20-Hydroxyecdysone activates the protective arm of the renin angiotensin system via Mas receptor
Summary (3 min read)
INTRODUCTION
- Steroids in animal and plant kingdoms Steroid hormones are sterol derivatives widespread in animals and plants, where they are involved in the control of plenty of physiological processes.
- They include for example vertebrate sex hormones (progestagens, estrogens, androgens), insect moulting hormones , as well as plant growth hormones .
- This means that the rigid carbon skeleton of sterols is particularly suitable to generate a very large number of derivatives, which differ by the carbon number and/or the position of various substituents (mainly hydroxyl or keto groups) 2 (1).
- 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(7,8).
Ecdysteroid effects on vertebrates
- Ecdysteroids are a large family of steroids initially discovered in arthropods and later in plants (9).
- They are present in many plant species where they can reach concentrations of up to 2-3 % of the plant dry weight, and they are expected to protect plants against phytophagous insects.
- With the aim to use these molecules for crop protection, toxicological studies were performed on mammals, which unexpectedly concluded to both their lack of toxicity (oral LD50 > 9 g/kg) and their “beneficial” effects, e.g. anti-diabetic and anabolic properties (10).
- Such effects have to be linked with the presence of large amounts of phytoecdysteroids in several plants used worldwide by traditional medicine.
- At the moment, numerous effects have been reported, allowing to consider ecdysteroids as some kind of “universal remedy”(11).
How do ecdysteroids work?
- In spite of more than 40 years of research, the mechanism of action of these molecules on mammals/humans has not been elucidated, as only diverging reports are available for the moment.
- Some evidence for a direct binding was provided by in silico modelling (22), but this certainely 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. (23).
- More recently, Gorelick-Feldman et al. (17) used a pharmacological approach with various inhibitors (e.g. pertussis toxin).
- The above pharmacological arguments appear strong enough to consider that the cell membrane is (maybe not exclusively) a site of action of 20E.
- 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.
Chemicals
- Except otherwise mentioned, all the reagents and chemicals were from Sigma (Saint-Quentin Fallavier, France).
- Peptides such as angiotensin-(1-7), A779 (Asp-Arg-Val-Tyr-IleHis-D-Ala) and A1 (Asp-Arg-Val-Tyr-Ile-HisD-Pro) 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
- Analyses were performed in a 4700 MALDI TOF/TOF proteomics analyzer (Applied Biosystems).
- Laser acceleration is set at 20kV and the default laser fluency was set at 2,000 and modified according to the signal-to-noise quality.
- The matrix used was α-cyano-4-hydrocinnamic Acid (HCCA).
- The mass shift of HSA around 5 kDa after coupling indicates that 9 molecules of 20E derivatives are coupled to each albumin molecule.
Cell culture
- Except otherwise mentioned, culture media, serum, antibiotics and supplements were from Life technologies (Villebon-sur-Yvette, France).
- 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 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)
- The cell soluble fraction-associated radioactivity was then counted using 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.
- 4 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.
- RNAs were converted into cDNAs with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, ThermoFisher, Villebon-surYvette, France) before performing a quantitative PCR using iTaq SybrGreen (Biorad, Marnes-la Coquette, France).
- Primer sequences used are described in Table 1.
SiRNA MAS assay
- After 3 days of differentiation, cells were transfected either with scramble SiRNA (10 nM) or MAS-1 SiRNA (10 nM) according to 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 affinity of 20E for human nuclear steroid receptors such as androgen receptor (AR), estrogen receptors alpha and beta (ERα, ERβ) and glucocorticoid receptor (GR) was determined by radioligand binding assays (CEREP/Eurofins).
- The selective ligands [3H]methyltrienolone, [3H]-estradiol, [3H]dexamethasone were employed on cells expressing either human endogenous or recombinant AR, ERα or β, or GR, respectively.
- Inhibition of control specific binding was determined, and IC50 and Ki were calculated when possible.
- Radioligand binding assays were performed according to manufacturer instructions employing 3H- or 125I-labelled 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 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 ttest or non-parametric Mann-Whitney test was based on the normality or non-normality of data distribution, respectively (D’Agostino & Pearson test).
RESULTS
- 20-Hydroxyecdysone stimulates muscle anabolism 20-Hydroxyecdysone (20E) effects were investigated on pre-established murine myotubes (following 6 days of differentiation).
- The inhibition of myostatin gene expression was then employed as a readout for 20E activity.
- In a similar way to what was observed with antagonists (Fig. 3A), down regulation of Mas receptor reversed 20E or Ang1-7 effects on myostatin gene expression (Fig. 3B).
- Thus, while the above binding studies seem to exclude canonical nuclear forms of ERs, the question remains open for the membrane ones.
DISCUSSION
- The authors 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 nonnuclear estradiol receptor.
- This allows us to conclude that 20E does not bind the concerned ER receptor, and that the activation of this estrogen receptor by 20E is secondary to Mas activation.
- Experiments on C2C12 cells treated with diarylheptanoid compounds (HPPH) provide very important informations(50,51).
- Interestingly, the authors would thus be in 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 and displaying opposite physiological effects.
CONCLUSION:
- Based on these above findings, a pharmaceutical grade preparation of 20E (BIO101) has been developed and is presently being assayed in a phase 2 clinical trial for treating sarcopenia, (a double-blind, placebo controlled, randomized interventional clinical trial (SARA-INT), ClinicalTrials #NCT03452488).
- The authors are confident that the beneficial effects of 20E/BIO101 will also be further established on e.g. lungs, kidneys and cardiovascular pathologies and could offer new therapeutic strategies.
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"20-Hydroxyecdysone activates the pr..." refers background in this paper
...It is however difficult to consider that this receptor corresponds to a canonical nuclear receptor of estradiol, given that several binding studies to nuclear ERs were unsuccessful (12,13,17)....
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...There is in fact no direct evidence for the binding of 20E to nuclear estrogen (or androgen) receptors (12,13)....
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...While many effects have been described on animals (10,12,13), the clinical evidence for 20E effectiveness in humans remains limited at the moment (14-16)....
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