupeptin) at 10 × 106cells/ml, and the post-nuclear lysate was precleared with 25 μl of protein G-Sepharose for 1 h then preincubated with 5 μg of monoclonal anti-p85α (U5) and further with 25 μl of protein G-Sepharose for the final 1 h. The pellets were washed three times in 0.5 ml of PI 3-kinase assay buffer. The pellet was then resuspended in 25 μl of kinase assay buffer. To this, 10 μl of a 1 mg/ml mixture of phosphatidylinositol and phosphatidylserine (made up in 100 mm HEPES, pH 7.5, and sonicated just before use) was added. The mixture was then preincubated at room temperature for 10 min, and the reaction was started by the addition of 15 μl of ATP mixture (340 μl of water, 4.2 μl of 1 mMgCl2, 16 μl of 100 mm ATP) supplemented with 5 μCi of [γ-32P]ATP. The reaction proceeded for 15 min and was stopped by the addition of 100 μl of 1 m HCl and vortexing, adding a further 200 μl of a 1:1 chloroform:methanol and vortexing again, and microfuge-spinning the tubes for 5 min. The lower layer was removed and dried in vacuo (or at 60 °C on dry block) then redissolved in 10 μl of 4:1 chloroform:methanol before spotting onto silica plates. The plate was developed in a preequilibrated vertical tank with chloroform, methanol, 28% ammonium hydroxide, water (180:140:10.8:27.5) for 3 h (or overnight) followed by phosphorimaging analysis (Fuji FLA-2000). Cells were lysed in 0.5 ml of p70S6 kinase lysis buffer (10 mm potassium phosphate, pH 7.05, 0.5% Triton X-100, 1 mm EDTA, 5 mm EGTA, 1 mm Na3VO4, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin), and the postnuclear lysate was precleared with 20 μl of protein A-agarose for 30 min. The precleared supernatant was then preincubated with 5 μl of rabbit antiserum to p70S6 kinase for 1 h and additionally with 25 μl of protein A-agarose with mixing for a further 1 h, all at 4 °C. The final immune complex was washed twice in 0.5 ml of lysis buffer and twice in 0.5 ml of kinase assay buffer (50 mm MOPS, pH 7.2, 1 mm DTT, 30 mm ATP, 5 mmMgCl2, 10 mm p-nitrophenylphosphate). To washed immune complex pellet was added 45 μl of assay mixture (made up of 35 μl of kinase assay buffer, 5 μl of 125 mm substrate peptide (KKRNRTLTK), 5 μl of 50 μm protein kinase A inhibitor, 5 μCi of [γ-32P]ATP), and the reaction was allowed to continue for 30 min at room temperature. The reaction was stopped by the addition of reducing sample buffer and boiling for 5 min. After separation on a peptide gel as described before (15.Crawley J.B. Williams L.M. Mander T. Brennan F.M. Foxwell B.M.J. J. Biol. Chem. 1996; 271: 16357-16362Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), radioactivity incorporated into peptide was quantitated by phosphorimaging. Lipid vesicles were made by drying down a mixture of phosphatidylcholine and phosphatidylserinein vacuo and reconstituting with lipid buffer (0.2m NaCl, 20 mm HEPES, 2 mm EGTA) to a final 5 times working stock (500 μmphosphatidylcholine, 500 μm phosphatidylserine, and 100 μm phosphatidylinositol 3,4,5-trisphosphate (PIP3) and sonicated before use. EE-tagged recombinant PDK1 and PKBα (both >98% pure) were prediluted in enzyme dilution buffer (1 mm DTT, 0.1 m NaCl, 1 mm EGTA, 20 mm HEPES). PDK1 assays were performed with 1 μm EE-PKB and 50 nm EE-PDK1 in the presence of appropriately diluted lipid vesicles, 0.5 μm ATP, and 1 μCi of [γ-32P]ATP in assay buffer (8 mmMgCl2, 0.12 m NaCl, 1.2 mm DTT, 1.2 mm EGTA, 0.01% azide) supplemented with protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin) in a final volume of 5 μl. The reaction was allowed to continue for 5 min at 30 °C and stopped by boiling with 10 μl of 1.5 times SDS sample buffer (with 5 mm EDTA). The PKB autokinase assays were performed as above for PDK1 but in the absence of PDK1 and PIP3. Samples were then resolved on a 10% SDS-polyacrylamide electrophoresis gel and quantitated by phosphorimaging (Fuji FLA-2000). After separation by SDS-polyacrylamide gel electrophoresis, proteins were transferred onto nitrocellulose or polyvinylidene difluoride membranes that were then blocked with 5% (w/v) milk powder in Tris-buffered saline/Tween 20 (0.1%) for 1 h at room temperature. The blocked membranes were then incubated in 1:1000 dilution of first-stage antibody (made up in Tris-buffered saline Tween, 2% bovine serum albumin supplemented with 0.1% azide) overnight, then washed and incubated with 1:1000 horseradish peroxidase-conjugated secondary antibody (made up in Tris-buffered saline Tween, 5% (w/v) milk powder) for 1 h at room temperature before developing with ECL. As we have shown previously (4.Crawley J.B. Rawlinson L. Lali F.V. Page T.H. Saklatvala J. Foxwell B.M.J. J. Biol. Chem. 1997; 272: 15023-15027Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 5.Hunt A.E. Lali F.V. Lord J.D. Nelson B.H. Miyazaki T. Tracey K.J. Foxwell B.M.J. J. Biol. Chem. 1999; 274: 7591-7597Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), the IL-2-induced proliferation of primary human T cells, murine CT6 T cells, or BAF F7 B cells is prevented by the p38 MAP kinase inhibitor with an IC50 of 3–5 μm (Fig. 1). However, as our recent studies (5.Hunt A.E. Lali F.V. Lord J.D. Nelson B.H. Miyazaki T. Tracey K.J. Foxwell B.M.J. J. Biol. Chem. 1999; 274: 7591-7597Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) showed that IL-2-induced proliferation and the inhibitory effects of SB203580 on this event were independent of p38 MAP kinase or even p54 MAP kinase activity in both T cells and B cells, we endeavored to identify other possible targets involved in mediating the anti-proliferative effects. To do this, we investigated events associated with cell cycle progression. SB203580 had no effect on Myc expression, except for a small reduction at 30 μm only (Fig. 2 a). Furthermore, nuclear staining of SB203580-treated CT6 cells with propidium iodide showed no evidence of apoptosis after stimulation with IL-2 for 20 h (data not shown). The expression of hyperphosphorylated Rb and degradation of p27kip1 were also measured as markers of S-phase entry. The addition of IL-2 to resting CT6 cells caused the hyperphosphorylation of Rb as detected by Western blotting (Fig.2 b). The presence of SB203580 in the antiproliferative (0–30 μm) range resulted in a dose-dependent reduction in the hyperphosphorylated form (Fig. 2 b). There also appeared to be some reduction in the total levels of Rb protein. Similar inhibitory effects on Rb hyperphosphorylation and protein levels were obtained with wortmannin and LY294002, both inhibitors of PI 3-kinase. The decrease in Rb protein is likely to be due to the IL-1-converting enzyme (ICE)-mediated proteolysis of the hypophosphorylated form, which has been previously reported (16.Janicke R.U. Walker P.A. Lin X.Y. Porter A.G. EMBO J. 1996; 15: 6969-6978Crossref PubMed Scopus (222) Google Scholar, 17.Dou Q.P. An B. Antoku K. Johnson D.E. J. Cell. Biochem. 1997; 64: 586-594Crossref PubMed Scopus (49) Google Scholar, 18.Gottlieb E. Oren M. EmBO J. 1998; 17: 3587-3596Crossref PubMed Scopus (74) Google Scholar). These results would also agree with previous studies on the role of PI 3-kinase in IL-2-induced Rb hyperphosphorylation and protein by Brennanet al. (10.Brennan P. Babbage J.W. Burgering B.M. Groner B. Reif K. Cantrell D.A. Immunity. 1997; 7: 679-689Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar) and would support previous indications that PI 3-kinase is a proximal regulator of Rb (10.Brennan P. Babbage J.W. Burgering B.M. Groner B. Reif K. Cantrell D.A. Immunity. 1997; 7: 679-689Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 19.Bacqueville D. Casagrande F. Perret B. Chap H. Darbon J.M. Breton-Douillon M. Biochem. Biophys. Res. Commun. 1998; 244: 630-636Crossref PubMed Scopus (40) Google Scholar, 20.Belham C.M. Scott P.H. Twomey D.P. Gould G.W. Wadsworth R.M. Plevin R. Cell. Signal. 1997; 9: 109-116Crossref PubMed Scopus (16) Google Scholar). The effects of SB203580 on Rb hyperphosphorylation were confirmed in similar studies on activated primary human T cells (Fig. 2 d).Figure 2Effect of SB203580 on cell cycle proteins. Resting CT6 cells (a, b,c) and PBMC (d and e) were preincubated with the indicated doses of SB203580, wortmannin, LY294002, or vehicle control for 1 h before stimulating with 20 ng/ml IL-2 or vehicle control for 3 h (c-Myc) or 20 h (Rb and p27kip). Cells were then lysed and prepared for total cellular c-Myc (a) Rb (b and d) or p27kip (c and e) measurement by Western blotting as described under “Experimental Procedures.” The data are representative of three separate experiments. pRb, hypophosphorylated; ppRb, hyperphosphorylated Rb.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also investigated a second cell cycle-regulated protein, p27kip1. The addition of IL-2 to resting CT6 cells induces the degradation of p27kip1(Fig. 2 c). This degradation was unaffected by SB203580, which if anything, further reduced levels of the protein. Wortmannin and LY294002 similarly had no inhibitory effect on p27kip1 degradation. Again, these studies on p27kip1 degradation were repeated in activated primary human T cells, with no significant inhibition observed with SB203580 or wortmannin, although LY294002 had some inhibitory effect (Fig. 2 e). The characteristic, S-phase hyperphosphorylation of Rb induced by IL-2 has been reported to be mediated by the PI 3-kinase pathway via the distal effector PKB (10.Brennan P. Babbage J.W. Burgering B.M. Groner B. Reif K. Cantrell D.A. Immunity. 1997; 7: 679-689Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Furthermore, both the mitogenic and survival functions of the PI 3-kinase pathway have, in several reports, been attributed to PKB (10.Brennan P. Babbage J.W. Burgering B.M. Groner B. Reif K. Cantrell D.A. Immunity. 1997; 7: 679-689Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 15.Crawley J.B. Williams L.M. Mander T. Brennan F.M. Foxwell B.M.J. J. Biol. Chem. 1996; 271: 16357-16362Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 19.Bacqueville D. Casagrande F. Perret B. Chap H. Darbon J.M. Breton-Douillon M. Biochem. Biophys. Res. Commun. 1998; 244: 630-636Crossref PubMed Scopus (40) Google Scholar, 20.Belham C.M. Scott P.H. Twomey D.P. Gould G.W. Wadsworth R.M. Plevin R. Cell. Signal. 1997; 9: 109-116Crossref PubMed Scopus (16) Google Scholar, 21.Ahmed N.N. Grimes H.L. Bellacosa A. Chan T.O. Tsichlis P.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3627-3632Crossref PubMed Scopus (487) Google Scholar, 22.Haas-Kogan D. Shalev N. Wong M. Mills G. Yount G. Stokoe D. Curr. Biol. 1998; 8: 1195-1198Abstract Full Text Full Text PDF PubMed Google Scholar). We were therefore interested in investigating the possibility that SB203580 mediates its effects on Rb by inhibiting these kinases, especially as wortmannin and LY294002 displayed similar effects. The activation of PKB requires the PI 3-kinase-generated second messenger PIP3 as well as phosphorylation on Thr308 and Ser473 mediated by the PIP3-dependent kinases, PDK1 and PDK2, respectively (23.Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 24.Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). We investigated the effect of SB203580 on PKB activation by looking at IL-2-induced phosphorylation of residue Ser473 of PKB in whole cell lysates using a phospho-specific antibody. In both CT6 and activated human T cells, SB203580 inhibited the phosphorylation of Ser473 in a dose-dependent manner (Fig.3, a and b). Similar studies on the IL-2-responsive BA/F3 F7 B cells gave the same result (Fig. 3 c). The approximate IC50 for the effect of SB203580 on this parameter is ∼5 μm, similar to the concentration required for the inhibitory effects on proliferation (Fig. 1). As expected, wortmannin (Fig. 3) and LY294002 (not shown) also inhibited Ser473 phosphorylation, whereas rapamycin (not shown) had no effect. The phosphorylation of PKB on Thr308 was similarly investigated. As the antibody was not so effective, PKB was first immunoprecipitated, and phospho-Thr308 was detected by Western blotting. SB203580 inhibited Thr308 phosphorylation in CT6 cells with similar efficacy to the Ser473 phosphorylation (Fig.4). Wortmannin, as expected, also inhibited this Thr308 phosphorylation. To confirm that the effects of SB203580 on PKB phosphorylation correlated with kinase activity, assays were performed on immunoprecipitated PKB from IL-2-stimulated CT6 cells (Fig. 5). The drug inhibited PKB activation with an IC50 of 3–10 μm, in agreement with its effects on phosphorylation of the kinase and cell proliferation.Figure 4Effect of SB203580 on phosphorylation of threonine-308 on PKB. Rested CT6 were preincubated with the indicated doses of SB203580 or vehicle control for 1 h or 100 nm wortmannin for 15 min before stimulating with 20 ng/ml IL-2 or vehicle control for 5 min. Cells were then lysed, and PKB was immunoprecipitated (IP) with anti-PKBα antiserum or irrelevant control antibody. Threonine 308 phosphorylation was assayed by Western blotting (upper panel). Membranes were stripped and reprobed for total PKB to confirm equal loading (lower panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5SB203580 inhibits IL-2-induced PKB activity. Rested CT6 cells per sample were preincubated with indicated concentrations of SB203580, wortmannin, or vehicle control before stimulating with 20 ng/ml IL-2 or vehicle control for 5 min. Immunokinase assays were performed as described under “Experimental Procedures.” Panel a is representative of three separate experiments. b, the accumulated data from the three experiments is represented graphically showing inhibition of PKB activity as a percentage of uninhibited, IL-2-activated controls.Error bars equal ± S.D. (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although the above results showed that PKB activation is inhibited, it is still possible that PKB is one of several mitogenic effector molecules downstream of the actual SB203580 target. Therefore the effect of SB203580 on IL-2-induced activation of the PI 3-kinase/PKB pathway was examined. Exposure of CT6 cells to IL-2 leads to a reproducible 2-fold increase in anti-p85-precipitable PI 3-kinase activity. This was unaffected by preincubating the cells with SB203580. In contrast, wortmannin totally inhibited this activity (Fig.6). Furthermore, direct addition of SB203580 to PI 3-kinase assays did not have any effect (results not shown), indicating that PI 3-kinase is not the target of the drug. The effect of SB203580 on PI 3-kinase/PKB pathway was also examined indirectly. Several studies have shown that p70S6 kinase is a distal mediator of PI 3-kinase activity in several systems (15.Crawley J.B. Williams L.M. Mander T. Brennan F.M. Foxwell B.M.J. J. Biol. Chem. 1996; 271: 16357-16362Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 25.Calvo V. Crews C.M. Vik T.A. Bierer B.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7571-7575Crossref PubMed Scopus (167) Google Scholar). As expected, wortmannin (Fig. 6 b) and LY294002 (not shown) inhibited the activation of p70S6 kinase by IL-2, as measured in immunokinase assays. We observed that SB203580 could also inhibit IL-2-induced p70S6 kinase activation, althou
Abstract The study of cytokines as a discrete topic has existed for approximately two decades. Initial studies focused on the identification of new cytokines, a process that is still continuing, with approximately 150 having been identified to date. The subsequent characterization of cytokine receptors led to the identification of several supramolecular cytokine receptor families (see Figure 1). As a consequence of these studies the complex nature of cytokine receptor structure also became apparent, with receptors often composed of multiple components. These observations gave an early indication of the complexity of cytokine signalling mechanisms. A general overview of cytokine and cytokine receptor structures can be found in the Cytokinefacts book (1).
Interleukin-2 (IL-2) is a potent T cell mitogen. However, the signaling pathways by which IL-2 mediates its mitogenic effect are not fully understood. One of the members of the mitogen-activated protein kinase (MAPK) family, p42/44MAPK (ERK2/1), is known to be activated by IL-2. We have now investigated the response to IL-2 of two other members of the MAP kinase family, p54MAP kinase (stress-activated protein kinase (SAPK)/Jun-N-terminal kinase (JNK)) and p38MAP kinase (p38/Mpk2/CSBP/RK), which respond primarily to stressful and inflammatory stimuli (e.g. tumor necrosis factor-alpha, IL-1, and lipopolysaccharide). Here we show that IL-2, and another T cell growth factor, IL-7, activate both SAPK/JNK and p38MAP kinase. Furthermore, inhibition of p38MAP kinase activity with a specific pyrinidyl imidazole inhibitor SB203580 that prevents activation of its downstream effector, MAPK-activating protein kinase-2, correlated with suppression of IL-2- and IL-7-driven T cell proliferation. These data indicate that in T cells p38MAP kinase has a role in transducing the mitogenic signal.
We retrospectively analysed concentrations of chromium and cobalt ions in samples of synovial fluid and whole blood taken from a group of 92 patients with failed current-generation metal-on-metal hip replacements. We applied acid oxidative digestion to our trace metal analysis protocol, which found significantly higher levels of metal ion concentrations in blood and synovial fluid than a non-digestive method. Patients were subcategorised by mode of failure as either 'unexplained pain' or 'defined causes'. Using this classification, chromium and cobalt ion levels were present over a wider range in synovial fluid and not as strongly correlated with blood ion levels as previously reported. There was no significant difference between metal ion concentrations and manufacturer of the implant, nor femoral head size below or above 50 mm. There was a moderately positive correlation between metal ion levels and acetabular component inclination angle as measured on three-dimensional CT imaging. Our results suggest that acid digestion of samples of synovial fluid samples is necessary to determine metal ion concentrations accurately so that meaningful comparisons can be made between studies.
Fibroblasts have been shown to play an increasingly important role within diabetic wounds. While several in vitro models of diabetic wound fibroblasts have been reported, none replicate the natural progression of the disease over time, recapitulating the acquisition of the diseased phenotype. Therefore, this study aimed to establish an in vitro model of the diabetic wound fibroblast through sustained exposure of healthy dermal fibroblasts to hyperglycaemia.Primary human fibroblasts were isolated from discarded healthy skin tissue and were either exposed to normoglycaemic (control 5.5mM glucose) media or hyperglycaemic (25mM glucose) media for four weeks. Quantitative polymerase chain reaction was performed to measure the expression of inflammatory cytokines and chemokines.In the hyperglycaemia model, stromal cell-derived factor (SDF)-1 expression remained consistently downregulated across all four weeks (p<0.01), while monocyte chemoattractant protein (MCP)-1 (p<0.001), interleukin (IL)-8 (p=0.847) and chemokine (C-X-C motif) ligand 1 (CXCL1) (p=0.872) were initially downregulated at one week followed by subsequent upregulation between 2-4 weeks.This hyperglycaemia model may serve as a useful tool to characterise pathological changes in the diabetic wound fibroblast and help identify candidate therapeutic targets, such as SDF-1, that may reverse the pathology.
The use of decellularised matrices as scaffolds offers the advantage of great similarity with the tissue to be replaced. Moreover, decellularised tissues and organs can be repopulated with the patient’s own cells to produce bespoke therapies. Great progress has been made in research and development of decellularised scaffolds, and more recently, these materials are being used in exciting new areas like hydrogels and bioinks. However, much effort is still needed towards preserving the original extracellular matrix composition, especially its minor components, assessing its functionality and scaling up for large tissues and organs. Emphasis should also be placed on developing new decellularisation methods and establishing minimal criteria for assessing the success of the decellularisation process. The aim of this review is to critically review the existing literature on decellularised scaffolds, especially on the preparation of these matrices, and point out areas for improvement, finishing with alternative uses of decellularised scaffolds other than tissue and organ reconstruction. Such uses include three-dimensional ex vivo platforms for idiopathic diseases and cancer modelling.
Metal-on-metal (MOM) hip resurfacings release chromium and cobalt wear debris into the surrounding joint. The hip tissue taken from failed MOM hips shows specific histological features including a subsurface band-like infiltrate of macrophages with particulate inclusions, perivascular lymphocytic infiltrate and fibrin exudation. This tissue response has been called Aseptic Lymphocytic Vasculitis Associated Lesion (ALVAL). There is a recognised carcinogenic potential associated with hexavalent chromium and epidemiological data from first generation MOM arthroplasties may suggest an increased incidence of haematological malignancy. The ALVAL type reaction includes a marked proliferation of lymphocytes in the perivascular space and thorough investigation of this lymphocytic response is warranted. This study aims to further characterise the lymphocytic infiltrate using immunohistochemistry and to test clonality using polymerase chain reaction (PCR). Tissues from revised all cause failed MOM hip arthroplasties (n=77) were collected and analysed initially using routine H&E staining. Those that met the diagnostic criteria of ALVAL described above (n=34) were further stained with a panel of immunohistochemical markers (CD3, CD4, CD8 (T-cell markers) and CD20 (B-cell marker)). 10 representative ALVAL cases were selected and sent for gene rearrangement studies using PCR to determine whether the lymphocytes were polyclonal or monoclonal in nature. The analysis of the lymphocytic aggregates in ALVAL, showed a mixed population of B and T cells. Within the aggregates, there was a predominance of B cells (CD20) over T cells (CD3). Of the 10 cases which were analysed by PCR, 7 were suitable for interpretation. None of these cases showed evidence of monoclonal lymphocyte proliferation. The carcinogenic potential of wear debris from MOM hips, particularly affecting the haematopoietic system should be investigated. This study has shown a predominantly B-lymphocyte response in tissues surrounding MOM hips which is polyclonal. Although the numbers are small, the study suggests an immune mediated response in MOM hip tissue and excludes a neoplastic proliferation. However, long term follow up of patients with MOM hips may be prudent.
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