Inhibition of early platelet-derived growth factor responses in BALB/c-3T3 cells by interferon

Stimulation of total inositol phosphate production, alteration of cytosolic free calcium [( Ca++]i), vinculin disruption from adhesion plaques, and DNA synthesis caused by PDGF were examined in normal and INF pretreated density arrested BALB/c-3T3 fibroblasts. In normal cells, PDGF caused an increase in total inositol phosphates, a rapid, transient increase in [Ca++]i, disappearance of vinculin from adhesion plaques, and stimulation of DNA synthesis. Pretreatment of cells with INF inhibited PDGF-stimulated increases in [Ca++]i, vinculin disruption from adhesion plaques, and DNA synthesis, but had no effect on PDGF-induced increase in total inositol phosphate levels. These findings suggest that INF prevents entry of quiescent BALB/c-3T3 cells into G1 by inhibiting PDGF-induced release of Ca++ from intracellular stores.     

Platelet-derived growth factor-induced c-myc RNA expression. Analysis of an inducible pathway independent of protein kinase C

Platelet-derived growth factor (PDGF) is generally considered to stimulate phosphoinositide turnover resulting in activation of protein kinase C and increased cytoplasmic [Ca2+]. We have examined the role of these secondary effects in regulation of c-myc mRNA accumulation in the MG-63 human osteogenic sarcoma line. Treatment of quiescent cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA) to down-regulate protein kinase C inhibited TPA-stimulated c-myc expression but did not affect the PDGF-modulated process. When cytoplasmic [Ca2+] was increased by addition of a Ca2+ ionophore (A23187 or ionomycin), no stimulation of c-myc RNA was seen; furthermore, these agents did not enhance the PDGF-modulated c-myc expression. Addition of EGTA to cultures treated with both PDGF and a Ca2+ ionophore did not inhibit c-myc induction but rather caused a superinduction of c-myc RNA accumulation. Superinduction occurred only if the [EGTA] was greater than [Ca2+] in the medium. This superinduction was distinct from the increased induction caused by inhibition of protein synthesis. Because PDGF-induced c-myc expression is independent of protein kinase C and increased cytoplasmic [Ca2+], the evidence suggests that PDGF modulates c-myc RNA accumulation in MG-63 cells via a novel pathway, seemingly uncoupled from the classic action of increased phosphoinositide metabolism.     

Platelet-derived growth factor-induced alterations in vinculin and actin distribution in BALB/c-3T3 cells

Exposure of BALB/c-3T3 cells (clone A31) to platelet-derived growth factor (PDGF) results in a rapid time- and dose-dependent alteration in the distribution of vinculin and actin. PDGF treatment (6-50 ng/ml) causes vinculin to disappear from adhesion plaques (within 2.5 min after PDGF exposure) and is followed by an accumulation of vinculin in punctate spots in the perinuclear region of the cell. This alteration in vinculin distribution is followed by a disruption of actin-containing stress fibers (within 5 to 10 min after PDGF exposure). Vinculin reappears in adhesion plaques by 60 min after PDGF addition while stress fiber staining is nondetectable at this time. PDGF treatment had no effect on talin, vimentin, or microtubule distribution in BALB/c-3T3 cells; in addition, exposure of cells to 5% platelet-poor plasma (PPP), 0.1% PPP, 30 ng/ml epidermal growth factor (EGF), 30 ng/ml somatomedin C, or 10 microM insulin also had no effect on vinculin or actin distribution. Other competence-inducing factors (fibroblast growth factor, calcium phosphate, and choleragen) and tumor growth factor produced similar alterations in vinculin and actin distribution as did PDGF, though not to the same extent. PDGF treatment of cells for 60 min followed by exposure to EGF (0.1-30 ng/ml for as long as 8 h after PDGF removal), or 5% PPP resulted in the nontransient disappearance of vinculin staining within 10 min after EGF or PPP additions; PDGF followed by 0.1% PPP or 10 microM insulin had no effect. Treatment of cells with low doses of PDGF (3.25 ng/ml), which did not affect vinculin or actin organization in cells, followed by EGF (10 ng/ml), resulted in the disappearance of vinculin staining in adhesion plaques, thus demonstrating the synergistic nature of PDGF and EGF. These data suggest that PDGF-induced competence and stimulation of cell growth in quiescent fibroblasts are associated with specific rapid alterations in the cellular organization of vinculin and actin.     

Persistence of the mitogenic response to platelet-derived growth factor (competence) does not reflect a long-term interaction between the growth factor and the target cell

Quiescent BALB/c-3T3 cells exposed briefly to platelet-derived growth factor (PDGF) become "competent" to replicate their DNA even if PDGF is removed from cell culture medium prior to the onset of DNA synthesis. We have suggested that persistence of the PDGF-induced competent state reflects a rapidly induced and relatively stable biochemical change within the target cells. Others suggest that the phenomenon reflects a long-term association between PDGF and its target cells or perhaps between PDGF and the cell culture dish. This controversy has been addressed (a) by examining the effect of anti-PDGF antibodies on PDGF- induced competence and (b) by studying the chemical fate of 125I- labeled PDGF. Anti-PDGF antibodies inactive both soluble and surface- bound PDGF. However, if quiescent 3T3 cells are exposed to PDGF for as little as 30 min, subsequent addition of these antibodies to the culture medium does not prevent the mitogenic response. Under conditions where the PDGF-induced competent state decays stochastically with a t1/2 of 18-20 h, cell-associated 125I-PDGF decays with a t1/2 of approximately 50 min. These data do not support the concept that persistence of the PDGF-induced competent state reflects a long-term association between PDGF and the target cells or between PDGF and the culture dish.     

In vivo phosphorylation and dephosphorylation of the platelet-derived growth factor receptor studied by immunoblot analysis with phosphotyrosine antibodies

Antibodies against the synthetic hapten azobenzyl phosphonate which specifically crossreact with phosphotyrosine have been produced and used to detect the proteins phosphorylated in tyrosine following exposure of intact quiescent Swiss 3T3 fibroblasts to the platelet-derived growth factor (PDGF). Western blotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated proteins followed by decoration with phosphotyrosine antibodies and 125I-labeled protein A have been used. The major tyrosine-phosphorylated component was a 170 kDa protein. The following lines of evidence suggest that this protein is the PDGF receptor in its tyrosine-phosphorylated form: (a) both proteins have the same (170 kDa) molecular weight; (b) the phosphorylated 170 kDa protein was detectable only in cell lines bearing the PDGF receptor; (c) the phosphorylation of the 170 kDa protein required PDGF and was dose-dependent. Kinetic studies showed that the phosphorylation of the receptor was maximal after 5-10 min at 37 degrees C and was followed by a rapid decrement of the band. The loss of the 170 kDa component was not prevented by inhibitors of membrane internalization and of lysosomal proteinases, while it was inhibited by lowering the temperature to 5 degrees C. In PDGF-stimulated cells, phosphotyrosine antibodies detected also a minor 36 kDa component phosphorylated at tyrosine.     

Platelet-derived growth factor elicits cyclic AMP accumulation in Swiss 3T3 cells: role of prostaglandin production

Partially purified porcine PDGF or purified human PDGF in the presence of phosphodiesterase inhibitors caused marked accumulation of cAMP in Swiss 3T3 cells. The responses were time- and dose-dependent; half-maximal effect was obtained at 0.6 nM PDGF. Indomethacin prevented the increase of cAMP levels in a dose-dependent manner; half-maximal effect was obtained at about 10 nM. Addition of PDGF increased (at least 25-fold) the production of E-type prostaglandins; PGE reached a concentration in the medium of 26 ng/ml 1 hr after treatment with human PDGF. This concentration of PGE produced a similar level of cAMP to that found with PDGF, suggesting that the PDGF-induced increase in cAMP is mediated by E-type prostaglandins released in the culture medium. Increased cAMP levels promoted by PDGF acting through stimulation of E-type prostaglandin synthesis may contribute to signal the initiation of cell proliferation in 3T3 cells.     

Different sensitivity to phorbol esters and pertussis toxin of bombesin- and platelet-derived growth factor-induced, phospholipase C-mediated hydrolysis of phosphoinositides in NIH/3T3 cells

Incubation of the serum-deprived cultures of NIH/3T3 cells with bombesin or platelet-derived growth factor (PDGF) induced the phospholipase C-mediated hydrolysis of phosphoinositides. Protein kinase C-activating 12-O-tetradecanoylphorbol 13-acetate (TPA) and pertussis toxin inhibited the bombesin-induced phospholipase C reactions. AlF4-, a direct activator of GTP-binding proteins (G proteins), also induced the phospholipase C reactions and TPA inhibited the AlF4- -induced reactions. These results suggest that a pertussis toxin-sensitive G protein is involved in the coupling of the bombesin receptor to the phospholipase C and that the coupling of the G protein to the phospholipase C is inhibited by protein kinase C. In contrast, neither TPA nor pertussis toxin inhibited the PDGF-induced phospholipase C reactions, indicating that a pertussis toxin-sensitive G protein is not involved in the coupling of the PDGF receptor to the phospholipase C and that this coupling is insensitive to protein kinase C. These results suggest that the regulatory mechanism of the PDGF receptor for the phospholipase C activation is different from that of the bombesin receptor.     

NHERF2 increases platelet-derived growth factor-induced proliferation through PI-3-kinase/Akt-, ERK-, and Src family kinase-dependent pathway

Platelet-derived growth factor (PDGF) has multiple functions including inhibition of apoptosis and promotion of cell proliferation. In this study, we show that Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) binds to the carboxyl-terminal PDZ domain-binding motif of the PDGF receptor through a PDZ domain-mediated interaction, and evaluate the consequence on PDGF-induced proliferation. Stable transfection with NHERF2 increased the PDGF-induced phosphorylation of ERK and Akt in Rat1 embryonic fibroblasts. The phosphorylation of Akt was blocked by pretreatment with LY294002, a PI-3-kinase inhibitor, in both Rat1/NHERF2 and Rat1/vector cells. In Rat1/vector cells, PDGF-induced phosphorylation of ERK was completely inhibited by pretreatment with PD98059, a MEK inhibitor. In contrast, the NHERF2-dependent increase of ERK phosphorylation was not affected by pretreatment with PD98059 in Rat1/NHERF2 cells. Thus, the NHERF2-dependent increase of ERK phosphorylation occurs in a MEK-independent fashion. Pretreatment with PP2, a specific inhibitor of Src family tyrosine kinase, completely blocked the NHERF2-dependent increase of the phosphorylation of ERK and Akt, suggesting that NHERF2 up-regulates Erk phosphorylation through a Src family kinase-dependent pathway. Consistent with these results, the PDGF-induced thymidine incorporation was increased in Rat1/NHERF2 cells, and the NHERF2-dependent increase of thymidine incorporation was prevented by treatment with LY294002 and PP2 but not with PD98059. These results suggest that NHERF2 stimulates PDGF-induced proliferation by increasing PI-3-kinase/Akt, MEKindependent ERK, and Src family kinase-mediated signaling pathways.     

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

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.     

Inactivation of platelet-derived growth factor receptor by the tumor suppressor PTEN provides a novel mechanism of action of the phosphatase

PTEN, mutated in a variety of human cancers, is a dual specificity protein phosphatase and also possesses D3-phosphoinositide phosphatase activity on phosphatidylinositol 3,4,5-tris-phosphate (PIP(3)), a product of phosphatidylinositol 3-kinase. This PIP(3) phosphatase activity of PTEN contributes to its tumor suppressor function by inhibition of Akt kinase, a direct target of PIP(3). We have recently shown that Akt regulates PDGF-induced DNA synthesis in mesangial cells. In this study, we demonstrate that expression of PTEN in mesangial cells inhibits PDGF-induced Akt activation leading to reduction in PDGF-induced DNA synthesis. As a potential mechanism, we show that PTEN inhibits PDGF-induced protein tyrosine phosphorylation with concomitant dephosphorylation and inactivation of tyrosine phosphorylated and activated PDGF receptor. Recombinant as well as immunopurified PTEN dephosphorylates autophosphorylated PDGF receptor in vitro. Expression of phosphatase deficient mutant of PTEN does not dephosphorylate PDGF-induced tyrosine phosphorylated PDGF receptor. Rather its expression increases tyrosine phosphorylation of PDGF receptor. Furthermore, expression of PTEN attenuated PDGF-induced signal transduction including phosphatidylinositol 3-kinase and Erk1/2 MAPK activities. Our data provide the first evidence that PTEN is physically associated with platelet-derived growth factor (PDGF) receptor and that PDGF causes its dissociation from the receptor. Finally, we show that both the C2 and tail domains of PTEN contribute to binding to the PDGF receptor. These data demonstrate a novel aspect of PTEN function where it acts as an effector for the PDGF receptor function and negatively regulates PDGF receptor activation.     

Guanosine 5'-O-(3-thiotrisphosphate) potentiates both thrombin- and platelet-derived growth factor-induced inositol phosphate release in permeabilized vascular smooth muscle cells. Signaling mechanisms distinguished by sensitivity to pertussis toxin and phorbol esters

We compared the mechanisms by which thrombin and platelet-derived growth factor (PDGF) activate phospholipase C in cultured vascular smooth muscle cells. Thrombin caused a transient (less than 5 min) increase in inositol trisphosphate (IP3) while PDGF caused a sustained (greater than 10 min) increase. Both pertussis toxin and phorbol 12-myristate 13-acetate (PMA) inhibited the thrombin-induced increase in IP3 but neither agent affected the PDGF-induced increase in IP3. To examine the role of GTP binding (G) proteins in the activation of phospholipase C by these two hormones, GTP analogues were introduced into saponin-permeabilized cells. In the absence of hormones, guanosine 5'-O-(3-thiotrisphosphate) (GTP gamma S) caused a progressive increase in IP3 release which was inhibited 55% by PMA (200 ng/ml). In the presence of thrombin, GTP gamma S caused synergistic increase in IP3 release. The synergism between GTP gamma S and thrombin was virtually eliminated by 10 min prior exposure to PMA (200 ng/ml). When PDGF was the hormonal agonist, GTP gamma S also caused synergistic increase in IP3 release and guanosine 5'-O-(2-thiodiphosphate) blunted PDGF-induced IP3 release. However, in contrast to thrombin, the synergism between GTP gamma S and PDGF was unaffected by PMA. Thus, thrombin and PDGF activate phospholipase C by signal transduction systems which differ in kinetic properties and in sensitivity to PMA and pertussis toxin. Despite these differences, both systems appear to involve GTP binding proteins at some step.     

The Rab4A effector protein Rabip4 is involved in migration of NIH 3T3 fibroblasts

The small GTP-binding protein Rab4 has been involved in the recycling of alphavbeta3 integrins in response to platelet-derived growth factor (PDGF) stimulation suggesting a role for Rab4 in cell adhesion and migration. In this study, we explored the role of Rabip4 and Rabip4', two Rab4 effector proteins, in migration of NIH 3T3 fibroblasts. In these cells, Rabip4 and Rabip4', collectively named Rabip4s, were partially co-localized with the early endosomal marker EEA1. PDGF treatment re-distributed endogenous Rabip4s toward the cell periphery where they colocalized with F-actin. In cells expressing green fluorescent protein (GFP)-Rabip4 or GFP-Rabip4', constitutive appearance of GFP-Rabip4s at the cell periphery was accompanied by local increase in cortical F-actin in membrane ruffles at the leading edge. The expression of GFP-Rabip4 induced an increased migration compared with control cells expressing GFP alone, even in the absence of PDGF stimulation. On the contrary, in cells expressing a mutated form of Rabip4s unable to interact with Rab4, lack of typical leading edge was observed. Furthermore, PDGF treatment did not stimulate the migration of these cells. Furthermore, down-regulation of the expression of Rabip4s inhibited PDGF-stimulated cell migration. Endogenous Rabip4s were localized with alphav integrins at the leading edge following PDGF treatment, whereas in cells expressing GFP-Rabip4s, alphav integrins, together with GFP-Rabip4s, were constitutively localized at the leading edge. In contrast, reduction in Rabip4s expression levels using small interfering RNA was associated with impaired PDGF-induced translocation of alphav integrins toward the leading edge. Taken together, our data provide evidence that Rabip4s, possibly via their interaction with Rab4, regulate integrin trafficking and are involved in the migration of NIH 3T3 fibroblasts.     

Suramin binds to platelet-derived growth factor and inhibits its biological activity

The polyanion suramin was recently found to inhibit binding of 125I-PDGF (platelet-derived growth factor) to Balb/c 3T3 cell membranes. Cultured Swiss 3T3 cells were used to investigate the mode of action of suramin and to monitor its effect on the biological activity of PDGF. Evidence is presented that suramin inhibits cellular binding of PDGF by binding to PDGF itself, thereby preventing it from binding to its cell surface receptor: First, while suramin inhibited 125I-PDGF binding with a half maximum inhibition concentration of approximately 60 microM or 90 micrograms/ml in a simultaneous competition assay, it was inactive in a sequential radioreceptor assay, in which an inhibitor is expected to be active if it interacts with the receptor (even with relatively low affinity) but to be inactive if it interacts with PDGF. Second, suramin prevented immunoprecipitation of 125I-PDGF in a dose-dependent manner, with a half maximum effective concentration of approximately 50 microM. Furthermore, suramin efficiently dissociated 125I-PDGF bound to its cell surface receptor, whereas unlabeled PDGF even in large excess was virtually inactive. This is also in line with the proposed direct interaction between PDGF and suramin, since such an interaction can be envisaged to induce a conformational change in the PDGF-receptor complex, resulting in an increased off-rate of the complex. Reduced 125I-PDGF binding in the presence of suramin correlated directly with a suramin dose-dependent inhibition of PDGF-induced incorporation of 3H-thymidine into quiescent Swiss 3T3 cells and of the proliferation of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)