Corticotropin releasing factor-induced ERK phosphorylation in AtT20 cells occurs via a cAMP-dependent mechanism requiring EPAC2

• Published in: Neuropharmacology Vol. 58; no. 1; pp. 135 - 144
• Main Authors: Van Kolen, Kristof, Verstraeten, Karin, Royaux, Ines, De Hoogt, Ronald, Gutknecht, Eric, Peeters, Pieter J.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 2010
• Subjects: Animals; AtT20; Calcium - metabolism; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Line; Chelating Agents - pharmacology; Corticotropin-Releasing Hormone - pharmacology; CRF; Cyclic AMP - metabolism; Cyclic AMP-Dependent Protein Kinases - metabolism; Egtazic Acid - analogs & derivatives; Egtazic Acid - pharmacology; Enzyme Inhibitors - pharmacology; EPAC; ERK phosphorylation; Extracellular Signal-Regulated MAP Kinases - metabolism; GTP-Binding Protein alpha Subunits, Gi-Go - metabolism; Guanine Nucleotide Exchange Factors - genetics; Guanine Nucleotide Exchange Factors - metabolism; Humans; Mice; Pertussis Toxin - pharmacology; Phosphorylation - drug effects; Receptors, Corticotropin-Releasing Hormone - metabolism; RNA, Small Interfering - pharmacology; Signal Transduction - drug effects; Signal Transduction - physiology; Time Factors; Transfection - methods; HTRF; PMA; H-89; EPAC; PKI; POMC; MEK; CREB; ERK phosphorylation; PTX; CRF; AtT20; 8-pMeOPT-2′-O-Me-cAMP; HPA; B2m; BAPTA-AM; ERK
• ISSN: 0028-3908; 1873-7064
• DOI: 10.1016/j.neuropharm.2009.06.022

CRF-induced ERK phosphorylation has been shown to be an important mechanism underlying expression of pro-opiomelanocortin, a key precursor molecule in the hypothalamic pituitary adrenal axis. In AtT20 cells, CRF signalling has been investigated but the mechanism behind CRF-induced ERK activity is not fully understood. This paper elucidates the signalling cascade involved in this phenomenon.
Involvement of CRF
1 receptor on ERK phosphorylation was shown by using CRF and urocortin 1. The lack of inhibitory effect of pertussis toxin and BAPTA-AM excluded involvement of G
i-coupling and calcium mobilization respectively. In contrast, the process is suggested to be driven by cAMP since treatment of AtT20 cells with forskolin triggered strong ERK phosphorylation. Treatment with PKA inhibitors had a minor effect on CRF-induced ERK signalling while phosphorylation of CREB was completely abolished. This ruled out involvement of PKA and suggested a role for exchange protein directly activated by cAMP (EPAC). Moreover, an activator of EPACs 8-(4-methoxyphenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate mimicked CRF-induced ERK phosphorylation. Gene expression analysis showed high levels of EPAC2 mRNA and protein but low levels of EPAC1. Knockdown of EPAC2 expression by the use of specific siRNAs abolished CRF- and forskolin-induced ERK phosphorylation. The current study demonstrates a clear cAMP-dependent but PKA-independent mechanism underlying CRF-induced ERK activity that proceeds via EPAC2 signalling. Further research will provide more insight in the role of EPAC2 in CRF signalling.