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The Biochemical Journal Aug 1985Both protein kinase C and Ca2+ may act in concert to bring about activation of secretion. This study examined the actions on pancreatic acini of ionomycin and phorbol...
Both protein kinase C and Ca2+ may act in concert to bring about activation of secretion. This study examined the actions on pancreatic acini of ionomycin and phorbol dibutyrate, which selectively stimulate one or the other of these pathways; their stimulatory effects were compared with those of receptor agonists, such as carbachol and caerulein, which activate phospholipase C. The Ca2+ ionophore ionomycin produced a dose-dependent increase in amylase secretion and intracellular free Ca2+ (as measured by quin-2). The increase in amylase secretion elicited by carbachol or caerulein was accompanied by a small sustained increase in intracellular free Ca2+, following an initial peak. However, the elevation in intracellular free Ca2+ produced by these receptor agonists for a given level of amylase secretion was less than that observed with ionomycin. Phorbol dibutyrate stimulated amylase secretion by a mechanism that was independent of extracellular Ca2+, and no change in intracellular free Ca2+ was observed. Synergistic stimulatory effects of phorbol dibutyrate and ionomycin were observed, whether the phorbol ester was present before, or in combination with, ionomycin. Diacylglycerols containing unsaturated fatty acids (1,2-dioleoylglycerol and 1,3-dioleoylglycerol) also stimulated amylase secretion and exhibited synergistic effects on secretion with ionomycin. These findings suggest that complete activation of amylase secretion from the pancreas requires stimulation of both Ca2+-dependent and protein kinase C-activated pathways.
Topics: Amylases; Animals; Calcium; Carbachol; Ceruletide; Diglycerides; Dose-Response Relationship, Drug; Drug Synergism; Ethers; Ionomycin; Ionophores; Male; Pancreas; Phorbol 12,13-Dibutyrate; Phorbol Esters; Rats; Rats, Inbred Strains
PubMed: 2413839
DOI: 10.1042/bj2300151 -
Cell Cycle (Georgetown, Tex.) Mar 2022Exocytosis of large dense core vesicles is responsible for hormone secretion in neuroendocrine cells. The population of primed vesicles ready to release upon cell...
Exocytosis of large dense core vesicles is responsible for hormone secretion in neuroendocrine cells. The population of primed vesicles ready to release upon cell excitation demonstrates large heterogeneity. However, there are currently no models that clearly reflect such heterogeneity. Here, we develop a novel model based on single vesicle release events from amperometry recordings of PC12 cells using carbon fiber microelectrode. In this model, releasable vesicles can be grouped into two subpopulations, namely, SP1 and SP2. SP1 vesicles replenish quickly, with kinetics of ~0.0368 s, but likely undergo slow fusion pore expansion (amperometric signals rise at ~2.5 pA/ms), while SP2 vesicles demonstrate slow replenishment (kinetics of ~0.0048 s) but prefer fast dilation of fusion pore, with an amperometric signal rising rate of ~9.1 pA/ms. Phorbol ester enlarges the size of SP2 partially via activation of protein kinase C and conveys SP1 vesicles into SP2. Inhibition of Rho GTPase-dependent actin rearrangement almost completely depletes SP2. We also propose that the phorbol ester-sensitive vesicle subpopulation (SP2) is analogous to the subset of superprimed synaptic vesicles in neurons. This model provides a meticulous description of the architecture of the readily releasable vesicle pool and elucidates the heterogeneity of the vesicle priming mechanism.
Topics: Animals; Dense Core Vesicles; Exocytosis; PC12 Cells; Phorbol Esters; Rats; Synaptic Vesicles
PubMed: 35067177
DOI: 10.1080/15384101.2022.2026576 -
Blood Mar 2009Despite the relevant therapeutic progresses made in these last 2 decades, the prognosis of acute myeloid leukemia (AML) remains poor. Phorbol esters are used at very low...
Despite the relevant therapeutic progresses made in these last 2 decades, the prognosis of acute myeloid leukemia (AML) remains poor. Phorbol esters are used at very low concentrations as differentiating agents in the therapy of myeloid leukemias. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in turn, is a death ligand that spares normal cells and is therefore currently under clinical trials for cancer therapy. Emerging evidence, however, suggests that TRAIL is also involved in nonapoptotic functions, like cell differentiation. PKCepsilon is differentially modulated along normal hematopoiesis, and its levels modulate the response of hematopoietic precursors to TRAIL. Here, we investigated the effects of the combination of phorbol esters (phorbol ester 4-beta-phorbol-12,13-dibutyrate [PDBu]) and TRAIL in the survival/differentiation of AML cells. We demonstrate here that PDBu sensitizes primary AML cells to both the apoptogenic and the differentiative effects of TRAIL via PKCepsilon down-modulation, without affecting TRAIL receptor surface expression. We believe that the use of TRAIL in combination with phorbol esters (or possibly more specific PKCepsilon down-modulators) might represent a significative improvement of our therapeutic arsenal against AML.
Topics: Antineoplastic Agents; Apoptosis; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 3; Caspase 8; Cell Culture Techniques; Cell Differentiation; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Leukemic; Humans; K562 Cells; Leukemia, Myeloid, Acute; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C-epsilon; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured
PubMed: 18988868
DOI: 10.1182/blood-2008-03-143784 -
Biochimica Et Biophysica Acta Oct 2000The roles of PKC in iNOS induction by IFN-gamma have been shown in some cell types. The effect of a PKC activator, phorbol ester, in iNOS induction is thought to be due... (Comparative Study)
Comparative Study
The roles of PKC in iNOS induction by IFN-gamma have been shown in some cell types. The effect of a PKC activator, phorbol ester, in iNOS induction is thought to be due to multiple mechanisms, and it is necessary to examine the involvement of phorbol ester on IFN-gamma-induced iNOS in detail. In the present study, we investigated the mechanisms of phorbol ester on IFN-gamma-induced iNOS in RAW 264.7 cells. PMA synergistically increased iNOS activity, protein and mRNA levels in IFN-gamma-treated RAW 264.7 cells. PMA together with IFN-gamma increased iNOS mRNA without affecting the iNOS mRNA degradation, suggesting that the synergistic effect of PMA on IFN-gamma-induced iNOS mRNA production may depend on the elevation of the transcription rate rather than a prolongation of mRNA stability. The DNA binding proteins that are involved in the regulation of iNOS expression are mainly NF-kappa B and IRF-1. IRF-1 transcriptionally regulates many IFN-inducible genes such as iNOS whose promoter contains an IRF-1 binding site. PMA might modulate iNOS induction as a cosignal with IFN-gamma in RAW 264.7 cells because the synergistic effect of PMA was mediated through IRF-1, rather than NF-kappa B. Ro 31-8220, a PKC inhibitor, decreased iNOS activity, protein, mRNA levels and IRF-1 activity, indicating that the effect of PMA on iNOS induction might occur via the PKC pathway. It is evidence that PKC plays an important role in IRF-1 activation and that phorbol ester has a synergistic effect on iNOS induction through IRF-1 activation in IFN-gamma-treated RAW 264.7 cells. The synergistic effect of PMA on IFN-gamma-induced IRF-1 binding activity was observed in macrophage cell line J774 cells as well as RAW 264.7 cells, but not in thioglycollate-elicited peritoneal macrophages.
Topics: Animals; Cell Line; DNA-Binding Proteins; Drug Synergism; Enzyme Inhibitors; Indoles; Interferon Regulatory Factor-1; Interferon-gamma; Mice; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phorbol Esters; Phosphoproteins; Protein Kinase C; RNA, Messenger
PubMed: 11042347
DOI: 10.1016/s0167-4889(00)00072-0 -
Biochimica Et Biophysica Acta May 1995Objectives were to identify the PKC isoforms in cultured muscle cells, to examine roles of Ca(2+)-dependent proteinases (calpains) in processing of various muscle PKC...
Objectives were to identify the PKC isoforms in cultured muscle cells, to examine roles of Ca(2+)-dependent proteinases (calpains) in processing of various muscle PKC isozymes and to obtain a mechanistic description of the processing of PKCs by examining the temporal relationships between phorbol ester-dependent translocation of muscle PKCs and calpains between cytosolic and membrane compartments. Using six isoform (alpha, beta, gamma, delta, epsilon, zeta)-specific polyclonal antibodies, PKC alpha, delta and zeta were detected in rat skeletal muscle and in L8 myoblasts and myotubes. PKC alpha and zeta were primarily localized in the cytosolic fraction of L8 myotubes whereas PKC delta was more abundant in the membrane fraction. Phorbol ester (TPA) caused rapid depletion of myotube PKC alpha and PKC alpha and PKC delta isoforms from the cytosolic compartment and rapid appearance of these isoforms in the membrane fraction. However, long-term exposure of myotubes to TPA eventually caused down-regulation of PKCs in the membrane compartment. Down-regulation of PKCs in the membrane fraction was partially blocked by calpain inhibitor II. However, the rapid TPA-dependent cytosolic depletion of PKCs was unaffected by calpain inhibitor. This suggests that calpains may be responsible for membrane-associated down-regulation of PKCs but not for cytosolic depletion. In the final study we assessed the effects of phorbol ester on compartmentation of m-calpain with PKCs in muscle cells. Like the PKCs, TPA caused rapid association of m-calpain with the membrane fraction followed by down-regulation. This demonstrates that phorbol esters cause translocation of both PKCs and calpains to membranes where processing of PKCs may occur via the limited proteolysis exerted by calpains.
Topics: Amino Acid Sequence; Animals; Calpain; Cells, Cultured; Down-Regulation; Endopeptidases; Isoenzymes; Molecular Sequence Data; Muscles; Phorbol Esters; Protein Kinase C; Rats; Signal Transduction; Subcellular Fractions
PubMed: 7779868
DOI: 10.1016/0167-4889(95)00024-m -
The Journal of Biological Chemistry Aug 1993Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester....
Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester. Treatment of wild-type EL4 cells with phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in phorbol ester-treated wild-type cells, but not in phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.
Topics: Amino Acid Sequence; Animals; Drug Resistance; Enzyme Activation; Ethers, Cyclic; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase Kinases; Molecular Sequence Data; Okadaic Acid; Phorbol Esters; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Thymoma; Tumor Cells, Cultured
PubMed: 8344897
DOI: No ID Found -
Journal of Experimental & Clinical... Feb 2019The differentiation-based therapy for acute promyelocytic leukemia (APL) is an inspiring example for the search of novel strategies aimed at treatment of other subtypes...
BACKGROUND
The differentiation-based therapy for acute promyelocytic leukemia (APL) is an inspiring example for the search of novel strategies aimed at treatment of other subtypes of acute myeloid leukemia (AML). Thus, the discovery of new molecular players in cell differentiation becomes a paramount research area to achieve this goal. Here, the involvement of the protein tyrosine phosphatases SHP1 and SHP2 on leukemic cells differentiation is shown, along with the therapeutic possibilities of their targeting to enhance the differentiation induction effect of phorbol esters.
METHODS
The oxidation status and enzymatic activity of SHP1 and SHP2 during PMA-induced differentiation of HEL cells was evaluated. Additionally, the effects of RNAi-mediated downregulation of these phosphatases on cell differentiation was studied. Afterwards, the impact of chemical inhibition of SHP1 and SHP2 on differentiation both in the presence and absence of phorbol esters was tested. Finally, the anti-leukemic potential of phorbol esters and chemical inhibitors of SHP1 and SHP2 was addressed in several AML model cell lines, a xenograft mouse model and AML primary cells in vitro.
RESULTS
An increase of oxidation with a concomitant decrease of activity was observed for both phosphatases at the onset of PMA-induced differentiation. Consistently, silencing of these proteins favored the process, with an enhanced effect upon their simultaneous downregulation. Moreover, the proteins SRC and β-catenin were identified as downstream targets of SHP1 and SHP2 in this context. In agreement with these findings, chemical inhibition of the phosphatases promoted cell differentiation itself and enhanced the effect of phorbol esters. Interestingly, treatment with the phorbol ester prostratin and the dual inhibitor of SHP1 and SHP2 NSC87877 synergistically hampered the proliferation of AML cell lines, prolonged the survival of xenografted mice and reduced the clonogenic potential of AML primary cells.
CONCLUSIONS
SHP1 and SHP2 are relevant mediators of differentiation in AML cells and their inhibition either alone or in combination with prostratin seems a promising differentiation-based therapeutic strategy against different subtypes of AML beyond APL.
Topics: Animals; Cell Differentiation; Female; Humans; Leukemia, Myeloid, Acute; Male; Mice, Inbred NOD; Mice, SCID; Phorbol Esters; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Xenograft Model Antitumor Assays
PubMed: 30764849
DOI: 10.1186/s13046-019-1097-z -
The Journal of Biological Chemistry Nov 1988We have examined the effect of phorbol esters and cAMP elevating compounds on tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) secretion....
Cyclic AMP potentiates phorbol ester stimulation of tissue plasminogen activator release and inhibits secretion of plasminogen activator inhibitor-1 from human endothelial cells.
We have examined the effect of phorbol esters and cAMP elevating compounds on tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) secretion. Phorbol esters induce a time- and dose-dependent increase in tPA release from endothelial cells, while forskolin, isobutylmethylxanthine, dibutyryl cAMP, and 8-bromo-cAMP had no significant stimulatory effect on tPA secretion. However, elevation of cAMP simultaneously with phorbol ester treatment potentiated the phorbol ester-induced release of tPA 6 times from 22.2 ng/ml with phorbol myristate acetate (PMA) alone to 122.1 ng/ml (PMA and forskolin). Potentiation was dose-dependent (half-maximal potentiation = 4 microM forskolin), and tPA release was enhanced at all stimulatory concentrations of PMA with no change in the PMA concentrations causing half-maximal or maximum tPA release. The kinetics of release was also similar in PMA versus PMA-forskolin-treated cells. A 4-h delay was observed, enhanced release was transient, and was followed by the onset of a refractory period. In contrast, elevation of cAMP reduced constitutive secretion of PAI-1 by 30-40% and prevented the increase in PAI-1 secretion stimulated by PMA. Elevated cAMP also decreased the rate of PAI-1 deposition into the endothelial substratum. These studies indicate that activation of a cAMP-dependent pathway(s) in coordination with phorbol ester-induced responses plays a central role in modifying the tPA and PAI-1 secretion from endothelial cells, leading to a profibrinolytic state in the endothelial environment.
Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Bucladesine; Cells, Cultured; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Drug Synergism; Endothelium, Vascular; Glycoproteins; Humans; Phorbol Esters; Plasminogen Inactivators; Tetradecanoylphorbol Acetate; Tissue Plasminogen Activator
PubMed: 2460455
DOI: No ID Found -
The Journal of Biological Chemistry May 2003Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A,...
Synthesis and phorbol ester binding of the cysteine-rich domains of diacylglycerol kinase (DGK) isozymes. DGKgamma and DGKbeta are new targets of tumor-promoting phorbol esters.
Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGKgamma-C1A and DGKbeta-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGKgamma-C1A, human-DGKgamma-C1A, and human-DGKbeta-C1A gave K(d) values of 3.6, 2.8, and 14.6 nm, respectively, suggesting that DGKgamma and DGKbeta are new targets of phorbol esters. An A12T mutation of human-DGKbeta-C1A enhanced the affinity to bind PDBu, indicating that the beta-hydroxyl group of Thr-12 significantly contributes to the binding. The K(d) value for PDBu of FLAG-tagged whole rat-DGKgamma (4.4 nm) was nearly equal to that of rat-DGKgamma-C1A (3.6 nm). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGKgamma and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGKgamma to cause its translocation.
Topics: Amino Acid Sequence; Animals; Binding, Competitive; Chromatography, High Pressure Liquid; Cricetinae; Cysteine; Diacylglycerol Kinase; Gene Deletion; Green Fluorescent Proteins; Humans; Kinetics; Luminescent Proteins; Molecular Sequence Data; Mutation; Neoplasms; Peptides; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Binding; Protein Conformation; Protein Folding; Protein Isoforms; Protein Structure, Tertiary; Protein Transport; Rats; Recombinant Fusion Proteins; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Threonine; Time Factors
PubMed: 12621060
DOI: 10.1074/jbc.M300400200 -
The Journal of Biological Chemistry May 2001The novel phorbol ester receptor beta2-chimaerin is a Rac-GAP protein possessing a single copy of the C1 domain, a 50-amino acid motif initially identified in protein...
The novel phorbol ester receptor beta2-chimaerin is a Rac-GAP protein possessing a single copy of the C1 domain, a 50-amino acid motif initially identified in protein kinase C (PKC) isozymes that is involved in phorbol ester and diacylglycerol binding. We have previously shown that, like PKCs, beta2-chimaerin binds phorbol esters with high affinity in a phospholipid-dependent manner (Caloca, M. J., Fernandez, M. N., Lewin, N. E., Ching, D., Modali, R., Blumberg, P. M., and Kazanietz, M. G. (1997) J. Biol. Chem. 272, 26488-26496). In this paper we report that like PKC isozymes, beta2-chimaerin is translocated by phorbol esters from the cytosolic to particulate fraction. Phorbol esters also induce translocation of alpha1 (n)- and beta1-chimaerins, suggesting common regulatory mechanisms for all chimaerin isoforms. The subcellular redistribution of beta2-chimaerin by phorbol esters is entirely dependent on the C1 domain, as revealed by deletional analysis and site-directed mutagenesis. Interestingly, beta2-chimaerin translocates to the Golgi apparatus after phorbol ester treatment, as revealed by co-staining with the Golgi marker BODIPY-TR-ceramide. Structure relationship analysis of translocation using a series of PKC ligands revealed substantial differences between translocation of beta2-chimaerin and PKCalpha. Strikingly, the mezerein analog thymeleatoxin is not able to translocate beta2-chimaerin, although it very efficiently translocates PKCalpha. Phorbol esters also promote the association of beta2-chimaerin with Rac in cells. These data suggest that chimaerins can be positionally regulated by phorbol esters and that each phorbol ester receptor class has distinct pharmacological properties and targeting mechanisms. The identification of selective ligands for each phorbol ester receptor class represents an important step in dissecting their specific cellular functions.
Topics: Animals; COS Cells; Caenorhabditis elegans Proteins; Carrier Proteins; Isoenzymes; Neoplasm Proteins; Phorbol Esters; Protein Kinase C; Receptors, Drug; Signal Transduction
PubMed: 11278894
DOI: 10.1074/jbc.M011368200