Corticotropin releasing factor-induced ERK phosphorylation in AtT20 cells occurs via a cAMP-dependent mechanism requiring EPAC2.
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.
Van Kolen
Kristof
K
CNS Discovery Research, Johnson & Johnson Pharmaceutical Research and Development, Division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium. kvkolen@its.jnj.com
Dautzenberg
Frank M
FM
Verstraeten
Karin
K
Royaux
Ines
I
De Hoogt
Ronald
R
Gutknecht
Eric
E
Peeters
Pieter J
PJ
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D018019:Receptors, Corticotropin-Releasing Hormone / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013997:Time Factors; D014162:Transfection / Q000379:methods; D000099052:CRF Receptor, Type 1
D002352:Carrier Proteins; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D034741:RNA, Small Interfering; C486707:Rapgef4 protein, mouse; D018019:Receptors, Corticotropin-Releasing Hormone; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D000099052:CRF Receptor, Type 1; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D037342:Pertussis Toxin; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium