scholarly journals Differential processing of colony-stimulating factor 1 precursors encoded by two human cDNAs.

1988 ◽  
Vol 8 (11) ◽  
pp. 5026-5034 ◽  
Author(s):  
C W Rettenmier ◽  
M F Roussel
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Biologically Active ◽  
Colony Stimulating Factor ◽  
Acid Product ◽  
Human Cdna ◽  
3T3 Fibroblasts ◽  
Nih 3T3 ◽  
Stimulating Factor

The biosynthesis of macrophage colony-stimulating factor 1 (CSF-1) was examined in mouse NIH-3T3 fibroblasts transfected with a retroviral vector expressing the 554-amino-acid product of a human 4-kilobase (kb) CSF-1 cDNA. Similar to results previously obtained with a 1.6-kb human cDNA that codes for a 256-amino-acid CSF-1 precursor, the results of the present study showed that NIH-3T3 cells expressing the product of the 4-kb clone produced biologically active human CSF-1 and were transformed by an autocrine mechanism when cotransfected with a vector containing a human c-fms (CSF-1 receptor) cDNA. The 4-kb CSF-1 cDNA product was synthesized as an integral transmembrane glycoprotein that was assembled into disulfide-linked dimers and rapidly underwent proteolytic cleavage to generate a soluble growth factor. Although the smaller CSF-1 precursor specified by the 1.6-kb human cDNA was stably expressed as a membrane-bound glycoprotein at the cell surface and was slowly cleaved to release the extracellular growth factor, the cell-associated product of the 4-kb clone was efficiently processed to the secreted form and was not detected on the plasma membrane. Digestion with glycosidic enzymes indicated that soluble CSF-1 encoded by the 4-kb cDNA contained both asparagine(N)-linked and O-linked carbohydrate chains, whereas the product of the 1.6-kb clone had only N-linked oligosaccharides. Removal of the carbohydrate indicated that the polypeptide chain of the secreted 4-kb cDNA product was longer than that of the corresponding form encoded by the smaller clone. These differences in posttranslational processing may reflect diverse physiological roles for the products of the two CSF-1 precursors in vivo.

Differential processing of colony-stimulating factor 1 precursors encoded by two human cDNAs

1988 ◽  
Vol 8 (11) ◽  
pp. 5026-5034
Author(s):  
C W Rettenmier ◽  
M F Roussel
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Biologically Active ◽  
Colony Stimulating Factor ◽  
Acid Product ◽  
Human Cdna ◽  
3T3 Fibroblasts ◽  
Nih 3T3 ◽  
Stimulating Factor

The biosynthesis of macrophage colony-stimulating factor 1 (CSF-1) was examined in mouse NIH-3T3 fibroblasts transfected with a retroviral vector expressing the 554-amino-acid product of a human 4-kilobase (kb) CSF-1 cDNA. Similar to results previously obtained with a 1.6-kb human cDNA that codes for a 256-amino-acid CSF-1 precursor, the results of the present study showed that NIH-3T3 cells expressing the product of the 4-kb clone produced biologically active human CSF-1 and were transformed by an autocrine mechanism when cotransfected with a vector containing a human c-fms (CSF-1 receptor) cDNA. The 4-kb CSF-1 cDNA product was synthesized as an integral transmembrane glycoprotein that was assembled into disulfide-linked dimers and rapidly underwent proteolytic cleavage to generate a soluble growth factor. Although the smaller CSF-1 precursor specified by the 1.6-kb human cDNA was stably expressed as a membrane-bound glycoprotein at the cell surface and was slowly cleaved to release the extracellular growth factor, the cell-associated product of the 4-kb clone was efficiently processed to the secreted form and was not detected on the plasma membrane. Digestion with glycosidic enzymes indicated that soluble CSF-1 encoded by the 4-kb cDNA contained both asparagine(N)-linked and O-linked carbohydrate chains, whereas the product of the 1.6-kb clone had only N-linked oligosaccharides. Removal of the carbohydrate indicated that the polypeptide chain of the secreted 4-kb cDNA product was longer than that of the corresponding form encoded by the smaller clone. These differences in posttranslational processing may reflect diverse physiological roles for the products of the two CSF-1 precursors in vivo.

Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes

1987 ◽  
Vol 7 (7) ◽  
pp. 2378-2387 ◽  
Author(s):  
C W Rettenmier ◽  
M F Roussel ◽  
R A Ashmun ◽  
P Ralph ◽  
K Price ◽  
...  
Keyword(s):  
Growth Factor ◽  
Disulfide Bonds ◽  
Mononuclear Phagocyte ◽  
Transformed Cells ◽  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Membrane Bound ◽  
Nih 3T3 ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

NIH 3T3 cells cotransfected with the human c-fms proto-oncogene together with a 1.6-kilobase cDNA clone encoding a 256-amino-acid precursor of the human mononuclear phagocyte colony-stimulating factor CSF-1 (M-CSF) undergo transformation by an autocrine mechanism. The number of CSF-1 receptors on the surface of transformed cells was regulated by ligand-induced receptor degradation and was inversely proportional to the quantity of CSF-1 produced. A tyrosine-to-phenylalanine mutation at position 969 near the receptor carboxyl terminus potentiated its transforming efficiency in cells cotransfected by the CSF-1 gene but did not affect receptor downmodulation. CSF-1 was synthesized as an integral transmembrane glycoprotein that was rapidly dimerized through disulfide bonds. The homodimer was externalized at the cell surface, where it underwent proteolysis to yield the soluble growth factor. Trypsin treatment of viable cells cleaved the plasma membrane form of CSF-1 to molecules of a size indistinguishable from that of the extracellular growth factor, suggesting that trypsinlike proteases regulate the rate of CSF-1 release from transformed cells. The data raise the possibility that this form of membrane-bound CSF-1 might stimulate receptors on adjacent cells through direct cell-cell interactions.

scholarly journals Hematopoietic progenitors in cyclic neutropenia: effect of granulocyte colony-stimulating factor in vivo

Blood ◽  
1990 ◽  
Vol 75 (10) ◽  
pp. 1951-1959 ◽  
Author(s):  
AR Migliaccio ◽  
G Migliaccio ◽  
DC Dale ◽  
WP Hammond
Keyword(s):  
Growth Factor ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Cyclic Neutropenia ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Stimulating Factor

Abstract The number and growth factor requirements of committed progenitor cells (colony-forming units-granulocyte/macrophage and burst-forming units- erythroid) in three patients with cyclic neutropenia (two congenital, one acquired) were studied before and during therapy with recombinant human granulocyte colony-stimulating factor (G-CSF; 3 to 10 micrograms/kg/d). When the patients with congenital disease were treated with G-CSF, the cycling of blood cells persisted, but the cycle length was shortened from 21 days to 14 days, and the amplitude of variations in blood counts increased. There was a parallel shortening of the cycle and increase of the amplitude of variations (from two- to three-fold to 10- to 100-fold) in the number of both types of circulating progenitor cells in these two patients. In the patient with acquired cyclic neutropenia, cycling of both blood cells and progenitors could not be seen. In cultures deprived of fetal bovine serum, erythroid and myeloid bone marrow progenitor cells from untreated patients and from normals differed in growth factor responsiveness. As examples, maximal growth of granulocyte/macrophage (GM) colonies was induced by granulocyte/macrophage (GM)-CSF plus G-CSF in the patients, whereas a combination of GM-CSF, G-CSF and interleukin- 3 (IL-3) was required in the normals, and erythropoietin alone induced fourfold more erythroid bursts from cyclic neutropenic patients than from normal donors (46% versus 11% of the maximal colony number, respectively). The growth factor responsiveness of marrow progenitor cells slightly changed during the treatment toward the values observed with normal progenitors. These results indicate that treatment with G- CSF not only ameliorated the neutropenia, but also increased the amplitude and the frequency of oscillation of circulating progenitor cell numbers. These data are consistent with the hypothesis that G-CSF therapy affects the proliferation of the hematopoietic stem cell.

scholarly journals Direct stimulation of cells expressing receptors for macrophage colony- stimulating factor (CSF-1) by a plasma membrane-bound precursor of human CSF-1

Blood ◽  
1990 ◽  
Vol 76 (7) ◽  
pp. 1308-1314 ◽  
Author(s):  
J Stein ◽  
GV Borzillo ◽  
CW Rettenmier
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Cell Surface ◽  
Mononuclear Phagocytes ◽  
Biologically Active ◽  
Colony Stimulating Factor ◽  
Membrane Bound ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Secreted forms of macrophage colony-stimulating factor (M-CSF or CSF-1) are generated by proteolytic cleavage of membrane-bound glycoprotein precursors. Alternatively spliced transcripts of the human CSF-1 gene encode at least two different transmembrane precursors that are differentially processed in mammalian expression systems. The larger precursor rapidly undergoes proteolysis to yield the secreted growth factor and does not give rise to forms of CSF-1 detected on the cell surface. By contrast, the smaller human CSF-1 precursor is stably expressed on the plasma membrane where it is inefficiently cleaved to release a soluble molecule. To determine whether the smaller precursor is biologically active on the cell surface, mouse NIH-3T3 fibroblasts expressing the different forms of human CSF-1 were killed by chemical fixation and tested for their ability to support the proliferation of cells that require this growth factor. Only fixed cells expressing human CSF-1 precursors on their surface stimulated the growth in vitro of a murine macrophage cell line or normal mouse bone marrow-derived mononuclear phagocytes. The ability of these nonviable fibroblasts to induce the proliferation of CSF-1-dependent cells was not mediated by release of soluble growth factor, required direct contact with the target cells, and was blocked by neutralizing antiserum to CSF-1. These results demonstrate that the cell surface form of the human CSF-1 precursor is biologically active and indicate that plasma membrane- bound growth factors can functionally interact with receptor-bearing targets by direct cell-cell contact.

scholarly journals Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes.

1987 ◽  
Vol 7 (7) ◽  
pp. 2378-2387 ◽  
Author(s):  
C W Rettenmier ◽  
M F Roussel ◽  
R A Ashmun ◽  
P Ralph ◽  
K Price ◽  
...  
Keyword(s):  
Growth Factor ◽  
Disulfide Bonds ◽  
Mononuclear Phagocyte ◽  
Transformed Cells ◽  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Membrane Bound ◽  
Nih 3T3 ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

NIH 3T3 cells cotransfected with the human c-fms proto-oncogene together with a 1.6-kilobase cDNA clone encoding a 256-amino-acid precursor of the human mononuclear phagocyte colony-stimulating factor CSF-1 (M-CSF) undergo transformation by an autocrine mechanism. The number of CSF-1 receptors on the surface of transformed cells was regulated by ligand-induced receptor degradation and was inversely proportional to the quantity of CSF-1 produced. A tyrosine-to-phenylalanine mutation at position 969 near the receptor carboxyl terminus potentiated its transforming efficiency in cells cotransfected by the CSF-1 gene but did not affect receptor downmodulation. CSF-1 was synthesized as an integral transmembrane glycoprotein that was rapidly dimerized through disulfide bonds. The homodimer was externalized at the cell surface, where it underwent proteolysis to yield the soluble growth factor. Trypsin treatment of viable cells cleaved the plasma membrane form of CSF-1 to molecules of a size indistinguishable from that of the extracellular growth factor, suggesting that trypsinlike proteases regulate the rate of CSF-1 release from transformed cells. The data raise the possibility that this form of membrane-bound CSF-1 might stimulate receptors on adjacent cells through direct cell-cell interactions.

Incomplete restoration of colony-stimulating factor 1 (CSF-1) function in CSF-1–deficient Csf1op/Csf1op mice by transgenic expression of cell surface CSF-1

Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 1114-1123 ◽  
Author(s):  
Xu-Ming Dai ◽  
Xiao-Hua Zong ◽  
Vonetta Sylvestre ◽  
E. Richard Stanley
Keyword(s):  
Cell Surface ◽  
Biologically Active ◽  
Surface Glycoprotein ◽  
Long Bone ◽  
Kidney Cortex ◽  
Colony Stimulating Factor ◽  
Cell Surface Glycoprotein ◽  
Transgenic Expression ◽  
Stimulating Factor

AbstractThe primary macrophage growth factor, colony-stimulating factor 1 (CSF-1), is expressed as a secreted glycoprotein or proteoglycan found in the circulation or as a biologically active cell surface glycoprotein (csCSF-1). To investigate the in vivo roles of csCSF-1, we created mice that exclusively express csCSF-1, in a normal tissue-specific and developmental manner, by transgenic expression of csCSF-1 in the CSF-1-deficient osteopetrotic (Csf1op/Csf1op) background. The gross defects of Csf1op/Csf1op mice, including growth retardation, failure of tooth eruption, and abnormal male and female reproductive functions were corrected. Macrophage densities in perinatal liver, bladder, sublinguinal salivary gland, kidney cortex, dermis, and synovial membrane were completely restored, whereas only partial or no restoration was achieved in adult liver, adrenal gland, kidney medulla, spleen, peritoneal cavity, and intestine. Residual osteopetrosis, significantly delayed trabecular bone resorption in the subepiphyseal region of the long bone, and incomplete correction of the hematologic abnormalities in the peripheral blood, bone marrow, and spleens of CSF-1-deficient mice were also found in mice exclusively expressing csCSF-1. These data suggest that although csCSF-1 alone is able to normalize several aspects of development in Csf1op/Csf1op mice, it cannot fully restore in vivo CSF-1 function, which requires the presence of the secreted glycoprotein and/or proteoglycan forms. (Blood. 2004;103:1114-1123)

scholarly journals Implantation of fibroblasts transfected with human granulocyte colony- stimulating factor cDNA into mice as a model of cytokine-supplement gene therapy

Blood ◽  
1989 ◽  
Vol 74 (4) ◽  
pp. 1274-1280
Author(s):  
K Tani ◽  
K Ozawa ◽  
H Ogura ◽  
T Takahashi ◽  
A Okano ◽  
...  
Keyword(s):  
Blot Analysis ◽  
Marker Gene ◽  
Diffusion Chamber ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
3T3 Fibroblasts ◽  
Endogenous Expression ◽  
C3h Mice ◽  
Stimulating Factor

A fibroblast-mediated gene delivery method was used for the endogenous expression of human granulocyte colony-stimulating factor (G-CSF) as a model for cytokine supplement therapy. Human G-CSF cDNA was inserted into the plasmid expression vector BMGNeo, which contains a partial sequence of bovine papilloma virus and a selectable marker gene. The recombinant plasmid (BMGNeo-GCSF) was transfected into NIH/3T3 fibroblasts by the calcium phosphate coprecipitation method, and the stably transformed cells were isolated by G418 selection. An appropriate clone producing a large amount of G-CSF was selected by enzyme immunoassay of the culture supernatants. Southern blot analysis suggested that the BMGNeo-GCSF plasmid replicated mainly as an episome, and Northern blot analysis demonstrated the high expression of human G- CSF mRNA in the cells. After the implantation of the G-CSF-producing fibroblasts into nude mice, prominent neutrophilia, about 30-fold the level of normal control, was observed within seven days. Moreover, the number of hematopoietic progenitor cells in spleen remarkably increased for all cell lineages in these mice. To regulate the in vivo expression of G-CSF, we designed a subcutaneous diffusion chamber apparatus that contains the G-CSF-producing fibroblasts. The leukocytosis (neutrophilia) induced in C3H mice after embedding the device quickly disappeared after ethanol treatment of the chamber. Furthermore, reinjection of the G-CSF-producing fibroblasts into the chamber caused a second neutrophilia.

scholarly journals Implantation of fibroblasts transfected with human granulocyte colony- stimulating factor cDNA into mice as a model of cytokine-supplement gene therapy

Blood ◽  
1989 ◽  
Vol 74 (4) ◽  
pp. 1274-1280 ◽  
Author(s):  
K Tani ◽  
K Ozawa ◽  
H Ogura ◽  
T Takahashi ◽  
A Okano ◽  
...  
Keyword(s):  
Blot Analysis ◽  
Marker Gene ◽  
Diffusion Chamber ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
3T3 Fibroblasts ◽  
Endogenous Expression ◽  
C3h Mice ◽  
Stimulating Factor

Abstract A fibroblast-mediated gene delivery method was used for the endogenous expression of human granulocyte colony-stimulating factor (G-CSF) as a model for cytokine supplement therapy. Human G-CSF cDNA was inserted into the plasmid expression vector BMGNeo, which contains a partial sequence of bovine papilloma virus and a selectable marker gene. The recombinant plasmid (BMGNeo-GCSF) was transfected into NIH/3T3 fibroblasts by the calcium phosphate coprecipitation method, and the stably transformed cells were isolated by G418 selection. An appropriate clone producing a large amount of G-CSF was selected by enzyme immunoassay of the culture supernatants. Southern blot analysis suggested that the BMGNeo-GCSF plasmid replicated mainly as an episome, and Northern blot analysis demonstrated the high expression of human G- CSF mRNA in the cells. After the implantation of the G-CSF-producing fibroblasts into nude mice, prominent neutrophilia, about 30-fold the level of normal control, was observed within seven days. Moreover, the number of hematopoietic progenitor cells in spleen remarkably increased for all cell lineages in these mice. To regulate the in vivo expression of G-CSF, we designed a subcutaneous diffusion chamber apparatus that contains the G-CSF-producing fibroblasts. The leukocytosis (neutrophilia) induced in C3H mice after embedding the device quickly disappeared after ethanol treatment of the chamber. Furthermore, reinjection of the G-CSF-producing fibroblasts into the chamber caused a second neutrophilia.

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