Estradiol induces membrane localization of the estrogen receptor and association with Shc in neurons

SCERBO, M.J.; GOROSITO, S.V.; GUTIERREZ, S.; MIR, F.; TORRES, A.; CAMBIASSO, M.J.

Torino

Congreso; VI Meeting Steroids and Nervous System; 2011

Introduction: 17-b estradiol (E2) has important physiological effects in central nervous system; it participates in neuronal plasticity, synaptic organization and neuritogenesis. Previous studies from our laboratory have demonstrated that E2 induces axonal growth on male hypothalamic neurons of embryonic stage 16 (E16) by a mechanism that does not involve the classical intracellular pathway of nuclear estrogen receptor alfa (ERa). Instead this axogenic effect depends on a membrane-initiated mechanism involving increment of intracellular levels of Ca2+, activation of PKC and MAPK as MEK and ERK and the phosphorilation of transcription factors as CREB (Cambiasso, 2001; Gorosito and Cambiasso, 2008). We have also found that a truncated form of the ERá can be detected from the cell exterior as a biotinylated cell-surface protein. Also, the presence of E2 in culture media of E16 hypothalamic neurons increases the levels of insulin-like growth factor 1 receptor (IGF-1R) and tyrosine kinase receptor type B (TrkB) (Cambiasso et al 2000; Brito VI et al 2004). Blocking the expression of TrKB with antisense oligonucleotids revert the axonal growth induced by E2. These evidences indicate a convergence of the signaling pathways for E2 and neurotrophins. The exact mechanism by which the ERa is translocated from the citosol from the membrane is not entirely clear. Many growth factor receptors on the cell membrane, such as nerve growth factor, and IGF, activate MAPK through a Shc-mediated pathway. It has been reported that adapter proteins as Shc (Src homology and collagen homology) are involved in this process in breast cancer cells (Song RX et al 2004). The adapter protein Shc has no intrinsic kinase domain and transduces signals dependent on the association with membrane receptors. In this scenario, the adaptor protein Shc could serve as a traslocator by binding to ERa and then carrying this receptor to Shc binding sites of trophic factor receptors located at the membrane as TrKB and/or IGF-1R. Objetives: ·        Corroborate the presence of ERá in the cell-surface of intact hypothalamic neurons by flow citometry and immuno-electron-microscopy in order to study the membrane distribution. ·        Determine if the ERá might mediate the effect of axonal growth induced by E2 through the direct association with the Shc adapter protein. Methods: Cell culture: E16 male hypothalamic neurons were culture with or without 10 nM of E2 in astroglia-conditioned medium for 48 hr. Immuno-electro-miscroscopy: Intact male hypothalamic neurons treated with E2 were incubated with anti-ERa (MC-20, Santa Cruz) 1/50 dilution for 1 hr. After that the immunoreactives sites were labeled with protein A-colloidal gold complex and processed for electron microscopy. FACS: Male hypothalamic neurons treated with E2 were incubated with 1 ug/106 cells of anti-ERa (HC-20, Santa Cruz) for 1 hr, then washed and incubated with an anti-rabit IgG conjugated to Alexa Fluor 488 (molecular probes) secondary antibody for 1 hr. Finally 10000 events were analyzed in a flow cytometer. All the steps of this technique where done at 4 °C to avoid receptor internalization. Immuno precipitation (IP) and western Blot: total homogenates (TH) of male hypothalamic neurons treated or not with E2, were incubated with 1 ug/500 ug of protein of anti-Shc (PG-979 Santa Cruz) and then immunoprecipitated with A/G sepharosa (Santa Cruz). HT and IP samples were subjected to SDS-PAGE electrophoresis and transferred to nitrocellulose membranes. Western blot analysis where performed with anti-ERa and anti-Shc and analyzed by infrared techniques. Results: Immuno-electron-microscopy method showed gold-particles adhered on the surface of the plasma membrane and attached to cytoplasmic vesicles membranes confirming that ERá is expressed on the cell-surface. The results obtained by FACS analysis also reinforced the previous observation, since it revealed that when cells are treated with E2 the amount of cells expressing ERa on the cell surface raiced from a 32 % (cells incubated with CM alone) to a 45 % of the total population. IP experiments showed that the full length and a truncated isoform of the ERá is associated to Shc in control conditions, but in the presence of E2 the amount of both isoforms of the ERá co-immpunoprecipitated with Shc increases two folds, as well as the quantity of Shc. These results go in the same way than those obtained by FACS analysis. Conclusion: Together our results confirm that E2 promotes an increase in the expression of ERa on the cell surface of hypothalamic neurons and that it is directly attached to the adaptor molecule Shc, suggesting a possible linkage of rapid E2 action to MAPK activation. Bibliography Cambiasso MJ, Colombo JA, Carrer HF. Differential effect of oestradiol and astroglia-conditioned media on the growth of hypothalamic neurons from male and female rat brains. Eur J Neurosci. 2000;12(7):2291-8. Cambiasso MJ, Carrer HF. Nongenomic mechanism mediates estradiol stimulation of axon growth in male rat hypothalamic neurons in vitro. J Neurosci Res. 2001 Nov 1;66(3):475-81. Brito VI, Carrer HF, Cambiasso MJ. Inhibition of tyrosine kinase receptor type B synthesis blocks axogenic effect of estradiol on rat hypothalamic neurones in vitro. Eur J Neurosci. 2004;20(2):331-7. Gorosito SV, Cambiasso MJ. 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