Bile acid deoxycholate induces differential subcellular localisation of the PKC isoenzymes beta 1, epsilon and delta in colonic epithelial cells in a sodium butyrate insensitive manner.

Elevated levels of bile acids have been implicated in the abnormal morphogenesis of the colonic epithelium thus contributing to colorectal cancer (CRC). Alternatively sodium butyrate (NaB) produced by anaerobic fermentation of dietary fibre is regarded as being protective against colon cancer. Bile acids such as deoxycholic acid (DCA) are thought to mediate some of their actions by differentially activating protein kinase C (PKC). We examined the effects of DCA on the subcellular localisation of PKC-beta(1), -epsilon and -delta and whether these responses could be modulated by NaB. HCT116 cells endogenously express PKC-epsilon and -delta but not PKC-beta. DCA treatment results in endogenous PKC-epsilon translocation but not PKC-delta after 1 hr. To study the subcellular localisation of PKC isoforms in response to DCA in real time, PKC-beta(1), PKC-epsilon and PKC-delta functionally intact green fluorescent protein (GFP) fusion constructs were used. Stimulation with 300 microM DCA induces rapid translocation of PKC-beta(1)-GFP and PKC-epsilon-GFP but not PKC-delta-GFP from the cytosol to the plasma membrane in 15 min. Interestingly, pretreatment with 4mM NaB does not modify the response of the PKC isoenzymes to DCA as PKC-beta(1)-GFP and PKC-epsilon-GFP translocates to the plasma membrane in 15 min whereas PKC-delta-GFP localisation remains unaltered. Immunofluorescence shows that PKC-beta(1)-GFP and PKC-epsilon-GFP cells treated with DCA colocalise with the cytoskeletal elements actin and tubulin adjacent to the plasma membrane. Our findings demonstrate that the differential activation of the PKC isoenzymes by DCA may be of critical importance for the functional responses of colonic epithelial cells. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.

PKC theta mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia.

Protein kinase (PK)C theta is a critical regulator of mature T-cell activation and proliferation, being implicated in TCR-triggered nuclear factor (NF)-kappa B activation and providing important survival signals to leukemic T cells. We previously showed that overexpression of pT alpha/pre-TCR and constitutive activation of NF-kappa B characterize the T-cell leukemia/lymphoma developing in Notch3-IC transgenic mice. We report here that PKC theta is a downstream target of Notch3 signaling and that its activation and membrane translocation require a functional pre-TCR in order to trigger NF-kappa B activation in thymocytes and lymphoma cells of transgenic mice. Furthermore, deletion of PKC theta in Notch3-IC transgenic mice reduces the incidence of leukemia, correlating with decreased NF-kappa B activation. This paper therefore suggests that PKC theta mediates the activation of NF-kappa B by pre-TCR in immature thymocytes and contributes to the development of Notch3-dependent T-cell lymphoma.

P38 mitogen-activated protein kinase mediates hexosamine-induced TGFbeta1 mRNA expression in human mesangial cells.

AIMS/HYPOTHESIS: The hexosamine pathway has been implicated in the induction of TGFbeta1 expression and in the pathophysiology of diabetic glomerulopathy. Glucose-induced TGFbeta1 expression is mediated by p38 mitogen-activated-protein-kinase (p38-MAPK) and this kinase is activated in the diabetic glomeruli. We examined whether the p38-MAPK is implicated in hexosamine-induced TGFbeta1 mRNA expression in human mesangial cells. GFAT overexpression induced an increase in p38-MAPK activation after 6 and 12 h incubation in normal glucose, and this was prevented by the GFAT inhibitor azaserine. Furthermore, high glucose enhanced p38-MAPK activation in GFAT tranfected cells ( p

Recombinant human activated protein C for use in severe sepsis.

OBJECTIVE: To review the efficacy and safety of drotrecogin alfa (recombinant human activated protein C) in the treatment of sepsis. DATA SOURCES: Literature was identified through a MEDLINE search (1966-January 2002), the product manufacturer, and the Food and Drug Administration. STUDY SELECTION/DATA EXTRACTION: All relevant information identified from the data sources was evaluated. DATA SYNTHESIS: Drotrecogin alfa reduces coagulation and inflammation in septic patients. A large placebo-controlled clinical trial (n = 1690) of drotrecogin alfa in severely septic patients demonstrated a reduction in mortality (24.7% vs. 30.8%; p = 0.005), with increased bleeding risks (24.9% vs. 17.7%; p <0.001). Patients with more severe sepsis appeared to gain the most benefit. The complete clinical and economic impact of this agent requires further analysis. CONCLUSIONS: Drotrecogin alfa offers a significant advance in the treatment of severe sepsis. Judicious use in appropriate patients is necessary to control cost and maximize clinical benefits.

Signal transduction pathways underlying the expression of tissue factor and thrombomodulin in promyelocytic cells induced to differentiate by retinoid acid and dibutyryl cAMP.

Acute promyelocytic leukaemia (APL) may be associated with disseminated intravascular coagulation, as a result of increased tissue factor (TF) expression and reduced thrombomodulin (TM) expression by APL blast cells. During retinoid acid (RA)- and dibutyryl cAMP (dbcAMP)-induced differentiation of the APL cells, there is a marked up-modulation of both the protein kinase A (PKA) and C (PKC) activities. In order to further assess whether these kinases are intimately associated with both the differentiation process and the regulation of TF and TM expression, we have correlated the modulation of their respective pathways with the extent of differentiation and modulation of these cellular receptors. NB4 cells were incubated with all-trans-RA (ATRA) or dbcAMP for up to 48 h. The contribution of phospholipase C (PLC), inositol phosphate (IP), PKC and PKA in the expression of CD11b, TF and TM was studied by the use of specific inhibitors. Myo-inositol uptake and PKC activity increased in cells induced to differentiate by ATRA but the retinoid did not affect cAMP levels or PKA activity. Under treatment with dbcAMP, PKA activity was increased while inositol uptake and PKC activity remained unchanged. Our results show that the effects of ATRA and dbcAMP on promyelocytic cells are closely related, respectively, to the PLC/IP/PKC and the cAMP/PKA pathways. In cells induced to differentiate by ATRA, CD11b expression seems more closely related to inositol uptake than to PKC activity while the expression of TF and TM show the opposite pattern, which suggests cellular events regulated at a different level within a common signal transduction pathway.

Uncoupling of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells.

1. Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca2+]i peak in a concentration-dependent manner. 2. Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min attenuated the BK-induced IPs formation and Ca2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h. 3. The concentrations of PMA that gave half-maximal (pEC50) inhibition of BK-induced IPs accumulation and an increase in [Ca2+]i were 7.07 M and 7.11 M, respectively. Inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate at 1 microM, did not inhibit these responses. Prior treatment of TECs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 4. In parallel with the effect of PMA on the BK-induced IPs formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-alpha, betaI, betaII, gamma, delta, epsilon, theta and zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, betaI, betaII, gamma, delta, epsilon and theta from cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 6 h treatment caused a partial down-regulation of these PKC isozymes. PKC-zeta was not significantly translocated and down-regulated at any of the times tested. 5. Treatment of TECs with 1 microM PMA for either 30 min or 6 h did not significantly change the KD, and Bmax receptor for BK binding (control: KD=1.7+/-0.3 nM; Bmax=50.5+/-4.9 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. 6. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit independently both responses to BK. The translocation of pKC-alpha, betaI, betaII, delta, epsilon, gamma, and theta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilization in TECs.

Hypoxia alters the subcellular distribution of protein kinase C isoforms in neonatal rat ventricular myocytes.

Cardiac myocytes coexpress multiple protein kinase C (PKC) isoforms which likely play distinct roles in signaling pathways leading to changes in contractility, hypertrophy, and ischemic preconditioning. Although PKC has been reported to be activated during myocardial ischemia, the effect of ischemia/hypoxia on individual PKC isoforms has not been determined. This study examines the effect of hypoxia on the subcellular distribution of individual PKC isoforms in cultured neonatal rat ventricular myocytes. Hypoxia induces the redistribution of PKC alpha and PKC epsilon from the soluble to the particulate compartment. This effect (which is presumed to represent activation of PKC alpha and PKC epsilon) is detectable by 1 h, sustained for up to 24 h, and reversible within 1 h of reoxygenation. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate (D609) prevents the hypoxia-induced redistribution of PKC alpha and PKC epsilon, whereas chelation of intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) blocks the redistribution of PKC alpha, but not PKC epsilon; D609 and BAPTA do not influence the partitioning of PKC alpha and PKC epsilon in normoxic myocytes. Hypoxia, in contrast, decreases the membrane association of PKC delta via a mechanism that is distinct from the hypoxia-induced translocation/activation of PKC alpha/PKC epsilon, since the response is slower in onset, slowly reversible upon reoxygenation, and not blocked by D609 or BAPTA. The hypoxia-induced shift of PKC delta to the soluble compartment does not prevent subsequent 4-beta phorbol 12-myristate-13-acetate-dependent translocation/activation of PKC delta. Hypoxia does not alter the abundance of any PKC isoform nor does it alter the subcellular distribution of PKC lambda. The selective hypoxia-induced activation of PKC isoforms through a pathway involving phospholipase C (PKC alpha/PKC epsilon) and intracellular calcium (PKC alpha) may critically influence cardiac myocyte contractility, gene expression, and/or tolerance to ischemia.

Stimulation of mitogen-activated protein kinase by thyrotropin in astrocytes.

We have recently reported the expression of the thyrotropin (TSH) receptor and the stimulation by TSH of type-II iodothyronine 5'-deiodinase in astrocytes. In these cells, TSH stimulated arachidonate release, but neither cAMP production, nor phosphatidylinositolbisphosphate hydrolysis, as described in the human thyroid gland. Here we report, in contrast to a recent observation made in dog thyroid cells, that TSH stimulates mitogen-activated protein kinase (MAP kinase) in astrocytes. Indeed, TSH increases the tyrosine phosphorylation of the two isoforms of MAP kinase expressed in these cells, in correlation with both a slower electrophoretic migration of the tyrosine phosphorylated species and an enhanced enzymic activity measured on a specific substrate peptide. This stimulation of MAP kinase by TSH was specifically inhibited by incubation of astrocytes in the presence of human blocking anti-(TSH receptor) IgG, and by immunoprecipitation of TSH with monoclonal anti-TSH IgG. In astrocytes, TSH was neither mitogenic by itself, nor modified significantly the basic-fibroblast-growth-factor-induced mitogenesis. The stimulation of MAP kinase by TSH was not affected by treatment with pertussis toxin, suggesting guanine-nucleotide-binding-regulatory protein i/o was not implicated in this TSH effect. Our model will allow the study of the stimulation of MAP kinase by TSH without interference either from cAMP or from phosphoinositide signalling pathways.

Staphylococcus albus-induced protein kinase C translocation in human neutrophils: the effect of opsonization, cytochalasin B, pertussis toxin, intra- and extracellular calcium, and R59022.

Membrane-associated protein kinase C has been proposed to be essential in transmembrane signalling systems activating neutrophils. A main function of the neutrophil is phagocytosis and killing of microorganisms. Nevertheless, previously published reports mainly have described the effect of artificial or soluble stimulators upon neutrophil protein kinase C activity. Therefore, membrane-associated protein kinase C was studied in neutrophils stimulated by Staphylococcus albus. The bacteria were found to induce a striking increase in membrane-associated protein kinase C, an effect which depended upon a previous opsonization of the bacteria. Cytochalasin B, which inhibits phagocytosis, was shown to abrogate S. albus-induced protein kinase C translocation. Chelation of intracellular calcium totally abolished S. albus-induced protein kinase C translocation, a phenomenon that could not exclusively be ascribed to chelation of extracellular calcium. The diacylglycerol kinase inhibitor R59022, which has been reported to increase endogenous diacylglycerol accumulation, nearly doubled the effect of S. albus upon membrane-associated protein kinase C. Pertussis toxin in concentrations which completely inhibited fLMP-induced superoxide generation did not affect S. albus-induced protein kinase C translocation. It is concluded that phagocytosis of S. albus is accompanied by a translocation of protein kinase C to the cell membrane, a phenomenon that relies upon enhanced diacylglycerol production and calcium transients and occurs independently of pertussis toxin-inhibitable G-proteins.

Purification and characterization of rabbit pancreas protein kinase C.

Protein kinase C was purified 6,900-fold from rabbit pancreas with a total yield of 15% by a procedure involving ammonium sulfate fractionation, diethyl aminoethyl ion exchange chromatography, hydroxylapatite chromatography, and finally protamine-agarose affinity chromatography. After these purification steps the protein kinase C preparation contained two major protein bands as judged by silver staining after SDS-polyacrylamide gel electrophoresis: 80 and 69-kDa bands. Monoclonal antibodies directed against bovine brain protein kinase C (alpha- and beta-subtype) recognized only the 80-kDa band. On the other hand, both the 80 and 69-kDa proteins were recognized by a polyclonal monospecific antibody directed against rat brain protein kinase C. Analysis of rabbit pancreas protein kinase C subtypes by means of hydroxylapatite chromatography showed the presence of the III (alpha) subtype as the major subtype. The enzyme depended absolutely on the presence of both phosphatidylserine and Ca2+ for its activity, with apparent Ka values of 3.1 micrograms/ml and 247 microM for phosphatidylserine and Ca2+, respectively. When dioctanoylglycerol or the phorbol ester 12-O-tetradecanoyl-phorbol 13 acetate (TPA) was present, the Ka value for Ca2+ decreased to 10 and 18 microM, respectively. In the presence of the phorbol ester, pancreatic protein kinase C could be activated without added Ca2+. The enzyme also required Mg2+ for its activity. The Ka value was 3.6 mM and maximal activity was reached at 10 mM Mg2+. Pancreatic protein kinase C activity showed a broad pH dependence, with optimal activity at pH 6.75. The Km value for ATP and for histone-H1 was 8.5 microM and 20.4 micrograms/ml, respectively. The present study shows that the kinetic properties of protein kinase C purified from rabbit pancreas closely resemble those found in other tissues.

Regulation of phospholipase A2 activation and arachidonic acid metabolism in an interleukin-3-dependent macrophage-like cell line.

An interleukin 3 (IL-3)-dependent macrophage-like cell line, 11-1-B3, was newly established from CBA/J mouse bone marrow cell cultures. Assay of eicosanoids in the culture supernatants of the intact and [3H]arachidonic acid (AA)-prelabeled cells showed that, after stimulation with the Ca2+ ionophore A23187, the 11-1-B3 cells synthesized and released relatively large amounts of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) but not LTC4. In addition, 11-1-B3 cells showed Ca(2+)-dependent and alkaline pH-optimal phospholipase A2 (PLA2) activity that preferentially hydrolyzed cleavage of sn-2-arachidonyl- but not sn-2-oleoylphosphatidylcholine. The cellular enzyme was distributed with 90% of the activity in the cytosol and 10% in the membrane fraction. Treatment of cells with A23187 for 5-10 min resulted in five- to sevenfold increases in the membrane-associated PLA2 but activity in the cytosol was unchanged. This increase in membrane-associated enzyme activity was transient, returning to the pretreatment distribution after 30 min. In sharp contrast, phorbol myristate acetate (PMA) stimulation failed to induce either eicosanoid release or PLA2 activation, although PMA induced translocation of protein kinase C (PKC) to the membrane fraction within 10 min. The data suggest that increases in cellular Ca2+ directly activate membrane-associated PLA2 and consequently initiate AA metabolism; PKC activation by PMA requires additional steps to activate PLA2, a mechanism that is apparently deficient in the IL-3-dependent M phi-like cells.

Protein kinase C isoforms in human neutrophil cytoplasts.

Granule-poor human neutrophil cytoplasts, prepared without heat or cytochalasin B treatment so as to preserve both motile function and activatable respiratory burst oxidase, were investigated for their content of several isoforms of protein kinase C (PKC). Immunoblotting with isoform-specific rabbit antibodies (Abs) to PKC revealed that both the alpha-specific and the beta(I and II)- specific Abs recognized a protein band of 78 kd comigrating with PKC from rat brain cytosol. The gamma-specific antiserum did not detect any protein of this molecular mass. The cytoplast beta-PKC band was more readily detected than the cytoplast alpha-PKC band. Antibodies to beta I- or beta-II- specific PKC sequences showed the beta II subtype to be the predominant form of beta-PKC, although some beta I was also found. The identity of the 78-kd cytoplast bands as PKC was established by the fact that phorbol ester treatment of intact cytoplasts induced translocation of the bands from cytosol to membrane fractions. However, whereas PKC specific activity was similar in cytoplast lysates and brain cytosol, immunoreactivity of cytoplast alpha- and beta-PKC bands was considerably less than that of rat brain. Hydroxylapatite chromatography of partially purified cytoplast PKC revealed two major peaks of PKC activity precisely coeluting with brain alpha- and beta-PKC and displaying comparable enzymatic activities despite the relatively weak immunoreactivity of cytoplast alpha- and beta-PKC. To our knowledge, this is the first demonstration that human neutrophil-derived cytoplasts contain alpha, beta I, and beta II forms of PKC and that each isoform translocates from cytosol to membrane upon exposure to phorbol ester at concentrations that induce superoxide production. In addition, our evidence raises the possibility that cytoplasts may also possess other isoforms of PKC that we are unable to detect with our alpha, beta, and gamma antibodies. Finally, the granule-poor cytoplasts seem a particularly useful preparation in which to examine the role of individual PKC isoforms in neutrophil activation.

Diminished specific activity of cytosolic protein kinase C in sciatic nerve of streptozocin-induced diabetic rats and its correction by dietary myo-inositol.

The impaired Na(+)-K(+)-ATPase activity in peripheral nerve from diabetic rats is prevented by dietary myo-inositol (MI) supplementation in vivo and corrected by protein kinase C (PKC) agonists in vitro, suggesting that PKC may mediate the effects of nerve MI depletion on Na(+)-K(+)-ATPase activity. However, little is known about the effect of diabetes on PKC activity or peptide in rat peripheral nerve. Therefore, the effect of streptozocin-induced diabetes and dietary MI supplementation on the activity and distribution of PKC in rat sciatic nerve homogenates and cytosolic and particulate fractions was explored with histone phosphorylation assay and Western-blot analysis. PKC activity but not peptide was selectively decreased in the cytosolic fraction by streptozocin-induced diabetes, and this abnormality was partially corrected by dietary MI supplementation. These results suggest that altered MI metabolism may affect nerve PKC specific activity, and this alteration may play a role in reduced Na(+)-K(+)-ATPase activity and blunted regenerative response in diabetic nerve.

Isolation and characterization of a T lymphocyte mutant defective in the protein kinase C signal transduction pathway.

The phorbol ester TPA is a potent protein kinase C (PKC) activator and a cofactor in the activation of the human Jurkat leukemic T cell line. We have studied the implication of the PKC signaling pathway in the process of T cell activation by generating TPA resistant mutants of Jurkat. These mutants were obtained by recovery of cells that survived a growth arrest induced by TPA. Several cellular phenomena dependent on TPA were dramatically altered in the mutated cells. The mutants were unable to form homoaggregates upon TPA stimulation. Moreover, they did not produce interleukin-2 after activation through engagement of the T cell receptor, in the presence of TPA. These results suggest that the PKC signaling pathway activated by TPA is defective in these cells. In an attempt to define and locate the defect present in the mutants, we have analysed the biochemical properties of PKC, the cellular receptor of TPA. The increase in kinase activity and the translocation of the enzyme to the plasma membrane after stimulation by TPA appeared to be normal in the mutants. We hypothesize that a metabolic step, critical for the completion of T cell activation, distinct from protein kinase C, is impaired in the mutant cells.

Cyclic AMP stimulates luteinizing-hormone (lutropin) exocytosis in permeabilized sheep anterior-pituitary cells. Synergism with protein kinase C and calcium.

Sheep anterior-pituitary cells permeabilized with Staphylococcus aureus alpha-toxin were used to investigate the role of cyclic AMP (cAMP) in exocytosis of luteinizing hormone (lutropin, LH) under conditions where the intracellular free Ca2+ concentration ([Ca2+]free) is clamped by Ca2+ buffers. At resting [Ca2+]free (pCa 7), cAMP rapidly stimulated LH exocytosis (within 5 min) and continued to stimulate exocytosis for at least 30 min. When cAMP breakdown was inhibited by 3-isobutyl-1-methylxanthine (IBMX), the concentration giving half-maximal response (EC50) for cAMP-stimulated exocytosis was 10 microM. cAMP-stimulated exocytosis required millimolar concentrations of MgATP, as has been found with Ca2(+)- and phorbol-ester-stimulated LH exocytosis. cAMP caused a modest enhancement of Ca2(+)-stimulated LH exocytosis by decreasing in the EC50 for Ca2+ from pCa 5.6 to pCa 5.9, but had little effect on the maximal LH response to Ca2+. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) dramatically enhanced cAMP-stimulated LH exocytosis by both increasing the maximal effect 5-7-fold and decreasing the EC50 for cAMP to 3 microM. This synergism between cAMP and PMA was further augmented by increasing the [Ca2+]free. Gonadotropin-releasing hormone (gonadoliberin, GnRH) stimulated cAMP production in intact pituitary cells. Since GnRH stimulation is reported to activate PKC and increase the intracellular [Ca2+]free, our results suggest that a synergistic interaction of the cAMP, PKC and Ca2+ second-messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis.