RNA-binding proteins regulate aldosterone homeostasis in human steroidogenic cells

Abstract

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.

Small-molecule Ro-08-2750 interacts with many RNA-binding proteins and elicits MUSASHI2-independent phenotypes

“…MSI2 is critical for the development and maintenance of embryonic stem cells, hematopoietic stem cells, and neural stem and progenitor cells ( Fox et al 2015 ). It is also important for fertility in steroid-producing mouse gonadal tissue, which is consistent with our previous findings that MSI2 promotes aldosterone production in human adrenocortical cells ( Sutherland et al 2014 , 2015 ; Fu et al 2021 ). MSI2 is a prognostic marker associated with clinical outcomes in several types of cancer including myeloid leukemia, breast cancer, and ovarian cancer among others ( Ito et al 2010 ; Kharas et al 2010 ; Li et al 2015 ; Lee et al 2016 ; Kang et al 2017 ).…”

Small molecule inhibition of multiple RNA binding proteins underlies Musashi-2 independent phenotypes

“…Angiotensin II (AngII) stimulation of immortalized adrenocortical H295R cells is a robust model for human steroidogenesis 23,24 . We previously found that siRNA depletion of MSI2 in AngII-stimulated H295R cells led to decreased aldosterone production 22 , indicating that MSI2 promotes steroidogenesis. Therefore, we hypothesized that Ro-mediated inhibition of MSI2 would also reduce aldosterone production.…”

“…MSI2 is important for fertility in steroid producing mouse gonadal tissue, as well as, steroidogenesis in human adrenocortical cells [20][21][22] . Here we show that Ro is a potent inhibitor of human adrenocortical steroidogenesis.…”

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.…”