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• W2018221796 abstract "Deficiency of the interferon consensus sequence-binding protein (ICSBP) is associated with increased myeloid cell proliferation in response to hematopoietic cytokines. However, previously identified ICSBP target genes do not indicate a mechanism for this “cytokine hypersen-sitivity.” In these studies, we identify the gene encoding neurofibromin 1 (Nf1) as an ICSBP target gene, by chromatin immunoprecipitation. Additionally, we find decreased Nf1 expression in bone marrow-derived myeloid cells from ICSBP–/– mice. Since Nf1 deficiency is also associated with cytokine hypersensitivity, our results suggested that NF1 is a functionally significant ICSBP target gene. Consistent with this, we find that the hyper-sensitivity of ICSBP–/– myeloid cells to granulocyte monocyte colony-stimulating factor (GM-CSF) is reversed by expression of the Nf1 GAP-related domain. We also find that treatment of ICSBP-deficient myeloid cells with monocyte colony-stimulating factor (M-CSF) results in sustained Ras activation, ERK phosphorylation, and proliferation associated with impaired Nf1 expression. These M-CSF effects are reversed by ICSBP expression in ICSBP–/– cells. Consistent with this, we find that ICSBP activates the NF1 promoter in myeloid cell line transfectants and identify an ICSBP-binding NF1 cis element. Therefore, the absence of ICSBP leads to Nf1 deficiency, impairing down-regulation of Ras activation by GM-CSF or M-CSF. These results suggest that one mechanism of increased myeloid proliferation, in ICSBP-deficient cells, is decreased NF1 gene transcription. This novel ICSBP function provides insight into regulation of myelopoiesis under normal conditions and in myeloproliferative disorders. Deficiency of the interferon consensus sequence-binding protein (ICSBP) is associated with increased myeloid cell proliferation in response to hematopoietic cytokines. However, previously identified ICSBP target genes do not indicate a mechanism for this “cytokine hypersen-sitivity.” In these studies, we identify the gene encoding neurofibromin 1 (Nf1) as an ICSBP target gene, by chromatin immunoprecipitation. Additionally, we find decreased Nf1 expression in bone marrow-derived myeloid cells from ICSBP–/– mice. Since Nf1 deficiency is also associated with cytokine hypersensitivity, our results suggested that NF1 is a functionally significant ICSBP target gene. Consistent with this, we find that the hyper-sensitivity of ICSBP–/– myeloid cells to granulocyte monocyte colony-stimulating factor (GM-CSF) is reversed by expression of the Nf1 GAP-related domain. We also find that treatment of ICSBP-deficient myeloid cells with monocyte colony-stimulating factor (M-CSF) results in sustained Ras activation, ERK phosphorylation, and proliferation associated with impaired Nf1 expression. These M-CSF effects are reversed by ICSBP expression in ICSBP–/– cells. Consistent with this, we find that ICSBP activates the NF1 promoter in myeloid cell line transfectants and identify an ICSBP-binding NF1 cis element. Therefore, the absence of ICSBP leads to Nf1 deficiency, impairing down-regulation of Ras activation by GM-CSF or M-CSF. These results suggest that one mechanism of increased myeloid proliferation, in ICSBP-deficient cells, is decreased NF1 gene transcription. This novel ICSBP function provides insight into regulation of myelopoiesis under normal conditions and in myeloproliferative disorders. The interferon consensus sequence-binding protein (ICSBP 1The abbreviations used are: ICSBP, interferon consensus sequence-binding protein; Nf1, neurofibromin 1; GM-CSF, granulocyte monocyte colony-stimulating factor; M-CSF, monocyte colony-stimulating factor; IFNγ, interferon γ; GRD, GAP-related domain; IRF, interferon regulatory factor; IL, interleukin; CML, chronic myeloid leukemia; EMSA, electrophoretic mobility shift assay; DMEM, Dulbecco's modified Eagle's medium; RBD, Ras binding domain; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; GAP, GTPase-activated protein.1The abbreviations used are: ICSBP, interferon consensus sequence-binding protein; Nf1, neurofibromin 1; GM-CSF, granulocyte monocyte colony-stimulating factor; M-CSF, monocyte colony-stimulating factor; IFNγ, interferon γ; GRD, GAP-related domain; IRF, interferon regulatory factor; IL, interleukin; CML, chronic myeloid leukemia; EMSA, electrophoretic mobility shift assay; DMEM, Dulbecco's modified Eagle's medium; RBD, Ras binding domain; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; GAP, GTPase-activated protein.; also referred to as IRF8) is a member of the interferon regulatory factor (IRF) family of transcription factors (1Driggers P.U. Ennist D.L. Gleason S.L. Maki W.H. Marks M.S. Levi B.Z. Flanagan J.R. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3743-3747Crossref PubMed Scopus (314) Google Scholar). ICSBP is expressed exclusively in myeloid and B-cells (1Driggers P.U. Ennist D.L. Gleason S.L. Maki W.H. Marks M.S. Levi B.Z. Flanagan J.R. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3743-3747Crossref PubMed Scopus (314) Google Scholar) and activates genes involved in the inflammatory response. For example, in phagocytic cells, ICSBP activates genes encoding the respiratory burst oxidase proteins gp91PHOX (2Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) and p67PHOX (3Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar), the Toll-like receptor 4 (4Rehli M. Poltorak A. Schwarzfischer L. Krause S.W. Andreesen R. Beutler B. J. Biol. Chem. 2000; 275: 9773-9781Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar), and the IL-12 receptor (5Wang I.M. Contursi C. Masumi A. Ma X. Trichieri G. Ozato K. J. Immunol. 2000; 165: 271-279Crossref PubMed Scopus (160) Google Scholar). To activate transcription of these genes, ICSBP participates in a multiprotein complex, which includes PU.1 and interferon regulatory factor 1 (IRF1) (2Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 3Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). Recruitment of ICSBP to these target genes requires binding of PU.1 to a composite Ets/IRF sequence in the proximal promoter (2Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Interaction of ICSBP with PU.1 is increased by ICSBP-tyrosine phosphorylation, which occurs during myeloid differentiation (6Kautz B. Kakar R. David E. Eklund E.A. J. Biol. Chem. 2001; 276: 37868-37878Abstract Full Text Full Text PDF PubMed Google Scholar). ICSBP also activates gene transcription by interaction with PRDI-consensus sequences, present in some promoters. Activation of an artificial promoter construct with the PRDI consensus sequence involves interaction between ICSBP and IRF1, which requires ICSBP tyrosine phosphorylation (7Sharf R. Meraro D. Azreil A. Thornton A.M. Ozato K. Petricoin E.F. Larner A.C. Schaper R. Hauser H. Levi B-Z. J. Biol. Chem. 1997; 272: 9785-9792Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Therefore, cytokine-dependant post-translational modification of ICSBP restricts transcription of some target genes to mature phagocytes (7Sharf R. Meraro D. Azreil A. Thornton A.M. Ozato K. Petricoin E.F. Larner A.C. Schaper R. Hauser H. Levi B-Z. J. Biol. Chem. 1997; 272: 9785-9792Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). In addition to transcriptional activation, ICSBP also has transcriptional repression activity. Non-tyrosine-phosphorylated ICSBP impacts transcription via direct, IRF1-independent interaction with PRDI consensus sequences (7Sharf R. Meraro D. Azreil A. Thornton A.M. Ozato K. Petricoin E.F. Larner A.C. Schaper R. Hauser H. Levi B-Z. J. Biol. Chem. 1997; 272: 9785-9792Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Only non-tyrosine-phosphorylated ICSBP binds directly to DNA (7Sharf R. Meraro D. Azreil A. Thornton A.M. Ozato K. Petricoin E.F. Larner A.C. Schaper R. Hauser H. Levi B-Z. J. Biol. Chem. 1997; 272: 9785-9792Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), suggesting that post-translational modification also regulates ICSBP repression activity. The only known genuine ICSBP repression target is the gene encoding the antiapoptotic protein, BclXL (8Gabriele L. Phung J. Fukumoto J. Segal D. Wang I.M. Giannakakou P. Giese N.A. Ozato K. Morse H.C. J. Exp. Med. 1999; 190: 411-421Crossref PubMed Scopus (101) Google Scholar). BclXL is expressed in mature myeloid cells in response to inflammatory mediators, such as lipopolysaccharide and interferon-γ (9Chatterjee D. Han Z. Mendoza J. Goodglick L. Hendrickson E.A. Pantazis P. Wyche J.H. Cell Growth Differ. 1997; 8: 1083-1089PubMed Google Scholar). In this context, BclXL expression increases survival of activated phagocytes (9Chatterjee D. Han Z. Mendoza J. Goodglick L. Hendrickson E.A. Pantazis P. Wyche J.H. Cell Growth Differ. 1997; 8: 1083-1089PubMed Google Scholar). Based upon these identified target genes, one would anticipate ICSBP deficiency to be characterized by terminal differentiation block and immune dysfunction. Indeed, this is the phenotype of the IRF1-deficient mouse (10Fehr T. Schoedon G. Odermatt B. Holtschke T. Schneemann M. Bachmann M.F. Mak T.W. Horak I. Zinkernagel R.M. J. Exp. Med. 1997; 185: 921-931Crossref PubMed Scopus (136) Google Scholar). Instead, 100% of mice with the ICSBP gene disruption develop a myeloproliferative disorder, resembling human chronic myeloid leukemia (CML) (11Holtschke T. Lohler J. Kanno J. Fehr T. Giese N. Rosenbauer F. Lou J. Knobeloch K-P. Gabriele L. Waring J.F. Bauchman M.F. Zinkernagel R.M. Morse H.C. Ozato K. Horak I. Cell. 1996; 87: 307-315Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar). Similar to CML, the majority of ICSBP–/– mice develop blast crisis, fatal in 35% of the mice at 4 months (11Holtschke T. Lohler J. Kanno J. Fehr T. Giese N. Rosenbauer F. Lou J. Knobeloch K-P. Gabriele L. Waring J.F. Bauchman M.F. Zinkernagel R.M. Morse H.C. Ozato K. Horak I. Cell. 1996; 87: 307-315Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar). Consistent with these observations, ICSBP expression is decreased in human CML blast crisis (12Schmidt M. Nagel S. Proba J. Thiede C. Ritter M. Waring J.F. Rosenbauer F. Huhn D. Wittig B. Horak I. Neubauer A. Blood. 1998; 91: 22-29Crossref PubMed Google Scholar), and forced overexpression of ICSBP delays blast crisis in a Bcr/Abl-expressing murine model (13Hao S.X. Ren R. Mol. Cell. Biol. 2000; 20: 1149-1161Crossref PubMed Scopus (127) Google Scholar). Such findings suggest that ICSBP is a leukemia anti-oncogene but do not indicate the mechanisms for this function. Bone marrow myeloid progenitor cells from ICSBP–/– mice exhibit greater than normal colony formation, in response to low doses of GM-CSF or granulocyte colony-stimulating factor (referred to as “hypersensitivity”) (14Scheller M. Foerster J. Heyworth C.M. Waring J.F. Lohler J. Gilmore G.L. Shadduck R.K. Dexter T.M. Horak I. Blood. 1999; 94: 3764-3771Crossref PubMed Google Scholar). Similar hypersensitivity to cytokines (including GM-CSF, IL-3, and stem cell factor) is also seen in bone marrow myeloid progenitor cells from mice with NF1 gene disruption (15Zhang Y.Y. Vik T.A. Ryder J.W. Srour E.F. Jacks T. Shannon K. Clapp D.W. J. Exp. Med. 1998; 187: 1893-1902Crossref PubMed Scopus (128) Google Scholar). NF1 encodes the Ras-GAP neurofibromin 1 (Nf1), which inactivates Ras in hematopoietic cells (16Boguski M. McCorkick F. Nature. 1990; 366: 643-653Crossref Scopus (1756) Google Scholar). Mutations that inactivate Nf1 or activate Ras have been described in the myeloproliferative disorder, juvenile chronic myelomonocytic leukemia (17Bollag G. Clapp D.W. Shih S. Adler F. Zhang Y. Thompson P. Lange B.J. Freedman M.H. McCormick F. Jacks T. Shannon K. Nat. Genet. 1996; 12: 144-148Crossref PubMed Scopus (467) Google Scholar). This disorder is also characterized by GM-CSF and stem cell factor hypersensitivity (18Ingram D.A. Wenning M.J. Shannon K. Clapp D.W. Blood. 2003; 101: 1984-1986Crossref PubMed Scopus (15) Google Scholar). In Nf1-deficient hematopoietic cells, increased Ras activity increases ERK and Akt activation, increasing proliferation (19Birnbaum R.A. O'Marcaigh A. Wardak Z. Zhang Y.Y. Dranoff G. Jacks T. Clapp D.W. Shannon K.M. Mol. Cell. 2000; 5: 189-195Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) and decreasing apoptosis (20Donovan S. See W. Bonifas J. Stokoe D. Shannon K.M. Cancer Cell. 2002; 2: 507-514Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Therefore, ICSBP and Nf1 deficiency states are both characterized by a myeloproliferative disorder, which includes an increased response to hematopoietic cytokines. These results suggest the possibility of a functional association between ICSBP and Nf1. Consistent with this, our current investigations identify the NF1 gene as an ICSBP activation target. Therefore, our results suggest that one mechanism of myeloproliferation in ICSBP-deficiency is impaired regulation of cytokine-induced Ras activity. Plasmids—The cDNA for human ICSBP was obtained from Dr. BenZion Levi (Technion, Haifa, Israel), and the full-length cDNA was generated by PCR and subcloned into the mammalian expression vector pcDNAamp for U937 transfection experiments. The cDNA was also subcloned into the pMSCVpuro vector for retroviral production (Stratagene, La Jolla, CA). Constructs with the cDNA for the Nf1 GAP-related domain (GRD; amino acids 1204–1535) and a GAP-activity mutant Nf1-GRD (R1276K) in the pMSCVpuro vector were a kind gift of Dr. D. Wade Clapp (Indiana University, Indianapolis). The proximal 973 bp of the human NF1 5′-flank was a kind gift of Dr. Meena Upadhyaya (Institute of Medical Genetics, Cardiff, UK). The promoter fragment was subcloned into the pCATE reporter vector (Stratagene). A 230-bp NF1 promoter truncation mutant was generated by taking advantage of a SmaI site in the 5′-flank. Truncation mutants were also generated by PCR and subcloned into the pCATE vector. Mutants were generated including the proximal 337 and 315 bp of the human NF1 promoter. All PCR products were sequenced to ensure no unintentional mutations had been introduced. Oligonucleotides—Oligonucleotides were custom synthesized by MWG Biotech (Piedmont, NC). Oligonucleotides that were used for analysis of co-immunoprecipitated chromatin were used to amplify the human promoter regions of the following genes: CYBB (F, 5′-tcagttgaccaatgattattagccatt-3′; R, 5′-ctatgcttcttcttccaatgaccaaat-3′) (2Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar); NCF2 (F, 5′-gcagaagcattttggggaactgatcct-3′; R, 5′-aaaatccacaggaaatgtcccaccttt-3′) (3Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar); TOLL-like receptor 4 (F, 5′-gatgactaattgggataaaagccaact-3′; R, 5′-tggagaggaagtgaaagcggcaacctta-3′) (4Rehli M. Poltorak A. Schwarzfischer L. Krause S.W. Andreesen R. Beutler B. J. Biol. Chem. 2000; 275: 9773-9781Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar); and NF1 (F, 5′-cagaggaaaagctgggcttaaat-3′; R, 5′-agatctgtcctccccgcggccgggg-3′) (21Cooper O.M. Upadhyaya M. Clin. Genet. 2000; 57: 221-224Crossref PubMed Scopus (7) Google Scholar). Oligonucleotide primers were also designed to amplify a conserved region of the proximal murine NF1 promoter, including the putative ICSBP-binding site, homologous to the PRDI-IRF-binding consensus (mNF1; F, 5′-ggatcccacttccggtggggtgtcatggcggcc-3′; R, 5′-gtcctcccggcgacccgggg-3′) (22Hajra A. Martin-Gallardo A. Tarle S.A. Freedman M. Wilson-Gunn S. Bernards A. Collins F.S. Genomics. 1994; 21: 649-652Crossref PubMed Scopus (43) Google Scholar). All oligonucleotides were designed with BamHI half-sites on the ends to facilitate subcloning into plasmid vectors for sequence verification of the PCR products. Oligonucleotides used to generate truncation mutants of the human NF1 promoter were designed as follows: NF1-337 F (5′-ggatcccacttccggtggggtgtcatggc-3′); NF1-315 F (5′-catggcggcgtctcggactgtgatggctgt-3′); NF1-3′ R(5′-gtcctccccgcggccgggg-3′) (22Hajra A. Martin-Gallardo A. Tarle S.A. Freedman M. Wilson-Gunn S. Bernards A. Collins F.S. Genomics. 1994; 21: 649-652Crossref PubMed Scopus (43) Google Scholar). These primers were designed with BglII half-sites to facilitate subcloning into plasmid vectors. Oligonucleotides used in quantitative real time PCR were designed to amplify the following messages: gp91PHOX (F, 5′-gagagccagatgcaggaaag-3′; R, 5′-ggtgcacagcaaagtgattg-3′); actin (F, 5′-gatgagattggcatggcttt-3′; R, 5′-caccttcaccgttccagttt-3′); Nf1 (F, 5′-agccaccacctagaatcgaa-3′; R, 5′-ggccgcatatgttcttcttt-3′); and 18 S rRNA (F, 5′-accgcggttctattttgttg-3′; R, 5′-cggtccaagaatttcacctc-3′). Double-stranded synthetic oligonucleotides were used in EMSA. Oligonucleotides were designed to the NF1 promoter sequence with homology to the IRF-binding, PRDI consensus (NF1-337 5′-ggatcccacttccggtggggtgtcatggc-3′) for use as a DNA-labeled probe in EMSA. Double-stranded oligonucleotide competitors were the PRDI consensus (5′-tcactttcactttcactt-3′) (7Sharf R. Meraro D. Azreil A. Thornton A.M. Ozato K. Petricoin E.F. Larner A.C. Schaper R. Hauser H. Levi B-Z. J. Biol. Chem. 1997; 272: 9785-9792Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) and the CCAAT box from the α-globin gene as an irrelevant competitor (5′-ggcggcgcttcattggctggcgcggagcccg-3′) (6Kautz B. Kakar R. David E. Eklund E.A. J. Biol. Chem. 2001; 276: 37868-37878Abstract Full Text Full Text PDF PubMed Google Scholar). The PRDI-like sequence and the PRDI-consensus in these oligonucleotides are underlined. These oligonucleotides were all designed with BamHI half-sites on the ends to facilitate subcloning into plasmid vectors. Cell Lines and Culture Conditions—The human myelomonocytic cell line U937 (23Larrick J.W. Fischer D.G. Anderson S.J. Koren H.A. J. Immunol. 1980; 125: 6-12Crossref PubMed Google Scholar) was obtained from Andrew Kraft (University of Colorado, Denver). Cells were maintained and differentiated as described (2Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). For differentiation experiments, U937 cells were treated for 48 h with 400 units/ml human recombinant IFNγ (Roche Applied Science) (2Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 3Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). Chromatin Immunoprecipitation and Cloning—U937 cells were cultured with or without IFNγ for 48 h, as described (2Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 3Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). Cells for chromatin immunoprecipitation were incubated with formaldehyde prior to lysis, and lysates were sonicated to generate chromatin fragments with an average size of 2.0 kb, as described (24Weinmann A.S. Bartley S.M. Zhang T. Zhang M.Q. Farnham P.J. Mol. Cell. Biol. 2001; 21: 6820-6832Crossref PubMed Scopus (331) Google Scholar). Lysates underwent two rounds of immunoprecipitation with either antiserum to ICSBP (a kind gift of Dr. Stephanie Vogel) or preimmune serum, as described (24Weinmann A.S. Bartley S.M. Zhang T. Zhang M.Q. Farnham P.J. Mol. Cell. Biol. 2001; 21: 6820-6832Crossref PubMed Scopus (331) Google Scholar). Some immunoprecipitated chromatin was treated with Klenow fragment to create blunt ends and ligated into a plasmid vector. Plasmids were transformed into Escherichia coli, and transformants with inserts were identified and sequenced according to standard techniques. The sequences of chromatin inserts were analyzed by searching GenBank™ and the National Center for Biological Information (NCBI) Human Genome Data base. The remainder of the U937 immunoprecipitated chromatin was analyzed by PCR for ICSBP antibody-specific co-precipitation of the CYBB, NCF2, TLR4, and NF1 genes. For these experiments, input chromatin was a positive control, and chromatin precipitated by preimmune serum was a negative control. PCR products were analyzed by acrylamide gel electrophoresis. The identity of the PCR product was verified by subcloning into a plasmid vector, followed by dideoxysequencing. In similar experiments, chromatin was co-immunoprecipitated with ICSBP antibody or irrelevant (glutathione S-transferase (GST)) control antibody from murine bone marrow mononuclear cells, cultured for 24 h in GM-CSF, followed by 72-h differentiation in M-CSF. For these experiments, wild type murine bone marrow was compared with ICSBP–/– bone marrow, which was a negative control in these experiments. Input (nonprecipitated chromatin) was a positive control, as above. PCRs were performed with primers to amplify the proximal NF1 promoter (see above). ICSBP Knock-out Mice—Mice with homologous disruption of the ICSBP gene were a generous gift of Dr. Keiko Ozato (National Institutes of Health, Bethesda, MD). Generation of these mice has been previously described (11Holtschke T. Lohler J. Kanno J. Fehr T. Giese N. Rosenbauer F. Lou J. Knobeloch K-P. Gabriele L. Waring J.F. Bauchman M.F. Zinkernagel R.M. Morse H.C. Ozato K. Horak I. Cell. 1996; 87: 307-315Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar). Homozygous ICSBP–/– mice and wild type litter mates were sacrificed at 8 weeks of age, when ICSBP–/– mice are in proliferative phase (11Holtschke T. Lohler J. Kanno J. Fehr T. Giese N. Rosenbauer F. Lou J. Knobeloch K-P. Gabriele L. Waring J.F. Bauchman M.F. Zinkernagel R.M. Morse H.C. Ozato K. Horak I. Cell. 1996; 87: 307-315Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar), and bone marrow was harvested from the femurs according to standard methods. Culture of ICSBP–/– Myeloid Cells—Bone marrow mononuclear cells were obtained from the femurs of ICSBP–/– and wild type litter mates. ScaI+ cells were separated using the Miltenyi magnetic bead system, according to the manufacturer's instructions (Miltenyi Biotechnology, Auburn, CA). These cells were cultured (at a concentration of 2 × 105 cells/ml) for 48 h in DMEM supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 10 ng/ml murine GM-CSF (R&D Systems Inc., Minneapolis, MN), and 5 ng/ml murine recombinant IL-3 (R&D Systems). Then cells were either maintained in GM-CSF + IL-3 (myeloid progenitor cells) or switched to DMEM supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 10 ng/ml murine recombinant M-CSF (R&D Systems) for 72 h (monocyte differentiation), with or without 200 units/ml recombinant murine IFNγ (Roche Applied Science) for the last 24 h. Cells were harvested, and cell lysates were used in Western blot experiments, as described below. Some ICSBP–/– bone marrow mononuclear cells were cultured long term in DMEM supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 10 ng/ml murine GM-CSF (R&D Systems), and 5 ng/ml murine recombinant IL-3 (R&D Systems), as described (25Tamura T. Nagamura-Inoue T. Shmeltzer Z. Kuwata T. Ozato K. Immunity. 2000; 13: 155-165Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). After 3 months of passage, these cells were evaluated by flow cytometry and found to be negative for the B-cell marker CD19 and the T-cell marker CD3, dimly positive for myeloid marker CD14, and dimly positive to negative for Mac1α (CD11b) and Gr1. Retroviral Transduction of ICSBP–/– Murine Bone Marrow Myeloid Cells—High titer murine stem cell retroviral supernatants were produced using the pMSCV vector and PT67 cell line, as per the manufacturer's instructions (Stratagene). Filtered retroviral supernatants were used immediately or stored at –80 °C. Transductions of long term cultured murine bone marrow myeloid cells from ICSBP–/– mice were performed as previously described (26Calvo K.R. Sykes D.B. Pasillas M. Kamps M.J. Mol. Cell. Biol. 2000; 20: 3274-3285Crossref PubMed Scopus (117) Google Scholar). Briefly, cells were harvested, and 4.0 × 106 cells were plated in 3 ml of DMEM, supplemented with 10% fetal calf serum, 10 ng/ml GM-CSF, and 5 ng/ml IL-3. An equal volume of retroviral supernatant was added to each dish, and Polybrene was added to a final concentration of 6 μg/ml. Cells were incubated for 8 h at 37 °C, 5% CO2, and then diluted 3-fold with media, supplemented as above. Cells were incubated overnight, and the procedure was repeated the next day. The day after transduction, puromycin was added to 1.2 ng/ml. Cells were selected in antibiotics for 96 h and then treated with cytokines as indicated. Each experiment was repeated at least three times. Expression of transduced proteins was independently verified by Western blots for each experiment. Western Blots, Immunoprecipitation, and Ras Activity Assays—For Western blots of wild type and ICSBP–/– murine bone marrow myeloid cells, cell pellets were lysed by boiling in 2× SDS sample buffer (without Coomassie Blue). Protein assays were performed by standard methods, and equivalent amounts of protein (50 μg) were separated by SDS-PAGE. Similar experiments were performed with long term cultured ICSBP–/– cells, transduced with murine stem cell retroviral vectors for protein expression, as indicated. In these experiments, the total amount of protein was either 30 μg (Fig. 2C) or 50 μg (Fig. 2D). Proteins were transferred to nitrocellulose, and Western blots were probed with antibodies to neurofibromin 1, phospho-ERK, and ERK1/2 (obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Proteins were detected by chemiluminescence using the ECL reagents from Amersham Biosciences according to the manufacturer's instructions. In other experiments, wild type murine bone marrow myeloid cells were lysed under denaturing conditions, as previously described (3Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). Cell lysates (1,000 μg) were immunoprecipitated with ICSBP antiserum (see above) or control preimmune serum. Immunoprecipitates were collected with Staph protein A-Sepharose and separated by SDS-PAGE. Proteins were transferred to nitrocellulose, and Western blots were sequentially probed with the 4G10 anti-phosphotyrosine antibody (Upstate, Waltham, MA) and ICSBP antibody (Santa Cruz Biotechnology). For Ras activity assays, murine bone marrow myeloid cells or long term cultured ICSBP–/– myeloid cells were lysed in MLB buffer (25 mm Hepes, pH 7.5, 150 mm NaCl, 1% Igepal, 10% glycerol, 10 mm MgCl2, 1 mm EDTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin). Lysates (150 μg) were precleared with recombinant GST conjugated to glutathione-agarose. Lysates were next incubated with glutathione-agarose conjugated to a GST fusion protein of the Ras binding domain (RBD) from Raf-1 (Upstate), or control GST-glutathione-agarose, according to the manufacturer's instructions. The beads were extensively washed in MLB buffer, and precipitated proteins were separated by SDS-PAGE and detected by probing Western blots with an anti-Ras antibody (pan-Ras; Santa Cruz Biotechnology). Total cell lysates (20 μg) were also separated by SDS-PAGE, and Western blots were probed with pan-Ras antibody to determine the abundance of total Ras protein. Cell Proliferation Assays—Long term cultured, transduced ICSBP–/– cells were selected for 96 h in puromycin prior to proliferation assays. Cells were harvested and plated at 105 cells/200 μl in a 96-well dish in DMEM supplemented with antibiotics and serial dilutions of GM-CSF or M-CSF (10-fold from 10 to 0.01 ng/ml or no cytokine). Cells were incubated for either 24 h (GM-CSF or M-CSF) or 72 h (M-CSF) at 37 °C, 5% CO2, with [3H]thymidine for the last 8 h. Cells were harvested, and TCA-precipitated DNA was counted by scintillation counting, according to standard techniques. RNA Isolation and Quantitative Real Time PCR—Total cellular RNA was isolated, as previously described (27Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63148) Google Scholar). Reverse transcription reactions were performed using the ImPromII reverse transcriptase kit, according to manufacturer's instructions (Promega). Quantitative real time PCR was performed using the Platinum SYBR Green qPCR SuperMix UDG kit, according to the manufacturer's instructions (Invitrogen). Real time PCR was performed using the ABI 7900 Thermocycler (Applied Biosystems, Foster City, CA), and results were analyzed using SDS version 2.1 software (Austin Biodiversity Web site gallery). Transfection and Reporter Gene Assays—U937 cells were cultured and transfected as previously described (2Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Cells (32 × 106 cells/sample) were transfected with a vector to express ICSBP (ICSBP/pcDNAamp) or empty vector control; the pCATE reporter vector with the proximal 973 bp of the human NF1 promoter (973NF1), 337 bp (337NF1), 315 bp (315NF1), 230 bp (230NF1), or empty pCATE vector control and p-CMVβgal (as a control for transfection efficiency). Transfectants were harvested 48 h after transfection, with and without incubation with recombinant human IFNγ (400 units/ml). Lysates were analyzed for CAT and β-galactosidase activity, as described (2Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 139" @default.
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