Caracterización funcional de MAP Quinasas del subgrupo C1 de plantas

RESUMEN En plantas, la señalización a través de las cascadas de MAP quinasas da lugar a un amplio número de respuestas celulares que incluyen la división y diferenciación celular, así como respuestas a estrés de origen abiótico o biótico (Mishra et al., 2006). Las MAP quinasas de plantas pueden ser clasificadas, en base a la similitud de la secuencia de aminoácidos, en cuatro grupos (A-D), cada grupo se ha clasificado a su vez en dos subgrupos (1 y 2). Se dispone de muy poca información acerca de los miembros del subgrupo C1 (Nakagami et al., 2005). Dentro de este grupo se encuentran Ntf3 de tabaco, PhMEK1 de petunia y PsMAPK2 de guisante. En Arabidopsis, el subgrupo C1 está constituido por dos genes de MAP quinasas: AtMPK1 (At1g10210) y AtMPK2 (At1g59580). La función de estos genes no se conoce, aunque hay algún dato que indica una posible relación entre esos genes y respuestas de estrés. Se ha descrito que los niveles del ARNm de AtMPK1 y AtMPK2 aumentan ligeramente tras un tratamiento de salinidad (Mizoguchi et al., 1996), y que disminuyen a las 24 horas tras un tratamiento a baja temperatura (Vogel et al., 2005). Además, resultados obtenidos mediante análisis de micromatrices indican que la expresión de AtMPK1 es mayor en plántulas crecidas en oscuridad que en plántulas crecidas en presencia de luz (Ma et al., 2005). El análisis de los datos de expresión depositados en bases de datos de acceso público indican que los niveles de expresión de estos genes son muy bajos y que no hay cambios relevantes tras las diferentes condiciones ensayadas (Zimmermann et al., 2004). No hay ningún dato sobre la regulación de la actividad quinasa de las MAP quinasas del subgrupo C1. La presente Tesis aborda el estudio de la función de PsMAPK2 (guisante), AtMPK1 y AtMPK2 (Arabidopsis), genes que codifican MAP quinasas del subgrupo C1. Para emprender dicho estudio, se han realizado diferentes aproximaciones: 1- Se ha analizado la expresión de estas MAPKs en distintos órganos de la planta; 2- Se han obtenido plantas transgénicas de Arabidopsis que expresan distintas versiones mutantes de PsMAPK2; 3- Se ha analizado la actividad quinasa de estas MAPKs en respuesta a distintas señales de estrés. Según resultados obtenidos por RT-PCR, PsMAPK2 se expresa en todos los órganos de guisante y principalmente en anteras. Por otro lado la expresión de las distintas versiones de PsMAPK2 en Arabidopsis da lugar a un fenotipo de esterilidad masculina, debido a que no se produce la dehiscencia de las anteras y la posterior liberación del polen. Estos resultados sugieren una posible función de PsMAPK2 en el desarrollo de las anteras. AtMPK1/2 se expresan en todos los órganos de Arabidopsis. Además, el análisis de la expresión de ambas MAPKs en plántulas indica que la luz disminuye su expresión tanto en plántulas crecidas con ciclos de luz/oscuridad como en plántulas etioladas cuando se transfieren a la luz. El estudio de la respuesta a la luz de plántulas etioladas del doble mutante Atmpk1 Atmpk2 revela que estas plántulas presentan una inhibición de la desetiolización con respecto a la línea silvestre, sugiriendo la participación de AtMPK1/2 en el proceso de desetiolización. Por último, en la presente Tesis se ha demostrado por primera vez la regulación de la actividad de PsMAPK2 y AtMPK1/2 en respuesta a una señal. Se ha detectado un aumento en la actividad quinasa de PsMAPK2 y AtMPK1/2 en respuesta al daño mecánico y al ácido jasmónico. Además, otras moléculas señalizadoras de estrés como el ácido abscísico y el peróxido de hidrógeno también regulan la actividad quinasa de PsMAPK2 y AtMPK1/2. __________________________________________________________________________________________________ SUMMARY Mitogen-activated protein kinase (MAPK) cascades link extracellular stimuli with several cellular responses. These cascades are evolutionary conserved signalling modules present in all eukaryotes. MAPKs are serine/threonine kinases that are activated by dual phosphorylation of the threonine and the tyrosine residues at a TXY activation motif. These phosphorylations are performed by a MAPK kinase (MAPKK), which is in turn activated by an upstream MAPKK kinase (MAPKKK). In Arabidopsis thaliana, twenty MAPKs have been identified and classified according to their sequence homology into four major groups (A to D). A large amount of information about MAPKs in group A and B is available but very little is known about MAPKs of subgroup C1. Specific changes in transcript levels during pollen or ovule development have been reported for two members of C1 subgroup, ntf3 from tobacco and PMEK1 from Petunia. In Arabidopsis, subgroup C1 is constituted by two MAPK genes: AtMPK1 (AT1G10210) and AtMPK2 (AT1G59580). The function of these genes remains mostly unknown, regardless of data which indicate a possible relationship between these genes and some stress responses. It has been reported that the mRNA levels of AtMPK1 and AtMPK2 increased slightly under salt stress and are downregulated after 24 h of cold treatment, respectively. Gene expression data deposited in public microarray repertories indicate a very low expression of the corresponding mRNAs and few and no relevant changes in their expression under a large variety of tissues and conditions. No data about the regulation of the kinase activity of subgroup C1 MAPKs is available. In this manuscript, we show that the two Arabidopsis MAPKs of subgroup C1 (AtMPK1 and AtMPK2) and one Pea MAPK of subgroup C1 (PsMAPK2) are activated by wounding, JA, ABA and H2O2. Meanwhile, transgenic Arabidopsis plants expressing PsMAPK2, PsMAPK2- LOF (loss-of-function mutation) or PsMAPK2-GOF (gain-of-function mutation) exhibited severe male sterility due to defects in anther development

Induction of CD36 and Thrombospondin-1 in Macrophages by Hypoxia-Inducible Factor 1 and Its Relevance in the Inflammatory Process

Inflammation is part of a complex biological response of vascular tissue to pathogens or damaged cells. First inflammatory cells attempt to remove the injurious stimuli and this is followed by a healing process mediated principally by phagocytosis of senescent cells. Hypoxia and p38-MAPK are associated with inflammation, and hypoxia inducible factor 1 (HIF-1) has been detected in inflamed tissues. We aimed to analyse the role of p38-MAPK and HIF-1 in the transcriptional regulation of CD36, a class B scavenger receptor, and its ligand thrombospondin (TSP-1) in macrophages and to evaluate the involvement of this pathway in phagocytosis of apoptotic neutrophils. We have also assessed HIF-1α, p38-MAPK and CD36 immunostaining in the mucosa of patients with inflammatory bowel disease. Results show that hypoxia increases neutrophil phagocytosis by macrophages and induces the expression of CD36 and TSP-1. Addition of a p38-MAPK inhibitor significantly reduced the increase in CD36 and TSP-1 expression provoked by hypoxia and decreased HIF-1α stabilization in macrophages. Transient transfection of macrophages with a miHIF-1α-targeting vector blocked the increase in mRNA expression of CD36 and TSP-1 during hypoxia and reduced phagocytosis, thus highlighting a role for the transcriptional activity of HIF-1. CD36 and TSP-1 were necessary for the phagocytosis of neutrophils induced by hypoxic macrophages, since functional blockade of these proteins undermined this process. Immunohistochemical studies revealed CD36, HIF-1α and p38-MAPK expression in the mucosa of patients with inflammatory bowel disease. A positive and significant correlation between HIF-1α and CD36 expression and CD36 and p38-MAPK expression was observed in cells of the lamina propria of the damaged mucosa. Our results demonstrate a HIF-1-dependent up-regulation of CD36 and TSP-1 that mediates the increased phagocytosis of neutrophils by macrophages during hypoxia. Moreover, they suggest that CD36 expression in the damaged mucosa of patients with inflammatory bowel disease depends on p38-MAPK and HIF-1 activity

M1 Macrophages Activate Notch Signalling in Epithelial Cells: Relevance in Crohn's Disease

Background: The Notch signalling pathway plays an essential role in mucosal regeneration, which constitutes a key goal of Crohn's disease (CD) treatment. Macrophages coordinate tissue repair and several phenotypes have been reported which differ in the expression of surface proteins, cytokines and hypoxia-inducible factors (HIFs). We analysed the role of HIFs in the expression of Notch ligands in macrophages and the relevance of this pathway in mucosal regeneration. Methods: Human monocytes and U937-derived macrophages were polarized towards the M1 and M2 phenotypes and the expression levels of HIF-1α, HIF-2α, Jagged 1 (Jag1) and delta-like 4 (Dll4) were evaluated. The effects of macrophages on the expression of hairy and enhancer of split-1 (HES1, the main target of Notch signalling) and intestinal alkaline phosphatase (IAP, enterocyte marker) in epithelial cells in co-culture were also analysed. Phenotype macrophage markers and Notch signalling were evaluated in the mucosa of CD patients. Results: M1 macrophages were associated with HIF-1-dependent induction of Jag1 and Dll4, which increased HES1 protein levels and IAP activity in co-cultured epithelial cells. In the mucosa of CD patients a high percentage of M1 macrophages expressed both HIF-1α and Jag1 while M2 macrophages mainly expressed HIF-2α and we detected a good correlation between the ratio of M1/M2 macrophages and both HES1 and IAP protein levels. Conclusion: M1, but not M2, macrophages are associated with HIF-1-dependent induction of Notch ligands and activation of epithelial Notch signalling pathway. In the mucosa of chronic CD patients, the prevalence of M2 macrophages is associated with diminution of Notch signalling and impaired enterocyte differentiation. Key Words: MacrophagesCrohn's diseasemucosal healingNotch signallin

M2 Macrophages Activate WNT Signaling Pathway in Epithelial Cells: Relevance in Ulcerative Colitis

Macrophages, which exhibit great plasticity, are important components of the inflamed tissue and constitute an essential element of regenerative responses. Epithelial Wnt signalling is involved in mechanisms of proliferation and differentiation and expression of Wnt ligands by macrophages has been reported. We aim to determine whether the macrophage phenotype determines the expression of Wnt ligands, the influence of the macrophage phenotype in epithelial activation of Wnt signalling and the relevance of this pathway in ulcerative colitis. Human monocyte-derived macrophages and U937-derived macrophages were polarized towards M1 or M2 phenotypes and the expression of Wnt1 and Wnt3a was analyzed by qPCR. The effects of macrophages and the role of Wnt1 were analyzed on the expression of β-catenin, Tcf-4, c-Myc and markers of cell differentiation in a co-culture system with Caco-2 cells. Immunohistochemical staining of CD68, CD206, CD86, Wnt1, β-catenin and c-Myc were evaluated in the damaged and non-damaged mucosa of patients with UC. We also determined the mRNA expression of Lgr5 and c-Myc by qPCR and protein levels of β-catenin by western blot. Results show that M2, and no M1, activated the Wnt signaling pathway in co-culture epithelial cells through Wnt1 which impaired enterocyte differentiation. A significant increase in the number of CD206+ macrophages was observed in the damaged mucosa of chronic vs newly diagnosed patients. CD206 immunostaining co-localized with Wnt1 in the mucosa and these cells were associated with activation of canonical Wnt signalling pathway in epithelial cells and diminution of alkaline phosphatase activity. Our results show that M2 macrophages, and not M1, activate Wnt signalling pathways and decrease enterocyte differentiation in co-cultured epithelial cells. In the mucosa of UC patients, M2 macrophages increase with chronicity and are associated with activation of epithelial Wnt signalling and diminution in enterocyte differentiation

Progastrin Represses the Alternative Activation of Human Macrophages and Modulates Their Influence on Colon Cancer Epithelial Cells

Macrophage infiltration is a negative prognostic factor for most cancers but gastrointestinal tumors seem to be an exception. The effect of macrophages on cancer progression depends on their phenotype, which may vary between M1 (pro-inflammatory, defensive) to M2 (tolerogenic, pro-tumoral). Gastrointestinal cancers often become an ectopic source of gastrins and macrophages present receptors for these peptides. The aim of the present study is to analyze whether gastrins can affect the pattern of macrophage infiltration in colorectal tumors. We have evaluated the relationship between gastrin expression and the pattern of macrophage infiltration in samples from colorectal cancer and the influence of these peptides on the phenotype of macrophages differentiated from human peripheral monocytes in vitro. The total number of macrophages (CD68+ cells) was similar in tumoral and normal surrounding tissue, but the number of M2 macrophages (CD206+ cells) was significantly higher in the tumor. However, the number of these tumor-associated M2 macrophages correlated negatively with the immunoreactivity for gastrin peptides in tumor epithelial cells. Macrophages differentiated from human peripheral monocytes in the presence of progastrin showed lower levels of M2-markers (CD206, IL10) with normal amounts of M1-markers (CD86, IL12). Progastrin induced similar effects in mature macrophages treated with IL4 to obtain a M2-phenotype or with LPS plus IFNγ to generate M1-macrophages. Macrophages differentiated in the presence of progastrin presented a reduced expression of Wnt ligands and decreased the number and increased cell death of co-cultured colorectal cancer epithelial cells. Our results suggest that progastrin inhibits the acquisition of a M2-phenotype in human macrophages. This effect exerted on tumor associated macrophages may modulate cancer progression and should be taken into account when analyzing the therapeutic value of gastrin immunoneutralization

Succinate activates EMT in intestinal epithelial cells through SUCNR1: a novel protagonist in fistula development

The pathogenesis of Crohn's disease-associated fibrostenosis and fistulas imply the epithelial-to-mesenchymal transition (EMT) process. As succinate and its receptor (SUCNR1) are involved in intestinal inflammation and fibrosis, we investigated their relevance in EMT and Crohn's disease (CD) fistulas. Succinate levels and SUCNR1-expression were analyzed in intestinal resections from non-Inflammatory Bowel Disease (non-IBD) subjects and CD patients with stenosing-B2 or penetrating-B3 complications and in a murine heterotopic-transplant model of intestinal fibrosis. EMT, as increased expression of Snail1, Snail2 and vimentin and reduction in E-cadherin, was analyzed in tissues and succinate-treated HT29 cells. The role played by SUCNR1 was studied by silencing its gene. Succinate levels and SUCNR1 expression are increased in B3-CD patients and correlate with EMT markers. SUCNR1 is detected in transitional cells lining the fistula tract and in surrounding mesenchymal cells. Grafts from wild type (WT) mice present increased succinate levels, SUCNR1 up-regulation and EMT activation, effects not observed in SUCNR1$^{-/-}$ tissues. SUCNR1 activation induces the expression of Wnt ligands, activates WNT signaling and induces a WNT-mediated EMT in HT29 cells. In conclusion, succinate and its receptor are up-regulated around CD-fistulas and activate Wnt signaling and EMT in intestinal epithelial cells. These results point to SUCNR1 as a novel pharmacological target for fistula prevention

Macrophages Modulate Hepatic Injury Involving NLRP3 Inflammasome: The Example of Efavirenz

Drug-induced liver injury (DILI) constitutes a clinical challenge due to the incomplete characterization of the mechanisms involved and potential risk factors. Efavirenz, an anti-HIV drug, induces deleterious actions in hepatocytes that could underlie induction of the NLRP3 inflammasome, an important regulator of inflammatory responses during liver injury. We assessed the potential of efavirenz to modulate the inflammatory and fibrogenic responses of major liver cell types involved in DILI. The effects of efavirenz were evaluated both in vitro and in vivo. Efavirenz triggered inflammation in hepatocytes, in a process that involved NF-κB and the NLRP3 inflammasome, and activated hepatic stellate cells (HSCs), thereby enhancing expression of inflammatory and fibrogenic markers. The NLRP3 inflammasome was not altered in efavirenz-treated macrophages, but these cells polarized towards the anti-inflammatory M2 phenotype and displayed upregulated anti-inflammatory mediators. Conversely, no evidence of damage was observed in efavirenz-treated animals, except when macrophages were depleted, which resulted in the in vivo manifestation of the deleterious effects detected in hepatocytes and HSCs. Efavirenz elicits a cell-specific activation of the NLRP3 inflammasome in hepatocytes and HSCs, but macrophages appear to counteract efavirenz-induced liver injury. Our results highlight the dynamic nature of the interaction among liver cell populations and emphasize the potential of targeting macrophage polarization as a strategy to treat NLRP3 inflammasome-induced liver injury

Diminished Vitamin D Receptor Protein Levels in Crohn’s Disease Fibroblasts: Effects of Vitamin D

Vitamin D (VD) deficiency has been associated to Crohn’s disease (CD) pathogenesis, and the exogenous administration of VD improves the course of the disease, but the mechanistic basis of these observations remains unknown. Vitamin D receptor (VDR) mediates most of the biological functions of this hormone, and we aim to analyze here the expression of VDR in intestinal tissue, epithelial cells, and fibroblasts from CD patients. The effects of VD on a fibroblast wound healing assay and murine intestinal fibrosis are also analyzed. Our data show diminished VDR protein levels in surgical resections and epithelial cells from CD patients. In intestinal fibroblasts isolated from damaged tissue of CD patients, we detected enhanced migration and decreased VDR expression compared with both fibroblasts from non-damaged tissue of the same CD patient or control fibroblasts. Treatment with VD increased VDR protein levels, avoided the accelerated migration in CD fibroblasts, and prevented murine intestinal fibrosis induced by the heterotopic transplant model. In conclusion, our study demonstrates diminished VDR protein levels associated with enhanced migration in intestinal fibroblasts from damaged tissue of CD patients. In these cells, VD accumulates VDR and normalizes migration, which supports that CD patients would benefit from the VD anti-fibrotic therapeutic value that we demonstrate in a murine experimental model

Influence of progastrin on the phenotype of human monocyte-derived macrophages.

<p>Human peripheral monocytes were derived to macrophages in the presence or absence of progastrin and the phenotype of the resultant macrophages evaluated by analyzing the following parameters: expression of CD86 (A, C, n = 3); expression of CD206 (B, D, n = 3); secretion of IL12 (F, n = 4); and secretion of IL10 (G, n = 4). Bars represent mean ±SEM. *P<0.05 and **P<0.01 vs corresponding value in vehicle-treated cells.</p