ABSTRACT
An estradiol metabolite, 2-methoxyestradiol (2ME), has emerged as an important regulator of ovarian physiology. 2ME is recognized as a potent anti-angiogenic agent in clinical trials and laboratory studies. However, little is known about its molecular actions and its endogenous targets. 2ME is produced by human ovarian cells during the normal menstrual cycle, being higher during regression of the corpus luteum, and is postulated to be involved in the anti-angiogenic process that plays out during luteolysis. We utilized cell biology techniques to understand the molecular mechanism of 2ME anti-angiogenic effects on human granulosa luteal cells. The principal effect of 2ME was to alter Hypoxia Inducible Factor 1A (HIF1A) sub-cellular localization. Molecular modelling and multiple bioinformatics tools indicated that 2ME impairs Hypoxia Inducible Factor complex (HIF) nuclear translocation by binding to a buried pocket in the HIF1A Per Arnt Sim (PAS)-B domain. Binding of 2ME to HIF1A protein is predicted to perturb HIF1A-Hypoxia Inducible Factor B (HIFB) interaction, a key step in HIF nuclear translocation, preventing the transcriptional actions of HIF, including Vascular Endotelial Growth Factor (VEGF) gene activation. To our knowledge, 2ME is the first putative HIF endogenous ligand characterized with anti-angiogenic activity. This postulate has important implications for reproduction, because angiogenic processes are critical for ovarian follicular development, ovulation and corpus luteum regression. The present research could contribute to the development of novel pharmacological approaches for controlling HIF activity in human reproductive diseases.
Subject(s)
2-Methoxyestradiol/metabolism , 2-Methoxyestradiol/pharmacology , Computational Biology , Hypoxia-Inducible Factor 1, alpha Subunit/chemistry , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Molecular Dynamics Simulation , Cell Line , Female , Humans , Luteal Cells/drug effects , Luteal Cells/metabolism , Protein Binding , Protein Domains , Protein Multimerization/drug effects , Protein Structure, QuaternaryABSTRACT
OBJECTIVE: To evaluate nuclear factor kappaB (NF-κB) activation and NF-κB-p65 subunit activation, immunolocalization, and expression in the endometrium of healthy women and endometriosis patients throughout the menstrual cycle. DESIGN: Prospective observational study. SETTING: Affiliated hospital and university research laboratory. PATIENT(S): Twenty-four healthy women and 24 endometriosis patients. INTERVENTION(S): Menstrual, proliferative, and secretory endometrial biopsies. MAIN OUTCOME MEASURE(S): Assessment of NF-κB and p65 activation by protein-DNA binding assays and p65 localization and expression by immunohistochemistry. RESULT(S): Total NF-κB-DNA binding was constitutive and variable in human endometrium accross the menstrual cycle. Healthy women (physiologic conditions) showed higher p65-DNA binding in proliferative than in menstrual and secretory endometrium. Conversely, in endometriosis patients, p65-DNA binding was higher in proliferative and secretory endometrium than in menstrual endometrium. Endometrial epithelial cells showed higher p65 expression level score than endometrial stromal cells. CONCLUSION(S): NF-κB activity is constitutive, physiologic, and variable in human endometrium. The physiologic cyclic p65 activation pattern was altered in endometriosis patients, showing no cyclic variation between the proliferative and secretory phase of the menstrual cycle. The absence of decreased p65 activity in secretory endometrium from endometriosis patients is concurrent with progesterone resistance and could participate in endometrial biologic alterations during the implantation window in endometriosis patients.
Subject(s)
Endometriosis/metabolism , Endometrium/metabolism , Menstrual Cycle/metabolism , NF-kappa B/metabolism , Adult , Binding Sites , Biopsy , Case-Control Studies , Chile , DNA/metabolism , Electrophoretic Mobility Shift Assay , Endometriosis/pathology , Endometrium/pathology , Epithelial Cells/metabolism , Female , Follicular Phase/metabolism , Humans , Immunohistochemistry , Luteal Phase/metabolism , Menstruation/metabolism , Middle Aged , Prospective Studies , Stromal Cells/metabolism , Transcription Factor RelA/metabolism , Young AdultABSTRACT
Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO(2). They are activated by Mn(2+), a metal ion that coordinates to the protein through the epsilon-amino group of a lysine residue, the N(epsilon-2)-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the epsilon-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the epsilon-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn(2+) in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn(2+) affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn(2+). In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.
