Abstract
The prototypical second messenger cAMP is a key regulator of immune and inflammatory responses. Its ability to inhibit interleukin (IL)-6 responses is due to induction of suppressor of cytokine signaling-3 (SOCS-3), a negative regulator of IL-6 receptor signaling. We have determined previously that SOCS-3 induction by cAMP occurs independently of cAMP-dependent protein kinase, instead requiring the recently identified cAMP sensor exchange protein activated by cAMP 1 (EPAC1). Here we present evidence to suggest that the C/EBP family of transcription factors link EPAC1 activation to SOCS-3 induction. Firstly, selective activation of EPAC in human umbilical vein endothelial cells increased C/EBP DNA binding activity and recruitment of C/EBPbeta to the SOCS-3 promoter. Secondly, knockdown of C/EBPbeta and -delta isoforms abolished both SOCS-3 induction and inhibition of IL-6 signaling in response to cAMP. Thirdly, overexpression of C/EBPalpha, -beta, or -delta potentiated EPAC-mediated accumulation of SOCS-3. Finally, these effects were not restricted to human umbilical vein endothelial cells, because similar phenomena were observed in murine embryonic fibroblasts in which C/EBPbeta or delta had been deleted. In summary, our findings constitute the first description of an EPAC-C/EBP pathway that can control cAMP-mediated changes in gene expression independently of protein kinase A.
Highlights
Persistent reports that certain signaling actions of cAMP are not mediated by protein kinase A (PKA), coupled with attempts to delineate the pathway by which cAMP activates the small GTPase Rap1, led to the discovery of a family of cAMP-activated guanine nucleotide exchange factors termed EPACs [5, 6]
3 The abbreviations used are: PKA, cAMP-dependent protein kinase; 8-pCPT2Ј-O-Me-cAMP, 8-(4-chlorophenylthio)-2Ј-O-methyladenosine-3Ј,5Ј-cyclic monophosphate; CCAAT/enhancer-binding proteins (C/EBPs), CCAAT/enhancer-binding protein; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; EPAC, exchange protein directly activated by cAMP; HUVEC, human umbilical vein endothelial cell; Suppressor of cytokine signaling 3 (SOCS-3), suppressor of cytokine signaling 3; siRNA, small interfering RNA; STAT, signal transducers and activators of transcription; cAMP-response elements (CREs), cAMP-response element; CBP, cAMP response element-binding protein (CREB)-binding protein; CREB, cAMP-response element-binding protein; Wild type (WT), wild type; MEF, mouse embryonic fibroblast; DIG, digoxigenin; IL, interleukine; forskolin and M rolipram (F/R), forskolin and rolipram
EPAC Is Necessary and Sufficient for cAMP-mediated SOCS-3 Accumulation in MEFs—We have recently identified a novel anti-inflammatory pathway in HUVECs, whereby selective activation of the cAMP sensor exchange protein activated by cAMP 1 (EPAC1), a guanine nucleotide exchange factor for Rap1, is necessary and sufficient to expression was undetectable in MEF cell lines
Summary
Until very recently it was generally considered that the intracellular effects of cAMP are mediated solely by protein kinase A (PKA).3 persistent reports that certain signaling actions of cAMP are not mediated by PKA, coupled with attempts to delineate the pathway by which cAMP activates the small GTPase Rap1, led to the discovery of a family of cAMP-activated guanine nucleotide exchange factors termed EPACs (exchange protein directly activated by cAMP) [5, 6]. To investigate a potential role of C/EBP isoforms in the control of SOCS-3 expression in response to cAMP, we decided initially to use isoform-specific C/EBP knock-out MEFs. The importance of EPAC in cAMP-mediated SOCS-3 induction in these cells was determined by stimulation of wild-type (WT) MEFs with a combination of the cAMP-elevating agents, forskolin and rolipram (F/R), which produced a robust induction of SOCS-3 protein (Fig. 2A).
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