Metabolites of an Epac-Selective cAMP Analog Induce Cortisol Synthesis by Adrenocortical Cells through a cAMP-Independent Pathway Judith A. Enyeart, John J. Enyeart* Department of Neuroscience, The Ohio State University, College of Medicine and Public Health, Columbus, Ohio, United States of America Abstract Adrenal zona fasciculata (AZF) cells express a cAMP-activated guanine nucleotide exchange protein (Epac2) that may function in ACTH-stimulated cortisol synthesis. Experiments were done to determine whether cAMP analogs that selectively activate Epacs could induce cortisol synthesis and the expression of genes coding for steroidogenic proteins in bovine AZF cells. Treatment of AZF cells with the Epac-selective cAMP analog (ESCA) 8CPT-29-OMe-cAMP induced large (.100 fold), concentration-dependent, delayed increases in cortisol synthesis and the expression of mRNAs coding for the steroid hydroxylases CYP11a1, CYP17, CYP21, and the steroid acute regulatory protein (StAR). However, a non-hydrolyzable analog of this ESCA, Sp-8CPT-29-OMe-cAMP, failed to stimulate cortisol production even at concentrations that activated Rap1, a downstream effector of Epac2. Accordingly, putative metabolites of 8CPT-29-OMe-cAMP, including 8CPT-29-OMe-59AMP, 8CPT-29-OMe-adenosine, and 8CPT-adenine all induced cortisol synthesis and steroid hydroxylase mRNA expression with a temporal pattern, potency, and effectiveness similar to the parent compound. At concentrations that markedly stimulated cortisol production, none of these metabolites significantly activated cAMP-dependent protein kinase (PKA). These results show that one or more metabolites of the ESCA 8CPT-29-OMe-cAMP induce cortico-steroidogenesis by activating a panel of genes that code for steroidogenic proteins. The remarkable increases in cortisol synthesis observed in this study appear to be mediated by a novel cAMP-, Epac- and PKA-independent signaling pathway. Citation: Enyeart JA, Enyeart JJ (2009) Metabolites of an Epac-Selective cAMP Analog Induce Cortisol Synthesis by Adrenocortical Cells through a cAMP- Independent Pathway. PLoS ONE 4(6): e6088. doi:10.1371/journal.pone.0006088 Editor: Damien Keating, Flinders University, Australia Received March 17, 2009; Accepted May 28, 2009; Published June 30, 2009 Copyright: 2009 Enyeart, Enyeart. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Award Number R01-DK47875 (to J. J. E.) from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (http://www2.niddk.nih.gov/). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Diabetes and Digestive and Kidney Diseases or the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: enyeart.1@osu.edu Introduction Cortisol is synthesized from cholesterol and secreted by adrenal zona fasciculata (AZF) cells in a diurnal rhythm under the control of the pituitary peptide adrenocorticotropic hormone (ACTH) [1]. Superimposed on this oscillatory pattern of secretion, stress- induced activation of the hypothalamic-pituitary-adrenal axis leads to additional ACTH-stimulated cortisol production [2]. In AZF cells, the effects of ACTH are mediated through an MC2R melanocortin receptor whose activation is coupled to cAMP synthesis through Gs [3–5]. ACTH stimulates corticosteroid synthesis through two tem- porally distinct cAMP-dependent processes. The acute phase occurs within minutes, is independent of gene transcription, and is initiated by the mobilization and delivery of cholesterol to intracellular sites where steroidogenic enzymes are located [1,6,7]. The delayed increase in cortisol synthesis is mediated through the enhanced transcription of genes coding for steroidogenic proteins, including the steroidogenic acute regula- tory protein (StAR) which transfers cholesterol from the outer to the inner mitochondrial membrane, and the steroidogenic enzymes that mediate the stepwise conversion of cholesterol to cortisol [1,6,8–10]. Most of these enzymes belong to the cytochrome P450 family of mixed function oxidases [11]. cAMP increases mRNA coding for these proteins within hours, with a temporal pattern resembling that produced by ACTH [1,6,8,9,12,13]. Until recently, nearly all of the actions of cAMP in eukaryotic cells were believed to be mediated through the activation of cAMP-dependent protein kinase (PKA). However, two cAMP- activated guanine nucleotide exchange factors (Epac1 and Epac2, also known as cAMP-GEFI and cAMP-GEFII) that activate the small GTPases Rap1 and Rap2 have been identified [14,15]. Epac1 and Epac2 function in various cellular processes, including the secretion of peptide hormones and neurotransmitters [16–18]. While Epac1 is widely expressed mammals, the expression of Epac2 is restricted to a few tissues including the adrenal gland [14,15]. In this regard, the adrenal gland consists of an inner medulla composed of neural crest-derived chromaffin cells and an outer cortex that includes corticosteroid-secreting cells of mesodermal origin [2]. The adrenal cortex includes aldosterone-secreting zona glomerulosa cells and cortisol-secreting cells of the zona fasciculata (AZF). Although Epac2 is highly expressed in AZF cells [19], it isn’t known if cAMP-dependent activation of Epac2 might contribute to ACTH-stimulated cortisol production. PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e6088