The importance of the rapid vascular effects of aldosterone is increasingly appreciated. Through these rapid pathways, aldosterone has been shown to regulate vascular contractility, cell growth, and ...apoptosis. In our most recent studies, we demonstrated the effects of aldosterone on cell growth and contractility in vascular smooth muscle cells. We showed that these effects could occur via activation of the classic mineralocorticoid receptor, as well the recently characterized G protein-coupled estrogen receptor (GPER), initially characterized as an estrogen-specific receptor. However, the mechanisms underlying aldosterone's endothelium-dependent actions are unknown. Furthermore, the ERK regulatory and proapoptotic effects of aldosterone mediated by GPER activation in cultured vascular smooth muscle cells were only apparent when GPER was reintroduced into these cells by gene transfer. Whether GPER activation via aldosterone might be an important regulator in native vascular cells has been questioned. Therefore, to determine the role of GPER in mediating aldosterone's effects on cell growth and vascular reactivity in native cells, we examined rat aortic vascular endothelial cells, a model characterized by persistent robust expression of GPER, but without detectable mineralocorticoid receptor expression. In these endothelial cells, the GPER agonist G1 mediates a rapid increase in ERK phosphorylation that is wholly GPER-dependent, paralleling the actions of aldosterone. The effects of G1 and aldosterone to stimulate ERK phosphorylation paralleled their proapoptotic and antiproliferative effects. In previous studies, we reported that aldosterone mediates a rapid endothelium-dependent vasodilatory effect, antagonistic to its direct vasoconstrictor effect in endothelium-denuded preparations. Using a rat aortic ring/organ bath preparation to determine the GPER dependence of aldosterone's endothelium-dependent vasodilator effects, we demonstrate that aldosterone inhibits phenylephrine-mediated contraction. This vasodilator effect parallels the actions of the GPER agonist G1. Furthermore, the effects of aldosterone were completely ablated by the GPER antagonist G15. These data support an important role of GPER activation in aldosterone-mediated regulation of endothelial cell growth, as well as in aldosterone's endothelium-mediated regulation of vasoreactivity.
It has been increasingly appreciated that steroids elicit acute vascular effects through rapid, so-called nongenomic signaling pathways. Though aldosterone, for example, has been demonstrated to ...mediate rapid vascular effects via both mineralocorticoid receptor-dependent and -independent pathways, the mechanism(s) of this mineralocorticoid receptor-independent effect of aldosterone is yet to be determined. For estrogen, its rapid effects have been reported to be, at least in part, mediated via the 7-transmembrane-spanning, G protein-coupled receptor GPR30. Previous studies have demonstrated common response outcomes in response to both aldosterone and estrogen on GPR30 expression, ie, activation of phosphatidylinositol 3-kinase-dependent contraction and extracellular signal-regulated kinase activation in vascular smooth muscle cells. The present studies were undertaken to test the hypothesis that the rapid response to aldosterone in smooth muscle is dependent on the availability of a GPR30-dependent signaling pathway. These findings not only reconcile differences in the literature for aldosterone response in freshly isolated versus cultured aortic smooth muscle cells but also suggest alternative therapeutic strategies for modulating aldosterone actions on the vasculature in vivo.
Recent studies suggest that the rapid and nongenomic effects of estradiol may be mediated through the G protein-coupled receptor dubbed GPR30 receptor. The present study examines the role of GPR30 ...versus a classical estrogen receptor (ERalpha) in mediating the growth regulatory effects of estradiol. GPR30 is readily detectable in freshly isolated vascular tissue but barely detectable in cultured vascular smooth muscle cells (VSMC). In freshly isolated aortic tissue, estradiol stimulated extracellular signal-regulated kinases (ERK) phosphorylation. In contrast, in cultured VSMC, where GPR30 expression is significantly reduced, estradiol inhibits ERK phosphorylation. Transfer of the genes encoding GPR30 led to estradiol stimulation of ERK phosphorylation, which is opposite the effects of estradiol in the primary culture of VSMCs. Transduction of the mineralocorticoid receptor (MR) had no effect on estradiol effects on ERK. Estradiol-mediated stimulation of ERK subsequent to heterologous GPR30 expression was pertussis toxin sensitive and phosphoinositide 3-kinase (PI3 kinase) dependent; under these conditions, estradiol also inhibited protein kinase A (PKA). In contrast, in the absence of GPR30 expression in cultured VSMC, estradiol stimulated PKA activity and inhibited ERK phosphorylation. To determine the functional effect of GPR30 (vs. estrogen receptor expression), we assessed estradiol-mediated apoptosis. In the absence of GPR30 expression, estradiol inhibited apoptosis. This effect was enhanced with ERalpha expression. In contrast, with GPR30 expression, estradiol stimulated apoptosis in an ERK-dependent manner. Thus the effect of estradiol on vascular smooth muscle cell apoptosis is likely dependent on the balance between ER-mediated PKA activation and GPR30-mediated PKA inhibition and PI3 kinase activation. Taken together, we postulate that modulation of GPR30 expression or activity may be an important determinant of the effects of estradiol in the vasculature.
Aldosterone exerts some of its effects not by binding to mineralocorticoid receptors, but rather by acting via G protein‐coupled estrogen receptors (GPER). To determine if aldosterone binds directly ...to GPER, we studied the ability of aldosterone to compete for the binding of 3H 2‐methoxyestradiol (3H 2‐ME), a high potency GPER‐selective agonist. We used GPER gene transfer to engineer Sf9‐cultured insect cells to express GPER. We chose insect cells to avoid interactions with any intrinsic mammalian receptors for aldosterone. 3H 2‐ME binding was saturable and reversible to a high‐affinity population of receptors with Kd = 3.7 nM and Bmax = 2.2 pmol/mg. Consistent with agonist binding to G Protein‐coupled receptors, 3H 2‐ME high‐affinity state binding was reduced in the presence of the hydrolysis‐resistant GTP analog, GppNHp. 3H 2‐ME binding was competed for by the GPER agonist G1, the GPER antagonist G15, estradiol (E2), as well as aldosterone (Aldo). The order of potency for competing for 3H 2‐ME binding, namely 2ME > Aldo > E2 ≥ G1, paralleled the orders of potency for inhibition of cell proliferation and inhibition of ERK phosphorylation by ligands acting at GPER. These data confirm the ability of aldosterone to interact with the GPER, consistent with the interpretation that aldosterone likely mediates its GPER‐dependent effects by direct binding to the GPER.
Significance statement
Despite the growing evidence for aldosterone's actions via G protein‐coupled estrogen receptors (GPER), there remains significant skepticism that aldosterone can directly interact with GPER. The current studies are the first to demonstrate directly that aldosterone indeed is capable of binding to the GPER and thus likely mediates its GPER‐dependent effects by direct binding to the receptor.
This graph summarizes the EC50 values obtained from Figures 2 and 4 and the Ki values from Figure 6. Table 1 shows the values. The symbols show the fit value of the Ki or EC50; the lines show the 95%CIs.
Compartmentation of cAMP signaling been demonstrated to be attributable to the structural association of protein kinase A (PKA) (via association with A-kinase anchoring proteins AKAPs) with ...phosphodiesterase and AKAP-dependent effector molecules. However, other mechanisms contributing to compartmentalization have not been rigorously explored, including the possibility that different isoforms of adenylyl cyclase (AC) may be functionally "compartmentalized" because of differential association with tethering or signaling molecules. To this end, we examined the effect of adenoviral transduction of representative AC isoforms (AC1, AC2, AC5, and AC6) on cellular cAMP production, PKA activation, extracellular signal-regulated kinase (ERK) activation, cell doubling and proliferation, as well as arborization responses (an index of cAMP-mediated cytoskeletal re-organization) in vascular smooth muscle cells. When isoforms were expressed at levels to achieve comparable forskolin-stimulated AC activity, only gene transfer of AC6 significantly enhanced PKA-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation and arborization responses. Treatment of control cells, which express AC6 endogenously, as well as vascular smooth overexpressing the AC6 isoform with small interfering RNA directed against AC6, significantly suppressed both isoproterenol-stimulated cAMP accumulation and arborization. Notably, the selective effects of AC6 expression were abrogated in the presence of phosphodiesterase suppression. In contrast, only the expression of AC1 enhanced forskolin-stimulated association of ERK with AC, demonstrated by coimmuno-isolation of ERK with Flag-tagged AC1, but not with Flag-tagged AC6. To determine whether these isoform-selective effects of AC were unique to differentiated and morphologically compartmentalized vascular smooth muscle cells or were a general property of these isoforms, we examined the consequence of expression of these various isoforms in human embryonic kidney (HEK) cells. Indeed, we observed similar isoform-dependent association of AC1 with ERK, activation of ERK by stimulation of AC1 with forskolin, and AC1-dependent lengthening of doubling time, indicating that these properties of AC1 are cell autologous and likely result from AC1-dependent protein-protein interactions. In aggregate, these findings suggest that isoform-selective signaling complexes likely contribute to various functional consequences of cAMP elevation in vascular smooth muscle cells.
Abstract only
Aldosterone is a well‐established regulator of renal and cardiovascular functions. However, its role in regulation of cancer growth and spread has only begun to be appreciated. Studies ...from our laboratory have demonstrated that aldosterone stimulates renal cancer cell migration in
vitro
and metastatic spread
in vivo
(Feldman et. al. FASEB J. 2016 30(6)2086–96). However, the generalizability of these findings to other cancers is unknown. Therefore, we assessed the effect of aldosterone on prostate cancer cell migration and the receptor mechanism involved utilizing three widely‐used prostate cancer cell lines, viz. TRAMP C1, LNCaP and DU 145 cells.
RT‐PCR analysis detected robust expression of the mineralocorticoid receptor (MR) in all cell lines. GPER expression was detectable in TRAMP C1 and LNCaP cells but not in DU145 cells. In LNCaP and TRAMPC1 cells, aldosterone mediated a concentration‐dependent increase in migration to a maximum of 141±7% of control at 1000 nM (n=5) in LNCaP cells and 125±3 % at 10 nM (n=6) in TRAMP C1 cells, similar to the maximal extent of stimulation by testosterone 146±9% (n=9) in LNCaP cells and 136±9% (n=3) in TRAMP C1 cells. The effects of aldosterone were inhibited by the MR antagonist eplerenone, but NOT by the GPER antagonist G15. In contrast, the GPER agonist, G1, INHIBITED migration in both cell lines‐effects that were blocked by G15. In DU 145, cells neither testosterone nor aldosterone stimulated migration.
In TRAMP C1 cells, infection with an adeno shMR vector blocked aldosterone‐mediated stimulation of proliferation/migration without effects on G1‐mediated inhibition, whereas infection with an adeno shGPER vector selectively blocked G1‐mediated inhibition of proliferation and migration, without effects on aldosterone’s actions.
These findings suggest that in TRAMP C1 and LNCaP cells aldosterone stimulates migration via an MR‐dependent pathway. In DU145 cells, resistance to the effect of aldosterone paralleled resistance to the effect of testosterone. These data support the hypothesis that inhibiting aldosterone secretion/effects may be a novel adjunctive therapeutic approach‐‐at least in some forms of prostate cancer.
Support or Funding Information
Heart and Stroke Foundation of Canadaand Molson Foundation
MR‐independent effects of aldosterone acting through GPER have been appreciated since 2011. However, whether this related to direct aldosterone‐GPER binding has been disputed. Two previous ...radioligand studies failed to demonstrate direct aldosterone binding to a putative GPER. However, in neither study was it demonstrated that the radioligand used, bound to a physiological receptor. Further, both utilized 3H estradiol‐ whose low potency for GPER binding would not be expected to allow GPER receptor identification using vacuum filtration methods related to its high off‐rates. Therefore to determine if aldosterone did bind to GPER, we studied its displacement of 3H 2‐methoxyestradiol binding (3H2ME ‐ a higher affinity GPER‐selective agonist) in SF9 cells with heterologous GPER expression mediated by baculovirus.
In this model system, following GPER gene transfer, 3H2ME demonstrated saturable and reversible binding to a high affinity population of receptors with a Kd = 3.4±0.4 nM and Bmax = 645±49 fmol/mg. In the absence of baculovirus GPER gene transduction, no high affinity 3H2ME binding was demonstrable in SF9 cells. 3H2ME binding was guanine‐nucleotide sensitive with a 43±8% reduction in high affinity binding in the presence of GppNHp (100μM). High affinity 3H2ME binding was displaceable by 2ME with an IC50 comparable to the determined Kd (1.3±0.4 nM), the GPER agonist G1, the GPER antagonist G15, estradiol (E2) and aldosterone (Aldo) but not by hydrocortisone (HC) with an order of potency of (2ME>Aldo>E2=G1>G15>>HC).
In summary, we demonstrate high affinity 3H2ME binding to GPER with an order of potency for aldosterone vs. estradiol (vs. hydrocortisone) that parallels the order of potency of their functional effects in previously studied models. These data support the hypothesis that aldosterone mediates its GPER‐dependent effects by direct binding to the receptor.
Support or Funding Information
Heart and Stroke Foundation of Canada
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Estrogens are important regulators of cardiovascular function. Some of estrogen's cardiovascular effects are mediated by a G-protein-coupled receptor mechanism, namely, G-protein-coupled estrogen ...receptor (GPER). Estradiol-mediated regulation of vascular cell programmed cell death reflects the balance of the opposing actions of GPER versus estrogen receptor α (ERα). However, the significance of these opposing actions on the regulation of vascular smooth muscle cell proliferation or migration in vitro is unclear, and the significance in vivo is unknown. To determine the effects of GPER activation in vitro, we studied rat aortic vascular smooth muscle cells maintained in primary culture. GPER was reintroduced using adenoviral gene transfer. Both estradiol and G1, a GPER agonist, inhibited both proliferation and cell migration effects that were blocked by the GPER antagonist, G15. To determine the importance of the GPER-ERα balance in regulating vascular remodeling in a rat model of carotid ligation, we studied the effects of upregulation of GPER expression versus downregulation of ERα. Reintroduction of GPER significantly attenuated the extent of medial hypertrophy and attenuated the extent of CD45 labeling. Downregulation of ERα expression comparably attenuated the extent of medial hypertrophy and inflammation after carotid ligation. These studies demonstrate that the balance between GPER and ERα regulates vascular remodeling. Receptor-specific modulation of estrogen's effects may be an important new approach in modifying vascular remodeling in both acute settings like vascular injury and perhaps in longer term regulation like in hypertension.
Oligomerization plays an important role in endoplasmic reticulum processing and membrane insertion (and ultimately in regulation of function) of a number of transmembrane spanning proteins. ...Furthermore, it is known that adenylyl cyclases (ACs), critical regulators of cellular functions, associate into higher order (dimeric) forms. However, the importance of these higher order aggregates in regulating adenylyl cyclase activity or trafficking to the cell membrane is unclear. Therefore, we examined the potential role of oligomerization in the membrane trafficking of adenylyl cyclase. For this purpose, the ability of full-length adenylyl cyclase and various truncation mutants to self-assemble and to be targeted to the cell membrane was assessed. A truncation mutant comprised of the initial six transmembrane spanning domains and half of the C1 catalytic domain coimmunoprecipitated with full-length AC VI. Using both biotinylation assays and assessment of enzyme distribution using sucrose density gradients, we demonstrate that expression of this mutant in human embryonic kidney 293 cells impaired the ability of AC VI to traffic to the plasma membrane. Furthermore, mutant expression resulted in a significant reduction in adenylyl cyclase activity. The decrease in AC VI membrane expression was not caused by alterations in enzyme transcription. The effect of the mutant was specific for the AC V and VI isoforms and expression of the transmembrane M1 domain but not the C1a domain was required for the mutant to affect adenylyl cyclase activity. In aggregate, these data suggest that alterations in the ability of adenylyl cyclases to form higher order forms regulate both enzyme trafficking and enzyme activity.
Abstract only
Estrogens are important physiological and pathophysiological regulators of cardiovascular function. The traditional view of the mechanism for these cardiovascular effects has focused on ...the activation of “classical” steroid receptors (i.e., estrogen receptors‐ERs). However, recent studies have elucidated the mechanism of estrogen's cardiovascular effects mediated by an alternate GPCR‐based mechanism viz., via GPER (aka GPR30), formerly characterized as an “orphan receptor”. We are beginning to appreciate that to understand the effect of estradiol in cardiovascular regulation one must understand the balance between GPER‐ and ER‐mediated effects. In vascular smooth muscle cells, estrogen‐mediated regulation of apoptotic cell death and proliferation is divergently regulated by activation of ER vs. GPER. However, the significance of this divergence in the in vivo regulation of vascular growth processes was unknown. To determine the role of GPER in regulating vascular remodeling we studied the effects of altering GPER expression vs. altering the expression of ERα in a rat model of carotid ligation. Under baseline conditions, 1 week following carotid ligation and endothelial disruption with distilled water installation there was a 51±7.6% (n=3) increase in medial thickness, paralleling neointimal proliferation in female rats. This medial hypertrophy paralleled down‐regulation of GPER protein content (26±5% of control; n=3) and mRNA expression (29±13% of control; n=3). Re‐introduction of GPER, via abluminal adenoviral delivery, significantly attenuated the extent of medial hypertrophy (adeno‐GPER: 28±2.2% increase; n=10 vs. adeno‐GFP/control: 48±3.3% increase; n=16, p<0.05). Similarly, inhibition of ERα expression using an adeno‐shERαRNA construct resulted in an attenuation of injury‐mediated medial hypertrophy. In these studies we show that in vivo the balance between GPER and ERα is a significant regulator in the extent of vascular remodeling. Receptor‐specific modulation of estrogen's growth regulatory effects may be an important new approach in modifying the extent of vascular remodeling in both acute settings like vascular injury and perhaps in longer term models of regulation like in hypertension.
Support or Funding Information
These studies were supported by grants from the Heart and Stroke Foundation of Canada (RG and RDF).
PDF
