Abstract
The ability of prototypical second messenger cyclic AMP (cAMP) to positively control transcription of the somatostatin gene was pivotal to the original identification of the transcription factor cAMP response element-binding protein. However, it is now clear that alternative intracellular cAMP sensors, of which the exchange protein directly activated by cAMP (Epac) proteins have been studied most intensively, also initiate transcription of key genes in response to cAMP elevation. For example, we have demonstrated in vascular endothelial cells that activation of Epac1 is necessary for cAMP-mobilizing agents to trigger the induction of the gene-encoding suppressor of cytokine signaling-3 (SOCS-3), a potent inhibitor of interleukin (IL)-6 signaling. This is achieved through the recruitment of CCAAT/enhancer-binding protein (C/EBP) transcription factors to the SOCS-3 promoter. Here, we describe in detail how to identify and measure cAMP-mediated recruitment of a specific C/EBP isoform to a candidate regulator region of the SOCS-3 promoter in vascular endothelial cells in vitro. We also describe the RNA interference strategies with which we identified a role for Epac1 and SOCS-3 in being responsible for mediating the inhibitory effect of cAMP elevation on IL-6 signaling.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Hansson, G.K., and Libby, P. (2006) The immune response in atherosclerosis: a double-edged sword. Nat. Rev. Immunol. 6, 508–519.
Schouten, M., Wiersinga, W.J., Levi, M, and van der Poll, T. (2008) Inflammation, endothelium, and coagulation in sepsis. J. Leukoc. Biol. 83, 536–545.
Hansson, G.K., Robertson, A.K., and Söderberg-Nauclér, C. (2006) Inflammation and atherosclerosis. Annu. Rev. Pathol. 1, 297–329.
Choi EY, Santoso S, and Chavakis T. (2009) Mechanisms of neutrophil transendothelial migration. Front. Biosci. 14, 1596–1605.
Sands, W.A., Woolson, H.D., Milne, G.R., Rutherford, C., and Palmer T.M. (2006) Exchange protein activated by cyclic AMP (Epac)-mediated induction of suppressor of cytokine signaling 3 (SOCS-3) in vascular endothelial cells. Mol. Cell Biol. 26, 6333–6346.
Yarwood, S.J., Borland, G., Sands, W.A., and Palmer, T.M. (2008) Identification of CCAAT/enhancer-binding proteins as exchange protein activated by cAMP-activated transcription factors that mediate the induction of the SOCS-3 gene. J. Biol. Chem. 283, 6843–6853.
Borland, G., Bird, R.J., Palmer, T.M., and Yarwood, S.J. (2009) 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. J Biol Chem. 284, 17391–17403.
Woolson, H.D., Thomson, V.S., Rutherford, C., Yarwood, S.J., and Palmer, T.M. (2009) Selective inhibition of cytokine-activated extracellular signal-regulated kinase by cyclic AMP via Epac1-dependent induction of suppressor of cytokine signalling-3. Cell. Signal. 21, 1706–1715.
Enserink, J.M., Christensen, A.E., de Rooij, J., van Triest, M., Schwede, F., Genieser, H.G., Døskeland, S.O., Blank, J.L., and Bos, J.L. (2002) A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK. Nat. Cell Biol. 4, 901–906.
Johnson, D.S., Mortazavi, A., Myers, R M., and Wold, B. (2007) Genome-wide mapping of in vivo protein-DNA interactions. Science 316, 1497–502.
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., and Klenk, D.C. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem. 150, 76–85.
Sledz, C.A., and Williams, B.R. (2004) RNA interference and double-stranded-RNA-activated pathways. Biochem. Soc. Trans. 32, 952–956.
Acknowledgments
This work was supported by project grants from the British Heart Foundation, the UK Biotechnology and Biological Sciences Research Council, and Chest Heart and Stroke Scotland to TMP and SJY and a PhD studentship from the British Heart Foundation to HDW.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Sands, W.A., Woolson, H.D., Yarwood, S.J., Palmer, T.M. (2012). Exchange Protein Directly Activated by Cyclic AMP-1-Regulated Recruitment of CCAAT/Enhancer-Binding Proteins to the Suppressor of Cytokine Signaling-3 Promoter. In: Vancura, A. (eds) Transcriptional Regulation. Methods in Molecular Biology, vol 809. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-376-9_14
Download citation
DOI: https://doi.org/10.1007/978-1-61779-376-9_14
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-61779-375-2
Online ISBN: 978-1-61779-376-9
eBook Packages: Springer Protocols