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.     

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.     

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.     

Interleukin 1 stimulates prostaglandin synthesis and cyclic AMP accumulation in Swiss 3T3 fibroblasts: interactions between two second messenger systems

Biochemical events elicited by interleukin 1 (IL-1) were studied in Swiss 3T3 fibroblasts. One hour after its addition, IL-1 stimulated synthesis of prostaglandin E2 (PGE2), which continued to accumulate for 4 days. IL-1 also stimulated cAMP accumulation. Indomethacin blocked cAMP accumulation in response to IL-1, suggesting that PGE2 was responsible for the increase. Addition of exogenous PGE2 to indomethacin-treated cells restored cAMP accumulation. IL-1 enhanced thymidine incorporation, and indomethacin attenuated responses to lower concentrations. Thus, PGE2 appeared to play a role in the ability of low concentrations of IL-1 to stimulate thymidine incorporation. PGE2 augmented thymidine incorporation by increasing cAMP accumulation because in the presence of indomethacin addition of exogenous cAMP enhanced thymidine incorporation in response to low concentrations of IL-1. Elevated cAMP further stimulated PGE2 synthesis. Thus, PGE2 and cAMP interacted to potentiate their mutual accumulation. In summary, IL-1 stimulates PGE2 synthesis. PGE2, in turn, stimulates cAMP accumulation which potentiates IL-1-stimulated PGE2 synthesis and thymidine incorporation.     

Bombesin enhancement of cAMP accumulation in Swiss 3T3 cells: evidence of a dual mechanism of action

Addition of bombesin in the presence of either forskolin or cholera toxin caused a marked (4-6 fold) enhancement of cAMP accumulation in Swiss 3T3 cells. This effect was time and concentration dependent, induced by various bombesin-like peptides and blocked by a bombesin antagonist. Enhancement of cAMP accumulation by bombesin was diminished by chronic pretreatment with phorbol dibutyrate implicating the involvement of protein kinase C in the activation. Pretreatment with pertussis toxin, which uncouples protein kinase C activation from cAMP accumulation (Proc. Natl. Acad. Sci. U.S.A., 84:2282, 1987) also inhibited bombesin enhancement of cAMP. Bombesin was also shown to release E type prostaglandins into the medium, an effect which was abolished by the cyclooxygenase inhibitor indomethacin. Low concentrations (100 nM) of indomethacin partially inhibited the accumulation of cAMP by bombesin in the presence of forskolin indicating that the release of E type prostaglandins into the medium is also involved in the accumulation of cAMP by bombesin. The additive nature of PBt2-mediated down-regulation and treatment with indomethacin suggests that activation of protein kinase C and the release of E type prostaglandins provide two distinct pathways involved in the enhancement of cAMP accumulation by bombesin. Finally, bombesin in the presence of forskolin stimulated the phosphorylation of the intermediate filament component vimentin, identified in the accompanying paper as a substrate for a cAMP dependent protein kinase in intact Swiss 3T3 cells.     

Possible involvement of cyclic AMP and calcium ion in prostaglandin E1-induced elevation of c-myc mRNA levels in Swiss 3T3 fibroblasts

Prostaglandin E1 (PGE1) caused a rapid and dose-dependent increase in cAMP levels, followed by elevation of c-myc mRNA levels and then increased DNA synthesis in quiescent cultures of Swiss 3T3 fibroblasts. The dose-response curves of PGE1 were nearly the same for each of these three processes. Both 8-bromo-cAMP and forskolin increased c-myc mRNA levels to 40-50% and DNA synthesis to 70-80% of those caused by a maximally effective dose of PGE1. Under the comparable conditions, PGE1 did not stimulate diacylglycerol formation or activate protein kinase C. However, PGE1 did elevate cytoplasmic free Ca2+ concentration as measured with the fluorescent Ca2+ indicator quin 2. 8-Bromo-cAMP and forskolin were inactive in this capacity. The Ca2+ ionophore A23187 increased the level of c-myc mRNA. Diacylglycerol and Ca2+ mediate the elevation of c-myc mRNA levels which is caused by platelet-derived growth factor and fibroblast growth factor (Kaibuchi, K., Tsuda, T., Kikuchi, A., Tanimoto, T., Yamashita, T., and Takai, Y. (1986) J. Biol. Chem. 261, 1187-1192). In contrast, the present results suggest that both cAMP and Ca2+ are involved in this PGE1-induced response in Swiss 3T3 cells.     

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.     

PGE2-induced cyclic AMP accumulation in human CD4+ T cells is strongly enhanced during the CD2-mediated activatory process

The effect of a mitogenic combination of two different anti-CD2 monoclonal antibodies on the PGE2-stimulated and basal cAMP production in human CD4+ T cell clones was investigated. The anti-CD2 stimulation strongly potentiates the PGE2-induced cAMP production while both PMA and A23187 produced a less potent effect. On the opposite the anti-CD2 treatment is without any effect on the basal cAMP level contrasting with a marked increase of intracellular cAMP concentrations with A23187 or the combination of A23187 and PMA. These results suggest that activation of CD4+ human T cells via the CD2 molecule significantly influences the cAMP-related transduction pathway. Although PMA and A23187 also modulate the activity of this pathway, their effect in this model is more likely mediated through an amplification of basal cAMP production.     

Bombesin stimulates the rapid activation of phospholipase A2-catalyzed phosphatidylcholine hydrolysis in Swiss 3T3 cells.

In Swiss 3T3 fibroblasts bombesin stimulated the release of arachidonic acid in a time- and dose-dependent manner. Arachidonate levels were significantly elevated after only a 2-s stimulation with the agonist. Furthermore, by measuring the arachidonate content of cellular phospholipids after cell activation, it was shown that there was selective depletion from phosphatidylcholine over the same time course. The corresponding production of lysophosphatidylcholine suggested the involvement of a phosphatidylcholine-specific phospholipase A2. Initial arachidonic acid release was not dependent on the presence of extracellular calcium, not activated by treatment of the cells with thapsigargin, and was unaffected by down-regulation of protein kinase C activity, or by treatment of the cells with the protein kinase C inhibitor staurosporine. These data strongly suggest that occupation of the bombesin receptor is closely coupled to activation of phospholipase A2 which results in the rapid release of arachidonic acid from phosphatidylcholine.     

Parathyroid hormone and prostaglandin E2 stimulate both inositol phosphates and cyclic AMP accumulation in mouse osteoblast cultures.

Parathyroid hormone (PTH) and prostaglandin E2 (PGE2) are physiological agonists which stimulate bone cells to resorb bone, a process by which the mineralized extracellular bone matrix is dissolved. Bone resorption has a key role in the maintenance of plasma calcium levels. It has been established that both PTH and PGE2 activate adenylate cyclase in osteoblasts, but it is apparent that (1) the two agents have qualitatively different effects on osteoblasts, and (2) the generation of cyclic AMP cannot account for all the effects of PTH on bone cell metabolism. Others have demonstrated that PTH and PGE2 may also elevate intracellular calcium levels, but the mechanism by which this is achieved has not been fully defined. Here we have investigated the effects of PTH on neonatal mouse osteoblasts in culture and shown that physiological concentrations of the hormone (50 nM) caused a small increase (22%) in total inositol phosphates accumulation, with a larger increase (40%) in inositol trisphosphate. We found that this activation occurred at lower concentration than was necessary to activate adenylate cyclase. PGE2 was a more effective activator of inositol phosphates accumulation than PTH, causing up to 300% increase in the total inositol phosphates after 30 min. Both PTH and PGE2 stimulated cyclic AMP accumulation, but the activation of adenylate cyclase by forskolin did not enhance inositol phosphates production. We conclude that both PTH and PGE2 stimulate phosphoinositide turnover in mouse osteoblasts and suggest that this mechanism may contribute to their elevation of intracellular calcium in bone cells.     

Prolonged prostaglandin E1 stimulation of cyclic AMP production in transformed and normal WI-38 fibroblasts

Summary Long-term (48-hr) incubations of either the fibroblast strain WI-48 or its SV40-transformed counterpart, WI-38-VA13-2RA, in growth medium containing 1 μm prostaglandin E₁ (PGE₁) resulted in a sustained production and release of cyclic AMP from the cells into the medium. Despite the steady production, intracellular levels of the nucleotide decreased, reaching steady-state values within 4 hr of the initial exposure to PGE₁. These values were maintained for the remainder of the 48-hr experimental period. The steadystate levels of intracellular cyclic AMP were higher than those observed in unstimulated cells, and cyclic AMP-dependent protein phosphokinase was in a highly activated state as compared to controls. Under these conditions little change in the growth or morphology of either the normal or transformed cells was observed. In contrast, inhibition of growth, apparent cell death, and unusual morphological changes were observed in both normal and transformed cells when high concentrations of either PGE₁ (10 μm) or the phosphodiesterase inhibitor 1-methyl, 3-isobutylxanthine (0.5mm to 2mm) were used, which was indicative of toxic effects of the drugs. It was concluded that cyclic AMP-mediated activation of protein phosphokinase does not completely inhibit growth in WI-38 cells or restore normal growth and morphology to the SV40-transformed cells. This work was supported by Grants AM 13904 and CA 21612 from the National Institutes of Health, Department of Health, Education and Welfare.     

Comparative effects of prostaglandin E2 and prostaglandin E3 on water flow and cyclic AMP in the urinary bladder of the frog, Rana pipiens

Prostaglandins (PGs) modulate osmotic water flow in amphibian urinary bladders. Gas chromatographic analysis of prostaglandin precursors in bladders showed that arachidonic acid represented 13.0 +/- 0.6% and eicosapentaenoic acid 4.3 +/- 0.1% of the total fatty acid content. The effects of PGE2 and PGE3 on basal and arginine vasotocin (AVT) stimulated water flow were compared. Control water flow (1.1 +/- 0.2 mg/min) was increased to 4.6 +/- 0.3 mg/min with AVT (10(-6)M) present. PGE2 (10(-6)M) inhibited both basal and AVT stimulated water flow. In contrast, PGE3 (10(-6)M) stimulated basal water flow and further increased AVT stimulated water flow. Basal adenylate cyclase activity (ACA, 59 +/- 0.3 pmol cyclic AMP/mg protein/10 min) was stimulated by the addition of AVT in the absence or presence of exogenous guanosine 5' triphosphate (GTP, 10(-5)M). Both PGE2 and PGE3 stimulated basal ACA in the absence, but not in the presence of GTP. In the absence of exogenous GTP, PGE2 increased AVT stimulated ACA, whereas PGE3 decreased it. Both prostaglandins inhibited AVT stimulation when GTP was added. The effects of PGE2, PGE3 and AVT on tissue cyclic AMP levels in whole urinary bladders were similar to the effects seen on ACA in the absence of exogenous GTP. The contrasting effects of PGE2 and PGE3 on control water flow appear distinct from their similar effects on ACA. However, PGE2 and PGE3 may regulate AVT stimulation through mechanisms involving cyclic AMP.     

Synergistic stimulation of DNA synthesis by cyclic AMP derivatives and growth factors in mouse 3T3 cells

Addition of the cAMP derivatives butcAMP or 8BrcAMP to quiescent cultures of Swiss 3T3 causes synergistic stimulation of DNAk synthesis with insulin, phorbol esters, vasopressin, epidermal growth factor, or fetal bovine serum (2-5%). In the presence of insulin, 8BrcAMP, and butcAMP stimulate [3H]-thymidine incorporation into acid-precipitable material in a dose-dependent manner. The effect of these agents is specific since 8Br5'AMP, 5'AMP, butyrate, or 8BrcGMP fail to stimulate DNA synthesis under identical experimental conditions. Furthermore, the mitogenic effects of the cAMP derivatives were markedly potentiated by 1-methyl-3-isobutyl xanthine and 4-(3-butoxy-4-methoxy benzyl)-2-imidazolidine, both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. The growth-promoting effects of the cAMP derivatives were demonstrated by [3H]-thymidine incorporation (either by scintillation counting or by autoradiography), by flow cytofluorometric analysis, and by increase in cell number. When quiescent Swiss 3T3 cells were exposed to butcAMP and insulin, DNA synthesis began after a lag of 17h. The result of sequential additions of cAMP derivatives and insulin to quiescent 3T3 cells suggest that these agents must act simultaneously in G0/G1 to stimulate entry into DNA synthesis in these cells. The findings support the proposition that an increase in cellular levels of cAMP (but not cGMP) act sas a mitogenic stimulus for confluent and quiescent Swiss 3T3 cells.