RNA-binding proteins regulate aldosterone homeostasis in human steroidogenic cells

Abstract

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.

Transcriptomic Response Dynamics of Human Primary and Immortalized Adrenocortical Cells to Steroidogenic Stimuli

“…To understand how this immortalized cell line compared to normal adrenocortical cells, we asked how well the changes induced by steroidogenic stimuli were recapitulated in primary cells. We previously performed an RNA-seq time course of H295R cells stimulated with AngII and we established 12 groups of genes exhibiting distinct temporal response profiles [ 25 ]. Likewise, we stimulated H295R cells using forskolin, an activator of adenylyl cyclase, which resulted in five temporal response profiles.…”

“…However, steroidogenesis involves interdependent molecular pathways that rely on multiple levels of gene regulation. Previous studies in H295R have delineated the temporal coordination of gene expression in response to AngII crucial for eliciting the appropriate production of aldosterone [ 25 ]. Unfortunately, gene expression dynamics in primary cells stimulated with either ACTH or AngII is limited to a handful of genes.…”