Phorbol ester-induced phosphoinositide hydrolysis in rat aorta: role of cyclooxygenase products

This study investigates whether phorbol esters increase phosphoinositide hydrolysis in intact vascular smooth muscle, and the mechanism underlying the hydrolysis. Phorbol myristate acetate induced time- and concentration-dependent increases in phosphoinositide hydrolysis, as demonstrated by elevated inositol monophosphate levels, in deendothelialized rat aorta. The phorbol ester-elevated inositol monophosphate levels were abolished by indomethacin, a cyclooxygenase inhibitor, but were only partially decreased by SQ29548, a thromboxane A2/prostaglandin H2 receptor antagonist. SQ29548 also only partially decreased elevated inositol monophosphate levels due to prostaglandin E2, prostaglandin F2alpha, prostaglandin I2 and carbacyclin, a stable prostaglandin I2 analog. SQ29548 abolished elevated inositol monophosphate levels due to U46619, a stable thromboxane A2/prostaglandin H2 receptor agonist. These studies demonstrate that phorbol esters increase phosphoinositide hydrolysis in intact vascular smooth muscle, and that the increase is due, at lease in part, to endogenously released prostaglandins other than prostaglandin H2.     

Stimulation of 86Rb+ and 32Pi movements in 3T3 cells by prostaglandins and phorbol esters

The potent tumor promoter tetradecanoyl phorbol acetate (TPA) induces early changes in ion movements analogous to those induced by prostaglandins E1 and F 2alpha. Among the earliest changes induced by TPA is a significant increase in 32Pi incorporation within 15 minutes incubation of TPA (10(-8)-10(-6) M) with post-confluent Swiss 3T3 mouse embryonic fibroblasts. Similarly, the active phorbol ester homolog 4-beta-OH phorbol didecanoate but not the inactive stereoisomeric 4-alpha-OH phorbol didecanoate stimulated 32Pi incorporation. Also, TPA at the above concentrations stimulated 86Rb+ influx shortly after administration. Both fluxes were ouabain-sensitive in accord with the idea that an early effect of TPA is to alter (Na+ + K+)-ATPase activity. Further, prostaglandin E1 (10(-7)-10(-6) M) and prostaglandin F 2alpha (3 X 10(-9)-10(-7) M) caused a similar stimulation of 86Rb+ and 32Pi uptake. The finding that water-soluble prostaglandin F 2alpha also exhibited stimulatory effects indicated that those hormone-induced responses are not mediated by solvent interactions. The similar responses of phorbol esters and prostaglandin derivatives suggests that phorbol esters and prostaglandin derivatives may act at common membrane sites. The finding that stimulatory effects were observed at discrete times in the logarithmic phase of growth suggests that the activation of membrane receptors may be cell-cycle dependent.     

Phorbol esters and diacylglycerols amplify bradykinin-stimulated prostaglandin synthesis in Swiss 3T3 fibroblasts. Possible independence from protein kinase C

When Swiss 3T3 fibroblasts were incubated with bradykinin, prostaglandin E2 (PGE2) synthesis was stimulated. Phorbol esters or the diacylglycerol analog 1-oleoyl-2-acetylglycerol (OAG), by themselves, did not acutely stimulate PGE2 synthesis. However, when cells were preincubated with phorbol esters or OAG, bradykinin-stimulated PGE2 synthesis was potentiated markedly. When phorbol esters and OAG were added together, bradykinin-stimulated PGE2 synthesis was potentiated in an additive manner. When cells were preincubated for 48 h with phorbol esters, then bradykinin added, amplification of bradykinin-stimulated PGE2 synthesis by phorbol ester or OAG was still apparent, even though prolonged pretreatment with phorbol esters abolished protein kinase C (Ca2+/phospholipid-dependent enzyme) activity in cell-free preparations. Further, the protein kinase C antagonist, H-7, only slightly inhibited phorbol ester or OAG amplification of bradykinin-stimulated PGE2 synthesis. The possibility is raised that diacylglycerol, formed in response to many receptors, may serve as a transducer of receptor-receptor interactions. Since desensitization or inhibition of protein kinase C only partially reduced the amplification of bradykinin-stimulated PGE2 synthesis by phorbol esters or OAG, the possibility is raised that diacylglycerol mimetics may have actions in addition to activation of protein kinase C.     

Dexamethasone inhibits mitogen induction of the TIS10 prostaglandin synthase/cyclooxygenase gene.

Glucocorticoids block the induced secretion of prostaglandins in a variety of biological contexts. We have identified a primary response gene, TIS10, which encodes a mitogen-inducible prostaglandin synthase/cyclooxygenase in Swiss 3T3 cells. TIS10 is distinct from prostaglandin synthase/cyclooxygenase. (EC 1.14.99.1), previously cloned from mouse, man, and sheep. Dexamethasone blocks prostaglandin E2 synthesis by 3T3 cells in response to tetradecanoylphorbol acetate. Dexamethasone also blocks both phorbol ester- and forskolin-induced TIS10 mRNA accumulation. In contrast, phorbol esters, forskolin, and dexamethasone have little or no effect on the levels of prostaglandin synthase/cyclooxygenase mRNA in 3T3 cells. Moreover, dexamethasone does not inhibit induction of TIS8/egr-1, another primary response gene. Inhibition of the synthesis of TIS10 prostaglandin synthase/cyclooxygenase may be a principal mechanism by which glucocorticoids block prostaglandin synthesis and secretion.     

Agonist but not phorbol ester induced desensitization of human lymphocyte prostaglandin receptor is dependent on tubulin polymerization

The regulation of prostaglandin stimulated cAMP accumulation in cells of the human T-cell leukemia line Jurkat was examined. Pretreatment with PGE2 (0.1-10 nM) for 2 hour caused a concentration dependent desensitization of the prostaglandin receptor. Tumor promoting phorbol esters (1-1000 nM) could also inhibit PGE2 stimulated cAMP production dose dependently. Inhibition of tubulin polymerization with colchicine or nocodazole (1 microM) eliminated prostaglandin but not phorbol ester induced desensitization of the receptor. It is concluded that agonist and phorbol ester induced desensitization are mediated by two distinct mechanisms and that tubulin polymerization appear to be required only for agonist induced desensitization of the prostaglandin receptor.     

Action of phorbol esters in cell culture: Mimicry of transformation,altered differentiation,and effects on cell membranes

The carcinogenic process is usually multifactor in its causation and multistep in its evolution. It is likely that entirely different molecular mechanisms underlie the many steps in this process. In contrast t o initiating carcinogens, the action of the tumor-promoting phorbol esters does not appear t o involve covalent binding t o cellular DNA and they are not mutagenic. Recent studies in cell culture have revealed two interesting biologic effects of the phorbol esters and related macrocyclic plant diterpenes. The first is that at nanomolar concentrations they induce several changes that resemble those seen in cells transformed by chemical carcinogens or tumor viruses. These include altered morphology and increased saturation density, altered cell surface fucose-glycopeptides, decrease in the LETS protein, increased transport of deoxyglucose, and increased levels of plasminogen activator and ornithine decarboxylase. In transformed cells exposed to phorbol esters the expression of these features is further accentuated. Phorbol esters do not induce normal cells to grow in agar but they do enhance the growth in agar of certain transformed cells. The second effect of the phorbol esters is inhibition of terminal differentiation. This effect extends to a variety of programs of differentiation and is reversible when the agent is removed. With certain cell culture systems induction of differentiation, rather than inhibition, is observed. Both the transformation mimetic and the differentiation effects are exerted by plant diterpenes that have tumor-promoting activity but not by congeners that lack such activity. The primary target of phorbol esters appears to be the cell membrane. Early membrane-related effects include enhanced uptake of 2-deoxyglucose and other nutrients, altered cell adhesion, induction of arachidonic acid release and prostaglandin synthesis, inhibition of the binding of epidermal growth factor t o cell surface receptors, altered lipid metabolism, and modifications in the activities of other cell surface receptors. A model of “two stage” carcinogenesis encompassing the known molecular and cellular effects of initiating carcinogens and tumor promoters is presented. According to this model, initiating carcinogens induce stable alterations in the cellular genome but these are not manifested until tumor promoters modulate programs of gene expression and induce the clonal outgrowth of the initiated cell.     

The regulation of decidual prostaglandin biosynthesis by growth factors, phorbol esters, and calcium

The production of prostaglandins by decidua may play a significant part in the mechanisms of human parturition. We have evaluated the regulation of prostaglandin production in human decidual cells in monolayer culture. Both epidermal growth factor and transforming growth factor-alpha induced concentration-related increases in decidual prostaglandin production with significant changes occurring at concentrations of 1 to 10 ng/ml. Two active phorbol esters elicited concentration-related increases in decidual prostaglandin production whereas an inactive phorbol analogue was without effect. The calcium ionophores, ionomycin and A-23187, also stimulated prostaglandin production by human decidual cell cultures. Hence prostaglandin production by human decidua is regulated by hormones and agents in a manner similar to that of other cells including amnion and may play a significant part in the mechanism of parturition.     

Glucose transport by cultured human fibroblasts: regulation by phorbol esters and insulin.

The regulation of 3-O-methyl-D-glucose (OMG) uptake by insulin and phorbol esters was studied in cultured human skin fibroblasts. Insulin rapidly stimulated OMG uptake through a mechanism independent of new protein synthesis. Maximal insulin effect was reached in 30 min and remained constant up to 12 h. The protein kinase C activators 12-O-tetradecanoyl phorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PdBU) promoted an initial rapid stimulation followed by a secondary long-term rise of OMG influx. This latter effect of phorbol esters on OMG influx began after 1 h, reached a maximum in 12-15 h, and was prevented by the simultaneous addition of protein synthesis inhibitors, suggesting that phorbol esters increased the synthesis of new glucose transporters. In accord with this interpretation, phorbol esters, but not insulin, increased mRNA levels for two distinct glucose transporters (GLUT1 and GLUT3) in human fibroblasts. Both the rapid and the long-term effects of phorbol esters on OMG influx were dose-dependent and half-maximal stimulations occurred at 15 nM for both PdBU and TPA. Kinetic analysis of OMG uptake indicated that both effects of phorbol esters were associated with an increase in the Vmax of the transport process, with no significant changes of the Km (4-6 mM). These results suggest that, in human fibroblasts, phorbol esters, unlike insulin, produce a long-term stimulation of OMG uptake, which is dependent upon protein synthesis and is associated with increased levels of GLUT1 and GLUT3 mRNA.     

Stimulation of prostaglandin E2 production by phorbol esters and epidermal growth factor in porcine thyroid cells

Effects of phorbol esters and epidermal growth factor (EGF) on prostaglandin E2 production by cultured porcine thyroid cells were examined. Both phorbol 12-myristate 13-acetate (PMA) and EGF stimulated prostaglandin E2 production by the cells in dose related fashion. PMA stimulated prostaglandin E2 production over fifty-fold with the dose of 10(-7) M compared with control. EGF (10(-7) M) also stimulated it about ten-fold. The ED50 values of PMA and EGF were respectively around 1 X 10(-9) M and 5 X 10(-10) M. Thyroid stimulating hormone (TSH), however, did not stimulate prostaglandin E2 production from 1 to 24-h incubation. The release of radioactivity from [3H]-arachidonic acid prelabeled cells was also stimulated by PMA and EGF, but not by TSH. These results indicate that both PMA and EGF are potent stimulators of prostaglandin E2 production, associated with the activity to stimulate arachidonic acid release in porcine thyroid cells.     

Activation of protein kinase C is coupled to prostaglandin E2 synthesis in swine granulosa cells

We have examined the role of phospholipid-sensitive calcium-dependent protein kinase (protein kinase C) in prostaglandin E2 synthesis by monolayer cultures of swine granulosa cells. Specific phorbol ester derivatives known to activate protein kinase C significantly augmented the production of prostaglandin E2. These stimulatory actions were dose and time-dependent, and could be abolished by the cyclooxygenase inhibitor, indomethacin, or the protein synthesis inhibitor, cycloheximide. Moreover, the rank order of potency of phorbol esters in enhancing prostaglandin E2 production was concordant with that demonstrated for activation of protein kinase C. Phorbol ester in conjunction with the divalent cation ionophore, A23187, increased prostaglandin E2 production synergistically. In addition, a non-phorbol stimulator of protein kinase C, 1-octanoyl-2-acetylglycerol, also significantly enhanced prostaglandin E2 biosynthesis. The stimulated synthesis of prostaglandin E2 was confirmed by high-pressure liquid chromatographic purification of this radiolabeled metabolite of 3H-arachidonic acid, and by capillary gas chromatography high-resolution mass spectrometry. Thus, the present studies indicate that the protein kinase C effector pathway is functionally coupled to prostaglandin E2 production in the swine granulosa cell.     

Phorbol ester blocks the coupling of GTP-binding protein with receptor-controlled calcium channels

Using the intracellular Ca2+-specific indicator, Quin 2, it was demonstrated that an addition to platelet suspensions of the GTP-binding protein activator, sodium fluoride, stimulates the Ca2+ and Ba2+ influx from the incubation medium into the cytoplasm via receptor-operated Ca2+ channels (Ca-ROC). The fluoride-induced Ca2+ influx is blocked by the protein kinase C activator, phorbol myristate acetate as well as by the platelet adenylate cyclase activator, prostaglandin E1. A two-dimensional electrophoretic analysis of platelet phosphoproteins revealed that the phorbol ester enhances the phosphorylation of proteins with molecular masses of about 20 and 40 kDa. The experimental results suggest that the participation of the GTP-binding protein in the receptor coupling to Ca-ROC. The mechanism of the blocking effect of phorbol esters and prostaglandin E1 on Ca-ROC consists in an impaired coupling of these channels to the GTP-binding protein that activates them.     

Cholera toxin and pertussis toxin regulate the Fc receptor-mediated phagocytic response of human neutrophils in a manner analogous to regulation by monoclonal antibody 1C2

Data presented in this paper indicate that polymorphonuclear leukocyte (PMN) Fc receptor-mediated phagocytosis can be markedly augmented and that this augmentation is under regulatory control. Stimulation of PMN with either a low m.w., heat-labile cytokine(s) (the culture supernatant effluent from a YM-10 Centricon unit, YM-10E), phorbol esters (phorbol dibutyrate), or the polyene antibiotic, amphotericin B, enhances Fc-mediated ingestion in a dose-dependent manner. YM-10 effluent- and amphotericin B-stimulated ingestion is completely abrogated by treating the PMN with either pertussis toxin (PT), cholera toxin (CT), or a monoclonal antibody (mAb), 1C2. However, neither toxin nor mAb 1C2 affects nonstimulated ingestion or phagocytosis stimulated by phorbol esters or synthetic diacylglycerol. Increasing intracellular cyclic adenosine monophosphate levels by stimulation with prostaglandin E1 and the phosphodiesterase inhibitor, isobutylmethylxanthine, does not mimic the effect of either toxin or mAb 1C2. In addition, toxin-mediated inhibition is not due to loss of either the Fc receptor recognized by mAb 3G8 or the antigen recognized by mAb 1C2. These data indicate that both CT and PT regulate the phagocytic response of PMN, in a manner like mAb 1C2, probably by affecting a guanosine 5'-triphosphate-binding protein distinct from those that regulate adenylate cyclase. Since phorbol ester-stimulated ingestion is not inhibited by either PT, CT, or mAb 1C2 and phorbol esters activate protein kinase C directly, phagocytosis amplification regulated by PT, CT, and mAb 1C2 may involve protein kinase C activation.     

Involvement of protein kinase C in prostaglandin D2 synthesis by cultured astrocytes

The role of protein kinase C (PKC) and calcium in the stimulation of prostaglandin D₂ (PGD₂) synthesis was investigated in primary rat astroglial cultures using the phorbol esters phorbol 12-myristate, 13-acetate (PMA), phorbol 12,13-dibutyrate (PDB) and the calcium ionophore A₂₃₁₈₇. Both phorbol esters and the ionophore were able to stimulate PGD₂ synthesis in a concentration dependent manner. The inactive stereoisomers of PMA and PDB had no significant effect. Combinations of subthreshold concentrations of phorbol esters (10 nM PMA or 10 nM PBD) potentiated PG formation induced by 100 nM A₂₃₁₈₇. An even more pronounced effect was observed when phorbol ester concentrations were increased to 100nM. The contribution of extra- and intracellular calcium in phorbol ester or A₂₃₁₈₇ stimulated PGD₂ synthesis was evaluated by carrying out experiments with calcium-free media plus EGTA or with the intracellular calcium-chelating agent TMB-8. Ionophore stimulated PGD₂ release was shut down to basal values upon removal of extracellular calcium, whereas phorbol ester stimulated PGD₂ formation persisted at a reduced level. It was unabated also upon further addition of EGTA. In the presence of TMB-8, however, phorbol ester stimulated PGD₂ synthesis was completely suppressed. These data strongly suggest that PKC has an additional effect on the activation of phospholipase A₂ and subsequent prostanoid synthesis, which is independent from extracellular calcium and, thus, support the concept of more than one metabolic pathway in astrocytes that synergistically regulate phospholipase A₂ activity.     

Keloid fibroblasts are refractory to inhibition of DNA synthesis by phorbol esters. Altered response is accompanied by reduced sensitivity to prostaglandin E2 and altered down-regulation of phorbol ester binding sites.

To investigate abnormal growth regulation in keloid fibroblasts, responses to phorbol esters were examined. Treatment of quiescent cultures with phorbol 12-myristate 13-acetate (PMA) blocked a normally occurring (20-24 h) peak of serum-stimulated thymidine incorporation in normal and keloid cells. In keloid fibroblasts PMA induced a delayed peak of DNA synthesis. When indomethacin was added with PMA the delayed peak appeared in normal fibroblasts. The ED50 for inhibition of the 20-24-h peak was 1 nM, whereas the delayed peak required a 50-fold-higher PMA concentration. In both cell types PMA induced prostaglandin E2 (PGE2) synthesis, and exogenous PGE2 caused 50% inhibition of the 20-24-h peak. When PMA and indomethacin were added with PGE2 the delayed peak was inhibited 90% in normal fibroblasts, whereas inhibition of keloid cells was the same as with PGE2 alone. Normal and keloid fibroblasts had the same number of phorbol ester binding sites. However, in normal cells, phorbol 12,13-dibutyrate bound with greater affinity, and down-regulation of phorbol ester binding occurred to a greater extent. These findings suggest that altered expression of protein kinase C isozymes or another molecule that binds phorbol esters may play a role in abnormal growth regulation of keloid cells.     

Interactive regulation of signalling pathways in bone cells: possible modulation of PGE2-stimulated adenylyl cyclase activity by protein kinase C

We have reported previously that tumour-promoting phorbol esters modulate both basal and vasoactive intestinal polypeptide (VIP)-stimulated adenylyl cyclase activity in GH3 (an established pituitary cell line). Here, we probe the receptor and cell specificity of this response. Experiments were performed in the presence of isobutylmethylxanthine. Unlike the response in GH3 cells, the tumour-promoting phorbol ester (tetradecanoyl phorbol acetate (TPA] did not affect either basal adenylyl cyclase activity nor VIP-stimulated activity in the rat osteosarcoma subclones UMR 106-01 and UMR 106-06. In addition, the cyclase responses to parathyroid hormone (PTH), and, in the case of UMR 106-06, to calcitonin were unaffected by tumour-promoting phorbol ester. However, prostaglandin E2-stimulated cyclase activity in both of these subclones was attenuated in a dose-dependent manner.     

Phorbol esters inhibit the binding of low-density lipoproteins (LDL) to U-937 monocytelike cells

The present study demonstrates that U-937 monocytelike human cells possess specific LDL receptors. 125I-LDL binds at 4 degrees C on the cell surface. The bound molecules are releasable by heparin. The reaction requires Ca2+ and the binding sites are sensitive to proteolysis. Unlabeled LDL compete with 125I-LDL, whereas HDL are ineffective. At 37 degrees C, LDL are internalized and degraded by a chloroquine-sensitive pathway. Tumor-promoting phorbol esters inhibit the binding of 125I-LDL to its receptor on U-937 cells. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, inhibition is 50% at 5 X 10(-9) M of TPA. After removal of phorbol esters, treated cells recover their 125I-LDL-binding activity in 60 min. The inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the number of available LDL receptors rather than a decrease in receptor affinity.     

Tumor-promoting phorbol esters inhibit the binding of colony-stimulating factor (CSF-1) to murine peritoneal exudate macrophages

L-cell colony-stimulating factor (CSF-1) is a sialoglycoprotein of molecular weight 70,000 daltons that specifically stimulates macrophage colony formation by single committed cells from normal mouse bone marrow and by various classes of more differentiated tissue-derived mononuclear phagocyte colony-forming cells (Stanley et al., 1978). CSF-1 interacts with target cells by direct and specific binding to membrane receptors (CSF-1 receptors) that are present only on cells of the mononuclear phagocyte series and their precursors. We studied the effect of tumor-promoting phorbol esters on the binding of 125I-labeled CSF-1 (125I-CSF-1) to murine peritoneal exudate macrophages (PEM). Biologically active TPA (12-O-tetradecanoyl phorbol-13-acetate) inhibits the binding of 125I-CSF-1 to its receptor on PEM. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, maximum inhibition occurred at about 10(-7) M; inhibition was 50% at 5 X 10(-9) M. At 0 degrees C, the inhibitory activity of TPA is diminished. The action of TPA on PEM is transient. Treated cells recover their 125I-CSF-1-binding activity whether TPA is later removed or not. The process of recovering CSF-1-binding activity is completely blocked by the addition of cycloheximide. When several phorbol derivatives were tested for their inhibitory activities, only biologically active phorbol esters were found to possess such activities. Furthermore, the inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the total number of available CSF-1 receptors rather than a decrease in receptor affinity.     

Activation of protein kinase C is coupled to prostaglandin E2 synthesis in swine granulosa cells

We have examined the role of phospholipid-sensitive calcium-dependent protein kinase (protein kinase C) in prostaglandin E₂ synthesis by monolayer cultures of swine granulosa cells. Specific phorbol ester derivatives known to activate protein kinase C significantly augmented the production of prostaglandin E₂. These stimulatory actions were dose and time-dependent, and could be abolished by the cyclooxygenase inhibitor, indomethacin, or the protein synthesis inhibitor, cycloheximide. Moreover, the rank order of potency of phorbol esters in enhancing prostaglandin E₂ production was concordant with that demonstrated for activation of protein kinase C. Phorbol ester in conjunction with the divalent cation ionophore, A23187, increased prostaglandin E₂ production synergistically. In addition, a non-phorbol stimulator of protein kinase C, 1-octanoyl-2-acetylglycerol, also significantly enhanced prostaglandin E₂ biosynthesis. The stimulated synthesis of prostaglandin E₂ was confirmed by high-pressure liquid chromatographic purification of this radiolabeled metabolite of ³H-arachidonic acid, and by capillary gas chromatography high-resolution mass spectrometry. Thus, the present studies indicate that the protein kinase C effector pathway is functionally coupled to prostaglandin E₂ production in the swine granulosa cell.