RESUMEN
Experimental and epidemiological data suggest a neuroprotective role for estrogen (E(2)). We have recently shown that, in PC12 cells, non-permeable estradiol conjugated to bovine serum albumin (BSA) prevent serum-deprivation induced apoptosis through activation of specific membrane estrogen receptors (mER). In the present study, we explored in detail the early signaling events involved in this anti-apoptotic action, downstream to activation of mER. Our findings suggest that mER is associated to G-proteins, and its activation with non-permeable E(2)-BSA results in the activation of the following downstream pro-survival kinases pathways: (1) the PKB/Akt pathway, (2) the Src-->MEK-->ERK kinases and finally (3) the MAPK-->ERK kinases. Activation of these pro-survival signals leads to CREB phosphorylation and NFkappaB nuclear translocation, two transcription factors controlling the expression of anti-apoptotic Bcl-2 proteins. These data suggest that major pro-survival kinases are involved in the mER-mediated anti-apoptotic effects of estrogen. This is further supported by experiments with specific kinases inhibitors, which partially but significantly reversed the mER-mediated anti-apoptotic effect of E(2)-BSA. Our findings suggest that estrogen act via mER as potent cytoprotective factors, downstream activating pro-survival kinases, assuring thus an efficient and multipotent activation of the anti-apoptotic machinery.
Asunto(s)
Membrana Celular/metabolismo , Supervivencia Celular/fisiología , Quinasas Quinasa Quinasa PAM/metabolismo , Receptores de Estrógenos/metabolismo , Activación Transcripcional/efectos de los fármacos , Familia-src Quinasas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Apoptosis/efectos de los fármacos , Bovinos , Membrana Celular/efectos de los fármacos , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Estrógenos/metabolismo , Estrógenos/farmacología , Proteínas de Unión al GTP/metabolismo , Mitocondrias/metabolismo , Modelos Biológicos , Óxido Nítrico Sintasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Albúmina Sérica Bovina/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Quinasa de Factor Nuclear kappa BRESUMEN
The neuroprotective role of estrogen (E2) is supported by a multitude of experimental and epidemiological data, although its mode of action is not fully understood. The present work was conducted to study the underlying mechanisms of its neuroprotective action, using the rat cell line PC12, an established model for neuronal cell apoptosis and survival. Our results show that E2 (but not androgens or progestins) prevent growth inhibition and apoptosis of PC12 cells, induced by serum deprivation. Several mechanisms of action were investigated: 1) intracellular estrogen receptors (ERs) have been identified but do not appear to mediate the protective effect of E2. 2) The antioxidant properties of E2 cannot explain their protective actions at the concentrations used (10(-12)-10(-6) M). 3) Finally, membrane sites for E2 have been identified, and the underlying initial signaling cascade (2-30 min after E2) has been tested, showing Ca(2+) mobilization-->PI3K activation-->Akt phosporylation-->NOS activation. Inhibition of PI3K or NOS completely reversed the anti-apoptotic effect of E2. These results suggest a new mechanism of neuroprotective action of estrogen.
Asunto(s)
Membrana Celular/metabolismo , Estrógenos/farmacología , Fármacos Neuroprotectores/farmacología , Óxido Nítrico Sintasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Activación Enzimática/efectos de los fármacos , Células PC12 , Unión Proteica , Proteínas Proto-Oncogénicas c-akt , Ratas , Receptores de Estrógenos/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
The neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester DHEA sulfate (DHEAS), and allopregnanolone (Allo), produced by the CNS and the adrenals, appear to exert a protective effect in hippocampal and cortical neuron ischemia- and excitotoxicity-induced injury. We hypothesized that they may also play a protective role on the adrenal medulla, an important part of the sympathetic nervous system, and the tissue adjacent to their primary site of production. DHEA, DHEAS, and Allo protected rat chromaffin cells and the rat pheochromocytoma PC12 cell line, an established model for the study of adrenomedullary cell apoptosis and survival, against serum deprivation-induced apoptosis. Their effects were time- and dose-dependent, with EC50 1.8, 1.1, and 1.5 nM, respectively. The antiapoptotic effect of DHEA DHEAS and Allo was compared to that of a long list of structurally related compounds and was found to be structure-specific, confined mainly to conformation 3beta-OH-Delta5 for androstenes and 3alpha-OH for pregnanes. Indeed, 3-keto, Delta4, or C7 hydroxylated androstenes and 3beta pregnanes were ineffective. The prosurvival effect of DHEA(S) and Allo was N-methyl-D-aspartate-, GABAA-, sigma1-, or estrogen receptor-independent. It involved the antiapoptotic Bcl-2 proteins, their role being sine qua non for their action because Bcl-2 antisense oligonucleotides reversed their effects. Finally, DHEA(S) and Allo activated cAMP response element-binding protein and NF-kappaB, upstream effectors of antiapoptotic Bcl-2 protein expression. They also activated the antiapoptotic kinase PKCalpha/beta, a posttranslational activator of Bcl-2 protein. Our findings suggest that decline of DHEA(S) and Allo during aging or stress may leave the adrenal medulla unprotected against proapoptotic challenges.