Activation of protein kinase Calpha by EPAC1 is required for the ERK- and CCAAT/enhancer-binding protein beta-dependent induction of the SOCS-3 gene by cyclic AMP in COS1 cells.
We recently found that induction of the anti-inflammatory SOCS-3 gene by cyclic AMP occurs through novel cyclic AMP-dependent protein kinase-independent mechanisms involving activation of CCAAT/enhancer-binding protein (C/EBP) transcription factors, notably C/EBPbeta, by the cyclic AMP GEF EPAC1 and the Rap1 GTPase. In this study we show that down-regulation of phospholipase (PL) Cepsilon with small interfering RNA or blockade of PLC activity with chemical inhibitors ablates exchange protein directly activated by cyclic AMP (EPAC)-dependent induction of SOCS-3 in COS1 cells. Consistent with this, stimulation of cells with 1-oleoyl-2-acetyl-sn-glycerol and phorbol 12-myristate 13-acetate, both cell-permeable analogues of the PLC product diacylglycerol, are sufficient to induce SOCS-3 expression in a Ca2+-dependent manner. Moreover, the diacylglycerol- and Ca2+-dependent protein kinase C (PKC) isoform PKCalpha becomes activated following cyclic AMP elevation or EPAC stimulation. Conversely, down-regulation of PKC activity with chemical inhibitors or small interfering RNA-mediated depletion of PKCalpha or -delta blocks EPAC-dependent SOCS-3 induction. Using the MEK inhibitor U0126, we found that activation of ERK MAPKs is essential for SOCS-3 induction by either cyclic AMP or PKC. C/EBPbeta is known to be phosphorylated and activated by ERK. Accordingly, we found ERK activation to be essential for cyclic AMP-dependent C/EBP activation and C/EBPbeta-dependent SOCS-3 induction by cyclic AMP and PKC. Moreover, overexpression of a mutant form of C/EBPbeta (T235A), which lacks the ERK phosphorylation site, blocks SOCS-3 induction by cyclic AMP and PKC in a dominant-negative manner. Together, these results indicate that EPAC mediates novel regulatory cross-talk between the cyclic AMP and PKC signaling pathways leading to ERK- and C/EBPbeta-dependent induction of the SOCS-3 gene.
Division of Molecular and Cellular Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
Gillian
G
Borland
Rebecca J
RJ
Bird
Timothy M
TM
Palmer
Stephen J
SJ
Yarwood
D000818:Animals; D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015870:Gene Expression; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D000089804:Shelterin Complex; D015398:Signal Transduction; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins / Q000235:genetics* / Q000378:metabolism; D034501:Telomere-Binding Proteins / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D022782:CCAAT-Enhancer-Binding Protein-beta; D020662:Guanine Nucleotide Exchange Factors; C116122:RAPGEF3 protein, human; D034741:RNA, Small Interfering; C106644:SOCS3 protein, human; D000089804:Shelterin Complex; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; C440809:TERF2IP protein, human; D034501:Telomere-Binding Proteins; D000242:Cyclic AMP; D008156:Luciferases; D051571:Protein Kinase C-alpha; D048049:Extracellular Signal-Regulated MAP Kinases; D054801:Phosphoinositide Phospholipase C; C423320:phospholipase C epsilon; D013755:Tetradecanoylphorbol Acetate