BALB/c-3T3 fibroblasts resistant to growth inhibition by beta interferon exhibit aberrant platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor signal transduction.

Several lines of evidence now exist to suggest an interaction between the platelet-derived growth factor (PDGF) growth-stimulatory signal transduction pathway and the beta interferon (IFN-beta) growth-inhibitory signal transduction pathway. The most direct examples are inhibition of PDGF-mediated gene induction and mitogenesis by IFN-beta and the effects of activators and inhibitors of the IFN-inducible double-stranded RNA-dependent eIF2 kinase on expression of PDGF-inducible genes. To further investigate the nature of this PDGF/IFN-beta interaction, we selected BALB/c-3T3 cells for resistance to growth inhibition by IFN-beta and analyzed the phenotypes of resulting clonal lines (called IRB cells) with respect to PDGF signal transduction. Although selected only for IFN resistance, the IRB cells were found to be defective for induction of growth-related genes c-fos, c-myc and JE in response to PDGF. This block to signal transduction was not due to loss or inactivation of PDGF receptors, as immunoprecipitation of PDGF receptors with antiphosphotyrosine antibodies showed them to be present at equal levels in the BALB/c-3T3 and IRB cells and to be autophosphorylated normally in response to PDGF. Furthermore, treatment with other peptide growth factors (PDGF-AA, fibroblast growth factor, and epidermal growth factor) also failed to induce c-fos, c-myc, or JE expression in IRB cells. All of these growth factors, however, were able to induce another early growth-related gene, Egr-1. The block to signaling was not due to a defect in inositol phosphate metabolism, as PDGF treatment induced normal calcium mobilization and phosphotidylinositol-3-kinase activation in these cells. Activation of protein kinase C by phorbol esters did induce c-fos, c-myc, and JE in IRB cells, indicating that signalling pathways distal to this enzyme remained intact. We have previously shown that IFN-inducible enzyme activities, including double-stranded RNA-dependent eIF2 kinase and 2',5'-oligoadenylate synthetase, are normal in IRB cells. The finding that the induction of multiple growth-related genes by several independent growth factors is inhibited in these IFN-resistant cells suggests that there is a second messenger common to both growth factor and IFN signaling pathways and that this messenger is defective in these cells.     

Inhibition of platelet-derived growth factor-mediated signal transduction by transforming ras. Suppression of receptor autophosphorylation.

The function of ras protein and its relationship to growth-factor mediated signal transduction remain unclear. The demonstration that the expression of transforming ras (v-ras) can block the stimulation of growth-related gene expression and cell division mediated by the platelet-derived growth factor (PDGF) may provide a model for the functional interation of ras with growth factor receptors. In the current studies, we have demonstrated that this blockade by v-ras of PDGF-BB signal transduction occurs very early in signal transduction, at the level of PDGF receptor autophosphorylation. Although the expression of PDGF receptor as detected by Western blot with anti-PDGF receptor antibody was not diminished in v-ras-transformed murine Balb/c 3T3 fibroblasts, the autophosphorylation of PDGF receptor in response to ligand (recombinant PDGF-BB homodimer) stimulation was profoundly suppressed. This same phenomenon of v-ras-mediated PDGF receptor autophosphorylation inhibition was also demonstrated in normal rat kidney fibroblasts. Further, factor(s) present in v-ras-expressing fibroblasts found in the membrane fractions of these cells can dominantly inhibit the autophosphorylation of the PDGF receptor obtained from normal fibroblasts. These findings suggest a role for ras in one of the earliest steps of the signal transduction pathway.     

Platelet-derived growth factor regulation of fos stability correlates with growth induction.

A brief exposure of quiescent, density-arrested Balb/c 3T3 fibroblasts to platelet-derived growth factor (PDGF) results in expression of the proto-oncogene c-fos; furthermore, the translation product of c-fos, p55fos, was shown to have increased stability in cells upon continued exposure to PDGF. The induction of competence or growth initiation requires a longer exposure to PDGF than that necessary for the induction of the immediate-early, growth-related genes. The need for the continued presence of PDGF for growth initiation beyond the time required for the induction of immediate-early gene expression may be due, in part, to PDGF-dependent post-translational stabilizations of gene products. We speculate that a PDGF-mediated event increases p55fos stability, resulting in a continued elevated level of Fos protein, which in turn allows a continued Fos-mediated activity required by Balb/c 3T3 cells to become competent to enter the cell cycle.     

Membrane-anchored form of v-sis/PDGF-B induces mitogenesis without detectable PDGF receptor autophosphorylation

The v-sis protein is structurally and functionally related to PDGF. Forms of the v-sis protein which are anchored to the cell membrane via the transmembrane domain of the vesicular stomatitis virus G protein have been previously described (Hannink, M., and D.J. Donoghue. 1986. J. Cell Biol. 103:2311-2322). Several of these fusion proteins were shown to interact productively with the PDGF receptor (PDGFR) based on their ability to transform NIH 3T3 cells. In this report, we further characterized one of these membrane-anchored v-sis proteins, designated v-sis239-G. The gene encoding v-sis239-G was placed under control of the Drosophila melanogaster hsp70 promotor and synthesis of this protein was shown to induce a mitogenic response in NIH 3T3 cells. Unexpectedly, v-sis239-G did not induce detectable autophosphorylation of the PDGFR, in contrast to a similarly expressed secreted form of the v-sis protein. Thus, it appears that a PDGFR-mediated mitogenic response may be dissociated from detectable receptor autophosphorylation. Furthermore, induced synthesis of v-sis239-G was shown to lead to c-fos expression even in the absence of detectable receptor autophosphorylation. Interestingly, a nonmitogenic membrane-anchored form of the v-sis protein, designated v-sis239-G338, also induced c-fos without receptor autophosphorylation. These results raise interesting questions regarding the roles of autophosphorylation and c-fos induction in PDGFR-mediated signal transduction and suggest the possibility of an autophosphorylation-independent signal transduction pathway.     

Platelet-derived growth factor does not induce c-fos in NIH 3T3 cells expressing the EJ-ras oncogene.

Platelet-derived growth factor (PDGF), the calcium ionophore A23187, and the tumor promoter phorbol myristate acetate stimulated c-fos mRNA levels in control NIH 3T3 cells. However, NIH 3T3 cells transformed by EJ-ras DNA transfection, which have diminished PDGF-stimulated phospholipase C activity, showed a 95% reduction in PDGF-stimulated c-fos mRNA levels. The responses to A23187 and phorbol myristate acetate were also attenuated, but not as severely as the PDGF-mediated induction. The reduction in PDGF-stimulated c-fos induction did not appear to be a general result of cellular transformation, since src-transformed NIH 3T3 cells displayed a strong PDGF-stimulated c-fos induction. Despite the reduction in PDGF-stimulated c-fos induction, EJ-ras-transformed cells still responded mitogenically to PDGF. These data suggest that the magnitude of c-fos induction cannot be directly correlated with PDGF-stimulated mitogenesis in EJ-ras-transformed NIH 3T3 cells.     

Quantitative dissociation between EGF effects on c-myc and c-fos gene expression, DNA synthesis, and epidermal growth factor receptor tyrosine kinase activity.

The exact relationship between EGF-stimulated tyrosine phosphorylation, induction of the cellular proto-oncogenes c-myc and c-fos, and DNA synthesis remains uncertain. Madin-Darby Canine Kidney (MDCK) cells possess EGF receptor sites with high binding capacity, and in contrast to A431 cells, respond to EGF by increasing DNA synthesis. Following EGF stimulation of intact MDCK cells, there was a rapid and marked increase in the autophosphorylation of the EGF receptor. This was associated with an increase in the tyrosine phosphorylation of a 120 kDa phosphoprotein believed to be an endogenous substrate of this receptor kinase. The ED50 for stimulation of phosphorylation of pp120 was approximately 0.05 nM versus 1.0 nM for receptor autophosphorylation, consistent with amplification of signalling at this step in EGF action. Stimulation of DNA synthesis occurred after 12 to 24 hours and revealed even further amplification with an ED50 of about 0.1 nM. Intermediate between these events was a time-dependent activation of c-fos and c-myc gene expression. However, the ED50 for these processes was approximately 10 nM, indicating a relatively lower sensitivity of EGF for stimulation of proto-oncogene expression. Tyrphostin (RG 50864), a compound reported to inhibit specifically the EGF receptor kinase, completely blocked EGF stimulation of proto-oncogene induction. Interestingly, under the same experimental conditions, EGF receptor autophosphorylation was decreased only 60%. These data, along with the dose-response studies, indicate that proto-oncogene induction requires near maximal stimulation of EGF receptor autophosphorylation. They also suggest that, in MDCK cells, the EGF dependent induction of the c-fos and c-myc genes is not strictly correlated to the extent of EGF receptor autophosphorylation or EGF-stimulated DNA synthesis, and that EGF stimulation of DNA synthesis likely involves additional rate-limiting intermediate steps.     

Autocrine stimulation by the v-sis gene product requires a ligand- receptor interaction at the cell surface

Autocrine expression of a growth factor and its receptor in the same cell raises the possibility of an intracellular receptor-ligand interaction within the cell, in addition to a receptor-ligand interaction at the cell surface. We have constructed a NIH3T3 cell line which contains the v-sis gene under the inducible control of the Drosophila melanogaster hsp70 promoter. Expression of both v-sis RNA and protein is rapidly induced by a short period of heat-shock. We have analyzed the cellular site of interaction between the v-sis protein and the platelet-derived growth factor receptor in these cells. Autophosphorylation of the PDGF receptor and induction of the c-fos gene were found to occur at 45 and 50 min, respectively, after heat-induced synthesis of the v-sis protein. Monensin treatment of the heat-induced cells prevented autophosphorylation of the mature PDGF receptor and also prevented subsequent induction of c-fos. Autophosphorylation of the PDGF receptor and c-fos induction were also prevented by the addition of suramin to the medium. These results demonstrate that autocrine stimulation, as monitored by c-fos induction and by PDGF receptor autophosphorylation, requires an interaction between the v-sis protein and the PDGF receptor that occurs at the cell surface, rather than an intracellular location.     

Insulin stimulation of gene expression mediated by p21ras activation.

In fibroblasts, insulin is a weak mitogen and does not induce expression of c-fos, c-jun or p33. However, increasing the expression levels of either normal p21Hras or the insulin receptor, but not mutant p21Hras, enables insulin to induce the expression of these genes. In cells expressing elevated levels of insulin receptor, this process involves a rapid increase in p21rasGTP levels (from 20% to 70% GTP as a percentage of total guanine nucleotides). No increase in p21rasGTP levels was observed after PDGF and EGF stimulation of cells expressing high levels of the cognate receptor, stressing the specificity of the insulin-induced increase. We conclude that in fibroblasts, p21ras is an intermediate of the insulin signal transduction pathway involved in the regulation of gene expression and mitogenicity.     

Contrasting role of phospholipase C-gamma1 in the expression of immediate early genes induced by epidermal or platelet-derived growth factors

While significant progress has been achieved in identifying the signal transduction elements that operate downstream of activated receptor tyrosine kinases, it remains unclear how different receptors utilize these signaling elements to achieve a common response. This study compares the capacity of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) to elicit the induction of immediate early gene (IEG) mRNAs in the presence or absence of phospholipase C-gamma1 (PLC-gamma1). The results show that while PDGF induction of nearly all IEG mRNAs is abrogated in plcg1 null cells, EGF induction of the same genes is variable in the null cells and exhibits three distinct responses. Five IEG mRNAs (Nup475, Cyr61, TF, Gly, TS7) are completely inducible by EGF in the presence or absence of PLC-gamma1, while three others (JE, KC, FIC) exhibit a stringent requirement for the presence of PLC-gamma1. The third type of response is exhibited by c-fos and COX-2. While these mRNAs are completely induced by EGF in the absence of PLC-gamma1, the time course of their accumulation is significantly delayed. No IEG was identified as completely inducible by EGF and PDGF in the absence of PLC-gamma1. Electrophoretic mobility shift assays (EMSA) demonstrate that PLC-gamma1 is necessary for nuclear extracts from PDGF-treated cells, but not EGF-treated cells, to interact with probes for AP-1 or NF-kappaB.     

Transforming growth factor beta 1 is a powerful modulator of platelet-derived growth factor action in vascular smooth muscle cells.

We have studied the effect of transforming growth factor beta 1 (TGF-beta 1) on vascular smooth muscle cell (SMC) mitogenesis and expression of thrombospondin and other growth related genes. We found that TGF-beta 1 treatment of vascular SMC induced a prolonged increase in steady-state mRNA levels of thrombospondin as well as alpha 1 (IV) collagen. The increase began at approximately 2 h, peaked by 24 h, and remained considerably elevated 48 h after growth factor addition. There was a corresponding increase in thrombospondin protein as well as increased expression of several other secreted polypeptides. The increase in thrombospondin contrasted sharply with that observed for platelet-derived growth factor (PDGF) which induced a rapid and transient increase in thrombospondin mRNA level. Although TGF-beta 1 was able to directly enhance expression of thrombospondin as well as the growth-related genes c-fos and c-myc, and induced c-fos expression with identical kinetics as PDGF, it was unable to elicit [3H]thymidine incorporation into DNA in three independent smooth muscle cell strains. However, TGF-beta 1 was able to strongly increase the mitogenic response of SMC to PDGF. Addition of both TGF-beta 1 and PDGF to SMC also caused a synergistic increase in the expression of thrombospondin as well as c-myc. Interestingly, in one other smooth muscle cell strain, a weak and delayed mitogenic response to TGF-beta 1 alone was observed. Our results strongly suggest that induction of thrombospondin expression by TGF-beta 1 and by PDGF occurs by distinct mechanisms. In addition, that TGF-beta 1 can enhance PDGF-induced mitogenesis may be due to the ability of TGF-beta 1 to directly induce the expression of thrombospondin, c-fos, c-myc, and the PDGF beta-receptor.     

Oncogenic ras induces an inhibitor of double-stranded RNA-dependent eukaryotic initiation factor 2 alpha-kinase activation.

The interferon-inducible 68-kDa dsRNA-dependent eIF2 alpha-kinase (dsI) is a potent cellular antiviral enzyme which is activated by autophosphorylation in response to double-stranded RNA (dsRNA). Activated dsI has also been implicated as a second messenger for gene induction by platelet-derived growth factor (PDGF) and interferon (IFN). We have shown previously that introduction of a transforming ras gene into BALB/c-3T3 fibroblasts blocks induction of responsive genes by PDGF and IFN. We therefore investigated the effect of transforming ras genes on dsI activity in these cells. We report here that dsRNA-mediated activation of dsI is blocked in v-ras-containing cells in a manner specific to ras and not attributable to the transformed phenotype since: 1) a dexamethasone-inducible v-Ha-ras gene produced the effect within 18 h of induction; 2) morphologic reversion of ras-transformed cells with cAMP or the Krev-1 gene restored potential for dsI activation; and 3) transformation by v-mos or v-abl had no effect on dsI activation. Latent dsI levels were unaffected by v-ras. A heat-sensitive dsI inhibitory activity could be demonstrated in v-ras-containing cells which functioned in trans when mixed with untransformed cell extracts prior to stimulation with dsRNA. The inhibitory activity, which was destroyed by phenol-chloroform extraction, did not bind dsRNA.     

Antisense-fos RNA causes partial reversion of the transformed phenotypes induced by the c-Ha-ras oncogene.

Several lines of evidence have suggested that c-fos may act downstream from c-Ha-ras in a growth-regulatory signal transduction pathway. We used antisense RNA to inhibit c-fos gene expression and investigated the effects of diminished c-fos expression on the phenotypes induced by the EJ c-Ha-ras oncogene in NIH 3T3 cells. Immunofluorescent staining demonstrated that the antisense RNA caused a marked reduction in the amount of c-fos protein expressed following serum stimulation. EJ cells containing antisense-fos RNA continued to overexpress ras and remained capable of proliferating in vitro. However, the antisense-fos RNA caused a partial reversion of the major transformed phenotypes of EJ cells, including a restoration of both density-dependent growth arrest and the ability to be rendered quiescent by serum deprivation, a reversion to a flat morphology, inhibition of anchorage-independent growth, and inhibition of tumorigenicity in nude mice. Our results indicate that inhibition of c-fos expression, to a level still supporting in vitro proliferation, prevents the transforming effects of the ras oncogene; they thus provide additional evidence for the participation of c-fos in ras-regulated signal transduction pathways.