Reconstituted human granulocyte-macrophage colony-stimulating factor receptor transduces growth-promoting signals in mouse NIH 3T3 cells: comparison with signalling in BA/F3 pro-B cells

1993 ◽  
Vol 13 (3) ◽  
pp. 1440-1448
Author(s):  
S Watanabe ◽  
A L Mui ◽  
A Muto ◽  
J X Chen ◽  
K Hayashida ◽  
...  
Keyword(s):  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Gm Csf ◽  
High Affinity ◽  
Macrophage Colony Stimulating Factor ◽  
Growth Promoting ◽  
Macrophage Colony ◽  
Nih 3T3 ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical role in growth and differentiation of myeloid cells. We previously reconstituted high-affinity human GM-CSF receptor (hGM-CSFR) in a pro-B cell line, BA/F3, by cotransfecting alpha- and beta-chain cDNA clones and showed that the reconstituted receptor could transduce growth-promoting signals. The high-affinity hGM-CSFR was also reconstituted in mouse NIH 3T3 cells, but its ability to transduce signals in fibroblasts remained undetermined. In the present study, we further characterized signal transduction by the reconstituted hGM-CSFR in both NIH 3T3 cells and BA/F3 cells. We found that the reconstituted hGM-CSFR transduces signals in NIH 3T3 fibroblasts and BA/F3 cells in response to hGM-CSF to activate transcription of the c-fos, c-jun, and c-myc proto-oncogenes. hGM-CSF also induces protein tyrosine phosphorylation and DNA synthesis in both cell types. These results indicated that hGM-CSFR is functional in fibroblasts, that signal transduction via hGM-CSFR in fibroblasts involves tyrosine kinase(s), and that association of hGM-CSFR with a factor(s) specific to hematopoietic cell lineage is not essential to transduce growth-promoting signals.

scholarly journals Reconstituted human granulocyte-macrophage colony-stimulating factor receptor transduces growth-promoting signals in mouse NIH 3T3 cells: comparison with signalling in BA/F3 pro-B cells.

1993 ◽  
Vol 13 (3) ◽  
pp. 1440-1448 ◽  
Author(s):  
S Watanabe ◽  
A L Mui ◽  
A Muto ◽  
J X Chen ◽  
K Hayashida ◽  
...  
Keyword(s):  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Gm Csf ◽  
High Affinity ◽  
Macrophage Colony Stimulating Factor ◽  
Growth Promoting ◽  
Macrophage Colony ◽  
Nih 3T3 ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical role in growth and differentiation of myeloid cells. We previously reconstituted high-affinity human GM-CSF receptor (hGM-CSFR) in a pro-B cell line, BA/F3, by cotransfecting alpha- and beta-chain cDNA clones and showed that the reconstituted receptor could transduce growth-promoting signals. The high-affinity hGM-CSFR was also reconstituted in mouse NIH 3T3 cells, but its ability to transduce signals in fibroblasts remained undetermined. In the present study, we further characterized signal transduction by the reconstituted hGM-CSFR in both NIH 3T3 cells and BA/F3 cells. We found that the reconstituted hGM-CSFR transduces signals in NIH 3T3 fibroblasts and BA/F3 cells in response to hGM-CSF to activate transcription of the c-fos, c-jun, and c-myc proto-oncogenes. hGM-CSF also induces protein tyrosine phosphorylation and DNA synthesis in both cell types. These results indicated that hGM-CSFR is functional in fibroblasts, that signal transduction via hGM-CSFR in fibroblasts involves tyrosine kinase(s), and that association of hGM-CSFR with a factor(s) specific to hematopoietic cell lineage is not essential to transduce growth-promoting signals.

scholarly journals Differential activation of leukotriene biosynthesis by granulocyte- macrophage colony-stimulating factor and interleukin-5 in an eosinophilic substrain of HL-60 cells

Blood ◽  
1995 ◽  
Vol 86 (9) ◽  
pp. 3507-3516 ◽  
Author(s):  
KA Scoggan ◽  
AW Ford-Hutchinson ◽  
DW Nicholson
Keyword(s):  
Binding Sites ◽  
Affinity Binding ◽  
Colony Stimulating Factor ◽  
High Affinity Binding ◽  
Interleukin 5 ◽  
Gm Csf ◽  
High Affinity ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Cytokines can stimulate eosinophils to produce cysteinyl leukotrienes (LTs) in the lung that provoke tissue destruction associated with asthma. Priming of an eosinophilic substrain of HL-60 cells (HL-60#7) with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) before ionophore challenge was found to produce an apparent 45% increase in total LT production in a dose-dependent manner (ED50 = 150 pmol/L) that could be accounted for by a decrease in the time required for maximal formation of LTs. GM-CSF had no effect on the kinetic parameters of LTC4 synthase and therefore probably acts upstream of this catalytic event. Incubation with interleukin-5 (IL-5), however, had no effect on LT biosynthesis. This differential priming ability was not a consequence of different receptor populations or differences in the affinity or stability of the ligand-receptor complexes of GM-CSF and IL-5. GM-CSF and IL-5 each displayed similar populations of high-affinity binding sites and neither GM-CSF nor IL-5 were able to cross-compete for the other's receptor binding sites. Analysis of phosphotyrosine patterns suggest that IL-5 is incapable of transducing a signal in eosinophilic HL-60#7 cells even though IL-5 and GM-CSF receptors mediate signal transduction via a common beta-chain component that is also necessary for high-affinity binding. Overall, this unique system may permit the dissection of distinct events responsible for specific intracellular signals transduced separately by GM-CSF or IL-5.

Simultaneous Antagonism of Interleukin-5, Granulocyte-Macrophage Colony-Stimulating Factor, and Interleukin-3 Stimulation of Human Eosinophils by Targetting the Common Cytokine Binding Site of Their Receptors

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 1943-1951 ◽  
Author(s):  
Q. Sun ◽  
K. Jones ◽  
B. McClure ◽  
B. Cambareri ◽  
B. Zacharakis ◽  
...  
Keyword(s):  
Affinity Binding ◽  
Colony Stimulating Factor ◽  
High Affinity Binding ◽  
Receptor Dimerization ◽  
Interleukin 5 ◽  
Gm Csf ◽  
High Affinity ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Human interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 are eosinophilopoietic cytokines implicated in allergy in general and in the inflammation of the airways specifically as seen in asthma. All 3 cytokines function through cell surface receptors that comprise a ligand-specific  chain and a shared subunit (βc). Although binding of IL-5, GM-CSF, and IL-3 to their respective receptor  chains is the first step in receptor activation, it is the recruitment of βc that allows high-affinity binding and signal transduction to proceed. Thus, βc is a valid yet untested target for antiasthma drugs with the added advantage of potentially allowing antagonism of all 3 eosinophil-acting cytokines with a single compound. We show here the first development of such an agent in the form of a monoclonal antibody (MoAb), BION-1, raised against the isolated membrane proximal domain of βc. BION-1 blocked eosinophil production, survival, and activation stimulated by IL-5 as well as by GM-CSF and IL-3. Studies of the mechanism of this antagonism showed that BION-1 prevented the high-affinity binding of125I–IL-5, 125I–GM-CSF, and125I–IL-3 to purified human eosinophils and that it bound to the major cytokine binding site of βc. Interestingly, epitope analysis using several βc mutants showed that BION-1 interacted with residues different from those used by IL-5, GM-CSF, and IL-3. Furthermore, coimmunoprecipitation experiments showed that BION-1 prevented ligand-induced receptor dimerization and phosphorylation of βc, suggesting that ligand contact with βc is a prerequisite for recruitment of βc, receptor dimerization, and consequent activation. These results demonstrate the feasibility of simultaneously inhibiting IL-5, GM-CSF, and IL-3 function with a single agent and that BION-1 represents a new tool and lead compound with which to identify and generate further agents for the treatment of eosinophil-dependent diseases such as asthma.

Simultaneous Antagonism of Interleukin-5, Granulocyte-Macrophage Colony-Stimulating Factor, and Interleukin-3 Stimulation of Human Eosinophils by Targetting the Common Cytokine Binding Site of Their Receptors

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 1943-1951 ◽  
Author(s):  
Q. Sun ◽  
K. Jones ◽  
B. McClure ◽  
B. Cambareri ◽  
B. Zacharakis ◽  
...  
Keyword(s):  
Affinity Binding ◽  
Colony Stimulating Factor ◽  
High Affinity Binding ◽  
Receptor Dimerization ◽  
Interleukin 5 ◽  
Gm Csf ◽  
High Affinity ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Human interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 are eosinophilopoietic cytokines implicated in allergy in general and in the inflammation of the airways specifically as seen in asthma. All 3 cytokines function through cell surface receptors that comprise a ligand-specific  chain and a shared subunit (βc). Although binding of IL-5, GM-CSF, and IL-3 to their respective receptor  chains is the first step in receptor activation, it is the recruitment of βc that allows high-affinity binding and signal transduction to proceed. Thus, βc is a valid yet untested target for antiasthma drugs with the added advantage of potentially allowing antagonism of all 3 eosinophil-acting cytokines with a single compound. We show here the first development of such an agent in the form of a monoclonal antibody (MoAb), BION-1, raised against the isolated membrane proximal domain of βc. BION-1 blocked eosinophil production, survival, and activation stimulated by IL-5 as well as by GM-CSF and IL-3. Studies of the mechanism of this antagonism showed that BION-1 prevented the high-affinity binding of125I–IL-5, 125I–GM-CSF, and125I–IL-3 to purified human eosinophils and that it bound to the major cytokine binding site of βc. Interestingly, epitope analysis using several βc mutants showed that BION-1 interacted with residues different from those used by IL-5, GM-CSF, and IL-3. Furthermore, coimmunoprecipitation experiments showed that BION-1 prevented ligand-induced receptor dimerization and phosphorylation of βc, suggesting that ligand contact with βc is a prerequisite for recruitment of βc, receptor dimerization, and consequent activation. These results demonstrate the feasibility of simultaneously inhibiting IL-5, GM-CSF, and IL-3 function with a single agent and that BION-1 represents a new tool and lead compound with which to identify and generate further agents for the treatment of eosinophil-dependent diseases such as asthma.

Receptor Clearance Obscures the Magnitude of Granulocyte-Macrophage Colony-Stimulating Factor Responses in Mice to Endotoxin or Local Infections

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1579-1585 ◽  
Author(s):  
Donald Metcalf ◽  
Nicos A. Nicola ◽  
Sandra Mifsud ◽  
Ladina Di Rago
Keyword(s):  
Intravenous Injection ◽  
Intraperitoneal Injection ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
High Affinity ◽  
Csf Levels ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Marrow cells from mice lacking high-affinity receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF; βc−/− mice) were shown to bind and internalize much less GM-CSF than cells from normal (βc+/+) mice. βc−/− mice were used to determine the effect of negligible receptor-mediated clearance on detectible GM-CSF responses to the intravenous injection of endotoxin or the intraperitoneal injection of casein plus microorganisms. Unlike the minor serum GM-CSF responses to endotoxin seen in βc+/+ mice, serum GM-CSF levels rose 30-fold to 9 ng/mL in βc−/− mice even though loss of GM-CSF in the urine was greater than in βc+/+ mice. Organs from βc−/− and βc+/+ mice had a similar capacity to produce GM-CSF in vitro, as did peritoneal cells from both types of mice when challenged in vitro by casein. However, when casein was injected intraperitoneally, βc−/− mice developed higher and more sustained levels of GM-CSF than did βc+/+ mice. The data indicated that receptor-dependent removal of GM-CSF masks the magnitude of GM-CSF responses to endotoxin and local infections. Because of this phenomenon, serum GM-CSF concentrations can be a misleading index of the occurrence or nonoccurrence of GM-CSF responses to infections.

scholarly journals Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3298-3306 ◽  
Author(s):  
M Weiss ◽  
C Yokoyama ◽  
Y Shikama ◽  
C Naugle ◽  
B Druker ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Beta Subunits ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Gm Csf ◽  
High Affinity ◽  
Alpha Subunits ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high- affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high- affinity; the cytoplasmic domain of beta was unnecessary for this high- affinity conversion. Proliferation assays showed that the membrane- proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.

scholarly journals Expression of Granulocyte-Macrophage Colony-Stimulating Factor Receptors in Human Prostate Cancer

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 1037-1043 ◽  
Author(s):  
Coralia I. Rivas ◽  
Juan Carlos Vera ◽  
Fernando Delgado-López ◽  
Mark L. Heaney ◽  
Victor H. Guaiquil ◽  
...  
Keyword(s):  
Human Prostate ◽  
Β Subunit ◽  
Colony Stimulating Factor ◽  
Lncap Cells ◽  
Α Subunit ◽  
Gm Csf ◽  
High Affinity ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

We studied the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in the human prostate carcinoma cell line LNCaP and looked for its presence in normal and neoplastic human prostatic tissue. The GM-CSF receptor is composed of two subunits, α and β. While the isolated α subunit binds GM-CSF at low-affinity, the isolated β subunit does not bind GM-CSF by itself; but complexes with the α subunit to form a high-affinity receptor. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed expression of mRNAs encoding the α and β subunits of the GM-CSF receptor in LNCaP cells, and the presence of the α and β proteins was confirmed by immunolocalization with anti-α and anti-β antibodies. Receptor binding studies using radiolabeled GM-CSF showed that LNCaP cells have about 150 high-affinity sites with a kd of 40 pmol/L and approximately 750 low-affinity sites with a kd of 2 nmol/L. GM-CSF signaled, in a time- and dose-dependent manner, for protein tyrosine phosphorylation and induced the proliferation of the LNCaP cells. Immunolocalization studies showed low level expression of GM-CSF α and β subunits in normal prostate tissue, with substantial expression in benign prostatic hyperplasia and prominent expression in neoplastic prostate tissue. Maximal expression of both subunits was observed in prostatic carcinomas metastatic to lymph node and bone. Tumor cells that stained positively with anti-α subunit antibodies were also reactive with anti-β subunit antibodies, indicating that they express high-affinity GM-CSF receptors. Our data show that the LNCaP cells express functional GM-CSF receptors and that prostatic carcinomas have prominent GM-CSF receptor expression. These findings imply that both hyperplastic and neoplastic prostatic tissues may be responsive to GM-CSF.

scholarly journals Differential regulation of early response genes and cell proliferation through the human granulocyte macrophage colony-stimulating factor receptor: selective activation of the c-fos promoter by genistein.

1993 ◽  
Vol 4 (10) ◽  
pp. 983-992 ◽  
Author(s):  
S Watanabe ◽  
A Muto ◽  
T Yokota ◽  
A Miyajima ◽  
K Arai
Keyword(s):  
Amino Acid ◽  
Early Response ◽  
Beta Subunit ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
High Affinity ◽  
Macrophage Colony Stimulating Factor ◽  
Early Response Genes ◽  
Macrophage Colony ◽  
Stimulating Factor

Granulocyte macrophage colony-stimulating factor (GM-CSF) binds to the high-affinity GM-CSF receptor (GMR) consisting of alpha and beta subunits and induces rapid tyrosine phosphorylation, activation of early response genes, and proliferation of hematopoietic cells. The alpha subunit is the primary cytokine binding component and the beta subunit is required for high-affinity binding as well as for signal transduction. Using tyrosine kinase inhibitors and cytoplasmic deletion mutants of the beta subunit, we obtained evidence that there are at least two distinct pathways downstream of the GMR in BA/F3 cell, one which is essential for proliferation, leads to the c-myc gene activation, and is sensitive to herbimycin and genistein. Activation of this pathway depends on the cytoplasmic region between amino acid positions 455 and 517 of the beta subunit. The second pathway, which leads to activation of c-fos and c-jun genes, is only partially sensitive to herbimycin, is resistant to genistein and depends on the region between amino acid positions 626 and 763 of the beta subunit. Unexpectedly, the c-fos mRNA induction was augmented by genistein. The enhanced expression of c-fos mRNA by genistein also occurred with stimulation with cAMP, PMA, or EGF in NIH3T3 cells. It thus seems likely that genistein affects a common pathway downstream of these signals.

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