Functional roles of protein kinase A (PKA) and exchange protein directly activated by 3',5'-cyclic adenosine 5'-monophosphate (cAMP) 2 (EPAC2) in cAMP-mediated actions in adrenocortical cells.
In the adrenal cortex, the biosynthesis of steroid hormones is controlled by the pituitary-derived hormone ACTH. The functions of ACTH are principally relayed by activating cAMP-dependent signaling pathways leading to the induction of genes encoding enzymes involved in the conversion of cholesterol to steroid hormones. Previously, protein kinase A (PKA) was thought to be the only direct effector of cAMP. However, the discovery of the cAMP sensors, exchange proteins directly activated by cAMP (EPAC1 and 2), has led to a reevaluation of this assumption. In the present study, we demonstrate the occurrence of the EPAC2 splicing variant EPAC2B in adrenocortical cancer cells. Immunocytochemistry demonstrated that EPAC2B is localized predominantly in the nucleus. EPAC2B is functional because it activates Rap1 in these cells. Using the cAMP analogs 8-p-chlorophenylthio-2'-O-methyl-cAMP and N6-benzoyl-cAMP, which specifically activate EPAC1/2 and PKA, respectively, we evaluated the contribution of these factors in steroid hormone production, cell morphology, actin reorganization, and migration. We demonstrate that the expression of cAMP-inducible factors involved in steroidogenesis (steroidogenic acute regulatory protein, cytochrome P450 11A1 and 17, and nerve growth factor-induced clone B) and the cAMP-induced biosynthesis of steroid hormones (cortisol and aldosterone) are mediated by PKA and not by EPAC2B. In contrast, both PKA- and EPAC-specific cAMP analogs induced cell rounding, loss of stress fibers, and blocked migration. Taken together, the presented data confirm PKA as the central cAMP mediator in steroid hormone production and reveal the involvement of EPAC2B in cAMP-induced effects on cytoskeleton integrity and cell migration.
Department of Biomedicine, University of Bergen, Bergen, Norway.
Linda
L
Aumo
Marte
M
Rusten
Gunnar
G
Mellgren
Marit
M
Bakke
Aurélia E
AE
Lewis
D000302:Adrenal Cortex / Q000378:metabolism / Q000473:pathology; D000450:Aldosterone / Q000096:biosynthesis; D017398:Alternative Splicing; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism*; D048430:Cell Shape / Q000187:drug effects; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D006854:Hydrocortisone / Q000096:biosynthesis; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; D020033:Protein Isoforms; C442864:RAPGEF4 protein, human; D000450:Aldosterone; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D020827:rap1 GTP-Binding Proteins; D006854:Hydrocortisone