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.

Endogenous inhibitors of the dsRNA-dependent eIF-2 alpha protein kinase PKR in normal and ras-transformed cells.

The serine/threonine kinase PKR is activated by autophosphorylation in response to nanomolar concentrations of double-stranded RNA and other polyanions. We have previously shown that expression of an oncogenic ras gene induces an endogenous protein inhibitor of PKR activation in murine fibroblasts, as measured by a greatly reduced ability of PKR to autophosphorylate in response to double-stranded RNA in lysates from these ras-expressing cells. Immunoprecipitation of PKR away from the ras-transformed cell lysate restored the ability of PKR to become autophosphorylated. However, the autophosphorylation was no longer dsRNA-dependent. In the present work, PKR immobilized either by immunoprecipitation or by affinity precipitation on Agpoly(I) poly(C) was found to autophosphorylate in a dsRNA-independent manner when incubated in the presence of a detergent lysis buffer and ATP. When lysis buffer was replaced by cytoplasmic extract from normal or ras-transformed cells, autophosphorylation of the immobilized PKR was inhibited in the presence or absence of dsRNA, even though it could be shown that PKR remained bound and intact in the precipitate, and able to autophosphorylate if rewashed with lysis buffer. These findings suggest that PKR activation is regulated by an endogenous inhibitor in murine fibroblasts as well as by dsRNA or other polyanions.

Platelet-derived growth factor signal transduction through the interferon-inducible kinase PKR. Immediate early gene induction.

The interferon-inducible, double-stranded RNA (dsRNA)-dependent eukaryotic initiation factor-2 alpha kinase PKR has primarily been characterized as a component of the interferon-mediated cellular antiviral response. Several lines of evidence now exist that suggest that PKR plays a role in the regulation of growth in uninfected cells. The most direct examples are the finding of an oncogenic variant of PKR and the effects of activators and inhibitors of PKR phosphorylation on the expression of platelet-derived growth factor (PDGF)-inducible genes. Previous reports have shown that 1) dsRNA, a direct activator of PKR, induces the genes c-myc, c-fos, and JE; 2) 2-aminopurine, a chemical inhibitor of PKR, blocks the induction of these genes by serum; and 3) activated p21ras induces a cellular inhibitor of PKR. We report here that activation of PKR was correlated with the induction of the immediate early genes c-fos, c-myc, and JE by PDGF in the following situations: 1) PDGF induction of these genes, also inducible by dsRNA, was blocked by two inhibitors of PKR activation: 2-aminopurine and v-ras; 2) PDGF induction of another immediate early gene, egr-1, which could not be induced by dsRNA, was not blocked by 2-aminopurine or v-ras; 3) agents that reverse v-ras inhibition of PKR activation also reversed the v-ras block of PDGF induction of c-myc, c-fos, and JE; 4) down-regulation of PKR protein levels by antisense inhibition of translation blocked the induction of c-myc, c-fos, and JE by PDGF, but had no effect on egr-1 induction; and finally, 5) PKR was autophosphorylated in vivo in response to PDGF. These results provide direct evidence that PKR activation functions as a second messenger in a growth factor signal transduction pathway. Thus, PKR may serve as a common mediator of growth-promoting and growth inhibitory signals.

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.

Platelet-derived growth factor (PDGF) induction of egr-1 is independent of PDGF receptor autophosphorylation on tyrosine.

Autophosphorylation of the platelet-derived growth factor (PDGF) receptor on tyrosine, which is dependent upon and occurs immediately after ligand binding, has been linked to the activation of second messenger pathways thought to be necessary for the induction of gene expression, DNA synthesis, and mitogenesis. We have investigated PDGF signal transduction in Balb/c3T3 and NIH-3T3 cells at the level of immediate-early gene induction under three conditions in which PDGF receptor autophosphorylation in response to PDGF binding is blocked: cells transformed by v-rasKi, cells transformed by v-mos, and cells treated with genistein, a specific inhibitor of tyrosine kinases. PDGF induction of immediate-early genes c-myc, c-fos, and JE is blocked in these systems. Induction of another immediate-early gene, egr-1, occurs normally despite the absence of measurable tyrosine kinase activity. The same results were obtained when cells were stimulated with PDGF-AA or PDGF-BB. It is not yet clear if this receptor tyrosine kinase-independent signal utilizes known PDGF second messengers, but these results demonstrate a new arm of the PDGF signal transduction pathway which operates in the absence of, and independently from, autophosphorylation of the receptor on tyrosine.

Dissociation of platelet-derived growth factor (PDGF) receptor autophosphorylation from other PDGF-mediated second messenger events.

Activated p21ras alters the platelet-derived growth factor (PDGF) signal transduction pathway in fibroblasts by inhibiting autophosphorylation of the receptor as well as by inhibiting the induction of the growth-related genes c-myc, c-fos, and JE. To elucidate the cause and effect relationships between receptor autophosphorylation and other second messenger events in the PDGF signaling pathway we created revertants of v-ras transformed cells by two methods: 1) the use of cAMP analogues, and 2) the introduction of a gene, Krev-1, which has been reported previously to revert ras transformed cells to normal morphology. Analysis of the revertants shows that the PDGF-mediated tyrosine phosphorylation of the 180-kDa PDGF receptor remains inhibited; however, the PDGF-mediated activation of phospholipase C and the induction of the growth-related genes c-myc, c-fos, and JE have been restored. These data suggest the presence of parallel pathways for PDGF signal transduction which are not dependent on autophosphorylation of the PDGF receptor.

v-mos suppresses platelet-derived growth factor (PDGF) type-beta receptor autophosphorylation and inhibits PDGF-BB-mediated signal transduction.

The function of mos protein in somatic cells, the mechanisms underlying its ability to transform cells, and its relationship to growth factor autonomy and growth factor-mediated signal transduction are not well defined. This report demonstrates that the expression of transforming mos (v-mos) can block the stimulation of growth-related gene expression mediated by the platelet-derived growth factor (PDGF-BB). This blockade by v-mos of PDGF-BB signal transduction occurs very early in the signalling pathway, at the level of PDGF type-beta receptor autophosphorylation. Although the expression of PDGF type-beta receptor, as detected by Western blot with anti-PDGF type-beta receptor antibody,was not diminished in v-mos transformed BALB/c-3T3 murine fibroblasts, the autophosphorylation of PDGF-beta receptor in response to ligand (recombinant PDGF-BB homodimer) stimulation was profoundly suppressed. This same phenomenon of v-mos-mediated PDGF type-beta receptor autophosphorylation inhibition was also demonstrated in NIH-3T3 fibroblasts. A v-mos mutant gene, which was incapable of binding ATP and was kinase-defective, did not block ligand-mediated receptor autophosphorylation. Factor(s) present in v-mos expressing fibroblasts, and found in the membrane fractions of these cells, dominantly inhibit the autophosphorylation of the PDGF type-beta receptor obtained from normal fibroblasts. This trans-acting factor does not appear to be a protein-tyrosine phosphatase. These findings suggest a role for mos, or a similar serine/threonine kinase, as a control mechanism in one of the earliest steps of the PDGF signal transduction pathway, and may provide a model for the functional interaction of mos with growth factor receptors.