STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF A NOVEL ENDOGENOUS STEROID, ESTRADIENOLONE-LIKE STRUCTURE (ED), IN HUMAN PREGNANCY URINE

F. Ghanbari, V. Giguere and A. Philip

Division of Plastic Surgery, Department of Surgery, McGill University, Montreal Quebec, Canada

Abstract

Introduction: Estrogen-receptor related receptors (ERRs) belong to the orphan nuclear receptor NR3B subfamily, and it consists of 3 isoforms: ERRa, ERRb and ERRg. Based on the recent studies ERRs have emerged as potential therapeutic targets for treatment of breast, prostate, ovarian cancer, osteoporosis and obesity in human. Despite the many attempts made by researchers to identify ERRs’ natural ligand in order to manipulate ERRs’ pathway and control their activities, no natural ligand has been identified for them so far. Therefore, the discovery of natural ligands that bind to these orphan receptors will be helpful for the identification of novel hormonal therapeutic strategies for cancer treatments. We have previously reported the identification of a novel steroid, with an estradienolone-like structure (ED) in human pregnancy serum, urine, and placenta, which increases during pregnancy and decreases in association with premature labour. We have also found that although ED is structurally similar to estradiol, it does not bind to estrogen receptor (ER). It was therefore of interest to examine if ED interacts with orphan receptors called estrogen related receptors (ERRs). The objectives of this study are to isolate and purify ED in large-scale quantities in order to determine the fine chemical structure of ED by establishing the position of double bonds, using liquid chromatography tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR). (ii) To identify ED as a natural ligand of ERRs. Methods: (i) ED purification: The unconjugated and conjugated form of ED was isolated and purified from pregnancy urine, using column chromatography, high pressure liquid chromatography (HPLC). The fractionation of ED during chromatography was monitored using a Sex Hormone Binding Globulin binding assay (SHBG), in order to determine which fractions contained ED. Preliminary studies using LC-MS/MS suggested that it has an estradienolone-like structure. To further determine its fine structure, we will use LC-MS/MS combined with SHBG assay of each fraction. Once the large quantity of ED is purified, the final structure of ED will be determined by 1H NMR and 13C NMR. (ii) ED binding to ERR: In order to determine whether ED interacts with ERRs, we determined the effect of ED on the association of ERRα/γ with co-activator, PGC-1, using a luciferase reporter assay. COS-1 cells were transfected the pS2-LUC reporter plasmid and along with ERRα or ERR γ expression vector, with or without the co-activator PGC-1, and the cells were left untreated or treated with 100 nM ED. (The pS2-Luc plasmid consists of the promoter region of the pS2 gene that contains the ERR binding element, coupled to the luciferase gene). Alterations in ED-induced luciferase activity were then measured using a luminometer. Results: Based on elution pattern of SHBG assay, ED was eluted in the region of low polarity close to androstenedione and before testosterone. Our preliminary LC-MS/MS results show that it has a estradieonolone-like structure. Our results also show that ED interacts with ERRα/γ as an inverse agonist, as detected by interference by ED of ERRα/γ-PGC-1 interaction. Conclusion: Our findings suggest that ED can act as an inverse agonist for ERRα/γ, implicating that it may be a possible natural ligand for ERRs.