Dipartimento di Biologia, Ecologia e Scienze della Terra

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<> ruolo del GPER nelle arterie uterine e mesenteriche in gravidanza
(2015-12-16) Tropea, Teresa; Canonaco, Marcello; Mandalà, Maurizio
Low uteroplacental blood flow has been implicated as a cause of pregnancy hypertension and intrauterine fetal growth retardation. A successful pregnancy outcome requires a sufficient uteroplacental blood flow, which increases of many fold compared to the non pregnant levels. This significant increase is achieved in part by vasodilation of the uterine vasculature because pregnancy induces a physiological remodelling of the maternal uterine circulation. Estrogen may contribute to this effect although the mechanisms involved remain unclear. It is known that estrogen receptor-α and estrogen receptor-β in the uterine vascular endothelium are both functionally implicated in the regulation of the uterine blood flow but so far nothing is known about the novel G-protein coupled estrogen receptor (GPER) in the uterine vasculature. GPER has been identified in many vascular bed exerting its potential vasorelaxing effect. The aim of this study was to investigate the function of GPER in the regulation of the uterine vascular tone during pregnancy. Experiments were carried out on mesenteric arterioles and uterine radial arteries isolated from both non-pregnant (NP) and age-matched pregnant (P) Sprague Dawley rats to compare reproductive and systemic vasculature effect. Arterial segments were pressurized to 50 mmHg in the pressure myography, preconstricted of 40% with phenylephrine and then incubated with incremental doses (10-12-10-6 M) of the specific GPER agonist, G-1. A dose-response curve was obtained for G-1 in the 1 pM – 1 μM range with a maximum vasodilation of uterine arteries of 97,8 ± 2,5% in P vs 66,5 ± 3,7% in NP, p<0.001. G-1 vasorelaxing effect was similar in pregnant (45,5 ± 6,1%) and non-pregnant (53,6 ± 2,3%) mesenteric arteries. Pregnancy induced a significantly higher G-1 vasodilation only in uterine artery and its protein expression was also increased. G-1 effect was significantly reduced by the GPER antagonist, G-15. The NOS inhibitors, L-NAME+L-NNA or endothelium removal reduced the G-1 induced relaxation of uterine artery, suggesting an endothelium-dependent mechanism, involving cGMP pathway but not BKca channels. Immunohistochemistry revealed the GPER expression in the main uterine artery of both eNOS-/- and wild type pregnant mice. This receptor was functionally activated in a dose response manner with a significant less effect in knock out mice, showing the relevance of the NO in GPER pathway. GPER is also present in human chorionic plate arteries and its activation induces a slight vasodilation. These results demonstrate for the first time that GPER may have a role in regulating vascular tone, placental perfusion and normal fetal development, suggesting a potential therapeutic target in pregnant diseases.
Il Recettore Estrogenico alfa e IRS-1 (Insulin Receptor Substate-1) come target dell’azione del Recettore Estrogenico beta
(2013-10-21) Palma,Maria Grazia; Canonaco,Marcello; Panno,Maria Luisa
Estrogen signalling through ERα plays a central role in many diseases such as breast and endometrial cancer, osteoporosis and cardiovascular disease. In 1996 it has been discovered a second estrogen receptor in rat prostate which has been named ERβ. Both receptors are co-expressed in many tissues and, surprisingly, ERβ was shown to sometimes mediate opposite effects to ERα. In the human, ERα is encoded at 6q25.1 and ERβ at 14q23.2, they have a high degree of homology in the DNA-binding domain (96%), but differ in the ligand-binding domain (53%). Estradiol is a natural ligand of ERα, while DPN (diarylpropionitrile) is a selective agonist of ERβ. The activation of ERα through estradiol increases cell proliferation and positively influences breast tumorogenesis and cell invasion, on the contrary, ERβ, also in the absence of the ligand, appears to oppose the action of ERα, suggesting that the estrogen receptor β could play an important role in carcinogenesis. To better define the action of ERβ, we used an estrogen receptor α positive MCF-7 breast cancer cell line overexpressing, through transient transfection, ERβ. In these cells the ectopic expression of ERβ induces a decrease of cell proliferation and a block of cell cycle in G2/M phase. Taking into account these data, we focused the attention on two proteins involved in the transduction of mitotic signallings: IRS-1 and ERα. The first one, is the major substrate of the tyrosine kinase activity of IR (Insulin Receptor) and IGF-R (Insulin- Like Growth Factor Receptors). This protein exerts a key role in the functional synergism between the growth factor/insulin and estradiol in the mammary gland. In fact, ERα is important to sustain IRS-1 protein expression. and to transactivate IRS-1 gene. On the basis of this finding, we wanted to study the possible influence of ERβ on IRS-1 by monitoring mRNA and protein contents in MCF-7 breast cancer cells. In this context, our results show that the ectopic expression of ERβ is able to negatively regulate the transactivation of IRS-1 promoter thus leading to a decrease of IRS-1 signalling. Further investigations indicate that ERβ appears to act as a dominant2 negative regulator of ERα expression in these cells. Indeed, ERβ represses the transcriptional activity of ERα gene promoter . To better investigate the molecular mechanism underlying the negative regulation of ERβ on ERα gene, we performed EMSA (Electrophoretic Mobility Shift Assay) experiments, by using ERE labelled probes present in ERα promoter. Our results show a decreased binding of the nuclear extracts, obtained from MCF-7 cells overexpressing ERβ, to the ERE labelled nucleotide. In addition, chromatin immunoprecipitation analysis reveals in MCF-7 cells overexpressing ERβ, the specific recruitment of the nuclear corepressor NCoR to these ERE sequences of ERα promoter. The recruitment of this corepressor weakens the binding between ERβ and ERE sequence, thus producing a repression of ERα transcriptional activity. This study is the first evidence to demonstrate that ERβ acts as a dominant negative regulator of ERα gene in breast cancer cells. Our data, in agreement with other findings, tend to better justify how Estrogen Receptor β overexpression in many tissues reduces ERα-regulated gene transcription and, in this way, inhibits the proliferative responses in breast cancer cells.

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