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• W2130486085 abstract "Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors with distinct modes of action on EpoR signaling in primary human erythroblasts and in a recently established murine erythroid cell line. Active PKC appeared essential for Epo-induced phosphorylation of the Epo receptor itself, STAT5, Gab1, Erk1/2, AKT, and other downstream targets. Under the same conditions, stem cell factor-induced signal transduction was not impaired. LY294002, a specific inhibitor of phosphoinositol 3-kinase, also suppressed Epo-induced signal transduction, which could be partially relieved by activators of PKC. PKC inhibitors or LY294002 did not affect membrane expression of the EpoR, the association of JAK2 with the EpoR, or the in vitro kinase activity of JAK2. The data suggest that PKC controls EpoR signaling instead of being a downstream effector. PKC and phosphoinositol 3-kinase may act in concert to regulate association of the EpoR complex such that it is responsive to ligand stimulation. Reduced PKC-activity inhibited Epo-dependent differentiation, although it did not effect Epo-dependent “renewal divisions” induced in the presence of Epo, stem cell factor, and dexamethasone. Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors with distinct modes of action on EpoR signaling in primary human erythroblasts and in a recently established murine erythroid cell line. Active PKC appeared essential for Epo-induced phosphorylation of the Epo receptor itself, STAT5, Gab1, Erk1/2, AKT, and other downstream targets. Under the same conditions, stem cell factor-induced signal transduction was not impaired. LY294002, a specific inhibitor of phosphoinositol 3-kinase, also suppressed Epo-induced signal transduction, which could be partially relieved by activators of PKC. PKC inhibitors or LY294002 did not affect membrane expression of the EpoR, the association of JAK2 with the EpoR, or the in vitro kinase activity of JAK2. The data suggest that PKC controls EpoR signaling instead of being a downstream effector. PKC and phosphoinositol 3-kinase may act in concert to regulate association of the EpoR complex such that it is responsive to ligand stimulation. Reduced PKC-activity inhibited Epo-dependent differentiation, although it did not effect Epo-dependent “renewal divisions” induced in the presence of Epo, stem cell factor, and dexamethasone. erythropoietin receptor erythropoietin stem cell factor protein kinase C phosphoinositol 3-kinase Src homology signal transducer and activator of transcription extracellular signal-regulated kinase protein kinase B phorbol 12-myristate 13-acetate phosphate-buffered saline fetal calf serum The erythropoietin receptor (EpoR)1 is essential for the regulation of proliferation, differentiation, and survival of erythroid cells (1Lin C.S. Lim S.K. D'Agati V. Costantini F. Genes Dev. 1996; 10: 154-164Crossref PubMed Scopus (345) Google Scholar, 2Wu H. Liu X. Jaenisch R. Lodish H.F. Cell. 1995; 83: 59-67Abstract Full Text PDF PubMed Scopus (842) Google Scholar). It does not contain a kinase domain and depends on associated kinases for its function. Association of JAK2 with the EpoR is crucial for erythropoiesis (3Miura O. Nakamura N. Quelle F.W. Witthuhn B.A. Ihle J.N. Aoki N. Blood. 1994; 84: 1501-1507Crossref PubMed Google Scholar, 4Witthuhn B.A. Quelle F.W. Silvennoinen O. Yi T. Tang B. Miura O. Ihle J.N. Cell. 1993; 74: 227-236Abstract Full Text PDF PubMed Scopus (997) Google Scholar). In addition, Src family kinases such as LYN appear to function in EpoR signaling (5Tilbrook P.A. Ingley E. Williams J.H. Hibbs M.L. Klinken S.P. EMBO J. 1997; 16: 1610-1619Crossref PubMed Scopus (114) Google Scholar, 6Chin H. Arai A. Wakao H. Kamiyama R. Miyasaka N. Miura O. Blood. 1998; 91: 3734-3745Crossref PubMed Google Scholar). Following ligand binding the receptor conformation changes (7Livnah O. Stura E.A. Middleton S.A. Johnson D.L. Jolliffe L.K. Wilson I.A. Science. 1999; 283: 987-990Crossref PubMed Scopus (532) Google Scholar, 8Remy I. Wilson I.A. Michnick S.W. Science. 1999; 283: 990-993Crossref PubMed Scopus (532) Google Scholar), subsequently inducing phosphorylation and activation of associated kinases (9Damen J.E. Krystal G. Exp. Hematol. 1996; 24: 1455-1459PubMed Google Scholar). Phosphorylation of cytoplasmic tyrosine residues of the EpoR creates docking sites for SH2 domain-containing signaling intermediates, which are in turn phosphorylated. Molecules recruited to the tyrosine-phosphorylated EpoR include SHP1, SHP2, Grb2, SHC, Gab-1, the p85 subunit of phosphoinositol 3-kinase (PI3K), and STAT5 (10Damen J.E. Cutler R.L. Jiao H. Yi T. Krystal G. J. Biol. Chem. 1995; 270: 23402-23408Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 11Damen J.E. Wakao H. Miyajima A. Krosl J. Humphries R.K. Cutler R.L. Krystal G. EMBO J. 1995; 14: 5557-5568Crossref PubMed Scopus (263) Google Scholar, 12Damen J.E. Liu L. Cutler R.L. Krystal G. Blood. 1993; 82: 2296-2303Crossref PubMed Google Scholar, 13He T.C. Zhuang H.M. Jiang N. Waterfield M.D. Wojchowski D.M. Blood. 1993; 82: 3530-3538Crossref PubMed Google Scholar, 14Miura O. Nakamura N. Ihle J.N. Aoki N. J. Biol. Chem. 1994; 269: 614-620Abstract Full Text PDF PubMed Google Scholar, 15Quelle F.W. Wang D. Nosaka T. Thierfelder W.E. Stravopodis D. Weinstein Y. Ihle J.N. Mol. Cell. Biol. 1996; 16: 1622-1631Crossref PubMed Scopus (242) Google Scholar, 16Klingmuller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (836) Google Scholar, 17Yi T. Zhang J. Miura O. Ihle J.N. Blood. 1995; 85: 87-95Crossref PubMed Google Scholar, 18Tauchi T. Damen J.E. Toyama K. Feng G.S. Broxmeyer H.E. Krystal G. Blood. 1996; 87: 4495-4501Crossref PubMed Google Scholar, 19Lecoq-Lafon C. Verdier F. Fichelson S. Chretien S. Gisselbrecht S. Lacombe C. Mayeux P. Blood. 1999; 93: 2578-2585Crossref PubMed Google Scholar). Active PKC is important for the development of erythroid cells (20Myklebust J.H. Smeland E.B. Josefsen D. Sioud M. Blood. 2000; 95: 510-518Crossref PubMed Google Scholar). In the presence of PKC inhibitors, Epo-induced mitogenesis is abrogated (21Spivak J.L. Fisher J. Isaacs M.A. Hankins W.D. Exp. Hematol. 1992; 20: 500-504PubMed Google Scholar, 22Haslauer M. Baltensperger K. Porzig H. Blood. 1999; 94: 114-126Crossref PubMed Google Scholar), as is the ability of bone marrow cells to form erythroid colonies (colony-forming units-erythroid; Ref. 23Jenis D.M. Johnson C.S. Furmanski P. Int. J. Cell Cloning. 1989; 7: 190-202Crossref PubMed Scopus (16) Google Scholar). Inhibition of PKC also blocks Epo-induced activation of RAF1, ERK1 and -2, and AP-1 (24Patel H.R. Sytkowski A.J. Exp. Hematol. 1995; 23: 619-625PubMed Google Scholar,25Devemy E. Billat C. Haye B. Cell Signal. 1997; 9: 41-46Crossref PubMed Scopus (30) Google Scholar) as well as up-regulation of Bcl-X, GATA-2, and c-Myc (26Li Y. Davis K.L. Sytkowski A.J. J. Biol. Chem. 1996; 271: 27025-27030Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 27Tsushima H. Urata Y. Miyazaki Y. Fuchigami K. Kuriyama K. Kondo T. Tomonaga M. Cell Growth Differ. 1997; 8: 1317-1328PubMed Google Scholar). Therefore, PKC appears to have a major role in EpoR signal transduction. However, the signaling pathway(s) in which PKC is involved has not been established. Activation of PKC involves its translocation to the plasma membrane (28Blobe G.C. Stribling S. Obeid L.M. Hannun Y.A. Cancer Surv. 1996; 27: 213-248PubMed Google Scholar, 29Hofmann J. FASEB J. 1997; 11: 649-669Crossref PubMed Scopus (331) Google Scholar), where it may come in close contact with receptor complexes. PKC has been implicated in functional regulation of several growth factor receptors. The tyrosine kinase receptors specific for SCF, epidermal growth factor, and insulin are phosphorylated on serine residues by activated PKC, which suppresses their tyrosine kinase activity (30Blume-Jensen P. Wernstedt C. Heldin C.H. Ronnstrand L. J. Biol. Chem. 1995; 270: 14192-14200Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 31Chen P. Xie H. Wells A. Mol. Biol. Cell. 1996; 7: 871-881Crossref PubMed Scopus (98) Google Scholar). PKC also affects signaling from the high affinity IgE receptor (FcεRI) on mast cells (32Kawakami Y. Yao L. Tashiro M. Gibson S. Mills G.B. Kawakami T. J. Immunol. 1995; 155: 3556-3562PubMed Google Scholar). This receptor recruits SYK, Src-like kinases, and the Tec family kinase Emt, which requires phosphorylation through PKC to be fully functional (33Kawakami Y. Yao L. Han W. Kawakami T. Immunol Lett. 1996; 54: 113-117Crossref PubMed Scopus (15) Google Scholar). Similarly, PKC phosphorylation of Bruton's tyrosine kinase, another Tec family kinase, is thought to be important for B-cell receptor signaling (34Kurosaki T. Curr. Opin. Immunol. 1997; 9: 309-318Crossref PubMed Scopus (181) Google Scholar, 35Leitges M. Schmedt C. Guinamard R. Davoust J. Schaal S. Stabel S. Tarakhovsky A. Science. 1996; 273: 788-791Crossref PubMed Scopus (412) Google Scholar). PKC activation produces pleiotropic effects in cells, which is partly explained by the large variety of PKC isoforms. PKCα, -βI, -βII, and -γ are classical, Ca2+-dependent kinases, while PKCδ, -ε, -η, -θ, and -μ function independent of Ca2+. PKC can be activated by phorbol ester, which results in degradation and depletion of certain isoforms (PKCα, -δ, and -ε). The atypical PKC isoforms (PKCζ, -λ, and -ι) function independent of phorbol esther (28Blobe G.C. Stribling S. Obeid L.M. Hannun Y.A. Cancer Surv. 1996; 27: 213-248PubMed Google Scholar). Certain isoforms are ubiquitously expressed (PKCα and -δ); others are selectively expressed in specific cell types (28Blobe G.C. Stribling S. Obeid L.M. Hannun Y.A. Cancer Surv. 1996; 27: 213-248PubMed Google Scholar, 29Hofmann J. FASEB J. 1997; 11: 649-669Crossref PubMed Scopus (331) Google Scholar). The large variety of PKC isoforms and the multitude of effects described have hampered studies concerning the biological role of PKC in proliferation and differentiation of hematopoietic cells. We recently showed that primary, human erythroid progenitors have the capacity to undergo up to 20 renewal divisions in vitro in the presence of Epo, SCF, and dexamethasone resulting in a 106-fold increase of cell numbers (36von Lindern M. Zauner W. Mellitzer G. Steinlein P. Fritsch G. Huber K. Löwenberg B. Beug H. Blood. 1999; 94: 550-559Crossref PubMed Google Scholar). These erythroid precursors develop to mature erythrocytes during 3–5 cell divisions as soon as the “renewal factors” Epo, SCF, and dexamethasone are replaced by “differentiation factors” Epo and insulin. Renewal divisions are characterized by size control (maintenance of a constant cell size) and sustained low levels of hemoglobin. Differentiation divisions are characterized by a loss of size control, resulting in a ∼4-fold reduction in cell volume and shortening of the G1phase of the cell cycle, while cellular hemoglobin increases (37Dolznig H. Bartunek P. Nasmyth K. Mullner E.W. Beug H. Cell Growth Differ. 1995; 6: 1341-1352PubMed Google Scholar). Epo is required for both renewal and differentiation divisions, while SCF plus dexamethasone are essential to sustain renewal divisions with no or minimal differentiation (36von Lindern M. Zauner W. Mellitzer G. Steinlein P. Fritsch G. Huber K. Löwenberg B. Beug H. Blood. 1999; 94: 550-559Crossref PubMed Google Scholar, 38Muta K. Krantz S.B. Bondurant M.C. Dai C.H. Blood. 1995; 86: 572-580Crossref PubMed Google Scholar). Since PKC was reported to be essential for at least some effects of EpoR activation and appeared to inhibit SCF signaling, we set out to further define the role of PKC in erythropoiesis, particularly with respect to regulation of proliferation and differentiation of erythroid cells. In this paper, we demonstrate that constitutive PKC activity is essential for phosphorylation and activation of the EpoR and multiple Epo-induced pathways. Our data suggest that PKC may control EpoR function directly or indirectly at the receptor level. In addition, LY294002, a specific inhibitor of phosphatidylinositide 3-kinase (PI3K), also reduced Epo-induced phosphorylation of multiple substrates, which could be partially reversed by activators of PKC. Primary erythroid progenitors were grown from human bone marrow as described previously (36von Lindern M. Zauner W. Mellitzer G. Steinlein P. Fritsch G. Huber K. Löwenberg B. Beug H. Blood. 1999; 94: 550-559Crossref PubMed Google Scholar) in presence of recombinant human Epo (0.5 unit/ml, a kind gift from Janssen-Cilag, Tilburg, The Netherlands), recombinant human SCF (100 ng/ml, a kind gift from Amgen) and dexamethasone (5 × 10−7m, Sigma). Cells were cultured at 1.5 - 3 × 106/ml through daily dilutions or medium changes with fresh medium containing factors. Cells were counted on an electronic cell counter (CASY-1, Schärfe-System, Germany). To induce terminal differentiation, cells were washed and transferred to medium with recombinant human Epo (5 units/ml), human insulin (1 unit/ml Actrapid, Bayer-Leverkusen) and a high concentration of iron-loaded transferrin (0.7 mg/ml). The murine erythroid cell line LK-I/11 2E. Deiner, M. von Lindern, and H. Beug, submitted for publication. was cultured in StemPro medium (Life Technologies, Inc.) supplemented with Epo (0.5 unit/ml), SCF (100 ng/ml), and dexamethasone (5 × 10−7m). Low molecular weight inhibitors GF109203X, chelerythrine, calphostin C, LY294002, and rapamycin were purchased from Biomol, phorbol 12-myristate 13-acetate (PMA) was from Sigma, and AG490 was a kind gift of Dr. A. Levitsky (Hebrew University, Jerusalem, Israel). All inhibitors were dissolved in Me2SO and diluted at least 500-fold in medium to obtain the final concentrations as indicated in the results. To determine hemoglobin accumulation, three 50-μl aliquots of the cultures were removed and processed for photometric determination of hemoglobin (39Kowenz E. Leutz A. Doderlein G. Graf T. Beug H. Neth R. Gallo R.C. Greaves M.F. Kabish H. Modern Trends in Human Leukemia VII. Springer-Verlag, Heidelberg1987: 199-209Google Scholar). To analyze cell morphology, cells were cytocentrifuged onto slides and stained with histological dyes and neutral benzidine for hemoglobin (40Beug H. Palmieri S. Freudenstein C. Zentgraf H. Graf T. Cell. 1982; 28: 907-919Abstract Full Text PDF PubMed Scopus (142) Google Scholar). Images were taken using a CCD camera and processed with Adobe Photoshop. Cultured cells were washed once with PBS and incubated in plain Iscove's medium without FCS or growth factors for 4 h at a density of 4 × 106/ml. If cells were factor-depleted for more than 4 h, they were incubated in serum-free medium containing BSA (1% w/v) as described previously (41Hoefsloot L.H. van Amelsvoort M.P. Broeders L.C.A.M. van der Plas D.C. van Lom K. Hoogerbrugge H. Touw I.P. Lowenberg B. Blood. 1997; 89: 1690-1700Crossref PubMed Google Scholar). Cells were concentrated to 20–40 × 106/ml, stimulated 10 min at 37 °C with either 10 units/ml rhEpo or 1 μg/ml SCF. To stop the reaction, 10 volumes of ice-cold phosphate-buffered saline (PBS) supplemented with 10 μmNa3VO4 were added. Cells were pelleted and lysed for 30 min at 4 °C in lysis buffer (20 mmTris-HCl, pH 8.0, 137 mm NaCl, 10 mm EDTA, 100 mm NaF, 1% (v/v) Nonidet P-40, 10% (v/v) glycerol, 2 mm Na3VO4, 1 mmPefabloc SC, 50 μg/ml aprotinin, 50 μg/ml leupeptin, 50 μg/ml bacitracin, and 50 μg/ml iodoacetamide). Lysates used for JAK2 immunoprecipitation were prepared in a JAK2 buffer (50 mmTris-HCl, pH 8.0, 200 mm NaCl, 0.5% (v/v) Triton X-100, 10% (v/v) glycerol, 2 mm Na3VO4, 1 mm Pefabloc SC, 50 μg/ml aprotinin, 50 μg/ml leupeptin, 50 μg/ml bacitracin, and 50 μg/ml iodoacetamide). Lysates were cleared by centrifugation at 4 °C for 15 min at 15,000 ×g. Immunoprecipitations and Western blots were performed as described previously (42Ward A.C. Hermans M.H. Smith L. van Aesch Y.M. Schelen A.M. Antonissen C. Touw I.P. Blood. 1999; 93: 113-124Crossref PubMed Google Scholar). In some instances, membranes were stripped in 62.5 mm Tris-HCl, pH 6.7, 2% (w/v) SDS, and 100 mm β-mercaptoethanol at 50 °C for 30 min; reblocked; washed; and reprobed. Antibodies used were mouse monoclonal antibodies recognizing Grb2, SHP1, SHP2, LYN, or SHC (Transduction Laboratories, Lexington, KY); rabbit antisera recognizing JAK2, EpoR, or Gab1(Upstate Biotechnology, Lake Placid, NY); rabbit antisera recognizing the mouse EpoR, c-Kit, or p44/p42 mitogen-activated protein kinase (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit antisera recognizing phospho-PKB (Ser473) or PKB, mouse monoclonal antibodies recognizing phospho-p44/p42 mitogen-activated protein kinase (Thr202/Tyr204; New England Biolabs, Beverly, MA); and the anti-phosphotyrosine mouse monoclonal antibody 4G10 (Upstate Biotechnology). STAT5 antiserum was a kind gift of Dr. T. Decker (University of Vienna, Vienna, Austria). Subtype-specific PKC antibodies and the peptides against which the antibodies were raised were obtained from Life Technologies, Inc. (Paisley, Scotland). Human recombinant Epo was biotinylated using biotin-aminocaproyl-hydrazide as described previously (43Wognum A.W. Lansdorp P.M. Humphries R.K. Krystal G. Blood. 1990; 76: 697-705Crossref PubMed Google Scholar). Cultured cells were washed and incubated in plain Iscove's medium without FCS or growth factors for 4 h in presence or absence of LY294002 (30 μm) or GF109203X (20 μm), or incubated in serum-free medium with BSA for 12 h in presence or absence of PMA (50 nm). Subsequently cells were stimulated with Epo (5 units/ml) or left untreated. The EpoR was detected using biotinylated Epo essentially as described previously (44de Jong M.O. Westerman Y. Wagemaker G. Wognum A.W. Stem Cells. 1997; 15: 275-285Crossref PubMed Scopus (15) Google Scholar). Cells were washed with PBS containing 1% FCS and 0.02% sodium azide and incubated in PBS/FCS/azide plus biotinylated Epo for 1 h at room temperature in presence or absence of a 100-fold excess of unlabeled Epo. Cells were washed, incubated with phycoerythrin-labeled streptavidin, washed, incubated with biotin-labeled anti-streptavidin, washed, incubated again with phycoerythrin-labeled streptavidin, and washed. Subsequently cells were analyzed by flow cytometry using fluorescence-activated cell sorting. Excess unlabeled Epo fully suppressed the signal obtained with biotinylated Epo, indicating specific detection of the EpoR. Nuclear extracts and STAT5 mobility shift assay were performed as described (41Hoefsloot L.H. van Amelsvoort M.P. Broeders L.C.A.M. van der Plas D.C. van Lom K. Hoogerbrugge H. Touw I.P. Lowenberg B. Blood. 1997; 89: 1690-1700Crossref PubMed Google Scholar). The oligonucleotide probe used in this study was the β-casein probe (5′-AGATTTCTAGGAATTCAATCC; Ref. 45Gouilleux F. Pallard C. Dusanter-Fourt I. Wakao H. Haldosen L.A. Norstedt G. Levy D. Groner B. EMBO J. 1995; 14: 2005-2013Crossref PubMed Scopus (332) Google Scholar). The DNA-protein complexes were separated by electrophoresis on 5% polyacrylamide gels containing 5% glycerol in 0.25× TBE. The gels were dried analyzed by autoradiography, and shifted probe was quantified on the ImageQuant PhosphorImager. Sepharose beads with immune complexes were washed twice in lysis buffer and once in kinase buffer (10 mm HEPES, pH 7.4, 50 mm NaCl, 5 mmMgCl, 5 mm MnCl2, 0.5 mmdithiothreitol) at 4 °C. Beads were resuspended in 100 μl of kinase buffer supplemented with protease inhibitors (see above), 1 mm ATP (unlabeled), and 10 μCi of [γ-32P]ATP (>5000 Ci/mmol, Amersham Pharmacia Biotech). The kinase reaction was incubated for 20 min at 22 °C. The reaction was stopped with an excess of ice-cold lysis buffer, and beads were washed twice in lysis buffer and once in PBS at 4 °C. Immune complexes were eluted by boiling for 5 min in sodium dodecyl sulfate (SDS) sample buffer. Following SDS-polyacrylamide gel electrophoresis, gels were dried and analyzed by autoradiography. To investigate whether inhibition of PKC impairs EpoR signaling, we first tested the effect of various PKC inhibitors on Epo-induced STAT5 DNA binding. Primary erythroid progenitors were factor-deprived in the presence of increasing concentrations of the PKC inhibitors GF109203X (blocking the ATP binding site; Ref. 46Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar), chelerythrine (blocking the catalytic site; Ref. 47Herbert J.M. Augereau J.M. Gleye J. Maffrand J.P. Biochem. Cell Biol. 1990; 172: 993-999Google Scholar), calphostin C (blocking the regulatory site; Ref. 48Dubyak G.R. Kertesy S.B. Arch. Biochem. Biophys. 1997; 341: 129-139Crossref PubMed Scopus (17) Google Scholar), and Gö6976 (specific for Ca2+-dependent PKC subtypes; Ref. 49Wenzel-Seifert K. Schachtele C. Seifert R. Biochem. Cell Biol. 1994; 200: 1536-1543Google Scholar). Cells were stimulated with Epo, and nuclear extracts were tested for Epo-induced STAT5 DNA binding. All four inhibitors completely blocked Epo-induced STAT5 DNA binding in a dose-dependent manner (Fig. 1 A). In control incubations the addition of solvent, Me2SO, had no effect on Epo-induced STAT5 DNA binding (data not shown). Cytological analysis indicated that incubation of the cells with the inhibitors did not induce any apparent cell death. We then tested the contribution of other pathways to Epo-induced Stat5 phosphorylation. First, PKC activity is often associated with PI3K activity. Addition of the PI3K inhibitor LY294002 inhibited Epo-induced STAT5 DNA binding as well (Fig. 1 B). This was not due to inhibition of TOR (Target of Rapamycin) kinases (50Brunn G.J. Williams J. Sabers C. Wiederrecht G. Lawrence Jr., J.C. Abraham R.T. EMBO J. 1996; 15: 5256-5267Crossref PubMed Scopus (615) Google Scholar), as addition of rapamycin had no effect on Epo-induced STAT5 DNA binding (Fig.1 B). Second, Epo-induced STAT5 phosphorylation was shown to depend on active JAK2 (45Gouilleux F. Pallard C. Dusanter-Fourt I. Wakao H. Haldosen L.A. Norstedt G. Levy D. Groner B. EMBO J. 1995; 14: 2005-2013Crossref PubMed Scopus (332) Google Scholar). Addition of the specific JAK2 inhibitor AG490 (51Meydan N. Grunberger T. Dadi H. Shahar M. Arpaia E. Lapidot Z. Leeder J.S. Freedman M. Cohen A. Gazit A. Levitzki A. Roifman C.M. Nature. 1996; 379: 645-648Crossref PubMed Scopus (845) Google Scholar) resulted in concentration-dependent inhibition of STAT5 activation to an extent comparable to that induced by the PKC inhibitors (Fig. 1 C). PMA is a pleiotropic modulator of PKC activity. PMA activates PKC, but activation is followed by a rapid cleavage and depletion of certain PKC subtypes (29Hofmann J. FASEB J. 1997; 11: 649-669Crossref PubMed Scopus (331) Google Scholar). We therefore evaluated the Epo response upon increasing exposure of the cells to PMA. Erythroid progenitors were factor-depleted for 8 h and subsequently stimulated with Epo. At various time points during starvation, GF109203X (20 μm) or PMA (50 nm) were added. Addition of GF109203 1–2 h before Epo stimulation of the cells largely inhibited Epo-induced STAT5 DNA binding (Fig. 2 A). In contrast, short term exposure of the cells to PMA enhanced Epo-induced STAT5 DNA binding to 150% of control values. Upon longer exposure of the cells to PMA, Epo-induced STAT5 DNA binding was suppressed (Fig.2 A). In conclusion, four different inhibitors that block PKC activity via distinct mechanisms all prevented Epo-induced STAT5 DNA binding, while activation of PKC promoted Epo-induced STAT5 DNA binding followed by down-modulation. Addition of the PI3K inhibitor LY294002 or the JAK2 inhibitor AG490 also inhibited Epo-induced STAT5 DNA binding. To verify that the inhibitors had no subtle effects on cell viability, we examined whether the inhibition of Epo-induced STAT5 DNA binding was reversible. Erythroid progenitors were factor-depleted in the presence of 20 μm chelerythrine or LY294002. Subsequently, cells were washed three times and re-seeded in medium lacking both inhibitor and Epo. At successive intervals, Epo-induced STAT5 DNA binding was assessed (Fig. 2, B and C). At 2–4 h after washing, Epo-induced STAT5 DNA binding appeared to be fully recoverable. Since PKC inhibitors and LY294002 both resulted in a virtually complete suppression of EpoR signaling (Fig. 1), PI3K is likely to act within the same pathway as PKC. To examine whether PI3K would act up- or downstream of PKC, cells treated with LY294002 were subsequently stimulated with the PKC activators PMA and bryostatin (52Hess A.D. Silanskis M.K. Esa A.H. Pettit G.R. May W.S. J. Immunol. 1988; 141: 3263-3269PubMed Google Scholar). Both PKC activators restored Epo-induced STAT5 DNA binding in the continuing presence of LY294002 to near 100% of control values (Fig.2 C). However, in the same experiment, the PKC activators boosted Epo-induced STAT5 DNA binding to 180% of control levels in the absence of LY294002 (data not shown). Thus, activation of PKC can partially compensate a lack of PI3K activity. This would suggest that PKC and PI3K may act in concert to control Epo responsiveness. We next examined whether the PKC inhibitors altered the time kinetics of Epo-induced STAT5 activation. Cells were pretreated with 20 μm GF109203X or 100 nmGö6976 and Epo-induced STAT5 DNA binding was analyzed at successive time intervals. In the presence of PKC inhibitor, the increment of STAT5 DNA binding was reduced as compared with untreated control cells. In both cases, maximal activation was reached at 20 min following the onset of Epo stimulation, indicating an effect on the magnitude rather than on the duration of the response (Fig.3). In addition we examined whether the tyrosine phosphatase inhibitor sodium orthovanadate (Na3VO4) would restore Epo-induced STAT5 activation. Although Na3VO4 caused a general increase of DNA-bound STAT5 both in the presence and absence of PKC inhibitors, 10 μm Na3VO4 did not relieve suppression of Epo-induced STAT5 by PKC inhibitors (data not shown). Thus, reduction of Epo-induced STAT5 activation following inhibition of PKC does not result from accelerated down-regulation of EpoR signaling. Inhibition of PKC could specifically affect Epo-induced STAT5 activation, or it could interfere with signaling from the EpoR in general. Human erythroid progenitors were preincubated with the PKC inhibitor GF109203X (20 μm), which suppressed Epo-induced phosphorylation of STAT5, the EpoR, and JAK2 in response to Epo (Fig.4 A). GF109203X had no effects in absence of Epo. Similar to erythroid progenitors from human bone marrow, murine erythroid progenitors from fetal liver can be expanded in presence of Epo, SCF, and dexamethasone. These cells are grown in serum-free medium and are a much more reproducible and clean system for signal transduction studies in erythroid progenitors. Moreover, using p53−/− fetal liver cells, indefinite cultures of erythroblasts could be established reproducibly. Like human erythroblasts, the cells retain the capacity to differentiate in presence of Epo plus insulin.2 In a cloned culture of these mouse erythroid progenitors, LK-I/11, STAT5 was similarly inhibited by PKC inhibitors (data not shown). To examine which pathways are controlled by PKC and PI3K, the cells were pre-incubated with the JAK2 inhibitor AG490 (50 μm), GF109203X (20 μm), or LY294002 (30 μm). All three inhibitors suppressed Epo-induced tyrosine phosphorylation of the EpoR, STAT5, and Gab1 (Fig. 4 B; data not shown). Furthermore, the inhibitors suppressed Epo-induced phosphorylation of ERK on Thr202/Tyr204 and Epo-induced tyrosine phosphorylation of several proteins detected in whole cell lysate (Fig. 4 B). Exceptionally, Epo-induced JAK2 phosphorylation was reduced to a lesser extent (Fig.4 B). In conclusion, active PKC is required not only for Epo-induced STAT5 DNA binding, but also for Epo-induced phosphorylation of the EpoR, and downstream targets both in human and mouse erythroid progenitors. Subsequently, we wanted to examine whether other receptors present in the same cells are similarly controlled by PKC. Murine erythroid progenitors were preincubated with increasing concentrations of the PKC inhibitor Gö6976 and subsequently stimulated with Epo or SCF. Ligand-induced phosphorylation of ERK1/2 and PKB was analyzed using phosphospecific antibodies on Western blots. Gö6976 inhibited Epo-induced phosphorylation of ERK and PKB, but it did not affect SCF-induced phosphorylation of these proteins (Fig.5 A). Similarly, Epo- and SCF-induced phosphorylation of ERK1/2 and PKB was assessed in LK-I/11 cells preincubated with increasing concentrations of the PI3K inhibitor LY294002. At 30 μm, LY294002 suppressed phosphorylation of ERK by Epo, while it did not affect SCF-induced ERK phosphorylation (Fig. 5 B). Thus, active PKC and PI3K are required to render the EpoR permissive for ligand-induced signaling, but they do not control c-Kit signaling. Phosphorylation of PKB by Epo or SCF was completely suppressed at 7.5 μm LY294002. This efficient inhibition of Epo- and SCF-induced phosphorylation of PKB represents inhibition of Epo- or SCF-induced PI3K activity, as previously documented (10Damen J.E. Cutler R.L. Jiao H. Yi T. Krystal G. J. B" @default.
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• W2130486085 title "Protein Kinase C α Controls Erythropoietin Receptor Signaling" @default.
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