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

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.     

Participation of phospholipase D and alpha/beta-protein kinase C in growth factor-induced signalling in C3H10T1/2 fibroblasts

We have studied phospholipase D (PLD) activation in relation to protein kinase C (PKC) and the involvement of PLD in extracellularly regulated kinase 1 (MAPK) (ERK1) activation and c-fos mRNA expression in C3H/10T1/2 (Cl8) fibroblasts. In these cells, the PLD activity was significantly increased by porcine platelet-derived growth factor (PDGF-BB), phorbol 12-myristate 13-acetate (PMA), and epidermal growth factor (EGF). PLD activation by PDGF-BB and PMA, but not EGF, was inhibited in Cl8 cells expressing the HAbetaC2-1 peptide (Cl8 HAbetaC2-1 cells), with a sequence (betaC2-1) shown to bind receptor for activated C kinase 1 (RACK1) and inhibit c-PKC-mediated cell functions [Science 268 (1995) 247]. A role of alpha-PKC in PLD activation is further underscored by co-immunoprecipitation of alpha-PKC with PLD1 and PLD2 in non-stimulated as well as PMA- and PDGF-BB-stimulated Cl8 cells. However, only PKC in PLD1 precipitates was activated by these agonists, while the PKC in the PLD2 precipitates was constitutively activated. The c-fos mRNA levels in Cl8 cells increased more than 30-fold in response to either PDGF-BB, EGF, or PMA. Approximately 60% inhibition of this increase in c-fos mRNA levels was observed in Cl8 HAbetaC2-1 cells. Formation of phosphatidylbutanol (PtdBut) at the expense of phosphatidic acid (PtdH) in the presence of n-butanol inhibited ERK1 activation and c-fos mRNA expression in PDGF-BB-treated Cl8 cells. ERK activation by PMA was unaffected by n-butanol in Cl8 cells but almost abolished by n-butanol in Cl8 HAbetaC2-1 cells, showing that ERK activation by PMA is heavily dependent on PKC and PLD1. In contrast, ERK activation by EGF in both cell types was not sensitive to n-butanol. These results indicate (1) a role of a functional interaction between the RACK1 scaffolding protein and a alphaPKC-PLD complex for achieving full PLD activity in PDGF-BB- and PMA-stimulated Cl8 cells; (2) PLD-mediated PtdH formation is needed for optimal ERK1 activation by PDGF-BB and maximal increase in c-fos mRNA expression. These findings place PLD as an important component in PDGF-BB- and PMA-stimulated intracellular signalling leading to gene activation in Cl8 cells, while EGF does not require PLD.     

Involvement of three intracellular messenger systems, protein kinase C, calcium ion and cyclic AMP, in the regulation of c-fos gene expression in Swiss 3T3 cells

In quiescent cultures of Swiss 3T3 cells, platelet-derived growth factor or fibroblast growth factor known to induce both protein kinase C activation and Ca2+ mobilization raised c-fos mRNA. This action of the growth factors was mimicked by the specific activators for protein kinase C, such as phorbol esters and a membrane-permeable synthetic diacylglycerol, and also by the Ca2+ ionophores, such as A23187 and ionomycin. Prostaglandin E1 known to elevate cyclic AMP also raised c-fos mRNA, and this action was mimicked by 8-bromo-cyclic AMP, dibutyryl cyclic AMP and forskolin. These results suggest that expression of the c-fos gene is regulated by three different intracellular messenger systems, protein kinase C, Ca2+ and cyclic AMP, in Swiss 3T3 cells.     

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.     

Bombesin induces c-fos and c-myc expression in quiescent Swiss 3T3 cells. Comparative study with other mitogens

We have studied the effect of the potent mitogen bombesin on the expression of c-fos and c-myc genes in quiescent mouse fibroblasts. We have demonstrated that bombesin rapidly induces a transient expression of c-fos mRNA followed by a more protracted elevation in c-myc mRNA levels. The intensity of the induction of expression of both proto-oncogenes depended on the dose of bombesin used. Prolonged treatment of the cells with TPA, which causes a selective decrease in protein kinase C activity, partially inhibited the induction of c-fos and c-myc gene expression by bombesin, similar to what has been observed with PDGF. However, a dramatic inhibition of the mitogenic response to bombesin--but not to PDGF--was found in TPA-treated cells. In contrast, TPA-treated cells showed an increased response to EGF with regard to proto-oncogene expression. The role of protein kinase C and Ca2+-dependent pathways in proto-oncogene induction by bombesin is discussed.     

Bombesin induction of c-fos and c-myc proto-oncogenes in Swiss 3T3 cells: significance for the mitogenic response

Bombesin is a potent mitogen for Swiss 3T3 cells and acts synergistically with insulin and other growth factors. We show here that addition of bombesin to quiescent Swiss 3T3 cells causes a striking increase in the levels of c-fos and c-myc mRNAs. Enhanced expression of c-fos (122 +/- 14-fold) occurred within minutes of peptide addition followed by increased expression of c-myc (82 +/- 16-fold). The concentrations of peptide required for half-maximal increase in the levels of c-fos and c-myc mRNAs were 1.0 and 0.9 nM, respectively. The peptide [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P which inhibits the binding of bombesin to its receptor and bombesin-stimulated DNA synthesis in Swiss 3T3 cells blocked the increase in c-fos and c-myc mRNA levels promoted by bombesin. Down-regulation of protein kinase C by long-term exposure to phorbol esters prevented c-fos and c-myc induction by bombesin. This and other results indicate that the induction of these proto-oncogenes by bombesin could be mediated by the coordinated effects of protein kinase C activation and Ca2+ mobilization. The marked synergistic effect between bombesin and insulin was used to assess whether the increase in the induction of c-fos and c-myc is an obligatory event in cell activation. In the presence of insulin, bombesin stimulated DNA synthesis at subnanomolar concentrations but had only a small effect on c-fos and c-myc mRNA levels. This apparent dissociation of mitogenesis from proto-oncogene induction was even more dramatic in 3T3 cells with down-regulated protein kinase C. In these cells bombesin stimulated DNA synthesis in the presence of insulin but failed to enhance c-fos and c-myc mRNA levels at comparable concentrations. Thus, the induction of c-fos and c-myc may be a necessary step in the mitogenic response initiated by ligands that act through activation of protein kinase C but the expression of these proto-oncogenes may not be an obligatory event in the stimulation of mitogenesis in 3T3 cells by mitogens that utilise other signalling pathways.     

Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells.

We investigated the molecular mechanisms underlying the ability of heparin to inhibit vascular smooth muscle cell (VSMC) growth. Previous experiments have shown that heparin inhibits induction of c-fos and c-myc protooncogene mRNA in rat VSMC stimulated by phorbol 12-myristate 13-acetate (PMA) but not when stimulated by epidermal growth factor (EGF) (Pukac, L. A., Castellot, J. J., Wright, T. C., Caleb, B. L., and Karnovsky, M. J. (1990) Cell Regul. 1, 435-443). The present experiments show that these mitogens activate distinct second messenger pathways in VSMC, because PMA but not EGF induction of c-fos and c-myc mRNA was suppressed in protein kinase C (PKC) down-regulated VSMC; this suggests that EGF does not act through a PKC-dependent pathway for induction of these genes. Heparin inhibited serum stimulation of c-fos mRNA in control VSMC, but heparin did not inhibit the smaller but significant serum stimulation of c-fos mRNA in PKC down-regulated VSMC, indicating that heparin may selectively inhibit PKC-dependent, but not PKC-independent, stimulation of gene expression. To further determine if heparin inhibits non-PKC pathways, VSMC were treated with dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187; stimulation of c-fos mRNA by this treatment was not inhibited by heparin. DNA synthesis and cell proliferation were inhibited in rat VSMC exposed briefly to heparin during the G0/G1 phase of the cell cycle. These experiments indicate heparin can act early in the cell cycle and suggest PKC-dependent but not PKC-independent signaling pathways for gene expression are selectively sensitive to heparin inhibition.     

Two independent growth factor-generated signals regulate c-fos and c-myc mRNA levels in Swiss 3T3 cells

Polypeptide growth factors that stimulate cell proliferation bind to cell surface receptors and activate intracellular signal transduction pathways. One major signalling pathway, initiated by phosphatidylinositol (PI) turnover, involves activation of protein kinase C. Some polypeptide growth factors, including mitogens that activate protein kinase C, induce a rapid increase in expression of the proto-oncogenes, c-myc and c-fos. In order to characterize the signal transduction pathways responsible for proto-oncogene activation, we treated Swiss 3T3 cells with the tumor promoter phorbol dibutyrate to generate cells deficient in protein kinase C. These cells were then stimulated with platelet extract, bombesin, or epidermal growth factor (EGF) and the levels of c-myc and c-fos mRNA were determined. Platelet extract or bombesin, which stimulate PI turnover, were substantially weaker inducers of c-myc and c-fos mRNA levels in the protein kinase C-depleted cells, although some variability with platelet extract was noted. EGF, which does not stimulate PI turnover in several cell systems, was by contrast a potent inducer of both proto-oncogenes whether or not the cells were deficient in protein kinase C. Pretreatment of cells with phorbol dibutyrate caused little or no change in the basal levels of c-myc or c-fos mRNA, but led to a small but significant increase in basal levels of ornithine decarboxylase mRNA. These results demonstrate that EGF and growth factors that activate PI turnover induce expression of the c-myc and c-fos proto-oncogenes through different pathways.     

Arachidonic acid and cyclic adenosine monophosphate stimulation of c-fos expression by a pathway independent of phorbol ester-sensitive protein kinase C

The stimulation of cell proliferation by platelet-derived and other growth factors is associated with a rapid increase in the expression of the c-fos protooncogene. We and others have shown that phosphosphoinositide turnover and protein kinase C play a role in the activation of this gene by growth factors, but that a second, kinase C-independent pathway(s) exist. Because cAMP potentiates the actions of a number of growth factors and is elevated in platelet-derived growth factor-stimulated Swiss 3T3 cells, we examined the ability of cAMP to stimulate c-fos expression in this cell type. Forskolin, a direct activator of adenylate cyclase, elicited marked increases in c-fos mRNA levels. Receptor-mediated activation of adenylate cyclase by prostaglandin E1 and stimulation with the cAMP analog 8-bromo-cAMP also enhanced c-fos expression. In cells made protein-kinase C deficient, c-fos induction by phorbol ester was abolished; by contrast, c-fos was still induced by cAMP-elevating agents in protein kinase C-depleted cells. Platelet-derived growth factor causes cAMP accumulation by stimulating arachidonic acid release and the formation of prostaglandins capable of activating adenylate cyclase. The addition of arachidonic acid and the arachidonate metabolite prostaglandin E2 to Swiss 3T3 cultures stimulated c-fos expression. These data suggest the existence of a pathway from growth factor receptor to gene induction that is mediated by cAMP and does not depend on a phorbol ester-sensitive protein kinase C.     

Effects of epidermal growth factor and platelet-derived growth factor on c-fos and c-myc mRNA levels in normal human fibroblasts

The mRNA levels of two proto-oncogenes, c-fos and c-myc, were determined in human foreskin fibroblasts exposed to epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) in a serum-free, defined medium (MCDB 104). Untreated, quiescent cells were found to have low or undetectable levels of c-fos and c-myc mRNA. Within 10 min after the addition of EGF or PDGF the c-fos mRNA level increased, reached a peak at 30 min, and then declined to the control level after 60 min. The level of c-myc mRNA increased somewhat later and peaked after 8 h in cultures treated with either of the growth factors. The c-myc mRNA level remained elevated throughout the 24 h of investigation. The concentrations of EGF and PDGF required for a maximal effect on c-fos or c-myc expression were found to be similar to those that give maximal effect on cell proliferation. Both c-fos and c-myc mRNA expression were super-induced by the addition of cycloheximide. The addition of neutralizing PDGF antibodies to cultures that had received PDGF 4 h earlier inhibited the subsequent increase in the c-myc mRNA level, indicating that the effect of PDGF on c-myc expression is not caused by a "hit and run," mechanism. Density-inhibited cells responded to EGF and PDGF by an increase in c-fos and c-myc mRNA levels in the absence of any mitogenic response. The present results conform to the view that the c-fos and c-myc proto-oncogenes may be important (or necessary) but not sufficient for the initiation of DNA synthesis. Moreover, the finding that both EGF and PDGF increase c-fos and c-myc expression supports our previous suggestion that these two growth factors may in part act via a common intracellular pathway in the prereplicative phase of human fibroblasts.     

Possible involvement of protein kinase C and calcium ion in growth factor-induced expression of c-myc oncogene in Swiss 3T3 fibroblasts

The addition of platelet-derived growth factor and fibroblast growth factor to quiescent cultures of Swiss 3T3 fibroblasts rapidly induced protein kinase C activation and Ca2+ mobilization and afterwards markedly increased c-myc mRNA levels. 1-Oleoyl-2-acetylglycerol, a membrane-permeable synthetic diacylglycerol, and 12-O-tetradecanoylphorbol 13-acetate, a tumor-promoting phorbol ester, stimulated protein kinase C activation without Ca2+ mobilization. Inversely, Ca2+ ionophores, A23187 and ionomycin, elicited Ca2+ mobilization without protein kinase C activation. Both protein kinase C-activating and Ca2+-mobilizing agents were able to increase c-myc mRNA levels in an additive manner. Prolonged treatment of the cells with phorbol 12,13-dibutyrate, another protein kinase C-activating phorbol ester, led to the down-regulation and complete disappearance of protein kinase C. In these cells, 1-oleoyl-2-acetylglycerol and 12-O-tetradecanoylphorbol 13-acetate did not increase c-myc mRNA levels, but platelet-derived growth factor, fibroblast growth factor, and the Ca2+ ionophores, all of which still induced Ca2+ mobilization, stimulated the increase of c-myc mRNA levels. These results strongly suggest that both protein kinase C and Ca2+ may be involved in platelet-derived growth factor- as well as fibroblast growth factor-induced expression of the c-myc oncogene in Swiss 3T3 cells.