Phorbol myristate acetate stimulates microtubule and 10-nm filament extension and lysosome redistribution in mouse macrophages

Phorbol myristate acetate (PMA) stimulates cell spreading and fluid- phase pinocytosis in mouse peritoneal macrophages. Colchicine (10(-5) M) and cytochalasin B (10(-5) M) abolish PMA stimulated pinocytosis but have little effect on cellular spreading (Phaire-Washington et al., 1980, J. Cell Biol., 86:634-640). We report here that PMA also alters the organization of the cytoskeleton and the distrubution of organelles in these cells. Neither control nor PMA-treated macrophages contain actin cables. PMA-treated resident thioglycolate-elicited macrophages exhibit beneath their substrate-adherent membranes many randomly distributed punctate foci that stain brightly for actin. The appearance and distribution of these actin-containing foci are not altered by colchicine (10(-5) M) or cytochalasin B (10(-5) M). In thioglycolate- elicited macrophages PMA causes the extension and radial organization of microtubules and 10-nm filaments and promotes the movement of secondary lysosomes from their perinuclear location to the peripheral cytoplasm. Depending upon the concentration of PMA used, 45-71% of thioglycolate-elicited macrophages and 32-44% of proteose-peptone- elicited macrophages and numerous lysosomes, radiating from the centrosphere region, arranged linearly along microtubule and 10-nm filament bundles. Colchicine (10(-5) M) and podophyllotoxin (10(-5) M) prevent the radial redistribution of microtubules, 10-nm filaments, and lysosomes in these cells. Cytochalasins B and D (10(-5) M) have no inhibitory effects on these processes. These findings indicate that microtubules and 10-nm filaments respond in a coordinated fashion to PMA and to agents that inhibit microtubule function; they suggest that these cytoskeletal elements regulate the movement and distribution of lysosomes in the macrophage cytoplasm.     

Phorbol myristate acetate induces the secretion of an elastase by populations of resident and elicited mouse peritoneal macrophages

Cultured thioglycollate-elicited mouse peritoneal macrophages secrete an enzyme which hydrolyzes [³H]elastin prepared by NaB³H₄ reduction of bovine ligamentum nuchae elastin. Over a 24 h culture period, 3.5 · 10⁶ thioglycollate-elicited cells secrete sufficient enzyme to solubilize 200–350 μg [³H]elastin in 18 h. Secretion at this rate continues for at least 6 days in culture. Secretion of the enzyme is stimulated 3-fold by exposure of the cultured cells to 10^?7 M phorbol myristate acetate, whereas the parent alcohol 4α-phorbol is inactive in this respect. Enzyme activity is linearly related to the amount of conditioned medium assayed and is linear over incubation times up to 30 h. Unlabelled elastin competitively inhibits the solubilization of [³H]elastin. The solubilization rate is doubled if the substrate is pretreated with sodium dodecyl sulfate, but the rate of solubilization of this pretreated substrate increases with time. Resident peritoneal macrophages secrete barely detectable amounts of elastase, but phorbol myristate acetate (10^?7 M) stimulates its secretion in amounts comparable to those secreted by phorbol myristate acetate-stimulated thioglycollate-elicited cells. Dexamethasone (10^?9 M) inhibits phorbol myristate acetate-induced secretion by 50%, but 10^?6 M indomethacin is without effect. The secreted enzyme has the characteristics of a metalloproteinase.     

Phorbol myristate acetate stimulates RNA and casein synthesis in cultured mouse mammary gland tissues

Prolactin and phorbol myristate acetate (TPA) stimulate the rate of [3H]uridine incorporation in cultured mouse mammary gland explants in a similar fashion. Both the time-courses and magnitude of responses were the same; in addition, maximum stimulatory concentrations of TPA and prolactin elicited a nonadditive response when tested together. Nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, abolished both the TPA and prolactin effects on [3H]uridine incorporation. TPA also effected an enhanced rate of [3H]leucine incorporation into a casein-rich phosphoprotein fraction, but only if the explants were also cultured with spermidine.     

Interferon suppresses pinocytosis but stimulates phagocytosis in mouse peritoneal macrophages: related changes in cytoskeletal organization

Treatment of thioglycolate-elicited macrophages with mouse beta-interferon markedly reduces pinocytosis of horseradish peroxidase and fluorescein isothiocyanate (FITC)-dextran but stimulates phagocytosis of IgG-coated sheep erythrocytes. Experiments with FITC-dextran have revealed that the overall decrease in pinocytosis is due to a nearly complete inhibition of pinocytosis in a large fraction of interferon-treated macrophages. In the remaining cells pinocytosis continues at a rate similar to that in untreated control cells. A considerable reduction in the number of cells pinocytosing FITC-dextran was observed within 12 h from the beginning of interferon treatment. Measurement of the overall level of pinocytic activity with horseradish peroxidase showed a progressive decline through 72 h of treatment. In the interferon-sensitive subpopulation, there were marked changes in cytoskeletal organization. Microtubules and 10-nm filaments were aggregated in the perinuclear region while most of the peripheral cytoplasm became devoid of these cytoskeletal structures as observed by fluorescence and electron microscopy. In addition, interferon treatment of macrophages appeared to disrupt the close topological association between bundles of 10-nm filaments and organelles such as mitochondria, lysosomes, and elements of the Golgi apparatus and endoplasmic reticulum. Such alterations in the distribution of microtubules and 10-nm filaments were not seen in the interferon-insensitive subpopulation. We have investigated the mechanism of the interferon-induced enhancement of phagocytic activity by binding IgG-coated sheep erythrocytes to mouse peritoneal macrophages at 4 degrees C and then initiating a synchronous round of ingestion by warming the cells to 37 degrees C. Thioglycolate-elicited macrophages that had been treated with mouse beta-interferon ingested IgG-coated erythrocytes faster and to a higher level than control cells in a single round of phagocytosis. In interferon-treated cultures, phagocytic cups became evident within 30 s of the shift of cultures from 4 degrees to 37 degrees C, whereas in control cultures, they appeared in 2 min. Cytochalasin D, an inhibitor of actin assembly and polymerization, abolished phagocytic activity in both control and beta-interferon-treated macrophages. However, to inhibit phagocytosis completely in thioglycolate-elicited interferon-treated macrophages, twice as much cytochalasin D was required in the treated as in control cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phorbol myristate acetate induced neutrophil autotoxicity

The viability of neutrophils in the condition under which they kill neoplastic cells was studied. In the presence of phorbol myristate acetate (PMA) the ⁵¹Cr-release by human neutrophils was markedly stimulated. The PMA-induced ⁵¹Cr-release by neutrophils correlated well with the number of nonviable neutrophils as determined by the uptake of trypan blue. Phorbol myristate acetate had no effect on the ⁵¹Cr-release by lymphocytes, LPC-1 myeloma cells, ovarian ascites tumor cells, or neutrophils from a patient with chronic granulomatous disease. This suggests that the effect of PMA is not due to its nonspecific toxic effect; instead, it is dependent on the reactive oxygen species produced by the normal neutrophils. Catalase, cytochrome C, histidine, and methionine inhibited the PMA-induced ⁵¹Cr-release by human neutrophils, whereas superoxide dismutase, myeloperoxidase inhibitors, and some hydroxyl radical scavengers or singlet oxygen quenchers had no effect. The clumping of neutrophils induced by PMA was also important in the PMA-induced ⁵¹Cr-release by human neutrophils.     

Phorbol myristate acetate receptors in human polymorphonuclear neutrophils

Resting or phorbol myristate acetate (PMA)-pretreated neutrophils were disrupted by nitrogen cavitation and were fractionated on Percoll density gradients to identify the subcellular location of PMA receptors. Receptors were found in the cytoplasm of resting cells; neither primary nor secondary granules bound [3H]PMA, and the few binding sites located in non-granule membrane fractions appeared to reflect cytosolic contamination. Contrastingly, PMA-pretreated cells lost cytosolic receptors; greater than 80% of PMA-binding sites were associated with non-granule membranes. Protein kinase C activity similarly shifted from cytosol to membranes after PMA treatment. Indeed, protein kinase C and PMA receptors co-sedimented on Percoll gradients, co-eluted from Ultragel AcA 44 columns loaded with neutrophil cytoplasm, and were identically influenced by various phospholipids. Finally, PMA, mezerein, diacylglycerol, and dialkylglycerol activated protein kinase C with potencies that paralleled their respective abilities to stimulate neutrophil aggregation responses and inhibit [3H]PMA binding to whole cells or cytosol. These results fit a model of stimulus-response coupling wherein exogenous PMA or endogenous diacylglycerol solvate in cellular membranes. Cytosolic protein kinase C binds to the intramembranous ligand, forming an active, membrane-associated complex that phosphorylates nearby elements involved in triggering aggregation and other responses.     

Phorbol myristate acetate inhibits increases in membrane fluidity induced by anti-IgM in B cells

Anti-IgM or anti-IgD stimulates B cells to induce increases in inositol phospholipid metabolism and intracellular free calcium concentration [( Ca2+]i). Anti-IgM also causes increases in membrane fluidity that occur more promptly than those in [Ca2+]i in resting B cells as well as BAL17 B lymphoma cells. However, other B cell activators such as LPS or PMA did not induce the membrane fluidity changes. Furthermore, sodium fluoride, which is considered to be an activator of the guanine nucleotide-binding protein, caused increases in membrane fluidity as well as increased [Ca2+]i or inositol phospholipid metabolism. Anti-IgM- or sodium fluoride-induced increases in membrane fluidity were inhibited by 20-min pretreatment of cells with PMA, but not by 24-h pretreatment. These results indicate that membrane fluidity changes are closely associated with increased [Ca2+]i after cross-linkage of membrane Ig and are regulated by protein kinase C in B cells.     

Phorbol myristate acetate activates protein kinase C, stimulates the phosphorylation of endogenous proteins and inhibits phosphate transport in mouse renal tubules

Calcium-activated, phospholipid-dependent protein kinase (protein kinase C) has been implicated in the regulation of transport processes in a variety of tissues and cell lines. To establish whether protein kinase C participates in the regulation of renal phosphate transport, we examined the effect of phorbol myristate acetate (PMA), a potent activator of protein kinase C, on phosphate uptake in fresh preparations of mouse renal tubules, and we correlated the changes in transport activity with protein kinase C activation and phosphorylation of endogenous proteins. PMA inhibited Na+-dependent phosphate transport, elicited a rapid translocation of protein kinase C from the cytosolic to the particulate fraction and stimulated the phosphorylation of endogenous substrates in the cytosolic and brush border membrane fractions. Effects of PMA were maximal after a 10 min incubation of the tubules with the activator. 4 alpha-Phorbol, an inert analogue of PMA, did not elicit any of these effects. The present results demonstrate a temporal correlation between inhibition of Na+-dependent phosphate transport, translocation and activation of protein kinase C, and phosphorylation of endogenous proteins in mouse renal tubules. These data suggest that protein kinase C may play a regulatory role in phosphate transport in mammalian kidney.     

Phorbol myristate acetate stimulation of lymphocyte guanylate cyclase

Human lymphocyte guanylate cyclase activities are increased in a dose-dependent fashion by incubation of intact cells with phorbol myristate acetate, a tumor promoter and lymphocyte mitogen. Increased activity is detectable after 1 minute, and peak membrane-bound and soluble forms of guanylate cyclase occur after 10- and 30-minute exposure to phorbol myristate acetate, respectively. The soluble form is stimulated much more than the membrane form. Enzyme activities measured in the presence of either Ca²⁺, Mg²⁺, or Mn²⁺ are elevated to similar degrees. Comparisons of phorbol and a series of its diesters revealed a good correlation between the capacities for guanylate cyclase stimulation, lymphocyte mitogenesis, and tumor promotion.     

The structure and function of mouse thrombomodulin. Phorbol myristate acetate stimulates degradation and synthesis of thrombomodulin without affecting mRNA levels in hemangioma cells

Thrombomodulin is an endothelial membrane anticoagulant protein that is a cofactor for protein C activation. We have evaluated the expression of thrombomodulin in cultured mouse hemangioma cells before and after treatment with phorbol myristate acetate (PMA), an agent that stimulates protein kinase C. We also isolated a cDNA encoding 481 amino acids of mouse thrombomodulin and the entire 3'-untranslated portion of its mRNA. The deduced amino acid sequence of mouse thrombomodulin is similar to those determined for human and bovine thrombomodulin. An S1 nuclease protection assay was used to measure thrombomodulin mRNA in hemangioma cells. The half-life for thrombomodulin mRNA was 8.9 +/- 1.8 h (S.D.) in cells treated with actinomycin D. Treatment with PMA had no effect on thrombomodulin mRNA levels. Thrombomodulin turnover was evaluated by immunoprecipitation of [35S]methionine-labeled thrombomodulin. The t1/2 was 19.8 +/- 3.9 h (S.D.); PMA treatment decreased the t1/2 to 10.9 +/- 1.1 h (S.D.) while increasing the rate of synthesis to a maximum of 190% of control. Protein C cofactor activity on hemangioma cells was reduced 35 +/- 4% by treatment with PMA within 30 min. This decrease was associated with a parallel decline in cell surface thrombomodulin antigen and with enhanced phosphorylation of thrombomodulin on serine residues. We conclude that thrombomodulin is phosphorylated in response to treatment of hemangioma cells with PMA which leads to decreased protein C cofactor activity and both increased degradation and synthesis of thrombomodulin.     

Phorbol myristate acetate stimulates formation of phosphatidyl inositol 4-phosphate and phosphatidyl inositol 4,5-bisphosphate in human platelets

There are conflicting data in the literature as to whether or not the Ca2+ activation of phospholipase A2 is mediated by the calcium binding protein calmodulin. In the present study the membrane-bound phospholipase A2 enzymes in rat and human platelets were shown to be absolutely Ca2+ dependent but were not stimulated by the addition of calmodulin. A partially purified phospholipase A2 from rat platelet membrane, which contained little endogenous calmodulin, also was not stimulated by calmodulin addition. Both isolated and membrane-bound phospholipase A2 were inhibited by the non-specific calmodulin antagonist trifluoperazine but the inhibition was not overcome by adding calmodulin. There was thus no evidence from these studies that phospholipase A2 is calmodulin regulated.     

Lysophosphatidylcholine stimulates phospholipase D activity in mouse peritoneal macrophages.

Lysophosphatidylcholine (lysoPC) is a bioactive phospholipid that is involved in atherogenesis and inflammatory processes. However, the present understanding of mechanisms whereby lysophosphatidylcholine exerts its pathophysiological actions is incomplete. In the present work, we show that lysoPC stimulates phospholipase D (PLD) activity in mouse peritoneal macrophages. PLD activation leads to the generation of important second messengers such as phosphatidic acid, lysophosphatidic acid, and diacylglycerol, all of which can regulate cellular responses involved in atherogenesis and inflammation. The activation of PLD by lysoPC was attenuated by down-regulation of protein kinase C activity with prolonged incubation with 100 nm of 4beta-phorbol 12-myristate 13-acetate (PMA). Preincubation of the macrophages with the tyrosine kinase inhibitor genistein also decreased the stimulation of PLD by lysoPC, while pretreatment with orthovanadate, which inhibits tyrosine phosphatases, enhanced basal and lysoPC-stimulated PLD activity. The activation of PLD by lysoPC was attenuated by the platelet activating factor (PAF) receptor antagonist WEB-2086, suggesting a role for PAF receptor activation in this process. Furthermore, acetylation of lysoPC substantially increased its potency in activating PLD, suggesting that a cellular metabolite of lysoPC such as 1-acyl 2-acetyl PC might be responsible for at least part of the effect of lysoPC on PLD.     

Phorbol myristate acetate enhances adenylate cyclase activity in human polymorphonuclear leukocytes

The putative role of protein phosphorylation in modulating adenylate cyclase activity in polymorphonuclear neutrophil membranes was assessed using phorbol myristate acetate (PMA) to stimulate the activity of protein kinase C. PMA was demonstrated to enhance the adenylate cyclase activity stimulated by isoproterenol.     

Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages

Lucifer Yellow CH (LY) is an excellent probe for fluid-phase pinocytosis. It accumulates within the macrophage vacuolar system, is not degraded, and is not toxic at concentrations of 6.0 mg/ml. Its uptake is inhibited at 0 degree C. Thioglycollate-elicited mouse peritoneal macrophages were found to exhibit curvilinear uptake kinetics of LY. Upon addition of LY to the medium, there was a brief period of very rapid cellular accumulation of the dye (1,400 ng of LY/mg protein per h at 1 mg/ml LY). This rate of accumulation most closely approximates the rate of fluid influx by pinocytosis. Within 60 min, the rate of LY accumulation slowed to a steady-state rate of 250 ng/mg protein per h which then continued for up to 18 h. Pulse-chase experiments revealed that the reduced rate of accumulation under steady-state conditions was due to efflux of LY. Only 20% of LY taken into the cells was retained; the remainder was released back into the medium. Efflux has two components, rapid and slow; each can be characterized kinetically as a first-order reaction. The kinetics are similar to those described by Besterman et al. (Besterman, J. M., J. A. Airhart, R. C. Woodworth, and R. B. Low, 1981, J. Cell Biol. 91:716-727) who interpret fluid-phase pinocytosis as involving at least two compartments, one small, rapidly turning over compartment and another apparently larger one which fills and empties slowly. To search for processes that control intracellular fluid traffic, we studied pinocytosis after treatment of macrophages with horseradish peroxidase (HRP) or with the tumor promoter phorbol myristate acetate (PMA). HRP, often used as a marker for fluid-phase pinocytosis, was observed to stimulate the rate of LY accumulation in macrophages. PMA caused an immediate four- to sevenfold increase in the rate of LY accumulation. Both HRP and PMA increased LY accumulation by stimulating influx and reducing the percentage of internalized fluid that is rapidly recycled. A greater proportion of endocytosed fluid passes into the slowly emptying compartment (presumed lysosomes). These experiments demonstrate that because of the considerable efflux by cells, measurement of marker accumulation inaccurately estimates the rate of fluid pinocytosis. Moreover, pinocytic flow of water and solutes through cytoplasm is subject to regulation at points beyond the formation of pinosomes.     

Effects of prostaglandins on the spreading, adhesion and migration of mouse peritoneal macrophages

The effects of prostaglandins on the properties of mouse peritoneal macrophages namely spreading, adhesion and migration were investigated. PGE₁ and PGE₂ inhibit the spreading and adhesion of complete Freund's Adjuvant induced peritoneal macrophages significantly at concentrations of 1 ng per ml and above whereas they enhance the migration of these cells at concentrations of 100 ng per ml and above. PGA₂ and PGB₂ are less potent as they inhibit spreading and adhesion only at a concentration of 1 μg per ml. At this concentration PGB₂ enhances migration whereas PGA₂ has no effect. PGF_2α has no effect on the spreading, adhesion and migration of macrophages in the concentration range of 0.1 ng to 1,000 ng per ml.     

Phorbol myristate acetate induces changes on F-actin and vinculin content in immature rat Sertoli cells

Actin and vinculin are two of the most abundant cytoskeletal proteins, widely expressed in nearly all types of eukaryotic cells. It has been well established that long-term exposure to the tumor promoter phorbol myristate acetate (PMA) affects Sertoli cell morphology, as well as F-actin and vinculin organization in vitro. To analyze in a quantitative manner the F-actin and vinculin content of rat immature Sertoli cells in vitro in response to PMA exposure, cytoskeletal fractions were prepared following extraction with Triton X-100. Analysis of the isolated cytoskeletal fractions by immunoblotting showed that exposure of immature rat Sertoli cells to PMA for 8h has an appreciable effect on the cellular level of both the actin and vinculin. Interestingly, as revealed by using calphostin C, a specific protein kinase C inhibitor, the observed F-actin and vinculin changes are most probably mediated by a mechanism that depends on protein kinase activity. A discussion is made concerning PKC modulation by PMA and the subsequent actin and vinculin quantitative changes and reorganization, phenomena that have been closely related to cell transformation.     

Receptor domains in the plasma membrane of cultured mouse peritoneal macrophages

Using the platinum-carbon surface replication technique, the distribution of receptors for two colloidal gold labeled lipoproteins (acetylated low density lipoprotein and high density lipoprotein), iron-saturated transferrin and bovine serum albumin in the plasma membrane of cultured mouse peritoneal macrophages was mapped. The plasma membrane surface of cultured peritoneal macrophages exhibits three morphologically distinct regions which have shown differences in the distribution, density and dynamics of these receptors. The flat border of the plasma membrane surfaces are the domains that appear to have the highest concentration of transferrin receptors, whereas the intermediate regions contain most of the receptors for acetylated low density lipoprotein and high density lipoprotein. Bovine serum albumin receptors are concentrated at the edge of the cells and had a uniform distribution on the rest of the cell surface. The functional significance and the mechanisms by which these regional differences in the distribution of the receptors are generated and then maintained are not yet known.     

Phorbol myristate acetate inhibits growth in S49 cells: isolation of resistant variants

We have used S49 mouse lymphoma cells to study phorbol ester effects on growth. Treatment of wild-type (wt) cells with phorbol 12-myristate 13-acetate (PMA) results in growth arrest within 72 hr. We have selected variants that are resistant to PMA-induced growth arrest, based on a selection in the presence of 10 nM PMA. We have characterized one of these variants, termed 21.1, in detail. The 21.1 and wt cells contain similar levels of protein kinase C (PKC) as determined by [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding. Treatment of both wt and 21.1 cells with PMA results in translocation of PKC to the membrane, suggesting that the coupling between PKC and an immediate biological response is intact. PMA treatment leads to the phosphorylation of many similar proteins in wild-type and 21.1 cells. However, in the 21.1 cells there is a prominent substrate of approximately 70 kilodaltons (kD) which is no longer phosphorylated after PMA treatment. In wild-type cells ornithine decarboxylase (ODC) activity and mRNA levels are decreased within 1 hr of PMA treatment. Likewise, ODC levels are decreased in the 21.1 cells after exposure to PMA even though PMA only slightly modulates the growth of these cells. The 21.1 cells represent a unique line with a dominant phenotype in which ODC expression is uncoupled from the growth state of the cell. These cells may represent a good model system in which to examine the steps involved in phorbol ester growth regulation in S49 cells.