Overview
The major goal of the studies carried out in the Sewer lab is to define the factors that regulate the metabolism of steroid hormones. Steroid hormones are key regulators of a diverse array of physiological processes, including the maintenance of carbohydrate metabolism, sodium and fluid homeostasis, reproduction, and the development of secondary sex characteristics. These molecules allow tissues to respond in a coordinated manner to changes in the internal and external environments by functioning as ligands for both nuclear and plasma membrane receptors. Steroid hormones are synthesized from cholesterol by members of the cytochrome P450 superfamily of monooxygenases and steroid dehydrogenases. Because steroid hormones control the expression of numerous genes in virtually all cell types, steroidogenic cells utilize multiple mechanisms that ensure tight control of the synthesis of these molecules. Further, the central role that these molecules play in facilitating communication between different organs and tissues necessitates multiple regulatory mechanisms. One of the main goals of our research program is to elucidate the mechanisms by which the peptide hormone adrenocorticotropin (ACTH) regulates steroid hormone production in the human adrenal cortex. In response to hypothalamic stimulation, ACTH is released from the anterior pituitary and acts to modulate cortisol and androgen secretion from the adrenal cortex by activating numerous signaling pathways, which integrate to maintain optimal steroidogenic capacity. The actions of ACTH in the adrenal cortex are mediated by cAMP and the cAMP-dependent protein kinase (PKA) via two temporally distinct pathways. The acute response leads to uptake and mobilization of cholesterol, the initial substrate for all steroidogenic pathways, to the site of the first enzymatic reaction. The slower, chronic response of ACTH in the adrenal cortex directs transcription of steroidogenic genes.
CYP17 Transcription.
We have spent the past several years examining the mechanism by which ACTH signaling controls the transcription of the steroidogenic enzyme CYP17. CYP17 encodes P450c17, which catalyzes both the 17α-hydroxylation of pregnenolone and progesterone required for cortisol biosynthesis and the 17,20-lyase reaction on 17α-hydroxylated steroids to produce androgens. ACTH/cAMP signaling regulates the transcription of CYP17 by promoting the assembly of a macromolecular complex containing steroidogenic factor-1 (SF-1), p54nrb, SRC-1 (steroid receptor coactivator0-1), and GCN5. The affinity of the complex for the CYP17 promoter is stimulated by cAMP and dependent on phosphatase activity. In the absence of ACTH/cAMP stimulation, the complex interacts with the corepressor mSin3A and histone deacetylases. Since these initial studies, we have carried out exhaustive analyses of the proteins that dynamically interact with the CYP17 promoter in response to ACTH/cAMP (Dammer et al, Mol Endo 2007). These studies demonstrated that SF-1 bound to the CYP17 promoter in a cyclical, transient manner and that this binding occurred in protein complexes with coactivator proteins such as GCN5 and SRC-1. The periodic binding of coactivator complexes is followed by the recruitment of corepressor proteins such as histone deacetylases and the carboxy terminal binding proteins (CtBPs).
Since ACTH induces nuclear accumulation of NADH and NADH regulates the corepressor activity of CtBPs, we predicted that changes in the nuclear concentrations of NADH are a part of the cyclic and periodic assembly and disassembly of transcription coregulator complexes on the CYP17 promoter. ACTH/cAMP stimulates a burst of NAD+ reduction and rapid cytoplasmic to nuclear shuttling of CtBPs concomitant with CtBP oligomerization.
The Nucleus is a Hub for Lipid Metabolism
Like most members of the nuclear receptor superfamily, SF-1 exerts its effects on a wide variety of cellular processes by increasing the transcription of target genes. The classical mechanism by which nuclear receptors increase target gene transcription involves binding to a ligand in the cytoplasm, translocating to the nucleus, forming a hetero- or homodimer with another nuclear receptor, and binding to DNA. However, SF-1 resides primarily in the nucleus, even in the absence of trophic hormone stimulation and binds to DNA as a monomer. We carried out mass spectrometric analysis of SF-1 that was isolated from the H295R human adrenocortical cell line and identified sphingosine (SPH) as an endogenous antagonist. SPH bound to SF-1 in unstimulated H295R cells and dissociated from the receptor in response to ACTH/cAMP stimulation. SPH inhibits the ability of SF-1 to activate CYP17 gene transcription by promoting the binding of corepressor complexes to the receptor. Significantly, in vitro assays demonstrated that SF-1 bound to several sphingolipids and phospholipids. Structural studies carried out by three different laboratories found various phospholipids in the ligand binding pocket of the crystallized receptor.
To identify agonists for SF-1, we once again performed mass spectrometric analysis of the purified receptor and identified phosphatidic acid (PA) as the predominant phospholipid that bound to SF-1. Unlike SPH, PA preferentially bound to the receptor in response to ACTH/cAMP stimulation and was an agonist. PA activated the transcription of CYP17 and several other steroidogenic genes. Stimulation of the ACTH/cAMP signaling pathway increased nuclear PA concentrations by activating diacylglycerol kinase theta (DGKθ). Interestingly, DGKθ binds to SF-1, indicating that ligand binding is facilitated by a direct interaction between the nuclear receptor and DGKθ. Based on these findings, we proposed a mechanism by which SPH maintains low levels of steroid hormone production in the absence of ACTH/cAMP stimulation by keeping SF-1 in an inactive conformation, thereby stabilizing interactions between the receptor and corepressor proteins. Upon ACTH/cAMP stimulation, SPH dissociates from the receptor and PA binds to the ligand binding pocket, thus promoting the interaction with coactivator proteins, including SRC-1 and the histone acetyltransferase GCN5. Ongoing and future studies entail determining the molecular mechanism by which ligand exchange occurs, characterizing the mechanism by which ACTH/cAMP signaling regulates SPH and PA biosynthesis availability, and determining the role of sphingosine kinases (SK) in mediating the dissociation of SPH from SF-1 by phosphorylating SPH. Given that SF-1 is predominantly expressed in nuclei, we are particularly interested in establishing the importance of local lipid metabolism in the nuclei of adrenocortical cells. We hypothesize that the nucleus is a hub for lipid metabolism and that enzymes that metabolize phospholipids and sphingolipids represent a novel class of coregulatory proteins.
Dynamic ACTH-Stimulated Mitochondrial Trafficking Regulates Steroidogenesis
We have recently found that ACTH/cAMP increases the rate of movement of mitochondria in H295R human adrenocortical cells. This rapid mitochondrial trafficking is dependent on microtubules, the RhoA GTPase, and the RhoA effector protein diaphanous 1 (DIAPH1). Further, mitochondrial movement, RhoA, and DIAPH1 are required for cortisol production. Intriguingly, impairing mitochondrial movement or mutating RhoA (or DIAPH1) increase adrenal androgen secretion. These data provide support for a key role for ACTH/cAMP-stimulated mitochondrial trafficking in facilitating inter-organelle substrate delivery. The first enzymatic reaction, conversion of cholesterol to pregnenolone, occurs in the inner mitochondrial membrane. However, the next steps occur in the ER and the final step in cortisol biosynthesis requires that the 11-deoxycortisol produced in the ER be acted on by P450 11β-hydroxylase in mitochondria.
We hypothesize that increased mitochondrial movement in response to ACTH/cAMP signaling allows for substrate delivery by bringing mitochondria in close proximity to regions of the ER that are enriched in steroidogenic enzymes. We also postulate that RhoA, DIAPH1, and intermediate sterol binding proteins form complexes on microtubules that promote substrate exchange between mitochondria and ER. The increase in adrenal androgens observed in adrenal cells expressing RhoA and DIAPH1 mutants suggest that mutations in these proteins may contribute to endocrine disorders such as congenital adrenal hyperplasia. Future studies are also planned to investigate the role of single nucleotide polymorphisms in these genes in states of adrenal androgen excess.
Post-Translational Modifications Control Steroidogenic Capacity
Optimal steroid hormone output requires unique tuning of adrenal cortex cellular metabolism, particularly the balance of reducing equivalents of NADPH and NADH to NADP+ and NAD+, which enable sterol oxidation by cytochromes P450, and hydroxysteroid dehydrogenase activity, while regulating the transcription rate of genes required for cortisol biosynthesis, exemplified by CYP17. When carrying out our studies on the mechanism by which CtBPs repress CYP17 transcription, we stumbled onto a role for NAD+-dependent sirtuin (SIRT) deacetylases in regulating the expression and activity of mitochondrial steroidogenic P450s. SIRT3, a mitochondrial deacetylase targets P450scc and increases the stability of the enzyme, thus resulting in increased cortisol production. We are currently using mass spectrometry to map acetylation sites on P450scc and carrying out studies to explore the role of SIRT-mediated deacetylation in controlling the function of other mitochondrial P450s. Future research in this area will entail identifying the acetyltransferase that targets mitochondrial proteins and characterizing the mechanism by which ACTH/cAMP signaling regulates its activity.
