Temporal relationship between inositol polyphosphate formation and increases in cytosolic Ca2+ in quiescent 3T3 cells stimulated by platelet-derived growth factor, bombesin and vasopressin.

ABSTRACT The GTPase-deficient, activated mutant of Gα12 (Gα12Q229L, or Gα12QL) induces neoplastic growth and oncogenic transformation of NIH 3T3 cells. Using microarray analysis, we have previously identified a role for platelet-derived growth factor receptor α (PDGFRα) in Gα12-mediated cell growth (R. N. Kumar et al., Cell Biochem. Biophys. 41:63-73, 2004). In the present study, we report that Gα12QL stimulates the functional expression of PDGFRα and demonstrate that the expression of PDGFRα by Gα12QL is dependent on the small GTPase Rho. Our results indicate that it is cell type independent as the transient expression of Gα12QL or the activation of Gα12-coupled receptors stimulates the expression of PDGFRα in NIH 3T3 as well as in human astrocytoma 1321N1 cells. Furthermore, we demonstrate the presence of an autocrine loop involving PDGF-A and PDGFRα in Gα12QL-transformed cells. Analysis of the functional consequences of the Gα12-PDGFRα signaling axis indicates that Gα12 stimulates the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway through PDGFR. In addition, we show that Gα12QL stimulates the phosphorylation of forkhead transcription factor FKHRL1 via AKT in a PDGFRα- and PI3K-dependent manner. Since AKT promotes cell growth by blocking the transcription of antiproliferative genes through the inhibitory phosphorylation of forkhead transcription factors, our results describe for the first time a PDGFRα-dependent signaling pathway involving PI3K-AKT-FKHRL1, regulated by Gα12QL in promoting cell growth. Consistent with this view, we demonstrate that the expression of a dominant negative mutant of PDGFRα attenuated Gα12-mediated neoplastic transformation of NIH 3T3 cells.

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Effect of Indapamide on the Proliferation of Balb/C 3T3 Cells Induced by Platelet-Derived Growth Factor.

The Japanese Journal of Pharmacology ◽

10.1254/jjp.57.609 ◽

1991 ◽

Vol 57 (4) ◽

pp. 609-612 ◽

Cited By ~ 2

Author(s):

Hisanobu Shimaji ◽

Hiroaki Shirahase ◽

Shigeyoshi Morishita ◽

Seimei Osumi

Keyword(s):

Growth Factor ◽

Platelet Derived Growth Factor ◽

3T3 Cells

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Differential mechanisms of inositol phosphate generation at the receptors for bombesin and platelet-derived growth factor

Biochemical Journal ◽

10.1042/bj2620665 ◽

1989 ◽

Vol 262 (2) ◽

pp. 665-668 ◽

Cited By ~ 22

Author(s):

M G Cattaneo ◽

L M Vicentini

Keyword(s):

Growth Factor ◽

Kinase Activity ◽

Inositol Phosphate ◽

Growth Factor Receptor ◽

Platelet Derived Growth Factor ◽

3T3 Cells ◽

Tyrosine Kinase Activity ◽

Independent Manner ◽

Streptolysin O ◽

Bombesin Receptor

We investigated the mechanism(s) whereby activation of a growth-factor receptor typically endowed with tyrosine kinase activity, such as the platelet-derived growth factor (PDGF) receptor, triggers phosphoinositide hydrolysis. In Swiss 3T3 cells permeabilized with streptolysin O, an analogue of GTP, guanosine 5′-[gamma-thio]triphosphate, was found to potentiate the coupling of the bombesin receptor to phospholipase C. In contrast, the activation of the enzyme by PDGF occurred in a GTP-independent manner. Moreover, the inactive analogue of GTP, guanosine 5′-[beta-thio]diphosphate, significantly inhibited the bombesin-induced InsP3 generation, whereas it did not decrease the same effect when stimulated by PDGF.

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Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-gamma in quiescent BALB/c 3T3 cells

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.2934-2943.1989 ◽

1989 ◽

Vol 9 (7) ◽

pp. 2934-2943

Author(s):

M I Wahl ◽

N E Olashaw ◽

S Nishibe ◽

S G Rhee ◽

W J Pledger ◽

...

Keyword(s):

Growth Factor ◽

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Hormonal Regulation ◽

Growth Factor Receptor ◽

Fold Increase ◽

Platelet Derived Growth Factor ◽

3T3 Cells ◽

Pdgf Stimulation

Platelet-derived growth factor (PDGF) stimulates the proliferation of quiescent fibroblasts through a series of events initiated by activation of tyrosine kinase activity of the PDGF receptor at the cell surface. Physiologically significant substrates for this or other growth factor receptor or oncogene tyrosine kinases have been difficult to identify. Phospholipase C (PLC), a key enzyme of the phosphoinositide pathway, is believed to be an important site for hormonal regulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces the intracellular second-messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Treatment of BALB/c 3T3 cells with PDGF led to a rapid (within 1 min) and significant (greater than 50-fold) increase in PLC activity, as detected in eluates of proteins from a phosphotyrosine immunoaffinity matrix. This PDGF-stimulated increase in phosphotyrosine-immunopurified PLC activity occurred for up to 12 h after addition of growth factor to quiescent cells. Interestingly, the PDGF stimulation occurred at 3 as well as 37 degrees C and in the absence or presence of extracellular Ca2+. Immunoprecipitation of cellular proteins with monoclonal antibodies specific for three distinct cytosolic PLC isozymes demonstrated the presence of a 145-kilodalton isozyme, PLC-gamma (formerly PLC-II), in BALB/c 3T3 cells. Furthermore, these immunoprecipitation studies showed that PLC-gamma is rapidly phosphorylated on tyrosine residues after PDGF stimulation. The results suggest that mitogenic signaling by PDGF is coincident with tyrosine phosphorylation of PLC-gamma.

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Regulation of the transcript for a lysosomal protein: evidence for a gene program modified by platelet-derived growth factor

Molecular and Cellular Biology ◽

10.1128/mcb.5.10.2582-2589.1985 ◽

1985 ◽

Vol 5 (10) ◽

pp. 2582-2589

Author(s):

K K Frick ◽

P J Doherty ◽

M M Gottesman ◽

C D Scher

Keyword(s):

Growth Factor ◽

De Novo ◽

Platelet Derived Growth Factor ◽

Tumor Promoter ◽

3T3 Cells ◽

Somatomedin C ◽

Epidermal Growth ◽

Rna Accumulation ◽

De Novo Protein Synthesis ◽

Lysosomal Protein

Platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize MEP, a lysosomal protein. This enhanced synthesis appears to be largely regulated by the PDGF-modulated accumulation of MEP mRNA, a 1.8-kilobase species. The increase in the MEP transcript, which is dependent on the PDGF concentration, begins 3 to 4 h after PDGF addition and is maximal at 12 h. The accumulation of the MEP transcript is growth-factor specific: PDGF and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, an agent which acts like PDGF, induce MEP RNA accumulation, whereas epidermal growth factor, somatomedin C, insulin, and whole plasma do not. A spontaneously transformed BALB/c-3T3 cell line (ST2-3T3), which does not require PDGF for growth, optimally expresses MEP RNA in the absence of PDGF. The PDGF-modulated increase in MEP RNA is unlike PDGF-modulated c-myc and c-fos RNA accumulation because it is blocked by cycloheximide, suggesting a requirement for de novo protein synthesis. It appears that PDGF modulates a program of gene expression with the accumulation of some transcripts, typified by MEP, being dependent upon the translation of others.

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Platelet-derived growth factor stimulates formation of active p21ras.GTP complex in Swiss mouse 3T3 cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.87.15.5993 ◽

1990 ◽

Vol 87 (15) ◽

pp. 5993-5997 ◽

Cited By ~ 139

Author(s):

T. Satoh ◽

M. Endo ◽

M. Nakafuku ◽

S. Nakamura ◽

Y. Kaziro

Keyword(s):

Growth Factor ◽

Platelet Derived Growth Factor ◽

Swiss Mouse ◽

3T3 Cells

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Bombesin and platelet-derived growth factor stimulate formation of inositol phosphates and Ca2+ mobilization in Swiss 3T3 cells by different mechanisms

Biochemical Journal ◽

10.1042/bj2580177 ◽

1989 ◽

Vol 258 (1) ◽

pp. 177-185 ◽

Cited By ~ 29

Author(s):

D M Blakeley ◽

A N Corps ◽

K D Brown

Keyword(s):

Protein Kinase C ◽

Growth Factor ◽

Protein Kinase ◽

Phorbol Esters ◽

Inositol Phosphates ◽

Platelet Derived Growth Factor ◽

3T3 Cells ◽

Kinase C ◽

Swiss 3T3 ◽

Stimulation Of

Highly purified platelet-derived growth factor (PDGF) or recombinant PDGF stimulate DNA synthesis in quiescent Swiss 3T3 cells. The dose-response curves for the natural and recombinant factors were similar, with half-maximal responses at 2-3 ng/ml and maximal responses at approx. 10 ng/ml. Over this dose range, both natural and recombinant PDGF stimulated a pronounced accumulation of [3H]inositol phosphates in cells labelled for 72 h with [3H]inositol. In addition, mitogenic concentrations of PDGF stimulated the release of 45Ca2+ from cells prelabelled with the radioisotope. However, in comparison with the response to the peptide mitogens bombesin and vasopressin, a pronounced lag was evident in both the generation of inositol phosphates and the stimulation of 45Ca2+ efflux in response to PDGF. Furthermore, although the bombesin-stimulated efflux of 45Ca2+ was independent of extracellular Ca2+, the PDGF-stimulated efflux was markedly inhibited by chelation of external Ca2+ by using EGTA. Neither the stimulation of formation of inositol phosphates nor the stimulation of 45Ca2+ efflux in response to PDGF were affected by tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In contrast, TPA inhibited phosphoinositide hydrolysis and 45Ca2+ efflux stimulated by either bombesin or vasopressin. Furthermore, whereas formation of inositol phosphates in response to both vasopressin and bombesin was increased in cells in which protein kinase C had been down-modulated by prolonged exposure to phorbol esters, the response to PDGF was decreased in these cells. These results suggest that, in Swiss 3T3 cells, PDGF receptors are coupled to phosphoinositidase activation by a mechanism that does not exhibit protein kinase C-mediated negative-feedback control and which appears to be fundamentally different from the coupling mechanism utilized by the receptors for bombesin and vasopressin.

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Identification of a BALB/c-3T3 cell protein modulated by platelet-derived growth factor.

Molecular and Cellular Biology ◽

10.1128/mcb.3.1.70 ◽

1983 ◽

Vol 3 (1) ◽

pp. 70-81 ◽

Cited By ~ 34

Author(s):

C D Scher ◽

R L Dick ◽

A P Whipple ◽

K L Locatell

Keyword(s):

Growth Factor ◽

Dna Synthesis ◽

Cell Lines ◽

Actinomycin D ◽

Platelet Derived Growth Factor ◽

Cell Protein ◽

Transformed Cells ◽

Antigenic Determinants ◽

3T3 Cells ◽

Constitutive Synthesis

The platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize a protein (pII; Mr, 35,000) that is constitutively synthesized by spontaneously transformed BALB/c-3T3 (ST2-3T3) cells which do not require PDGF for growth. Antisera against a major excreted protein family (MEP) of retrovirus-transformed cells quantitatively precipitated cellular pII. PDGF-stimulated pII has the same molecular weight, a similar charge, and similar antigenic determinants as authentic MEP isolated from ST2-3T3 or retrovirus-transformed cells. MEP represented about 2% of the nonnuclear proteins synthesized by ST2-3T3 cells and 0.3 to 0.6% of the proteins synthesized by PDGF-treated BALB/c-3T3 cells, a three- to sixfold increase over the background. In BALB/c-3T3 cells, less PDGF was required for pII (MEP) synthesis than for DNA synthesis. PDGF induced a selective increase in pII (MEP) within 40 min. Such preferential synthesis was inhibited by brief treatment with actinomycin D, suggesting a requirement for newly formed RNA. The constitutive synthesis of pII (MEP) by ST2-3T3 cells was not inhibited by actinomycin D. Five spontaneously or chemical carcinogen-transformed tumorigenic BALB/c-3T3 cell lines were studied; they neither required PDGF for growth nor responded to it. These cell lines became arrested at confluence with a G1 DNA content. Each of these independently isolated lines synthesized pII (MEP) constitutively. Thus, the synthesis of pII (MEP) may be required, but is not sufficient, for PDGF-modulated DNA synthesis.

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Antisense RNA of proto-oncogene c-fos blocks renewed growth of quiescent 3T3 cells

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.639-649.1987 ◽

1987 ◽

Vol 7 (2) ◽

pp. 639-649

Author(s):

K Nishikura ◽

J M Murray

Keyword(s):

Growth Factor ◽

Antisense Rna ◽

Platelet Derived Growth Factor ◽

3T3 Cells ◽

Mammary Tumor Virus ◽

Quiescent Cells ◽

Mouse Mammary Tumor ◽

Tumor Virus ◽

Virus Promoter ◽

Stimulation Of

Mouse 3T3 cells were transformed with an antisense c-fos gene fused to a mouse mammary tumor virus promoter. In transformants that integrated a large number of antisense c-fos sequences, the usual large increase in c-fos mRNA and protein following stimulation of quiescent cells by platelet-derived growth factor was blocked in the presence of dexamethasone. These cells subsequently also failed to show the stimulation of DNA synthesis normally induced by platelet-derived growth factor. Appropriate expression of c-fos appears to be a prerequisite for reentry of quiescent cells into the cell cycle.

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