Insulin Inhibits Platelet-derived Growth Factor-induced Cell Proliferation

Cellular behavior can be considered to be the result of a very complex spatial and temporal integration of intracellular and extracellular signals. These signals arise from serum-soluble factors as well as from cell–substrate or cell–cell interactions. The current approach in mitogenesis studies is generally to analyze the effect of a single growth factor on serum-starved cells. In this context, a metabolic hormone such as insulin is found to be a mitogenic agent in many cellular types. In the present study, we have considered the effect of insulin stimulation in platelet-derived growth factor (PDGF)-activated NIH-3T3 and C2C12 cells. Our results show that insulin is able to inhibit strongly both NIH-3T3 and C2C12 cell growth induced by PDGF, one of the most powerful mitotic agents for these cell types. This inhibitory effect of insulin is due primarily to a premature down-regulation of the PDGF receptor. Thus, when NIH-3T3 or C2C12 cells are stimulated with both PDGF and insulin, we observe a decrease in PDGF receptor phosphorylation with respect to cells treated with PDGF alone. In particular, we find that costimulation with insulin leads to a reduced production of H2O2 with respect to cell stimulation with PDGF alone. The relative low concentration of H2O2 in PDGF/insulin-costimulated cell leads to a limited down-regulation of protein tyrosine phosphatases, and, consequently, to a reduced PDGF receptor phosphorylation efficiency. The latter is very likely to be responsible for the insulin-dependent inhibition of PDGF-receptor mitogenic signaling.

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Hypoxia Enhances Platelet-derived Growth Factor Signaling in the Pulmonary Vasculature by Down-Regulation of Protein Tyrosine Phosphatases

American Journal of Respiratory and Critical Care Medicine ◽

10.1164/rccm.200911-1663oc ◽

2011 ◽

Vol 183 (8) ◽

pp. 1092-1102 ◽

Cited By ~ 50

Author(s):

Henrik ten Freyhaus ◽

Markus Dagnell ◽

Maike Leuchs ◽

Marius Vantler ◽

Eva M. Berghausen ◽

...

Keyword(s):

Growth Factor ◽

Protein Tyrosine Phosphatases ◽

Platelet Derived Growth Factor ◽

Pulmonary Vasculature ◽

Growth Factor Signaling ◽

Tyrosine Phosphatases ◽

Down Regulation ◽

Protein Tyrosine

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Down-regulation of cellular platelet-derived growth factor receptors induced by an activated neu receptor tyrosine kinase.

Cell Regulation ◽

10.1091/mbc.2.8.651 ◽

1991 ◽

Vol 2 (8) ◽

pp. 651-661 ◽

Cited By ~ 3

Author(s):

L Lehtola ◽

M Nistér ◽

E Hölttä ◽

B Westermark ◽

K Alitalo

Keyword(s):

Growth Factor ◽

Tyrosine Kinase ◽

Receptor Expression ◽

Platelet Derived Growth Factor ◽

Target Cells ◽

Mrna Levels ◽

Pdgf Receptor ◽

Down Regulation ◽

Beta Receptors ◽

Neu Oncogene

The functional integration of growth factor signaling occurs at several levels in target cells. One of the most proximal mechanisms is receptor transmodulation, by which one activated receptor can regulate the expression of other receptors in the same cells. Well-established transregulatory loops involve platelet-derived growth factor (PDGF) down-regulation of epidermal growth factor (EGF) receptors and beta-type transforming growth factors modulation of PDGF receptors. We have studied the relationship between neu tyrosine kinase activation and the expression of the PDGF receptors in transfected NIH/3T3 cells. Expression of the neu oncogene, but not of the neu proto-oncogene, was associated with a decrease of PDGF alpha- and beta-receptors on the cell surface, as measured by [125-I]PDGF-AA and -BB binding. These results were corroborated by metabolic labeling and immunoprecipitation of the PDGF beta-receptors. PDGF alpha- and beta-receptor mRNAs were strongly decreased in the neu oncogene-transformed cells in comparison with control cells expressing the neu proto-oncogene. Down-regulation of the PDGF receptors and their mRNAs was also observed after EGF treatment of cells expressing a chimeric EGF receptor/neu receptor, where the neu tyrosine kinase is activated by EGF binding. These results show that the neu tyrosine kinase can down-modulate PDGF receptor expression, and the effect is mediated via decreased PDGF receptor mRNA levels.

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Transactivation of Platelet-Derived Growth Factor Receptor α by the GTPase-Deficient Activated Mutant of Gα12

Molecular and Cellular Biology ◽

10.1128/mcb.26.1.50-62.2006 ◽

2006 ◽

Vol 26 (1) ◽

pp. 50-62 ◽

Cited By ~ 17

Author(s):

Rashmi N. Kumar ◽

Ji Hee Ha ◽

Rangasudhagar Radhakrishnan ◽

Danny N. Dhanasekaran

Keyword(s):

Growth Factor ◽

Cell Growth ◽

Signaling Pathway ◽

Growth Factor Receptor ◽

Platelet Derived Growth Factor ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

3T3 Cells ◽

Nih 3T3 ◽

Nih 3T3 Cells

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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Involvement of a phosphotyrosine protein phosphatase in the suppression of platelet-derived growth factor receptor autophosphorylation in ras-transformed cells

Biochemical Journal ◽

10.1042/bj2930215 ◽

1993 ◽

Vol 293 (1) ◽

pp. 215-221 ◽

Cited By ~ 9

Author(s):

L Tomáska ◽

R J Resnick

Keyword(s):

Growth Factor ◽

Phosphatase Activity ◽

Receptor Tyrosine Kinase ◽

Protein Phosphatase ◽

Kinase Activity ◽

Platelet Derived Growth Factor ◽

Transformed Cells ◽

Pdgf Receptor ◽

Tyrosine Kinase Activity ◽

Phosphotyrosine Protein Phosphatase

The nature of the suppression of platelet-derived growth factor (PDGF) receptor autophosphorylation in ras-transformed NIH 3T3 fibroblasts was investigated. The PDGF receptor from ras-transformed cells that had been purified by wheatgerm-lectin affinity chromatography displayed normal PDGF-induced autophosphorylation, indicating that the receptor is not irreversibly modified. Various phosphotyrosine-protein-phosphatase inhibitors did not reverse the inhibition of PDGF-receptor kinase in crude membrane preparations from ras-transformed cells. However, treatment of intact ras-transformed cells both with 2 mM sodium orthovanadate and with 20 microM phenylarsine oxide restored PDGF-receptor tyrosine-kinase activity to a level similar to that observed in normal cells. Direct measurement of the phosphatase activities in crude cellular fractions revealed a 2.5-fold higher membrane-associated phosphotyrosine-protein-phosphatase activity in ras-transformed cells, whereas phosphoserine-protein-phosphatase activity remained unchanged between the cell lines. These data suggest that the suppression of the PDGF-receptor tyrosine-kinase activity in ras-transformed cells is mediated via an inhibitory component, distinct from the receptor, that may be positively regulated by the dephosphorylation of tyrosine residue(s).

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The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2040-2048.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2040-2048

Author(s):

F Fazioli ◽

U H Kim ◽

S G Rhee ◽

C J Molloy ◽

O Segatto ◽

...

Keyword(s):

Growth Factor ◽

Dna Synthesis ◽

Phospholipase C ◽

Signaling Pathway ◽

Tyrosine Phosphorylation ◽

Platelet Derived Growth Factor ◽

Mitogenic Signaling ◽

Gtpase Activating Protein ◽

3T3 Fibroblasts ◽

Nih 3T3

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.

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A small v-sis/platelet-derived growth factor (PDGF) B-protein domain in which subtle conformational changes abrogate PDGF receptor interaction and transforming activity

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5496-5501.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5496-5501

Author(s):

N Giese ◽

W J LaRochelle ◽

M May-Siroff ◽

K C Robbins ◽

S A Aaronson

Keyword(s):

Monoclonal Antibody ◽

Growth Factor ◽

Conformational Changes ◽

Protein Domain ◽

Platelet Derived Growth Factor ◽

Receptor Interaction ◽

Pdgf Receptor ◽

Amino Acid Residues ◽

Disulfide Linkages ◽

Transforming Activity

Deletion scanning mutagenesis within the transforming region of the v-sis oncogene was used to dissect structure-function relationships. Mutations affecting codons within a domain encoding amino acids 136 through 148 had no effect upon homodimer formation or recognition by antisera which detect determinants dependent upon native intrachain disulfide linkages, yet the same mutations completely abolished transforming activity. A platelet-derived growth factor B (PDGF B) monoclonal antibody that prevents its interaction with PDGF receptors recognized v-sis, delta 142 (deletion of codon 142), and delta 148 but not delta 136, delta 137, or delta 139 mutants. These findings mapped the epitope recognized by this monoclonal antibody to include amino acid residues 136 to 139. Furthermore, mutations in the codon 136 to 148 domain caused markedly impaired ability to induce PDGF receptor tyrosine phosphorylation. Thus, subtle conformational alterations in this small domain critically affect PDGF receptor recognition and/or functional activation.

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Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2359-2366.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2359-2366

Author(s):

D K Morrison ◽

D R Kaplan ◽

S G Rhee ◽

L T Williams

Keyword(s):

Growth Factor ◽

Tyrosine Phosphorylation ◽

Serine Phosphorylation ◽

Platelet Derived Growth Factor ◽

Pdgf Receptor ◽

Wild Type ◽

Receptor Proteins ◽

Signaling Complex

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.

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