DESCRIPTION (provided by applicant): Steroid hormones are key regulators of a diverse array of physiological processes, including sodium 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. 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. A major goal of our research is to understand the mechanisms by which the pituitary-derived hormone adrenocorticotropin (ACTH) regulates cortisol production by the adrenal cortex. Our research has identified a key role for bioactive sphingolipids and phospholipids. These molecules control hormone production by controlling the transcription of steroidogenic genes. We hypothesize that ACTH controls steroid hormone biosynthesis by modulating the availability of bioactive sphingolipids and phospholipids in the nuclei of adrenocortical cells. Specific Aim 1 will define the capacity for nuclear sphingolipid and phospholipid metabolism. Preliminary data has established that bioactive phospholipids and sphingolipids control steroidogenic gene transcription by serving as ligands for the nuclear receptor steroidogenic factor-1 (SF-1). Since is primarily localized in the nucleus, we hypothesize that locally synthesized bioactive lipids are key for controlling SF-1 function. Lipid profiling will be used to gain a comprehensive and quantitative assessment of the concentrations of sphingolipid and phospholipid species in the nucleus and the effect of ACTH on the amounts of these lipids. Specific Aim 2 determine the mechanism by which acid ceramidase (encoded by ASAH1b) regulates nuclear SPH concentrations and SF-1 function. ASAH1b is one of three ceramidases that produces sphingosine (SPH). We have evidence to support a role for direct interaction between ASAH1b and SF-1 and for the regulation of ASAH1 function by phosphorylation. We will investigate the role of this enzyme as a SF-1 coregulatory protein. Specific Aim 3 will define the mechanism by which activation of cAMP signaling promotes ligand (PA)-dependent activation of steroidogenic gene expression. Increased steroidogenic gene expression occurs with the binding agonist ligand (phosphatidic acid) to SF-1 and the subsequent activation of transcription. Tandem mass spectrometry and enzymatic assays will be used to define the role of posttranslational modification in controlling the activity of enzymes that synthesize phosphatidic acid. In sum this proposal addresses major questions about the mechanisms underlying gene regulation by locally produced bioactive lipids. The knowledge gained from these studies will provide valuable information about the metabolic capacity of the nucleus and the key role that dynamic flux of bioactive lipids plays in the control of gene expression.

Relevance Understanding how genes are regulated in cells that make steroid hormones will provide insight into the mechanisms by which pathophysiological concentrations of cortisol and adrenal androgens are produced. This work will provide insight into multiple endocrine disorders, including adrenal hyperplasia, polycystic ovary syndrome, and Cushing's disease.