Involvement of two intracellular messenger systems, protein kinase C and cyclic AMP, in the regulation of c-fos gene expression in human promyelocytic leukemia (HL-60) cells

Incubation of human promyelocytic leukemia (HL-60) cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, caused a marked increase in c-fos mRNA in a dose-dependent manner. Phorbol-12,13-dibutyrate and 1-oleoyl-2-acetyl-glycerol, other protein kinase C-activating agents, were also active in this capacity. 4 alpha-Phorbol-12,13-didecanoate, known to be inactive for protein kinase C, was ineffective. 8-Bromo-cyclic AMP (8-Br-cAMP) also increased c-fos mRNA in a dose-dependent manner. This action of 8-Br-cAMP was mimicked by prostaglandin E2, which is known to raise the cyclic AMP level in HL-60 cells. c-fos mRNA increased within 15 min and reached a maximal level 45 min after the stimulation of the cells by TPA or 8-Br-cAMP. The simultaneous stimulation of the cells by TPA and 8-Br-cAMP at the respective doses giving maximal elevation of c-fos mRNA increased this mRNA in an additive manner. These results suggest that in HL-60 cells expression of the c-fos gene is regulated independently by two different intracellular messenger systems, protein kinase C and cyclic AMP.     

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.     

Epidermal growth factor increases c-myc mRNA without eliciting phosphoinositide turnover, protein kinase C activation, or calcium ion mobilization in Swiss 3T3 fibroblasts

Incubation of quiescent cultures of Swiss 3T3 cells with epidermal growth factor (EGF) caused an increase in c-myc mRNA. Under these conditions, EGF did not induce phosphoinositide turnover, formation of diacylglycerol, formation of inositol tris-, bis-, and monophosphates, protein kinase C activation, or Ca2+ mobilization. Although it has been reported that both protein kinase C and Ca2+ may be responsible for the platelet-derived growth factor- and fibroblast growth factor-induced increases in c-myc mRNA in Swiss 3T3 cells (Kaibuchi, K., Tsuda, T., Kikuchi, A., Tanimoto, T., Yamashita, T., & Takai, Y. (1986) J. Biol. Chem. 261, 1187-1192), these results indicate that neither protein kinase C nor Ca2+ is involved in the EGF-induced increase in c-myc mRNA, and that an unidentified system may be involved in this reaction.     

A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts

Endothelin, a novel vasoactive peptide derived from endothelial cells (Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988) Nature 332, 411-415), acts as a potent mitogen in Swiss 3T3 fibroblasts. The effect is dose-dependent with a half-maximal effect obtained at approximately 3 x 10(-11) M and is synergistically enhanced by a low concentration of insulin-like growth factor-I. Endothelin specifically binds to a single class of high affinity receptors in intact Swiss 3T3 cells and stimulates phospholipase C with the production of second messengers inositol trisphosphate and 1,2-diacylglycerol, leading to biphasic increases in the intracellular free Ca2+ concentration, as measured with a fluorescent indicator fura-2, phosphorylation of a putative cellular substrate of 80 kDa for protein kinase C, and transient expression of cellular protoonocogenes, c-fos and c-myc. Mitogenic effect of endothelin is markedly attenuated in phorbol ester-pretreated, protein kinase C-depleted cells. Endothelin-induced inositol phosphates production is not affected by removal of extracellular Ca2+, suggesting that endothelin-induced phospholipase C activation is not the result of stimulation of Ca2+ influx across the plasma membrane. These composite results indicate that the inositol lipid signaling pathway plays an important role in endothelin-induced mitogenesis in Swiss 3T3 fibroblasts. The mitogenic effect of endothelin is considerably smaller than that of bombesin, another well characterized mitogen acting through the inositol lipid pathway, despite comparable potencies in eliciting initial second messenger signals. In endothelin-treated cells, an increase in cellular 1,2-diacylglycerol content is transient, and cellular cyclic AMP content is reduced. By contrast, bombesin induces a more prolonged increase in cellular 1,2-diacylglycerol content and a slight increase in cellular cyclic AMP content. Because both 1,2-diacylglycerol and cyclic AMP are thought to serve as signals for promoting DNA synthesis in Swiss 3T3 fibroblasts, these differences in the signal generation may contribute to the differences in potencies between the two mitogens.     

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.     

Inhibition of prostaglandin E1-induced elevation of cytoplasmic free calcium ion by protein kinase C-activating phorbol esters and diacylglycerol in Swiss 3T3 fibroblasts

In quiescent cultures of Swiss 3T3 cells, prostaglandin E1 (PGE1) known to elevate cAMP increased rapidly cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured with the fluorescent Ca2+ indicator quin2. The primary source of the PGE1-induced elevation of [Ca2+]i was extracellular. Pretreatment of the cells with various doses of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C-activating phorbol ester, inhibited the PGE1-induced elevation of [Ca2+]i in a dose-dependent manner. Inversely, TPA enhanced slightly the PGE1-induced increase of cAMP. TPA alone did not affect the basal level of [Ca2+]i or cAMP in the absence of PGE1. The inhibitory action of TPA on the PGE1-induced elevation of [Ca2+]i was mimicked by other protein kinase C-activating agents such as phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate known to be inactive for protein kinase C was ineffective in this capacity. Prolonged treatment of the cells with phorbol 12,13-dibutyrate resulted in the down-regulation and disappearance of protein kinase C. In these protein kinase C-deficient cells, PGE1 still elevated [Ca2+]i to the same extent as that in the control cells, but TPA did not inhibit the PGE1-induced elevation of [Ca2+]i. These results strongly suggest that protein kinase C serves as an inhibitor for PGE1-induced Ca2+ influx 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 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.     

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.     

4 beta-Phorbol 12-myristate 13-acetate attenuates the glucagon-induced increase in cytoplasmic free Ca2+ concentration in isolated rat hepatocytes.

Hepatocytes were isolated from rats and then loaded with the fluorescent Ca2+ indicator quin2. Glucagon caused a sustained increase (at least 5 min) in the fluorescence of the quin2-loaded cells; the increase was much greater than that observed with control, non-quin2-loaded, cells. These observations indicate that glucagon caused an increase in cytoplasmic free Ca2+ concentration [( Ca2+]c). The effects of glucagon were mimicked if forskolin (to activate adenylate cyclase), dibutyryl cyclic AMP or bromo cyclic AMP were added directly to the cells. Thus an increase in cyclic AMP concentration may mediate the effect of glucagon on [Ca2+]c. If 4 beta-phorbol 12-myristate 13-acetate (PMA; an activator of protein kinase C) was added to the cells before glucagon, the magnitude of the increase in [Ca2+]c was greatly diminished. If PMA was added after glucagon it caused a lowering of [Ca2+]c. These effects of PMA on the glucagon-induced increase in [Ca2+]c could not be mimicked if [Ca2+]c was increased by the Ca2+-ionophore ionomycin. Thus an event involved in the mechanism by which glucagon increases [Ca2+]c appears to be required for the action of PMA. If [Ca2+]c was increased by forskolin, dibutyryl cyclic AMP or bromo cyclic AMP, the effect of PMA on [Ca2+]c was similar to that observed when glucagon was used to elevate [Ca2+]c. When [Ca2+]c was raised by dibutyryl cyclic AMP the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine did not prevent the subsequent addition of PMA from causing [Ca2+]c to decrease. These observations suggest that PMA can inhibit the cyclic AMP-induced increase in [Ca2+]c independently of any changes in cyclic AMP concentration. Glucagon appears to increase [Ca2+]c by releasing intracellular stores of Ca2+ and stimulating net influx of Ca2+ into the cell; PMA greatly diminishes both of these effects.     

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.     

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.     

2-Aminopurine inhibits RNA and protein synthesis and reduces catecholamine desensitization in C6-2B rat glioma cells.

We previously proposed that intracellular cyclic AMP accumulation induces a putative, rapidly turning over protein inhibitory to further hormone activation of adenylate cyclase. In the present study, 2-aminopurine, which has been reported to selectively block c-fos gene expression, was used to test the hypothesis that c-fos protein might be involved in the desensitization to catecholamines was observed in 2-aminopurine-treated C6-2B rat glioma cells. However, we found 2-aminopurine to inhibit, in a concentration-dependent manner, total cellular RNA and protein synthesis in C6-2B, HeLa, Swiss 3T3 and BALB/c cells. mRNA synthesis was also markedly reduced in 2-aminopurine-treated cells. These unexpected findings, while supporting our hypothesis of a protein synthesis-sensitive step in the development of refractoriness, raise concern about the specificity of action of 2-aminopurine to inhibit c-fos induction and thus any cellular process, including desensitization, which might be regulated by c-fos gene expression.     

Tumor promoters enhance basal and growth hormone releasing factor stimulated cyclic AMP levels in anterior pituitary cells

Tumor promoters, such as phorbol esters and teleocidin, amplified the ability of growth hormone releasing factor to increase pituitary cyclic AMP levels. This effect of tumor promoters was concentration-dependent, could be observed in 5 minutes, and was over by 4 hours. Inactive tumor promoters (i.e., 4-alpha-didecanoate) had no effect on this system, whereas a synthetic diacylglycerol (i.e., 1-oleoly-2-acetyl glycerol), mimicked the action of tumor promoters. Due to the known stimulation of protein kinase C by both tumor promoters and diacylglycerols, we suggest that this calcium and phospholipid dependent protein kinase C can enhance the ability of the growth hormone releasing factor receptor to activate the cyclic AMP generating system.     

Regulation of protooncogenes c-fos and c-myc expressions by protein tyrosine kinase, protein kinase C, and cyclic AMP mitogenic pathways in dog primary thyrocytes: a positive and negative control by cyclic AMP on c-myc expression

The proliferation of dog thyrocytes in primary culture is stimulated by three distinct intracellular signaling pathways: (1) the thyrotropin or forskolin-cyclic AMP-mediated cascade which is compatible with the differentiated state of the cell; (2) the protein kinase C pathway activated by diacylglycerol and phorbol esters; and (3) a protein tyrosine kinase system activated by epidermal growth factor. The two latter pathways also induce dedifferentiation. The activation of the three cascades induced the expression of the protooncogenes c-fos and c-myc with dose-response curves similar to those for DNA synthesis. After TPA and EGF, the time courses of stimulation of c-fos and c-myc were the same as those for mitogenically stimulated fibroblasts. However, after the cyclic AMP stimulation, c-myc expression was biphasic with an enhancement at 1 h followed by a down-regulation. A similar inhibition by cyclic AMP was also observed on the increased c-myc expression induced by EGF. This down-regulation is suppressed by cycloheximide, which suggests the involvement of a neosynthesized or a labile protein intermediate. The action of cyclic AMP on c-myc mRNA levels could be related to the opposite requirements of the stimulation of both proliferation and differentiation expression by the cyclic AMP pathway in the differentiated thyrocytes.     

Phosphorylation of an acidic mol. wt. 80 000 cellular protein in a cell-free system and intact Swiss 3T3 cells: a specific marker of protein kinase C activity.

Activation of the endogenous Ca2+-activated phospholipid-dependent protein kinase (protein kinase C) by Ca2+, phosphatidylserine (PS) and phorbol dibutyrate (PBt2) in detergent-solubilized extracts of Swiss 3T3 cells resulted in a very rapid increase (detectable within seconds) in the phosphorylation of an 80 000 mol. wt. protein (termed 80 K). Neither cyclic AMP nor Ca2+ had any effect on 80 K phosphorylation. The 80 K phosphoproteins generated after activation of protein kinase C, both in cell-free conditions and in intact fibroblasts, are identical as judged by one and two-dimensional polyacrylamide slab gel electrophoresis and peptide mapping. Prolonged treatment of cells with phorbol esters causes a selective decrease in protein kinase C activity and prevents the stimulation of 80 K phosphorylation in intact fibroblasts. We now show that extracts from PBt2-treated cultures fail to stimulate 80 K phosphorylation after the addition of the protein kinase C activators. This effect was due to the lack of protein kinase C activity since the addition of exogenous protein kinase C from mouse brain stimulated 80 K phosphorylation in both control and PBt2-treated preparations. The 80 K phosphoprotein generated by activation of endogenous and exogenous protein kinase C yielded similar phosphopeptide fragments after peptide mapping by limited proteolysis. We conclude that the detection of changes in the phosphorylation of 80 K provides a useful approach to ascertain which extracellular ligands activate protein kinase C in intact cells.     

Ca2+ and pH responses to sequential additions of mitogens in single 3T3 fibroblasts: correlations with DNA synthesis

The progression of Swiss 3T3 fibroblasts from the quiescent state (G0) through G1 to DNA synthesis in S phase generally requires the synergistic action of two mitogens. The main aim of this study was to compare systematically the early Ca2+ and pH responses in quiescent cells to all of the pair combinations of eight mitogens (bombesin, platelet-derived growth factor, vasopressin, prostaglandin F2 alpha, epidermal growth factor, 12-O-tetradecanoyl phorbol-13-acetate, insulin, 8-bromo-cAMP) with their subsequent effects on DNA synthesis. Each of the mitogens which caused inositol phosphate accumulation (bombesin, platelet-derived growth factor, vasopressin, prostaglandin F2 alpha) also activated Ca2+- and phospholipid-dependent protein kinase (protein kinase C) and generated both the Ca2+ and pH responses, although epidermal growth factor also generated the ionic responses without causing release of inositol phosphates or activation of protein kinase C. For sequential mitogen additions the ionic signals were measured in single cells as well as in cell populations to avoid ambiguities due to heterogeneity in the responses of the cells to the various mitogens. The modulating effects of the mitogens on the [Ca2+]i responses to subsequent mitogen additions varied widely, but detailed comparisons showed that the pattern of blocking effects could not be attributed solely to the effect of the first mitogen causing either maximal breakdown of phosphatidylinositol 4,5-bisphosphate or complete depletion of the intracellular Ca2+ pool or activation of protein kinase C. From these analyses it was concluded that the requirement for two mitogens for effective DNA synthesis could not be attributed to the summation to a critical threshold of either the ionic signals or phosphatidylinositol 4,5-bisphosphate breakdown, and that these responses are insufficient by themselves to cause the cells to progress to DNA synthesis in S phase.