Abstract
To understand the effects of the immunosuppressant cyclosporin A (CsA) on Ca2+-mediated intracellular signalling pathways in human peripheral blood mononuclear cells (PBMCs), we investigated its effects on the activity profiles of mitogen-activated protein kinase (MAPK) cascades. PBMCs, or subpopulations thereof, were simultaneously stimulated with a phorbol ester and the calcium ionophore ionomycin, in the presence or absence of therapeutic concentrations of CsA. In these primary human cells, CsA significantly inhibited PMA/ionomycin-mediated and ionomycin-mediated activation of the MAPK kinase MKK6, as well as its downstream kinases SAPK2a (p38alpha) and MAPKAP-K2. PMA/ionomycin treatment also mediated activation of SAPK1 (JNKs) which was inhibited by CsA. Treatment with ionomycin alone also resulted in CsA-sensitive activation of SAPK1. With regard to transcription factors targeted by the Ca2+-induced MAPK signalling network, we found CsA to inhibit the ionomycin-mediated phosphorylation of ATF2 at Thr71. We identified the heterodimeric transcription factor ATF2/CREB as constitutively binding to the essential cAMP response element (CRE) site within the Ca2+-regulated DNA polymerase beta promoter and contributing to the activation of this promoter. Our data implicate ATF2 phosphorylation status as a nuclear sensor within PBMCs that monitors converging intracellular Ca2+-signalling pathways.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Activating Transcription Factor 2
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Base Sequence
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Binding Sites
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Calcineurin / metabolism
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Calcium Signaling / drug effects*
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Calcium-Calmodulin-Dependent Protein Kinases*
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Cyclic AMP Response Element-Binding Protein / metabolism
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Cyclosporine / pharmacology*
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DNA / genetics
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Enzyme Activation / drug effects
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Genes, pol / drug effects
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Humans
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Immunosuppressive Agents / pharmacology*
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In Vitro Techniques
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Intracellular Signaling Peptides and Proteins
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Ionomycin / pharmacology
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JNK Mitogen-Activated Protein Kinases
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Leukocytes, Mononuclear / drug effects*
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Leukocytes, Mononuclear / enzymology
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Leukocytes, Mononuclear / metabolism*
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MAP Kinase Kinase 6
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Mitogen-Activated Protein Kinase Kinases / metabolism
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Mitogen-Activated Protein Kinases / metabolism*
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Phosphorylation
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Promoter Regions, Genetic
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Protein Serine-Threonine Kinases / metabolism
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Transcription Factors / metabolism
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p38 Mitogen-Activated Protein Kinases
Substances
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ATF2 protein, human
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Activating Transcription Factor 2
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Cyclic AMP Response Element-Binding Protein
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Immunosuppressive Agents
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Intracellular Signaling Peptides and Proteins
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Transcription Factors
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Ionomycin
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Cyclosporine
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DNA
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MAP-kinase-activated kinase 2
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Protein Serine-Threonine Kinases
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Calcium-Calmodulin-Dependent Protein Kinases
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JNK Mitogen-Activated Protein Kinases
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Mitogen-Activated Protein Kinases
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p38 Mitogen-Activated Protein Kinases
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MAP Kinase Kinase 6
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MAP2K6 protein, human
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Mitogen-Activated Protein Kinase Kinases
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Calcineurin