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Darlene A Dartt - One of the best experts on this subject based on the ideXlab platform.
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effect of opc 12759 on egf receptor activation p44 P42 mapk activity and secretion in conjunctival goblet cells
Experimental Eye Research, 2008Co-Authors: David J Rios, Marie A Shatos, Hiroki Urashima, Darlene A DarttAbstract:The purpose of the study was to determine if OPC-12759 stimulates secretion from conjunctival goblet cells in culture and if it activates the EGF receptor (EGFR) and p44/P42 mitogen-activated protein kinase (MAPK) to cause mucin secretion. Conjunctival goblet cells were cultured from pieces of male rat conjunctiva. OPC-12759 was added at increasing concentrations and for varying times to the cultured cells. The cholinergic agonist carbachol was used as a positive control. In selected experiments an inhibitor of the EGFR, AG1478, or an inhibitor of the kinase that activates MAPK, U0126, were added before OPC-12759. Goblet cell secretion of high molecular weight glycoconjugates was measured by an enzyme-linked lectin assay using the lectin UEA-1. Activation of the EGFR and MAPK were determined with Western blotting analysis using antibodies specific to the phosphorylated and the total amounts of these proteins. We found that OPC-12759 induced goblet cell secretion in a time- and concentration-dependent manner. Inhibition of the EGFR with AG1478 blocked secretion stimulated by OPC-12759. Inhibition of MAPK with U0126 also blocked secretion stimulated by OPC-12759. OPC-12759 increased the phosphorylation of the EGFR and MAPK in a time-dependent manner. We concluded that OPC-12759 stimulates secretion from cultured conjunctival goblet cells by activating the EGFR, which then induces MAPK activity.
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effect of protein kinase c and ca 2 on P42 p44 mapk pyk2 and src activation in rat conjunctival goblet cells
Experimental Eye Research, 2007Co-Authors: Robin R Hodges, Marie A Shatos, Jose D Rios, Yoshitaka Horikawa, Darlene A DarttAbstract:Conjunctival goblet cells synthesize and secrete mucins onto the ocular surface to lubricate it and protect it from bacterial infections. Mucin secretion is under neural control, and cholinergic agonists released from parasympathetic nerves are major stimuli of this secretion. The signal transduction pathways these agonists use to stimulate secretion involve activating protein kinase C (PKC) and increasing intracellular [Ca(2+)] to activate the non-receptor kinases Pyk2 and p60Src (Src) to transactivate the EGF receptor. Transactivation of the EGF receptor activates a kinase cascade culminating in the activation of P42/p44 MAPK (MAPK) and ultimately that leads to secretion of high molecular weight glycocongujates (HMWGC), including mucins. To further examine the roles of PKC and Ca(2+) in the activation of MAPK, Pyk2, and Src in mucin secretion, rat conjunctival pieces and cultured goblet cells were incubated with the PKC activator phorbol myristate acid (PMA), the cholinergic agonist carbachol, or the calcium ionophore, ionomycin for varying times. Conjunctival pieces were preincubated with PKC inhibitors 10min prior to addition of carbachol (10(-4)M) for 10min. The amount of phosphorylated (activated) MAPK, Pyk2 and Src was determined by Western blotting techniques using antibodies specific to the phosphorylated forms of each kinase. PMA significantly increased the activation of MAPK, Pyk2, and Src in a time and concentration-dependent manner. PMA-stimulated MAPK activity was completely inhibited by the EGF receptor inhibitor AG1478 (10(-7)M). Carbachol-stimulated MAPK activity was inhibited by three PKC inhibitors, calphostin C, chelethyrine, and staurosporine. Ionomycin (10(-6)M)-stimulated MAPK activity was inhibited 66% by AG1478 (10(-7)M). Ionomycin also significantly increased Pyk2 and Src in time dependent manner. PKC and ionomycin also activated P42/p44 MAPK, Pyk2, and Src in cultured conjunctival goblet cells. We conclude that PKC and intracellular Ca(2+) activate Pyk2 and Src and phosphorylate the EGF receptor leading to stimulation of MAPK in conjunctival goblet cells.
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role of camp inhibition of p44 P42 mitogen activated protein kinase in potentiation of protein secretion in rat lacrimal gland
American Journal of Physiology-cell Physiology, 2007Co-Authors: Chika Funaki, Robin R Hodges, Darlene A DarttAbstract:We previously found that addition of cAMP and a Ca2+/PKC-dependent agonist causes synergism or potentiation of protein secretion from rat lacrimal gland acini. In the present study we determined wh...
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roles of protein kinase c ca2 pyk2 and c src in agonist activation of rat lacrimal gland P42 p44 mapk
Investigative Ophthalmology & Visual Science, 2006Co-Authors: Robin R Hodges, Jose D Rios, Joanna Vrouvlianis, Isao Ota, Driss Zoukhri, Darlene A DarttAbstract:Purpose Although P42/p44 mitogen-activated protein kinase (MAPK) negatively modulates protein secretion stimulated by cholinergic and alpha(1D)-adrenergic agonists, it does not play a role in epidermal growth factor (EGF)-stimulated protein secretion. Therefore, this study was conducted to determine the roles that protein kinase C (PKC), intracellular Ca(2+) ([Ca(2+)](i)), and nonreceptor tyrosine kinases Pyk2 and Src play in the activation of agonist- and EGF-stimulated MAPK activation. Methods Lacrimal gland acini were isolated by collagenase digestion and incubated with phorbol 12-myristate 13-acetate (PMA) to activate PKC or ionomycin, a Ca(2+) ionophore. Acini were preincubated with the PKC inhibitors calphostin C or Ro-31-8220, EGTA to chelate Ca(2+), or the c-Src inhibitor PP1 before stimulation with the cholinergic agonist carbachol, the alpha(1D)-adrenergic agonist phenylephrine, or EGF. Activated MAPK, Pyk2, and c-Src amounts were measured by Western blot analysis. Results PMA and ionomycin significantly increased the activation of MAPK in a time- and concentration-dependent manner. Inhibition of PKC partially inhibited carbachol-stimulated MAPK activation while completely inhibiting phenylephrine- and EGF-stimulated MAPK activation. Chelation of Ca(2+) also partially inhibited carbachol-stimulated MAPK with no effect on phenylephrine- and EGF-stimulated MAPK activation. Carbachol increased the phosphorylation of Pyk2 on tyrosine 402 and c-src on tyrosine 416 in a time-dependent manner. The c-src inhibitor PP1 inhibited carbachol-stimulated phosphorylation of Pyk2. Conclusions It was concluded that cholinergic agonists use Ca(2+) and PKC to phosphorylate Pyk2 and c-Src, which subsequently stimulate MAPK activity. In contrast, alpha(1D)-adrenergic agonists and EGF do not use Pyk2 and Src but do use PKC to activate MAPK.
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nitric oxide and cgmp mediate α1d adrenergic receptor stimulated protein secretion and P42 p44 mapk activation in rat lacrimal gland
Investigative Ophthalmology & Visual Science, 2005Co-Authors: Robin R Hodges, Marie A Shatos, Joanna Vrouvlianis, Rachel S Tarko, Darlene A DarttAbstract:Nitric oxide (NO) is a small, diffusible gaseous molecule that has been identified as a mediator in a variety of cellular functions including secretion, inflammation, and blood flow 1,2,3. NO is synthesized from arginine and molecular oxygen resulting in NO and L-citrulline by nitric oxide synthase (NOS). Nitric oxide synthase is a family of enzymes consisting of three known isoforms, endothelial nitric oxide synthase (eNOS or NOS-3), neuronal nitric oxide synthase (nNOS or NOS-1), and inducible nitric oxide synthase (iNOS or NOS-2). eNOS was first identified in endothelial cells as the enzyme which produced endothelial-derived relaxing factor, later identified as NO 4. It has since been shown to be present in a variety of tissues. nNOS was originally identified in neuronal cells, but has also been shown to be present in many different tissues. eNOS and nNOS are constitutively expressed hence they are referred to as constitutive enzymes. These two isoforms are activated by intracellular calcium and calmodulin. iNOS is not present in resting cells and is induced in a variety of cells by cytokines, infection, or lipopolysaccaride. This isoform is calcium independent and constitutively active 5. Induction of iNOS results in a large, rapid increase in NO that can have detrimental effects to surrounding tissue. In contrast, activation of eNOS and nNOS results in a smaller, slower increase in NO that interacts with a variety of signaling pathways. The effects of NO can be classified as either cGMP-dependent or cGMP-independent. NO interacts with many different types of proteins however interactions with heme-containing proteins such as hemoglobin, cytochrome P450, ryanodine receptors, or guanylate cyclase (GC) are well-documented 4,6. Ryanodine receptors are sarcoplasmic reticulum calcium-release channels whereas GCs convert GTP to cGMP. The family of GCs is divided into soluble GCs and particulate GCs 7. NO is a potent activator of soluble GCs but not of particulate GC 7. Signal transduction by cGMP is dependent upon its synthesis by GCs, its targeting, and its degradation by cGMP-dependent phosphodiesterases (PDEs). Once produced, cGMP interacts with protein kinase G (PKG) to phosphorylate downstream proteins, many which have yet to be identified that activate a variety of cellular functions. The lacrimal gland is an exocrine gland responsible for producing the majority of the aqueous portion of the tear film 8. Acinar cells are the major cell type present in the lacrimal gland. In addition, myoepithelial and ductal epithelial cells are also present. The function of the lacrimal gland is to secrete water, electrolytes and protein, which maintain, nourish, and protect the cells of the cornea and conjunctiva. If lacrimal gland secretion is altered in either amount or composition, a spectrum of diseases called dry eye syndromes results. Therefore secretion from the lacrimal gland is tightly regulated. As a result, parasympathetic and sympathetic nerves extensively innervate the lacrimal gland. Stimulation of the afferent sensory nerves in the cornea triggers tear secretion through the efferent parasympathetic and sympathetic nerves that innervate the lacrimal gland 8. Receptors for these neurotransmitters are located on the basolateral side of the acinar cells8. Activation of these receptors initiates signal transduction pathways that culminate in secretion of proteins, electrolytes, and water across the apical membranes into the lumen and onto the cornea and conjunctiva. We have previously shown that cholinergic agonists released from parasympathetic nerves and α1-adrenergic agonists released from sympathetic nerves are potent stimuli of protein secretion from the lacrimal gland. The signal transduction pathways used by cholinergic agonists have been well characterized 8. These agonists bind to the M3 muscarinic receptor to activate Gαq/11, which in turn activates phospholipase C (PLC). PLC causes the production of inositol trisphosphate, which causes the release of intracellular Ca2+. Diacylglycerol is also produced by PLC, which activates protein kinase C (PKC)α,-δ, and -ɛ9. In contrast, the pathways utilized by α1-adrenergic agonists have remained elusive. We have also shown that α1-adrenergic receptors mediate both protein secretion and activation of P42/p44 MAPK 10. In spite of this information, the G protein(s) involved in transduction of this signal are unknown, though Meneray et al have shown that inhibition Gαq is responsible for approximately 32% of the α1-adrenergic agonist-induced protein secretion 11. The phospholipase involved has also not been identified though it is known that neither phospholipase C nor D are involved 12,13. It is known that phenylephrine (an α1-adrenergic agonist in the lacrimal gland) activates PKC-ɛ, which stimulates protein secretion and PKCα and –δ to inhibit secretion. We have also shown that α1-adrenergic agonists transactivate the EGF receptor, recruiting Shc and Grb2 leading to activation of P42/p44 MAPK. Activation of P42/p44 MAPK inhibits protein secretion and may act to attenuate overall protein secretion from the lacrimal gland or to terminate stimulated secretion. It is known that the lacrimal gland contains many of the proteins necessary for the production of NO and cGMP. nNOS has been shown to be present in the lacrimal gland surrounding ducts, blood vessels, and acinar cells 14,15. Exogenous addition of NO, through the use of NO donors, has been shown to increase total protein secretion, which is inhibited by guanylate cyclase inhibitors in cultured lacrimal gland acinar cells 3,16. In addition, NO donors which increase cGMP induced a rise in intracellular [Ca2+] ([Ca2+]i) through the release of Ca2+ from intracellular stores 17. Jorgensen et al demonstrated that α1-adrenergic agonist-induced increase in [Ca ]i was inhibited by inhibitors of GC and cGMP-dependent protein kinase Ia 18. Although it is not known if activation of muscarinic receptors increases NO, it is known that when total IgGs and purified autoantibodies against M3 muscarinic receptors from patients suffering from the autoimmune disease Sjogren’s syndrome were added to freshly prepared rat lacrimal gland slices, NOS activity was increased. This activity was mediated via the M3 muscarinic receptor as the M3 muscarinic inhibitor, 4-DAMP, inhibited NOS activity 19. Thus evidence supporting a role for α1-adrenergic, β-adrenergic, or muscarinic agonists in stimulating lacrimal gland protein secretion via the NO/cGMP pathway is lacking. In the present study, we confirm the presence of eNOS and nNOS in the lacrimal gland and demonstrate that the α1-adrenergic agonist phenylephrine, using α1D-adrenergic receptors, activates eNOS, but not nNOS, to increase NO production. NO, in turn, activates GC to increase cGMP levels. α1-Adrenergic agonist activation of the NO/cGMP pathway leads to an increase in protein secretion and P42/p44 MAPK induction from freshly isolated rat lacrimal gland acinar cells.
Jacques Pouysségur - One of the best experts on this subject based on the ideXlab platform.
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signaling angiogenesis via P42 p44 map kinase cascade
Annals of the New York Academy of Sciences, 2006Co-Authors: Gilles Pages, Julie Milanini, Darren E Richard, Edurne Berra, Emmanuel Gothie, Francesc Vinals, Jacques PouysségurAbstract:Vascular endothelial growth factor (VEGF), a potent agonist secreted by virtually all cells, controls migration and division of vascular endothelial cells. Disruption of one VEGF allele in mice has revealed a dramatic lethal effect in early embryogenesis, suggesting a key role in vasculogenesis. We analyzed the regulation of VEGF mRNA in normal and transformed CCL39 fibroblasts and then dissected the VEGF promoter to identify the signaling pathway(s) controlling the activation of this promoter in response to growth factors, oncogenes, and hypoxic stress. We demonstrated that the P42/p44 MAP kinase signaling cascade controls VEGF expression at least at two levels. In normoxic conditions, MAPKs activate the VEGF promoter at the proximal (-88/-66) region where Sp-1/AP-2 factors bind. Activation of P42/p44 MAPKs is sufficient to turn on VEGF mRNA. At low O2 tension, hypoxia inducible factor-1 alpha (HIF-1 alpha), a limiting factor rapidly stabilized and phosphorylated, plays a key role in the expression of several genes including VEGF. We demonstrated that P42/p44MAPKs stoichiometrically phosphorylate HIF-1 alpha in vitro and that HIF-1-dependent VEGF gene expression is strongly enhanced by the exclusive activation of P42/p44MAPKs. Finally, we demonstrated that the regulation of P42/p44MAPK activity is critical for controlling proliferation and growth arrest of vascular endothelial cells at confluency. These results point to at least three major targets of angiogenesis where P42/p44 MAP kinases exert a determinant action.
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P42 p44 mapks are intracellular targets of the cdk inhibitor purvalanol
Oncogene, 2002Co-Authors: Marie Knockaert, Philippe Lenormand, Jacques Pouysségur, Nathanael S. Gray, Laurent MeijerAbstract:: Chemical inhibitors of cyclin-dependent kinases (CDKs) have a great therapeutic potential against various proliferative and neurodegenerative disorders. Intensive screening of a combinatorial chemistry library of 2,6,9-trisubstituted purines has led to the identification of purvalanol, one of the most potent and selective CDK inhibitors to date. In preliminary studies, this compound demonstrates definite anti-mitotic properties, consistent with its nanomolar range efficiency towards purified CDK1 and CDK2. However, the actual intracellular targets of purvalanol remain to be identified, and a method for the determination of its in vivo selectivity was developed. In this technique, cell extracts were screened for purvalanol-interacting proteins by affinity chromatography on immobilized inhibitor. In addition to CDK1, P42/p44 MAPK were found to be two major purvalanol-interacting proteins in five different mammalian cell lines (CCL39, PC12, HBL100, MCF-7 and Jurkat cells), suggesting the generality of the purvalanol/P42/p44 MAPK interaction. The Chinese hamster lung fibroblast cell line CCL39 was used as a model to investigate the anti-proliferative properties of purvalanol. The compound inhibited cell growth with a GI(50) value of 2.5 microM and induced a G2/M block when added to exponentially growing cells. It did not appear to trigger massive activation of caspase. We next tested whether CDKs and P42/p44 MAPK were actually targeted by the compound in vivo. P42/p44 MAPK activity was visualized using an Elk-Gal4 luciferase reporter system and CDK1 activity was detected by the phosphonucleolin level. When cells were treated with purvalanol, P42/p44 MAPK and CDK1 activities were inhibited in a dose-dependent manner. Furthermore, purvalanol inhibited the nuclear accumulation of P42/p44 MAPK, an event dependent on the catalytic activity of these kinases. We conclude that the anti-proliferative properties of purvalanol are mediated by inhibition of both P42/p44 MAPK and CDKs. These observations highlight the potency of moderate selectivity compounds and encourage the search for new therapeutics which simultaneously target distinct but relevant pathways of cell proliferation.
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identification of two sp1 phosphorylation sites for P42 p44 mitogen activated protein kinases their implication in vascular endothelial growth factor gene transcription
Journal of Biological Chemistry, 2002Co-Authors: Julie Milaninimongiat, Jacques Pouysségur, Gilles PagesAbstract:Abstract Sp1 regulates activation of many genes implicated in tumor growth and cell cycle progression. We have previously demonstrated its implication in the up-regulation of vascular endothelial growth factor (VEGF) gene transcription following growth factor stimulation of quiescent cells, a situation where P42/p44 mitogen-activate protein kinase (MAPK) activity is dramatically increased. Here we show that P42/p44 MAPK directly phosphorylates Sp1 on threonines 453 and 739 both in vitro and in vivo. Mutation of these sites to alanines decreases by half the MAPK-dependent transcriptional activity of Sp1, in the context of the VEGF promoter, in SL2 Drosophila cells devoid of the endogenous Sp1 protein. Moreover, inducible overexpression of the (T453A,T739A) Sp1 double mutant compromises MAPK-driven VEGF mRNA transcription in fibroblasts. These results highlight Sp1 as a key molecular link between elevated activation of the Ras ≫ P42/p44MAPK signaling pathway and increased VEGF expression, two major steps deregulated in tumor cells.
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the nucleus a site for signal termination by sequestration and inactivation of P42 p44 map kinases
Journal of Cell Science, 2001Co-Authors: Veronique Volmat, Jacques Pouysségur, Montserrat Camps, Steve Arkinstall, Philippe LenormandAbstract:We previously reported that nuclear translocation is essential for P42/p44 MAPKs (ERKs) mitogenic signaling. Here we show that, during long-term stimulation, P42/p44 MAPKs become inactive while they accumulate in the nucleus. This inactivation was monitored by phospho-specific immunostaining and dephosphorylation of a nuclear P42/p44 MAPKs substrate, HIF-1α. The phosphatases responsible for P42/p44 MAPKs nuclear inactivation are neo-synthesized, show tyrosine or dual specificity, and interact with P42/p44 MAPKs via a specific docking site. Likely candidates are MKP1/2 phosphatases. In addition, P42/p44 MAPKs permanently shuttle between the cytoplasm and the nucleus in quiescent as well as in serum stimulated cells. Hence, the nucleus is a critical site for mitogenic signal termination by: (1) nuclear sequestration of P42/p44 MAPKs away from MEK, their cytoplasmic activator; and (2) dephosphorylation by specific nuclear phosphatases.
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signaling angiogenesis via P42 p44 map kinase and hypoxia
Biochemical Pharmacology, 2000Co-Authors: Edurne Berra, Gilles Pages, Julie Milanini, Darren E Richard, Emmanuel Gothie, Francesc Vinals, Maude Le Gall, Daniele Roux, Jacques PouysségurAbstract:Angiogenesis is associated with a number of pathological situations. In this study, we have focused our attention on the role of P42/p44 MAP (mitogen-activated protein) kinases and hypoxia in the control of angiogenesis. We demonstrate that P42/p44 MAP kinases play a pivotal role in angiogenesis by exerting a determinant action at three levels: i) persistent activation of P42/p44 MAP kinases abrogates apoptosis; ii) P42/p44 MAP kinase activity is critical for controlling proliferation and growth arrest of confluent endothelial cells; and iii) P42/p44 MAP kinases promote VEGF (vascular endothelial growth factor) expression by activating its transcription via recruitment of the AP-2/Sp1 (activator protein-2) complex on the proximal region (-88/-66) of the VEGF promoter and by direct phosphorylation of hypoxia-inducible factor 1 alpha (HIF-1 alpha). HIF-1 alpha plays a crucial role in the control of HIF-1 activity, which mediates hypoxia-induced VEGF expression. We show that oxygen-regulated HIF-1 alpha protein levels are not affected by intracellular localization (nucleus versus cytoplasm). Finally, we propose a model which suggests an autoregulatory feedback mechanism controlling HIF-1 alpha and therefore HIF-1-dependent gene expression.
Robin R Hodges - One of the best experts on this subject based on the ideXlab platform.
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effect of protein kinase c and ca 2 on P42 p44 mapk pyk2 and src activation in rat conjunctival goblet cells
Experimental Eye Research, 2007Co-Authors: Robin R Hodges, Marie A Shatos, Jose D Rios, Yoshitaka Horikawa, Darlene A DarttAbstract:Conjunctival goblet cells synthesize and secrete mucins onto the ocular surface to lubricate it and protect it from bacterial infections. Mucin secretion is under neural control, and cholinergic agonists released from parasympathetic nerves are major stimuli of this secretion. The signal transduction pathways these agonists use to stimulate secretion involve activating protein kinase C (PKC) and increasing intracellular [Ca(2+)] to activate the non-receptor kinases Pyk2 and p60Src (Src) to transactivate the EGF receptor. Transactivation of the EGF receptor activates a kinase cascade culminating in the activation of P42/p44 MAPK (MAPK) and ultimately that leads to secretion of high molecular weight glycocongujates (HMWGC), including mucins. To further examine the roles of PKC and Ca(2+) in the activation of MAPK, Pyk2, and Src in mucin secretion, rat conjunctival pieces and cultured goblet cells were incubated with the PKC activator phorbol myristate acid (PMA), the cholinergic agonist carbachol, or the calcium ionophore, ionomycin for varying times. Conjunctival pieces were preincubated with PKC inhibitors 10min prior to addition of carbachol (10(-4)M) for 10min. The amount of phosphorylated (activated) MAPK, Pyk2 and Src was determined by Western blotting techniques using antibodies specific to the phosphorylated forms of each kinase. PMA significantly increased the activation of MAPK, Pyk2, and Src in a time and concentration-dependent manner. PMA-stimulated MAPK activity was completely inhibited by the EGF receptor inhibitor AG1478 (10(-7)M). Carbachol-stimulated MAPK activity was inhibited by three PKC inhibitors, calphostin C, chelethyrine, and staurosporine. Ionomycin (10(-6)M)-stimulated MAPK activity was inhibited 66% by AG1478 (10(-7)M). Ionomycin also significantly increased Pyk2 and Src in time dependent manner. PKC and ionomycin also activated P42/p44 MAPK, Pyk2, and Src in cultured conjunctival goblet cells. We conclude that PKC and intracellular Ca(2+) activate Pyk2 and Src and phosphorylate the EGF receptor leading to stimulation of MAPK in conjunctival goblet cells.
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role of camp inhibition of p44 P42 mitogen activated protein kinase in potentiation of protein secretion in rat lacrimal gland
American Journal of Physiology-cell Physiology, 2007Co-Authors: Chika Funaki, Robin R Hodges, Darlene A DarttAbstract:We previously found that addition of cAMP and a Ca2+/PKC-dependent agonist causes synergism or potentiation of protein secretion from rat lacrimal gland acini. In the present study we determined wh...
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roles of protein kinase c ca2 pyk2 and c src in agonist activation of rat lacrimal gland P42 p44 mapk
Investigative Ophthalmology & Visual Science, 2006Co-Authors: Robin R Hodges, Jose D Rios, Joanna Vrouvlianis, Isao Ota, Driss Zoukhri, Darlene A DarttAbstract:Purpose Although P42/p44 mitogen-activated protein kinase (MAPK) negatively modulates protein secretion stimulated by cholinergic and alpha(1D)-adrenergic agonists, it does not play a role in epidermal growth factor (EGF)-stimulated protein secretion. Therefore, this study was conducted to determine the roles that protein kinase C (PKC), intracellular Ca(2+) ([Ca(2+)](i)), and nonreceptor tyrosine kinases Pyk2 and Src play in the activation of agonist- and EGF-stimulated MAPK activation. Methods Lacrimal gland acini were isolated by collagenase digestion and incubated with phorbol 12-myristate 13-acetate (PMA) to activate PKC or ionomycin, a Ca(2+) ionophore. Acini were preincubated with the PKC inhibitors calphostin C or Ro-31-8220, EGTA to chelate Ca(2+), or the c-Src inhibitor PP1 before stimulation with the cholinergic agonist carbachol, the alpha(1D)-adrenergic agonist phenylephrine, or EGF. Activated MAPK, Pyk2, and c-Src amounts were measured by Western blot analysis. Results PMA and ionomycin significantly increased the activation of MAPK in a time- and concentration-dependent manner. Inhibition of PKC partially inhibited carbachol-stimulated MAPK activation while completely inhibiting phenylephrine- and EGF-stimulated MAPK activation. Chelation of Ca(2+) also partially inhibited carbachol-stimulated MAPK with no effect on phenylephrine- and EGF-stimulated MAPK activation. Carbachol increased the phosphorylation of Pyk2 on tyrosine 402 and c-src on tyrosine 416 in a time-dependent manner. The c-src inhibitor PP1 inhibited carbachol-stimulated phosphorylation of Pyk2. Conclusions It was concluded that cholinergic agonists use Ca(2+) and PKC to phosphorylate Pyk2 and c-Src, which subsequently stimulate MAPK activity. In contrast, alpha(1D)-adrenergic agonists and EGF do not use Pyk2 and Src but do use PKC to activate MAPK.
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nitric oxide and cgmp mediate α1d adrenergic receptor stimulated protein secretion and P42 p44 mapk activation in rat lacrimal gland
Investigative Ophthalmology & Visual Science, 2005Co-Authors: Robin R Hodges, Marie A Shatos, Joanna Vrouvlianis, Rachel S Tarko, Darlene A DarttAbstract:Nitric oxide (NO) is a small, diffusible gaseous molecule that has been identified as a mediator in a variety of cellular functions including secretion, inflammation, and blood flow 1,2,3. NO is synthesized from arginine and molecular oxygen resulting in NO and L-citrulline by nitric oxide synthase (NOS). Nitric oxide synthase is a family of enzymes consisting of three known isoforms, endothelial nitric oxide synthase (eNOS or NOS-3), neuronal nitric oxide synthase (nNOS or NOS-1), and inducible nitric oxide synthase (iNOS or NOS-2). eNOS was first identified in endothelial cells as the enzyme which produced endothelial-derived relaxing factor, later identified as NO 4. It has since been shown to be present in a variety of tissues. nNOS was originally identified in neuronal cells, but has also been shown to be present in many different tissues. eNOS and nNOS are constitutively expressed hence they are referred to as constitutive enzymes. These two isoforms are activated by intracellular calcium and calmodulin. iNOS is not present in resting cells and is induced in a variety of cells by cytokines, infection, or lipopolysaccaride. This isoform is calcium independent and constitutively active 5. Induction of iNOS results in a large, rapid increase in NO that can have detrimental effects to surrounding tissue. In contrast, activation of eNOS and nNOS results in a smaller, slower increase in NO that interacts with a variety of signaling pathways. The effects of NO can be classified as either cGMP-dependent or cGMP-independent. NO interacts with many different types of proteins however interactions with heme-containing proteins such as hemoglobin, cytochrome P450, ryanodine receptors, or guanylate cyclase (GC) are well-documented 4,6. Ryanodine receptors are sarcoplasmic reticulum calcium-release channels whereas GCs convert GTP to cGMP. The family of GCs is divided into soluble GCs and particulate GCs 7. NO is a potent activator of soluble GCs but not of particulate GC 7. Signal transduction by cGMP is dependent upon its synthesis by GCs, its targeting, and its degradation by cGMP-dependent phosphodiesterases (PDEs). Once produced, cGMP interacts with protein kinase G (PKG) to phosphorylate downstream proteins, many which have yet to be identified that activate a variety of cellular functions. The lacrimal gland is an exocrine gland responsible for producing the majority of the aqueous portion of the tear film 8. Acinar cells are the major cell type present in the lacrimal gland. In addition, myoepithelial and ductal epithelial cells are also present. The function of the lacrimal gland is to secrete water, electrolytes and protein, which maintain, nourish, and protect the cells of the cornea and conjunctiva. If lacrimal gland secretion is altered in either amount or composition, a spectrum of diseases called dry eye syndromes results. Therefore secretion from the lacrimal gland is tightly regulated. As a result, parasympathetic and sympathetic nerves extensively innervate the lacrimal gland. Stimulation of the afferent sensory nerves in the cornea triggers tear secretion through the efferent parasympathetic and sympathetic nerves that innervate the lacrimal gland 8. Receptors for these neurotransmitters are located on the basolateral side of the acinar cells8. Activation of these receptors initiates signal transduction pathways that culminate in secretion of proteins, electrolytes, and water across the apical membranes into the lumen and onto the cornea and conjunctiva. We have previously shown that cholinergic agonists released from parasympathetic nerves and α1-adrenergic agonists released from sympathetic nerves are potent stimuli of protein secretion from the lacrimal gland. The signal transduction pathways used by cholinergic agonists have been well characterized 8. These agonists bind to the M3 muscarinic receptor to activate Gαq/11, which in turn activates phospholipase C (PLC). PLC causes the production of inositol trisphosphate, which causes the release of intracellular Ca2+. Diacylglycerol is also produced by PLC, which activates protein kinase C (PKC)α,-δ, and -ɛ9. In contrast, the pathways utilized by α1-adrenergic agonists have remained elusive. We have also shown that α1-adrenergic receptors mediate both protein secretion and activation of P42/p44 MAPK 10. In spite of this information, the G protein(s) involved in transduction of this signal are unknown, though Meneray et al have shown that inhibition Gαq is responsible for approximately 32% of the α1-adrenergic agonist-induced protein secretion 11. The phospholipase involved has also not been identified though it is known that neither phospholipase C nor D are involved 12,13. It is known that phenylephrine (an α1-adrenergic agonist in the lacrimal gland) activates PKC-ɛ, which stimulates protein secretion and PKCα and –δ to inhibit secretion. We have also shown that α1-adrenergic agonists transactivate the EGF receptor, recruiting Shc and Grb2 leading to activation of P42/p44 MAPK. Activation of P42/p44 MAPK inhibits protein secretion and may act to attenuate overall protein secretion from the lacrimal gland or to terminate stimulated secretion. It is known that the lacrimal gland contains many of the proteins necessary for the production of NO and cGMP. nNOS has been shown to be present in the lacrimal gland surrounding ducts, blood vessels, and acinar cells 14,15. Exogenous addition of NO, through the use of NO donors, has been shown to increase total protein secretion, which is inhibited by guanylate cyclase inhibitors in cultured lacrimal gland acinar cells 3,16. In addition, NO donors which increase cGMP induced a rise in intracellular [Ca2+] ([Ca2+]i) through the release of Ca2+ from intracellular stores 17. Jorgensen et al demonstrated that α1-adrenergic agonist-induced increase in [Ca ]i was inhibited by inhibitors of GC and cGMP-dependent protein kinase Ia 18. Although it is not known if activation of muscarinic receptors increases NO, it is known that when total IgGs and purified autoantibodies against M3 muscarinic receptors from patients suffering from the autoimmune disease Sjogren’s syndrome were added to freshly prepared rat lacrimal gland slices, NOS activity was increased. This activity was mediated via the M3 muscarinic receptor as the M3 muscarinic inhibitor, 4-DAMP, inhibited NOS activity 19. Thus evidence supporting a role for α1-adrenergic, β-adrenergic, or muscarinic agonists in stimulating lacrimal gland protein secretion via the NO/cGMP pathway is lacking. In the present study, we confirm the presence of eNOS and nNOS in the lacrimal gland and demonstrate that the α1-adrenergic agonist phenylephrine, using α1D-adrenergic receptors, activates eNOS, but not nNOS, to increase NO production. NO, in turn, activates GC to increase cGMP levels. α1-Adrenergic agonist activation of the NO/cGMP pathway leads to an increase in protein secretion and P42/p44 MAPK induction from freshly isolated rat lacrimal gland acinar cells.
Nigel J. Pyne - One of the best experts on this subject based on the ideXlab platform.
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the effect of rgs12 on pdgfβ receptor signalling to P42 p44 mitogen activated protein kinase in mammalian cells
Cellular Signalling, 2006Co-Authors: Balwinder Sambi, David P Siderovski, Melinda D. Hains, Catherine M. Waters, Michelle Connell, Francis S. Willard, Adam J. Kimple, Susan Pyne, Nigel J. PyneAbstract:We have previously shown that the PDGFβ receptor uses a classical GPCR-mediated pathway in order to induce efficient activation of P42/p44 MAPK in response to PDGF. We therefore, considered the possibility that GTPase accelerating proteins (RGS proteins), which regulate GPCR signalling, modulate PDGFβ receptor-mediated signal transmission. Several lines of evidence were obtained to support functional interaction between the PDGFβ receptor and RGS12 in HEK 293 and airway smooth muscle cells. Firstly, the over-expression of the RGS12 PDZ/PTB domain N-terminus or RGS12 PTB domain reduced the PDGF-induced activation of P42/p44 MAPK. Secondly, the RGS12 PDZ/PTB domain N-terminus and RGS12 PDZ domain can form a complex with the PDGFβ receptor. Therefore, the results presented here provide the first evidence to support the concept that the PDZ/PTB domain N-terminus and/or the PTB domain of RGS12 may modulate PDGFβ receptor signalling. In airway smooth muscle cells, over-expressed recombinant RGS12 and the isolated PDZ/PTB domain N-terminus co-localised with PDGFβ receptor in cytoplasmic vesicles. To provide additional evidence for a role of the PDZ/PTB domain N-terminus, we used RGS14. RGS14 has the same C-terminal domain architecture of an RGS box, tandem Ras-binding domains (RBDs) and GoLoco motif as RGS12, but lacks the PDZ/PTB domain N-terminus. In this regard, RGS14 exhibited a different sub-cellular distribution compared with RGS12, being diffusely distributed in ASM cells. These findings suggest that RGS12 via its PDZ/PTB domain N-terminus may regulate trafficking of the PDGFβ receptor in ASM cells.
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nerve growth factor signaling involves interaction between the trk a receptor and lysophosphatidate receptor 1 systems nuclear translocation of the lysophosphatidate receptor 1 and trk a receptors in pheochromocytoma 12 cells
Cellular Signalling, 2004Co-Authors: Noreen A Moughal, Balwinder Sambi, Catherine M. Waters, Susan Pyne, Nigel J. PyneAbstract:We report here that the nerve growth factor (NGF) and lysophosphatidate (LPA) receptor signaling systems interact to regulate the P42/p44 MAPK pathway in PC12 cells. This is based upon several lines of evidence. First, the treatment of PC12 cells, which express LPA(1) receptors, with a sub-maximal concentration of LPA and NGF induced synergistic activation of P42/p44 MAPK. Second, the transfection of PC12 cells with LPA(1) receptor anti-sense construct, which reduced the expression of LPA(1), abrogated both LPA- and NGF-stimulated activation of P42/p44 MAPK. Third, the over-expression of recombinant LPA(1) receptor potentiated LPA- and NGF-dependent activation of P42/p44 MAPK. Fourth, the over-expression of C-terminal GRK2 peptide (which sequesters G-protein betagamma subunits) or beta-arrestin I clathrin binding domain (amino acids: 319-418) or pre-treatment of cells with pertussis toxin reduced the LPA- and NGF-dependent stimulation of P42/p44 MAPK. These findings support a model in which the Trk A receptor uses a G-protein-mediated mechanism to regulate the P42/p44 MAPK pathway. Such G-protein-mediated signaling is activated by the LPA(1) receptor as a means of cross-talk regulation with the Trk A receptor. Fifth, the treatment of cells with LPA induced the transactivation of the Trk A receptor. Sixth, LPA and/or NGF stimulated the translocation of tyrosine phosphorylated Trk A receptor and LPA(1) receptor to the nucleus. Taken together, these findings suggest that NGF and LPA exert cross-talk regulation both at the level of P42/p44 MAPK signaling and in the nuclear translocation of LPA(1) and Trk A receptors.
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nerve growth factor stimulation of P42 p44 mitogen activated protein kinase in pc12 cells role of g i o g protein coupled receptor kinase 2 beta arrestin i and endocytic processing
Molecular Pharmacology, 2001Co-Authors: Soma Rakhit, Susan Pyne, Nigel J. PyneAbstract:In this study, we have shown that nerve growth factor (NGF)dependent activation of the P42/p44 mitogen-activated protein kinase (P42/p44 MAPK) pathway in PC12 cells can be partially blocked by pertussis toxin (which inactivates the G proteins Gi/o). This suggests that the Trk A receptor may use a G protein-coupled receptor pathway to signal to P42/p44 MAPK. This was supported by data showing that the NGF-dependent activation of P42/p44 MAPK is potentiated in cells transfected with G protein-coupled receptor kinase 2 (GRK2) or b-arrestin I. Moreover, GRK2 is constitutively bound with the Trk A receptor, whereas NGF stimulates the pertussis toxin-sensitive binding of b-arrestin I to the TrkA receptor-GRK2 complex. Both GRK2 and b-arrestin I are involved in clathrin-mediated endo
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platelet derived growth factor stimulation of the P42 p44 mitogen activated protein kinase pathway in airway smooth muscle role of pertussis toxin sensitive g proteins c src tyrosine kinases and phosphoinositide 3 kinase
Biochemical Journal, 1999Co-Authors: Annmarie Conway, Susan Pyne, Soma Rakhit, Nigel J. PyneAbstract:The mechanism used by the platelet-derived growth factor receptor (PDGFR) to activate the mitogen-activated- protein-kinase (P42/p44 MAPK) pathway was investigated in cultured airway smooth muscle (ASM) cells. We have found that pertussis toxin (PTX, which was used to inactivate the heterotrimeric G-protein Gi) induced an approx. 40-50% decrease in the activation of c-Src and P42/p44 MAPK by PDGF. An essential role for c-Src was confirmed using the c-Src inhibitor, PP1, which abolished P42/p44 MAPK activation (PP1 and PTX were without effect on PDGFR tyrosine phosphorylation). Furthermore, the PTX-dependent decrease in c-Src and P42/p44 MAPK activation appeared correlated. These findings suggest that the PDGFR can utilize the PTX-sensitive G-protein, Gi, to regulate c-Src and subsequent P42/p44 MAPK activation. Phosphoinositide 3-kinase (PI3K) has been shown by others to be involved in P42/p44 MAPK activation. This is confirmed here by experiments which showed that PI3K inhibitors (wortmannin and LY294002) reduced the activation of P42/p44 MAPK by PDGF. PI3K activity was increased in Grb-2 immunoprecipitates from PDGF-stimulated cells and was decreased by pretreating these cells with PTX. These findings show that Gi might also promote Grb-2-PI3K complex formation and that Grb-2 may be a site at which PI3K is integrated into the P42/p44 MAPK cascade. In conclusion, our results demonstrate that Gi enables the PDGFR to signal more efficiently to P42/p44 MAPK, and this appears to be achieved through the regulation of c-Src and Grb-2/PI3K, which are intermediates in the P42/p44 MAPK cascade.
Gilles Pages - One of the best experts on this subject based on the ideXlab platform.
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signaling angiogenesis via P42 p44 map kinase cascade
Annals of the New York Academy of Sciences, 2006Co-Authors: Gilles Pages, Julie Milanini, Darren E Richard, Edurne Berra, Emmanuel Gothie, Francesc Vinals, Jacques PouysségurAbstract:Vascular endothelial growth factor (VEGF), a potent agonist secreted by virtually all cells, controls migration and division of vascular endothelial cells. Disruption of one VEGF allele in mice has revealed a dramatic lethal effect in early embryogenesis, suggesting a key role in vasculogenesis. We analyzed the regulation of VEGF mRNA in normal and transformed CCL39 fibroblasts and then dissected the VEGF promoter to identify the signaling pathway(s) controlling the activation of this promoter in response to growth factors, oncogenes, and hypoxic stress. We demonstrated that the P42/p44 MAP kinase signaling cascade controls VEGF expression at least at two levels. In normoxic conditions, MAPKs activate the VEGF promoter at the proximal (-88/-66) region where Sp-1/AP-2 factors bind. Activation of P42/p44 MAPKs is sufficient to turn on VEGF mRNA. At low O2 tension, hypoxia inducible factor-1 alpha (HIF-1 alpha), a limiting factor rapidly stabilized and phosphorylated, plays a key role in the expression of several genes including VEGF. We demonstrated that P42/p44MAPKs stoichiometrically phosphorylate HIF-1 alpha in vitro and that HIF-1-dependent VEGF gene expression is strongly enhanced by the exclusive activation of P42/p44MAPKs. Finally, we demonstrated that the regulation of P42/p44MAPK activity is critical for controlling proliferation and growth arrest of vascular endothelial cells at confluency. These results point to at least three major targets of angiogenesis where P42/p44 MAP kinases exert a determinant action.
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identification of two sp1 phosphorylation sites for P42 p44 mitogen activated protein kinases their implication in vascular endothelial growth factor gene transcription
Journal of Biological Chemistry, 2002Co-Authors: Julie Milaninimongiat, Jacques Pouysségur, Gilles PagesAbstract:Abstract Sp1 regulates activation of many genes implicated in tumor growth and cell cycle progression. We have previously demonstrated its implication in the up-regulation of vascular endothelial growth factor (VEGF) gene transcription following growth factor stimulation of quiescent cells, a situation where P42/p44 mitogen-activate protein kinase (MAPK) activity is dramatically increased. Here we show that P42/p44 MAPK directly phosphorylates Sp1 on threonines 453 and 739 both in vitro and in vivo. Mutation of these sites to alanines decreases by half the MAPK-dependent transcriptional activity of Sp1, in the context of the VEGF promoter, in SL2 Drosophila cells devoid of the endogenous Sp1 protein. Moreover, inducible overexpression of the (T453A,T739A) Sp1 double mutant compromises MAPK-driven VEGF mRNA transcription in fibroblasts. These results highlight Sp1 as a key molecular link between elevated activation of the Ras ≫ P42/p44MAPK signaling pathway and increased VEGF expression, two major steps deregulated in tumor cells.
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signaling angiogenesis via P42 p44 map kinase and hypoxia
Biochemical Pharmacology, 2000Co-Authors: Edurne Berra, Gilles Pages, Julie Milanini, Darren E Richard, Emmanuel Gothie, Francesc Vinals, Maude Le Gall, Daniele Roux, Jacques PouysségurAbstract:Angiogenesis is associated with a number of pathological situations. In this study, we have focused our attention on the role of P42/p44 MAP (mitogen-activated protein) kinases and hypoxia in the control of angiogenesis. We demonstrate that P42/p44 MAP kinases play a pivotal role in angiogenesis by exerting a determinant action at three levels: i) persistent activation of P42/p44 MAP kinases abrogates apoptosis; ii) P42/p44 MAP kinase activity is critical for controlling proliferation and growth arrest of confluent endothelial cells; and iii) P42/p44 MAP kinases promote VEGF (vascular endothelial growth factor) expression by activating its transcription via recruitment of the AP-2/Sp1 (activator protein-2) complex on the proximal region (-88/-66) of the VEGF promoter and by direct phosphorylation of hypoxia-inducible factor 1 alpha (HIF-1 alpha). HIF-1 alpha plays a crucial role in the control of HIF-1 activity, which mediates hypoxia-induced VEGF expression. We show that oxygen-regulated HIF-1 alpha protein levels are not affected by intracellular localization (nucleus versus cytoplasm). Finally, we propose a model which suggests an autoregulatory feedback mechanism controlling HIF-1 alpha and therefore HIF-1-dependent gene expression.
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P42 p44 map kinase module plays a key role in the transcriptional regulation of the vascular endothelial growth factor gene in fibroblasts
Journal of Biological Chemistry, 1998Co-Authors: Julie Milanini, Jacques Pouysségur, Francesc Vinals, Gilles PagesAbstract:Abstract Vascular EndothelialGrowth Factor (VEGF) is a potent mitogen for vascular endothelial cells that has been implicated in tumor neovascularization. We show that, in hamster fibroblasts (CCL39 cells), VEGF mRNAs are expressed at low levels in serum-deprived or exponentially growing cells, whereas it is rapidly induced after stimulation of quiescent cells with serum. CCL39 derivatives, transformed with Polyoma virus or with active members of the P42/p44 mitogen-activated protein (MAP) kinase pathway, Gly/Val point mutant of Ras at position 12 (Ras-Val12), MKK1 in which Ser218 and Ser222 were mutated to Asp (MKK1-SS/DD)), express very high levels of VEGF mRNA. To analyze the contribution of the P42/p44MAP kinase in this induction, we used the CCL39-derived cell line (Raf-1:ER) expressing an estradiol-activable Raf-1. We show a time and an estradiol dose-dependent up-regulation of VEGF mRNA clearly detectable after 2 h of stimulation. The induction of VEGF mRNA in response to conditioned activation of Raf-1 is reverted by an inhibitor of MKK1, PD 098059, highlighting a specific role for the P42/p44 MAP kinase pathway in VEGF expression. Interestingly, hypoxia has an additive effect on VEGF induction in CCL39 cells stimulated by serum or in Raf-1:ER cells stimulated by estradiol. In contrast to VEGF, the isoforms VEGF-B and VEGF-C are poorly regulated by growth and oncogenic factors. We have identified a GC-rich region of the VEGF promoter between −88 and −66 base pairs which contains all the elements responsible of its up-regulation by constitutive active Ras or MKK1-SS/DD. By mutation of the putative binding sites and electrophoretic mobility supershift experiments, we showed that the GC-rich region constitutively binds Sp1 and AP-2 transcription factors. Furthermore, following activation of the P42/p44 MAP kinase module, the binding of Sp1 and AP-2 is increased in the complexes formed in this region of the promoter. Altogether, these data suggest that hypoxia and P42/p44 MAP kinase independently play a key role in the regulation of the VEGF expression.
