Biomedical subjects

V Duronio

Publications and source records attributed to V Duronio.

At least 55 records · Page 3Linked to original sources

PubMed1995

Selective inhibition of phosphatidylinositol 3-kinase by phosphatidic acid and related lipids.

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) is necessary for stimulation of cell division and inhibition of apoptosis in several cell types. We report that a synthetic phosphonolipid, 4-(hexadecyloxy)-3-(S)-methoxybutyl phosphonic acid (PoA), as well as the naturally occurring lipids, phosphatidic acid and lyso-phosphatidic acid, are potent and specific inhibitors of PI 3-kinase. The IC50's for inhibition using phosphatidylinositol as substrate ranged from 10-20 microM. PoA is also the putative primary intracellular metabolite following phospholipase D hydrolysis of the anti-tumour ether lipid, 2'-(trimethylammonio) ethyl-4-(hexadecyloxy)-3-(S)-methoxybutanephosphonate. These results suggests that inhibition of PI 3-kinase following metabolic degradation of ether lipids by phospholipase D may contribute to the cytotoxicity of these compounds. The sensitivity of PI 3-kinase to PA and lyso-PA could imply cross-talk between the phospholipase D and PI 3-kinase signal transduction pathways in vivo.

Animals↗

PubMed1995

Molecular mechanisms of sepsis: molecular biology of the cell.

Complex and interrelated biological processes are at work in the expression of the host response to sepsis. To a large degree, these processes reflect drastic changes in the molecular workings of cells of the body. The protean nature of sepsis reflects this molecular adaptation. Studies are continuing to accrue that describe aspects of this process in tissue culture, animal models, and man. However, without an understanding of the basic mechanisms of molecular biology, the understanding of this important and expanding literature is limited. This review describes the basic molecular processes involved in replication of deoxyribonucleic acid (DNA) and transcription of DNA to ribonucleic acid (RNA) in the nucleus, translation of messenger RNA into proteins and the posttranslational modifications of these proteins in the cytoplasm. It uses the process of endotoxin-induced cellular activation as its model and highlights important aspects of DNA promoter and enhancer processes in this activation. Specific examples of known promoter genes and genomic translation are described. This review serves as a "primer" for the subsequent three review articles in this series that will follow it in preceding issues.

Animals↗

PubMed1994

Characterization of the activation-associated isoform of CD43 on murine T lymphocytes.

A rat mAb termed 1B11 recognizes a 130-kDa cell surface glycoprotein expressed on T lymphocytes. Transfection studies using the Cd43 gene transfected into murine L cells, and immunoblots using anti-peptide Abs specific for the CD43 polypeptide identified the 1B11 Ag as the 130-kDa isoform of murine CD43. mAb 1B11 fails to recognize the other major CD43 isoform, 115-kDa CD43, either by Western blotting or by FACS analysis, thus differing from the previously characterized anti-CD43 mAb S7 that recognizes only the CD43 115-kDa isoform and not the CD43 130-kDa isoform. CD43 130-kDa recognized by mAb 1B11 is differentially expressed on T lymphocytes. Whereas most CD4-8-, CD4+8+, and CD4-8+ thymocytes express 130-kDa CD43 constitutively, the Ag is expressed by less than 20% of CD4+ T cells in immature and mature populations. On activation, expression of 130-kDa CD43 is up-regulated dramatically on CD4+ T lymphocytes, and to a lesser extent on CD8+ T lymphocytes. In contrast, T cell activation resulted in only minor up-regulation of 115-kDa CD43. CD43 130-kDa contains sialylated O-linked carbohydrate; however, recognition by mAb 1B11 is not dependent on the presence of sialic acid. Interestingly, removal of sialic acid by neuraminidase treatment of 1B11-negative CD4+ T lymphocytes or 1B11-negative EL4 cells confers 1B11 reactivity, suggesting that the 1B11 epitope is masked by sialic acid residues on the CD43 115-kDa isoform. The isoelectric point (pl) of 130-kDa CD43 was determined to be 6.0, which is higher than the pl reported for 115-kDa CD43. Different molecular properties of 115-kDa and 130-kDa CD43 and their differential expression in T cell subsets may indicate specific roles for these CD43 isoforms in T cell ontogeny and/or T cell function.

Animals↗

PubMed1994

Multiple hemopoietins, with the exception of interleukin-4, induce modification of Shc and mSos1, but not their translocation.

Shc, grb2, and Son-of-sevenless (mSos1) proteins are potential upstream regulators of p21ras. We show that p52Shc and p46Shc comprise a significant portion of two of the major protein substrates phosphorylated on tyrosine in response to interleukin-2 (IL-2), IL-3, granulocyte-macrophage colony stimulating factor (GM-CSF), Steel factor (SLF), and colony-stimulating factor-1 (CSF-1). Once tyrosine phosphorylated, p52Shc and p46Shc associated with grb2. However, in contrast to published results with epidermal growth factor, treatment with GM-CSF, IL-3, and SLF failed to induce significant biochemically detectable translocation of Shc, grb2, or mSos1 from the cytosol to the plasma membrane. In addition, we did not observe significant epidermal growth factor-induced translocation of Sos1 to the membrane in Rat-1 cells. Treatment with SLF or IL-3 did increase tyrosine phosphorylation of membrane-localized p52Shc, which could then associate with grb2, although the majority of tyrosine-phosphorylated Shc was located in the cytosol. SLF, IL-3, and phorbol ester induced a decrease in the electrophoretic mobility of mSos1. This occurred with slower kinetics than p21ras activation and unlike hemopoietin-induced activation of p21ras was partially inhibited by a specific protein kinase C inhibitor. Thus, growth factor-induced modification of mSos1 may represent a downstream event, subsequent to p21ras activation. Significantly, IL-4, a cytokine that fails to activate p21ras, also failed to induce significant tyrosine phosphorylation of Shc or a shift in mSos1 mobility for the first time correlating these events with the ability of a growth factor to activate p21ras. Together, these data suggest that the current model for regulation of p21ras, which proposes a stable association of Shc-grb2-Sos1 complexes at the plasma membrane, may be an oversimplification.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Interleukin-4-dependent proliferation dissociates p44erk-1, p42erk-2, and p21ras activation from cell growth.

The activation of erk/mitogen-activated protein kinases and p21ras is strongly associated with progression through the cell cycle. Cell growth induced by the cytokine interleukin-4 (IL-4) effectively dissociates the activation of p44erk-1 and p42erk-2 mitogen-activated protein kinases and p21ras from cell proliferation. In two cell lines of T lymphocyte and myeloid origin that were dependent upon IL-4 for continuous growth, IL-4 failed to detectably activate or induce tyrosine phosphorylation of p44erk-1 and p42erk-2. The activation of p21ras was also not detectably affected by IL-4 treatment of these cells. Treatment of the same cells with other growth factors (colony-stimulating factor-1 and Steel factor) or phorbol esters induced the tyrosine phosphorylation and activation of p44erk-1 and p42erk-2 and stimulated p21ras activity. The presence of IL-4 neither diminished nor enhanced the activation of p44erk-1 and p42erk-2 by colony-stimulating factor-1, Steel factor, or 12-O-tetradecanoylphorbol-13-acetate. Furthermore, IL-4 also failed to activate p44erk-1, p42erk-2, and p21ras in normal T lymphocytes and mast cells derived from spleen and bone marrow, respectively. Significantly, these findings demonstrate that IL-4-induced cell growth may be dissociated from the activation of p44erk-1, p42erk-2, and p21ras, suggesting that their activation may not be an absolute requirement for growth factor-stimulated mitogenesis.

Bone Marrow↗

PubMed1994

Multiple cytokines activate phosphatidylinositol 3-kinase in hemopoietic cells. Association of the enzyme with various tyrosine-phosphorylated proteins.

Activation of phosphatidylinositol (PI) 3-kinase is a common sequel to tyrosine kinase activation and appears to be essential for tyrosine kinases to induce proliferation. Since multiple hemopoietic growth factors activate tyrosine kinases, we investigated whether these growth factors activate PI 3-kinase. We show that interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), granulocyte-macrophage colony stimulating factor (GM-CSF), and steel factor (SLF) all activate PI 3-kinase. These cytokines increased the amount of PI 3-kinase activity that could be immunoprecipitated with anti-phosphotyrosine antibodies from the MC-9 mast cell line or from the hemopoietic progenitor cell line FDC-P1. Increases in this assay frequently correlate with PI 3-kinase activation in vivo. To determine directly whether these factors activate PI 3-kinase in vivo, we measured the levels of 3-phosphorylated inositol phospholipids in intact 32P-labeled MC-9 cells. IL-3, IL-4, IL-5, GM-CSF, and SLF all caused increased synthesis of the PI 3-kinase products phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate with a relative potency of SLF >> IL-3 > IL-5, GM-CSF > IL-4. In contrast, IL-4 caused the largest increase in the in vitro anti-phosphotyrosine immune complex PI 3-kinase assay. Thus, the in vitro assay does not accurately reflect in vivo activation of PI 3-kinase. Cytokine treatment did not stimulate tyrosine phosphorylation of either the 85-kDa regulatory subunit or the 110-kDa catalytic subunit of PI 3-kinase and is therefore not required for activation of PI 3-kinase by these factors. Cytokine treatment did induce PI 3-kinase to associate with other tyrosine-phosphorylated proteins in a cytokine-specific manner. PI 3-kinase associated with c-kit after SLF stimulation, a 170-kDa protein after IL-4 stimulation, and a 70-kDa protein after treatment with IL-3 or GM-CSF. Thus, multiple hemopoietic growth factors that act through different types of receptors activate PI 3-kinase in vivo and induce factor-specific interactions of PI 3-kinase with other tyrosine-phosphorylated proteins.

Animals↗

PubMed1992

Tyrosine phosphorylation of receptor beta subunits and common substrates in response to interleukin-3 and granulocyte-macrophage colony-stimulating factor.

The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) consist of two polypeptides each belonging to a new class of molecules referred to as the hemopoietin receptor family. When expressed alone, receptor polypeptides of this family often bind their respective factors with lower affinity than the receptors identified in whole cells. Despite the lack of structural evidence for any enzymatic activity of the receptor polypeptides, both IL-3 and GM-CSF stimulate tyrosine phosphorylation of multiple intracellular substrates. We investigated IL-3 and GM-CSF receptor structure and signaling in a myeloid cell line, FDC-P1, which is dependent on either IL-3 or GM-CSF for growth. Antiphosphotyrosine antibodies were used to immunoprecipitate tyrosine-phosphorylated proteins from 32P-labeled cells or to probe immunoblots. Both IL-3 and GM-CSF stimulated the phosphorylation of a similar pattern of polypeptides on tyrosine. One tyrosine phosphorylated polypeptide migrated with M(r) = 135,000 and increased to 150,000 over a period of 10 min following stimulation of cells with IL-3 or GM-CSF. The M(r) = 135,000-150,000 polypeptide phosphorylated in response to IL-3 was shown to be primarily the Aic-2A polypeptide, the low affinity IL-3 receptor. Phosphatase treatment showed that the dramatic IL-3-induced shift in apparent molecular weight from M(r) = 125,000 in unstimulated cells was entirely due to phosphorylation. The closely related receptor, Aic-2B, was also tyrosine phosphorylated in response to IL-3, although to a lesser extent than Aic-2A. Treatment with GM-CSF resulted in tyrosine phosphorylation of the Aic-2B polypeptide exclusively. It was intriguing that GM-CSF treatment did affect the mobility of the Aic-2A polypeptide on polyacrylamide gels. Together, these results suggest that the Aic-2A polypeptide is part of the IL-3 receptor complex, but not the GM-CSF receptor. In contrast, the Aic-2B polypeptide is a component of the GM-CSF receptor, but it can also be utilized in an IL-3 receptor.

Animals↗

PubMed1992

Multiple hemopoietic growth factors stimulate activation of mitogen-activated protein kinase family members.

Stimulation of hemopoietic cells with IL-3, IL-4, IL-5, granulocyte-macrophage-CSF and Steel factor-(SLF) induced tyrosine phosphorylation of a number of protein substrates. Two of these proteins, designated p42 and p44, were tyrosine phosphorylated rapidly in response to treatment with IL-3, IL-5, granulocyte-macrophage-CSF and SLF, but not IL-4. We demonstrate that these common substrates are members of the mitogen-activated protein kinase (MAP kinase) family of protein serine/threonine kinases. Ion-exchange chromatography yielded a peak of MAP kinase activity eluting at 0.3 to 0.32 M NaCl. Immunoblotting of column fractions with antiphosphotyrosine antibodies showed coelution of the peak of MAP kinase enzyme activity with the p42 and p44 tyrosine phosphorylated species, and with two proteins of 42 and 44 kDa which were immunoreactive with anti-MAP kinase antibodies. Moreover, a characteristic shift in mobility of the p42 and p44 species was observed after factor treatment. Time-course analyses and subsequent ion-exchange chromatography demonstrated SLF activation of MAP kinase activity was maximal after 2 min of factor treatment and decreased to basal levels after 30 min stimulation. By contrast, activation of MAP kinase after IL-5 treatment was not as rapid. Maximal activity was observed 15 min after stimulation and remained elevated for up to 60 min after IL-5 addition. Investigation of the role of protein kinase C in the mechanism of activation by these growth factors demonstrated that specific inhibition of protein kinase C led to a reduction, but not ablation, of the SLF and IL-3 induced stimulation of MAP kinase activity. The use of synthetic peptide substrates confirmed SLF and IL-5 activate isoforms of MAP kinases. These results demonstrate that members of the MAP kinase family are involved in common signal transduction events elicited by IL-3, IL-5, granulocyte-macrophage-CSF and Steel factor, but not those involving IL-4.

Amino Acid Sequence↗

PubMed1992

p21ras activation via hemopoietin receptors and c-kit requires tyrosine kinase activity but not tyrosine phosphorylation of p21ras GTPase-activating protein.

Products of the ras gene family, termed p21ras, are GTP-binding proteins that have been implicated in signal transduction via receptors encoding tyrosine kinase domains. Recent findings have defined a superfamily of hemopoietin receptors that includes receptors for a number of interleukins and colony-stimulating factors. The intracellular portions of these receptors show only restricted homologies, have no tyrosine kinase domain, and provide no clues to the mode of signal transduction. However, in most cases the factors stimulate tyrosine phosphorylation. We demonstrate here that ligand-induced activation of the interleukin (IL)-2, IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptors resulted in activation of p21ras in various hemopoietic cell lines. The only cytokine tested that binds to a hemopoietin receptor and that did not activate p21ras was IL-4. Activation of p21ras was also observed in response to Steel factor, which stimulates the endogenous tyrosine kinase activity of the c-kit receptor, as well as with phorbol esters, which activate protein kinase C. Experiments with protein kinase inhibitors implicated tyrosine kinase activity, but not protein kinase C activity, as the upstream signal in p21ras activation via these growth factor receptors. Attempts to demonstrate tyrosine phosphorylation of the p21ras GTPase-activating protein (GAP) were negative, suggesting that phosphorylation of GAP may not be the major mechanism for regulation of p21ras activity by tyrosine kinases.

Animals↗

PubMed1992

Inhibition of human neutrophil responses by alpha-cyano-3,4-dihydroxythiocinnamamide; a protein-tyrosine kinase inhibitor.

1. Activation of neutrophils results in increased tyrosine phosphorylation of several proteins that may have important roles in receptor/effector coupling. In this study, the effect of a protein tyrosine kinase inhibitor on receptor-mediated neutrophil activation by platelet-activating factor (PAF), leukotriene, B4 (LTB4) and N-formylmethionylleucylphenylalanine (FMLP) is investigated. 2. alpha-Cyano-3,4-dihydroxythiocinnamamide dose-dependently inhibited intracellular calcium release and superoxide generation from human neutrophils activated by 1 microM LTB4, PAF, and FMLP. 3. In the presence of cytochalasin B, FMLP stimulated elastase release from neutrophils was also inhibited to unstimulated levels by 5 min pretreatment with alpha-cyano-3,4-dihydroxythiocinnamamide. 4. The inhibitory action of alpha-cyano-3,4-dihydroxythiocinnamamide was found to be at or upstream of phospholipase C activation, blocking both phosphatidylinositol hydrolysis and protein kinase C activation. alpha-Cyano-3,4-dihydroxythiocinnamamide did not affect agonist receptor binding sites or receptor affinity in neutrophils. 5. Immunoblot analysis demonstrated the tyrosine phosphorylation of proteins of 41, 56, 66, and 104 kDa in neutrophils treated with agonists. Treatment of neutrophils with alpha-cyano-3,4-dihydroxythiocinnamamide prior to stimulation with chemoattractants reduced tyrosine phosphorylation of the above phosphoproteins. 6. These results indicate that alpha-cyano-3,4-dihydroxythiocinnamamide might be a useful agent in characterizing the essential proteins and biochemical pathways that regulate neutrophil activation.

Calcium↗

PubMed1992

Tyrosine protein kinase inhibitors block invasin-promoted bacterial uptake by epithelial cells.

The ability to enter into (invade) mammalian cells is an essential virulence determinant of many pathogenic bacteria and intracellular parasites. These organisms are internalized by host cells upon attachment to their surface. However, the mechanisms used by intracellular parasites to induce internalization into host cells have not been defined. We found that the protein kinase inhibitor staurosporine blocks invasion by some pathogenic bacteria, including Yersinia enterocolitica and Yersinia pseudotuberculosis. Using Escherichia coli containing the cloned Y. enterocolitica invasion gene inv (which codes for invasin, an integrin-binding protein), we found that staurosporine inhibits invasion by blocking bacterial internalization. Two specific tyrosine protein kinase inhibitors, genistein and tyrphostin, also block the internalization but not the binding of bacteria, suggesting that bacterial uptake may be dependent on the activity of this enzyme class in host HeLa cells. In contrast to invasion promoted by invasin, the invasion of HeLa cells by Salmonella typhimurium is not inhibited by any of these drugs.

Adhesins, Bacterial↗

PubMed1992

Two polypeptides identified by interleukin 3 cross-linking represent distinct components of the interleukin 3 receptor.

Two receptor polypeptides have been identified in several studies by using cross-linking with interleukin 3 (IL-3). It has been suggested that proteolytic degradation of the larger polypeptide yields the lower molecular weight fragment, but there is little proof that this is the case. We have used several different approaches to characterize the polypeptides cross-linked in R6X or FDC-P1 cells. Several bifunctional cross-linkers of various sizes were tested to determine their effectiveness in cross-linking IL-3 to its receptor. The longer cross-linker gave the highest proportion of labeling of the low molecular weight band. Incubation in the absence of protease inhibitors caused a decrease in labeling of both cross-linked polypeptides, but no indication of a significant increase in the lower molecular weight band. Direct comparison of the two cross-linked polypeptides by V8 protease mapping showed no common peptides that might be expected if they were related molecules, except those derived from the iodinated IL-3. Digestion with N-glycanase resulted in a decrease in apparent molecular weight of 5000 in the larger polypeptide but a decrease of 15,000 in the smaller polypeptide. These results suggest that the 70-kd polypeptide identified by cross-linking of IL-3 represents a second binding chain of the receptor. By analogy with some of the other hemopoietin receptors, the 70- and 125-kd polypeptides may form a complex necessary for high affinity binding.

Amidohydrolases↗

PubMed1991

Solubilization of a functionally active platelet-activating factor receptor from rabbit platelets.

Binding of platelet-activating factor (PAF) to a specific high-affinity membrane receptor has been demonstrated in numerous cell types, but very little is known about the molecular nature of this receptor. The receptor from rabbit platelets was solubilized using CHAPS, digitonin, octyl glucoside, Nonidet P-40 or sodium cholate, either with pre-bound [3H]PAF or in the absence of ligand. We have been able to demonstrate for the first time that the receptor solubilized with CHAPS, in the absence of ligand, could retain its binding activity. It migrated as a high molecular mass complex (greater than 350 kDa) on a Bio-Gel A-0.5 m gel filtration column. Binding to solubilized receptor rapidly reached an equilibrium at room temperature, but was much slower at 0 degrees C. Scatchard plots were used to calculate the number (approx. 100 per cell) and the affinity (Kd 2.5 +/- 1.4 nM) of the solubilized receptors. These values were comparable with those obtained from whole-cell binding experiments. Competition by PAF antagonists also verified that the assay was measuring PAF receptor binding activity. The presence of a protein in the receptor complex was demonstrated by heat and trypsin inactivation of binding activity. Trypsin had no effect on binding of PAF to whole cells, but was able to decrease binding activity in solubilized receptor preparations. Attempts to demonstrate the involvement of a glycoprotein by use of various lectin columns proved unsuccessful. The latter results are consistent with findings suggesting that the binding site of the PAF receptor may not be exposed at the cell surface.

Animals↗

PubMed1991

Antibodies to interleukin 3 as probes for the interaction of interleukin 3 with its receptor.

A series of antibodies, directed against murine interleukin-3 (IL-3) or synthetic peptides corresponding to portions of the IL-3 sequence, has been used to detect receptor-bound IL-3 on the surface of cells. An assay was developed in which the bound primary antibody was detected using a biotinylated secondary antibody and fluorescein isothiocyanate-labeled streptavidin, followed by analysis on a fluorescence-activated cell sorter. The fluorescence signal was shown to be specific for cells known to express IL-3 receptors and was dependent on the preincubation of cells with IL-3 under conditions that did not allow internalization of receptors. Antisera raised against full-length synthetic IL-3 or to the N-terminal 29 residues were found to give equivalent signals. On the other hand, antibodies to residues 91-118 showed no signal in this assay, despite being able to bind to IL-3 in solution and neutralize IL-3 bioactivity. When peptides corresponding to residues 30-43 and 123-140 were incubated with the anti-IL-3 antiserum, the majority of the fluorescence signal was abolished, indicating that these two peptides contained the primary epitopes being recognized by the antiserum in this assay. This antiserum also bound to the 91-118 peptide, but the corresponding peptide was not able to reduce the fluorescence signal in a similar competition assay. These results suggest that the 91-118 region is not accessible to antibody when IL-3 is bound to its receptor, whereas at least portions of epitopes 1-29, 30-43, and 123-140 remain accessible to antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1990

Insulin receptor is phosphorylated in response to treatment of HepG2 cells with insulin-like growth factor I.

1. Binding of insulin and insulin-like growth factor I (IGF-I) to HepG2 cells was analysed with regard to competition by both insulin and IGF-I. At concentrations of insulin that caused maximal phosphorylation of the insulin receptor, virtually no displacement of IGF-I binding was observed. Similarly, at concentrations of IGF-I that caused maximal phosphorylation of the IGF-I receptor, no displacement of insulin binding was observed. 2. When the phosphorylation of both receptors was examined individually by using specific monoclonal antibodies to immunoprecipitate the receptors, phosphorylation of the insulin receptor was found to increase on both serine and tyrosine residues in cells treated with 100 ng of IGF-I/ml. In contrast, no increased phosphorylation of IGF-I receptor was observed in cells treated with 100 ng of insulin/ml. 3. The increase in phosphorylation of insulin receptor in response to IGF-I correlated with the dose-response of IGF-I-stimulated phosphorylation of the IGF-I receptor. 4. The IGF-I-stimulated phosphorylation of the insulin receptor could be blocked by preincubation with a monoclonal antibody that blocks IGF-I binding to the IGF-I receptor.

Antibodies, Monoclonal↗

PubMed1990

Translocation-independent activation of protein kinase C by platelet-activating factor, thrombin and prostacyclin. Lack of correlation with polyphosphoinositide hydrolysis in rabbit platelets.

The relationship between polyphosphoinositide hydrolysis and protein kinase C (PKC) activation was explored in rabbit platelets treated with the agonists platelet-activating factor (PAF), thrombin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and with the anti-aggregant prostacyclin (PGI2). Measurement of the hydrolysis of radiolabelled inositol-containing phospholipids relied upon the separation of the products [3H]inositol mono-, bis- and tris-phosphates by Dowex-1 chromatography. PKC activity, measured in platelet cytosolic and Nonidet-P40-solubilized particulate extracts that were fractionated by MonoQ chromatography, was based upon the ability of the enzyme to phosphorylate either histone H1 in the presence of the activators Ca2+, diacylglycerol and phosphatidylserine, or protamine in the absence of Ca2+ and lipid. Treatment of platelets for 1 min with PAF (2 nM) or thrombin (2 units/ml) led to the rapid hydrolysis of inositol-containing phospholipids, a 2-3-fold stimulation of both cytosolic and particulate-derived PKC activity, and platelet aggregation. Exposure to TPA (200 nM) for 5 min did not stimulate formation of phosphoinositides, but translocated more than 95% of cytosolic PKC into the particulate fraction, and induced a slower rate of aggregation. PGI2 (1 microgram/ml) did not enhance phosphoinositide production, and at higher concentrations (50 micrograms/ml) it antagonized the ability of PAF, but not that of thrombin, to induce inositol phospholipid turnover, even though platelet aggregation in response to both agonists was blocked by PGI2. On the other hand, PGI2 alone also appeared to activate (by 3-5-fold) cytosolic and particulate PKC by a translocation-independent mechanism. The activation of PKC by PGI2 was probably mediated via cyclic AMP (cAMP), as this effect was mimicked by the cAMP analogue 8-chlorophenylthio-cAMP. It is concluded that this novel mechanism of PKC regulation by platelet agonists may operate independently of polyphosphoinositide turnover, and that activation of cAMP-dependent protein kinase represents another route leading to PKC activation.

Animals↗

PubMed1990

Erbstatin blocks platelet activating factor-induced protein-tyrosine phosphorylation, polyphosphoinositide hydrolysis, protein kinase C activation, serotonin secretion and aggregation of rabbit platelets.

The role of protein-tyrosine phosphorylation in the signal transduction of platelet activating factor (PAF) was investigated in rabbit platelets with a range of synthetic compounds that inhibit protein-tyrosine kinases. In particular, erbstatin (IC50 approximately 20 micrograms/ml) abrogated a wide range of platelet responses to PAF, including tyrosine phosphorylation of cellular proteins, polyphosphoinositide turnover, activation of membranous protein kinase C, platelet aggregation, and serotonin secretion. With about a third of the potency of erbstatin, compound RG50864 also inhibited many of these responses, whereas at 100 micrograms/ml, genistein, 670C88 and ST271 were without effect. Finally, the ability of thrombin to cause platelet aggregation and serotonin secretion was also compromised by erbstatin.

Animals↗

PubMed1990

Partial down-regulation of protein kinase C reverses the growth inhibitory effect of phorbol esters on HepG2 cells.

Phorbol ester treatment of HepG2, a human tumorigenic cell line, caused rapid morphological changes characterized by a flattening and spreading of the cells that coincided with a rapid inhibition of thymidine incorporation. Within 24 h, cell division was completely inhibited, suggesting the cells had entered a quiescent state. Continued incubation in the presence of phorbol esters resulted in the resumption of thymidine incorporation and cell division, but this coincided with only a partial down-regulation of PKC activity. Seventy-two hours of treatment was required to obtain down-regulation of greater than 80% of the PKC activity, but reversal of the inhibitory effects occurred between 24 and 48 h after the addition of phorbol esters, when a large proportion of the PKC activity was still present. Northern blot analysis of a number of transcripts showed that the steady-state levels of c-myc and transforming growth factor beta 1 (TGF-beta 1) messages increased only after 3 h of phorbol ester treatment and returned to normal levels after 24 h. C-fos, albumin, and alphafetoprotein messages were not affected, suggesting the differentiation state of the cells was not altered. Therefore, phorbol ester activation of PKC causes an inhibition of HepG2 cell growth initially, but this is unlike the promotion of differentiation seen in other systems. Partial down-regulation of PKC activity causes a reversal of the growth inhibition and the cells return to a normal growth rate. This effect is also clearly different from systems in which phorbol esters have been shown to have a mitogenic effect on cells.

Carcinoma, Hepatocellular↗