Helicobacter pylori induces interleukin-8 expression in endothelial cells and the signal pathway is protein tyrosine kinase dependent

Helicobacter pylori (HP) infection has been shown to increase gastric mucosal interleukin 8 (IL-8) expression, and whether HP or its toxin induces endothelial cell IL-8 expression is unknown. We aimed to compare the IL-8 expression in endothelial cells after stimulation with HP toxin, tumor necrosis factor alpha (TNF-alpha), and lipopolysaccharide (LPS) and to study their signal pathways. HP or its toxin induced significant IL-8 expression in endothelial cells. HP toxin, TNF-alpha, and LPS also showed a time- and dose-dependent increase in IL-8 expression over the control. Both protein kinase C (PKC) and protein kinase A (PKA) inhibitors had no effect on IL-8 response to these stimuli. Protein tyrosine kinase (PTK) inhibitor genistein at concentrations of 150, 300, and 450 microM dose-dependently reduced LPS- and TNF-alpha-induced IL-8 expression by 29.43, 43.8, and 47.3% and 20.5, 49.9, and 61.8% respectively, whereas HP toxin-induced IL-8 secretion could only be reduced at 450 microM by 35.7%. Geldanamycin, a more potent PTK inhibitor, at doses of 0.5, 1, and 2 microM dose-dependently reduced HP toxin induced endothelial cell IL-8 expression by 24.8, 26, and 44.3% respectively. It is concluded that HP and its toxin can increase IL-8 expression in endothelial cells, and the expression of IL-8 elicited by HP toxin, TNF-alpha, and LPS is partially dependent on PTK but not PKA or PKC activation.     

SPARC inhibits endothelial cell adhesion but not proliferation through a tyrosine phosphorylation-dependent pathway

SPARC, a counteradhesive matricellular protein, inhibits endothelial cell adhesion and proliferation, but the pathways through which these activities are blocked are not known. In this study, we used inhibitors of major signaling proteins to identify mediators through which SPARC exerts its counteradhesive and antiproliferative functions. Pretreatments with the general protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, protected against the inhibitory effect of SPARC on bovine aortic endothelial (BAE) cell spreading by more than 60%. Similar pretreatments with PTK inhibitors significantly blocked the diminishment of focal adhesions by SPARC in confluent BAE cell monolayers, as determined by the formation of actin stress-fibers and the distribution of vinculin in focal adhesion plaques. Inhibition of endothelial cell cycle progression by SPARC and a calcium-binding SPARC peptide, however, was not affected by PTK inhibitors. Inhibition of DNA synthesis by SPARC was not reversed by inhibitors of the activity of protein kinase C (PKC), or of cAMP-dependent protein kinase (PKA), but was sensitive to pertussis (and to a lesser extent, cholera) toxin. The counteradhesive effect of SPARC on endothelial cells is, therefore, mediated through a tyrosine phosphorylation-dependent pathway, whereas its antiproliferative function is dependent, in part, on signal transduction via a G protein-coupled receptor.     

Regulation of N-acetylglucosaminyltransferase V by protein kinases

When 7721 human hepatocarcinoma cells were treated with 100 nM phorbol-12-myristate-13-acetate (PMA), the activity of N-acetylglucosaminyltransferase V(GnT-V) in the cells varied in accordance with the activity of membranous protein kinase C (PKC), but not with that of cytosolic PKC. Quercetin, a non-specific inhibitor of Ser/Thr protein kinase, and D-sphingosine and staurosporine, two specific inhibitors of PKC, blocked the activation of membranous PKC and GnT-V by PMA. Among the three inhibitors, quercetin was least effective. The inhibitory rates of quercetin and staurosporine toward membranous PKC and GnTV were proportional to the concentrations of the two inhibitors. The activities of GnTV and membranous protein kinase A (PKA) were also induced in parallel by dibutyryl cAMP (db-cAMP) and this induction was blocked by a specific PKA inhibitor. When cell free preparations of 7721 cells and human kidney were treated with alkaline phosphatase (ALP) to remove the phosphate groups, the GnTV activities were decreased. These results suggest that GnTV may be activated by membranous PKC or PKA, indirectly or directly, via phosphorylation of Ser/Thr residues.     

Stimulation of cell elongation in teleost rod photoreceptors by distinct protein kinase inhibitors

The rod photoreceptors of teleost retinas elongate in the light. To characterize the role of protein kinases in elongation, pharmacological studies were carried out with rod fragments consisting of the motile inner segment and photosensory outer segment (RIS-ROS). Isolated RIS-ROS were cultured in the presence of membrane-permeant inhibitors that exhibit selective activity toward specific serine/threonine protein kinases. We report that three distinct classes of protein kinase inhibitors stimulated elongation in darkness: (1) cyclic-AMP-dependent protein kinase (PKA)-selective inhibitors (H-89 and KT5720), (2) a protein kinase C (PKC)-selective inhibitor (GF 109203X) that affects most PKC isoforms, and (3) a kinase inhibitor (H-85) that does not affect PKC and PKA in vitro. Other kinase inhibitors tested neither stimulated elongation in darkness nor inhibited light-induced elongation; these include the myosin light chain kinase inhibitors ML-7 and ML-9, the calcium-calmodulin kinase II inhibitor KN-62, and inhibitors or activators of diacylglycerol-dependent PKCs (sphingosine, calphostin C, chelerythrine, and phorbol esters). The myosin light chain kinase inhibitors as well as the PKA and PKC inhibitors H-89 and GF 109203X all enhanced light-induced elongation. These observations suggest that light-induced RIS-ROS elongation is inhibited by both PKA and an unidentified kinase or kinases, possibly a diacylglycerol-independent form of PKC.     

v-Abl protein tyrosine kinase (PTK) mediated suppression of apoptosis is associated with the up-regulation of Bcl-XL

We demonstrated previously that the activation of v-Abl protein tyrosine kinase (PTK) in IC.DP murine pre-mast cells resulted in suppression of apoptosis after withdrawal of interleukin 3 (IL-3), that protein kinase C (PKC) translocated to the nucleus 6 h after v-Abl PTK activation and that inhibition of PKC restored apoptosis after IL-3 deprivation in the presence of v-Abl PTK activity. Here we demonstrate that v-Abl PTK activation is followed by an approximately twofold increase in mRNA level of Bcl-XL by 6 h and a corresponding increase in Bcl-XL protein level by 24 h. Bcl-xL RNA and protein decreased in IL-3 deprived cells in the absence of v-Abl PTK activity. Exposure of cells with v-Abl PTK active to the PKC inhbitor calphostin C (125 ng/ml) prevented the increase in Bcl-xL protein and resulted in apoptosis. No changes in Bax or Bcl-2 protein level were noted after IL-3 withdrawal and/or activation of v-Abl PTK. Bak was barely detectable and Bad protein level decreased in cells undergoing apoptosis. The data suggest that suppression of apoptosis by v-Abl PTK in the absence of IL-3 is associated with PKC signalling and the upregulation of Bcl-xL in IC.DP cells.     

Functional modulation of P2X2 receptors by cyclic AMP-dependent protein kinase

It is generally believed that protein phosphorylation is an important mechanism through which the functions of voltage- and ligand-gated channels are modulated. The intracellular carboxyl terminus of P2X2 receptor contains several consensus phosphorylation sites for cyclic AMP (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC), suggesting that the function of the P2X2 purinoceptor could be regulated by the protein phosphorylation. Whole-cell voltage-clamp recording was used to record ATP-evoked cationic currents from human embryonic kidney (HEK) 293 cells stably transfected with the cDNA encoding the rat P2X2 receptor. Dialyzing HEK 293 cells with phorbol 12-myristate 13-acetate, a PKC activator, failed to affect the amplitude and kinetics of the ATP-induced cationic current. The role of PKA phosphorylation in modulating the function of the P2X2 receptor was investigated by internally perfusing HEK 293 cells with 8-bromo-cAMP or the purified catalytic subunit of PKA. Both 8-bromo-cAMP and PKA catalytic subunit caused a reduction in the magnitude of the ATP-activated current without affecting the inactivation kinetics and the value of reversal potential. Site-directed mutagenesis was also performed to replace the intracellular PKA consensus phosphorylation site (Ser431) with a cysteine residue. In HEK 293 cells expressing (S431C) mutant P2X2 receptors, intracellular perfusion of 8-bromo-cAMP or purified PKA catalytic subunit did not affect the amplitude of the ATP-evoked current. These results suggest that as with other ligand-gated ion channels, protein phosphorylation by PKA could play an important role in regulating the function of the P2X2 receptor and ATP-mediated physiological effects in the nervous system.     

Role of protein tyrosine kinase on regulation of trabecular meshwork and ciliary muscle contractility

PURPOSE: Trabecular meshwork and ciliary muscle express properties of smooth muscle cells. The contractility of trabecular meshwork and ciliary muscle is differently modulated by various agents. To reveal contractile regulatory processes, the effects of activation and inhibition of protein tyrosine kinases (PTKs) and their interaction with other protein kinases on contractility were measured. METHODS: Measurements of isometric tension were performed on isolated bovine trabecular meshwork and ciliary muscle strips using a custom-built, electromagnetic, force-length transducer. Protein tyrosine kinase (PTK) was stimulated by epidermal growth factor (EGF) and was inhibited by genistein or tyrphostin 51. Protein kinase C (PKC) was inhibited by chelerythrine or NPC-15437 and protein kinases A and G (PKA-PKG) by H8. RESULTS: Isolated strips were precontracted by applying carbachol 10(-6) M for 30 minutes (100% carbachol maximum contraction). Inhibition of PTK evoked a maximum relaxation of 79.2+/-4.2% in trabecular meshwork and of 38.1+/-3.1% in ciliary muscle (n=8). Inhibition of PKC or PKA-PKG induced relaxations only in trabecular meshwork. When PTK and PKC or PKA-PKG were inhibited, the relaxation induced by inhibition of PTK was additive to inhibition of the other protein kinases. Stimulation of a receptor with PTK activity by EGF induced a relaxation in trabecular meshwork and a contraction in ciliary muscle precontracted by carbachol. When trabecular meshwork and ciliary muscle were activated by EGF, inhibition of PTK by genistein relaxed the cell preparations. CONCLUSIONS: Inhibition of PTK induces more prominent relaxation in trabecular meshwork than in ciliary muscle. The effects of inhibition of PTK on relaxation are independent of inhibition of PKC and PKA-PKG. The signaling cascade after activation of a tyrosine kinase receptor by EGF is differently modulated in trabecular meshwork and ciliary muscle. The effect of genistein on relaxation is probably not directly related to the EGF receptor. PTK inhibitors are possible agents for the development of novel antiglaucoma drugs.     

Inhibition of eukaryote protein kinases and of a cyclic nucleotide-binding phosphatase by prenylated xanthones

A series of prenylated xanthones are variously potent inhibitors of the catalytic subunit (cAK) of rat liver cyclic AMP-dependent protein kinase (PKA), rat brain Ca2+ and phospholipid-dependent protein kinase C (PKC), chicken gizzard myosin light chain kinase (MLCK), wheat embryo Ca2+-dependent protein kinase (CDPK) and potato tuber cyclic nucleotide-binding phosphatase (Pase). The prenylated xanthones examined are mostly derivatives of alpha-mangostin in which the 3-hydroxyl and 6-hydroxyl are variously substituted with groups R or R', respectively, or derivatives of 3-isomangostin (mangostanol) in which the 9-hydroxyl is substituted with groups R' or the prenyl side chain is modified. The most potent inhibitors of cAK have non-protonatable and relatively small R' and R groups. Conversely, the most potent inhibitors of PKC and MLCK have bulkier and basic R' groups. Some prenylated xanthones are also potent inhibitors of CDPK. PKC and cAK are competitively inhibited by particular prenylated xanthones whereas the compounds that are the most potent inhibitors of MLCK and CDPK are non-competitive inhibitors. Prenylated xanthones having relatively small and non-protonatable R' and R groups inhibit a high-affinity cyclic nucleotide binding Pase in a non-competitive fashion.     

Modulation of TTX-R INa by PKC and PKA and their role in PGE2-induced sensitization of rat sensory neurons in vitro

A tetrodotoxin-resistant voltage-gated Na+ current (TTX-R INa) appears to be the current primarily responsible for action potential generation in the cell body and terminals of nociceptive afferents. Although other voltage-gated Na+ currents are modulated by the activation of protein kinase C (PKC), protein kinase A (PKA), or both, the second messenger pathways involved in the modulation of TTX-R INa are still being defined. We have examined the modulation of TTX-R INa in isolated sensory neurons with whole-cell voltage-clamp recording. Activation of either PKC or PKA increased TTX-R INa. PKA activation also produced a leftward shift in the conductance-voltage relationship of TTX-R INa and an increase in the rates of current activation, deactivation, and inactivation. Inhibitors of PKC decreased TTX-R INa, whereas inhibitors of PKA had no effect on the current. Investigating the interaction between PKC and PKA revealed that although inhibitors of PKA had little effect on PKC-induced modulation of TTX-R INa, inhibitors of PKC significantly attenuated PKA-induced modulation of the current. Finally, although PGE2-induced modulation of TTX-R INa was more similar to PKA-induced modulation of the current than to PKC-induced modulation, PGE2-induced effects were inhibited by inhibitors of both PKC and PKA. Thus, although TTX-R INa is a common target for cellular processes involving the activation of either PKA or PKC, PKC activity is necessary to enable subsequent PKA-mediated modulation of TTX-R INa.     

Pseudomonas pyocyanin increases interleukin-8 expression by human airway epithelial cells

Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1alpha. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease.     

Different hippocampal activity profiles for PKA and PKC in spatial discrimination learning.

Protein kinases are considered essential for the processing and storage of information in the brain. However, the dynamics of protein kinase activation in the hippocampus during spatial learning are poorly understood. In this study, rats were trained to learn a holeboard spatial discrimination task and the activity profiles for cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and Ca2+/ phospholipid-dependent protein kinase C (PKC) in the hippocampus were examined. Hippocampal PKA activity increased rapidly on Day 1 of spatial learning and remained moderately high at later stages of acquisition. In contrast, PKC activity increased in particulate fractions compared with cytosolic fractions after habituation training and was maximal at Day 3 of spatial acquisition. The results establish a temporal dissociation between PKA and PKC during acquisition of spatial discrimination learning. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Modulation of protein kinase C and cAMP-dependent protein kinase by delta-opioid

Modulation of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) activities by delta-opioid receptor specific agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) was investigated in neuroblastoma x glioma hybrid NG 108-15 cells. DPDPE activated PKC in a dose-dependent manner, with the maximal response at 5 min. The DPDPE-stimulated PKC activation could be blocked by naltrindole. The activation of PKC by DPDPE was dependent on Ca2+ and was inhibited by chelerythrine chloride (10 microM), but not by H89 (1 microM). Pretreatment of NG 108-15 cells with pertussis toxin (100 ng/ml for 24 h) completely abolished DPDPE-stimulated PKC activation. In contrast to the result from the acute treatment with DPDPE, which had no significant effect on PKA activity, chronic treatment of DPDPE (1 microM for 24 h) increased PKA activity, but reduced the basal activity of PKC. These results demonstrated that DPDPE differentially modulated PKC and PKA activities via a receptor-mediated, PTX sensitive pathway.     

Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1

Phosphorylation sites in members of the protein kinase A (PKA), PKG, and PKC kinase subfamily are conserved. Thus, the PKB kinase PDK1 may be responsible for the phosphorylation of PKC isotypes. PDK1 phosphorylated the activation loop sites of PKCzeta and PKCdelta in vitro and in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner in vivo in human embryonic kidney (293) cells. All members of the PKC family tested formed complexes with PDK1. PDK1-dependent phosphorylation of PKCdelta in vitro was stimulated by combined PKC and PDK1 activators. The activation loop phosphorylation of PKCdelta in response to serum stimulation of cells was PI 3-kinase-dependent and was enhanced by PDK1 coexpression.     

Satellite cell myogenesis is highly stimulated by the kinase inhibitor iso-H7: comparison with HA1004 and staurosporine effects

Differentiation of rat satellite cells, measured by cell fusion into myotubes and isozymic conversion of creatine kinase and phosphoglycerate mutase, was shown to be highly increased in the presence of 1-(5-isoquinolinylsulfonyl)-3-methylpiperazine (iso-H7). This substance inhibited both protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) activities with similar IC50 between 22 and 34 microM. Iso-H7, as well as the PKA inhibitor HA1004 increased myogenic differentiation without altering the proliferation of satellite cells, whereas the proliferation and the differentiation of these cells were inhibited by the PKC inhibitor staurosporine. Our results suggest a predominant negative control of PKA on satellite cell myogenesis.     

Regulation of L-type calcium channels by protein tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells

The effect of protein tyrosine kinases (PTKs) on L-type calcium channel currents was studied in cultured rat and human retinal pigment epithelial cells. Barium currents through L-type channels were measured in the perforated patch-clamp technique and identified by using the L-type calcium channel opener Bay K8644 (10(-6) M). Application of the PTK blockers genistein (5 x 10(-6) M) or lavendustin A (5 x 10(-6) M) led to a decrease of L-type currents. The inactive genistein analog daidzein (10(-5) M) showed no effect on calcium channels. Intracellular application of pp60(c-src) (30 U/ml) via the patch-pipette during the conventional whole-cell configuration led to an increase of L-type currents. The protein kinase A and protein kinase G blocker H9 (10(-6) M) showed no effect on L-type currents; genistein reduced the current in the presence of H9. The protein kinase C (PKC) blocker chelerythrine (10(-5) M) reduced the L-type current; additional inhibition of PTK by lavendustin showed an additional reduction of currents. Intracellular application of myristoylated PKC substrate (5 x 10(-5) M) for PKC inhibition led to a fast rundown of L-type current amplitudes. Intracellularly applied myristoylated PKC substrate (10(-4) M) together with pp60(c-src) showed no effect on L-type current. Up-regulation of PKC by 10(-6) M phorbol-12-myristate-13-acetate (PMA) had no effect on the L-type current amplitude. However, genistein in cells pretreated with PMA led to an increase of the L-type currents. Intracellular application of pp60(c-src) in PMA-treated cells led to a reduction of L-type currents. We conclude that in the resting cell, PTK and PKC regulate L-type calcium channels in an additive manner. L-type channels appeared as a site of integration of PTK activation and of PKC-dependent pathways. The activity of PKC determines whether PTK decreases or increases L-type channel activity.     

Multiple second-messenger system modulation of voltage-activated calcium currents in teleost retinal horizontal cells

Two voltage-activated calcium currents, a transient T-type and a PL-sustained type, have been measured in isolated, cultured white bass horizontal cells. These two voltage-activated calcium currents were found to be modulated by two independent second-messenger systems. Furthermore, activation of either second-messenger system led to similar changes in calcium current activity. Activation of the cyclic AMP second-messenger pathway or the sn-1,2-diacylglycerol (DAG) second-messenger system resulted in a significant decrease in the amplitude of the transient current and a simultaneous large increase in the amplitude of the sustained current. Both second-messenger systems achieved their effects through protein phosphorylation. The cyclic AMP pathway resulted in the activation of protein kinase A (PKA) and the DAG pathway worked to activate protein kinase C (PKC). Two protein kinase inhibitors were analyzed in this study for their ability to inhibit second-messenger activated protein kinase activity and separate the two pathways. The peptide cyclic AMP-dependent protein kinase inhibitor and staurosporine were found to be nonspecific at high concentrations and inhibited both second-messenger pathways. At low concentrations however, staurosporine specifically inhibited only PKC, whereas adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase inhibitor was selective for PKA. Both second-messenger systems were activated by the neuromodulator, dopamine. Thus one agonist can initiate multiple second-messenger systems leading to similar changes in voltage-activated calcium current activity. The modulatory action on calcium currents produced by one second-messenger system added to the modulatory action resulting from activation of the other second-messenger system. The effect is to alter the magnitude of the horizontal cell calcium currents.     

Atherogenic lipoproteins enhance mesangial cell expression of platelet-derived growth factor: role of protein tyrosine kinase and cyclic AMP-dependent protein kinase A

Mesangial cell proliferation and extracellular matrix accumulation are fundamental in the pathogenesis of glomerulosclerosis. Platelet-derived growth factor (PDGF) is a major cytokine involved in mesangial cell proliferation, and its increased expression is seen in glomerular injury. Atherogenic lipoproteins stimulate mesangial cell proliferation and induce glomerular injury in experimental animals. We examined the effect of low-density lipoprotein (LDL) and its more atherogenic oxidized forms, minimally modified LDL (mm-LDL) and oxidized LDL (ox-LDL) on mesangial cell PDGF mRNA expression. Incubation with 2.5 to 25 microg/ml LDL or mm-LDL for 1 to 4 hours stimulated mesangial cell PDGF mRNA expression (mm-LDL 2 to 3 times greater than LDL); ox-LDL had no effect. Similarly, both LDL and mm-LDL induced mesangial cell DNA synthesis (mm-LDL 1.5 to 2 times greater). In further studies evaluating key associated intracellular signal transduction mechanisms, the protein tyrosine kinase (PTK) inhibitors herbimycin and genistein markedly decreased basal and lipoprotein-induced PDGF mRNA expression. Both pertussis toxin and isoproterenol, cyclic AMP-generating substances, stimulated PDGF mRNA expression. Preincubation with H-8 or H-89, cyclic AMP-dependent protein kinase A (PKA) inhibitors, blocked the lipoprotein-induced PDGF message, whereas preincubation with calphostin C, a protein kinase C inhibitor, did not alter LDL- or mm-LDL-mediated PDGF mRNA expression. These data suggest that the accumulation of atherogenic lipoproteins and their endogenous oxidized forms within the glomerulus may regulate mesangial cell PDGF expression and related cellular responses. These events appear to be modulated by signal transduction pathways involving PTK and PKA.