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The Journal of Biological Chemistry May 1992To investigate abnormal growth regulation in keloid fibroblasts, responses to phorbol esters were examined. Treatment of quiescent cultures with phorbol 12-myristate...
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.
Topics: Binding Sites; Caenorhabditis elegans Proteins; Carrier Proteins; Cells, Cultured; DNA; Dinoprostone; Down-Regulation; Fibroblasts; Humans; Indomethacin; Isoenzymes; Keloid; Phorbol 12,13-Dibutyrate; Protein Kinase C; Receptors, Drug; Thymidine
PubMed: 1577738
DOI: No ID Found -
Scientific Reports Feb 2015We present a system-wide transcriptional network structure that controls cell types in the context of expression pattern transitions that correspond to cell type...
System-wide analysis of the transcriptional network of human myelomonocytic leukemia cells predicts attractor structure and phorbol-ester-induced differentiation and dedifferentiation transitions.
We present a system-wide transcriptional network structure that controls cell types in the context of expression pattern transitions that correspond to cell type transitions. Co-expression based analyses uncovered a system-wide, ladder-like transcription factor cluster structure composed of nearly 1,600 transcription factors in a human transcriptional network. Computer simulations based on a transcriptional regulatory model deduced from the system-wide, ladder-like transcription factor cluster structure reproduced expression pattern transitions when human THP-1 myelomonocytic leukaemia cells cease proliferation and differentiate under phorbol myristate acetate stimulation. The behaviour of MYC, a reprogramming Yamanaka factor that was suggested to be essential for induced pluripotent stem cells during dedifferentiation, could be interpreted based on the transcriptional regulation predicted by the system-wide, ladder-like transcription factor cluster structure. This study introduces a novel system-wide structure to transcriptional networks that provides new insights into network topology.
Topics: Cell Differentiation; Cell Line, Tumor; Cluster Analysis; Computational Biology; Gene Expression Regulation, Leukemic; Gene Regulatory Networks; Humans; Leukemia, Myelomonocytic, Acute; Models, Biological; Phorbol Esters; Transcription, Genetic
PubMed: 25655563
DOI: 10.1038/srep08283 -
The Journal of Neuroscience : the... Oct 2005Abnormalities in dendritic spines have long been associated with cognitive dysfunction and neurodevelopmental delay, whereas rapid changes in spine shape underlie... (Comparative Study)
Comparative Study
Abnormalities in dendritic spines have long been associated with cognitive dysfunction and neurodevelopmental delay, whereas rapid changes in spine shape underlie synaptic plasticity. The key regulators of cytoskeletal reorganization in dendrites and spines are the Rho GTPases, which modify actin polymerization in response to synaptic signaling. Rho GTPase activity is modulated by multiple regulatory proteins, some of which have been found to associate with proteins localized to spines. Here, we show that the nonkinase phorbol ester receptor alpha1-chimerin is present in dendrites and spines, where it binds to the NMDA receptor NR2A subunit in a phorbol ester-dependent manner. Alpha1-chimerin contains a GTPase activating (GAP) domain, with activity toward the Rho family member Rac1. Overexpression of alpha1-chimerin in cultured hippocampal neurons inhibits formation of new spines and removes existing spines. This reduction in spine density is mediated by Rac1 inhibition, because it depends critically on the presence of a functional GAP domain. Conversely, depletion of alpha1-chimerin leads to an increase in spine density, indicating that a basal inhibition of Rac1 maintains the number of spines at a submaximal level. The ability of alpha1-chimerin to modulate spine number requires an interaction with the NMDA receptor, because an alpha1-chimerin mutant that binds weakly to NR2A fails to decrease spine density. Together, these results suggest that alpha1-chimerin is able to modulate dendritic spine morphology by binding to synaptic NMDA receptors and locally inactivating Rac1.
Topics: Animals; Cell Count; Cell Line; Cells, Cultured; Chimerin 1; Dendritic Spines; Humans; Mice; Phorbol Esters; Protein Binding; Protein Subunits; Rats; Receptors, N-Methyl-D-Aspartate
PubMed: 16221859
DOI: 10.1523/JNEUROSCI.2450-05.2005 -
The Journal of Biological Chemistry May 1988Rat brain neuropeptide Y precursor (prepro-NPY) cDNA clones were isolated and sequenced in order to study regulation of the prepro-NPY gene. Rat prepro-NPY (98 amino...
Rat brain neuropeptide Y precursor (prepro-NPY) cDNA clones were isolated and sequenced in order to study regulation of the prepro-NPY gene. Rat prepro-NPY (98 amino acid residues) contains a 36-residue NPY sequence, followed by a proteolysis/amidation site Gly-Lys-Arg, followed by a 30-residue COOH-terminal sequence. The strong evolutionary conservation of rat and human sequences of NPY (100%) and COOH-terminal peptide (93%) suggests that both peptides have important biological functions. In the rat central nervous system, prepro-NPY mRNA (800 bases) is most abundant in the striatum and cortex and moderately abundant in the hippocampus, hypothalamus, and spinal cord. The rat adrenal, spleen, heart, and lung have significant levels of prepro-NPY mRNA. Regulation of the prepro-NPY mRNA abundance was studied in several rodent neural cell lines. PC12 rat pheochromocytoma and N18TG-2 mouse neuroblastoma cells possess low basal levels of prepro-NPY mRNA, while NG108-15 hybrid cells possess high levels. Treatment of PC12 cells with a glucocorticoid such as dexamethasone or elevation of cAMP by forskolin increased the prepro-NPY mRNA level 2-3-fold or 3-10-fold, respectively. In N18TG-2 cells dexamethasone and forskolin synergistically increased prepro-NPY mRNA 7-fold. Treatment of PC12 cells with the protein kinase C activator phorbol 12-myristate 13-acetate alone elevated prepro-NPY mRNA marginally, but the phorbol ester plus forskolin elicited 20-70-fold increases, which were further enhanced to over 200-fold by dexamethasone and the calcium ionophore A23187. These results indicate that NPY gene expression can be positively regulated by synergistic actions of glucocorticoids, cAMP elevation, and protein kinase C activation.
Topics: Animals; Base Sequence; Brain Chemistry; Calcimycin; Cell Line; Chromatography, High Pressure Liquid; Colforsin; Cyclic AMP; Dexamethasone; Gene Expression Regulation; Glucocorticoids; Humans; Molecular Sequence Data; Neuropeptide Y; Phorbol Esters; Protein Precursors; RNA, Messenger; Rats; Tetradecanoylphorbol Acetate; Tissue Distribution; Tumor Cells, Cultured
PubMed: 2834371
DOI: No ID Found -
Journal of Neurochemistry Jan 2006Rapid signal transduction pathways play a prominent role in mediating neuroprotective actions of estrogen in the CNS. We have previously shown that estrogen-induced...
Rapid signal transduction pathways play a prominent role in mediating neuroprotective actions of estrogen in the CNS. We have previously shown that estrogen-induced neuroprotection of primary cerebrocortical neurons from beta-amyloid peptide (Abeta) toxicity depends on activation of protein kinase C (PKC). PKC activation with phorbol-12-myristate-13-acetate (PMA) also provides neuroprotection in this paradigm. Because the PKC family includes several isoforms that have opposing roles in regulating cell survival, we sought to identify which PKC isoforms contribute to neuroprotection induced by PMA and estrogen. We detected protein expression of multiple PKC isoforms in primary neuron cultures, including conventional (alpha, betaI, betaII), novel (delta, epsilon, theta) and atypical (zeta, iota/lambda) PKC. Using a panel of isoform-specific peptide inhibitors and activators, we find that novel and atypical PKC isoforms do not participate in the mechanism of either PMA or estrogen neuroprotection. In contrast, a selective peptide activator of conventional PKC isoforms provides dose-dependent neuroprotection against Abeta toxicity. In addition, peptide inhibitors of conventional, betaI, or betaII PKC isoforms significantly reduce protection afforded by PMA or 17beta-estradiol. Taken together, these data provide evidence that conventional PKC isoforms mediate phorbol ester and estrogen neuroprotection of cultured neurons challenged by Abeta toxicity.
Topics: Amyloid beta-Peptides; Animals; Blotting, Western; Cell Fractionation; Cell Survival; Cells, Cultured; Enzyme Activators; Estradiol; Estrogens; Isoenzymes; Neuroprotective Agents; Phorbol Esters; Protein Kinase C; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate
PubMed: 16336227
DOI: 10.1111/j.1471-4159.2005.03545.x -
The Journal of Biological Chemistry Dec 1986One of the early events associated with the treatment of cells by tumor promotor phorbol esters is the tight association of protein kinase C to the plasma membrane. To...
Factors influencing chelator-stable, detergent-extractable, phorbol diester-induced membrane association of protein kinase C. Differences between Ca2+-induced and phorbol ester-stabilized membrane bindings of protein kinase C.
One of the early events associated with the treatment of cells by tumor promotor phorbol esters is the tight association of protein kinase C to the plasma membrane. To better understand the factors that regulate this process, phorbol ester-induced membrane binding of protein kinase C was studied using homogenates, as well as isolated membranes and purified enzyme. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to the homogenates of parietal yolk sac cells and NIH 3T3 cells in the presence of Ca2+ resulted in plasma membrane binding of protein kinase C which subsequently remained bound to the membrane independent of Ca2+. Although protein kinase C was activated by TPA in the absence of Ca2+ and by diolein in the presence of Ca2+, both these agents when added to homogenates under these respective conditions had no effect on membrane association of protein kinase C. However, under these conditions relatively weak binding of protein kinase C was found if purified protein kinase C was used with isolated membranes. Binding studies using purified protein kinase C and washed membranes showed that the binding of the TPA-kinase complex to membranes required phospholipids and reached saturation at 0.1 unit (24 ng of protein kinase C)/mg of parietal yolk sac cell membrane protein. Phorbol ester treatment of cells in media with and without Ca2+ showed that the TPA-induced increase in membrane-associated protein kinase C was regulated by Ca2+ levels even in intact cells. TPA-stabilized membrane binding of protein kinase C differs in several aspects from the previously reported Ca2+-induced reversible binding. TPA-stabilized binding of protein kinase C to isolated membranes is temperature dependent, relatively high in the plasma membrane-enriched fraction, saturable at physiological levels of protein kinase C, requires the presence of both membrane protein(s) and phospholipids, and further requires the addition of phospholipid micelles. In contrast, Ca2+-induced reversible binding is more rapid, not appreciably influenced by temperature, not selective for a particular subcellular fraction, not saturable with physiological amounts of protein kinase C, exhibits trypsin-insensitive membrane binding sites, and requires membrane phospholipids but not added phospholipid micelles.
Topics: Animals; Calcium; Calcium Chloride; Cell Line; Cell Membrane; Cells, Cultured; Detergents; Diglycerides; Glycerides; Organoids; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phospholipases; Protein Binding; Protein Kinase C; Tetradecanoylphorbol Acetate; Trypsin
PubMed: 3465725
DOI: No ID Found -
The Journal of Cell Biology Jul 1985The Na+/H+ antiport is stimulated by 12-O-tetradecanoylphorbol-13, acetate (TPA) and other phorbol esters in rat thymic lymphocytes. Mediation by protein kinase C is...
The Na+/H+ antiport is stimulated by 12-O-tetradecanoylphorbol-13, acetate (TPA) and other phorbol esters in rat thymic lymphocytes. Mediation by protein kinase C is suggested by three findings: (a) 1-oleoyl-2-acetylglycerol also activated the antiport; (b) trifluoperazine, an inhibitor of protein kinase C, blocked the stimulation of Na+/H+ exchange; and (c) activation of countertransport was accompanied by increased phosphorylation of specific membrane proteins. The Na+/H+ antiport is also activated by osmotic cell shrinking. The time course, extent, and reversibility of the osmotically induced and phorbol ester-induced responses are similar. Moreover, the responses are not additive and they are equally susceptible to inhibition by trifluoperazine, N-ethylmaleimide, and ATP depletion. The extensive analogies between the TPA and osmotically induced effects suggested a common underlying mechanism, possibly activation of a protein kinase. It is conceivable that osmotic shrinkage initiates the following sequence of events: stimulation of protein kinase(s) followed by activation of the Na+/H+ antiport, resulting in cytoplasmic alkalinization. The Na+ taken up through the antiport, together with the HCO3- and Cl- accumulated in the cells as a result of the cytoplasmic alkalinization, would be followed by osmotically obliged water. This series of events could underlie the phenomenon of regulatory volume increase.
Topics: Adenosine Triphosphate; Amiloride; Animals; Calcium; Carrier Proteins; Cytoplasm; Diglycerides; Enzyme Activation; Lymphocytes; Male; Phorbol Esters; Phorbols; Protein Kinase C; Protein Kinases; Rats; Rats, Inbred Strains; Sodium-Hydrogen Exchangers; Trifluoperazine; Water-Electrolyte Balance
PubMed: 2989300
DOI: 10.1083/jcb.101.1.269 -
Journal of Molecular and Cellular... Dec 1993Using a paced Lagendorff-perfused rabbit heart paradigm, we investigated the role of protein kinase C (PKC) in the development of ventricular fibrillation (VF) in hearts...
Using a paced Lagendorff-perfused rabbit heart paradigm, we investigated the role of protein kinase C (PKC) in the development of ventricular fibrillation (VF) in hearts subjected to hypoxia (12 min) and re-oxygenation (40 min). We studied the effect of putative activators and inhibitors of PKC on the incidence of VF. Hearts exposed to 4 beta-phorbol,12,13-dibutyrate (PDBu), isophorbol or the membrane permeant diacylglycerol analog, 1-oleoyl-2-acetyl-rac-glycerol (OAG), during the prehypoxic phase had an increased incidence of VF during the hypoxic and reoxygenation periods. The incidence of VF was 90%, 83% and 75% in hearts exposed to PDBu, isophorbol and OAG, respectively (P < 0.05 vs control). Perfusion of hearts with PDBu was associated with a significant increase in the membrane fraction of cardiac PKC activity. In the presence of the inactive phorbol ester 4 alpha-phorbol didecanoate, the incidence of VF was 17% (P > 0.05 vs control). PKC activators were profibrillatory at concentrations that did not affect cardiac function: neither left ventricular developed pressure nor coronary perfusion pressure were affected. The effect of PDBu was antagonized by staurosporine: the incidence of VF was 17% in PDBu+staurosporine treated hearts (P < 0.05 vs control). To further study the profibrillatory effect of PDBu, hearts were exposed to PDBu in the presence of the ATP-dependent potassium channel antagonist glibenclamide. The latter prevented PDBu-induced VF. The results show that under the conditions employed, PDBu-induced activation of PKC induces redistribution of PKC activity and is associated with the development of VF.
Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Diglycerides; Glyburide; Guanidines; Isoquinolines; Perfusion; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Pinacidil; Piperazines; Protein Kinase C; Rabbits; Staurosporine; Ventricular Fibrillation
PubMed: 8158662
DOI: 10.1006/jmcc.1993.1159 -
The Journal of Physiology Nov 19931. The effects of phorbol esters on evoked and spontaneous excitatory neurotransmission were studied in the CA1 area in the in vitro hippocampal slice preparation of the...
1. The effects of phorbol esters on evoked and spontaneous excitatory neurotransmission were studied in the CA1 area in the in vitro hippocampal slice preparation of the rat. Experiments were conducted using field potential recording and whole-cell voltage clamp of CA1 pyramidal neurons. 2. Pyramidal cells dialysed during whole-cell recording with EGTA-containing electrode solutions, unable to support the induction of long-term potentiation (LTP), still showed robust phorbol ester-induced potentiation of excitatory synaptic transmission. 3. Spontaneous miniature excitatory postsynaptic currents (EPSCs), recorded in whole-cell voltage clamp in the presence of tetrodotoxin and picrotoxin, had amplitudes ranging from 4 to 40 pA and occurred at an average frequency of 0.8-5 Hz. Neither the amplitude nor the frequency of spontaneous EPSCs was altered by cadmium, dihydropyridines, or omega-conotoxin GVIA. 4. The phorbol ester 4-beta-phorbol 12,13-diacetate increased the frequency of spontaneous miniature EPSCs without changing the shape of the EPSC amplitude distribution, suggesting that phorbol esters exert their potentiating effects presynaptically. 5. Blockade of voltage-dependent calcium channels with cadmium attenuated the phorbol-induced increase in spontaneous miniature EPSCs frequency. The phorbol ester-induced increase in miniature EPSC frequency was also attenuated by dihydropyridines, but not by omega-conotoxin GVIA. 6. Unlike spontaneous synaptic currents, stimulus-evoked synaptic currents were reduced by omega-conotoxin but not by nifedipine. 7. We conclude that the phorbol ester increases spontaneous release of glutamate by modulating an L-type channel that does not participate in stimulus-evoked neurotransmitter release.
Topics: Action Potentials; Animals; Cadmium; Calcium; Calcium Channel Blockers; Evoked Potentials; Hippocampus; In Vitro Techniques; Male; Peptides; Phorbol Esters; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Synaptic Transmission; omega-Conotoxins
PubMed: 8120806
DOI: 10.1113/jphysiol.1993.sp019900 -
Journal of Neurochemistry Jan 2009The potentiation of P2X(1) receptor currents by phorbol ester (PMA) treatment and stimulation of mGluR1alpha receptors was sensitive to inhibition of novel forms of...
The potentiation of P2X(1) receptor currents by phorbol ester (PMA) treatment and stimulation of mGluR1alpha receptors was sensitive to inhibition of novel forms of protein kinase C. Potentiation was also reduced by co-expression of an amino terminal P2X(1) receptor minigene. Cysteine point mutants of residues Tyr(16)-Gly(30) were expressed in Xenopus oocytes. Peak current amplitudes to ATP for Y16C, T18C and R20C mutants were reduced, however this did not result from a decrease in surface expression of the channels. The majority of the mutants showed changes in the time-course of desensitization of ATP evoked currents indicating the important role of this region in regulation of channel properties. PMA and mGluR1alpha potentiation was abolished for the mutants Y16C, T18C, R20C, K27C and G30C. Minigenes incorporating either Y16C, K27C, V29C or G30C still inhibited PMA responses. However D17C, T18C or R20C mutant minigenes were no longer effective suggesting that these residues are important for interaction with regulatory factors. These results demonstrate that the conserved YXTXK/R sequence and a region with a conserved glycine residue close to the first transmembrane segment contribute to PMA and GPCR regulation of P2X(1) receptors.
Topics: Adenosine Triphosphate; Animals; Cysteine; Electric Stimulation; Enzyme Inhibitors; Glutamic Acid; Humans; Membrane Potentials; Mutagenesis; Oocytes; Patch-Clamp Techniques; Peptide Fragments; Phorbol Esters; Receptors, Metabotropic Glutamate; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Xenopus laevis
PubMed: 19046321
DOI: 10.1111/j.1471-4159.2008.05761.x