Inhibition of mitogenic activity of a platelet growth factor (platelet basic protein) in 3T3 cells by heparin

Because platelet-derived growth factor (PDGF) may be released at sites where neutrophil proteinases may also be released, we examined the effects of neutrophil elastase and cathepsin G upon the chemotactic and mitogenic activities of PDGF. Elastase abolished the chemotactic activity of PDGF for fibroblasts but had no effect on its chemotactic activity for monocytes, or on its mitogenic activity for 3T3 cells or its capacity to bind to 3T3 cells. Cathepsin G had no effect upon the chemotactic or mitogenic activities of PDGF. In contrast, trypsin eliminated the chemotactic activity of PDGF for monocytes and fibroblasts and the mitogenic activity of PDGF. After reduction and alkylation, PDGF retained full chemotactic activity for fibroblasts and monocytes but exhibited no mitogenic activity and only limited binding to 3T3 cells. These results indicate separate domains on PDGF for fibroblast chemotactic and mitogenic activity and for monocyte and fibroblast chemotactic activity and raise the possibility that the biological activities of PDGF may be modified selectively in vivo. The findings further suggest that the majority of PDGF receptors on fibroblasts mediate mitogenic activity and that only a minority of the PDGF receptors on fibroblasts are responsible for chemotactic activity.

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A partially purified serum fraction synergistically enhances the mitogenic activity of epidermal growth factor and insulin in quiescent cultures of 3T3 cells

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(78)91476-6 ◽

1978 ◽

Vol 83 (3) ◽

pp. 874-880 ◽

Cited By ~ 7

Author(s):

Karl Mierzejewski ◽

Enrique Rozengurt

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Mitogenic Activity ◽

3T3 Cells ◽

Epidermal Growth

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Evidence for an epidermal growth factor-stimulated protein kinase cascade in Swiss 3T3 cells. Activation of serine peptide kinase activity by myelin basic protein kinases in vitro.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)38424-8 ◽

1990 ◽

Vol 265 (20) ◽

pp. 11495-11501 ◽

Cited By ~ 11

Author(s):

N G Ahn ◽

E G Krebs

Keyword(s):

Growth Factor ◽

Protein Kinase ◽

Epidermal Growth Factor ◽

Kinase Activity ◽

Basic Protein ◽

3T3 Cells ◽

Kinase Cascade ◽

Epidermal Growth ◽

Protein Kinase Cascade

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The epidermal growth factor-like domain of recombinant human thrombomodulin exhibits mitogenic activity for Swiss 3T3 cells

Blood ◽

10.1182/blood.v86.1.225.bloodjournal861225 ◽

1995 ◽

Vol 86 (1) ◽

pp. 225-233 ◽

Cited By ~ 40

Author(s):

H Hamada ◽

H Ishii ◽

K Sakyo ◽

S Horie ◽

K Nishiki ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Dna Synthesis ◽

Cell Line ◽

Control Level ◽

Mitogenic Activity ◽

Thymidine Uptake ◽

3T3 Cells ◽

Epidermal Growth ◽

Swiss 3T3

Thrombomodulin (TM) is an anticoagulant endothelial cell surface glycoprotein containing six tandem epidermal growth factor (EGF)-like structures. We prepared a recombinant TM peptide (rTME1–6, from R214GHWA to DSGK466 of native TM) composed of these six EGF-like structures and investigated the effect of rTME1–6 peptide on the growth of the Swiss 3T3 fibroblast cell line. It was found that rTME1–6 induced proliferation of Swiss 3T3 cells and accelerated [3H]thymidine uptake into their DNA. [3H]Thymidine uptake increased in a dose- dependent manner, plateauing at 50 ng/mL rTME1–6, which was 1.8 times the control level. rTME1–6 peptide (50 ng/mL) also accelerated the DNA synthesis of human dermal fibroblasts (HDFs), A549 (a human lung cancer cell line), HepG2 (a human hepatocarcinoma cell line), and U937 cells (a human monocytic cell line) to 1.5, 1.6, 1.4, and 1.2 times the control level, respectively. The magnitude of the acceleration of DNA synthesis in Swiss 3T3 induced by rTME1–6 was approximately 20% of that of EGF on a molar basis. The uptake of [3H]thymidine was accelerated synergistically by coculture of the cells with rTME1–6 and insulin, similar to the coculture with EGF and insulin. The effects of rTME1–6 were abolished by addition of polyclonal antihuman TM IgG, whereas the actions of insulin and EGF were not influenced. Glucose uptake in Swiss 3T3 cells also increased 1.6 times over control levels by culture with 50 ng/mL rTME1–6 (1.25 nmol/L), compared with 2.7 times by 10 ng/mL EGF (1.66 nmol/L). Binding of [125I]EGF (0.5 ng/mL, 0.083 nmol/L) by the cells was inhibited by about 60% by addition of an eight-fold molar excess of nonlabeled EGF (0.664 nmol/L), whereas no inhibition of [125I]EGF binding was observed, even in the presence of a 1,000-fold molar excess (83 nmol/L) of rTME1–6. Specific binding of [125I]rTME1–6 on the cells showed a saturation curve, and the apparent concentration of rTME1–6 required for half maximum binding of the peptide on the cells was calculated to be 31.5 ng/mL. Thus, the overall results indicated that the rTME1–6 peptide had mitogenic activity for Swiss 3T3 cells, accelerated DNA synthesis and glucose uptake, and that the mitogenic activity might be mediated by binding of the peptide to a specific site different from the EGF receptor.

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Characterization of heparin-binding endothelial mitogen(s) produced by the ovine endometrium during early pregnancy

Biochemistry and Cell Biology ◽

10.1139/o98-011 ◽

1998 ◽

Vol 76 (1) ◽

pp. 89-96 ◽

Cited By ~ 9

Author(s):

Jing Zheng ◽

Dale A Redmer ◽

S Derek Killilea ◽

Lawrence P Reynolds

Keyword(s):

Growth Factor ◽

Affinity Chromatography ◽

Early Pregnancy ◽

Molecular Size ◽

Immunoblot Analysis ◽

Binding Activity ◽

Mitogenic Activity ◽

Heparin Binding ◽

3T3 Cells ◽

Heparin Affinity

To characterize mitogenic factors produced by ovine endometrium during early pregnancy, endometrial explant-conditioned media (ECM) were obtained from ewes on day 12, 18, 24, or 30 after mating. These ECM contained mitogenic activity for both endothelial and 3T3 cells across all days. The endothelial mitogenic activity was greatest on day 24, whereas mitogenic activity for 3T3 cells did not differ across days. By ultrafiltration, ion exchange, and heparin-affinity chromatography, the endothelial mitogenic activity was found to have a molecular mass greater than 100 kDa, to be anionic, and to be heparin binding, respectively. Three peaks of endothelial mitogenic activity were recovered from heparin-affinity chromatography. The major peak, H3, was mitogenic for endothelial but not for 3T3 cells. H3 was further purified, and the single peak of heparin-binding activity, designated H3b, represented a 681-fold purification of endothelial mitogenic activity from endometrial ECM. H3 and H3b were heat labile and trypsin sensitive, and their biological activity was heparin enhanced. The majority of the endothelial mitogenic activity was immunoneutralized by antibodies against acidic and basic FGF. Nevertheless, we were unable to detect bFGF in H3 or H3b by Western immunoblot analysis. Thus, in this study we have extended our previous observations and demonstrated that (i) during early pregnancy the ovine endometrium produces mitogenic activity for both endothelial and 3T3 cells, (ii) the endothelial mitogenic activity is greatest on day 24 after mating, which corresponds with the onset of endometrial vascular growth, and (iii ) the major endothelial mitogen has a high affinity for heparin, and although it is immunologically related to FGF, it differs from known FGF in its apparent molecular size and biological activities.Key words: heparin-binding growth factor, fibroblast growth factor, uterus, early pregnancy, ewe.

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