Angiotensin II (AngII) stimulates adrenocortical cells to produce aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in the precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding prosteroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. These data support a model in which coordinated increases in transcription and decay facilitates the major transcriptomic changes required to implement a pro-steroidogenic expression program that actively resolved to prevent aldosterone overproduction.

Adrenal steroid hormone production is a dynamic process stimulated by adrenocorticotropic hormone (ACTH) and angiotensin II (AngII). These ligands initialize a rapid and robust gene expression response required for steroidogenesis. Here, we compare the predominant human immortalized cell line model, H295R cell, with primary cultures of adult adrenocortical cells derived from human kidney donors. We performed temporally resolved RNA-seq on primary cells stimulated with either ACTH or AngII at multiple time points. The magnitude of the expression dynamics elicited by ACTH was greater than AngII in primary cells. This is likely due to the larger population of adrenocortical cells that are responsive to ACTH. The dynamics of stimulus-induced expression in H295R cells are mostly recapitulated in primary cells. However, there are some expression responses in primary cells absent in H295R cells. These data are a resource for the endocrine community and will help researchers determine whether H295R is an appropriate model for the specific aspect of steroidogenesis that they are studying.

Angiotensin II (AngII) binds to the type I angiotensin receptor in the adrenal cortex to initiate a cascade of events leading to the production of aldosterone, a master regulator of blood pressure and volume. Little is known about post-transcriptional regulation of this process. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII or ACTH, which are models for aldosterone and cortisol production respectively. Using a candidate siRNA screen, we identified a subset of RBPs that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of a mixed incoherent feedforward loop controlling aldosterone homeostasis. Together, these data support a model in which coordinated increases in transcription and RBP-regulated RNA decay facilitates the major transcriptomic changes required to implement a pro-steroidogenic gene expression program that is temporally restricted to prevent aldosterone overproduction.

Complex cellular resonses require the temporal coordination of stimulus-induced gene expression programs. Angiotensin II (AngII) is the active 8 amino acid peptide in the renin-angiotensin-aldosterone system that controls blood pressure and fluid balance. AngII binds to type I angiotensin receptor in the adrenal cortex to initiate a cascade of temporally coordinated events leading to the production of aldosterone, a master regulator of blood pressure and volume. We stimulated a steroidogenic human cell line (H295R) with AngII and performed RNA-seq at twelve points. We identified twelve distict temporally distinct groups of gene expression responses each encoding functionally related proteins important for various steps of aldosterone productionl. Interstingly, the shape of the impulse response suggested a key role for RNA decay. Indeed, RNA decay rates in unstimulated H295R cells strongly correlated with the amplitude and peakiness of the gene expression response for each group of genes. We also found evidence for increases in RNA decay during the AngII response. Next, we selected candidate RBPs based on motif finding, adrenal specific expression, and AngII responsiveness. We performed an siRNA knockdown screen on these 22 candidates to identify RBPs that regulate aldosterone levels. Eight of these RBPs exhibited statistically significant changes in aldosterone for at least two independent siRNAs. Interestingly, multiple RBPs that promote RNA decay were found to suppress aldosterone production and induced in response to AngII-stimulation. These RBPs could be responsible for our observed increases in RNA decay. Altogether, these data support a model in which RNA decay is a critical regulator of the timing and strength of AngII-induced gene expression and ultimately aldosterone production.

Human steroid hormones produced by the adrenal cortex control important physiology including metabolism, inflammation, blood pressure and volume, and sexual characteristics. While the signaling components, transcriptional regulators, and steroidogenic enzymes necessary for adrenocortical production of hormones have been identified, little to nothing is known about post-transcriptional regulation by RNA-binding proteins (RBPs). Recently technological advances have revolutionized our ability to investigate RBP-driven RNA regulation making it possible for the first time to investigate how this mechanism controls steroidogenesis. We have recently carried out an siRNA screen of RBPs regulating human aldosterone production that revealed a critical role for the tristetraprolin (ZFP36) family of RNA-binding protein. The ZFP36 family of RBPs binds to AU-rich elements in 3’UTRs and consequently destabilizes and/or translationally represses these ARE-containing mRNAs. Remarkably, depletion of either ZFP36L2 or ZFP36L1 significantly increased aldosterone levels. In addition, we have data demonstrating that: 1) mRNA stability controls the temporal pattern of RNA expression during steroidogenesis; 2) mRNAs with AU-rich elements (AREs) in their 3’ UTR are rapidly induced and cleared out in response to steroidogenic stimulation; 3) The ZFP36 family of RBPs are induced during steroidogenesis. We propose a model in which the ZFP36 family of proteins operate a negative feedback loop that prevent overproduction of aldosterone by destabilizing and/or translationally repressing ARE-containing mRNAs encoding steroidogenic proteins . Notably, over-production of aldosterone is a major cause of hypertension, suggesting that failure of this negative feedback loop could have important implications for human health. Our ongoing work will elucidate the mechanism underlying this negative feedback loop that controls aldosterone biosynthesis post-transcriptionally through the action of ZFP36 RNA binding proteins and yet to be discovered factors that they interact with. The adrenal cortex is amenable to the delivery of modified antisense oligonucleotides. Thus, our discoveries can facilitate the design of oligonucleotide therapeutics that can be used to precisely and specifically modulate human steroidogenesis. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.