Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human neutrophils isolated from the blood were incubated in vitro for 30 min with 25 ng/ml of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, to study its effect on the fine structure of granulocytes and on the subcellular distribution of lactoferrin (Lf). Flow cytometry analysis of the human neutrophils showed that PMA induced a decrease in size and granularity of the cells. By electron microscopy, the PMA-treated cells showed numerous empty vesicles, smoothing of the cell surface, and a marked decrease in the compactness of the cytoplasmic ground substance. High-resolution immunogold ultracytochemistry showed that neutrophils lost most of their content in Lf after the PMA incubation. In thin sections, the cytoplasmic compartment of PMA-treated neutrophils contained an average of 1.03 +/- 0.3 Lf-positive vesicles per microns 2, whereas control granulocytes showed an average of 5.72 +/- 1.49 Lf vesicles per microns 2 of cytoplasm. Most of PMA-treated neutrophils kept some Lf-positive vesicles; a significant minority (10-15%) of the granulocytes showed cytological features of cell death. We conclude that the PMA-induced neutrophil activation is associated with microanatomical changes that include i) exocytosis of most, but not all, of the Lf-bearing vesicles; ii) rounding up of the cell outline; and iii) decrease in the compactness of the cytoplasmic ground substance.
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PMID:Activation of human neutrophils by phorbol ester decreases the cytoplasm compactness and the lactoferrin content of the granulocytes. 174 40

Lymphotoxin (LT) can activate human neutrophils. Using a hemolytic plaque assay to detect secretion of lactoferrin and myeloperoxidase (MPO) from single adherent neutrophils, we showed that LT induced secretion from both primary and secondary granules. Incubation of cells with cytochalasin B was required for MPO secretion, and it enhanced lactoferrin secretion. Pertussis toxin, which blocks a G-protein in the plasma membrane, inhibited LT-induced exocytosis of MPO, but not of lactoferrin. Incubation with LT did not induce any detectable changes of the cytoplasmic free [Ca2+] in neutrophils. On the other hand, secretion of granule proteins from adherent neutrophils in response to LT was blocked by loading neutrophils with quin-2 in order to increase the intracellular calcium buffering capacity. This was achieved at a concentration of quin-2, at which the secretion induced by the phorbol ester PMA and the chemotactic peptide FMLP was unaffected. Trifluoroperazine (TFP), a dual protein kinase C and calmodulin inhibitor, significantly inhibited the LT-mediated secretion of lactoferrin from adherent granulocytes. The PMA effect was unaltered by TFP under these conditions, suggesting that the inhibitory effect was on a calcium-calmodulin dependent step. The secretion induced by TNF and GM-CSF was also blocked by buffering changes in the intracellular [Ca2+] and inhibited to a similar extent by TFP. Our results suggest that calmodulin and minute changes in the cytoplasmic free [Ca2+] may be involved in a common signal transduction pathway engaged in activation of adherent neutrophils by several cytokines.
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PMID:Lymphotoxin induces secretion of granule proteins from adherent neutrophils: possible role of intracellular free calcium. 216 92

We have examined the ability of bryostatin 1 (bryo), an activator of protein kinase C, to induce differentiation of chronic myelogenous leukemia (CML) cells obtained from peripheral blood. Bryo induced a prompt and persistent macrophage-like differentiation, as evidenced by functional, morphological, and immunological criteria. Differentiated cells remained viable for at least 21 days with little change in cell number. CML cell cultures treated in semisolid medium with bryo showed diffuse infiltration with single macrophages, as well as discrete macrophage, mixed, and granulocytic colonies. Supernatants of suspension cultures of bryo-treated CML cells contained granulocyte-macrophage colony-stimulating factor (GM-CSF) by enzyme-linked immunosorbent assay. Furthermore, colony formation could be significantly inhibited by the addition of antibodies to GM-CSF. Prolonged liquid culture of CML cells in bryo reduced colony-forming unit, granulocyte-macrophage content. Bryo-induced differentiation was associated with a decrease in lactoferrin, a marker of granulocyte differentiation, and an increase in both c-fms and interleukin-1 beta RNA, both of which are expressed by monocytes/macrophages. These data demonstrate that bryostatin 1 is capable of inducing macrophage-like differentiation in maturing CML cells. Furthermore, bryostatin induces secretion of GM-CSF by such cells in suspension and semisolid medium and also promotes clonal extinction of granulocyte-macrophage progenitors. Bryostatin may be a possible therapeutic agent for CML.
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PMID:Differentiation and growth modulation of chronic myelogenous leukemia cells by bryostatin. 238 56

Sphingoid long-chain bases (sphinganine and sphingosine) have recently been shown to inhibit protein kinase C both in vitro [Y. Hannun et al. (1986) J. Biol. Chem. 261, 12604-12609] and in intact human neutrophils, in which they block activation of the superoxide-generating respiratory burst [E. Wilson et al. (1986) J. Biol. Chem. 261, 12616-12623]. In the present study we have used sphingosine to investigate the pathways for agonist-induced secretion of neutrophil granule contents. Induction of secretion of the specific granule component lactoferrin by a variety of agonists [phorbol 12-myristate-13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore A23187] was completely inhibited by sphingosine with an ED50 of 6 to 10 microM. PMA-induced secretion of lysozyme (present in both the azurophilic and specific granules) was completely blocked with an ED50 of 10 microM, whereas fMLP-induced secretion was only about 50% inhibited. Secretion of the azurophilic granule proteins beta-glucuronidase and myeloperoxidase was activated by fMLP and A23187, but not by PMA, and was not affected by sphingosine. The use of A23187 in the presence of sphingosine allowed differentiation between calcium activation of protein kinase C-dependent versus-independent pathways. The effect of sphingosine was not mediated by neutralizing intracellular acidic compartments, since treatment of neutrophils with inhibitory concentrations of sphingosine did not significantly alter the uptake of labeled methylamine. We conclude that at least two mechanisms participate in the regulation of specific and azurophilic granule secretion, respectively: a protein kinase C-dependent pathway and a calcium-dependent pathway which does not involve protein kinase C.
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PMID:Protein kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils. 282 97

We have studied effects of ferric transferrin (FeTF), ferric lactoferrin (FeLF), ferric complexes of pyridoxal- or salicylaldehyde-isonicotinoyl hydrazone, (Fe-PIH, Fe-SIH), and ferric ammonium citrate (FAC) on expression of protein kinase C (PKC) mRNA transcripts in a variety of cultured cell lines. FeTF supported an increase of PKC-beta mRNA transcripts in T-lymphoblastoid (CCRF-CEM; Jurkat), B-lymphoblastoid (Daudi; Raji), promyelocyte (HL-60), erythroleukemia (K562), and monocyte (U937) cell lines. By contrast, FeLF, Fe-PIH, and Fe-SIH did not support an increase of PKC-beta mRNA transcripts in any of these cell lines. Furthermore, FAC supported an increase of PKC-beta mRNA transcripts in HL-60, K562, and U937 cells only. Preincubation of cells with desferrioxamine (DF), a cell-permeable iron chelator, abolished the increments of PKC-beta mRNA observed in response to FeTF or FAC. In contrast to results with PKC-beta, neither FeTF nor FAC caused an increase of PKC-alpha transcripts in any cell line. To locate iron-responsive DNA regulatory elements of the PKC-beta gene, we prepared genetic constructs containing various portions of the human PKC-beta 5'-flanking DNA linked to the firefly luciferase gene. Constructs were cotransfected with the neomycin resistance plasmid, Pwl-neo, into HRE H9 cells, and stable transfectants were selected in G418. Treatment with FeTF of transfectants bearing chimeric gene constructs with 2,200 bp of the PKC-beta 5'-flanking region increased luciferase activity and mRNA transcripts 2.5-fold. This increase was blocked by DF. Neither luciferase activity nor mRNA increased with FeTF in stable transfectants bearing constructs with 342 bp or 587 bp of the PKC-beta 5'-flanking region. These data provide direct confirmation that iron is involved in regulation of PKC-beta but not PKC-alpha gene expression in many cell lines. The form in which iron is presented to these cell lines appears to affect its availability for this function, and cells vary in their capabilities to use nontransferrin iron to support PKC-beta gene expression. Finally, transcriptional upregulation of PKC-beta by FeTF is mediated by DNA sequences located between -2200 bp and -587 bp in the 5'-flanking region of the human PKC-beta gene.
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PMID:Regulation of protein kinase C (PKC) expression by iron: effect of different iron compounds on PKC-beta and PKC-alpha gene expression and role of the 5'-flanking region of the PKC-beta gene in the response to ferric transferrin. 794 5

Lactoferrin is present in a variety of tissues and biological fluids; however, the amount differs significantly due to differential expressions. We have previously demonstrated that the mouse lactoferrin gene is regulated by estrogen through an estrogen-response DNA element located at -349, upstream from the transcription start site (+1). In this report, we characterized by deletion and mutation analyses a cluster of mitogen-response elements located between -80 and -40 of the mouse lactoferrin promoter. We demonstrated that the chimeric chloramphenicol acetyltransferase reporter constructs (the -103 to +1 sequence of the mouse lactoferrin gene) containing the mitogen-response unit of the lactoferrin gene were stimulated by cAMP, forskolin, 12-O-tetradecanoylphorbol-13-acetate, and epidermal growth factor/recombinant transforming growth factor-alpha (EGF/TGF-alpha) in a time- and dose-dependent manner. The sequence at position -52 to -40 (mLF-CRE) of the gene conferred transcriptional activation in the presence of forskolin, cyclic AMP, and 12-O-tetradecanoylphorbol-13-acetate in transiently transfected human endometrium carcinoma RL95-2 cells, whereas the region at -80 to -60 responded to EGF/TGF-alpha stimulation. Overexpression of the catalytic unit of protein kinase C or protein kinase A in the RL95-2 cells elevated the chloramphenicol acetyl-transferase activity of the reporter construct 5-6-fold. The mobility shift assay suggested that AP1 and CREB or related proteins participated in complex formation with the mLF-CRE, whereas different proteins bound to the EGF/TGF-alpha-response element.
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PMID:Characterization of a mitogen-response unit in the mouse lactoferrin gene promoter. 817 15

We studied the degranulation reaction of electropermeabilized human neutrophils induced by 1,2-didecanoyl-3-sn-phosphatidic acid (PA10). PA10 dose-dependently induced the release of beta-glucuronidase, an enzyme of azurophil granules, but did not induce the release of lactoferrin, a protein of specific granules. The enzyme release by PA10 absolutely required Ca2+, ATP, and Mg2+ and the concentrations for the half-maximal response were 2.5 microM, 60 microM, and 0.25 mM, respectively. Although Ca2+ alone at concentrations higher than 10 microM induced the release of both beta-glucuronidase and lactoferrin, the extents of the release were far less than that of the beta-glucuronidase release by PA10. Phorbol myristate acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol induced the release of lactoferrin alone at concentrations of Ca2+ below 0.5 microM while they induced the release of both beta-glucuronidase and lactoferrin at higher Ca2+ concentrations, indicating that the degranulation induced by PA10 is not mediated by diacylglycerol which might be formed from PA. The degranulation reactions induced by PA10 and PMA were dose-dependently inhibited by staurosporine and calphostin C, protein kinase C inhibitors, although no direct activation of protein kinase C by PA10 was observed. The extent of the beta-glucuronidase release by PA10 was not enhanced by the addition of PMA. Propranolol, which inhibits protein kinase C as well as phosphatidic acid phosphohydrolase, strongly inhibited the degranulation reactions induced by PA10 and PMA. Ethanol, a metabolic modulator of phospholipase D, and cyclic AMP did not affect the degranulation reactions by PMA and PA10.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphatidic acid induces the release of beta-glucuronidase but not lactoferrin from electropermeabilized human neutrophils. 820 72

The purpose of this study was to elucidate likely signal transduction pathways in activated bovine neutrophils, by comparing the effects of various inhibitors on the bovine neutrophil respiratory burst and degranulation in vitro. The protein kinase C(PKC) inhibitors staurosporine, and chelerythine, and the beta-adrenergic receptor antagonist DL-propranolol, markedly inhibited opsonized zymosan (OZ) stimulated luminol-dependent chemiluminescence (LDCL). The G-protein inhibitor pertussis toxin (PT), the protein tyrosine inhibitor genistein, and the calcium channel blocker verapamil also reduced LDCL in a dose-dependent manner. In contrast, the lipoxygenase inhibitor zileuton had only a slight effect, and the cyclooxygenase inhibitor indomethacin had no effect on LDCL. The effects of these inhibitors on degranulation was also examined. Staurosporine, propranolol, and pertussis toxin significantly decreased primary granule (beta-glucosaminidase) release in response to OZ. These inhibitors also significantly reduced both phorbol myristate acetate (PMA)-induced primary and secondary granule (lactoferrin) release. Regulation of secondary granule (lactoferrin) release was complex, as it was significantly depressed by propranolol, enhanced by PT and unaffected by staurosporine. These findings suggest that PKC, beta-adrenergic receptors, G-proteins, protein tyrosine kinase(s) and Ca(2+) uptake, may all be involved in some part of the process of bovine neutrophil activation. Moreover, stimulation of LDCL and degranulation may be mediated through distinct signal transduction pathways.
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PMID:Regulation of luminol-dependent chemiluminescence and degranulation by bovine neutrophils stimulated with opsonized zymosan. 915 84

Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin (alpha2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RI alpha of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RI alpha antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated alpha2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.
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PMID:Receptor-mediated endocytosis of urokinase-type plasminogen activator is regulated by cAMP-dependent protein kinase. 921 25

Polymorphonuclear neutrophils (PMN) adherent to integrin ligands respond to inflammatory mediators by reorganizing their cytoskeleton and releasing reactive oxygen intermediates. As Src family tyrosine kinases are implicated in these responses, we investigated their possible role in regulating degranulation. Human PMN incubated on fibrinogen released lactoferrin in response to TNF-alpha and this response was inhibited by PP1, a Src family tyrosine kinase inhibitor. This drug had no effect on lactoferrin secretion induced by PMA, an adhesion-independent agonist of PMN degranulation. However, PP1 blocked secretion in PMN plated on plain tissue culture plastic, a surface inducing PMN spreading in the absence of any stimulus. Double knockout hck-/- fgr-/- PMN adherent to collagen or fibrinogen failed to release lactoferrin in response to TNF-alpha but responded to PMA as wild-type PMN. Degranulation induced by spreading over tissue culture plastic was also defective in hck-/- fgr-/- PMN. Defective adhesion-dependent degranulation required the absence of both kinases, because single knockout fgr-/- or hck-/- PMN responded as wild-type cells. Analysis of lactoferrin secretion in hck-/- fgr-/- or PP1-treated, suspended PMN showed that Src kinases are not implicated in degranulation dependent on activation of protein kinase C or increase in intracellular free Ca2+ but may play a role in the response to FMLP of cytochalasin B-treated PMN. These findings identify a role for Src family kinases in a signaling pathway leading to granule-plasma membrane fusion and suggest that Fgr and Hck would be targets for pharmacological control of adhesion-dependent degranulation in the inflammatory site.
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PMID:Adhesion-dependent degranulation of neutrophils requires the Src family kinases Fgr and Hck. 991 42


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