Dolores Ortiz-Masiá

Diverse stress signals activate the C1 subgroup MAP kinases of Arabidopsis

Mitogen‐activated protein kinase (MAPK) cascades play an important role in mediating stress responses in plants. In Arabidopsis, 20 MAPKs have been identified and classified into four major groups (A–D). Little is known about the role of group C MAPKs. We have studied the activation of Arabidopsis subgroup C1 MAPKs (AtMPK1/AtMPK2) in response to mechanical injury. An increase in their kinase activity was detected in response to wounding that was blocked by cycloheximide. Jasmonic acid (JA) activated AtMPK1/AtMPK2 in the absence of wounding. Wound and JA‐induction of AtMPK1/2 kinase activity was not prevented in the JA‐insensitive coi1 mutant. Other stress signals, such as abscisic acid (ABA) and hydrogen peroxide, activated AtMPK1/2. This report shows for the first time that regulation of AtMPK1/2 kinase activity in Arabidopsis might be under the control of signals involved in different kinds of stress.

Identification and characterization of an ABA‐activated MAP kinase cascade in Arabidopsis thaliana

Abscisic acid (ABA) is a major phytohormone involved in important stress-related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA-triggered phosphoproteins as putative mitogen-activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA-activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3-1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA-dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR-SnRK2-PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA-induced MAPK pathway in plant stress signalling.

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.

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.

The activation of Wnt signaling by a STAT6-dependent macrophage phenotype promotes mucosal repair in murine IBD.

The complete repair of the mucosa constitutes a key goal in inflammatory bowel disease (IBD) treatment. The Wnt signaling pathway mediates mucosal repair and M2 macrophages that coordinate efficient healing have been related to Wnt ligand expression. Signal transducer and activator of transcription 6 (STAT6) mediates M2 polarization in vitro and we hypothesize that a STAT6-dependent macrophage phenotype mediates mucosal repair in acute murine colitis by activating the Wnt signaling pathway. Our results reveal an impaired mucosal expression of M2 macrophage-associated genes and delayed wound healing in STAT6−/− mice treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS). These mice also exhibited decreased mucosal expression of Wnt2b, Wnt7b, and Wnt10a, diminished protein levels of nuclear β-catenin that is mainly located in crypts adjacent to damage, and reduced mRNA expression of two Wnt/β-catenin target molecules Lgr5 and c-Myc when compared with wild-type (WT) mice. Murine peritoneal macrophages treated with interleukin-4 (IL-4) and polarized toward an M2a phenotype overexpressed Wnt2b, Wnt7b, and Wnt10a in a STAT6-dependent manner. Administration of a Wnt agonist as well as transfer of properly polarized M2a macrophages to STAT6−/− mice activated the Wnt signaling pathway in the damaged mucosa and accelerated wound healing. Our results demonstrate that a STAT6-dependent macrophage phenotype promotes mucosal repair in TNBS-treated mice through activation of the Wnt signaling pathway.

Characterization of PsMPK2, the first C1 subgroup MAP kinase from pea (Pisum sativum L.)

Mitogen-activated protein kinase (MAPK) cascades play a key role in plant growth and development as well as in biotic and abiotic stress responses. They are classified according to their sequence homology into four major groups (A–D). A large amount of information about MAPKs in groups A and B is available but few data of the C group have been reported. In this study, a C1 subgroup MAP kinase cDNA, PsMPK2, was isolated from Pisum sativum. PsMPK2 is expressed in vegetative (root and leaf) and reproductive (stamen, pistil and fruit) organs. Expression of PsMPK2 in Arabidopsis thaliana shows that mechanical injury and other stress signals as abscisic acid, jasmonic acid and hydrogen peroxide increase its kinase activity, extending previous results indicating that C1 subgroup MAPKs may be involved in the response to stress.

Hypoxic macrophages impair autophagy in epithelial cells through Wnt1: relevance in IBD

A defective induction of epithelial autophagy may have a role in the pathogenesis of inflammatory bowel diseases. This process is regulated mainly by extracellular factors such as nutrients and growth factors and is highly induced by diverse situations of stress. We hypothesized that epithelial autophagy is regulated by the immune response that in turn is modulated by local hypoxia and inflammatory signals present in the inflamed mucosa. Our results reveal that HIF-1α and Wnt1 were co-localized with CD68 in cells of the mucosa of IBD patients. We have observed increased protein levels of β-catenin, phosphorylated mTOR, and p62 and decreased expression of LC3II in colonic epithelial crypts from damaged mucosa in which β-catenin positively correlated with phosphorylated mTOR and negatively correlated with autophagic protein markers. In cultured macrophages, HIF-1 mediated the increase in Wnt1 expression induced by hypoxia, which enhanced protein levels of β-catenin, activated mTOR, and decreased autophagy in epithelial cells in co-culture. Our results demonstrate a HIF-1-dependent induction of Wnt1 in hypoxic macrophages that undermines autophagy in epithelial cells and suggest a role for Wnt signaling and mTOR pathways in the impaired epithelial autophagy observed in the mucosa of IBD patients.

iNOS-derived nitric oxide mediates the increase in TFF2 expression associated with gastric damage: role of HIF-1

Trefoil (TFF) peptides are involved in gastrointestinal mucosal restitution. An hypoxia inducible factor 1 (HIF‐l)‐dependent induction of TFF genes has been reported in gastric epithelial cells. Nitric oxide (NO) is associated with mucosal damage and modulates HIF‐1 activity. The aim of the present study was to analyze the role of iNOS‐derived NO in HIF‐1 α stabilization and TFF gene expression in damaged gastric mucosa. Aspirin caused gastric injury that peaked 6 h after dosing and returned to normality at 24 h. iNOS mRNA expression occurs in the corpus in parallel with damage. Blockade of iNOS activity did not modify gastric lesions induced by aspirin but delayed mucosal healing. Aspirin induced HIF‐1α stabilization and TFF2 mRNA up‐regulation in the mucosa, but these effects were diminished when iNOS activity was inhibited. Results obtained using a coculture setup showed that iNOS‐derived NO from activated mac‐rophages induced HIF‐1 α stabilization, TFF gene expression’ and accelerated wound healing in cultured epithelial cells. Finally, transient silencing of endogenous HIF‐1α in epithelial cells significantly undermined activated mac‐rophage‐induced TFF gene expression. Evidence suggests that the iNOS‐derived NO associated with NSAID‐induced gastric injury is implicated in mucosal restitution via the HIF‐1‐mediated induction of TFF genes.—Ortiz‐Masiá, D., Hernandez, C., Quintana, E., Velazquez, M., Cebrián, S., Riano, A., Calatayud, S., Esplugues, J. V., Barrachina, M. D. iNOS‐derived nitric oxide mediates the increase in TFF2 expression associated with gastric damage: role of HIF‐1. FASEB J. 24, 136–145 (2010). www.fasebj.org

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.

Autophagy stimulation prevents intestinal mucosal inflammation and ameliorates murine colitis

Defective autophagy contributes to the pathogenesis of inflammatory disorders such as inflammatory bowel disease and there are interactions between autophagy and inflammation. Here we have analysed the effects of autophagy stimulators on murine colitis.