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Exchange Protein Directly Activated by Cyclic AMP-1-Regulated Recruitment of CCAAT/Enhancer-Binding Proteins to the Suppressor of Cytokine Signaling-3 Promoter

  • William A. Sands
  • Hayley D. Woolson
  • Stephen J. Yarwood
  • Timothy M. PalmerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 809)

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.

Key words

Cyclic AMP Epac Chromatin immunoprecipitation Endothelial cells RNA interference 

Notes

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.

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • William A. Sands
    • 1
  • Hayley D. Woolson
    • 1
  • Stephen J. Yarwood
    • 2
  • Timothy M. Palmer
    • 1
    Email author
  1. 1.Institute for Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life SciencesUniversity of GlasgowGlasgowUK
  2. 2.Institute for Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life SciencesUniversity of GlasgowGlasgowUK

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