Publication: Implicación de AMPK en la señalización intracelular inducida por CCR₇ en las células dendríticas
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2017-06-19
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Universidad Complutense de Madrid
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Abstract
Las células dendríticas son células presentadoras de antígenos profesionales capaces de activar a los linfocitos T vírgenes e iniciar la respuesta inmune adaptativa. En su fase final de diferenciación, denominada fase de CD maduras (CDms), estos leucocitos pueden migrar a través de los vasos linfáticos aferentes hasta los ganglios linfáticos, donde presentan los antígenos capturados en las áreas de inflamación a los linfocitos T. Las CDms maduras expresan el receptor de quimioquinas CCR7 que es fundamental para que estas células se dirijan a los ganglios linfáticos. En trabajos previos se mostró que la activación de CCR7 aumenta la supervivencia de las CDms, aunque los mecanismos de señalización que regulan este proceso no se conocen por completo. AMPK (AMP-dependent Kinase) es una molécula considerada un “guardián energético” celular debido a que es capaz de detectar cambios en la proporción AMP/ATP. En respuesta a un aumento de los niveles de AMP, AMPK activa las rutas de señalización catabólicas e inhibe las anabólicas. En esta tesis se estudia la posible implicación de AMPK en la regulación de la supervivencia de las CDs mediada por CCR7. En primer lugar, analizamos si AMPK tiene un papel inductor de la supervivencia o de la apoptosis en las CDs. La reducción de los niveles de AMPK con un siRNA provoca un aumento de la supervivencia de las CDms, lo que indica que AMPK induce muerte en las CDs. Se sugiere que la activación de AMPK provoca una muerte de tipo apoptótico porque morfológicamente implica procesos de picnosis y de fragmentación del núcleo y bioquímicamente depende de caspasas, implicando específicamente a la caspasa-3. Los experimentos en los que se analizó la supervivencia de las células en los ganglios linfáticos mostraron que AMPK induce apoptosis de las CDs in vivo. Además, los resultados obtenidos indican que AMPK induce la apoptosis de las CDs mediante al menos dos mecanismos. Primero, la activación de AMPK induce la translocación del factor de transcripción FOXO1 del citoplasma al núcleo. Una vez en el núcleo, FOXO1 activa la transcripción de la proteína pro-apoptótica Bim, miembro de la familia Bcl2. Segundo, la activación de AMPK inhibe la actividad del complejo pro-supervivencia mTORC1 en las CDs...
Dendritic cells (DCs) are professional antigen presenting cells capable of initiating the immune response by activating naive T lymphocytes. In their last phase of differentiation called mature CD (mDCs), these cells present antigens captured in the areas of inflammation to lymphocytes in the lymph nodes. To carry out this role, mCDs must migrate through the afferent lymphatics to the lymph nodes. The chemokine receptor CCR7, which is expressed by mDCs, is key to direct these cells to the lymph nodes. Before, it has been shown that CCR7 relays intracellular signaling resulting in the extension of the survival of mDCs. However, the mechanisms used by this receptor to promote survival are not completely understood. AMPK (AMP-dependent Kinase) is a sensor of energy status that can detect changes in the AMP/ATP ratio. This enzyme responds to increased levels of AMP by inducing activation of catabolic and inhibition of anabolic pathways. In this thesis, it was investigated whether AMPK plays a role in CCR7-dependent regulation of survival in DCs. We first analyzed whether AMPK kinase played a pro-apoptotic or a pro-survival role in DCs. Reduction of AMPK levels using siRNA increases the survival of human mDCs. We suggest that AMPK induces specifically apoptosis because it promotes morphological and biochemical changes associated with this type of cell death. In this regard, AMPK induces nuclear pyknosis and fragmentation and as well as a caspase-dependent cell death that involves the specific activation of caspase-3. Activation of AMPK also induces apoptosis of DCs in vivo, as shown in experiments where apoptosis was analyzed in popliteal lymph nodes. The data presented indicate that AMPK may induce apoptosis in DCs through two mechanisms. First, AMPK may induce translocation of the transcription factor FOXO1 from the cytoplasm to the nucleus. Once in the nucleus, FOXO1 activates the transcription of the pro-apoptotic Bcl2 family member Bim. Second, AMPK inhibits the activity of the pro-survival kinase complex mTORC1...
Dendritic cells (DCs) are professional antigen presenting cells capable of initiating the immune response by activating naive T lymphocytes. In their last phase of differentiation called mature CD (mDCs), these cells present antigens captured in the areas of inflammation to lymphocytes in the lymph nodes. To carry out this role, mCDs must migrate through the afferent lymphatics to the lymph nodes. The chemokine receptor CCR7, which is expressed by mDCs, is key to direct these cells to the lymph nodes. Before, it has been shown that CCR7 relays intracellular signaling resulting in the extension of the survival of mDCs. However, the mechanisms used by this receptor to promote survival are not completely understood. AMPK (AMP-dependent Kinase) is a sensor of energy status that can detect changes in the AMP/ATP ratio. This enzyme responds to increased levels of AMP by inducing activation of catabolic and inhibition of anabolic pathways. In this thesis, it was investigated whether AMPK plays a role in CCR7-dependent regulation of survival in DCs. We first analyzed whether AMPK kinase played a pro-apoptotic or a pro-survival role in DCs. Reduction of AMPK levels using siRNA increases the survival of human mDCs. We suggest that AMPK induces specifically apoptosis because it promotes morphological and biochemical changes associated with this type of cell death. In this regard, AMPK induces nuclear pyknosis and fragmentation and as well as a caspase-dependent cell death that involves the specific activation of caspase-3. Activation of AMPK also induces apoptosis of DCs in vivo, as shown in experiments where apoptosis was analyzed in popliteal lymph nodes. The data presented indicate that AMPK may induce apoptosis in DCs through two mechanisms. First, AMPK may induce translocation of the transcription factor FOXO1 from the cytoplasm to the nucleus. Once in the nucleus, FOXO1 activates the transcription of the pro-apoptotic Bcl2 family member Bim. Second, AMPK inhibits the activity of the pro-survival kinase complex mTORC1...
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Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Bioquímica y Biología Molecular I, leída el 19-12-2016