Controlling angiotensin AT1 receptor function has been shown to be protective for many pathophysiological disorders. Although estrogen metabolite, 2-methoxyestradiol (2ME2) can down-regulate angiotensin AT1 receptor expression independently of nuclear receptors, no specific cellular targets have been identified. This study was focused on identification and validation of a cellular target responsible for 2ME2-mediated angiotensin AT1 receptor down-regulation in a continuously passaged rat liver epithelial cell line. Cell membranes were isolated and used to determine 2ME2 specific binding. Cell membranes exposed to [3H]2ME2 showed specific saturable binding, which was found to be pertussis toxin (PTx) sensitive. Under similar conditions, G-protein coupled receptor 30 (GPR30) agonist (G1) and antagonist (G15) inhibited 2ME2 specific binding. In these cells GPR30 was found localized to endoplasmic reticulum (ER) membranes. In intact cells, G1 down-regulated angiotensin AT1 receptor expression and this effect was reversed by G15. Furthermore, 2ME2 mediated activation of epidermal growth factor receptor (EGFR) followed by ERK1/2 phosphorylation, an essential signaling step in angiotensin AT1 receptor down-regulation, was abrogated by G15, suggesting that this signal is GPR30 dependent. Additionally, EGF was found to independently down-regulate angiotensin AT1 receptor in an ERK1/2-dependent manner. In summary, our results demonstrate for the first time that 2ME2 down-regulation of angiotensin AT1 receptor is dependent on ER membrane-associated GRP30. Moreover, this effect is facilitated by GPR30 dependent transactivation of EGFR and ERK1/2 phosphorylation. This study provides further understanding of the physiological significance of 2ME2 and its role in modulating angiotensin AT1 receptor expression.

Delayed onset of cardiovascular disease among females is not well understood, but could be in part due to the protective effect of estrogen before menopause. Experimental studies have identified the angiotensin type 1 receptor (AT1R) as a key factor in the progression of CVD. In this study, we examined the effects of the estrogen metabolite, 2-methoxyestradiol (2ME2), on AT1R expression. Rat liver cells were exposed to 2ME2 for 24 h and angiotensin II (AngII) binding and AT1R mRNA expressions were assessed. In the presence of 2ME2, cells exhibited significant down-regulation of AngII binding in a dose and time dependent manner, independent of estrogen receptors (ERα/ERβ). Down-regulation of AngII binding was AT1R specific with no change in receptor affinity. Under similar conditions, we observed lower expression of AT1R mRNA, significant inhibition of AngII mediated increase in intracellular Ca2+, and increased phosphorylation of ERK1/2. Pretreatment of cells with the MEK inhibitor PD98059 prevented 2ME2 induced ERK1/2 phosphorylation and down-regulation of AT1R expression, suggesting that the observed inhibitory effect is mediated through ERK1/2 signaling intermediate(s). Similar analyses in stably transfected CHO cell lines with a constitutively active cytomegalovirus (CMV) promoter showed no change in AT1R expression suggesting that 2ME2 mediated effects are through transcriptional regulation. The effect of 2ME2 on AT1R down-regulation through ERK1/2 were consistently reproduced in primary rat aortic smooth muscle cells. As AT1R plays a critical role in the control of cardiovascular diseases, 2ME2-induced changes in receptor expression may provide beneficial effects to the cardiovascular as well as other systems.

In the present study, we investigated the effects of tannic acid (TA), a hydrolysable polyphenol, on angiotensin type 1 receptor (AT1R) expression in continuously passaged rat liver epithelial cells. Under normal conditions, exposure of cells to TA resulted in the down-regulation of AT1R-specific binding in concentrations ranging from 12.5-100 μg/ml (7.34-58.78 μm) over a time period of 2-24 h with no change in receptor affinity to angiotensin II (AngII). The inhibitory effect of TA on AT1R was specific and reversible. In TA-treated cells, we observed a significant reduction in AngII-mediated intracellular calcium signaling, a finding consistent with receptor down-regulation. Under similar conditions, TA down-regulated AT1R mRNA expression without changing the rate of mRNA degradation, suggesting that TA's effect is mediated through transcriptional inhibition. Cells expressing recombinant AT1R without the native promoter show no change in receptor expression, whereas a pCAT reporter construct possessing the rat AT1R promoter was significantly reduced in activity. Furthermore, TA induced the phosphorylation of MAPK p42/p44. Pretreatment of the cells with a MAPK kinase (MEK)-specific inhibitor PD98059 prevented TA-induced MAPK phosphorylation and down-regulation of the AT1R. Moreover, there was no reduction in AngII-mediated intracellular calcium release upon MEK inhibition, suggesting that TA's observed inhibitory effect is mediated through MEK/MAPK signaling. Our findings demonstrate, for the first time, that TA inhibits AT1R gene expression and cellular response, suggesting the observed protective effects of dietary polyphenols on cardiovascular conditions may be, in part, through inhibition of AT1R expression.

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.

The role of 2-methoxyestradiol is becoming a major area of investigation because of its therapeutic utility, though its mechanism is not fully explored. Recent studies have identified the G-protein-coupled receptor 30 (GPR30, GPER) as a high-affinity membrane receptor for 2-methoxyestradiol. However, studies aimed at establishing the binding affinities of steroid compounds for specific targets are difficult, as the tracers are highly lipophilic and often result in nonspecific binding in lipid-rich membrane preparations with low-level target receptor expression. 2-Methoxyestradiol binding studies are essential to elucidate the underlying effects of this novel estrogen metabolite and to validate its targets; therefore, this competitive receptor-binding assay protocol was developed in order to assess the membrane receptor binding and affinity of 2-methyoxyestradiol.

1. Literature data suggest that the electron-donating enzyme, cytochrome P450 reductase (CPR), might act as a source of reactive oxygen species (ROS). However, the role of CPR in pathophysiological conditions associated with oxidative stress is unknown. The aim of the present study was to study the role of CPR in the generation of ROS and cellular injury under basal conditions, and after simulated in vitro ischaemia-reperfusion (IR). 2. Plasmid DNA or siRNA approaches were used to transiently overexpress or knockdown the human CPR gene in rat liver epithelial (WB-F344) or human hepatoblastoma (HepG2) cells, respectively. The generation of ROS and/or cellular injury was then studied under the basal conditions and after simulated IR (4 h of ischaemia plus 30 min of reoxygenation). 3. Under the basal conditions, transient overexpression of CPR protein in WB-F344 cells caused a 90% increase in the CPR activity, which was associated with a 100% increase in the ROS production. In contrast, after simulated IR, a 2.5-fold higher CPR activity did not significantly affect the magnitude of ROS generation or cell death. Similarly, although the knockdown of CPR protein resulted in a significant reduction (∼30%) in the CPR activity, the ROS production was not substantially altered after simulated IR in HepG2 cells. 4. Our data suggest that CPR plays a major role in the ROS generation by liver cells under the basal conditions. However, the role of CPR in the ROS generation during simulated in vitro IR injury in these cells is minimal, if any.

Recently, we have demonstrated that 13-cis retinoic acid (13cRA) down-regulates rat angiotensin type 1A receptor (AT1AR) gene transcription through a MAP Kinase (ERK1/2) dependent mechanism in rat liver epithelial and aortic smooth muscle cells. However, the exact mechanism remained unknown. In this study, we determined the signaling intermediates activated by ERK1/2 involved in 13cRA mediated AT1AR down-regulation. Serially deleted rat AT1AR promoter CAT constructs indicate fragments containing a region −2541 and −1836 bp upstream of the 5 prime possess an Sp1 consensus sequence (5′-TGGGGCGGGGCGGGG-3′) have reduced CAT activity. Mobility shift analysis using untreated nuclear extracts in the presence of mithramycin A suggest the trans-acting factor binding to this cis-acting element is Sp1. 13cRA significantly reduced specific binding without any change in Sp1 protein expression. Studies showed 13cRA maximally phosphorylates and tranlocates to the nucleus ERK1/2 within 5–10 minutes, activating Egr-1 mRNA expression at 20 minutes followed by de novo protein synthesis, leading to an Egr-1/Sp1 interaction. siRNA silencing of Egr-1 restored AT1AR mRNA and protein expression in 13cRA treated cells, and Sp1 silencing results in complete loss of AT1AR expression. Our study suggests that 13cRA mediated activation of ERK1/2, through Egr-1, is capable of disrupting Sp1, the requisite transactivator for AT1AR expression, providing a novel paradigm in AT1AR gene transcription.

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