13-cis-Retinoic acid specific down-regulation of angiotensin type 1 receptor in rat liver epithelial and aortic smooth muscle cells

Abstract

Transcriptional repression through cis-and trans-acting factors enabling an alternate approach to control angiotensin type 1 receptor (AT1 or AGTR1 as listed in the MGI database) expression has not been studied. In previous investigations, treatment with retinoic acid was found to be associated with enhanced insulin sensitivity. In our previous study, expression of AT1 was found to be inversely correlated with intracellular glucose concentrations. Therefore, we hypothesized that 13-cis-retinoic acid (13cRA), an antioxidant, enhances insulin-sensitive glucose-mediated downregulation of the AT1. In this study, we used continuously passaged rat liver epithelial cells. Our study shows that cells exposed to 13cRA specifically down-regulated the AT1 protein in a dose-and time-dependent manner, independently of any change in receptor affinity. Down-regulation of the AT1 expression leads to reduced AngII-mediated intracellular calcium release, a hallmark of receptor-mediated intracellular signaling. Similarly with receptor down-regulation, we observed a significant reduction in AT1 mRNA; however, the AT1 down-regulation was independent of insulin-sensitive glucose uptake and retinoic acid receptor activation (RAR/RXR). Treatment with 13cRA resulted in phosphorylation of p42/p44 MAP kinases in these cells. Subsequent studies using MEK inhibitor PD98059 prevented 13cRA-mediated AT1 down-regulation and restored AngII-mediated intracellular calcium response. Furthermore, 13cRA-mediated inhibitory effects on AT1 were validated in primary rat aortic smooth muscle cells. In summary, our results demonstrate for the first time that 13cRA has a glucose-and RAR/RXR-independent mechanism for transcriptional inhibition of AT1, suggesting its therapeutic potential in systems in which AT1 expression is deregulated in insulin-sensitive and -insensitive tissues.

Pharmacologic Effects of 2-Methoxyestradiol on Angiotensin Type 1 Receptor Down-Regulation in Rat Liver Epithelial and Aortic Smooth Muscle Cells

“…Results are presented as mean ± S.E.M and the value of P<0.05 was considered statistically significant. Analyses performed include parametric t-tests, first testing for Gaussian distribution, and one-way analysis of variance (ANOVA) with post-hoc Bonferroni analysis where appropriate according to previously established methods [47]. Values are normalized to milligrams of protein determined by Bio-Rad DC protein assay system based on the Bradford method [43].…”

Interplay between EGR1 and SP1 is critical for 13-cis retinoic acid-mediated transcriptional repression of angiotensin type 1A receptor

“…In our previous study, we have shown that exposure to 13cRA reduced the expression of rat AT1 A R protein via transcriptional down-regulation (Snyder & Thekkumkara, 2012). To further determine whether there was promoter activity of AT1 A R, and if so, whether down-regulation is involved with specific cis -acting elements, we investigated the involvement of the −3276 to +45-base pair promoter sequence in the regulation of the rat AT1 A R gene transcription.…”

“…Receptor binding studies were performed in triplicate on WB cells as described previously (Snyder & Thekkumkara, 2012). Briefly, cells were washed twice with PBS and incubated with [ 3 H]AngII (20 pM) at 22°C in a binding buffer containing 50 mM Tris HCl (pH 7.5), 120 mM NaCl, 4 mM KCl, 5 mM MgCl 2 , 1 mM CaCl 2 , 10 μg/ml bacitracin, 2 mg/ml dextrose, and 2.5 mg/ml bovine serum albumin.…”

“…Total RNA was isolated using guanidinium thiocyanate-phenol-chloroform method as described previously (Snyder & Thekkumkara, 2012). 5 μg total RNA was processed for cDNA template conversion using MLV-RT.…”

“…In a recently published study, we have demonstrated that in both rat liver epithelial and aortic smooth muscle cells, 13cRA is capable of down-regulating AT1 A R by inhibiting AT1 A R mRNA transcription (Snyder & Thekkumkara, 2012). Additionally, the study revealed that 13cRA induced AT1 A R down-regulation is dependent on the activation of MAPK p42/p44 (ERK1/2).…”