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Isolation of immediate early genes involved in phorbol ester-induced endothelial cell differentiation
84 / SECOND INTERNATIONAL
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ON CYTOKINES
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PHOSPHORYLATION OF THE IFNv RECEPTOR (IFNvR) IN COLO205. Guriit K. Khurana Hershev and Robert 6. Schreiber Mashinaton Univ. Sch. of Med.. St. Louis, MO. 63110. Phosphorylation of the human IFNyR was studied using radiophosphate CPO4) labeling techniques and immunoprecipitation with GIR-208, a receptor specific Mab. IFNyR on Colo-205 showed a low level of constitutive phosphorylation. However, phosphorylation was substantially increased following stimulation of the cells with rHuIFNy. Phosphorylated IFNyR migrated as a single component of Mr 90 kDa that was indistinguishable from the fully mature 35S-labeled Co10 205 IFNyR. No bands were detected when a nonspecific Mab was used. IFNy-mediated receptor phosphorylation was time dependent. Enhanced phosphorylation was first observed when cells were exposed to rHuIFNy for 10 min and reached maximal levels (5.3 fold enhancement) at 30 min. Thereafter, the amount of phosphorylated product diminished and approached baseline levels by 2 hr. Phosphorylation was also dependent on ligand dose. Maximal induction of 5.35 fold was achieved with 10 ng rHuIFNy, a dose sufficient to saturate IFNyR at the cell surface. At higher ligand doses, less receptor phosphorylation was observed. PMA (100 rig/ml) also induced IFNyR phosphorylation. The time course of PMA-induced phosphorylation was indistinguishable to that observed for ligand. Thus, the IFNyR on Colo-205 surfaces is phosphorylated in response to IFNy in a dose-dependent and time-dependent and reversible manner.
HUMAN GM-CSF AND G-CSF TRANSCRIPTION: MECHANISMS OF INDUCTION MEDIATED BY NUCLEAR FACTOR, NF-GMa. E. Kuczek. L. Pell, F. Occhiodoro, M. Vadas and M.F. Shannon. Division of Human Immunology Inst. of Med. and Vet. Science, Adelaide, South Australia 5000. Growth and differentiation of haemopoietic cells are regulated by a network of signals transmitted by growth factors, whose own production is tightly controlled and in most cases, induced by stimulation of cells with mitogens and other cytokines. We have previously identified a nuclear factor (NF-GM= which binds to the CK-1 sequence found conserved in the promocer region of a number of haemopoietic growth factor including G (granulocyte) and GM (granulocyte-macrophagej-CSF (colony stimulating factor) genes. This lo-base sequence acts as an enhancer following transient transfection assays in the human Jurkat T-cell line. Experiments have shown that the level of NF-GMa binding is enhanced 5 and lo-fold in fibroblasts and endothelial cells respectively following TNF-a treatment. MUimum levels of induction are achieved after 6hr treatment, which correlates with the time course of GM-CSF mRNA induction by TNF- a. Further to this, the CK-1 sequence from the G-CSF gene acts as a TNF-a responsive element following transfection into fibroblasts and we are currently investigating whether interleukin-1 (IL-l), another monokine like TNF-a, acts through the same mechanism as TNF-ato induce GM-CSF production in fibroblasts.
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JSOLATlONOFIMMEDlATEEARLYGENESINVOLVEDINPHORBOL ESI-ER-INDUCED ENDOTHELIAL CELL DIFFERENllA TION. Timothy Hla and Thomas Maciag Lab. Molec. Biol., J.H. Holland Lab. for Biomed. Sci.. American Red 00s;. 15601 Crabbs Branch Wav. Rockville. MD 20855 Phorbol Ester (PMAj inhibits the proliferatidd of endoihelial cells induced by heparin-binding growth factors in vitro. PMA also induces differentiation of endothelial cells into a capillary-like phenotype. The actions of PMA on endothelial cells are thought to mimic those of cytokines such as IL- 1, TNF and YIFN, which are potent inhibitors of endothelial cell proliferation. In addition, TNF is angiogenic in viva In order to study the molecular events involved in endothelial cell differentiation, we sought out to isolate genes induced by PMA in the presence of cycloheximide from human endothelial cells. Poly A+ RNA was isolated from cells treated for four hours with PMA and cycloheximide, reverse transcribed with dT 7adaptor, primer and subtracted with control mRNA to R t value of IgbO. Single-stranded fraction was purified by hydroxyapatite lhromatography, tailed with deoxycytosine residues and amplified in the polymerase chain reaction with dG1+tdaptors primer. An aliquot of the amplified material was labeled and used to screen a cDNA library from endothelial cells. We obtained >500 positives from 280,000 pfu of the library. Dot blot and Northern blot analysis confirmed PMA induction. Sequence analysis of selected clones revealed that collagenase type-1 and ribosomal protein L-l were among the mRNA transcripts induced by PMA. This cloning strategy has also yielded unknown cDNAs. One such cDNA, P4, is rapidly induced by PMA and IL-l but not by TNF and 7IFN. Sequence analysis of the 3’end of P4 revealed significant homology with the @-adrenergic receptor superfamily. Sequence analysis of full length cDNA is in progress. We anticipate that these data will help to defin the molecular events involved in the immediate early stage of endothelial cell differentiation. a necessary component of angiogenesis in viva
HUMAN IL 3 AND GM-CSF SIGNAL TRANSDUCTION THROUGH COACTIVATION OF SERINE AND TYROSINE K1NASES.D. Linnekin'. D, Michielz. D. Ferrisl. and W. Farrar' 'BCDP, Program Resources, Inc., NCI-FCRF, Frederick, Maryland 21701-1013, USA and 'Laboratory of Molecular Immunoregulation, BRMP, DCT, NCIFCRF, Frederick, Maryland 21701-1013, USA. IL 3 and GM-CSF activation of protein kinases was examined in the human myeloid cell line AML-193. AM-193 cells proliferated vigorously in response to recombinant human IL 3 and GM-CSF. Cells labeled with =P-ortho"hosohate were . . stimulated with IL 3, GM-CSF or PMA and both serine and tyrosine phosphoproteins examined by high resolution 2 dimensional NEPHGE analysis. Phosphorylation of a 68 kDa protein was observed in response to IL 3, GM-CSF and PMA. A 140.150 kDa phosphotyrosylprotein was observed 10 minutes after IL 3 and GM-CSF stimulation as assessed by immunoprecipitation with a monoclonal antibody against Depletion of PKC isozymes through high dose phosphotyrosine. phorbol ester pretreatment did not effect the IL 3- and GMCSF- induced tyrosine kinase activation. The PKC isozyme system is not apparently involved in tyrosine kinase activation by either of these cytokines, however, it may be involved in the phosphorylation of the p68 serine substrate. Thus. GM-CSF- and IL 3- induced activation of serine and tyrosine kinases are coordinate but appear to be regulated independently.
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72
IL-1 OR TNF INCRF4SE WHICH ARF: RELATED M
U.S. Lynn, CY'IOKINES AND RECEPTOR-MEDIATED CELL DEATH. UTME, Galveston, TX 77550 D. Mathews. Many receptors in lymphoid cells, when activated by their ligands, eg, INF, cortisone, HIV-l, retinoic interleukins, lead to chronic elevation of Ca %::8ce11These ularly with apparent decline in intracellular zinc. ionic alterations and the attendant chronic changes in cell permeability are usually associated with activation of a C&+-dependent, protein synthesis-independent endonuclease which Droduces double strand breaks in the DNA. I" all cases, inhibition of the endonuclease by the polyanion, aurintricarboxylate, 5Oum, prevents cell death. All growth is reestablished and intracellular cholesterol in maintained at normal levels. zn2+ and phorbol esters can, like AT, also partially prevent activation of the endonuclease. However. intracellular Ca*+ remains elevated. Inhibitors of proteases or poly(ADP-ribose) polymerases are ineffective in preventing Thus, cytokine or virallvcell death in these cell lines. mediated cell death appears to be mediated terminally by Ca2+-dependent endonucleases.
PHOSPHORYLATJON OF FIBWBMT HSP 28.
PROTEINS
We h&e previoky show" that Il.1 or TNF stimulated the phosphorylation of a triad of 27 klie proteins in fibroblasts (1). We now rqxwt further biochemical characterisation of this response. On stimulating lMH!Hc:-?jfibroblasts with activators of ltinase C IPMA, OAG), analogues of c&Q', or we wert- IllX,blC to detect. a EGF, Bombesin, and .ilwllin, specific increase in phosphorylation of ~27. However, aFGF iI 111nl,
WORKSHOP
ON CYTOKINES
61
70
PHOSPHORYLATION OF THE IFNv RECEPTOR (IFNvR) IN COLO205. Guriit K. Khurana Hershev and Robert 6. Schreiber Mashinaton Univ. Sch. of Med.. St. Louis, MO. 63110. Phosphorylation of the human IFNyR was studied using radiophosphate CPO4) labeling techniques and immunoprecipitation with GIR-208, a receptor specific Mab. IFNyR on Colo-205 showed a low level of constitutive phosphorylation. However, phosphorylation was substantially increased following stimulation of the cells with rHuIFNy. Phosphorylated IFNyR migrated as a single component of Mr 90 kDa that was indistinguishable from the fully mature 35S-labeled Co10 205 IFNyR. No bands were detected when a nonspecific Mab was used. IFNy-mediated receptor phosphorylation was time dependent. Enhanced phosphorylation was first observed when cells were exposed to rHuIFNy for 10 min and reached maximal levels (5.3 fold enhancement) at 30 min. Thereafter, the amount of phosphorylated product diminished and approached baseline levels by 2 hr. Phosphorylation was also dependent on ligand dose. Maximal induction of 5.35 fold was achieved with 10 ng rHuIFNy, a dose sufficient to saturate IFNyR at the cell surface. At higher ligand doses, less receptor phosphorylation was observed. PMA (100 rig/ml) also induced IFNyR phosphorylation. The time course of PMA-induced phosphorylation was indistinguishable to that observed for ligand. Thus, the IFNyR on Colo-205 surfaces is phosphorylated in response to IFNy in a dose-dependent and time-dependent and reversible manner.
HUMAN GM-CSF AND G-CSF TRANSCRIPTION: MECHANISMS OF INDUCTION MEDIATED BY NUCLEAR FACTOR, NF-GMa. E. Kuczek. L. Pell, F. Occhiodoro, M. Vadas and M.F. Shannon. Division of Human Immunology Inst. of Med. and Vet. Science, Adelaide, South Australia 5000. Growth and differentiation of haemopoietic cells are regulated by a network of signals transmitted by growth factors, whose own production is tightly controlled and in most cases, induced by stimulation of cells with mitogens and other cytokines. We have previously identified a nuclear factor (NF-GM= which binds to the CK-1 sequence found conserved in the promocer region of a number of haemopoietic growth factor including G (granulocyte) and GM (granulocyte-macrophagej-CSF (colony stimulating factor) genes. This lo-base sequence acts as an enhancer following transient transfection assays in the human Jurkat T-cell line. Experiments have shown that the level of NF-GMa binding is enhanced 5 and lo-fold in fibroblasts and endothelial cells respectively following TNF-a treatment. MUimum levels of induction are achieved after 6hr treatment, which correlates with the time course of GM-CSF mRNA induction by TNF- a. Further to this, the CK-1 sequence from the G-CSF gene acts as a TNF-a responsive element following transfection into fibroblasts and we are currently investigating whether interleukin-1 (IL-l), another monokine like TNF-a, acts through the same mechanism as TNF-ato induce GM-CSF production in fibroblasts.
68
71
JSOLATlONOFIMMEDlATEEARLYGENESINVOLVEDINPHORBOL ESI-ER-INDUCED ENDOTHELIAL CELL DIFFERENllA TION. Timothy Hla and Thomas Maciag Lab. Molec. Biol., J.H. Holland Lab. for Biomed. Sci.. American Red 00s;. 15601 Crabbs Branch Wav. Rockville. MD 20855 Phorbol Ester (PMAj inhibits the proliferatidd of endoihelial cells induced by heparin-binding growth factors in vitro. PMA also induces differentiation of endothelial cells into a capillary-like phenotype. The actions of PMA on endothelial cells are thought to mimic those of cytokines such as IL- 1, TNF and YIFN, which are potent inhibitors of endothelial cell proliferation. In addition, TNF is angiogenic in viva In order to study the molecular events involved in endothelial cell differentiation, we sought out to isolate genes induced by PMA in the presence of cycloheximide from human endothelial cells. Poly A+ RNA was isolated from cells treated for four hours with PMA and cycloheximide, reverse transcribed with dT 7adaptor, primer and subtracted with control mRNA to R t value of IgbO. Single-stranded fraction was purified by hydroxyapatite lhromatography, tailed with deoxycytosine residues and amplified in the polymerase chain reaction with dG1+tdaptors primer. An aliquot of the amplified material was labeled and used to screen a cDNA library from endothelial cells. We obtained >500 positives from 280,000 pfu of the library. Dot blot and Northern blot analysis confirmed PMA induction. Sequence analysis of selected clones revealed that collagenase type-1 and ribosomal protein L-l were among the mRNA transcripts induced by PMA. This cloning strategy has also yielded unknown cDNAs. One such cDNA, P4, is rapidly induced by PMA and IL-l but not by TNF and 7IFN. Sequence analysis of the 3’end of P4 revealed significant homology with the @-adrenergic receptor superfamily. Sequence analysis of full length cDNA is in progress. We anticipate that these data will help to defin the molecular events involved in the immediate early stage of endothelial cell differentiation. a necessary component of angiogenesis in viva
HUMAN IL 3 AND GM-CSF SIGNAL TRANSDUCTION THROUGH COACTIVATION OF SERINE AND TYROSINE K1NASES.D. Linnekin'. D, Michielz. D. Ferrisl. and W. Farrar' 'BCDP, Program Resources, Inc., NCI-FCRF, Frederick, Maryland 21701-1013, USA and 'Laboratory of Molecular Immunoregulation, BRMP, DCT, NCIFCRF, Frederick, Maryland 21701-1013, USA. IL 3 and GM-CSF activation of protein kinases was examined in the human myeloid cell line AML-193. AM-193 cells proliferated vigorously in response to recombinant human IL 3 and GM-CSF. Cells labeled with =P-ortho"hosohate were . . stimulated with IL 3, GM-CSF or PMA and both serine and tyrosine phosphoproteins examined by high resolution 2 dimensional NEPHGE analysis. Phosphorylation of a 68 kDa protein was observed in response to IL 3, GM-CSF and PMA. A 140.150 kDa phosphotyrosylprotein was observed 10 minutes after IL 3 and GM-CSF stimulation as assessed by immunoprecipitation with a monoclonal antibody against Depletion of PKC isozymes through high dose phosphotyrosine. phorbol ester pretreatment did not effect the IL 3- and GMCSF- induced tyrosine kinase activation. The PKC isozyme system is not apparently involved in tyrosine kinase activation by either of these cytokines, however, it may be involved in the phosphorylation of the p68 serine substrate. Thus. GM-CSF- and IL 3- induced activation of serine and tyrosine kinases are coordinate but appear to be regulated independently.
69
72
IL-1 OR TNF INCRF4SE WHICH ARF: RELATED M
U.S. Lynn, CY'IOKINES AND RECEPTOR-MEDIATED CELL DEATH. UTME, Galveston, TX 77550 D. Mathews. Many receptors in lymphoid cells, when activated by their ligands, eg, INF, cortisone, HIV-l, retinoic interleukins, lead to chronic elevation of Ca %::8ce11These ularly with apparent decline in intracellular zinc. ionic alterations and the attendant chronic changes in cell permeability are usually associated with activation of a C&+-dependent, protein synthesis-independent endonuclease which Droduces double strand breaks in the DNA. I" all cases, inhibition of the endonuclease by the polyanion, aurintricarboxylate, 5Oum, prevents cell death. All growth is reestablished and intracellular cholesterol in maintained at normal levels. zn2+ and phorbol esters can, like AT, also partially prevent activation of the endonuclease. However. intracellular Ca*+ remains elevated. Inhibitors of proteases or poly(ADP-ribose) polymerases are ineffective in preventing Thus, cytokine or virallvcell death in these cell lines. mediated cell death appears to be mediated terminally by Ca2+-dependent endonucleases.
PHOSPHORYLATJON OF FIBWBMT HSP 28.
PROTEINS
We h&e previoky show" that Il.1 or TNF stimulated the phosphorylation of a triad of 27 klie proteins in fibroblasts (1). We now rqxwt further biochemical characterisation of this response. On stimulating lMH!Hc:-?jfibroblasts with activators of ltinase C IPMA, OAG), analogues of c&Q', or we wert- IllX,blC to detect. a EGF, Bombesin, and .ilwllin, specific increase in phosphorylation of ~27. However, aFGF iI 111nl,