Phorbol esters inhibit agonist-induced [3H] inositol-1-phosphate accumulation in rat hippocampal slices

In rat hippocampal slices, carbachol and norepinephrine induce an accumulation of [3H]-inositol-1-phosphate which is markedly amplified in the presence of lithium. The tumor-promoting agents phorbol 12,13-dibutyrate (PDB) and 4 beta phorbol, 12 beta-myristate, 13 alpha-acetate (PMA) have no effect on [3H] inositol-1-phosphate accumulation alone, but when preincubated with hippocampal slices significantly inhibit the accumulation of [3H]-inositol-1-phosphate induced by carbachol and norepinephrine. The IC50 values for PDB and PMA are 0.2 microM and 25 microM respectively. In contrast, the weak tumor promoting agents 4-O-methylphorbol 12 myristate 13 acetate (MPMA) and phorbol 13,20-diacetate (P 13,20 DA) only slightly attenuate the agonist-induced response at concentrations less than or equal to 100 microM, whereas 4 alpha-phorbol (4 alpha-PHR), a biologically inactive phorbol, has no effect. These data suggest that phorbol ester receptor-mediated events may be negatively coupled to agonist-induced phosphatidylinositol hydrolysis.     

Stability of phospholipase D in primary astrocytes

Induction of expression and proteolytic breakdown of phospholipase D (PLD) isoforms in primary astrocyte cultures have been investigated. Astrocytes express both PLD1 and 2 and are dependent on PLD activity for cell proliferation [K. K?tter, J. Klein, J. Neurochem. 73 (1999) 2517]. Competitive RT-PCR analysis demonstrated a higher level of PLD1 mRNA than PLD2 mRNA (8.9 vs. 0.9amol/microg RNA, respectively). Treatment of astroglial cultures with the phorbol ester, 4beta-phorbol-12beta,13alpha-dibutyrate (0.1 microM), for 24-48h selectively induced PLD1b but not PLD1a or 2 expression as shown by PCR and Western blot; the effect was sensitive to G? 6976. In cells transiently permeabilized with streptolysin-O, antisense oligonucleotides directed against PLD1 or 2 entered the cytoplasm as shown by immunofluorescence experiments but did not affect astroglial proliferation within 2-6 days. Treatment of the cultures with cycloheximide revealed that PLD1 and 2 proteins had biological half-lives of 2-3 days (PLD2) and 4-6 days (PLD1), respectively. It has been concluded that astroglial PLD1b is up-regulated by phorbol esters via protein kinase C activation. Down-regulation of PLD isoforms is prevented by extended biological half-lives of the PLD proteins.     

Comparison of phorbol ester/calcium ionophore and phytohemagglutinin-induced signaling in human T lymphocytes. Demonstration of interleukin 2-independent transferrin receptor gene expression

The proliferation of human T lymphocytes is regulated, in part, by the coordinated expression of genes encoding T cell growth factor (interleukin 2 (IL-2), IL-2 receptors, and transferrin receptors (TFR). We examined the time course of accumulation of mRNA for these genes in T cells stimulated with the phorbol ester, phorbol 12,13-dibutyrate (PDB) and the calcium ionophore, ionomycin, and compared their expression to T cells stimulated with phytohemagglutinin. In cells treated with PDB/ionomycin, maximum expression was observed at 3 hr for IL-2 mRNA and at 6 hr for TFR mRNA, whereas the level of IL-2 receptor mRNA reached a peak 24 to 48 hr after stimulation. In phytohemagglutinin-stimulated T cells IL-2 mRNA was detectable within 3 hr but peaked later at 12 hr; the level of IL-2 receptor mRNA similarly peaked 24 to 48 hr later. Accumulation of TFR mRNA in phytohemagglutinin-stimulated T cells, however, was not detectable at 6 hr and reached a peak only between 12 to 24 hr. The early accumulation of TFR mRNA in PDB/ionomycin-stimulated T cells seemed, in part, independent of the interaction of IL-2 with its own receptor, because TFR mRNA was detectable as early as 1 hr after stimulation and addition of cycloheximide before addition of PDB/ionomycin did not abolish the PDB/ionomycin-induced accumulation of TFR mRNA. In addition, either PDB or ionomycin used alone induced the expression of TFR mRNA but not IL-2 mRNA. These results indicated that the combination of PDB/ionomycin accelerated the expression of IL-2 and TFR genes in T cells compared to phytohemagglutinin and triggered an IL-2-independent pathway for the induction of TFR mRNA.     

Ca2+ Regulates Hormone Secretion and Proopiomelanocortin Gene Expression in Melanotrope Cells via the Calmodulin and the Protein Kinase C Pathways

The mechanism by which Ca2+ regulates proopiomelanocortin (POMC)-derived peptide secretion and POMC mRNA levels was investigated in primary cultures of porcine intermediate lobe (IL) cells maintained in serum-free medium. POMC gene expression was evaluated by the dot blot hybridization assay with a 32P-labeled DNA probe complementary to the full-length sequence of porcine POMC mRNA. Treatment of IL cells for 24 h with the calmodulin (CAM) antagonists W7 and W13 reduced POMC mRNA levels by a maximum of 50% in a dose-dependent manner (ED50 approximately 10(-8) M). Accumulation of alpha-melanocyte-stimulating hormone (alpha-MSH) in the medium was also depressed by 50% after 8 h of treatment. The role of protein kinase C (PKC) was investigated by depleting the IL cell PKC content with phorbol ester treatment. Phorbol 12-myristate 13-acetate (PMA) at 5 X 10(-8) M induced a rapid translocation of cytoplasmic PKC activity toward the membrane. After 12 h of PMA treatment, PKC activity was undetectable in either the cytoplasmic or the particulate fractions. The same dose of PMA induced a time-dependent decrease in POMC mRNA levels (50% inhibition after 24 h). The same effect was seen with the phorbol ester phorbol 12,13-dibutyrate at 5 X 10(-8) M, whereas the inactive phorbol ester 4 alpha-phorbol at 5 X 10(-8) M was without effect after 24 h of treatment. PMA treatment had a biphasic effect on alpha-MSH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of protein kinase C in the regulation of ornithine decarboxylase mRNA by phorbol esters in rat hepatoma cells

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates a rapid increase in ornithine decarboxylase (EC 4.1.1.17; ODC) activity in target cells. Here we demonstrate that this process involves a rapid accumulation of ODC mRNA, which is maximal 3 h after treatment (three- to eightfold greater than control cells) and decays to control levels within 18 h. Stimulation of ODC mRNA by TPA is blocked by phorbol dibutyrate down-regulation of protein kinase C (PKC). ODC mRNA was also induced by the PKC activators, phospholipase C and 1-oleoyl-2-acetyl-rac-glycerol, and blocked by kinase inhibitors (trifluoroperazine, H7, and palmitoyl-L-carnitine), consistent with a requirement for PKC activation in the induction mechanism. However, the non-PKC-specific protein kinase inhibitor HA1004 also suppressed expression of ODC mRNA in response to TPA, under conditions where it did not inhibit PKC, suggesting that additional kinases may be involved in the intracellular signalling process. The stability of the ODC mRNA (control value = 6.2 +/- 1.6 h) is not significantly changed by either TPA (5.7 +/- 0.8 h) or by cycloheximide (6.0 h). These results are inconsistent with any contribution from altered mRNA half-life towards the accumulation of ODC mRNA following treatment with phorbol ester tumor promoters.     

Protein kinase C inhibits Ca2+ accumulation in cardiac sarcoplasmic reticulum

It is now recognized that phorbol esters are negative inotropic agents in mammalian heart which presumably act via stimulation of Ca2(+)-activated phospholipid-dependent protein kinase (PKC). The goal in the present study was to identify the underlying cellular processes. Digitonin-permeabilized cultured neonatal rat ventricular myocytes were used to study biochemical and functional effects of phorbol esters on cardiac sarcoplasmic reticulum (SR). These cells contracted spontaneously at 3 microM Ca2+. Beating was inhibited by 10 microM ryanodine and was insensitive to 1 microM nifedipine. Thus, beating behavior results from the phasic oscillation of Ca2+ transport by SR in this preparation. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), decreased frequency by 30%, suggesting that Ca2+ transport by SR had been reduced. Whereas cAMP stimulated the rate of oxalate-supported 45Ca2+ uptake 2-fold, phorbol esters, TPA, and phorbol 12,13-dibutyrate inhibited this process by about 45%. The effects of phorbols were specific: (a) the alpha-analogues of TPA and phorbol 12,13-dibutyrate were inactive; and (b) the phorbol esters had no effect on Ca2+ transport in cells that had been depleted of PKC. TPA decreased oxalate-stimulated Ca2+ uptake over the entire range of Ca2+ concentrations, from 0.1 to 10 microM, by at least 70% without shifting the half-maximal effective Ca2+ concentration. Taken together these results indicate that the effects of phorbol ester on cardiac contraction are due to decreased Ca2+ transport by the SR and that these responses are mediated by PKC. These studies support the interpretation that the negative inotropic effects of phorbol esters are due, in part, to decreased SR function.     

Phorbol ester-induced stimulation of prostaglandin biosynthesis in human amnion cells

Both phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (10(-8)-10(-6) M) induced concentration-dependent increases in prostaglandin E2 (PGE2) production by human amnion cells, with maximum stimulations of 10.8-fold and 5.9-fold, respectively. 4 alpha-Phorbol 12,13-didecanoate, an inactive phorbol ester analogue, had little or no effect on PGE2 production by amnion cells. PMA and phorbol 12,13-dibutyrate (10(-7) M) induced a maximal increase in the rate of PGE2 biosynthesis within 15 min of treatment. These results suggest that there is an active protein kinase C present in amnion cells that is linked to arachidonic acid release and/or metabolism.     

Up-regulation of fibroblast growth factor (FGF) receptor mRNA levels by basic FGF or testosterone in androgen-sensitive mouse mammary tumor cells.

Since we had previously shown that both basic fibroblast growth factor (bFGF) and testosterone stimulate the growth of mouse mammary carcinoma cells (SC-3) in serum-free culture, we tested the effect of bFGF or testosterone on FGF receptor mRNA levels. Northern blot analyses revealed that stimulation with bFGF resulted in a 5-fold increase in FGF receptor mRNA levels at 6-8 h followed by a decline to the unstimulated levels at 24 h. Simultaneous addition of cycloheximide blocked bFGF-induced accumulation of FGF receptor mRNA, although exposure of SC-3 cells to cycloheximide alone caused marginal increase in its basal level. Neither phorbol ester nor forskolin stimulated FGF receptor mRNA expression, but testosterone could raise FGF receptor mRNA levels. To obtain the maximum stimulation, however, testosterone required the longer stimulation period (12 h) than bFGF, suggesting that testosterone-induced FGF receptor mRNA accumulation is mediated through an induction of FGF-like growth factor.     

Requirements for the simultaneous presence of phorbol esters and calcium ionophores in the expression of human T lymphocyte proliferation-related genes

We have investigated the synergistic effects of phorbol ester and calcium ionophore on human T lymphocyte proliferation and the expression of the proliferation-related genes, c-myc, c-fos, interleukin 2 receptors (IL-2R) and interleukin 2 (IL-2). Incubation of T lymphocytes with both the phorbol ester, phorbol 12,13-dibutyrate (PDB), and the calcium ionophore, ionomycin, leads to the expression of a series of proliferation-related genes, followed by T cell proliferation. In contrast, stimulation of T cells sequentially with PDB and then ionomycin did not induce mitogenesis, demonstrating that simultaneous exposure to both agents is necessary for proliferation. Exposure of T cells to both agents together for different time periods resulted in a proliferative response in proportion to the duration of the exposure, with more than 6 hr required for maximum proliferation. In contrast, a 1-hr exposure to both drugs was sufficient for maximum expression of c-fos or c-myc proto-oncogene mRNA. The expression of IL-2R and the production of IL-2 were also dependent on the duration of simultaneous exposure to both phorbol ester and calcium ionophore. Levels of IL-2 mRNA became detectable at 1 hr and peaked at 3 hr after stimulation. The induction of IL-2 mRNA occurred only in the presence of both agents and became undetectable within 2 hr after the drugs were removed. In contrast, the expression of IL-2R mRNA became detectable at 1 hr, but was maintained even after the drugs were removed and reached a peak at 24 hr. Both IL-2 and IL-2R mRNA accumulated in proportion to the duration of the exposure. Augmentation of cell proliferation by exogenous IL-2 was observed in T cells exposed to the drugs for less than 3 hr. These data demonstrated that the induction of maximum expression of the nuclear proto-oncogenes c-myc and c-fos was not sufficient for PDB-ionomycin-induced T cell proliferation. The level of IL-2 mRNA accumulation and resultant IL-2 secretion is one of the limiting factors for proliferation of T cells exposed to the drugs for less than 3 hr, but not for longer exposures. Additional events such as accumulation of IL-2R mRNA and protein triggered by a long exposure to the drugs were obligatory for obtaining maximum proliferation.     

Protein kinase C isozyme expression in phorbol ester-sensitive and -resistant EL4 thymoma cells

To investigate whether differential protein kinase C isozyme expression in phorbol ester-sensitive and -resistant EL4 thymoma cells could account for the difference in phorbol ester responsiveness, we purified and characterized isozymes from the two cell lines. In both cell types, two peaks of protein kinase C activity were resolved on hydroxylapatite following DEAE-cellulose and phenyl-Superose chromatography. Western blot analysis showed that the first peak corresponded to protein kinase C-beta and the second to protein kinase C-alpha. Two-dimensional phosphotryptic mapping of the purified alpha and beta isozymes did not reveal any reproducible differences between sensitive and resistant EL4 cells. Nor were any differences between the cell types observed in the cytosolic versus membrane localization of alpha and beta protein kinase C. Northern blot analysis showed the expression of mRNA for protein kinase C-alpha, -beta, -delta and -epsilon in both cell lines, and the absence of mRNA for gamma or zeta. Although no major differences in expression of alpha, beta, or delta mRNA between sensitive and resistant EL4 cells were detectable, expression of protein kinase C-epsilon mRNA in resistant cells was only 20-25% of that in sensitive. Western blot analysis with anti-protein kinase C-epsilon antibodies showed the presence of the epsilon-isozyme in sensitive cells and the absence of detectable amounts in resistant cells. Although protein kinase C-epsilon constitutes only a small portion of the total protein kinase C in sensitive cells, the possibility is raised that decreased protein kinase C-epsilon expression may contribute to the failure of resistant EL4 cells to respond to phorbol esters.     

Two distinct pathways in the down-regulation of type-1 angiotension II receptor gene in rat glomerular mesangial cells.

The mRNA level of the type-1 angiotensin II receptor (AT1) was down-regulated by angiotensin II in cultured rat glomerular mesangial cells. The effect was maximum with 1 microM AII at 6 h, sensitive to cycloheximide, and specific to AT1 since this phenomenon was blocked by DuP753, an AT1 antagonist, but not by type-2 antagonist PD123319. Dibutyryl cAMP, forskolin, and cholera toxin also caused AT1 down-regulation. These effects were not altered by either the protein kinase A inhibitor H-8 or cycloheximide. Calcium ionophore A23187, pertussis toxin, protein kinase C inhibitor staurosporine, or prolonged incubation with phorbol ester were without effect. These results suggest that there are at least two pathways to down-regulate AT1 mRNA; one way is an angiotensin II-induced, protein kinase C-independent, and cycloheximide-sensitive pathway and the other is an angiotensin II-independent, cAMP-induced, and cycloheximide-insensitive pathway.     

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.     

Inhibition of DNA synthesis by protein kinase C-activating phorbol esters in NIH/3T3 cells

Fibroblast growth factor (FGF) plus insulin induced DNA synthesis in and proliferation of NIH/3T3 cells. The protein kinase C-activating phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibited both the DNA synthesis and cell proliferation induced by FGF plus insulin. The concentration of TPA required for 50% inhibition of the DNA synthesis was about 5 nM. Phorbol-12,13-dibutyrate, another protein kinase C-activating phorbol ester, also inhibited the DNA synthesis but 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme, was ineffective. DNA synthesis started at about 12 h after the addition of FGF plus insulin. The inhibitory action of TPA on the DNA synthesis was observed when it was added within 12 h after the addition of FGF plus insulin. These results suggest that phorbol esters exhibit an antiproliferative action through protein kinase C activation in NIH/3T3 cells, and that this action of phorbol esters is due to inhibition of the progression from the late G1 to the S phase of the cell cycle.