Identification of novel catabolic genes involved in 17β-estradiol degradation by Novosphingobium sp. ES2-1

Novosphingobium sp. ES2-1 is an efficient 17β-estradiol (E2)-degrading bacterium, which can convert E2 to estrone (E1), then to 4-hydroxyestrone (4-OH-E1) for subsequent oxidative cracking. In this study, the molecular bases for this process were elucidated. Two novel monooxygenase systems EstP and EstO were shown to catalyse the oxygenation of E1 and 4-OH-E1, respectively. EstP was a three-component cytochrome P450 monooxygenase system consisting of EstP1 (P450 monooxygenase), EstP2 (ferredoxin) and EstP3 (ferredoxin reductase). Ultraperformance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) analysis revealed that EstP catalysed the 4-hydroxylation of E1 to produce 4-OH-E1. The resultant 4-OH-E1 was further oxidized by a two-component monooxygenase system EstO consisting of EstO1 (flavin-dependent monooxygenases) and EstO2 (flavin reductase). UPLC-HRMS combined with ¹H-nuclear magnetic resonance analysis demonstrated that EstO catalysed the breakage of C9-C10 to yield a ring B-cleavage product. In addition, the oxygenase component genes estP1 and estO1 exhibited contrary inductive behaviours when exposed to different steroids, suggesting that EstP1-mediated 4-hydroxylation was E2-specific, whereas EstO1-mediated monooxygenation might be involved in the degradation of testosterone, androstenedione, progesterone and pregnenolone. This also implied that the mechanisms of the catabolism of different steroids by the same microorganism might be partially interlinked.     

Complex effects of 17β-estradiol on mitochondrial function

Existing literature on estradiol indicates that it affects mitochondrial functions at low micromolar concentrations. Particularly blockade of the permeability transition pore (PTP) or modulation of the enzymatic activity of one or more complexes of the respiratory chain were suspicious. We prepared mitoplasts from rat liver mitochondria (RLM) to study by single-channel patch-clamp techniques the PTP, and from rat astrocytes to study the potassium BK-channel said to modulate the PTP. Additionally, we measured respiration of intact RLM. After application of 17β-estradiol (βE) our single-channel results reveal a transient increase of activity of both, the BK-channel and the PTP followed by their powerful inhibition. Respiration measurements demonstrate inhibition of the Ca(2+)-induced permeability transition, as well, though only at higher concentrations (≥30μM). At lower concentrations, we observed an increase of endogenous- and state 2-respiration. Furthermore, we show that βE diminishes the phosphorylating respiration supported by complex I-substrates (glutamate/malate) or by the complex II-substrate succinate. Taken together the results suggest that βE affects mitochondria by several modes, including partial inhibition of the activities of ion channels of the inner membrane and of respiration. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Regulation of PTEN degradation and NEDD4–1 E3 ligase activity by Numb

The critical tumor suppressor PTEN is regulated by numerous post-translational modifications including phosphorylation, acetylation and ubiquitination. Ubiquitination of PTEN was reported to control both PTEN stability and nuclear localization. Notably, the HECT E3-ligase NEDD4–1 was identified as the ubiquitin ligase for PTEN, mediating its degradation and down-stream events. However, the mechanisms how NEDD4–1 is regulated by up-stream signaling pathways or interaction with other proteins in promoting PTEN degradation remain largely unclear. In the present study, we identified that the adaptor protein Numb, which is demonstrated to be a novel binding partner of NEDD4–1, plays important roles in controlling PTEN ubiquitination through regulating NEDD4–1 activity and the association between PTEN and NEDD4–1. Furthermore, we provided data to show that Numb regulates cell proliferation and glucose metabolism in a PTEN-dependent manner. Overall, our study revealed a novel regulation of the well-documented NEDD4–1/PTEN pathway and its oncogenic behavior.     

Regioselective hydroxylation of 17β-estradiol by mutants of CYP102A1 from Bacillus megaterium

A large set of mutants of CYP102A1 from Bacillus megaterium have human cytochrome P450-like activities and the ability to metabolize a number of marketed drugs and steroids. Here, we tested whether the CYP102A1 mutants could be used to produce hydroxylated human metabolites of 17β-estradiol (E₂). A set of the mutants, which were generated by site-directed and random mutagenesis, was used to produce hydroxylated human metabolites of E₂ in this study. Some of the tested mutants could regioselectively generate 2-OH E₂ as a major metabolite but not other hydroxylated products. These results suggest that CYP102A1 mutants would be useful for the bioconversion of steroid hormones to hydroxylated products, which can be used for industrial applications.     

Pro-survival effects of 17β-estradiol on osteocytes are mediated by nitric oxide/cGMP via differential actions of cGMP-dependent protein kinases I and II

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.     

Stimulation of phosphatidylcholine synthesis by 17β-estradiol in fetal rabbit lung

We investigated the mechanism by which estrogen stimulates pulmonary surfactant production in the fetal rabbit. Maternal administration of 17β-estradiol (5–75 μg) on day 25 of gestation resulted in a greater than twofold increase in the rate of choline incoporation into phosphatidylcholine in fetal lung slices on day 26 (full term = 31 days). Estrogen administration increased the activity of fetal lung cholinephosphate cytidylyltransferase by 62%. It had no effect on the liver enzyme. When assayed in the presence of phosphatidylglycerol fetal lung cholinephosphate cytidylyltransferase activity was increased 4.6-fold but it was not influenced by estrogen under these conditions. These findings suggest that estrogen stimulates cholinephosphate cytidyltransferase by increasing the activity of existing enzyme (possibly by increasing the amount of phosphatidylglycerol or other acidic phospholipid in the tissue) rather than by increasing the amount of enzyme-protein. Stimulation of fetal lung cholinephosphate cytidylyltransferase by estrogen as well as by glucocorticoids (Rooney, S.A., Gobran, L.I., Marino, P.A., Maniscalco, W.M., and Gross, I. (1979) Biochim. Biophys, Acta 572, 64–76) suggest that this enzyme may be rate-regulatory in the de novo biosynthesis of phosphatidylcholine.Estrogen administration also resulted in a 26% increase in the activity of pulmonary lysolecithin acyltransferase, an enzyme involved in the synthesis of disaturated, surface-active phosphatidylcholine. Lung choline kinase was slightly decreased following estrogen treatment bu ethanolaminephosphate cytidylyltransferase, cholinephosphotransferase, phosphatidate phosphatase and lysolecithin : lysolecithin acyltransferase were unaffected.     

Effects of 17β-estradiol replacement on the apoptotic effects caused by ovariectomy in the rat hippocampus

AimsThe aim of the present study was to investigate the effects of different periods of ovariectomy and 17β-estradiol replacement on apoptotic cell death and expression of members of the Bcl-2 family in the rat hippocampus.Main methodsHippocampi were obtained from rats in proestrus, ovariectomized (15 days, 21 days and 36 days), ovariectomized for 15 days and then treated with 17β-estradiol for 7 or 21 days, and rats ovariectomized and immediately treated with 17β-estradiol for 21 days.The expression of Bcl-2 and Bax and the number of apoptotic cells were determined.Key findingsOvariectomy decreased Bcl-2 expression and increased Bax expression and the number of apoptotic cells. Replacement with 17β-estradiol (21 days) throughout the post-ovariectomy period reduced the number of apoptotic cells to the control levels, and prevented the effects of ovariectomy on Bax expression, but only partially restored the Bcl-2 expression. After 15 days of ovariectomy, the replacement with 17β-estradiol for 21 days, but not for 7 days, restored the Bcl-2 and Bax expression and the percentage of apoptotic cells to the levels found in the proestrus control.SignificanceThe present results show that a physiological concentration of 17β-estradiol may help maintain long-term neuronal viability by regulating the expression of members of the Bcl-2 family. Even after a period of hormonal deprivation, treatment with 17β-estradiol is able to restore the expression of Bax and Bcl-2 to control levels, but the duration of the treatment is a key factor to obtain the desired effect. These data provide new understanding into the mechanisms contributing to the neuroprotective action of estrogen.     

Cellulose degradation by Sulfolobus solfataricus requires a cell-anchored endo-β-1-4-glucanase

A sequence encoding a putative extracellular endoglucanase (sso1354) was identified in the complete genome sequence of Sulfolobus solfataricus. The encoded protein shares signature motifs with members of glycoside hydrolases family 12. After an unsuccessful first attempt at cloning the full-length coding sequences in Escherichia coli, an active but unstable recombinant enzyme lacking a 27-residue N-terminal sequence was generated. This 27-amino-acid sequence shows significant similarity with corresponding regions in the sugar binding proteins AraS, GlcS, and TreS of S. solfataricus that are responsible for anchoring them to the plasma membrane. A strategy based on an effective vector/host genetic system for Sulfolobus and on expression control by the promoter of the S. solfataricus gene which encodes the glucose binding protein allowed production of the enzyme in sufficient quantities for study. In fact, the enzyme expressed in S. solfataricus was stable and highly thermoresistant and showed optimal activity at low pH and high temperature. The protein was detected mainly in the plasma membrane fraction, confirming the structural similarity to the sugar binding proteins. The results of the protein expression in the two different hosts showed that the SSO1354 enzyme is endowed with an endo-β-1-4-glucanase activity and specifically hydrolyzes cellulose. Moreover, it also shows significant but distinguishable specificity toward several other sugar polymers, such as lichenan, xylan, debranched arabinan, pachyman, and curdlan.     

Puerarin suppresses invasion and vascularization of endometriosis tissue stimulated by 17β-estradiol

Background

Puerarin, a phytoestrogen with a weak estrogenic effect, binds to estrogen receptors, thereby competing with 17β-estradiol (E2) and producing an anti-estrogenic effect. This study was to investigate whether puerarin could suppress the invasion and vascularization of E2-stimulated endometriotic tissue.

Methodology/Principal Findings

The endometriotic stromal cells (ESCs) were successfully established and their invasive ability under different treatments was assessed through a Transwell Assay. Simultaneously, matrix metallopeptidase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) were detected by western blotting. Vascularization of endometriotic tissues was observed by chicken chorioallantoic membrane (CAM) assay. The staining of MMP-9, intercellular adhesion molecule 1 (ICAM-1), TIMP-1, and vascular endothelial growth factor (VEGF) in grafted endometriotic tissues was examined using immunohistochemistry analysis. The purity of ESCs in isolated cells was >95%, as determined by the fluoroimmunoassay of vimentin. E2 (10⁻⁸ mol/L) promoted the invasiveness of ESCs by increasing MMP-9 accumulation and decreasing TIMP-1 accumulation. Interestingly, puerarin (10⁻⁹ mol/L) significantly reversed these effects (P<0.01). The CAM assay indicated that puerarin (10⁻⁹ mol/L) also inhibited the angiopoiesis of endometriotic tissue stimulated by the E2 (10⁻⁸ mol/L) treatment (P<0.05). Accordingly, immunohistochemistry showed that the accumulation of MMP-9, ICAM-1, and VEGF was reduced whereas that of TIMP-1 increased in the combination treatment group compared with the E2 treatment group.

Conclusions/Significance

This study demonstrated that puerarin could suppress the tissue invasion by ESCs and the vascularization of ectopic endometrial tissues stimulated by E2, suggesting that puerarin may be a potential drug for the treatment of endometriosis.     

Partial neuroprotection by 17-β-estradiol in neonatal γ-irradiated rat cerebellum

Acute and long-term complications can occur in patients receiving radiation therapy. It has been suggested that cytoprotection might decrease the incidence and severity of therapy-related toxicity in these patients. Developing cerebellum is highly radiosensitive and for that reason it is a useful structure to test potential neuroprotective substances to prevent radiation induced abnormalities. Recent studies have shown that estrogen can rapidly modulate intracellular signalling pathways involved in cell survival. Thus, it has been demonstrated that estrogens mediate neuroprotection by promoting growth, cell survival and by preventing axonal pruning. The aim of this work was to evaluate the effect of the treatment with 17-β-estradiol on the motor, structural and biochemical changes induced by neonatal ionizing radiation exposure, and to investigate the participation of nitric oxide and protein kinase C, two important intracellular messengers involved in neuronal activity. Our results show that perinatal chronic 17-β-estradiol treatment partially protects against radiation-induced cerebellar disorganization and motor abnormalities. PKC and NOS activities could be implicated in its neuroprotective mechanisms. These data provide new evidence about the mechanisms underlying estrogen neuroprotection, which could have therapeutic relevance for patients treated with radiotherapy.     

Effect of 17β-estradiol on the human osteosarcoma cell line MG-63

We present evidence that 17β-estradiol (17β-E₂) regulates 1,25(OH)₂D₃-induced alkaline phosphatase synthesis and osteocalcin secretion by the human osteosarcoma cell line MG-63. When cells were pre-treated with 17β-E₂ for 48 h prior to treatment with 1,25(OH)₂D₃ (50 nM) for another 48 h, alkaline phosphatase activity increased by 40% (P < 0.025) with 2 nM 17β-E₂ and plateaued at levels of 20 and 200 nM 17β-E₂. Under the same experimental conditions, osteocalcin secretion was enhanced by 37% (P < 0.005) with 2 nM E₂. However, 17β-E₂ had no effect on basal alkaline phosphatase or on osteocalcin secretion. Moreover, simultaneous addition of 17β-E₂ and 1,25(OH)₂D₃ to cells did not result in any additional effect over l,25(OH)₂D₃ treatment alone. Tamoxifen (10 nM) inhibited 17β-E₂-induced activities in l,25(OH)₂D₃-treated cells while not affecting control cells. Dexamethasone pretreat-ment (100 nM, 48 h) also stimulated alkaline phosphatase activity in MG-63 cells. Moreover, dexamethasone pretreatment followed by treatment with 17β-E₂ and l,25(OH)₂D₃ gave an additive effect for alkaline phosphatase activity. 17α-Estradiol (17α-E₂), a less active form of estrogen, failed to modify, at low concentrations, control or l,25(OH)₂D₃-induced alkaline phosphatase synthesis and osteocalcin secretion. In fact, a 100–1000-fold higher concentration of 17α-E₂ was necessary to reproduce the effects of 17β-E₂ on osteocalcin secretion. The addition of insulin-like growth factor I (IGF-I) for 24 h (1–50 ng/ml) to MG-63 cells did not modify 1,25(OH)₂D₃-induced osteocalcin release from these cells. However, longer incubations with 50 ng/ml IGF-I did reproduce some of the effects observed with 17β-E₂. Thus, the effects of 17β-E₂ are probably not related to IGF-I production in MG-63 cells since under these conditions the addition of IGF-I alone should have produced a response at shorter incubation times and in the presence of lower concentrations of IGF-I. Since 17β-E₂ pretreatment was necessary to observe any effects on l,25(OH)₂D₃-induced activities, we hypothesized that 17β-E₂ regulated 1,25(OH)₂D₃ receptors in MG-63 cells. When cells were treated with 100 nM 17β-E₂ for 48 h, the binding affinity was unchanged: 37.3 ± 1.9 versus 35.1 ± 0.4 pM for cells whether treated or not with l7β-E₂, respectively. In contrast, a significant increase in binding capacity (B_max) was noted (15 ± 3.5%; P < 0.025). These results suggest that the estrogen analogue 17β-E₂ induces the differentiation of MG-63 cells into a more osteoblastic-like phenotype while 17α-E₂ is without physiological effect. They also suggest that estrogens may regulate bone remodeling by modulating hormonal-induction of proteins involved in bone mineralization. This effect is indirect since it does not modify basal activities, but involves a regulation of 1,25(OH)₂D₃ receptor levels in these MG-63 cells.     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

The mitogenic effect of testosterone and 17β-estradiol on airway smooth muscle cells

Airway disease distribution and/or severity exhibit sex differences suggesting that sex hormones are involved in the respiratory system physiology and pathophysiology. The implication of airway smooth muscle cells (ASMCs) in the physiology of the airways and the pathogenetic mechanism of airway remodeling is of great interest. Therefore, we studied the effect of testosterone and 17β-estradiol on ASMC proliferation and the mechanisms involved.Cell proliferation was estimated using the methyl-[³H]thymidine incorporation and Cell Titer 96^® AQueous One Solution Assay methods. ASMC isolated from adult male or female rabbit trachea were incubated with testosterone (1 pM-1 μM) or 17β-estradiol (1 pM-1 μM), in the presence or absence of the androgen receptor antagonist flutamide (10 nM) or estrogen receptor antagonist ICI182780 (10 nM), as well as of the PI3K inhibitors LY294002 (20 μM) or wortmannin (1 μM), or the MAPK inhibitors PD98059 (100 μM) or U0126 (1 μM).After 24 h of incubation, testosterone and 17β-estradiol increased methyl-[³H]thymidine incorporation and cell number, in ASMC isolated from male or female animals. The induction of ASMC proliferation by testosterone or 17β-estradiol was inhibited by flutamide or ICI182780 respectively, as well as by LY294002, wortmannin, PD98059 or U0126.In conclusion, testosterone and 17β-estradiol have a mitogenic effect on ASMC, which is receptor-mediated and involves the MAPK and PI3K signaling pathways. Moreover, their effect is the same for ASMC from male and female animals. It is possible that gender-related differences in ASMC remodeling, may be influenced by the different patterns of sex steroid hormone secretion in males and females.     

The study of core-shell molecularly imprinted polymers of 17β-estradiol on the surface of silica nanoparticles

In this paper, highly selective core-shell molecularly imprinted polymers of 17β-estradiol on the surface of silica nanoparticles (SiO(2)@E2-MIPs) were prepared. The SiO(2)@E2-MIPs were characterized by Fourier transform infrared spectrometer (FT-IR), transmission electron microscope (TEM), dynamic adsorption and static adsorption tests. The sorption capacity of the SiO(2)@E2-MIPs were nearly 5 times that of the non-imprinted polymers (NIPs), and it only took 25 min to achieve the sorption equilibrium. It indicated that the SiO(2)@E2-MIPs exhibited a high selectivity, large adsorption capacity and fast kinetics. When the SiO(2)@E2-MIPs were used as dispersive solid-phase extraction (dSPE) absorbents to selectively enrich and determine estrogens in duck feed, the average recoveries of E2 and estriol (E3) were higher than 96.74% and 72.07%, respectively, and the relative standard deviations (RSD) of E2 and E3 were less than 1.61% and 3.28%, respectively. The study provides an effective method for the separation and enrichment of estrogens in the complex matrix samples by the SiO(2)@E2-MIPs.     

Effects of 17β-estradiol and bisphenol A on the formation of reproductive organs in planarians

Planarians have a remarkable capacity for regeneration after ablation, and they reproduce asexually by fission. However, some planarians can also reproduce and maintain their sexual organs. During the regenerative process, their existing sexual organs degenerate and new ones develop. However, little is known about hormonal regulation during the development of reproductive organs in planarians. In this study, we investigated the effects of 17β-estradiol (a steroid) and bisphenol A (an endocrine disrupter) on the formation of sexual organs in the hermaphroditic planarian Dugesia ryukyuensis. Under control conditions, all worm tissues regenerated into sexual planarians with sexual organs within 4 weeks after ablation. However, in the presence of bisphenol A or 17β-estradiol, although they apparently regenerated into sexual planarians, the yolk glands, which are one of the female sexual organs, failed to regenerate even 7 weeks after ablation. These data suggest that planarians have a steroid hormone system, which plays a key role in the formation and maturation of sexual organs.