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

SCERBO MARÍA J.; MIR FRANCO R.; GUTIÉRREZ SILVINA; GOROSITO SILVANA; TORRES ALICIA I; CAMBIASSO MARÍA J.

Torino

Congreso; 6th INTERNATIONAL MEETING STEROIDS AND NERVOUS SYSTEM; 2011

17-β 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 age 16 (E16) by a mechanism that does not involve the classical nuclear pathway of estrogen receptor (ER). Instead this axogenic effect depends on a membrane-initiated mechanism involving participation of a Ca2+ dependent PKC, the MEK-ERK signaling and the transcription factor CREB (Cambiasso, 2001; Gorosito and Cambiasso, 2008). We have also found a membrane truncated ERα as a biotinylated cell-surface protein. The exact mechanism by which the ERα is translocated from the citosol from the membrane is not entirely clear. E2 also increases the expression of trophic factor receptors such as IGF-1R and TrkB (Cambiasso et al 2000; Brito VI et al 2004)and blocking the expression of TrKB with an antisense oligonucleotide prevents the axonal growth induced by E2. These evidences indicate a convergence of the signaling pathways for E2 and neurotrophic factors at the membrane. Many growth factor receptors on the cell membrane activate MAPK through a shc-mediated pathway. The adapter protein Shc (Src homology and collagen homology) 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 ERα 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 ERα in the cell-surface of intact hypothalamic neurons. Determine if the ERα is in 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-microscopy: Intact male hypothalamic neurons treated with E2 were incubated with anti- ERα (MC-20, 1/50, Santa Cruz Biotechnology) for 1 hr. The immunoreactive sites were labeled with protein A-colloidal gold complex and processed for electron microscopy. FACS: Male hypothalamic neurons treated with or without E2 were incubated with 1 ug/106 cells of anti- ERα (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. Immunoprecipitation (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- ERα 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 few endocytic vesicles confirming that ERα is expressed on the cell-surface. The results obtained by FACS analysis also reinforced the previous observation, since it revealed that when cells were treated with E2 the amount of cells expressing ERα on the cell surface raised from 32 % (cells incubated with CM alone) to 45 % of the total population. IP experiments showed that the full length and a truncated isoform of the ERα is associated to Shc in control conditions, but in the presence of E2 the amount of both isoforms of the ERα co-immpunoprecipitated with Shc increases two folds, as well as the quantity of Shc. Conclusion: Together our results confirm that E2 promotes an increase in the expression of ERα on the cell surface of hypothalamic neurons and that it is directly bound to the adaptor molecule Shc, suggesting a possible linkage of rapid E2 action to MAPK activation. Reference List 1. Cambiasso MJ, Colombo JA, Carrer HF (2000) Differential effect of oestradiol and astrogliaconditioned media on the growth of hypothalamic neurons from male and female rat brains. Eur J Neurosci 12(7):2291-8. 2. Cambiasso MJ, Carrer HF (20001) Nongenomic mechanism mediates estradiol stimulation of axon growth in male rat hypothalamic neurons in vitro. J Neurosci Res 66(3):475-81. 3. Brito VI, Carrer HF, Cambiasso MJ (2004) Inhibition of tyrosine kinase receptor type B synthesis blocks axogenic effect of estradiol on rat hypothalamic neurones in vitro. Eur J Neurosci 20(2):331-7. 4. 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