Cortisol Secretion Pathway Figure 5. The 8CPT-29-OMe-cAMP Metabolite 8CPT-29-OMe-59AMP Increases Cortisol Secretion and CYP17 mRNA Expression. A) Time- and concentration-dependent effects of 8CPT-29-OMe-59AMP on cortisol secretion by AZF cells. AZF cells were treated with 8CPT-29-OMe- 59AMP at concentrations ranging from 0.01 to 100 mM. Cortisol was measured from media samples after 6, 24 and 48 h. B) Concentration-dependent effects of 8CPT-29-OMe-59AMP on CYP17 mRNA expression. AZF cells were incubated without (control, white bar) or with 0.01–100 mM 8CPT-29-OMe- 59AMP (black bars) for 48 hr before isolating total RNA. doi:10.1371/journal.pone.0006088.g005 CYP11a1 and CYP21 mRNAs were also increased similarly (data not shown). 8CPT-29-OMe-59AMP itself may be a substrate for 59 nucleotidases that would convert this molecule to 8-(4-chlorophe- nylthio)-29-O-methyladenosine (8CPT-29-OMe-Ado) (see Figure 4) [36]. Accordingly, 8CPT-29-OMe-Ado stimulated cortisol secre- tion and the expression of steroid hydroxylases with characteristics similar to the parent molecules. In contrast, adenosine was completely ineffective at stimulating cortisol synthesis or mRNA expression (Figure 6A,B). 8CPT-29-OMe-Ado may be further metabolized to 8CPT-29- adenine (8CPT-Ade) by purine nucleoside phosphorylase (see Figure 4) [36]. 8CPT-Ade also induced a delayed increase in both cortisol synthesis and expression of steroid hydroxylase mRNA with a temporal pattern, potency, and effectiveness similar to each of the previously described putative metabolites (Figure 6C, D). In summary, the Epac-selective cAMP activator 8CPT-29- OMe-cAMP stimulated cortico-steroidogenesis by bovine AZF cells through a mechanism which is likely independent of Epac2 activation. Rather, the steroidogenic effect appears to be mediated through one or more metabolites that, after several hours delay, induce the expression of mRNAs coding for steroid hydroxylases involved in the synthesis of cortisol from cholesterol. Stimulation of StAR Expression by 8CPT-29-OMe-59AMP and its Metabolites The rate-limiting step in ACTH-stimulated cortisol synthesis, the transfer of cholesterol from the outer to the inner mitochondrial membrane, is mediated by StAR [7,10,37]. We found that, in addition to increasing the expression of steroid hydroxylase genes, 8CPT-29-OMe-cAMP also induced the expression of StAR mRNA. The potency and temporal pattern were similar to those observed for increases in steroid hydroxylase mRNAs (Figure 7A,B). In contrast, Sp-8CPT-29-OMe-cAMP failed to induce the expression of StAR mRNA, indicating that this effect was again mediated by one or more metabolites of the hydrolyzable ESCA (Figure 7C). Accordingly, both 8CPT-29- OMe-59AMP and 8CPT-adenine stimulated large increases in the quantity of StAR mRNAs (Figure 7C,D). 8CPT-29-OMe-cAMP and 8CPT-Ade induced StAR mRNA similarly at concentrations from 10–50 mM (Figure 7A,D). Steroidogenic Effects of 8CPT-29-OMe-cAMP are PKA- independent Although 8CPT-29-OMe-cAMP is a relatively specific activator of Epac proteins, it is a weak and less effective activator of PKA [28,30,35]. It isn’t known whether the putative Epac metabolites activate PKA at the same concentrations that stimulate cortisol secretion. It was discovered that none of these agents increased PKA activity in AZF cells when they were applied at concentrations sufficient to induce large increases in cortisol synthesis (Figure 8A). These metabolites also failed to activate PKA when applied directly to AZF cell lysates (data not shown). Cycloheximide Sensitivity The delay of hours that precedes the 8CPT-29-OMe-cAMP- induced increase in the expression of genes coding for steroido- genic proteins suggests that this effect requires de novo protein synthesis. Accordingly, the protein synthesis inhibitor cyclohexi- mide (5 mg/ml) inhibited the expression of mRNAs coding for each of the three steroid hydroxylases and StAR (Figure 8B). Discussion The major findings of this study are that the Epac-selective cAMP analog 8CPT-29-OMe-cAMP induced large, delayed increases in the expression of mRNAs coding for steroidogenic proteins, including three steroid hydroxylases and StAR, leading to corresponding increases in cortisol synthesis. Importantly, these corticosteroidogenic actions are not mediated by the ESCA itself, but by one or more enzymatic products of this compound. These metabolites, including 8CPT-29-OMe-59AMP, 8CPT-29-OMe- Ado, and 8CTP-Ade, all induce mRNAs and cortisol synthesis with a potency and temporal pattern similar to the parent compound, but by an unknown Epac2- and PKA-independent PLoS ONE | www.plosone.org 7 June 2009 | Volume 4 | Issue 6 | e6088