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-2′-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-2′-OMe-cAMP, failed to stimulate cortisol production even at concentrations that activated Rap1, a downstream effector of Epac2. Accordingly, putative metabolites of 8CPT-2′-OMe-cAMP, including 8CPT-2′-OMe-5′AMP, 8CPT-2′-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-2′-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.
Highlights
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]
The delayed increase in cortisol synthesis is mediated through the enhanced transcription of genes coding for steroidogenic proteins, including the steroidogenic acute regulatory 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 [1,6,8,9,10]
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]
Summary
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, stressinduced activation of the hypothalamic-pituitary-adrenal axis leads to additional ACTH-stimulated cortisol production [2]. The delayed increase in cortisol synthesis is mediated through the enhanced transcription of genes coding for steroidogenic proteins, including the steroidogenic acute regulatory 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 [1,6,8,9,10]. 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]
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