Striatin Is a Novel Regulator Of Aldosterone-Mediated Endothelial Cell Activation
Authors: Machado-Fiallo Enrique D. , Vega Christopher , Ramos-Rivera Arelys , Benabe-Carlo Josue A. , Prado Gregory N. , Pojoga Luminita H. , Rivera Alicia , Romero Jose R. 
Published: October 2019

Abstract
Activation of the minerolocorticoid receptor (MR) by aldosterone (ALDO) has been shown to play an important role in inflammatory vascular responses in addition to its well described effects on sodium homeostasis. Steroid responses are mediated by well-known genomic and less known rapid/nongenomic responses. However, characterization of the mechanisms underlying ALDO’s rapid/nongenomic actions have been difficult to study and are not clearly understood. We recently reported that in vivo and in vitro activation of MR leads to increases of striatin levels in endothelial cells, aortas and heart tissue (Pojoga, Amer J Hypertens, 2012) and that MR forms a complex with caveolin-1 and striatin within caveolae in endothelial cells. We hypothesized that striatin is a critical intermediary of the rapid effects of ALDO and that striatin serves as a novel link for MR regulation in endothelial cells activation. Endothelial cell activation promotes, among other factors, increased levels of reactive oxygen species (ROS) and protein disulfide isomerase (PDI), a redox modifying enzyme that catalyze disulfide interchange reactions. We studied EA.hy926 cells (EA), a human endothelial cell line that expresses MR, striatin and maintains its caveolae while in culture. We incubated EA cells with ALDO (10–9–10–7M) for 60 min and observed a dose-dependent rise in ROS production (P<0.001, n=4) using the oxidative fluorescent indicator dye 5-6-chloromethyl-2ʼ,7ʼ-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) that peaked at around 10-8M ALDO, an event that was blocked by pre-incubation of EA cells for 30 mins with 1μM canrenoic acid (CA), an MR antagonist (P<0.03, n=3). Time course analyses showed ALDO stimulated ROS responses that increased for up to 3 hours following the addition of ALDO. As there are no known inhibitors for striatin we then used siRNA technology to down regulate striatin in these cells. EA cells were transfected with striatin siRNA and subsequently stimulated with ALDO and ROS production measured. The transfection process itself did not modify baseline levels of ROS significantly, as assessed in cells transfected with scrambled siRNA and non-transfected cells, which had nearly identical ROS levels, basally and in response to ALDO. In addition, in the presence of lower levels of striatin protein the ALDO-stimulated ROS response was abrogated, supporting the concept that striatin is necessary for the rapid effects of ALDO. We also measured phosphorylated ERK-1/2 (pERK) levels that peaked within 10 minutes in EA as estimated by western blot analyses. Consistent with these observations pre-incubation of EA cells with 10-6M PD0325901, a selective MEK-1/2 inhibitor was associated with greater than 90% reduction of the ALDO-stimulated ROS responses (7244.3±497 vs 4386.6±586 RFU, P<0.02, n=3). Qualitatively similar responses were observed using another MEK inhibitor, U0126 [10-5M] (P<0.01, n=3). We then tested the effects of ALDO on PDI secretion. Incubation of EA cells with ALDO (10-7M) led to PDI increases when compared to vehicle treated cells (P<0.01, n=3). We also tested the effects of low levels of striatin using siRNA on PDI activity in EA cells. We found that PDI secretion was reduced by 62% in striatin knockdown conditions. We then tested the effects of Methyl-β-cyclodextrin to disrupt caveolae in these cells and observed a blunted 10nM ALDO–stimulated PDI response (530±117 to 215±99 RFU/mg protein, n=3, P<0.01). We then isolated early cultures of mouse aortic endothelial cells (MAEC) from endothelial-specific caveolin-1 knockout mice and measured PDI activity following 24 hrs of incubation in 0.4% fetal bovine serum. Our results show that MAEC from caveolin-1 knockout mice had lower PDI secretion when compared to cells from WT mice (99.4±16 vs 129.9±35, n=5, P<0.03). These results suggest that striatin is a novel mediator for ALDO’s rapid effects on PDI and ROS, thereby suggesting a unique level of interaction between the MR and striatin in endothelial cell activation. Supported by NIH R01HL090632 (AR), R01HL104032 (LHP) and R01HL096518 (JRR).

Disclosures:
No relevant conflicts of interest to declare.