Abstract
20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysteroid receptors (EcR/RXR complex) and at least one membrane GPCR receptor (DopEcR). It also displays numerous pharmacological effects in mammals, where its mechanism of action is still debated, involving either an unidentified GPCR or the estrogen ERβ receptor. The goal of this study was to better understand 20E mechanism of action in mammals. A mouse myoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) were used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with angiotensin(1–7), the endogenous ligand of MAS. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using siRNA or pharmacological inhibitors. 17β-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abolished by angiotensin(1–7) antagonists. A mechanism involving cooperation between the MAS receptor and a membrane-bound palmitoylated estrogen receptor is proposed. The possibility to activate the MAS receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals and, indeed, the proposed mechanism may explain the close similarity between the effects of angiotensin(1–7) and 20E. Our findings open up many possible therapeutic developments involving stimulation of the protective arm of the renin–angiotensin–aldosterone system (RAAS) with 20E.
Introduction
Steroids in animal and plant kingdoms
Steroid hormones are (chole)sterol derivatives widespread in animals and plants, where they are involved in the control of many physiological processes. They include, for example, vertebrate sex hormones (progestagens, estrogens, androgens), insect moulting hormones (ecdysteroids), as well as plant growth hormones (brassinosteroids). The rigid carbon skeleton of sterols is particularly suitable to generate a very large number of derivatives, which differ in carbon number and/or the position of various substituents (mainly hydroxyl or keto groups) (Karlson 1983). In addition to hormones, sterols give rise to bile acids/alcohols, initially considered as emulsifiers facilitating lipid digestion but nowadays also known to be important signaling molecules acting on specific receptors (Chiang 2013).
Diversity of steroid mechanisms of action
Our concepts of (steroid) hormone mechanism of action have evolved. In the classical scheme, steroid hormones interact with nuclear receptors and the complex formed regulates the transcriptional activity of target genes, the promoters of which contain specific sequences (hormone-responsive elements). However, steroids also act at cell membrane level, where they elicit rapid non-transcriptional effects. Among the identified steroid membrane receptors, we may mention vertebrate GPER1/GPR30 (a membrane estrogen receptor (Prossnitz et al. 2008)), TGR5 (a bile acid membrane receptor (Thomas et al. 2009)), MARRS (a calcitriol receptor (Khanal & Nemere 2007)) or drosophila DopEcR (a dopamine and ecdysteroid membrane receptor (Evans et al. 2014)). All these receptors belong to the family of GPCR/7TD receptors. Moreover, intact or truncated forms or the steroid nuclear receptors are bound to the plasma membrane and do not act there as transcription factors (Meitzen et al. 2013, Schreihofer et al. 2018).
Ecdysteroid effects on vertebrates
We are especially interested by the pharmacological effects of ecdysteroids on mammals. Ecdysteroids are a large family of steroids initially discovered in arthropods (zooecdysteroids) and later in plants (phytoecdysteroids) (Dinan 2009). They are present in many plant species where they can reach concentrations of up to 2–3% of the plant dry weight and where they are expected to protect plants against phytophagous insects.
With the prospect of using these molecules for crop protection, toxicological studies were performed on mammals, which unexpectedly concluded as to both their lack of toxicity (oral LD50 >9 g/kg) and their ‘beneficial’ effects, for example, anti-diabetic and anabolic properties (Dinan & Lafont 2006). Such effects have been linked with the presence of large amounts of phytoecdysteroids in several plants used worldwide in traditional medicine. Up to now, numerous positive effects have been reported, such that ecdysteroids can be considered as some kind of ‘universal remedy’ (Sláma & Lafont 1995). While many effects have been described on animals (Sláma & Lafont 1995, Báthori et al. 2008, Seidlova-Wuttke et al. 2010), the clinical evidence for 20E effectiveness in humans remains limited at the moment (Simakin et al. 1988, Wuttke & Seidlova-Wuttke 2015, Isenmann et al. 2019).
How do ecdysteroids work?
In spite of more than 40 years of research, the mechanism(s) of action of these molecules in mammals/humans has not been clearly elucidated, as diverging hypotheses are currently available. Several studies favor an action on membranes through a GPCR receptor (Gorelick-Feldman et al. 2010), whereas others suggest the involvement of a nuclear receptor, the estrogen receptor ERβ (Parr et al. 2014, Parr & Müller-Schöll 2018).
There is in fact no direct evidence for the binding of 20E to nuclear estrogen (or androgen) receptors (Báthori et al. 2008, Seidlova-Wuttke et al. 2010). The evidence of ERβ involvement in 20E action is based on the use of specific pharmacological activators or inhibitors of ERs, the former being able to mimic and the latter to inhibit the effects of 20E on target cells such as osteoblasts (Gao et al. 2008) or myoblasts (Parr et al. 2014). These studies, however, do not provide proof for direct 20E binding, as ER receptor could be activated indirectly, and even in the absence of ligand, for example, by phosphorylation (Sanchez et al. 2010). Some supporting evidence for direct binding was provided by in silico modeling (Parr et al. 2015), but this certainly does not represent a definite proof, as the result may strongly depend on the model used, and an opposite conclusion was drawn by Lapenna et al. (2015).
Evidence for membrane effects of 20E is based on early studies showing the rapid modulation of several second messengers (cAMP, cGMP, IP3, DAG, Ca2+) in target cells (Kotsyuruba et al. 1995, 1998, 1999) and on the fact that 20E bound to metallic nanoparticles, preventing its entrance in target cells, is still active (Mykhaylyk et al. 2001). More recently, Gorelick-Feldman et al. (2010) used a pharmacological approach with various inhibitors (e.g.pertussis toxin). They concluded that the membrane 20E receptor belongs to the GPCR family and proposed a mechanism of transduction involving an unidentified GPCR and a membrane calcium channel (Supplementary Fig. 1, see section on supplementary materials given at the end of this article).
The above pharmacological arguments appear strong enough to consider that the cell membrane is (maybe not exclusively) a site of action of 20E in mammalian cells. The present experiments have been undertaken in an attempt to identify the/one GPCR involved in 20E effects and to understand its estrogen-like effects using gene silencing or different pharmacological approaches.
Materials and methods
Chemicals
Except where otherwise mentioned, all the reagents and chemicals were from Sigma. Peptides such as angiotensin(1–7) and A779 (Asp-Arg-Val-Tyr-Ile-His-D-Ala) were custom-prepared by the IBPS Peptide Synthesis Platform (Sorbonne University, Paris, France). 20-Hydroxyecdysone (20E) was obtained from Chemieliva Pharmaceutical (Chongqing, China) or from Patheon (Regensburg, Germany) and had a purity of 96.5–97%.
Preparation of HSA-conjugated 20-hydroxyecdysone
22-Succinyl-20E was prepared according to Dinan et al. (2003). Coupling the 20E derivative to human serum albumin (HSA) was performed either with the Pierce Protein Coupling Kit (Product 77672, Thermo Scientific, using 1 mg 22-succinyl-20E and 10 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling reagent per milligram HSA with a reaction time of 2 h at RT; batch 1) or according to a method provided by Dr J-P Delbecque (J-P Delbecque and M de Reggi, personal communication; batch 2) using ethylchloroformate as the coupling reagent. The 20E-HSA conjugate was repeatedly washed with H2O/DMSO (9:1, v/v) in an Amicon filter (Millipore; 30K cut-off) to remove non-covalently bound steroid, lyophilized and then analyzed by mass spectrometry to determine the number of 20E molecules coupled to each HSA molecule. Analyses were performed in a 4700 MALDI TOF/TOF proteomics analyzer (Applied Biosystems). The 20E conjugate was studied in linear mode, positive-ion mode. Laser acceleration was set at 20 kV and the default laser fluency was set at 2000 and modified according to the signal-to-noise quality. The matrix used was α-cyano-4-hydroxycinnamic acid (HCCA). Samples were prepared following the dried-droplet method, 1 µL of a mixture of 1 µL of matrix (10 mg/mL) and 1 µL of sample were spotted and dried with gaseous nitrogen. The mass shift, relative to HSA, after coupling indicates that 11 molecules of 20E derivative are coupled to each albumin molecule for batch 1 and 9 for batch 2.
Cell culture
The C2C12 mouse myoblast cell line (Yaffe & Saxel 1977) was purchased from ATCC (CRL-1772). Except where otherwise mentioned, culture media, serum, antibiotics and supplements were from Life Technologies. All cultures contained 100 U/mL of penicillin and 100 μg/mL of streptomycin and were maintained in a humidified 5% CO2/95% air atmosphere at 37°C. For C2C12 proliferation, cells were maintained in DMEM medium containing 4.5 g/L glucose, supplemented with 10% FBS. C2C12 cells were maintained at low passage (3–20 passages) for all experiments to maintain the differentiation potential of the cultures. Cell confluency was always kept below or equal to ~80%. For all experiments, cells were first seeded at 30,000 cells per well in 24-well plates. To induce differentiation, C2C12 cells at ~80% confluency in proliferation medium were shifted to DMEM medium supplemented with either 2% FBS or 2% horse serum.
Protein synthesis (3H-leucine incorporation)
C2C12 cells were grown in 24-well plates at a density of 30,000 cells/well in 0.5 mL of growth medium (DMEM + 4.5 g/L glucose supplemented with 10% FBS). Twenty-four hours after plating, the differentiation into multinucleated myotubes was induced in DMEM + 4.5 g/L glucose containing 2% FBS. After 5 days, cells were pre-incubated in Krebs medium for 1 h at 37°C before being incubated in DMEM media without serum for 2.5 h in the presence of radiolabeled leucine (5 µCi/mL) and DMSO (control condition) or insulin growth factor (IGF-1, 100 ng/mL) or 20E (0.1, 0.5, 1, 5 or 10 µM). At the end of the incubation, supernatants were discarded and cells were lysed in 0.1 N NaOH for 30 min. The radioactivity associated with the cell-soluble fraction was then counted using a Wallac Microbeta 1450-021 TriLux Luminometer Liquid Scintillation Counter (Wallac EG&G, Gaithersburg, MD, USA) and protein quantification was performed using the colorimetric Lowry method.
Myostatin and MAS gene expression assays
Cells were plated at a density of 30,000 cells per well in 24-well plates and were grown overnight in 5% CO2 at 37°C. On day 5 of differentiation, treatments were carried out for 6 h. At the end of the incubation, RNAs were extracted and purified using RNAzol (Eurobio, Les Ulis, France). RNAs were converted into cDNAs using a high-capacity cDNA RT Kit (Applied Biosystems, ThermoFisher) before performing quantitative PCR using iTaq SybrGreen (Biorad). Q-RT-PCRs were then performed using a 7900HT Fast Real-Time PCR detection system (Applied Biosystems) and a standard qPCR program (1 cycle at 95°C for 15 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min). QRT-PCR master mix contained the 100 ng cDNA samples and a set of primers at final concentration of 200 nM designed for two different exons and described below. The quality of RNA was checked using NanoDrop™ technology (ThermoFisher) when necessary.
The relative differences in gene expression levels between treatments were expressed as increases or decreases in cycle time (Ct) numbers compared to the control group where the Ct value of each gene was normalized to the beta-actin gene or hypoxanthine guanine phosphoribosyl transferase (HPRT) gene. Results of gene expression were expressed as 2−∆∆CT after normalization with house-keeping genes. Primer sequences used are described in Table 1.
Primers used for mRNA quantification by RT-QPCR.
| Gene | Sequence |
|---|---|
| Mstn | F 5’-GAGTCTGACTTTCTAATGCAAG-3’ |
| R 5’-TGTTGTAGGAGTCTTGACGG-3’ | |
| Mas1 | F 5’-TGCCTTGGTGACCACCATGG-3’ |
| R 5’-ACCAAGATGGTGCTGGACAC-3’ | |
| Actb | F 5’-CTCTAGACTTCGAGCAGGAG-3’ |
| R 5’-GGTACCACCAGACAGCACT-3’ | |
| Hprt | F 5’-TCCTCATGGACTGATTATGGA-3’ |
| R 5’-TCCAGCAGGTCAGCAAAGAA-3’ |
siRNA MAS assay
Cells were plated at a density of 10,000 cells per well in 24-well plates. After 3 days of differentiation, cells were transfected either with scramble siRNA (10 nM) or Mas1 siRNA (10 nM) according to the manufacturer’s instructions (Origene Technologies, Rockville, MD, USA). Two days after transfection, myotubes were treated with either DMSO or IGF-1 or 20E or angiotensin(1–7) for 6 h. At the end of incubation, RNA was extracted and analyzed by QRT-PCR as described above.
Binding studies
The relative affinities of 20E for human nuclear steroid receptors such as androgen receptor (AR), estrogen receptors alpha and beta (ERα, ERβ) and glucocorticoid receptor (GR) were determined by radioligand binding assays (CEREP/Eurofins). The selective ligands [3H]-methyltrienolone, [3H]-estradiol and [3H]-dexamethasone were employed with cells expressing either human endogenous or recombinant AR, ERα or β, or GR, respectively. 20E was used at concentrations up to 100 μM as a potential competitor. Inhibition of control specific binding was determined, and IC50 and Ki were calculated when possible. Additionally, a receptor screen was carried out on 45 GPCR and 5 nuclear receptors at a fixed concentration of 20E (10 μM). Radioligand binding assays were performed according to the manufacturer’s instructions employing 3H- or 125I-labeled specific ligands of each receptor (SafetyScreen87 Panel, Panlabs, Taipei, Taiwan).
Statistical analyses
Statistical analysis was performed using Graph Pad Prism® Software. ANOVA followed by a Dunnett t-test or a Kruskal–Wallis test followed by a Dunn’s test (when the variances significantly differed) have been performed. To evaluate the significance of differences between two groups, the choice of parametric Student’s t-test or non-parametric Mann–Whitney test was based on the normality or non-normality of data distribution, respectively (D’Agostino–Pearson test). The results are considered significant at P -value < 0.05 (*), <0.01(**), <0.001 (***).
Results
20-Hydroxyecdysone stimulates muscle anabolism
20-Hydroxyecdysone (20E) effects were investigated on pre-established murine myotubes (following 6 days of differentiation). An anabolic effect was investigated by means of a de novo protein synthesis assay. A dose-dependent increase in protein synthesis was observed in response to 20E treatment of C2C12 myotubes vs untreated conditions (Fig. 1A). IGF-1 (100 ng/mL), employed as positive control (Semsarian et al. 1999), displayed, as expected, an improvement in [3H] Leu incorporation (+18 %, P < 0.01). The 20E effect was significant from 1 to 5 μM. The maximal effect (+22 %, P < 0.001) was measured with 5 μM of 20E, while treatment with a higher concentration of 20E (10 μM) appeared to be notably less efficient (+14%, ns) than the previous concentration tested.
Effects of 20E on protein synthesis and myostatin gene expression in C2C12 cells. (A) Representative results from at least three distinct experiments displaying 20E effects on protein synthesis in differentiated myotubes detected by [3H]-leucine incorporation. (B) C2C12 mouse myoblasts were differentiated for 6 days into myotubes. They were then treated for 6 h with concentrations of 20E ranging from 0.001 to 10 μM. Myostatin gene expression was detected by qRT-PCR. Results are shown as means ± s.e.m. of at least seven independent experiments with ***P < 0.001, **P < 0.01, *P < 0.05 vs untreated control (Kruskal–Wallis one-way ANOVA followed by uncorrected Dunn’s test).
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
Effects of 20E on protein synthesis and myostatin gene expression in C2C12 cells. (A) Representative results from at least three distinct experiments displaying 20E effects on protein synthesis in differentiated myotubes detected by [3H]-leucine incorporation. (B) C2C12 mouse myoblasts were differentiated for 6 days into myotubes. They were then treated for 6 h with concentrations of 20E ranging from 0.001 to 10 μM. Myostatin gene expression was detected by qRT-PCR. Results are shown as means ± s.e.m. of at least seven independent experiments with ***P < 0.001, **P < 0.01, *P < 0.05 vs untreated control (Kruskal–Wallis one-way ANOVA followed by uncorrected Dunn’s test).
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
Effects of 20E on protein synthesis and myostatin gene expression in C2C12 cells. (A) Representative results from at least three distinct experiments displaying 20E effects on protein synthesis in differentiated myotubes detected by [3H]-leucine incorporation. (B) C2C12 mouse myoblasts were differentiated for 6 days into myotubes. They were then treated for 6 h with concentrations of 20E ranging from 0.001 to 10 μM. Myostatin gene expression was detected by qRT-PCR. Results are shown as means ± s.e.m. of at least seven independent experiments with ***P < 0.001, **P < 0.01, *P < 0.05 vs untreated control (Kruskal–Wallis one-way ANOVA followed by uncorrected Dunn’s test).
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
Myostatin is a major autocrine regulator that inhibits muscle growth in mammals. The myostatin transcript bioassay was developed and standardized in order to assess ecdysteroid activity (Zubeldia et al. 2012). IGF-1 (100 ng/mL), used as a positive control, demonstrated a significant inhibition of myostatin gene expression (60% of untreated control cells, P < 0.001, Fig. 1B). A dose-dependent and partial inhibition of myostatin gene expression was observed in response to 20E treatment at concentrations between 0.05 and 10 µM. This inhibition was significant from 1 µM 20E (Fig. 1B). The inhibition of myostatin gene expression was then employed as a readout for 20E activity.
20-Hydroxyecdysone acts on cell membranes
In order to confirm that 20E acts primarily at the cell membrane, or if it needs to penetrate into the cell to exert its effects, a membrane-impermeable derivative of 20E was produced (Fig. 2A). We compared the effects of free 20E and its 22S-HSA conjugate (batch 1) on myostatin gene expression at the concentration of 10 µM 20E-equivalents (Fig. 2B). We observed that the membrane-impermeable 20E-derivative retained an activity similar to that of free 20E. Similar results were obtained with another batch of 20E-22S-HSA conjugate prepared with another coupling method (batch 2) and also when using 20E-6-CMO coupled to albumin (data not shown). This result demonstrates that covalent attachment to a bulky protein does not prevent 20E activity and is an additional argument for the interaction of 20E with a membrane receptor, as proposed by Gorelick-Feldman et al. (2010).
20E acts on C2C12 myotubes from outside of the cell. (A) Structure of 20E 22-succinate coupled with human serum albumin. (B) C2C12 mouse myoblasts were differentiated for 6 days into myotubes. They were then treated for 6 h with IGF-1 (100 nM), 20E (10 μM) or 20E-HSA (10 μM 20E-equivalent). Myostatin gene expression was detected by qRT-PCR. Results are shown as means ± s.e.m. of four independent experiments with *P < 0.05, **P < 0.01 vs untreated control (Kruskal–Wallis one-way ANOVA followed by uncorrected Dunn’s test).
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
20E acts on C2C12 myotubes from outside of the cell. (A) Structure of 20E 22-succinate coupled with human serum albumin. (B) C2C12 mouse myoblasts were differentiated for 6 days into myotubes. They were then treated for 6 h with IGF-1 (100 nM), 20E (10 μM) or 20E-HSA (10 μM 20E-equivalent). Myostatin gene expression was detected by qRT-PCR. Results are shown as means ± s.e.m. of four independent experiments with *P < 0.05, **P < 0.01 vs untreated control (Kruskal–Wallis one-way ANOVA followed by uncorrected Dunn’s test).
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
20E acts on C2C12 myotubes from outside of the cell. (A) Structure of 20E 22-succinate coupled with human serum albumin. (B) C2C12 mouse myoblasts were differentiated for 6 days into myotubes. They were then treated for 6 h with IGF-1 (100 nM), 20E (10 μM) or 20E-HSA (10 μM 20E-equivalent). Myostatin gene expression was detected by qRT-PCR. Results are shown as means ± s.e.m. of four independent experiments with *P < 0.05, **P < 0.01 vs untreated control (Kruskal–Wallis one-way ANOVA followed by uncorrected Dunn’s test).
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
20-Hydroxyecdysone acts via a GPCR-type receptor
The GPCR hypothesis is based on the inhibition of 20E effects by pertussis toxin, but there are still numerous possible GPCR candidates. We selected a set of GPCR receptors based on available literature and using different criteria corresponding to well-established effects of 20E: (i) involvement in the control of muscle cells activity and glycemia/insulin sensitivity and (ii) ability to reduce fat mass gain in high-fat fed animals (Kizelsztein et al. 2009, Foucault et al. 2012). A set of GPCRs was thus selected including TGR5 (bile acid receptor: Thomas et al. 2009), GPER/GPR30 (estradiol, aldosterone receptors: Prossnitz et al. 2008, Evans et al. 2014), LPA1 (lysophosphatidic acids receptor: Jean-Baptiste et al. 2005), APJ (apelin receptor: Bertrand et al. 2013), OXTR (oxytocin receptor: Breton et al. 2002), AVPR1 (vasopressin receptor: Nervi et al. 1995), MrgD (alamandine receptor: Lautner et al. 2013), MARRS (vitamin D3 receptor: Khanal & Nemere 2007) and MAS (angiotensin(1–7) receptor: Santos et al. 2003, Muñoz et al. 2010). The possible interaction of 20E with those receptors was assayed using different approaches according to available methodologies: (1) in silico binding when 3D structures were available (Lapenna et al. 2015), (2) in vitro direct binding studies by competition with a radioactive ligand (e.g. Supplementary Table 1), (3) comparison of the effects of known receptor agonists with those of 20E on C2C12 cells or (4) effect of known antagonists of those receptors on the response of C2C12 cells to 20E. Using this approach, the only receptor which gave positive data was MAS, the receptor of angiotensin(1–7).
20-Hydroxyecdysone and angiotensin(1–7) act via MAS receptor activation
Using the myostatin gene expression assay, a pharmacological approach was employed to compare the effects of the endogenous MAS receptor agonist (angiotensin(1–7)) with those of 20E on C2C12 cells in the presence or absence of a known antagonist.
Angiotensin(1–7) (Fig. 3A), as well as 20E (Fig. 1B), partially inhibits myostatin gene expression in a dose-dependent manner. As expected, this inhibition by angiotensin(1–7) (10 µM) was totally abolished by specific angiotensin(1–7) antagonist (A779; 10 µM) (Fig. 3B). Interestingly, 20E inhibitory effects on myostatin gene expression were also fully reversed by the same antagonist (Fig. 3B), suggesting that inhibition of myostatin gene expression by 20E (or angiotensin(1–7)) is mediated by the receptor of angiotensin(1–7). In contrast, the effect of IGF-1, which acts through its own receptor (insulin-like growth factor 1 receptor; IGF1R) remains unchanged in the presence or in the absence of angiotensin(1–7) antagonist (not shown).
20-Hydroxyecdysone effects are mediated through Mas receptor activation. C2C12 mouse myoblasts were differentiated for 6 days into myotubes. (A) They were then treated for 6 h with concentrations of angiotensin(1–7) ranging from 0.001 to 10 μM. Myostatin gene expression was determined using qRT-PCR. (B) Effect of MAS antagonist (A779, 10 µM) on 20E- and angiotensin(1–7)-induced myostatin gene expression inhibition. (C) Effect of MAS siRNA on 20E- and angiotensin(1–7)-induced myostatin gene expression inhibition. Results are shown as means ± s.e.m. of at least three independent experiments with *P < 0.05; **P < 0.01; ***P < 0.001. Kruskal–Wallis one-way ANOVA followed by uncorrected Dunn’s test compared to untreated control.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
20-Hydroxyecdysone effects are mediated through Mas receptor activation. C2C12 mouse myoblasts were differentiated for 6 days into myotubes. (A) They were then treated for 6 h with concentrations of angiotensin(1–7) ranging from 0.001 to 10 μM. Myostatin gene expression was determined using qRT-PCR. (B) Effect of MAS antagonist (A779, 10 µM) on 20E- and angiotensin(1–7)-induced myostatin gene expression inhibition. (C) Effect of MAS siRNA on 20E- and angiotensin(1–7)-induced myostatin gene expression inhibition. Results are shown as means ± s.e.m. of at least three independent experiments with *P < 0.05; **P < 0.01; ***P < 0.001. Kruskal–Wallis one-way ANOVA followed by uncorrected Dunn’s test compared to untreated control.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
20-Hydroxyecdysone effects are mediated through Mas receptor activation. C2C12 mouse myoblasts were differentiated for 6 days into myotubes. (A) They were then treated for 6 h with concentrations of angiotensin(1–7) ranging from 0.001 to 10 μM. Myostatin gene expression was determined using qRT-PCR. (B) Effect of MAS antagonist (A779, 10 µM) on 20E- and angiotensin(1–7)-induced myostatin gene expression inhibition. (C) Effect of MAS siRNA on 20E- and angiotensin(1–7)-induced myostatin gene expression inhibition. Results are shown as means ± s.e.m. of at least three independent experiments with *P < 0.05; **P < 0.01; ***P < 0.001. Kruskal–Wallis one-way ANOVA followed by uncorrected Dunn’s test compared to untreated control.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
In order to further assess the involvement of MAS receptor in 20E activity on myotubes, we designed a gene interference experiment using silencing RNA directed against MAS receptor (siRNA Mas1). The efficiency of MAS receptor downregulation by siRNA was tested first. A significant decrease of MAS gene expression by a factor 2 in all transfected groups by directed siRNA vs scramble siRNA was observed (data not shown). In a similar way to what was observed with antagonists (Fig. 3B), downregulation of MAS receptor reversed 20E or angiotensin(1–7) effects on myostatin gene expression (Fig. 3C). As expected, and consistent with the pharmacological approach, downregulation of MAS receptor had no impact on the effect of IGF-1 on myostatin gene expression (data not shown). Finally, we employed MCF-7 human breast cancer cells which highly and constitutively express MAS receptor (Luo et al. 2015; Supplementary Fig. 2A) to assess NO production in response to MAS receptor activation by 20E. As shown in Supplementary Fig. 2B, the DAF-FM relative fluorescence of 20E-treated MCF7 cells was significantly enhanced at all concentrations used when compared to untreated control, indicating that 20E enhanced NO production. Similar levels of NO production in the presence of 10 µM 20E or angiotensin(1–7) were observed (+38% (P < 0.05) and +34% (P < 0.01), respectively). NO production induced by 20E and angiotensin(1–7) was inhibited by A779 treatment (Supplementary Fig. 2B). These results are consistent with our data obtained on myostatin gene expression (Fig. 3) and further demonstrate the role of MAS receptor activation in 20E-mediated effects. These data are in concordance with previously published studies by Heitsch et al. (2001) and Sampaio et al. (2007).
20-Hydroxyecdysone does not bind to a set of nuclear receptors
Parr et al. (2014, 2015) have proposed that 20E effects are explained by its binding to estrogen receptor ERβ. It is however difficult to consider that this receptor corresponds to a canonical nuclear receptor of estradiol, given that several previous binding studies of 20E to nuclear ERs were unsuccessful (Báthori et al. 2008, Gorelick-Feldman et al. 2010, Seidlova-Wuttke et al. 2010). We too performed competitive binding tests of 20E for AR, ERα and ERβ which were all negative at up to 100 µM (data not shown). Similarly, an off-target safety screen for 87 receptors was equally negative for ERα and AR (Supplementary Table 1). Nevertheless, Parr’s results are not unique, since Gao et al. (2008) showed that 20E can activate several ERβ target genes, and indeed there are multiple similarities between the effects of 20E and 17β-estradiol (E2) on muscles (Velders et al. 2012) or skin cells (Ehrhardt et al. 2011). Thus, while the above binding studies seem to exclude canonical nuclear forms of ERs, the question remains open for the membrane ones.
Estradiol effects on C2C12 myotubes
In an attempt to explain this discrepancy, we engaged in a set of experiments to characterize estradiol effects on C2C12 cells and to identify which type of E2 receptor could be involved in 20E effects. We first checked if, like 20E, E2 was able to impact myostatin gene expression in C2C12 cells. Myotubes were treated with increasing doses of E2 during 6 h and myostatin expression was then determined at transcriptional level. E2 significantly inhibits myostatin gene expression from 0. 1 to 1 µM (Fig. 4A). To determine if E2 activity relies on an interaction with a plasma membrane receptor, we employed the same strategy as the one presented above for 20E (Fig. 2). We used a membrane-impermeable conjugate of E2 made of 6-hydroxy-E2 attached via a 6-carboxymethyloxime (CMO) linker to BSA. E2-6-CMO itself significantly inhibited myostatin gene expression (−34%, P < 0.01) with the same trend as IGF-1 positive control (−44%, P < 0.01). By contrast, the E2-6-CMO-BSA conjugate was inactive (Fig. 4B) and did not significantly decrease myostatin expression (−13%, ns). This experiment allows the exclusion of an interaction of E2 with a transmembrane receptor but rather could possibly fit with a receptor bound to the cytoplasmic side of the cell membrane by a lipid anchor.
17β-Estradiol-mediated myostatin inhibition is not linked with a transmembrane receptor and is not blunted by a MAS antagonist. (A) Differentiated C2C12 cells were treated with IGF-1 (100 ng/mL) or 17β-estradiol (E2; 0.01, 0.1 and 1 µM) for 6 h. Myostatin gene expression was detected by qRT-PCR. (B) Effect of E2-CMO (0.1 µM) and E2-CMO-BSA (0.1 µM) on myostatin gene expression (qRT-PCR). (C) Effect of E2 (0.02 µM) in combination with Mas antagonist A779 (10 µM) on myostatin gene expression. Results are shown as means ± s.e.m. of at least five independent experiments with *P < 0.05, **P < 0.01 (Kruskal–Wallis one-way ANOVA followed by uncorrected Dunn’s test) compared to untreated control.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
17β-Estradiol-mediated myostatin inhibition is not linked with a transmembrane receptor and is not blunted by a MAS antagonist. (A) Differentiated C2C12 cells were treated with IGF-1 (100 ng/mL) or 17β-estradiol (E2; 0.01, 0.1 and 1 µM) for 6 h. Myostatin gene expression was detected by qRT-PCR. (B) Effect of E2-CMO (0.1 µM) and E2-CMO-BSA (0.1 µM) on myostatin gene expression (qRT-PCR). (C) Effect of E2 (0.02 µM) in combination with Mas antagonist A779 (10 µM) on myostatin gene expression. Results are shown as means ± s.e.m. of at least five independent experiments with *P < 0.05, **P < 0.01 (Kruskal–Wallis one-way ANOVA followed by uncorrected Dunn’s test) compared to untreated control.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
17β-Estradiol-mediated myostatin inhibition is not linked with a transmembrane receptor and is not blunted by a MAS antagonist. (A) Differentiated C2C12 cells were treated with IGF-1 (100 ng/mL) or 17β-estradiol (E2; 0.01, 0.1 and 1 µM) for 6 h. Myostatin gene expression was detected by qRT-PCR. (B) Effect of E2-CMO (0.1 µM) and E2-CMO-BSA (0.1 µM) on myostatin gene expression (qRT-PCR). (C) Effect of E2 (0.02 µM) in combination with Mas antagonist A779 (10 µM) on myostatin gene expression. Results are shown as means ± s.e.m. of at least five independent experiments with *P < 0.05, **P < 0.01 (Kruskal–Wallis one-way ANOVA followed by uncorrected Dunn’s test) compared to untreated control.
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
Sobrino et al. (2017) showed that the vasodilatory effect of E2 was blunted by a MAS antagonist. This encouraged us to check if a MAS antagonist would inhibit the effect of E2 on myostatin gene expression by C2C12 cells. E2’s effect on myostatin gene expression was tested in the presence of a MAS antagonist (A779), but, unexpectedly, the effect of E2 was not inhibited by A779 (Fig. 4C).
17α-E2is an epimer of estradiol which does not bind nuclear ERα or ERβ with high affinity and is known to bind only membrane forms of ER (Dykens et al. 2005). This compound proved active for the inhibition of myostatin gene activity (Supplementary Fig. 3). This result provides an additional argument for the involvement of a non-nuclear ER.
Discussion
Our data combined with those previously available from the literature allow us to conclude that the effects of 20E on C2C12 cells involve both Mas receptor and a non-nuclear estradiol receptor. Although these results do not allow the unambiguous identification of its primary target, they allow a reconciliation of the findings of Gorelick-Feldman et al. (2010) and Parr et al. (2014) by invoking a mixed mechanism of action.
Indeed, of paramount importance is our finding that a membrane-impermeable form of E2 is inactive, whereas a membrane-impermeable form of 20E still remains active. This allows us to conclude that 20E does not bind to the concerned ER receptor and that the activation of this estrogen receptor by 20E is secondary to MAS activation.
Five different E2 receptors (not including splice variants) have been described including two GPCRs (Micevych & Christensen 2012), GPR30 (Filardo et al. 2000) and Gq-mER (Lagrange et al. 1997), plus possibly another plasma membrane-associated receptor, ER-X (Toran-Allerand 2005). Our experiments with GPR30 agonists and antagonists exclude GPR30 from the candidates. In addition, nuclear canonical receptors being excluded, the question remains open regarding which membrane E2 receptor form is involved in the effects of E2 and 20E on muscle cells.
Different membrane forms of the nuclear estrogen receptors (ERα and ERβ), produced by alternative splicing, have been described. These may correspond to either palmitoylated full forms or truncated forms unmasking a potential transmembrane alpha-helix sequence (Meitzen et al. 2013, Maneix et al. 2015, Schreihofer et al. 2018). Non-truncated ERs may reversibly bind to the cytoplasmic face of the membrane by a S-palmitoyl anchor (Marino & Ascenzi 2006).
In this respect, experiments on C2C12 cells treated with diarylheptanoid compounds (HPPH) provide very important information (Tipbunjong et al. 2017, 2019). These molecules display growth- and differentiation-promoting effects on C2C12 cells that involve a membrane form of ERα receptor bound by a palmitoyl anchor, and they were abolished by 2-bromohexadecanoic acid, an inhibitor of palmitoylation (Tipbunjong et al. 2019).
Interestingly, Garratt et al. (2019) have shown that 17αE2, an E2 epimer that does not bind nuclear forms of estrogen receptors, has beneficial effect on muscles, notably during sarcopenia. These results demonstrate that nuclear and membrane forms of E2 receptors display different ligand specificities. Therefore, the use of ‘selective’ inhibitors based on their effects on nuclear ERs does not allow unambiguous conclusions about whether ERα or ERβ membrane forms are targeted, and specific silencing experiments have to be preferred.
Membrane forms of ERs can associate with a GPCR. For example, in neuronal membranes, ER is associated with mGLUR1a (a glutamate receptor) and in this system, E2 effects are blunted by a mGLUR1a antagonist (Pastore et al. 2019).
A functional interaction between ER and MAS receptor has already been observed by Sobrino et al. (2017) in HUVEC cells. These authors observed that an E2 effect (increased NO synthesis) was abolished by an antagonist of MAS receptor. An association between MAS and a non-nuclear form of ER seemed therefore highly probable.
Taken together, our data best fit with the presence of a complex associating MAS and a estrogen membrane receptor (Fig. 5), likely together with a caveolin and/or striatin, as well as additional transduction effectors (e.g. Gq, NOS). The demonstration of such a functional association will require membrane fractionation combined with various immunoprecipitation techniques. Whether a symmetrical interaction of this ER and IGFR exists is an attractive possibility, which is presently under investigation.
Proposed mechanism of myostatin gene control by angiotensin(1–7), 20E and E2. According with this scheme, myostatin and protein synthesis would be controlled directly by ER and indirectly by MAS, while NO synthesis would be controlled directly by angiotensin(1–7) (Tirupula et al. 2015) or 20E (Omanakuttan et al. 2016), and indirectly by E2 (Sobrino et al. 2017).
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
Proposed mechanism of myostatin gene control by angiotensin(1–7), 20E and E2. According with this scheme, myostatin and protein synthesis would be controlled directly by ER and indirectly by MAS, while NO synthesis would be controlled directly by angiotensin(1–7) (Tirupula et al. 2015) or 20E (Omanakuttan et al. 2016), and indirectly by E2 (Sobrino et al. 2017).
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
Proposed mechanism of myostatin gene control by angiotensin(1–7), 20E and E2. According with this scheme, myostatin and protein synthesis would be controlled directly by ER and indirectly by MAS, while NO synthesis would be controlled directly by angiotensin(1–7) (Tirupula et al. 2015) or 20E (Omanakuttan et al. 2016), and indirectly by E2 (Sobrino et al. 2017).
Citation: Journal of Molecular Endocrinology 68, 2; 10.1530/JME-21-0033
The case of ER is not unique. Ruhs et al. (2017) described an association between aldosterone receptor (MR) and GPER/GPR30. Accordingly, some effects of aldosterone are inhibited by G15, a GPER inhibitor (Feldman et al. 2016) and, most interestingly, they also showed an association between MR and the angiotensin II receptor AT1. Interestingly, there would thus be the presence of both a complex between aldosterone and angiotensin II AT1 receptors and, symmetrically, of a complex between estradiol and angiotensin(1–7) MAS receptors, displaying opposite and competing physiological effects.
It is worth mentioning that 20E will only activate a particular membrane form of ER, whereas E2 would in addition bind nuclear receptor(s). Thus 20E is devoid of any feminizing activity (Prabhu & Nayar 1974, Seidlova-Wuttke et al. 2010) and is probably inactive on estrogen-dependent cancer cells.
Conclusion
It is noteworthy that angiotensin(1–7), E2 and 20E display similar pleiotropic effects on many different organs/functions (Supplementary Table 2). During recent years, the number of identified beneficial effects of the protective arm of the renin–angiotensin–aldosterone system has been continuously increasing, and from the available data, it is expected that 20E will provide similar beneficial effects on several types of diseases (sarcopenia, diabetes, metabolic syndrome, etc.).
Based on the above findings, a pharmaceutical grade preparation of 20E (BIO101) has been developed and has been assayed in a phase 2 clinical trial for treating sarcopenia (a double-blind, placebo controlled, randomized interventional clinical trial (SARA-INT), ClinicalTrials #NCT03452488). We are confident that the beneficial effects of 20E/BIO101 will also be further established on, for example, lungs, kidneys and cardiovascular pathologies and could offer new therapeutic strategies.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JME-21-0033.
Declaration of interest
R L, M S, B D B, S R, L G, L D, S V, M L, W D and P J D are, or were, employees of Biophytis. They declare, however, that the company’s potential commercial interests had no impact on the conduct of this research work.
Funding
This work was supported by Biophytis.
Author contribution statement
R L and P J D performed formal analysis, conceptualization, supervision, validation, writing review and editing and provided methodology. W D performed formal analysis, conceptualization, supervision and provided methodology; L D performed conceptualization, writing review and editing; M L performed formal analysis, conceptualization, writing review and editing and provided methodology; M S performed investigation, formal analysis, writing review and editing and provided methodology. S R and B D B performed investigation, formal analysis and provided methodology; L G performed investigation; S V performed funding acquisition, validation and provided resources.
Acknowledgements
The authors thank Dr J-P Delbecque (University of Bordeaux) for his help for the synthesis of 20E-HSA conjugate.
References
Báthori M, Tóth N, Hunyadi A, Marki A & Zador E 2008 Phytoecdysteroids and anabolic-androgenic steroids-structure and effects on humans. Current Medicinal Chemistry 15 75–91. (https://doi.org/10.2174/092986708783330674)
Bertrand C, Pignalosa A, Wanecq E, Rancoule C, Batut A, Deleruyelle S, Lionetti L, Valet P & Castan-Laurell I 2013 Effects of dietary eicosapentaenoic acid (EPA) supplementation in high-fat fed mice on lipid metabolism and apelin/APJ system in skeletal muscle. PLoS ONE 8 e78874. (https://doi.org/10.1371/journal.pone.0078874)
Breton C, Haenggeli C, Barberis C, Heitz F, Bader CR, Bernheim L & Tribollet E 2002 Presence of functional oxytocin receptors in cultured human myoblasts. Journal of Clinical Endocrinology and Metabolism 87 1415–1418. (https://doi.org/10.1210/jcem.87.3.8537)
Chiang JY 2013 Bile acid metabolism and signaling. Comprehensive Physiology 3 1191–1212. (https://doi.org/10.1002/cphy.c120023)
Dinan L 2009 The Karlson lecture. Phytoecdysteroids: what use are they? Archives of Insect Biochemistry and Physiology 72 126–141. (https://doi.org/10.1002/arch.20334)
Dinan L & Lafont R 2006 Effects and applications of arthropod steroid hormones (ecdysteroids) in mammals. Journal of Endocrinology 191 1–8. (https://doi.org/10.1677/joe.1.06900)
Dinan L, Bourne P, Whiting P, Tsitsekli A, Saatov Z, Dhadialla TS, Hormann RE, Lafont R & Coll J 2003 Synthesis and biological activities of turkesterone 11alpha-acyl derivatives. Journal of Insect Science 3 6. (https://doi.org/10.1093/jis/3.1.6)
Dykens JA, Moos WH & Howell N 2005 Development of 17alpha-estradiol as a neuroprotective therapeutic agent: rationale and results from a phase I clinical study. Annals of the New York Academy of Sciences 1052 116–135. (https://doi.org/10.1196/annals.1347.008)
Ehrhardt C, Wessels JT, Wuttke W & Seidlova-Wuttke D 2011 The effects of 20-hydroxyecdysone and 17beta-estradiol on the skin of ovariectomized rats. Menopause 18 323–327. (https://doi.org/10.1097/gme.0b013e3181f322e3)
Evans PD, Bayliss A & Reale V 2014 GPCR-mediated rapid, non-genomic actions of steroids: comparisons between DmDopEcR and GPER1 (GPR30). General and Comparative Endocrinology 195 157–163. (https://doi.org/10.1016/j.ygcen.2013.10.015)
Feldman RD, Ding Q, Hussain Y, Limbird LE, Pickering JG & Gros R 2016 Aldosterone mediates metastatic spread of renal cancer via the G protein-coupled estrogen receptor (GPER). FASEB Journal 30 2086–2096. (https://doi.org/10.1096/fj.15-275552)
Filardo EJ, Quinn JA, Blang KI & Frackelton AR 2000 Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Molecular Endocrinology 14 1649–1660. (https://doi.org/10.1210/mend.14.10.0532)
Foucault AS, Mathe V, Lafont R, Even P, Dioh W, Veillet S, Tome D, Huneau JF, Hermier D & Quignard-Boulange A 2012 Quinoa extract enriched in 20-hydroxyecdysone protects mice from diet-induced obesity and modulates adipokines expression. Obesity 20 270–277. (https://doi.org/10.1038/oby.2011.257)
Gao L, Cai G & Shi X 2008 β-Ecdysterone induces osteogenic differentiation in mouse mesenchymal stem cells and relieves osteoporosis. Biological and Pharmaceutical Bulletin 31 2245–2249. (https://doi.org/10.1248/bpb.31.2245)
Garratt M, Leander D, Pifer K, Bower B, Herrera JJ, Day SM, Fiehn O, Brooks SV & Miller RA 2019 17-Alpha estradiol ameliorates age-associated sarcopenia and improves late-life physical function in male mice but not in females or castrated males. Aging Cell 18 e12920. (https://doi.org/10.1111/acel.12920)
Gorelick-Feldman J, Cohick W & Raskin I 2010 Ecdysteroids elicit a rapid Ca2+ flux leading to Akt activation and increased protein synthesis in skeletal muscle cells. Steroids 75 632–637. (https://doi.org/10.1016/j.steroids.2010.03.008)
Heitsch H, Brovkovych S, Malinski T & Wiemer G 2001 Angiotensin-(1–7)-stimulated nitric oxide and superoxide release from endothelial cells. Hypertension 37 72–76. (https://doi.org/10.1161/01.hyp.37.1.72)
Isenmann E, Ambrosio G, Joseph JF, Mazzarino M, de la Torre X, Zimmer P, Kazlauskas R, Goebel C, Botre F & Diel P et al.2019 Ecdysteroids as non-conventional anabolic agent: performance enhancement by ecdysterone supplementation in humans. Archives of Toxicology 93 1807–1816. (https://doi.org/10.1007/s00204-019-02490-x)
Jean-Baptiste G, Yang Z, Khoury C & Greenwood MT 2005 Lysophosphatidic acid mediates pleiotropic responses in skeletal muscle cells. Biochemical and Biophysical Research Communications 335 1155–1162. (https://doi.org/10.1016/j.bbrc.2005.08.011)
Karlson P 1983 Eighth Adolf Butenandt lecture. Why are so many hormones steroids? Hoppe-Seyler’s Zeitschrift für Physiologische Chemie 364 1067–1087. (https://doi.org/10.1515/bchm2.1983.364.2.1067)
Khanal RN & Nemere I 2007 Membrane receptors for vitamin D metabolites. Critical Reviews in Eukaryotic Gene Expression 17 31–47. (https://doi.org/10.1615/critreveukargeneexpr.v17.i1.30)
Kizelsztein P, Govorko D, Komarnytsky S, Evans A, Wang Z, Cefalu WT & Raskin I 2009 20-Hydroxyecdysone decreases weight and hyperglycemia in a diet-induced obesity mice model. American Journal of Physiology: Endocrinology and Metabolism 296 E433–E439. (https://doi.org/10.1152/ajpendo.90772.2008)
Kotsyuruba AV, Bukchanevich OM, Tuganova AV & Tarakanov SS 1995 Mechanisms of the early effect of biologically active oxysterones calcitriol and ecdysterone, modulation of intracellular pools of arachidonic acid and products of its oxidative metabolism. Ukrainskii Biokhimicheskii Zhurnal 67 45–52.
Kotsyuruba AV, Bukchanevich OM, Berdyshev AG, Meged OF, Hula NM, Mykhailik OM & Bakaï EA 1998 In vitro study of the early membrane effects of C27-steroid hormone ecdysterone, immobilized on the nanodispersed magnetite surface. Ukrainskii Biokhimicheskii Zhurnal 70 22–32.
Kotsyuruba A, Bukchanevich O, Meged O, Tarakanov S, Berdyshev A & Tuganova A 1999 The C(27)-steroid hormones ecdysterone and calcitriol activate the phosphoinositide messenger cascade in its membrane phase of action. Ukrainskii Biokhimicheskii Zhurnal 71 27–32.
Lagrange AH, Rønnekleiv OK & Kelly MJ 1997 Modulation of G protein-coupled receptors by an estrogen receptor that activates protein kinase A. Molecular Pharmacology 51 605–612. (https://doi.org/10.1124/mol.51.4.605)
Lapenna S, Gemen R, Wollgast J, Worth A, Maragkoudakis P & Caldeira S 2015 Assessing herbal products with health claims. Critical Reviews in Food Science and Nutrition 55 1918–1928. (https://doi.org/10.1080/10408398.2012.726661)
Lautner RQ, Villela DC, Fraga-Silva RA, Silva N, Verano-Braga T, Costa-Fraga F, Jankowski J, Jankowski V, Sousa F & Alzamora A et al.2013 Discovery and characterization of alamandine: a novel component of the renin-angiotensin system. Circulation Research 112 1104–1111. (https://doi.org/10.1161/CIRCRESAHA.113.301077)
Luo Y, Tanabe E, Kitayoshi M, Nishiguchi Y, Fujiwara R, Matsushima S, Sasaki T, Sasahira T, Chihara Y & Nakae D et al.2015 Expression of MAS1 in breast cancer. Cancer Science 106 1240–1248. (https://doi.org/10.1111/cas.12719)
Maneix L, Antonson P, Humire P, Rochel-Maia S, Castaneda J, Omoto Y, Kim HJ, Warner M & Gustafsson JÅ 2015 Estrogen receptor beta exon 3-deleted mouse: the importance of non-ERE pathways in ERbeta signaling. PNAS 112 5135–5140. (https://doi.org/10.1073/pnas.1504944112)
Marino M & Ascenzi P 2006 Steroid hormone rapid signaling: the pivotal role of S-palmitoylation. IUBMB Life 58 716–719. (https://doi.org/10.1080/15216540601019485)
Meitzen J, Luoma JI, Boulware MI, Hedges VL, Peterson BM, Tuomela K, Britson KA & Mermelstein PG 2013 Palmitoylation of estrogen receptors is essential for neuronal membrane signaling. Endocrinology 154 4293–4304. (https://doi.org/10.1210/en.2013-1172)
Micevych P & Christensen A 2012 Membrane-initiated estradiol actions mediate structural plasticity and reproduction. Frontiers in Neuroendocrinology 33 331–341. (https://doi.org/10.1016/j.yfrne.2012.07.003)
Muñoz MC, Giani JF & Dominici FP 2010 Angiotensin-(1–7) stimulates the phosphorylation of Akt in rat extracardiac tissues in vivo via receptor Mas. Regulatory Peptides 161 1–7. (https://doi.org/10.1016/j.regpep.2010.02.001)
Mykhaylyk OM, Kotzuruba AV, Buchanevich OM, Korduban AM, Meged EF & Gulaya NM 2001 Signal transduction of erythrocytes after specific binding of ecdysterone and cholesterol immobilized on nanodispersed magnetite. Journal of Magnetism and Magnetic Materials 225 226–234. (https://doi.org/10.1016/S0304-8853(0001262-2)
Nervi C, Benedetti L, Minasi A, Molinaro M & Adamo S 1995 Arginine-vasopressin induces differentiation of skeletal myogenic cells and upregulation of myogenin and Myf-5. Cell Growth and Differentiation 6 81–89. (https://doi.org/10.3934/molsci.2018.2.131)
Omanakuttan A, Bose C, Pandurangan N, Kumar GB, Banerli A & Nair BG 2016 Nitric oxide and ERK mediates regulation of cellular processes by ecdysterone. Experimental Cell Research 346 167–175. (https://doi.org/10.1016/j.yexcr.2016.07.019)
Parr M & Müller-Schöll A 2018 Pharmacology of doping agents – mechanisms promoting muscle hypertrophy. AIMS Molecular Science 5 131–159.
Parr MK, Zhao P, Haupt O, Ngueu ST, Hengevoss J, Fritzemeier KH, Piechotta M, Schlorer N, Muhn P & Zheng WY et al.2014 Estrogen receptor beta is involved in skeletal muscle hypertrophy induced by the phytoecdysteroid ecdysterone. Molecular Nutrition and Food Research 58 1861–1872. (https://doi.org/10.1002/mnfr.201300806)
Parr MK, Botre F, Nass A, Hengevoss J, Diel P & Wolber G 2015 Ecdysteroids: a novel class of anabolic agents? Biology of Sport 32 169–173. (https://doi.org/10.5604/20831862.1144420)
Pastore MB, Landeros RV, Chen DB & Magness RR 2019 Structural analysis of estrogen receptors: interaction between estrogen receptors and cav-1 within the caveolaedagger. Biology of Reproduction 100 495–504. (https://doi.org/10.1093/biolre/ioy188)
Prabhu VK & Nayar KK 1974 Crustecdysone is without estrogenic or antiestrogenic activity in the rat. Experientia 30 821. (https://doi.org/10.1007/BF01924207)
Prossnitz ER, Arterburn JB, Smith HO, Oprea TI, Sklar LA & Hathaway HJ 2008 Estrogen signaling through the transmembrane G protein-coupled receptor GPR30. Annual Review of Physiology 70 165–190. (https://doi.org/10.1146/annurev.physiol.70.113006.100518)
Ruhs S, Nolze A, Hubschmann R & Grossmann C 2017 30 years of the mineralocorticoid receptor: nongenomic effects via the mineralocorticoid receptor. Journal of Endocrinology 234 T107–T124. (https://doi.org/10.1530/JOE-16-0659)
Sampaio WO, Souza dos Santos RA, Faria-Silva R, da Mata Machado LT, Schiffrin EL & Touyz RM 2007 Angiotensin-(1–7) through receptor Mas mediates endothelial nitric oxide synthase activation via Akt-dependent pathways. Hypertension 49 185–192. (https://doi.org/10.1161/01.HYP.0000251865.35728.2f)
Sanchez M, Picard N, Sauve K & Tremblay A 2010 Challenging estrogen receptor beta with phosphorylation. Trends in Endocrinology and Metabolism 21 104–110. (https://doi.org/10.1016/j.tem.2009.09.007)
Santos RA, Simoes e Silva AC, Maric C, Silva DM, Machado RP, de Buhr I, Heringer-Walther S, Pinheiro SV, Lopes MT & Bader M et al.2003 Angiotensin-(1–7) is an endogenous ligand for the G protein-coupled receptor Mas. PNAS 100 8258–8263. (https://doi.org/10.1073/pnas.1432869100)
Schreihofer DA, Duong P & Cunningham RL 2018 N-terminal truncations in sex steroid receptors and rapid steroid actions. Steroids 133 15–20. (https://doi.org/10.1016/j.steroids.2017.10.018)
Seidlova-Wuttke D, Christel D, Kapur P, Nguyen BT, Jarry H & Wuttke W 2010 β-Ecdysone has bone protective but no estrogenic effects in ovariectomized rats. Phytomedicine 17 884–889. (https://doi.org/10.1016/j.phymed.2010.03.021)
Semsarian C, Sutrave P, Richmond DR & Graham RM 1999 Insulin-like growth factor (IGF-I) induces myotube hypertrophy associated with an increase in anaerobic glycolysis in a clonal skeletal-muscle cell model. Biochemical Journal 339 443–451. (https://doi.org/10.1042/bj3390443)
Simakin S, Panyushkin V, Portugalov S, Kostina L & Martisorov E 1988 Combined application of preparation ecdysten and product bodrost during training in cyclic sports. Scientific Sports Bulletin 2 29–31.
Sláma K & Lafont R 1995 Insect hormones – ecdysteroids: their presence and actions in vertebrates. European Journal of Entomology 92 355–377.
Sobrino A, Vallejo S, Novella S, Lazaro-Franco M, Mompeon A, Bueno-Beti C, Walther T, Sanchez-Ferrer C, Peiro C & Hermenegildo C 2017 Mas receptor is involved in the estrogen-receptor induced nitric oxide-dependent vasorelaxation. Biochemical Pharmacology 129 67–72. (https://doi.org/10.1016/j.bcp.2017.01.012)
Thomas C, Gioiello A, Noriega L, Strehle A, Oury J, Rizzo G, Macchiarulo A, Yamamoto H, Mataki C & Pruzanski M et al.2009 TGR5-mediated bile acid sensing controls glucose homeostasis. Cell Metabolism 10 167–177. (https://doi.org/10.1016/j.cmet.2009.08.001)
Tipbunjong C, Kitiyanant Y, Chaturapanich G, Sornkaew N, Suksamrarn A, Kitiyanant N, Esser KA & Pholpramool C 2017 Natural diarylheptanoid compounds from Curcuma comosa Roxb. promote differentiation of mouse myoblasts C2C12 cells selectively via ER alpha receptors. Medicinal Chemistry Research 26 274–286. (https://doi.org/10.1007/s00044-016-1748-y)
Tipbunjong C, Khuituan P, Kitiyanant Y, Suksamrarn A & Pholpramool C 2019 Diarylheptanoid 1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-one enhances C2C12 myoblast differentiation by targeting membrane estrogen receptors and activates Akt-mTOR and p38 MAPK-NF-kappaB signaling axes. Journal of Natural Medicines 73 735–744. (https://doi.org/10.1007/s11418-019-01322-7)
Tirupula KC, Zhang D, Osbourne A, Chatterjee A, Desnoyer R, Willard B & Karnik SS 2017 Mas C-terminal tail interacting proteins identified by mass spectrometry-based proteomic approach. PLoS ONE 10 e0140872. (https://doi.org/10.1371/journal.pone.0140872)
Toran-Allerand CD 2005 Estrogen and the brain: beyond ER-alpha, ER-beta, and 17beta-estradiol. Annals of the New York Academy of Sciences 1052 136–144. (https://doi.org/10.1196/annals.1347.009)
Velders M, Schleipen B, Fritzemeier KH, Zierau O & Diel P 2012 Selective estrogen receptor-beta activation stimulates skeletal muscle growth and regeneration. FASEB Journal 26 1909–1920. (https://doi.org/10.1096/fj.11-194779)
Wuttke W & Seidlova-Wuttke D 2015 Eine neue alternative für die prävention und therapie postmenopausaler erkrankungen, insbesondere des metabolischen syndroms. Journal für Gynäkologische Endokrinologie 25 6–12.
Yaffe D & Saxel O 1977 Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270 725–727. (https://doi.org/10.1038/270725a0)
Zubeldia JM, Hernández-Santana A, Jiménez-del-Rio M, Pérez-López V, Pérez-Machín R & García-Castellano JM 2012 In vitro characterization of the efficacy and safety profile of a proprietary Ajuga turkestanica extract. Chinese Medicine 3 215–222. (https://doi.org/10.4236/cm.2012.34031)
