Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P(2U) receptors in MDCK-D1 cells: Involvement of MAP kinase-dependent and -independent pathways

Mingzhao Xing, Bonnie Firestein-Miller, Gregory H. Shen, Paul A. Insel

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74 Citations (Scopus)

Abstract

Defining the mechanism for regulation of arachidonic acid (AA) release is important for understanding cellular production of AA metabolites, such as prostaglandins and leukotrienes. Here we have investigated the differential roles of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase in the regulation of cytosolic phospholipase A2 (cPLA2)- mediated AA release by P(2U)-purinergic receptots in MDCK-D1 cells. Treatment of cells with the P(2U) receptor agonists ATP and UTP increased PLA2 activity in subsequently prepared cell lysates. PLA2 activity was inhibited by the cPLA2 inhibitor AACOCF3, as was AA release in intact cells. Increased PLA2 activity was recovered in anticPLA2 immunoprecipitates of lysates derived from nucleotide-treated cells, and was lost from the immunodepleted lysates. Thus, cPLA2 is responsible for AA release by P(2U) receptors in MDCK-D1 cells. P(2U) receptors also activated MAP kinase. This activation was PKC-dependent since phorbol 12-myristate 13- acetate (PMA) promoted down-regulation of PKC-eliminated MAP kinase activation by ATP or UTP. Treatment of cells with the MAP kinase cascade inhibitor PD098059, the PKC inhibitor GF109203X, or down-regulation of PKC by PMA treatment, all suppressed AA release promoted by ATP or UTP, suggesting that both MAP kinase and PKC are involved in the regulation of cPLA2 by P(2U) receptors. Differential effects of GF109203X on cPLA2- mediated AA release and MAP kinase activation, however, were observed: at low concentrations, GF109203X inhibited AA release promoted by ATP, UTP, or PMA without affecting MAP kinase activation. Since GF109203X is more selective for PKC(α), PKC(α) may act independently of MAP kinase to regulate cPLA2 in MDCK-D1 cells. This conclusion is further supported by data showing that PMA-promoted AA release, but not MAP kinase activation, was suppressed in cells in which PKC(α) expression was decreased by antisense transfection. Based on these data, we propose a model whereby both MAP kinase and PKC are required for cPLA2-mediated AA release by P(2U) receptors in MDCK-D1 cells. PKC plays a dual role in this process through the utilization of different isoforms: PKC(α) regulates cPLA2-mediated AA release independently of MAP kinase, while other PKC isoforms act through MAP kinase activation. This model contrasts with our recently demonstrated mechanism (J. Clin. Invest. 99:1302-1310.) whereby α1-adrenergic receptors in the same cell type regulate cPLA2-mediated AA release only through sequential activation of PKC and MAP kinase.

Original languageEnglish (US)
Pages (from-to)805-814
Number of pages10
JournalJournal of Clinical Investigation
Volume99
Issue number4
DOIs
StatePublished - Feb 15 1997

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Cytosolic Phospholipases A2
Madin Darby Canine Kidney Cells
Mitogen-Activated Protein Kinases
Arachidonic Acid
Protein Kinase C
Uridine Triphosphate
Acetates
Adenosine Triphosphate
Mitogen-Activated Protein Kinase Kinases
Protein Kinase Inhibitors
Protein Isoforms
Down-Regulation
Purinergic P2 Receptors
Protein C Inhibitor
Leukotrienes
Adrenergic Receptors

All Science Journal Classification (ASJC) codes

  • Medicine(all)

Keywords

  • ATP
  • eicosanoid
  • epithelial cells
  • phospholipase
  • purinergic receptors

Cite this

@article{accb11b6312d4c8b90e5de658154efc3,
title = "Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P(2U) receptors in MDCK-D1 cells: Involvement of MAP kinase-dependent and -independent pathways",
abstract = "Defining the mechanism for regulation of arachidonic acid (AA) release is important for understanding cellular production of AA metabolites, such as prostaglandins and leukotrienes. Here we have investigated the differential roles of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase in the regulation of cytosolic phospholipase A2 (cPLA2)- mediated AA release by P(2U)-purinergic receptots in MDCK-D1 cells. Treatment of cells with the P(2U) receptor agonists ATP and UTP increased PLA2 activity in subsequently prepared cell lysates. PLA2 activity was inhibited by the cPLA2 inhibitor AACOCF3, as was AA release in intact cells. Increased PLA2 activity was recovered in anticPLA2 immunoprecipitates of lysates derived from nucleotide-treated cells, and was lost from the immunodepleted lysates. Thus, cPLA2 is responsible for AA release by P(2U) receptors in MDCK-D1 cells. P(2U) receptors also activated MAP kinase. This activation was PKC-dependent since phorbol 12-myristate 13- acetate (PMA) promoted down-regulation of PKC-eliminated MAP kinase activation by ATP or UTP. Treatment of cells with the MAP kinase cascade inhibitor PD098059, the PKC inhibitor GF109203X, or down-regulation of PKC by PMA treatment, all suppressed AA release promoted by ATP or UTP, suggesting that both MAP kinase and PKC are involved in the regulation of cPLA2 by P(2U) receptors. Differential effects of GF109203X on cPLA2- mediated AA release and MAP kinase activation, however, were observed: at low concentrations, GF109203X inhibited AA release promoted by ATP, UTP, or PMA without affecting MAP kinase activation. Since GF109203X is more selective for PKC(α), PKC(α) may act independently of MAP kinase to regulate cPLA2 in MDCK-D1 cells. This conclusion is further supported by data showing that PMA-promoted AA release, but not MAP kinase activation, was suppressed in cells in which PKC(α) expression was decreased by antisense transfection. Based on these data, we propose a model whereby both MAP kinase and PKC are required for cPLA2-mediated AA release by P(2U) receptors in MDCK-D1 cells. PKC plays a dual role in this process through the utilization of different isoforms: PKC(α) regulates cPLA2-mediated AA release independently of MAP kinase, while other PKC isoforms act through MAP kinase activation. This model contrasts with our recently demonstrated mechanism (J. Clin. Invest. 99:1302-1310.) whereby α1-adrenergic receptors in the same cell type regulate cPLA2-mediated AA release only through sequential activation of PKC and MAP kinase.",
keywords = "ATP, eicosanoid, epithelial cells, phospholipase, purinergic receptors",
author = "Mingzhao Xing and Bonnie Firestein-Miller and Shen, {Gregory H.} and Insel, {Paul A.}",
year = "1997",
month = "2",
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doi = "https://doi.org/10.1172/JCI119227",
language = "English (US)",
volume = "99",
pages = "805--814",
journal = "The Journal of clinical investigation",
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publisher = "The American Society for Clinical Investigation",
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TY - JOUR

T1 - Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P(2U) receptors in MDCK-D1 cells

T2 - Involvement of MAP kinase-dependent and -independent pathways

AU - Xing, Mingzhao

AU - Firestein-Miller, Bonnie

AU - Shen, Gregory H.

AU - Insel, Paul A.

PY - 1997/2/15

Y1 - 1997/2/15

N2 - Defining the mechanism for regulation of arachidonic acid (AA) release is important for understanding cellular production of AA metabolites, such as prostaglandins and leukotrienes. Here we have investigated the differential roles of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase in the regulation of cytosolic phospholipase A2 (cPLA2)- mediated AA release by P(2U)-purinergic receptots in MDCK-D1 cells. Treatment of cells with the P(2U) receptor agonists ATP and UTP increased PLA2 activity in subsequently prepared cell lysates. PLA2 activity was inhibited by the cPLA2 inhibitor AACOCF3, as was AA release in intact cells. Increased PLA2 activity was recovered in anticPLA2 immunoprecipitates of lysates derived from nucleotide-treated cells, and was lost from the immunodepleted lysates. Thus, cPLA2 is responsible for AA release by P(2U) receptors in MDCK-D1 cells. P(2U) receptors also activated MAP kinase. This activation was PKC-dependent since phorbol 12-myristate 13- acetate (PMA) promoted down-regulation of PKC-eliminated MAP kinase activation by ATP or UTP. Treatment of cells with the MAP kinase cascade inhibitor PD098059, the PKC inhibitor GF109203X, or down-regulation of PKC by PMA treatment, all suppressed AA release promoted by ATP or UTP, suggesting that both MAP kinase and PKC are involved in the regulation of cPLA2 by P(2U) receptors. Differential effects of GF109203X on cPLA2- mediated AA release and MAP kinase activation, however, were observed: at low concentrations, GF109203X inhibited AA release promoted by ATP, UTP, or PMA without affecting MAP kinase activation. Since GF109203X is more selective for PKC(α), PKC(α) may act independently of MAP kinase to regulate cPLA2 in MDCK-D1 cells. This conclusion is further supported by data showing that PMA-promoted AA release, but not MAP kinase activation, was suppressed in cells in which PKC(α) expression was decreased by antisense transfection. Based on these data, we propose a model whereby both MAP kinase and PKC are required for cPLA2-mediated AA release by P(2U) receptors in MDCK-D1 cells. PKC plays a dual role in this process through the utilization of different isoforms: PKC(α) regulates cPLA2-mediated AA release independently of MAP kinase, while other PKC isoforms act through MAP kinase activation. This model contrasts with our recently demonstrated mechanism (J. Clin. Invest. 99:1302-1310.) whereby α1-adrenergic receptors in the same cell type regulate cPLA2-mediated AA release only through sequential activation of PKC and MAP kinase.

AB - Defining the mechanism for regulation of arachidonic acid (AA) release is important for understanding cellular production of AA metabolites, such as prostaglandins and leukotrienes. Here we have investigated the differential roles of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase in the regulation of cytosolic phospholipase A2 (cPLA2)- mediated AA release by P(2U)-purinergic receptots in MDCK-D1 cells. Treatment of cells with the P(2U) receptor agonists ATP and UTP increased PLA2 activity in subsequently prepared cell lysates. PLA2 activity was inhibited by the cPLA2 inhibitor AACOCF3, as was AA release in intact cells. Increased PLA2 activity was recovered in anticPLA2 immunoprecipitates of lysates derived from nucleotide-treated cells, and was lost from the immunodepleted lysates. Thus, cPLA2 is responsible for AA release by P(2U) receptors in MDCK-D1 cells. P(2U) receptors also activated MAP kinase. This activation was PKC-dependent since phorbol 12-myristate 13- acetate (PMA) promoted down-regulation of PKC-eliminated MAP kinase activation by ATP or UTP. Treatment of cells with the MAP kinase cascade inhibitor PD098059, the PKC inhibitor GF109203X, or down-regulation of PKC by PMA treatment, all suppressed AA release promoted by ATP or UTP, suggesting that both MAP kinase and PKC are involved in the regulation of cPLA2 by P(2U) receptors. Differential effects of GF109203X on cPLA2- mediated AA release and MAP kinase activation, however, were observed: at low concentrations, GF109203X inhibited AA release promoted by ATP, UTP, or PMA without affecting MAP kinase activation. Since GF109203X is more selective for PKC(α), PKC(α) may act independently of MAP kinase to regulate cPLA2 in MDCK-D1 cells. This conclusion is further supported by data showing that PMA-promoted AA release, but not MAP kinase activation, was suppressed in cells in which PKC(α) expression was decreased by antisense transfection. Based on these data, we propose a model whereby both MAP kinase and PKC are required for cPLA2-mediated AA release by P(2U) receptors in MDCK-D1 cells. PKC plays a dual role in this process through the utilization of different isoforms: PKC(α) regulates cPLA2-mediated AA release independently of MAP kinase, while other PKC isoforms act through MAP kinase activation. This model contrasts with our recently demonstrated mechanism (J. Clin. Invest. 99:1302-1310.) whereby α1-adrenergic receptors in the same cell type regulate cPLA2-mediated AA release only through sequential activation of PKC and MAP kinase.

KW - ATP

KW - eicosanoid

KW - epithelial cells

KW - phospholipase

KW - purinergic receptors

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