17β-estradiol promotes extracellular vesicle release and selective miRNA loading in ERα-positive breast cancer

The causes and consequences of abnormal biogenesis of extracellular vesicles (EVs) are not yet well understood in malignancies, including in breast cancers (BCs). Given the hormonal signaling dependence of estrogen receptor–positive (ER+) BC, we hypothesized that 17β-estradiol (estrogen) might influence EV production and microRNA (miRNA) loading. We report that physiological doses of 17β-estradiol promote EV secretion specifically from ER+ BC cells via inhibition of miR-149-5p, hindering its regulatory activity on SP1, a transcription factor that regulates the EV biogenesis factor nSMase2. Additionally, miR-149-5p downregulation promotes hnRNPA1 expression, responsible for the loading of let-7’s miRNAs into EVs. In multiple patient cohorts, we observed increased levels of let-7a-5p and let-7d-5p in EVs derived from the blood of premenopausal ER+ BC patients, and elevated EV levels in patients with high BMI, both conditions associated with higher levels of 17β-estradiol. In brief, we identified a unique estrogen-driven mechanism by which ER+ BC cells eliminate tumor suppressor miRNAs in EVs, with effects on modulating tumor-associated macrophages in the microenvironment

Implicación de las proteínas G y de la PKA en la regulación local de la vía de Sonic Hedgehog en el cilio primario

Tesis doctoral presentada para optar al grado de Doctora por la Universidad de Barcelona, Programa de Doctorado de Bilogía y Patología Celulares (Departamento de Biología Celular, Inmunología y neurociencias, Facultad de Medicina).-- Tesis realizada en el Departamento de Muerte y Proliferación Celular del IIBB-CSIC-IDIBAPS.El cerebelo es el órgano encargado de integrar las vías sensitivas y las motoras del sistema nervioso controlando, por ejemplo, los movimientos involuntarios del cuerpo. Su desarrollo comienza en la etapa embrionaria, en la que tiene lugar la formación del órgano y de sus principales capas. Conforme avanza el desarrollo, tiene lugar un crecimiento del órgano debido a una elevada proliferación de los precursores de las células granulares (PNGCs), localizados en su capa más externa. Su desarrollo finaliza en la etapa postnatal, en la que el cerebelo aumenta aun más su tamaño debido al mantenimiento de esta elevada actividad mitogénica. Alcanza su madurez cuando los PNGCs se diferencian y migran hacia la capa más interna. El morfógeno Sonic Hedgehog (Shh) es el responsable de la proliferación de los PNGCs. Esta proteína activa al complejo de receptores constituído por Patched (Ptch) y Smoothened (Smo), que desencadenaran una cascada de señalización dando lugar a la división celular. La actividad de Shh puede ser regulada negativamente por la proteína quinasa A dependiente de AMP cíclico (PKA). En su estado basal, la subunidad catalítica de PKA (PKA-C) se encuentra inhibida por su asociación con la subunidad reguladora (R-PKA). En la presente tesis se demuestra que en presencia de Shh la subunidad PKA-C inactiva se localiza en la base del cilio de PNGCs, estructura celular necesaria para que la señalización de Shh tenga lugar y donde se acumulan varias moléculas de la vía como Smo y Ptch.Este trabajo ha sido financiado por proyectos del Ministerio de Educación y Ciencia (MCI BFU2005-01599 y MCI BFU2008-024024BFI) y ha sido realizado mediante el soporte económico de una beca predoctoral del Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS).Peer Reviewe

Implicación de proteinas G y de PKA en la regulación local de la via de Sonic Hedgehog en el cilio primario

[spa] El cerebelo es el órgano encargado de integrar las vías sensitivas y las motoras del sistema nervioso controlando, por ejemplo, los movimientos involuntarios del cuerpo. Su desarrollo comienza en la etapa embrionaria, en la que tiene lugar la formación del órgano y de sus principales capas. Conforme avanza el desarrollo, tiene lugar un crecimiento del órgano debido a una elevada proliferación de los precursores de las células granulares (PNGCs), localizados en su capa más externa. Su desarrollo finaliza en la etapa postnatal, en la que el cerebelo aumenta aun más su tamaño debido al mantenimiento de esta elevada actividad mitogénica. Alcanza su madurez cuando los PNGCs se diferencian y migran hacia la capa más interna. El morfógeno Sonic Hedgehog (Shh) es el responsable de la proliferación de los PNGCs. Esta proteína activa al complejo de receptores constituído por Patched (Ptch) y Smoothened (Smo), que desencadenaran una cascada de señalización dando lugar a la división celular. La actividad de Shh puede ser regulada negativamente por la proteína quinasa A dependiente de AMP cíclico (PKA). En su estado basal, la subunidad catalítica de PKA (PKA-C) se encuentra inhibida por su asociación con la subunidad reguladora (R-PKA). En la presente tesis se demuestra que en presencia de Shh la subunidad PKA-C inactiva se localiza en la base del cilio de PNGCs, estructura celular necesaria para que la señalización de Shh tenga lugar y donde se acumulan varias moléculas de la vía como Smo y Ptch. Cuando los PNGCs son deprivados de Shh tiene lugar una activación y una parcial dispersión de PKA-C de la base del cilio. Mostramos que la subunidad reguladora RII (PKA-RII) de PKA es la responsable del anclaje de la holoenzima a través de su unión a las proteínas de anclaje de PKA (AKAPs). La disrupción de la interacción entre PKA y AKAPs inhibe la actividad de Shh bloqueando la proliferación de los PNGCs. Estos resultados, por lo tanto, demuestran que el pool de PKA localizado en la base del cilio juega un papel esencial en la integración de la transducción de la señal de Shh. En la segunda parte de la presente tesis estudiamos la activación de proteínas G heterotriméricas mediante Smo. Aunque en su secuencia Smo sugiere que pertenece a la familia de los receptores acoplados a proteína G (GPCR), únicamente en Drosophila melanogaster se demostró que la proteína Gαi es necesaria para la actividad de Hedgehog (Hh), mientras que las evidencias de la implicación de las proteínas G como efectoras de la vía de Shh de los vertebrados no son muy claras. En este trabajo demostramos que la inducción de la proliferación de PNGCs se potencia por la expresión de formas activas de proteínas de la clase Gαi/o (Gαi1, Gαi2, Gαi3 y Gαo) pero no por las de la clase Gα12 de proteínas G. Además, observamos que los miembros de la familia de Gαi poseen un patrón de expresión específico en el cerebelo en desarrollo: únicamente Gαi2 y Gαi3 se encuentran altamente expresados en la parte más externa de la capa granular externa, donde tiene lugar la proliferación de los PNGCs. En concordancia con este resultado observamos que la proliferación de los PNGCs se reduce de manera significativa mediante la eliminación de Gαi2 y Gαi3 pero no cuando silenciamos a otros miembros de la familia de Gαi/o. Finalmente, los resultados de la presente tesis demuestran que las subunidades Gαi2 y Gαi3 se localizan en el cilio primario cuando son sobre-expresados en cultivos de PCGCs. Por lo tanto, los efectos proliferativos de Shh en estas células están mediados por la activación combinada de las proteínas Gαi2 y Gαi3 de proteínas G.[eng] During cerebellum development, clonal expansion of cerebellar granular neuronal precursors (CGNPs) takes place in response to Sonic Hedgehog (Shh) signalling pathway. Shh transduces its signals through the Patched (Ptc) and Smoothened (Smo) receptor complex, delivering activated forms of Gli (Ci) transcription factors to the nucleus. PKA is a known negative regulator of the CGNPs mitogenic activity by inducing Gli2/3 phosphorylation and their subsequent degradation by the proteasome generating potent transcriptional repressors of the pathway. In mammals, Shh pathway requires and takes place at a specific cellular structure: the primary cilum, where many of the proteins of the pathway localize. In this thesis we demonstrated that PKA-C subunit accumulates at the base of the primary cilium through PKA-RII subunit that joins to an unknown AKAP. In the absence of Shh, PKA activates and spreads from the primary cilium base and phosphorylates its target proteins. PKA needs to be accumulated at this specific location, since the inhibition of PKA localization to the cilium base reduces CGNPs mitosis. In the second part of the present thesis we demonstrate that any active member of the Gai/o but not of the Ga12/13 family of heterotrimeric G proteins can induce CGNPs proliferation at low but non active doses of Shh. We also show that Gai2 and Gai3 are the only expressed proteins of this family in the external granular layer of the developing cerebellum (EGL), where proliferation takes place. These proteins are the specific members responsible for Shh activity as their ablation by RNAi has a synergic effect in reducing CGNPs mitogenic activity. Moreover, these subunits can exclusively accumulate at the cilium shaft. All together these results indicate that Gai2 and Gai3 are the two specific members that mediate Shh pathway in this cells

Probucol increases glutathione peroxidase-1 activity and displays long-lasting protection against methylmercury toxicity in cerebellar granule cells

El pdf del artículo es el manuscrito de autor.Methylmercury (MeHg) is an environmental neurotoxicant whose molecular mechanisms underlying toxicity remain elusive. Here we investigated molecular events involved in MeHg-induced neurotoxicity in cultured cerebellar granule cells (CGCs) as well as potential protective strategies for such toxicity. Glutathione peroxidase (GPx-1) activity was significantly (p = 0.0017) decreased at 24 h before MeHg-induced neuronal death (day in vitro 4). This event was related to enhanced susceptibilities to hydrogen- or tert-butyl-peroxides and increased lipid peroxidation. However, intracellular calcium levels, glutamate uptake and glutathione levels, as well as glutathione reductase and catalase activities, were not changed by MeHg exposure at this time-point. Probucol (PB), a lipid-lowering drug, displayed a long-lasting protective effect against MeHg-induced neurotoxicity. The beneficial effects of PB were correlated to increased GPx-1 activity and decreased lipid peroxidation. The protection afforded by PB was significantly higher when compared to the antioxidants ascorbic acid and trolox. In vitro studies with the purified GPx-1 proved that MeHg inhibits and PB activates the enzyme activity. Overexpression of GPx-1 prevented MeHg-induced neuronal death. These data indicate that (i) GPx-1 is an important molecular target involved in MeHg-induced neurotoxicity and (ii) PB, which increases GPx-1 activity in CGCs, induces enduring protection against such toxicity. The results bring out new insights on the potential therapeutic strategies for poisonings to MeHg and other pathological conditions related to increased production and/or decreased detoxification of peroxides.This study was supported by the Grant FIS PI061212 and 2005-SGR-00826 (Ministry of Health and Generalitat de Catalunya, respectively, Spain). Marcelo Farina was recipient of a post- doctoral fellowship (Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq/201362/2007-4) and received financial support from CNPq (479239/2007-0) and FAPESC (Jovens Pesquisadores - FAPESC/CNPq 04/2007).Peer reviewe

Bone morphogenetic protein 2 opposes Shh-mediated proliferation in cerebellar granule cells through a TIEG-1-based regulation of Nmyc

Nmyc is a potent regulator of cell cycle in cerebellar granular neuron precursors (CGNPs) and has been proposed to be the main effector of Shh (Sonic hedgehog) proliferative activity. Nmyc ectopic expression is sufficient to promote cell autonomous proliferation and can lead to tumorigenesis. Bone morphogenetic protein 2 (BMP2) antagonizes Shh proliferative effect by promoting cell cycle exit and differentiation in CGNPs. Here we report that BMP2 opposes Shh mitogenic activity by blocking Nmyc expression. We have identified TIEG-1 (KLF10) as the intermediary factor that blocks Nmyc expression through the occupancy of the Sp1 sites present in its promoter. We also demonstrate that TIEG-1 ectopic expression in CGNPs induces cell cycle arrest that can lead to apoptosis but fails to promote differentiation. Moreover, TIEG-1 synergizes with BMP2 activity to terminally differentiate CGNPs and independent differentiator signals such as dibutyryl cAMP and prevents apoptosis in TIEG-1 arrested cells. All together, these data strongly suggest that the BMP2 pathway triggers cell cycle exit and differentiation as two separated but coordinated processes, where TIEG-1 acts as a mediator of the cell cycle arrest. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported by Ministerio de Educación y Ciencia Grant BFU2005-01599.Peer Reviewe

Sonic-hedgehog-mediated proliferation requires the localization of PKA to the cilium base

Cerebellar granular neuronal precursors (CGNPs) proliferate in response to the mitogenic activity of Sonic hedgehog (Shh), and this proliferation is negatively regulated by activation of cAMP-dependent protein kinase (PKA). In the basal state, the PKA catalytic subunits (C-PKA) are inactive because of their association with the regulatory subunits (R-PKA). As the level of cAMP increases, it binds to R-PKA, displacing and thereby activating the C-PKA. Here we report that, in the presence of Shh, inactive C-PKA accumulates at the cilium base of proliferative CGNPs whereas removal of Shh triggers the activation of PKA at this particular location. Furthermore, we demonstrate that the anchoring of the PKA holoenzyme to the cilium base is mediated by the specific binding of the type II PKA regulatory subunit (RII-PKA) to the A-kinase anchoring proteins (AKAPs). Disruption of the interaction between RII-PKA and AKAPs inhibits Shh activity and, therefore, blocks proliferation of CGNP cultures. Collectively, these results demonstrate that the pool of PKA localized to the cilium base of CGNP plays an essential role in the integration of Shh signal transduction.Work in S. P.'s laboratory is supported by the Spanish Ministry of Education Grant BFU200802424/BFI.Peer Reviewe

In Situ Liver Expression of HBsAg/CD3-Bispecific Antibodies for HBV Immunotherapy

Current therapies against hepatitis B virus (HBV) do not reliably cure chronic infection, necessitating new therapeutic approaches. The T cell response can clear HBV during acute infection, and the adoptive transfer of antiviral T cells during bone marrow transplantation can cure patients of chronic HBV infection. To redirect T cells to HBV-infected hepatocytes, we delivered plasmids encoding bispecific antibodies directed against the viral surface antigen (HBsAg) and CD3, expressed on almost all T cells, directly into the liver using hydrodynamic tail vein injection. We found a significant reduction in HBV-driven reporter gene expression (184-fold) in a mouse model of acute infection, which was 30-fold lower than an antibody only recognizing HBsAg. While bispecific antibodies triggered, in part, antigen-independent T cell activation, antibody production within hepatocytes was non-cytotoxic. We next tested the bispecific antibodies in a different HBV mouse model, which closely mimics the transcriptional template for HBV, covalently closed circular DNA (cccDNA). We found that the antiviral effect was noncytopathic, mediating a 495-fold reduction in HBsAg levels at day 4. At day 33, bispecific antibody-treated mice exhibited 35-fold higher host HBsAg immunoglobulin G (IgG) antibody production versus untreated groups. Thus, gene therapy with HBsAg/CD3-bispecific antibodies represents a promising therapeutic strategy for patients with HBV

Targeting the Apoa1 locus for liver-directed gene therapy

Clinical application of somatic genome editing requires therapeutics that are generalizable to a broad range of patients. Targeted insertion of promoterless transgenes can ensure that edits are permanent and broadly applicable while minimizing risks of off-target integration. In the liver, the Albumin (Alb) locus is currently the only well-characterized site for promoterless transgene insertion. Here, we target the Apoa1 locus with adeno-associated viral (AAV) delivery of CRISPR-Cas9 and achieve rates of 6% to 16% of targeted hepatocytes, with no evidence of toxicity. We further show that the endogenous Apoa1 promoter can drive robust and sustained expression of therapeutic proteins, such as apolipoprotein E (APOE), dramatically reducing plasma lipids in a model of hypercholesterolemia. Finally, we demonstrate that Apoa1-targeted fumarylacetoacetate hydrolase (FAH) can correct and rescue the severe metabolic liver disease hereditary tyrosinemia type I. In summary, we identify and validate Apoa1 as a novel integration site that supports durable transgene expression in the liver for gene therapy applications