Malgré les récentes avancées réalisées dans le traitement du cancer du côlon, la résistance des tumeurs est une cause fréquente de l'échec des chimiothérapies. Cette thèse a pour objectif d'identifier les mécanismes moléculaires impliqués dans la résistance à l'Irinotécan, un agent couramment utilisé dans le traitement des cancers colorectaux. Nous avons montré l'implication de la MAPK p38 dans la résistance à l'Irinotécan et en particulier avons démontré que les isoformes α et β étaient impliquées dans cette résistance. De plus, nous avons corrélé la faible phosphorylation de p38 dans des tumeurs coliques primaires de patient sensibles au traitement à l'Irinotécan par rapport aux patients non répondeurs. Dans la suite du projet nous avons étudié le rôle de p38 dans les processus autophagiques et leur impact dans la réponse à l'Irinotécan. Nous avons démontré que p38 induisait une autophagie qui mène à la survie des cellules cancéreuses déficientes pour p53, et que l'inhibition de l'autophagie sensibilisait ces cellules au traitement au SN38, métabolite actif de l'Irinotécan. Enfin de manière préliminaire, nous avons étudié le rôle de p38 dans l'augmentation du métabolisme lipidique dans des cellules déficiente pour p53. Ces travaux ouvrent de nouvelles voies de recherche pour l'identification des mécanismes impliqués dans la résistance aux traitements anticancéreux et pour le développement d'approches pharmacologiques pour contourner la résistance. / Despite the recent advances achieved in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. This work was aimed to determine the molecular mechanisms involved in the resistance to Irinotecan, an anticancer agent widely used in colorectal cancer treatment. We have demonstrated that the α and β forms of p38 MAPK were involved in this resistance. Moreover, we have correlated less phospho-p38 in colon cancer primary tumor of patients sensitive to Irinotecan-based treatment, compared to non responder patients. During the project, we aimed to determine the role of p38 MAPK in the processes of autophagy in colorectal cancer cells, and their impact in Irinotecan cytotoxicity. We have shown that p38 induced survival autophagy in p53 deficient cells. Then, we have shown that autophagy inhibition increased the SN38 cytotoxicity (active metabolite of Irinotecan) in p53 deficient cell lines. Finally, we have studied the role of p38 MAPK in lipid metabolism in p53 deficient cells. All these findings highlight new ways of research to identify the molecular mechanisms involved in chemoresistance as well as new pharmacological approaches to overcome the resistance.

Amiodarone is a potent antidysrhythmic agent that can cause potentially life-threatening pulmonary fibrosis. Accumulating evidence has demonstrated that myofibroblast differentiation is related to the pathogenesis of pulmonary fibrosis. In the present study, we treated human embryonic lung fibroblasts (HELFs) with amiodarone, and investigated the relative molecular mechanism of amiodarone-induced pulmonary fibrosis and pathway determinants PD98059 (extracellular signal-regulated kinase (ERK) inhibitor) and SB203580 (p38 mitogen-activated protein kinase (MAPK) inhibitor). Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8). The secretion of collagen Ⅰ was detected by ELISA. The expressions of α-smooth muscle actin (α-SMA), vimentin, phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, phosphorylated p38 MAPK (p-p38), and p38 MAPK were investigated using Western blot analysis. The levels of α-SMA and vimentin were also determined by immunofluorescence and qRT-PCR. We report that amiodarone promoted cell proliferation and collagen Ⅰ secretion, induced α-SMA and vimentin protein and mRNA expression accompanied by increased phosphorylation of ERK1/2 and p38 MAPK, and furthermore, PD98059 and SB203580 remarkably reduced the proliferative response of HELFs compared with amiodarone group and greatly attenuated α-SMA, vimentin and collagen Ⅰ protein production induced by amiodarone. Taken together, our study suggests that amiodarone regulates cell proliferation and myofibroblast differentiation in HELFs through modulating ERK1/2 and p38 MAPK pathways, and these signal pathways may therefore represent an attractive treatment modality in amiodarone-induced pulmonary fibrosis.

Multiple Sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system, characterized by axonal demyelination and multifocal inflammation. Like many autoimmune diseases, it is a sexually dimorphic disease, being 3-4 times more common in females than in males. p38α MAP kinase (MAPK) has an integral role in modulating inflammatory processes in autoimmunity. Conditionally ablating p38α MAPK in myeloid cells in B6 mice shows a sex difference in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). In the absence of sex hormones, this sex difference was reversed, suggesting a role for sex hormones in modulating p38α MAPK signaling in EAE. Based on these findings, we hypothesized that pro-inflammatory functions in EAE is p38-indepdendent in the presence of androgens and p38-dependent in the presence of estrogens. For the purposes of this project, the role of androgens was evaluated. Both in vivo and in vitro techniques were used to assess how androgen receptor (AR) signaling: 1) impacts EAE pathogenesis, and 2) impacts the role of p38α in EAE pathogenesis and macrophage function. To this end, using Cre-Lox technology, we generated mice deficient in: 1) AR globally or conditionally in macrophages, as well as 2) mice doubly deficient in AR and p38α. In vivo results from p38α-sufficient global AR knockout mice show no effect of global AR deletion on EAE pathogenesis. Surprisingly, results from p38α-sufficient conditional AR knockout mice showed significant worsening in disease compared to WT counterparts, suggesting that AR signaling in myeloid cells has a protective role in EAE pathogenesis. These findings implicate a protective role for AR signaling in EAE. Studies with mice doubly deficient in p38α and AR to determine whether AR regulates the role of p38α in EAE are ongoing, but so far show no effect on AR deletion on the role of p38α MAPK. Further studies with larger cohorts of mice are needed elucidate the relationship between AR and p38α MAPK signaling in myeloid cells in EAE pathogenesis. In vitro studies using the immortalized macrophage cell line RAW 264.7 showed that pharmacologic inhibition of p38 MAPK after stimulation with LPS reduced the production of classic pro-inflammatory cytokines IL-6 and TNFα, and effect that was not affected by treatment with 5-dihydrotestosterone, suggesting that the AR does not modulate the role of p38α in cytokine production. These findings implicate no direct role of AR signaling on the functional role of p38α MAPK in the myeloid cell lineage in inflammatory and autoimmune responses.

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La supervivencia celular frente a los cambios ambientales requiere el mantenimiento de un equilibrio dinámico entre la síntesis y la degradación de proteínas. La degradación de proteínas, además de regular diferentes procesos celulares, tiene como función principal la eliminación de productos que no son útiles para la célula en determinadas situaciones o cuya acumulación puede ser tóxica. Los productos de esta degradación, es decir los aminoácidos, son reutilizados para la síntesis de nuevas moléculas o son metabolizados para la obtención de energía. La alteración de esta proteólisis intracelular puede llevar a la acumulación en el citoplasma de orgánulos defectuosos o de moléculas que se pueden agrupar en agregados insolubles y que pueden así desencadenar diferentes patologías. Aunque se ha avanzado bastante durante los últimos años en los conocimientos sobre la degradación intracelular de proteínas y de sus principales mecanismos, existen bastantes detalles moleculares todavía desconocidos. Por este motivo es necesario aportar nueva información sobre estos procesos que además podría ser relevante para identificar nuevas dianas terapéuticas y desarrollar tratamientos más eficaces para las enfermedades derivadas de alteraciones en los mismos. La degradación de proteínas ocurre por diferentes mecanismos que pueden clasificarse generalmente en dependientes o no de unos orgánulos citoplásmicos, los lisosomas. La macroautofagia (a la que se denomina generalmente con el término más simple de autofagia) y el sistema ubicuitina-proteasomas son, respectivamente, los más importantes de esos dos grupos. Básicamente, el sistema ubicuitina-proteasomas consiste en la poliubicuitinación de proteínas que son después degradadas por los proteasomas. La autofagia en cambio se inicia con el secuestro de porciones del citoplasma en estructuras de doble membrana que se cierran formando los autofagosomas. Posteriormente, los autofagosomas se fusionan con endosomas y con lisosomas dando lugar a los autolisosomas, en los que por la acción de las proteasas o catepsinas lisosomales se degrada el material encerrado. La autofagia está regulada por una amplia variedad de vías de señalización que responden a multitud de factores ambientales. Entre estos últimos, la situación de ayuno de nutrientes es la inductora más potente de la autofagia. Durante la privación de nutrientes como los aminoácidos, la célula sufre un estrés energético que debe tratar de reducir produciendo ATP a partir de nuevas fuentes. Para ello activa la autofagia para degradar los componentes de la célula, como las proteínas, hasta producir sus unidades básicas que después son metabolizadas. Por el contrario, se ha demostrado que cuando se proporcionan aminoácidos a la célula la autofagia es inhibida. Aunque el efecto sobre la autofagia de los aminoácidos ha sido estudiado ampliamente en muchos laboratorios, no estaba tan claro ese efecto en el caso de otro nutriente, la glucosa, ya que cuando planteamos ese estudio los datos eran contradictorios. En este trabajo hemos podido establecer claramente que la glucosa tiene un papel inductor sobre la autofagia empleando técnicas muy variadas que incluyen: la cuantificación por ¿Western-blot¿ de los niveles del marcador de autofagia LC3-II en presencia o en ausencia de inhibidores lisosomales, la cuantificación de la proteína degradada, total y por la vía autofágica, mediante experimentos de pulso y caza, la cuantificación morfométrica de estructuras autofágicas (equivalentes a autofagosomas y autolisosomas) por microscopia electrónica y la cuantificación de la masa lisosomal por fluorescencia. Además, hemos comprobado que la glucosa también induce la ubicuitinación de proteínas y la degradación de estas por los proteasomas. Con estos y otros datos obtenidos durante el desarrollo de esta tesis doctoral, hemos podido concluir que la glucosa induce la autofagia en todos los tipos celulares estudiados y en todas las condiciones ensayadas. Este efecto disminuye o se enmascara cuando están presentes a la vez otros factores que son inhibidores de la autofagia, como los aminoácidos o el suero bovino fetal, lo que podría explicar algunos de los datos contradictorios en la literatura. La glucosa aporta la energía necesaria para el correcto funcionamiento de la autofagia a partir de unos niveles mínimos de ATP. Un descenso en la disponibilidad energética a través de la inhibición de la glucólisis reprime la autofagia inducida por la glucosa. Sin embargo, la estimulación de la autofagia por glucosa no parece depender únicamente de la disponibilidad de ATP, sino que hemos identificado una vía de señalización en la que no interviene AMPK a pesar de responder al descenso de los niveles de ATP y al aumento de los niveles de calcio durante la incubación en un medio carente de glucosa. Esta vía tampoco implica a mTORC1 y en ella sí interviene en cambio la MAPK p38¿, como hemos comprobado con diferentes inhibidores de esta quinasa, con el uso de siRNAs o empleando MEFs p38-/-. Consideramos que estos resultados contribuyen a clarificar más la regulación de la autofagia por nutrientes y, más concretamente, por uno tan relevante como es la glucosa. / Moruno Manchón, JF. (2013). Regulación de la degradación intracelular de proteínas por glucosa [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34775

Yes / The molecular mechanisms and signalling cascades that trigger the induction of group I metabotropic glutamate receptor (GI-mGluR)-dependent long-term depression (LTD) have been the subject of intensive investigation for nearly two decades. The generation of genetically modified animals has played a crucial role in elucidating the involvement of key molecules regulating the induction and maintenance of mGluR-LTD. In this review we will discuss the requirement of the newly discovered MAPKAPK-2 (MK2) and MAPKAPK-3 (MK3) signalling cascade in regulating GI-mGluR-LTD. Recently, it has been shown that the absence of MK2 impaired the induction of GI-mGluR-dependent LTD, an effect that is caused by reduced internalization of AMPA receptors (AMPAR). As the MK2 cascade directly regulates tumour necrosis factor alpha (TNFα) production, this review will examine the evidence that the release of TNFα acts to regulate glutamate receptor expression and therefore may play a functional role in the impairment of GI-mGluRdependent LTD and the cognitive deficits observed in MK2/3 double knockout animals. The strong links of increased TNFα production in both aging and neurodegenerative disease could implicate the action of MK2 in these processes. / This work was supported by the BBSRC-BB/H018344/1 to S.A.L.C.

Yes / A key aim of Alzheimer disease research is to develop efficient therapies to prevent and/or delay the irreversible progression of cognitive impairments. Early deficits in long-term potentiation (LTP) are associated with the accumulation of amyloid beta in rodent models of the disease; however, less is known about how mGluR-mediated long-term depression (mGluR-LTD) is affected. In this study, we have found that mGluR-LTD is enhanced in the APPswe /PS1dE9 mouse at 7 but returns to wild-type levels at 13 months of age. This transient over-activation of mGluR signalling is coupled with impaired LTP and shifts the dynamic range of synapses towards depression. These alterations in synaptic plasticity are associated with an inability to utilize cues in a spatial learning task. The transient dysregulation of plasticity can be prevented by genetic deletion of the MAP kinase-activated protein kinase 2 (MK2), a substrate of p38 MAPK, demonstrating that manipulating the mGluR-p38 MAPK-MK2 cascade at 7 months can prevent the shift in synapse dynamic range. Our work reveals the MK2 cascade as a potential pharmacological target to correct the over-activation of mGluR signalling. / Wellcome Trust, Grant/Award Number: 200646/Z/16/Z

IL-12 family cytokines IL-23 and IL-27 play an important role linking innate and adaptive immunity, and regulating T-cell responses. The production of IL-12, a structurally similar cytokine, is decreased in chronic HIV infection; therefore IL-23 and IL-27 may also be influenced by HIV infection. I hypothesized that HIV inhibits LPS-induced IL-23 and IL-27 production in human MDMs by suppressing the activation of signalling pathways regulating their expression. In vitro HIV-infection of MDMs did not have any effect on basal secretion of IL-23 or IL-27; however, HIV inhibited LPS-induced production of IL-12/23 p40 and IL-23 p19, and IL-27 EBI3 and IL-27 p28 mRNA expression, and IL-23, IL-12/23 p40 and IL-27 secretion. In order to evaluate the molecular mechanisms by which HIV inhibits IL-23 and IL-27 in LPS-stimulated MDMs, the signalling pathways regulating their expression were evaluated. The PI3K, p38 MAPK, and JNK MAPK pathways were found to positively regulate LPS-induced IL-27 secretion. Interestingly, in vitro HIV infection inhibited LPS-induced p38 and JNK MAPK activation in MDMs. In summary, I have shown that HIV inhibits IL-23 and IL-27 production in LPS-stimulated MDMs and that HIV may inhibit LPS-induced IL-27 production through the inhibition of p38 and JNK MAPK activation. It is currently unknown whether PKCs regulate LPS-induced IL-23 or IL-27 in human monocytes/macrophages. I demonstrated that classical PKCs differentially regulate LPS-induced IL-23 and IL-27 secretion within THP-1 cells, primary monocytes, and MDMs. Classical PKCs were found to positively regulate LPS-induced IL-12/23 p40 and IL-27 p28 mRNA expression and IL-12/23 p40, IL-23, and IL-27 secretion in primary human monocytes. Similarly, the classical PKCs were found to positively regulate IL-27 p28 mRNA expression and IL-27 secretion in THP-1 cells. However, classical PKCs did not regulate LPS-induced IL-27 production in MDMs, or LPS-induced IL-23 production in THP-1 cells. Overall, this demonstrates that classical PKCs differentially regulate LPS-induced IL-23 and IL-27 production in different myeloid cells.