GF 109203X Research Articles

Hyperglycemia in vitro attenuates insulin-stimulated chemokinesis in normal human neutrophils. Role of protein kinase C activation.

This study was performed to test whether the inhibitory effect of elevated D-glucose concentrations on insulin-stimulated chemokinesis in normal human neutrophils is mediated by increase in protein kinase C (PKC) activity. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) at 0-100 nM dose-dependently inhibited neutrophil random locomotion in the absence of insulin. Sub-optimal concentrations of PMA (0.1-0.5 nM) inhibited the chemokinetic effect of 160 microU/mL insulin in a dose-dependent way. The specific PKC inhibitor bisindolylmaleimide (GF 109203x) did not affect the insulin-stimulated chemokinesis at 5 mM glucose but restored the chemokinetic effect of insulin at 15 mM glucose. These results therefore suggest that glucose-induced PKC activation may mediate the inhibitory effects of high glucose levels on insulin-stimulated chemokinesis in normal human neutrophils.

Source(s) of activator calcium for noradrenaline-induced vasoconstriction in the perfused rabbit isolated ovarian vascular bed A role for tyrosine kinase

The effects of Ca 2+ withdrawal and agents affecting Ca 2+ translocation on α 1-adrenoceptor-mediated vasoconstrictor responses in the perfused rabbit ovarian vascular bed were studied. Noradrenaline-induced vasoconstriction was lost in a Ca 2+-free Krebs’ solution, and the rate of loss of the response was accelerated by EGTA (2 mM). Noradrenaline-induced vasoconstriction and SDZ NVI 085-induced vasoconstriction were concentration-dependently inhibited by verapamil and nifedipine. These agents were, however, more effective against KCl-induced responses. Cyclopiazonic acid, an intracellular Ca 2+ depletor, concentration-dependently inhibited noradrenaline-induced responses and abolished the response in Ca 2+-free Krebs’ solution. GF 109203X and polymyxin B, inhibitors of protein kinase C (PKC), had no significant effect on noradrenaline-induced responses. Tyrosine kinase inhibitors, genistein and erbstatin, inhibited noradrenaline-induced vasoconstriction in the perfused rabbit ovarian vascular bed. The results would suggest that both extracellular Ca 2+ and intracellular Ca 2+ participate in noradrenaline-induced vasoconstrictor responses in the perfused rabbit ovarian vascular bed. The results would also suggest that tyrosine kinase and not protein kinase C activation has a role in such effects.

The levels of leukemia inhibitory factor mRNA in a Schwann cell line are regulated by multiple second messenger pathways.

Axotomy of sympathetic and sensory neurons leads to changes in their neuropeptide phenotypes. These changes are mediated in part by the induction of leukemia inhibitory factor (LIF) by nonneuronal cells. In the present study, we identified satellite/Schwann cells as a possible source of the injury-induced LIF. Using a Schwann cell line, SC-1 cells, we examined mechanisms of LIF induction. LIF mRNA levels increased rapidly when the cells were treated with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, phorbol 12-myristate 13-acetate (PMA), or A23187. Among these reagents, PMA was the most efficacious. Inhibition of protein kinase C (PKC) by GF-1 09203X significantly reduced the PMA-induced LIF mRNA levels. As PKC is known to activate the extracellular signal-regulated kinase (ERK) signaling pathway, the involvement of this pathway in the PMA-stimulated induction of LIF mRNA was examined. Phosphorylation of ERKs was increased following PMA treatment in SC-1 cells. Moreover, inhibition of ERK kinase activity by PD98059 dramatically reduced PMA-stimulated phosphorylation of ERKs and induction of LIF mRNA. These results indicate that LIF mRNA levels can be regulated by ERK activation via stimulation of PKC in Schwann cells.

Evidence that cytosolic phospholipase A 2 is down-regulated by protein kinase C in intact human platelets stimulated with fluoroaluminate

We reported that protein kinase C (PKC) inhibitors increase the release of arachidonic acid induced by fluoroaluminate (AlF 4 −), an unspecific G-protein activator, in intact human platelets. Now we demonstrate that this effect is independent of the extracellular Ca 2+ concentration and that AlF 4 −-induced release of AA is abolished by BAPTA, an intracellular Ca 2+ chelator, even in the presence of GF 109203X, a specific and potent PKC inhibitor. This compound also blocks the liberation of the secretory phospholipase A 2 in the extracellular medium, indicating that this enzyme is not involved in the potentiation of arachidonic acid by PKC inhibitors. On the other hand, the latter effect is completely abolished by treatment of platelets with AACOCF 3, a specific inhibitor of cytosolic phospholipase A 2 (cPLA 2). These observations indicate that cPLA 2 is responsible for the AlF 4 −-induced release of arachidonic acid by a mechanism that is down-regulated by PKC.

Interplay between the NO pathway and elevated [Ca2+]i enhances ciliary activity in rabbit trachea.

1. Average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were simultaneously measured in rabbit airway ciliated cells in order to elucidate the molecular events that lead to ciliary activation by purinergic stimulation. 2. Extracellular ATP and extracellular UTP caused a rapid increase in both [Ca2+]i and CBF. These effects were practically abolished by a phospholipase C inhibitor (U-73122) or by suramin. 3. The effects of extracellular ATP were not altered: when protein kinase C (PKC) was inhibited by either GF 109203X or chelerythrine chloride, or when protein kinase A (PKA) was inhibited by RP-adenosine 3', 5'-cyclic monophosphothioate triethylamine (Rp-cAMPS). 4. Activation of PKC by phorbol 12-myristate, 13-acetate (TPA) had little effect on CBF or on [Ca2+]i, while activation of PKA by forskolin or by dibutyryl-cAMP led to a small rise in CBF without affecting [Ca2+]i. 5. Direct activation of protein kinase G (PKG) with dibutyryl-cGMP had a negligible effect on CBF when [Ca2+]i was at basal level. However, dibutyryl-cGMP strongly elevated CBF when [Ca2+]i was elevated either by extracellular ATP or by ionomycin. 6. The findings suggest that the initial rise in [Ca2+]i induced by extracellular ATP activates the NO pathway, thus leading to PKG activation. In the continuous presence of elevated [Ca2+]i the stimulated PKG then induces a robust enhancement in CBF. In parallel, activated PKG plays a central role in Ca2+ influx via a still unidentified mechanism, and thus, through positive feedback, maintains CBF close to its maximal level in the continuous presence of ATP.

Tumor necrosis factor-alpha stimulates mucin secretion and cyclic GMP production by guinea pig tracheal epithelial cells in vitro.

Tumor necrosis factor (TNF)-alpha, a pluripotent cytokine implicated in the pathogenesis of airway inflammation, has been shown to provoke hypersecretion of mucin by airway epithelial cells in vitro. In this study, we investigated potential signaling pathways mediating TNF-alpha-induced mucin secretion using guinea pig tracheal epithelial (GPTE) cells in air-liquid interface culture. Exogenously applied TNF-alpha (human recombinant) stimulated mucin secretion in a concentration-dependent manner, with maximal effects at 10 to 15 ng/ml (286 to 429 U/ml). The pathway of stimulated secretion appeared to involve generation of intracellular nitric oxide (NO), activation of soluble guanylate cyclase (GC-S), production of cyclic guanosine monophosphate (cGMP), and activation of cGMP-dependent protein kinase (PKG). TNF-alpha increased production of nitrite and nitrate by GPTE cells; both mucin secretion and cGMP production were attenuated by NG-monomethyl-L-arginine (1 mM), a competitive inhibitor of nitric oxide synthase (NOS), or by the GC-S inhibitor LY83583 (50 microM); and mucin secretion in response to TNF-alpha or to the cGMP analogue dibutyryl cGMP (100 and 500 microM) was attenuated by the specific PKG inhibitor KT5823 (1 microM). Increased mucin secretion and increased cGMP production in response to TNF-alpha both appeared to be mediated by a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC), and by protein kinase C (PKC), since both responses were attenuated by either D609 (10 and 20 microg/ml), a specific PC-PLC inhibitor, or by each of three PKC inhibitors: Calphostin C (0.3 and 0.5 microM), bisindoylmaleimide (GF 109203X, Go 6850; 20 nM), or Ro31-8220 (10 microM). Collectively, the results suggest that TNF-alpha stimulates secretion of mucin by GPTE cells via a mechanism(s) dependent on PC-PLC and PKC, and involving activation of NOS, generation of NO, production of cGMP, and activation of PKG.

The essential role of phorbol ester-sensitive protein kinase C isoforms in activation-induced cell death of Th1 cells.

Th1 and Th2 cells, which were induced from naive T cells of TCR-transgenic mice, showed differential sensitivity to activation-induced cell death (AICD) triggered by stimulation with anti-CD3 monoclonal antibody. The Th1 cells showed more rapid AICD than Th2 cells. This accelerated AICD of Th1 cells was strongly blocked by protein kinase C (PKC) inhibitors (H-7 or GF 109203X). Moreover, long-term treatment of Th1 cells with phorbol 12-myristate 13-acetate (PMA) caused the abrogation of anti-CD3-induced AICD in parallel with the disappearance of PMA-sensitive PKC isoforms such as PKC alpha, gamma, epsilon and theta. Therefore, it was clearly demonstrated that PMA-sensitive PKC isoforms are essential for AICD of Th1 cells. The different susceptibility to AICD between Th1 and Th2 cells was not due to their differential expression levels of PMA-sensitive PKC isoforms but appeared to be due to their differential requirement for PMA-sensitive isoforms in the up-regulation of Fas ligand which is involved in suicide killing of activated Th1 cells.

The essential role of phorbol ester-sensitive protein kinase C isoforms in activation-induced cell death of Th1 cells

Th1 and Th2 cells, which were induced from naive T cells of TCR-transgenic mice, showed differential sensitivity to activation-induced cell death (AICD) triggered by stimulation with anti-CD3 monoclonal antibody. The Th1 cells showed more rapid AICD than Th2 cells. This accelerated AICD of Th1 cells was strongly blocked by protein kinase C (PKC) inhibitors (H-7 or GF 109203X). Moreover, long-term treatment of Th1 cells with phorbol 12-myristate 13-acetate (PMA) caused the abrogation of anti-CD3-induced AICD in parallel with the disappearance of PMA-sensitive PKC isoforms such as PKC alpha, gamma, epsilon and theta. Therefore, it was clearly demonstrated that PMA-sensitive PKC isoforms are essential for AICD of Th1 cells. The different susceptibility to AICD between Th1 and Th2 cells was not due to their differential expression levels of PMA-sensitive PKC isoforms but appeared to be due to their differential requirement for PMA-sensitive isoforms in the up-regulation of Fas ligand which is involved in suicide killing of activated Th1 cells.

Regulation of cell surface expression of Fas (CD95) ligand and susceptibility to Fas (CD95)-mediated apoptosis in activation-induced T cell death involves calcineurin and protein kinase C, respectively.

We show that an influenza hemagglutinin-specific CD4+ murine T cell hybridoma (IP-12-7) enters the apoptotic suicide program via the Fas ligand (FasL)/Fas-mediated pathway upon T cell receptor (TCR) stimulation. These cells express Fas and FasL mRNA, cell surface Fas and intracellular FasL, but do not enter apoptosis upon Fas ligation prior to TCR stimulation. TCR stimulation additionally results in protein synthesis-dependent cell surface expression of the preformed FasL. Addition of phorbol dibutyrate (PBu2) alone was sufficient to induce susceptibility to Fas ligation induced apoptosis, while addition of both PBu2 and calcium ionophore A23187 were required to induce FasL cell surface expression. Addition of cyclosporin A completely inhibited TCR-mediated death and FasL cell surface up-regulation, but had no effect on apoptosis induced directly by Fas ligation following TCR stimulation. Inhibitors of protein kinase C (PKC) (Gö 6976 and GF 109203X) completely inhibited TCR-induced susceptibility to Fas ligation, but only partially inhibited TCR-induced cell surface expression of FasL. PKC isoenzymes alpha, beta, delta and zeta were expressed by this cell line and only the alpha and betaI isoforms translocated to the membrane fraction upon TCR stimulation. Our data suggest that in activation-induced T cell apoptosis PKC is involved in pathways that mediate the acquisition of Fas susceptibility, while calcineurin is required for cell surface expression of the preformed FasL.

Mechanism of endothelial nitric oxide-dependent vasorelaxation induced by wine polyphenols in rat thoracic aorta.

The mechanisms by which red wine polyphenolic compounds (RWPCs) induced endothelium-dependent relaxation were investigated in rat thoracic aorta rings with endothelium. RWPCs produced relaxation that was prevented by the nitric oxide (NO) synthase inhibitor, N(omega)-nitro-L-arginine-methyl-ester. This relaxation was abolished in the absence of extracellular calcium in the medium or in the presence of the Ca2+ entry blocker, La3+, but it was not affected by the nonselective K+ channels blocker, tetrabutylammonium. N-Ethyl-maleimide (NEM), a sulfhydryl alkylating agent, abolished vasorelaxation produced by RWPCs and acetylcholine but not that produced either by the sarcoendoplasmic reticulum Ca2+-adenosine triphosphatase (ATPase) pump inhibitor, cyclopyazonic acid (CPA) or the calcium ionophore, ionomycin. Neither pertussis toxin (PTX) nor cholera toxin (CTX) inhibited the vasorelaxant effect of RWPC. The effect of RWPC was not affected by the phospholipase C (PLC) blocker, L-alpha-glycerophospho-D-myo-inositol 4-monophosphate (Gro-pip), and the phospholipase A2 pathway blockers, quinacrine and ONO-RS-082. Finally, the protein kinase C (PKC) inhibitor, GF 109203X, and tyrosine kinase inhibitors, tyrphostin A-23 and genistein, did not impair the response to RWPCs. These results suggest that RWPCs produce endothelium-NO-derived vasorelaxation through an extracellular Ca2+-dependent mechanism via an NEM-sensitive pathway. They also show that PTX- or CTX-sensitive G proteins, activation of PLC or PLA2 pathways, PKC, or tyrosine kinase may not be involved.

A2B Adenosine and P2Y2 Receptors Stimulate Mitogen-activated Protein Kinase in Human Embryonic Kidney-293 Cells: CROSS-TALK BETWEEN CYCLIC AMP AND PROTEIN KINASE C PATHWAYS

Mitogen-activated protein kinase (MAPK) cascades underlie long-term mitogenic, morphogenic, and secretory activities of purinergic receptors. In HEK-293 cells, N-ethylcarboxamidoadenosine (NECA) activates endogenous A2BARs that signal through Gs and Gq/11. UTP activates P2Y2 receptors and signals only through Gq/11. The MAPK isoforms, extracellular-signal regulated kinase 1/2 (ERK), are activated by NECA and UTP. H-89 blocks ERK activation by forskolin, but weakly affects the response to NECA or UTP. ERK activation by NECA or UTP is unaffected by a tyrosine kinase inhibitor (genistein), attenuated by a phospholipase C inhibitor (U73122), and is abolished by a MEK inhibitor (PD098059) or dominant negative Ras. Inhibition of protein kinase C (PKC) by GF 109203X failed to block ERK activation by NECA or UTP, however, another PKC inhibitor, Ro 31-8220, which unlike GF 109203X, can block the zeta-isoform, and prevents UTP- but not NECA-induced ERK activation. In the presence of forskolin, Ro 31-8220 loses its ability to block UTP-stimulated ERK activation. PKA has opposing effects on B-Raf and c-Raf-1, both of which are found in HEK-293 cells. The data are explained by a model in which ERK activity is modulated by differential effects of PKC zeta and PKA on Raf isoforms.

Regulation of cell surface expression of Fas (CD95) ligand and susceptibility to Fas (CD95)-mediated apoptosis in activation-induced T cell death involves calcineurin and protein kinase C, respectively

We show that an influenza hemagglutinin-specific CD4+ murine T cell hybridoma (IP-12-7) enters the apoptotic suicide program via the Fas ligand (FasL) / Fas-mediated pathway upon T cell receptor (TCR) stimulation. These cells express Fas and FasL mRNA, cell surface Fas and intracellular FasL, but do not enter apoptosis upon Fas ligation prior to TCR stimulation. TCR stimulation additionally results in protein synthesis-dependent cell surface expression of the preformed FasL. Addition of phorbol dibutyrate (PBu2) alone was sufficient to induce susceptibility to Fas ligation induced apoptosis, while addition of both PBu2 and calcium ionophore A23187 were required to induce FasL cell surface expression. Addition of cyclosporin A completely inhibited TCR-mediated death and FasL cell surface up-regulation, but had no effect on apoptosis induced directly by Fas ligation following TCR stimulation. Inhibitors of protein kinase C (PKC) (Gö 6976 and GF 109203X) completely inhibited TCR-induced susceptibility to Fas ligation, but only partially inhibited TCR-induced cell surface expression of FasL. PKC isoenzymes α, β, δ and ζ were expressed by this cell line and only the α and βI isoforms translocated to the membrane fraction upon TCR stimulation. Our data suggest that in activation-induced T cell apoptosis PKC is involved in pathways that mediate the acquisition of Fas susceptibility, while calcineurin is required for cell surface expression of the preformed FasL.

A high throughput system for the evaluation of protein kinase C inhibitors based on Elk1 transcriptional activation in human astrocytoma cells.

Protein kinase C (PKC) designates a family of kinases that regulate many essential functions including cell growth and differentiation. The tight regulation of PKC activity is crucial for maintaining normal cellular proliferation and excessive activity leads to abnormal or uncontrolled cell growth. Recent reports indicate that malignant glioma cell lines express 100 to 1000-fold higher PKC activity when compared to non-neoplastic astrocytes. This high activity correlates well with the proliferation of tumor cells in vitro. We recently reported on the anti-proliferative properties of selective PKC inhibitors on the growth of U-373MG human astrocytoma cell line, and their ability to block mitogen-activated protein (MAP) kinase pathway activated by substance P (SP) neuropeptide receptor signaling via a PKC-dependent mechanism. Therefore, inhibiting PKC activity by selective PKC inhibitors may present a promising approach for improving astroglial brain tumor therapy. For this purpose, we constructed a high throughput model cell system to evaluate the efficacy of PKC inhibitors. This system is based on the measurement of light production in U-373MG cells stably transfected with the luciferase reporter gene whose expression depends on the transcriptional activation of GAL4-Elk1 fusion protein by enzyme components of the MAP kinase pathway and the upstream activation of PKC (PKC activation-->MAP kinases-->GAL4-Elk1 phosphorylation-->luciferase expression-->luciferase activity). In brief, we have demonstrated that the PKC activator 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced luciferase activity in this cell system is mediated via the MAP kinase pathway and can be blocked in the presence of MEK1 selective inhibitors (PD 098059 or U0126). We also demonstrated that TPA-induced luciferase activity in U-373MG stable clones can be blocked by PKC inhibitors (CGP 41251, Go 6976, and GF 109203X) in a concentration dependent manner. In contrast, epidermal growth factor (EGF)-induced luciferase activity, which is independent of PKC activation (Ras-->Raf-1-->MEK1-->MAP kinases-->GAL4-Elk1 phosphorylation-->luciferase expression-->luciferase activity) can only be blocked using a selective EGF receptor inhibitor (AG 1478). In conclusion, we have constructed a model cell system for the high throughput screening and identification of PKC inhibitors potentially active against astrocytoma cells in culture.

Suppression Subtractive Hybridization Identifies High Glucose Levels as a Stimulus for Expression of Connective Tissue Growth Factor and Other Genes in Human Mesangial Cells

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu.

Pervanadate Activates NADPH Oxidase via Protein Kinase C-Independent Phosphorylation of p47-phox

We studied differences between the NADPH oxidase activation pathways triggered by pervanadate, a protein tyrosine phosphatase inhibitor, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, in guinea pig neutrophils. Previously, pervanadate has been shown to activate NADPH oxidase via the tyrosine kinase-dependent pathway (Yamaguchiet al. Arch. Biochem. Biophys.323, 382–386, 1995). Both pervanadate and PMA induced superoxide anion (O−2) production, translocation of the 47-kDa protein component of the phagocyte oxidase (p47-phox) to the plasma membrane, and phosphorylation of p47-phox in the membrane. A selective protein kinase C inhibitor, GF 109203X, markedly inhibited PMA-induced O−2production, p47-phox translocation, and p47-phox phosphorylation, but did not inhibit pervanadate-induced O−2production and only slightly suppressed pervanadate-induced translocation and phosphorylation. These results demonstrate that pervanadate activates NADPH oxidase independently of protein kinase C. Phosphopeptide mapping of p47-phox revealed differences in the mechanism between pervanadate-induced and PMA-induced phosphorylation. Furthermore, some protein kinases which phosphorylate p47-phox-derived peptide are activated by pervanadate. These results suggest the existence of novel protein kinases responsible for the phosphorylation of p47-phox and the activation of these protein kinases by tyrosine kinase.

Transcriptional Activation of Gonadotropin-Releasing Hormone (GnRH) Receptor Gene by GnRH: Involvement of Multiple Signal Transduction Pathways

Previous studies have shown that GnRH activates transcriptional activity of its own receptor (GnRHR) gene in part through the cAMP signal transduction pathway. In the present study we explored the possible involvement of multiple signal transduction pathways in GnRH regulation of GnRHR gene transcription; these studies relied upon a luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment (−1164 to +62, relative to the major transcription start site) of the mouse GnRHR gene in GGH3 cells (GH3 cells stably expressing rat GnRHR). Activation of protein kinase C (PKC) by phorbol myristic acid significantly stimulated GnRHR-luciferase reporter gene (GnRHR-Luc) activity, but did not potentiate the stimulation of GnRHR-Luc activity by the GnRH agonist, buserelin (GnRH-A). Inhibition of PKC by PKC inhibitor (GF 109203X) or depletion of PKC blocked phorbol myristic acid- or GnRH-A-stimulated GnRHR-Luc activity, but did not affect (Bu)2cAMP-stimulated GnRHR-Luc activity. In addition, GnRH-A-stimulated GnRHR-Luc activity was inhibited by preventing external Ca2+ influx with the external Ca2+ chelator EGTA or the Ca2+ ion channel antagonist, D600. Surprisingly, overexpression of the mitogen-activated protein kinase (MAPK) kinase kinase (Raf-1) inhibited GnRHR-Luc activity and partially blocked GnRH-A-stimulated GnRHR-Luc activity. In contrast, inhibition of MAPK activity by MAPK kinase inhibitor (PD 98059) or by overexpression of kinase-deficient MAPKs activated basal and GnRH-A-stimulated GnRHR-Luc activity. These results suggested that PKC- and Ca2+-dependent signal transduction pathways participate in the GnRH activation of GnRHR promoter activity, and that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRHR transcriptional activity conferred by the 1226-bp promoter fragment.

Transcriptional activation of gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: involvement of multiple signal transduction pathways.

Previous studies have shown that GnRH activates transcriptional activity of its own receptor (GnRHR) gene in part through the cAMP signal transduction pathway. In the present study we explored the possible involvement of multiple signal transduction pathways in GnRH regulation of GnRHR gene transcription; these studies relied upon a luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment (-1164 to +62, relative to the major transcription start site) of the mouse GnRHR gene in GGH3 cells (GH3 cells stably expressing rat GnRHR). Activation of protein kinase C (PKC) by phorbol myristic acid significantly stimulated GnRHR-luciferase reporter gene (GnRHR-Luc) activity, but did not potentiate the stimulation of GnRHR-Luc activity by the GnRH agonist, buserelin (GnRH-A). Inhibition of PKC by PKC inhibitor (GF 109203X) or depletion of PKC blocked phorbol myristic acid- or GnRH-A-stimulated GnRHR-Luc activity, but did not affect (Bu)2cAMP-stimulated GnRHR-Luc activity. In addition, GnRH-A-stimulated GnRHR-Luc activity was inhibited by preventing external Ca2+ influx with the external Ca2+ chelator EGTA or the Ca2+ ion channel antagonist, D600. Surprisingly, overexpression of the mitogen-activated protein kinase (MAPK) kinase kinase (Raf-1) inhibited GnRHR-Luc activity and partially blocked GnRH-A-stimulated GnRHR-Luc activity. In contrast, inhibition of MAPK activity by MAPK kinase inhibitor (PD 98059) or by overexpression of kinase-deficient MAPKs activated basal and GnRH-A-stimulated GnRHR-Luc activity. These results suggested that PKC- and Ca2+-dependent signal transduction pathways participate in the GnRH activation of GnRHR promoter activity, and that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRHR transcriptional activity conferred by the 1226-bp promoter fragment.

Involvement of desmoplakin phosphorylation in the regulation of desmosomes by protein kinase C, in HeLa cells.

In the present study, we have examined how modulation of protein kinase C (PKC) activity affected desmosome organization in HeLa cells. Immunofluorescence and electron microscopy showed that PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a reduction of intercellular contacts, splitting of desmosomes and dislocation of desmosomal components from the cell periphery towards the cytoplasm. As determined by immunoblot analysis of Triton X-100-soluble and -insoluble pools of proteins, these morphological changes were not correlated with modifications in the extractability of both desmoglein and plakoglobin, but involved almost complete solubilization of the desmosomal plaque protein, desmoplakin. Immunoprecipitation experiments and immunoblotting with anti-phosphoserine, antiphosphothreonine and anti-phosphotyrosine antibodies revealed that desmoplakin was mainly phosphorylated on serine and tyrosine residues in both treated and untreated cells. While phosphotyrosine content was not affected by PKC activation, phosphorylation on serine residues was increased by about two-fold. This enhanced serine phosphorylation coincided with the increase in the protein solubility, suggesting that phosphorylation of desmoplakin may be a mechanism by which PKC mediates desmosome disassembly. Consistent with the loss of PKC activity, we also showed that down-modulation of the kinase (in response to prolonged TPA treatment) or its specific inhibition (by GF 109203X) had opposite effects and increased desmosome formation. Taken together, these results clearly demonstrate an important role for PKC in the regulation ofdesmosomal junctions in HeLa cells, and identify serine phosphorylation of desmoplakin as a crucial event in this pathway.

Desensitization of the histamine H1-receptor and transcriptional down-regulation of histamine H1-receptor gene expression in bovine tracheal smooth muscle.

We have investigated the role of protein kinase C (PKC) in the desensitization of histamine H1-receptors and in the expression of the histamine H1-receptor gene in airway smooth muscle. Prolonged 4beta-phorbol 12,13 dibutyrate (PDBu) pretreatment (4 h, 100 nM-1 microM) of bovine trachealis caused a concentration-dependent loss of contraction in response to histamine H1-receptor stimulation, which was associated with a concentration-dependent decrease in histamine-induced total [3H]-inositol phosphates accumulation. In contrast, the responses to sodium fluoride, a direct G-protein activator, were unalterd by PDBu (100-300 nM) pre-incubation and only slightly reduced following incubation with 1 microM PDBu. A selective PKC inhibitor, GF 109203X, partially blocked the PDBu (1 microM)-induced desensitization and completely blocked the effect of 100 nM PDBu, confirming the involvement of PKC. Binding experiments using [3H]-pyrilamine revealed a class of high-affinity binding sites within the range for the histamine H1 receptor in airway smooth muscle. PDBu (1 microM) pretreatment for 4 h did not change the number of histamine H1 receptors. PDBu (1 microM) exposure caused a time-dependent reduction in the steady-state levels of histamine H1-receptor mRNA, which was inhibited by pre-incubation with GF 109203X and by cycloheximide, a protein synthesis inhibitor. Nuclear run-on assays revealed a 50% reduction in the rate of histamine H1-receptor gene transcription after 17 h PDBu pretreatment, whereas mRNA stability was not affected by PDBu pretreatment (17 h). In conclusion, we have shown a PKC-mediated desensitization of the histamine H1-receptor in BTSM and a transcriptional down-regulation of the histamine H1-receptor gene expression, which requires new protein synthesis.

Basic secretagogues activate protein tyrosine phosphorylation and release of arachidonic acid in mast cells via a novel protein kinase C and phosphatidylinositol 3-kinase-dependent mechanism.

Mast cells play a central role in inflammatory and immediate-type allergic reactions. These granulated cells release by a process of regulated exocytosis a variety of biologically active substances which are either preformed (e.g. histamine), or synthesized de novo following activation [e. g. metabolites of arachidonic acid (AA) and multifunctional cytokines]. Exocytosis in mast cells is activated either in response to aggregation of the receptors for immunoglobulin E (FcepsilonRI) or by the direct activation of pertussis toxin-sensitive G-proteins by a class of receptor mimetic agents, collectively known as basic secretagogues of mast cells. In the present study we show that compound 48/80 (c48/80), a synthetic member of the class of basic secretagogues, stimulates protein tyrosine phosphorylation of a number of as yet unidentified cellular substrates. These phosphorylations were inhibited by the tyrphostin AG-18, by the phosphatidylinositol 3-kinase inhibitor wortmannin and by the protein kinase C inhibitors K252a and GF1 09203X. These inhibitors also inhibited the release of AA induced by c48/80 but had no effect on exocytosis. Taken together, our findings suggest that basic secretagogues induce protein tyrosine phosphorylation as part of their parallel multiple signaling pathways which are presumably mediated by more than one G-protein. Both protein kinase C and phosphatidylinositol 3-kinase serve as intermediates in this signaling pathway. The protein tyrosine kinase signaling pathway, which mediates the activation of AA release, does not contribute to secretion of the preformed mediators such as histamine, but it might largely contribute to the de novo production of inflammatory mediators like leukotrienes and prostaglandins.