Platelet-derived growth factor-induced c-myc RNA expression. Analysis of an inducible pathway independent of protein kinase C

Platelet-derived growth factor (PDGF) is generally considered to stimulate phosphoinositide turnover resulting in activation of protein kinase C and increased cytoplasmic [Ca2+]. We have examined the role of these secondary effects in regulation of c-myc mRNA accumulation in the MG-63 human osteogenic sarcoma line. Treatment of quiescent cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA) to down-regulate protein kinase C inhibited TPA-stimulated c-myc expression but did not affect the PDGF-modulated process. When cytoplasmic [Ca2+] was increased by addition of a Ca2+ ionophore (A23187 or ionomycin), no stimulation of c-myc RNA was seen; furthermore, these agents did not enhance the PDGF-modulated c-myc expression. Addition of EGTA to cultures treated with both PDGF and a Ca2+ ionophore did not inhibit c-myc induction but rather caused a superinduction of c-myc RNA accumulation. Superinduction occurred only if the [EGTA] was greater than [Ca2+] in the medium. This superinduction was distinct from the increased induction caused by inhibition of protein synthesis. Because PDGF-induced c-myc expression is independent of protein kinase C and increased cytoplasmic [Ca2+], the evidence suggests that PDGF modulates c-myc RNA accumulation in MG-63 cells via a novel pathway, seemingly uncoupled from the classic action of increased phosphoinositide metabolism.     

PDGF induces c-myc mRNA expression in MG-63 human osteosarcoma cells but does not stimulate cell replication

The platelet-derived growth factor (PDGF) modulated growth response of the MG-63 human osteosarcoma cell line, which neither expresses c-sis mRNA nor secretes a PDGF analogue, was characterized. Scatchard analysis demonstrated that the MG-63 cells have 23,000 receptors per cell with a Kd of 5 X 10(-11) M. The receptor became phosphorylated, in a PDGF concentration-dependent manner, when 32P-orthophosphate-labeled cells were treated with PDGF for 3 h at 4 degrees C. The phosphorylated receptor was identified by autoradiography and gel electrophoresis after isolation of the 32P-labeled receptor using a solid-phase monoclonal antibody directed against phosphotyrosine. Binding of the receptor to the antibody was inhibited by 5 mM phenyl phosphate, further suggesting that PDGF stimulated tyrosine-specific receptor autophosphorylation. In addition, treatment of MG-63 cells with PDGF for 3 h at 37 degrees C induced a 7.5-fold increase in c-myc mRNA accumulation as analyzed on Northern gels. However, MG-63 cells grew equally well in either serum-(which contains PDGF) or plasma-(which does not) supplemented medium. Furthermore, PDGF did not stimulate DNA synthesis in growth arrested MG-63 cells, nor did it potentiate DNA synthesis modulated by somatomedin C. Thus MG-63 cells are a naturally occurring cell variant in which PDGF stimulates c-myc expression but does not modulate mitogenesis.     

Phorbol ester prevents the thyroid-stimulating-hormone-induced but not the forskolin-induced decrease of cAMP-dependent protein kinase activity in thyroid cell cultures

The potent tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) affects several thyroid cell functions and interacts with thyroid-stimulating hormone (TSH) either by inhibiting or potentiating its action on different cellular parameters. Since phorbol ester acts mainly through the activation of protein kinase C, which is its receptor, we studied this activation and its interaction with TSH and forskolin in suspension cultures of porcine thyroid cells. In thyroid cell cultures, TPA has a dual effect on protein kinase C activity: immediately (2-5 min) after exposure of cells to TPA, it began to be translocated from the cytosol to the particulate fraction. The transfer of the cytosolic enzyme was total and could occur with or without a loss of activity. The translocated enzyme still needed Ca2+ and phospholipids for its activation. The basal activity increased transiently (2-4 h) in both the cytosol and particulate fractions during translocation. The peak activity in the particulate fraction was reached 10-30 min after exposure of cells to TPA, and was followed by down-regulation of protein kinase C and almost complete disappearance of its activity. The residual activity was about 13% of control after a 2-day exposure to TPA. It was unequally distributed between cytosol (4%) and particulate fraction (9%). Prolonged exposure of cells to TPA did not affect either the activity or the subcellular distribution of the cAMP-dependent protein kinase activity. TPA interacted with TSH and prevented the decrease of this activity induced by prolonged exposure of cells to the hormone not only when it was introduced simultaneously with TSH, but also when it was added 24 h after TSH. However, the forskolin-induced decrease in cAMP-dependent protein kinase activity was not prevented by the presence of TPA. TPA also affected the increases in cAMP accumulation mediated by TSH and forskolin. The TSH-induced increase was significantly stimulated by TPA after short contacts (5-15 min), while longer preincubations of cells with TPA provoked a very strong inhibition of the TSH action. However, the forskolin-induced stimulation of the cAMP accumulation was maintained and even further increased in the presence of TPA. Consequently, the actions of TSH and TPA are apparently interdependent, while those of forskolin and TPA seem to be parallel and independent. Neither TSH nor forskolin prevented the TPA-induced down regulation of protein kinase C. The biologically inactive phorbol ester analogue 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity, and did not interact with either TSH or forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of protein kinase C inhibits prostaglandin- and potentiates adenosine receptor-stimulated accumulation of cyclic AMP in a human T-cell leukemia line

Accumulation of cAMP in the human T-cell leukemia cell line Jurkat was stimulated by the adenosine analogue 5'-N-ethylcarboxamidoadenosine (NECA) and by prostaglandin E2 (PGE2). Addition of two phorbol esters, PDiBu and TPA, markedly enhanced the NECA-stimulated accumulation of cAMP whereas the PGE2-stimulated cAMP accumulation was substantially reduced. The non-tumor-promoting phorbol ester, 4 alpha-PDD, had no effect on either NECA- or PGE2-stimulated cAMP accumulation. The ability of PDiBu to inhibit the effect of PGE2 and to stimulate the effect of NECA remained in the presence a low concentration of forskolin (0.3 microM), which per se increased both NECA- and PGE2-stimulated cAMP accumulation. Our results suggest that the effect of PK-C-activating drugs on receptor-mediated cAMP accumulation is entirely dependent on which receptor is being stimulated.     

Cyclic AMP agonists induce the phosphorylation of phospholipase C-tau and of a 76 kDa protein co-precipitated by anti-(phospholipase C-tau) monoclonal antibodies in BALB/c-3T3 cells. Relationship to inositol phosphate formation.

Previous studies have demonstrated enhanced phosphorylation of phospholipase C-tau (PLC-tau), a key regulatory enzyme in phosphoinositide metabolism, in cells treated with platelet-derived growth factor (PDGF) and epidermal growth factor, both of which act via specific receptor tyrosine kinases. Our studies on BALB/c-3T3 cells show that agents that promote cellular cyclic AMP accumulation also increase the phosphorylation, specifically the serine phosphorylation, of this enzyme. Increased phosphorylation of PLC-t (2-3-fold) was evident within 5-10 min of addition of isobutylmethylxanthine (IBMX) and either cholera toxin or forskolin to cells, and persisted for at least 3 h. Treatment of cells with cyclic AMP agonists also enhanced, with similar kinetics, the phosphorylation of a 76 kDa protein co-precipitated by anti-PLC-tau monoclonal antibodies. Brief exposure of cells to cholera toxin/IBMX or forskolin/IBMX decreased inositol phosphate formation induced by the GTP-binding protein (G-protein) activator aluminium fluoride by approx. 50%, but was without effect on PDGF-stimulated inositol phosphate formation. These findings suggest that PLC-tau, and perhaps the 76 kDa co-precipitated protein, are substrates of cyclic AMP-dependent protein kinase in BALB/c-3T3 cells: however, the lack of effect of cyclic AMP elevation on PDGF-stimulated inositol phosphate formation indicates that the intrinsic activity of PLC-tau is unaltered by cyclic AMP-mediated phosphorylation.     

Inhibitory effects of cAMP and protein kinase C on meiotic maturation and MAP kinase phosphorylation in porcine oocytes

The regulation of MAP kinase phosphorylation by cAMP and protein kinase C (PKC) modulators during pig oocyte maturation was studied by Western immunoblotting. We showed that both forskolin and IBMX inhibited MAP kinase phosphorylation and meiosis resumption in a dose-dependent manner, and this inhibitory effect was overcome by the protein phosphatase inhibitor, okadaic acid. Pharmacological PKC activator phorbol myristate acetate or physiological PKC activator diC8 also delayed MAP kinase phosphorylation and meiosis resumption, and their effect was abrogated by PKC inhibitors, staurosporine, and calphostin C. The results suggest that meiotic resumption is inhibited by elevation of cAMP or delayed by activation of PKC probably via down-regulation of MAP kinase activation, which is mediated by protein phosphatase, during pig oocyte maturation.     

Regulation of protein kinase C by cyclic adenosine 3':5'-monophosphate and a tumor promoter in skeletal myoblasts

The specific activity of protein kinase C in rat skeletal myoblasts decreased when they were exposed for very short periods to isoproterenol, forskolin, dibutyryl cyclic AMP (Bt2cAMP), or the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). In the presence of Bt2cAMP or forskolin only the cytosolic but not the membrane-bound kinase activity was found to decrease. Treatment with TPA, however, led to a decrease in the activity of the enzyme both in the cytosolic as well as the membrane fractions. The effects observed in vivo could be duplicated in crude extracts of myoblasts incubated with cAMP analogues or TPA. In the presence of ATP, protein kinase C activity decreased considerably in crude cytosolic fractions treated with the cAMP analogues, but a requirement for ATP was not evident for the decrease in activity brought about by TPA. For the cAMP analogues the decrease in protein kinase C was also prevented by incubation of the extracts with an inhibitor of cAMP-dependent protein kinase. The regulation of protein kinase C by Bt2cAMP (but not by TPA) was altered in Rous sarcoma virus-transformed myoblasts. It is considered likely that a component affected by cAMP (probably a substrate for cAMP-dependent protein kinase) participates in the regulation of protein kinase C activity, and it is altered in unknown ways in transformed myoblasts.     

Activation of protein kinase C potentiates cyclic AMP production and stimulates steroidogenesis in differentiated ovarian granulosa cells

Gonadotropin-stimulated steroidogenesis in the differentiating ovarian granulosa cell is mediated through the activation of cAMP-dependent protein kinase, and is also modulated by calcium-dependent mechanisms. Granulosa cells contain calcium-activated, phospholipid-dependent protein kinase (C kinase), and show an increase in phosphatidylinositol turnover in response to GnRH agonist analogs. To evaluate the role of C kinase in ovarian steroidogenesis, the potent phorbol ester, TPA, and the permeant diacylglycerol, OAG, were used to activate C kinase in granulosa cells from PMSG-treated immature rats. Both TPA and OAG caused dose-dependent stimulation of progesterone production without affecting intra- or extracellular cAMP levels. However, the maximum steroid responses to these compounds were less than those stimulated by cAMP. The ED50 for TPA-stimulated progesterone production was 3 nM, which is close to the known Km for activation of C kinase. Stimulation of steroidogenesis was only observed with biologically-active phorbol esters and permeant diacylglycerols such as OAG and DOG. Exposure of granulosa cells to phospholipase C also increased progesterone production in a dose-dependent manner without changing the cAMP content. Although TPA and OAG did not increase basal cAMP production, both agents enhanced the cAMP responses stimulated by hCG and forskolin; likewise, phospholipase C alone did not change cAMP production but caused a dose-dependent increase in the cAMP responses to hCG and forskolin. These results demonstrate that activation of C kinase promotes steroidogenesis in ovarian granulosa cells, and potentiates the activation of adenylate cyclase by hCG and forskolin. Such findings support the possibility that the calcium, phospholipid-dependent enzyme could be involved in the regulation of progesterone production by hormonal ligands such as gonadotropins and GnRH.     

Stimulation of protein kinase C or protein kinase A mediated signal transduction pathways shows three modes of response among serum inducible genes

Activation of the signal transduction pathways mediated by protein kinase A (PKA) or protein kinase C (PKC) led to different responses of several serum inducible genes including the jun gene family, c-fos, c-myc, krox 20 and krox 24. Whereas all of these genes were stimulated by the phorbol ester TPA, a chemical activator of protein kinase C, they were differently regulated upon cAMP stimulation of the PKA dependent pathway. The proto-oncogenes jun B, c-fos, and to a lesser extent jun D were stimulated by increasing the intracellular concentration of cAMP, whereas the TPA stimulation of c-jun and c-myc was inhibited under these conditions. Krox 20 and krox 24 were insensitive to this second messenger. This study allowed us to classify these growth stimulated genes into three distinct groups distinguished by their sensitivity to elevated concentrations of intracellular cAMP. The inhibition of c-jun and c-myc expression in the presence of increased cAMP levels may be at least partially responsible for the growth inhibitory effect of this agent in Balb/c-3T3 cells.     

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.     

Downregulation of c-myc RNA is not a prerequisite for reduced cell proliferation, but is associated with G1 arrest in B-lymphoid cell lines

We related the effects of c-myc expression on the ability of growth inhibitors to block the cells in the G0/G1 phase of the cell cycle. In two different B-cell lines, there was an association between the accumulation of cells in the middle to late G1 phase of the cell cycle and a rapid transient downregulation of c-myc mRNA levels. The phorbol ester TPA and the adenylate cyclase activator forskolin reduced the c-myc RNA, levels and after 3 days of treatment a proportion of the cells accumulated in G1. In contrast, neither interferon-gamma, tumor necrosis factor-alpha nor the monoclonal antibody 33-1 against DQ major histocompatibility antigens changed the cell-cycle distribution or regulated the c-myc RNA levels. Yet, all five growth inhibitors reduced the proliferation to approximately the same extent. The growth reduction was not accompanied by definite differentiation, as judged by the absence of the B-cell differentiation marker B1 (CD20).     

Cross-talk regulation between cyclic AMP production and phosphoinositide hydrolysis induced by prostaglandin E2 in osteoblast-like cells.

In cloned osteoblast-like MC3T3-E1 cells, PGE2 stimulated both cAMP accumulation and the formation of inositol trisphosphate (IP3) dose dependently. The cAMP accumulation showed the peak value at 5 min and decreased thereafter, whereas the IP3 formation reached a plateau almost within 10 min and sustained it up to 30 min. The effect of PGE2 on cAMP accumulation (EC50 was 80 nM) was more potent than that on IP3 formation (EC50 was 0.8 microM). 12-O-Tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, reduced the PGE2-induced cAMP accumulation, whereas 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, had little effect on the cAMP accumulation. 1-Oleoyl-2-acetyl-glycerol, a specific activator for PKC, inhibited PGE2-induced cAMP accumulation. TPA had little effect on cAMP accumulation induced by forskolin or NaF, a GTP-binding protein activator. So, the effect of TPA is presumed to be exerted at the point between the PGE2 receptor and Gs. On the other hand, forskolin and dibutyryl cAMP had little effect on the IP3 formation stimulated by PGE2. H-7, a PKC inhibitor, enhanced the PGE2-induced cAMP accumulation in comparison with HA1004, a control for H-7. Our data suggest that PGE2 regulates cAMP production through self-induced activation of PKC. These results strongly suggest that there is an autoregulatory mechanism in PGE2 signaling, and PGE2 modulates osteoblast functions through a cross-talk interaction between cAMP production and phosphoinositide hydrolysis in osteoblast-like cells.     

Pertussis Toxin Enhances Proenkephalin Synthesis in Bovine Chromaffin Cells

Abstract: The synthesis of the neuropeptide precursor proenkephalin was measured in bovine adrenal Chromaffin cells following radiolabeling with [³⁵S]methionine. Treatment of Chromaffin cells with pertussis toxin (100 ng/ml) approximately doubled proenkephalin synthesis without altering total protein synthesis. Pertussis toxin pretreat-ment also increased proenkephalin synthesis in chromaf-fin cells exposed to vasoactive intestinal peptide (VIP) and 3-isobutyl-1 -methylxanthine (IBMX). Combinations of IBMX plus nicotine, VIP, or histamine also synergistically enhanced proenkephalin synthesis, with no further elevation when the cells were also pretreated with pertussis toxin. The action of forskolin, a direct activator of adenyl-ate cyclase, on proenkephalin synthesis was similarly potentiated by pertussis toxin or IBMX, presumably reflecting the abilities of both the toxin and this phosphodiester-ase inhibitor to enhance the cyclic AMP response to forskolin. In contrast, increased synthesis of proenkephalin in response to phorbol esters was not affected by pertussis toxin treatment. These results suggest that pertussis toxin potentiates proenkephalin synthesis primarily through inactivation of guanine nucleotide-binding proteins that inhibit adenylate cyclase, although other signaling pathways may also be involved.     

Enhancement of adenylate cyclase activity in S49 lymphoma cells by phorbol esters. Putative effect of C kinase on alpha s-GTP-catalytic subunit interaction

Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to S49 lymphoma cells (wild type and a cyclic AMP-dependent protein kinase-lacking clone) has little effect alone but doubles accumulation of cyclic AMP in response to isoproterenol. The effect is immediate and has an apparent affinity and order of potency characteristic of the activation of protein kinase C by phorbol esters. Enhancement does not reflect an altered time course of the beta-adrenergic response, enhanced affinity of the cellular beta-receptor for agonist, or decreased degradation and export of cellular cyclic AMP. Reduction of the beta-adrenergic response by somatostatin does not remove the effect of TPA nor does TPA abolish the effect of somatostatin. Phorbol ester enhances cyclic AMP accumulation in response to cholera toxin in wild type and UNC clones but not in H21a or cyc-. TPA also enhances cAMP accumulation in response to forskolin in wild type cells. The effect of TPA is stable to rapid preparation of membranes. In adenylate cyclase assays on membranes from cells treated with TPA, the activation by guanosine 5'-(beta, gamma-imino)triphosphate is enhanced by 40% with no change in lag time; the effect of beta-agonist plus Gpp(NH)p is similarly enhanced; activation by Mn2+ is unchanged. We conclude that phorbol ester facilitates the productive interaction of the alpha subunit of the transducer protein Gs with the catalytic unit of adenylate cyclase, hypothetically via an action of protein kinase C.     

Studies on the Mechanism of Action of Estradiol in Regulating Follicular Progesterone Levels: Effects on cAMP Mediated Events and 3β-Hydroxysteroid Dehydrogenase

Previous studies demonstrated that estradiol interferes with pituitary-induced progesterone production and oocyte maturation in cultured amphibian ( Rana pipiens ) ovarian follicles. To elucidate the mode of action of estradiol in modulating follicular progesterone accumulation we have examined its effects on cAMP-induced progesterone production and enzymatic conversion of pregnenolone to progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD). Follicular cAMP levels were manipulated with forskolin (an adenylate cyclase activator), isobutyl methyl xanthine (IBMX-phosphodiesterase inhibitor) and exogenously added cAMP. Progesterone production induced by forskolin alone or forskolin in combination with frog pituitary homogenate (FPH) was inhibited by estrogen. Addition of estradiol to culture medium markedly inhibited follicular progesterone accumulation following treatment of follicles with cAMP and IBMX. In the presence of exogenous pregnenolone, non-FPH stimulated ovarian follicles effectively converted the 3β-HSD substrate to progesterone. Treatment of follicles with estradiol inhibited conversion of pregnenolone to progesterone. The results indicate that estradiol acts, following FPH stimulation, at one or more steps subsequent to elevation of cAMP levels to regulate intrafollicular progesterone accumulation and oocyte maturation. Estrogen appears to directly influence the enzymatic (3β-HSD) conversion of pregnenolone to progesterone.     

Locus of inhibitory action of cAMP-dependent protein kinase in the antigen receptor-triggered cytotoxic T lymphocyte activation pathway

The mechanism of the cAMP involvement in regulation of cellular functions was studied here using a novel functional assay (antigen receptor-triggered exocytosis of granules) of cloned cytotoxic T lymphocytes (CTL). We suggest that cAMP-dependent protein kinase, protein kinase A, counteracts the protein kinase C and Ca2+-mediated stimulatory T-cell antigen receptor (TcR)-triggered biochemical pathway. This suggestion is supported by experimental results which satisfy criteria for protein kinase A involvement in cellular functions. Pretreatment of CTL with cholera toxin induces cAMP accumulation in CTL, partially inhibits TcR-triggered "lethal hit" delivery to the target cell, and almost completely blocks TcR-triggered exocytosis of granules from CTL. Other agents that raise the intracellular level of cAMP, including forskolin and isobutylmethylxanthine (IBMX) also inhibit TcR-triggered CTL activation. Involvement of cAMP-dependent protein kinase in an inhibitory pathway is suggested by the synergistic effects of cyclic nucleotide analogs 8-bromo-cAMP and N6-benzoyl-cAMP in inhibition of TcR-triggered exocytosis. Forskolin and IBMX inhibited TcR-triggered phosphoinositide turnover in CTL, suggesting that cAMP affected very early events in signal transduction that follow TcR cross-linking by a ligand. The ability of IBMX to inhibit CTL activation when the TcR cross-linking step was by-passed by the combination of phorbol myristate acetate and ionophore A23187 suggests that the locus of inhibitory effect of cAMP is at both the early and late stages of the TcR-triggered transmembrane signaling pathway.