Bombesin stimulation of c-fos expression and mitogenesis in Swiss 3T3 cells: the role of prostaglandin E2-mediated cyclic AMP accumulation

Bombesin is a potent mitogen for Swiss 3T3 cells and can stimulate DNA synthesis in the absence of any other growth factor. This effect is mediated by multiple synergistic signaling pathways, including an accumulation of intracellular cyclic AMP (cAMP) and an increase in c-fos mRNA expression. The cyclooxygenase inhibitor indomethacin abolished prostaglandin E2 release and substantially depressed cAMP levels induced by bombesin (EC50 congruent to 10 nM). In contrast, indomethacin at 1 microM did not affect 80K phosphorylation or Ca2+ mobilization by bombesin, indicating that cAMP synthesis can occur through a phospholipase C-independent pathway. Indomethacin caused a 30 to 35% decrease in c-fos induction and DNA synthesis in cells treated with bombesin (EC50 congruent to 40 nM). Significantly, the inhibitory effect of indomethacin was reversed in the presence of forskolin, a direct activator of adenylate cyclase. We conclude that cAMP plays a regulatory role in c-fos induction and mitogenesis in Swiss 3T3 cells treated with bombesin.     

Mitogenic effect of prostaglandin E1 in Swiss 3T3 cells: role of cyclic AMP

Addition of prostaglandin E1 (PGE1) to quiescent cultures of Swiss 3T3 cells rapidly elevates the intracellular levels of cAMP and increases the activity of adenylate cyclase in particulate fractions of these cells. In the presence of insulin, PGE1 stimulates the reinitiation of DNA synthesis. Both effects (increase in cellular cAMP and stimulation of DNA synthesis) are markedly potentiated by 1-methyl-3-isobutyl xanthine (IBMX) or by 4-(3-butoxy-4 methoxy benzyl)-2-imidazolidine (Ro 20-1724), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. In the presence of 50 microM IBMX, PGE1 caused a dose-dependent increase in cAMP levels and in [3H]thymidine incorporation into acid-insoluble material at concentrations (5-50 ng/ml) that are orders of magnitude lower than those used in previous studies (50 micrograms/ml) to demonstrate growth-inhibitory effects. Thus, the inhibitory effects produced by adding high concentrations of PGE1 on the initiation of DNA synthesis in Swiss 3T3 cells are not mediated by cAMP and should be regarded as nonspecific. In contrast, the mitogenic activity of PGE1 parallels its ability to increase the intracellular levels of cAMP. The findings support the proposition that a sustained increase in the level of this cyclic nucleotide acts as a mitogenic signal for confluent and quiescent Swiss 3T3 cells.     

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.     

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.     

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.     

Bombesin stimulation of c-fos expression and mitogenesis in Swiss 3T3 cells: The role of prostaglandin E2-mediated cyclic AMP accumulation

Bombesin is a potent mitogen for Swiss 3T3 cells and can stimulate DNA synthesis in the absence of any other growth factor. This effect is mediated by multiple synergistic signaling pathways, including an accumulation of intracellular cyclic AMP (cAMP) and an increase in c-fos mRNA expression. The cyclooxygenase inhibitor indomethacin abolished prostaglandin E₂ release and substantially depressed cAMP levels induced by bombesin (EC₅₀ - 10 nM). In contrast, indomethacin at 1 μM did not affect 80K phosphorylation or Ca²⁺ mobilization by bombesin, indicating that cAMP synthesis can occur through a phospholipase C-independent pathway. Indomethacin caused a 30 to 35% decrease in c-fos induction and DNA synthesis in cells treated with bombesin (EC₅₀ - 40 nM). Significantly, the inhibitory effect of indomethacin was reversed in the presence of forskolin, a direct activator of adenylate cyclase. We conclude that cAMP plays a regulatory role in c-fos induction and mitogenesis in Swiss 3T3 cells treated with bombesin.     

Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells: II. A pathway involving arachidonic acid release, prostaglandin synthesis, and cyclic AMP accumulation.

We have previously shown that extracellular ATP acts as a mitogen via protein kinase C (PKC)-dependent and independent pathways (Wang, D., Huang, N., Gonzalez, F.A., and Heppel, L.A. Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells. I. Involvement of protein kinase C-dependent and independent pathways in the mitogenic response of mammalian cells to extracellular ATP. J. Cell. Physiol., 1991). The present aim was to determine if metabolism of arachidonic acid, resulting in prostaglandin E2 (PGE2) synthesis and elevation of cAMP levels, plays a role in mitogenesis mediated by extracellular ATP. Addition of ATP caused a marked enhancement of cyclic AMP accumulation in 3T3, 3T6, and A431 cells. Aminophylline, an antagonist of the adenosine A2 receptor, had no effect on the accumulation of cyclic AMP elicited by ATP, while it inhibited the action of adenosine. The accumulation of cyclic AMP was concentration dependent, which corresponds to the stimulation of DNA synthesis by ATP. The maximal accumulation was achieved after 45 min, with an initial delay period of about 15 min. That the activation of arachidonic acid metabolism contributed to cyclic AMP accumulation and mitogenesis stimulated by ATP in 3T3, 3T6, and A431 cells was supported by the following observations: (a) extracellular ATP stimulated the release of [3H]arachidonic acid and PGE2 into the medium; (b) inhibition of arachidonic acid release by inhibitors of phospholipase A2 blocked PGE2 production, cyclic AMP accumulation, and DNA synthesis activated by ATP, and this inhibition could be reversed by adding exogenous arachidonic acid; (c) cyclooxygenase inhibitors, such as indomethacin and aspirin, diminished the release of PGE2 and blocked cyclic AMP accumulation as well as [3H]thymidine incorporation in response to ATP; (d) PGE2 was able to restore [3H]thymidine incorporation when added together with ATP in the presence of cyclooxygenase inhibitors; (e) pertussis toxin inhibited ATP-stimulated DNA synthesis in a time- and dose-dependent fashion as well as arachidonic acid release and PGE2 formation. Other evidence for involvement of a pertussis toxin-sensitive G protein(s) in ATP-stimulated DNA synthesis as well as in arachidonic acid release is presented. In A431 cells, the enhancement of arachidonic acid and cyclic AMP accumulation by ATP was partially blocked by PKC down-regulation, implying that the activation of PKC may represent an additional pathway in ATP-stimulated metabolism of arachidonic acid. In all of these studies, ADP and AMP-PNP, but not adenosine, were as active as ATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Two signal transduction pathways mediate interleukin-1 receptor expression in Balb/c3T3 fibroblasts

Our previous studies showed that platelet-derived growth factor (PDGF) modulated interleukin-1 (IL-1) activity and IL-1 binding to Balb/c3T3 fibroblasts (Bonin, P. D., and Singh, J. P. (1988) J. Biol. Chem. 263, 11052-11055). Subsequent studies have demonstrated an action of PDGF at the level of IL-1 receptor (IL-1R) gene expression. PDGF treatment of Balb/c3T3 cells produces a 10-20-fold stimulation of mRNA for IL-1 receptor. Investigation of the signal transduction pathways shows that activation of either the protein kinase C pathway or the cAMP-mediated pathway leads to the stimulation of IL-1 receptor expression in Balb/c3T3 cells. Treatment of Balb/c3T3 cells with phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, produced an increased 125I-IL-1 binding to cells and stimulation of IL-1R mRNA. Staurosporine, an inhibitor of protein kinase C, blocked the induction of IL-1 binding by PDGF or PMA. Down-regulation of protein kinase C by pretreatment with PMA reduced the subsequent stimulation by PDGF. Chronic treatment with PMA, however, did not produce a complete inhibition of PDGF effect on IL-1R. Further studies showed that the agents that stimulate cAMP accumulation (isobutyl methylxanthine, dibutyryl), directly stimulate adenylate cyclase (forskolin), or activate G protein (choleragen) stimulated 125I-IL-1 binding and IL-1R mRNA accumulation in Balb/c3T3 cells. These studies suggest that potentially two signal transduction pathways mediate IL-1 receptor expression in Balb/c3T3 fibroblasts. Evidence is presented that suggests that stimulation of IL-1R through these two pathways (PMA/PDGF-stimulated and cAMP-stimulated) occurs independent of each other.     

Mastoparan transiently permeabilizes Swiss 3T3 cells and induces c-fos proto-oncogene expression. Role of calcium and G protein activation

Mastoparan, a widely used tetradecapeptide activator of Gi/Go G proteins, has been reported to be a potent co-mitogen for Swiss 3T3 fibroblasts. However, we have previously shown that the peptide promotes the release of lactate dehydrogenase from Swiss 3T3 cells and evokes only a modest and delayed increase in DNA. We suggested that the ability of the peptide to permeabilise these cells may account for its mitogenic action. Here we show that mastoparan caused a rapid release of fluorescein from cells which had been pre-incubated with fluorescein diacetate, indicating that the peptide increases membrane permeability to small molecules. Furthermore, the release of lactate dehydrogenase evoked by mastoparan was lost after prolonged (24 h) incubation of cells with the peptide. Together, these data indicate that mastoparan-induced cell permeabilisation is both rapid and transient. We have also shown that mastoparan increased c-fos mRNA accumulation and that this response was not influenced by pertussis toxin or indomethacin. Although mastoparan increased the intracellular calcium concentration, the removal of extracellular calcium had no effect on mastoparan stimulated c-fos mRNA accumulation. These data show that mastoparan-induced c-fos mRNA accumulation is not mediated by activation of a G protein and subsequent activation of phospholipase D nor by a non-selective increase in calcium influx. The data have significance for the interpretation of studies in which mastoparan is, or has been, used as an activator of Gi/Go.     

Demonstration of functional immaturity of signal transduction pathways in human cord T cells

We examined the nature of cytoplasmic signal transduction pathways in cord blood T cells by stimulating them with tumor promoter (TPA) and calcium ionophore (A23187). Costimulation of T cells with TPA and A23187 induced optimal proliferative responses on Day 2 in cord T cells but on Day 4 in adult T cells. The maximal responses observed in cord T cells were much less than those of adult T cells, whereas the Con A-induced proliferative responses of these cells showed no significant differences. The reduced responses of cord T cells were due to their lower efficiency in activating the cellular events in T cell activation and proliferation phase, because cord T cells have significantly less ability than adult T cells to express IL-2 receptor as well as HLA-DR and produce IL-2 molecules, thereby inducing proliferation. These data show immature characteristics of intracellular signal transduction pathways in cord T cells, which are directly related with the functional immaturity of cord T cells.     

The effects of p21N-ras expression in NIH-3T3 cells upon cyclic AMP metabolism

The effects of overexpression of p21N-ras upon cyclic AMP metabolism have been examined in the inducible T15 cell line. In cells overexpressing the N-ras gene product, beta-adrenergic stimulation of cyclic AMP generation was reduced. The reduction was more pronounced the longer the ras gene was expressed and in chronically transformed cells a reduction in forskolin-stimulated cyclic AMP generation was also observed. The transformed cells exhibited a reduction in beta-adrenergic binding sites, but no change in the apparent EC50 for agonist induced cyclic AMP generation. Treatment of the cells with dibutyryl cyclic AMP induced a dose-dependent inhibition of proliferation, with the transformed cells being more sensitive than the control cells.     

Regulation of pp90rsk phosphorylation and S6 phosphotransferase activity in Swiss 3T3 cells by growth factor-, phorbol ester-, and cyclic AMP-mediated signal transduction.

Somatic cell homologs to the Xenopus laevis S6 protein kinases (referred to collectively as pp90rsk) have recently been identified and partially characterized. Here we examine alterations in pp90rsk phosphorylation and S6 phosphotransferase activity in response to regulators of multiple signal transduction systems: purified growth factors, phorbol ester, changes in cyclic AMP (cAMP) levels, and sodium vanadate. All reagents tested increased pp90rsk serine and threonine phosphorylation, but only those agents that regulate cell proliferation and sodium vanadate activated its S6 kinase activity. In addition to the cAMP-stimulated phosphorylation of pp90rsk, a simple correlation between the extent of growth-regulated pp90rsk phosphorylation and S6 phosphotransferase activity was not observed. Quantitative phosphorylation of pp90rsk continued to increase after its S6 kinase activity began its return towards basal levels. However, a close correlation between the appearance and disappearance of a slow-mobility form of phosphorylated pp90rsk (by electrophoresis) and pp90rsk activity was observed. In addition, pp90rsk was regulated by both protein kinase C-independent and -dependent signaling mechanisms. The extent of protein kinase C participation, however, varied depending on which growth factor receptor was activated. Furthermore, growth factor-specific differences in the temporal regulation of pp90rsk S6 phosphotransferase activity were also observed. These results support the notion that the complex regulation of the rsk gene product constitutes one of the primary responses of animal cells to mitogenic signals.     

Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts.

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.