CXCR4-mediated glutamate exocytosis from astrocytes.

The role of astrocytes as structural and metabolic support for neurons is known since the beginning of the last century. Because of their strategic localization between neurons and capillaries they can monitor and control the level of synaptic activity by providing energetic metabolites to neurons and remove excess of neurotransmitters. During the last two decades number of papers further established that the astrocytic plasma-membrane G-protein coupled receptors (GPCR) can sense external inputs (such as the spillover of neurotransmitters) and transduce them as intracellular calcium elevations and release of chemical transmitters such as glutamate. The chemokine CXCR4 receptor is a GPCR widely expressed on glial cells (especially astrocytes and microglia). Activation of the astrocytic CXCR4 by its natural ligand CXCL12 (or SDF1 alpha) results in a long chain of intracellular and extracellular events (including the release of the pro-inflammatory cytokine TNFalpha and prostanglandins) leading to glutamate release. The emerging role of CXCR4-CXCL12 signalling axis in brain physiology came from the recent observation that glutamate in astrocytes is released via a regulated exocytosis process and occurs with a relatively fast time-scale, in the order of few hundred milliseconds. Taking into account that astrocytes are electrically non-excitable and thus exocytosis rely only on a signalling pathway that involves the release Ca(2+) from the internal stores, these results suggested a close relationship between sites of Ca(2+) release and those of fusion events. Indeed, a recent observation describes structural sub-membrane microdomains where fast ER-dependent calcium elevations occur in spatial and temporal correlation with fusion events.

In vivo activation of PPAR target genes by RXR homodimers.

The ability of a retinoid X receptor (RXR) to heterodimerize with many nuclear receptors, including LXR, PPAR, NGF1B and RAR, underscores its pivotal role within the nuclear receptor superfamily. Among these heterodimers, PPAR:RXR is considered an important signalling mediator of both PPAR ligands, such as fatty acids, and 9-cis retinoic acid (9-cis RA), an RXR ligand. In contrast, the existence of an RXR/9-cis RA signalling pathway independent of PPAR or any other dimerization partner remains disputed. Using in vivo chromatin immunoprecipitation, we now show that RXR homodimers can selectively bind to functional PPREs and induce transactivation. At the molecular level, this pathway requires stabilization of the homodimer-DNA complexes through ligand-dependent interaction with the coactivator SRC1 or TIF2. This pathway operates both in the absence and in the presence of PPAR, as assessed in cells carrying inactivating mutations in PPAR genes and in wild-type cells. In addition, this signalling pathway via PPREs is fully functional and can rescue the severe hypothermia phenotype observed in fasted PPARalpha-/- mice. These observations have important pharmacological implications for the development of new rexinoid-based treatments.

Inflammatory diseases: is ubiquitinated NEMO at the hub?

Many patients with Crohn's disease carry mutations in NOD2, a molecule that can both activate and attenuate the pro-inflammatory effects of NF-kappa B. Recent studies implicate NOD2-induced ubiquitination of the NF-kappa B regulator NEMO as a potential means of manipulating the NF-kappa B signal.

Maturation of lymph node fibroblastic reticular cells from myofibroblastic precursors is critical for antiviral immunity.

The stromal scaffold of the lymph node (LN) paracortex is built by fibroblastic reticular cells (FRCs). Conditional ablation of lymphotoxin-β receptor (LTβR) expression in LN FRCs and their mesenchymal progenitors in developing LNs revealed that LTβR-signaling in these cells was not essential for the formation of LNs. Although T cell zone reticular cells had lost podoplanin expression, they still formed a functional conduit system and showed enhanced expression of myofibroblastic markers. However, essential immune functions of FRCs, including homeostatic chemokine and interleukin-7 expression, were impaired. These changes in T cell zone reticular cell function were associated with increased susceptibility to viral infection. Thus, myofibroblasic FRC precursors are able to generate the basic T cell zone infrastructure, whereas LTβR-dependent maturation of FRCs guarantees full immunocompetence and hence optimal LN function during infection.

Proteomic analysis of membrane rafts of melanoma cells identifies protein patterns characteristic of the tumor progression stage.

The molecular mechanisms controlling the progression of melanoma from a localized tumor to an invasive and metastatic disease are poorly understood. In the attempt to start defining a functional protein profile of melanoma progression, we have analyzed by LC-MS/MS the proteins associated with detergent resistant membranes (DRMs), which are enriched in cholesterol/sphingolipids-containing membrane rafts, of melanoma cell lines derived from tumors at different stages of progression. Since membrane rafts are involved in several biological processes, including signal transduction and protein trafficking, we hypothesized that the association of proteins with rafts can be regulated during melanoma development and affect protein function and disease progression. We have identified a total of 177 proteins in the DRMs of the cell lines examined. Among these, we have found groups of proteins preferentially associated with DRMs of either less malignant radial growth phase/vertical growth phase (VGP) cells, or aggressive VGP and metastatic cells suggesting that melanoma cells with different degrees of malignancy have different DRM profiles. Moreover, some proteins were found in DRMs of only some cell lines despite being expressed at similar levels in all the cell lines examined, suggesting the existence of mechanisms controlling their association with DRMs. We expect that understanding the mechanisms regulating DRM targeting and the activity of the proteins differentially associated with DRMs in relation to cell malignancy will help identify new molecular determinants of melanoma progression.

NLRP3 inflammasome is required in murine asthma in the absence of aluminum adjuvant.

Background: Inflammasome activation with the production of IL-1 beta received substantial attention recently in inflammatory diseases. However, the role of inflammasome in the pathogenesis of asthma is not clear. Using an adjuvant-free model of allergic lung inflammation induced by ovalbumin (OVA), we investigated the role of NLRP3 inflammasome and related it to IL-1R1 signaling pathway.Methods: Allergic lung inflammation induced by OVA was evaluated in vivo in mice deficient in NLRP3 inflammasome, IL-1R1, IL-1 beta or IL-1 alpha. Eosinophil recruitment, Th2 cytokine, and chemokine levels were determined in bronchoalveolar lavage fluid, lung homogenates, and mediastinal lymph node cells ex vivo.Results: Allergic airway inflammation depends on NLRP3 inflammasome activation. Dendritic cell recruitment into lymph nodes, Th2 lymphocyte activation in the lung and secretion of Th2 cytokines and chemokines are reduced in the absence of NLRP3. Absence of NLRP3 and IL-1 beta is associated with reduced expression of other proinflammatory cytokines such as IL-5, IL-13, IL-33, and thymic stromal lymphopoietin. Furthermore, the critical role of IL-1R1 signaling in allergic inflammation is confirmed in IL-1R1-, IL-1 beta-, and IL-1 alpha-deficient mice.Conclusion: NLRP3 inflammasome activation leading to IL-1 production is critical for the induction of a Th2 inflammatory allergic response.

Role of salt-inducible kinase 1 in the activation of MEF2-dependent transcription by BDNF.

Substantial evidence supports a role for myocyte enhancer factor 2 (MEF2)-mediated transcription in neuronal survival, differentiation and synaptic function. In developing neurons, it has been shown that MEF2-dependent transcription is regulated by neurotrophins. Despite these observations, little is known about the cellular mechanisms by which neurotrophins activate MEF2 transcriptional activity. In this study, we examined the role of salt-inducible kinase 1 (SIK1), a member of the AMP-activated protein kinase (AMPK) family, in the regulation of MEF2-mediated transcription by the neurotrophin brain-derived neurotrophic factor (BDNF). We show that BDNF increases the expression of SIK1 in primary cultures of rat cortical neurons through the extracellular signal-regulated kinase 1/2 (ERK1/2)-signaling pathway. In addition to inducing SIK1 expression, BDNF triggers the phosphorylation of SIK1 at Thr182 and its translocation from the cytoplasm to the nucleus of cortical neurons. The effects of BDNF on the expression, phosphorylation and, translocation of SIK1 are followed by the phosphorylation and nuclear export of histone deacetylase 5 (HDAC5). Blockade of SIK activity with a low concentration of staurosporine abolished BDNF-induced phosphorylation and nuclear export of HDAC5 in cortical neurons. Importantly, stimulation of HDAC5 phosphorylation and nuclear export by BDNF is accompanied by the activation of MEF2-mediated transcription, an effect that is suppressed by staurosporine. Consistent with these data, BDNF induces the expression of the MEF2 target genes Arc and Nur77, in a staurosporine-sensitive manner. In further support of the role of SIK1 in the regulation of MEF2-dependent transcription by BDNF, we found that expression of wild-type SIK1 or S577A SIK1, a mutated form of SIK1 which is retained in the nucleus of transfected cells, is sufficient to enhance MEF2 transcriptional activity in cortical neurons. Together, these data identify a previously unrecognized mechanism by which SIK1 mediates the activation of MEF2-dependent transcription by BDNF.

The TRAF3-binding site of human molluscipox virus FLIP molecule MC159 is critical for its capacity to inhibit Fas-induced apoptosis.

Members of the viral Flice/caspase-8 inhibitory protein (v-FLIP) family prevent induction of apoptosis by death receptors through inhibition of the processing and activation of procaspase-8 and -10 at the level of the receptor-associated death-inducing signaling complex (DISC). Here, we have addressed the molecular function of the v-FLIP member MC159 of the human molluscum contagiosum virus. MC159 FLIP powerfully inhibited both caspase-dependent and caspase-independent cell death induced by Fas. The C-terminal region of MC159 bound TNF receptor-associated factor (TRAF)3, was necessary for optimal TRAF2 binding, and mediated the recruitment of both TRAFs into the Fas DISC. TRAF-binding-deficient mutants of MC159 showed impaired inhibition of FasL-induced caspase-8 processing and Fas internalization, and had reduced antiapoptotic activity. Our findings provide evidence that a MC159/TRAF2/TRAF3 complex regulates a new aspect of Fas signaling, and identify MC159 FLIP as a molecule that targets multiple features of Fas-induced cell death.

Cholesterol is the major component of native lipoproteins activating the p38 mitogen-activated protein kinases.

Elevated low-density lipoprotein (LDL) levels induce activation of the p38 mitogen-activated protein kinase (MAPK), a stress-activated protein kinase potentially participating in the development of atherosclerosis. The nature of the lipoprotein components inducing p38 MAPK activation has remained unclear however. We show here that both LDLs and high-density lipoproteins (HDLs) have the ability to stimulate the p38 MAPKs with potencies that correlate with their cholesterol content. Cholesterol solubilized in methyl-beta-cyclodextrin was sufficient to activate the p38 MAPK pathway. Liposomes made of phosphatidylcholine (PC) or sphingomyelin, the two main phospholipids found in lipoproteins, were unable to stimulate the p38 MAPKs. In contrast, PC liposomes loaded with cholesterol potently activated this pathway. Reducing the cholesterol content of LDL particles lowered their ability to activate the p38 MAPKs. Cell lines representative of the three main cell types found in blood vessels (endothelial cells, smooth muscle cells and fibroblasts) all activated their p38 MAPK pathway in response to LDLs or cholesterol-loaded PC liposomes. These results indicate that elevated cholesterol content in lipoproteins, as seen in hypercholesterolemia, favors the activation of the stress-activated p38 MAPK pathway in cells from the vessel wall, an event that might contribute to the development of atherosclerosis.

Molecular signaling in zebrafish development and the vertebrate phylotypic period

During development vertebrate embryos pass through a stage where their morphology is most conserved between species, the phylotypic period (approximately the pharyngula). To explain the resistance to evolutionary changes of this period, one hypothesis suggests that it is characterized by a high level of interactions. Based on this hypothesis, we examined protein-protein interactions, signal transduction cascades and miRNAs over the course of zebrafish development, and the conservation of expression of these genes in mouse development. We also investigated the characteristics of genes highly expressed before or during the presumed phylotypic period. We show that while there is a high diversity of interactions during the phylotypic period (protein-DNA, RNA-RNA, cell-cell, and between tissues), which is well conserved with mouse, there is no clear difference with later, more morphologically divergent, stages. We propose that the phylotypic period may rather be the expression at the morphological level of strong conservation of molecular processes earlier in development.

Reactive electrophile species activate defense gene expression in Arabidopsis.

Compounds containing alpha,beta-unsaturated carbonyl groups are increasingly implicated as potent regulators of gene expression; some are powerful cytotoxins known to accumulate at the site of lesion formation in host-pathogen interactions. We used a robust measurement of photosynthetic efficiency to quantify the toxicity of a variety of lipid derivatives in Arabidopsis leaves. Small alpha,beta-unsaturated carbonyl compounds (e.g. acrolein and methyl vinyl ketone) were highly active and proved to be potent stimulators of expression of the pathogenesis-related gene HEL (PR4). These small volatile electrophiles were far more active than larger alkenal homologs like 2(E)-hexenal, and activated HEL expression in a manner independent of salicylate, ethylene, and jasmonate production/perception. Electrophile treatment massively increased the levels of unesterified cyclopentenone jasmonates, which themselves are electrophiles. Patterns of gene expression in response to electrophile treatment and in response to avirulent bacteria were compared, which revealed strikingly similar transcript profiles. The results broaden the range of known biologic effects of reactive electrophile species to include the activation of a pathogenesis-related gene (HEL) and genes involved in metabolism. Electrophiles can act as mediators of both genetic and biochemical effects on core defense signal transduction.

The transcription factor NFATc2 controls IL-6-dependent T cell activation in experimental colitis.

The nuclear factor of activated T cells (NFAT) family of transcription factors controls calcium signaling in T lymphocytes. In this study, we have identified a crucial regulatory role of the transcription factor NFATc2 in T cell-dependent experimental colitis. Similar to ulcerative colitis in humans, the expression of NFATc2 was up-regulated in oxazolone-induced chronic intestinal inflammation. Furthermore, NFATc2 deficiency suppressed colitis induced by oxazolone administration. This finding was associated with enhanced T cell apoptosis in the lamina propria and strikingly reduced production of IL-6, -13, and -17 by mucosal T lymphocytes. Further studies using knockout mice showed that IL-6, rather than IL-23 and -17, are essential for oxazolone colitis induction. Administration of hyper-IL-6 blocked the protective effects of NFATc2 deficiency in experimental colitis, suggesting that IL-6 signal transduction plays a major pathogenic role in vivo. Finally, adoptive transfer of IL-6 and wild-type T cells demonstrated that oxazolone colitis is critically dependent on IL-6 production by T cells. Collectively, these results define a unique regulatory role for NFATc2 in colitis by controlling mucosal T cell activation in an IL-6-dependent manner. NFATc2 in T cells thus emerges as a potentially new therapeutic target for inflammatory bowel diseases.

New insights into the role of PPARs.

Peroxisome proliferator-activated receptors (PPARs) are fatty acid-activated transcription factors belonging to the nuclear hormone receptor family. While PPARs are best known as regulators of energy homeostasis, evidence also has accumulated recently for their involvement in basic cellular functions. We review novel insights into PPAR functions in skin wound healing and liver, with emphasis on PPARβ/δ and PPARα, respectively. Activation of PPARβ/δ expression in response to injury promotes keratinocyte survival, directional sensing, and migration over the wound bed. In addition, interleukin (IL)-1 produced by the keratinocytes activates PPARβ/δ expression in the underlying fibroblasts, which hinders the mitotic activity of keratinocytes via inhibition of IL-1 signaling. Initially, roles were identified for PPARα in fatty acid catabolism. However, PPARα is also involved in downregulating many genes in female mammals. We have elucidated the mechanism of this repression, which requires sumoylation of PPARα. Physiologically, this control confers protection against estrogen-induced intrahepatic cholestasis.

Detritivorous crustaceans become herbivores on jasmonate-deficient plants.

The jasmonate signal pathway is known to control defenses against herbivores, such as leaf eaters (folivores). Does the reach of the pathway extend to defense against other types of animal? Among the arthropods attracted to seed baits placed below flowering Arabidopsis thaliana plants are 2 largely nocturnal isopod crustaceans generally considered as detritivores: Porcellio scaber and Armadillidium vulgare. Parallel laboratory experiments identified the isopods as being capable of predation on intact plants. Isopod feeding was strongly facilitated in jasmonate-deficient Arabidopsis and rice plants. The feeding activity of isopods revealed potentially detritivore-sensitive, jasmonate-protected Achilles' heels in these architecturally different plants (petioles and inflorescence stems in Arabidopsis, and lower stem and mesocotyl in rice). The work addresses the question of what stops 2 detritivores from attacking living plants and provides evidence that it is, in part, the jasmonate signal pathway. Furthermore, senescent leaves from an Arabidopsis jasmonate mutant were consumed more rapidly than senescent wild-type leaves, suggesting that past activity of the jasmonate signal pathway in leaves may slow carbon recycling through detritivory.

Increased Wnt signaling triggers oncogenic conversion of human breast epithelial cells by a Notch-dependent mechanism.

Wnt and Notch signaling have long been established as strongly oncogenic in the mouse mammary gland. Aberrant expression of several Wnts and other components of this pathway in human breast carcinomas has been reported, but evidence for a causative role in the human disease has been missing. Here we report that increased Wnt signaling, as achieved by ectopic expression of Wnt-1, triggers the DNA damage response (DDR) and an ensuing cascade of events resulting in tumorigenic conversion of primary human mammary epithelial cells. Wnt-1-transformed cells have high telomerase activity and compromised p53 and Rb function, grow as spheres in suspension, and in mice form tumors that closely resemble medullary carcinomas of the breast. Notch signaling is up-regulated through a mechanism involving increased expression of the Notch ligands Dll1, Dll3, and Dll4 and is required for expression of the tumorigenic phenotype. Increased Notch signaling in primary human mammary epithelial cells is sufficient to reproduce some aspects of Wnt-induced transformation. The relevance of these findings for human breast cancer is supported by the fact that expression of Wnt-1 and Wnt-4 and of established Wnt target genes, such as Axin-2 and Lef-1, as well as the Notch ligands, such as Dll3 and Dll4, is up-regulated in human breast carcinomas.