Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule.

Cell death is achieved by two fundamentally different mechanisms: apoptosis and necrosis. Apoptosis is dependent on caspase activation, whereas the caspase-independent necrotic signaling pathway remains largely uncharacterized. We show here that Fas kills activated primary T cells efficiently in the absence of active caspases, which results in necrotic morphological changes and late mitochondrial damage but no cytochrome c release. This Fas ligand-induced caspase-independent death is absent in T cells that are deficient in either Fas-associated death domain (FADD) or receptor-interacting protein (RIP). RIP is also required for necrotic death induced by tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). In contrast to its role in nuclear factor kappa B activation, RIP requires its own kinase activity for death signaling. Thus, Fas, TRAIL and TNF receptors can initiate cell death by two alternative pathways, one relying on caspase-8 and the other dependent on the kinase RIP.

Novel Agents Targeting the IGF-1R/PI3K Pathway Impair Cell Proliferation and Survival in Subsets of Medulloblastoma and Neuroblastoma.

The receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) pathway is fundamental for cancer cell proliferation and is known to be frequently altered and activated in neoplasia, including embryonal tumors. Based on the high frequency of alterations, targeting components of the PI3K signaling pathway is considered to be a promising therapeutic approach for cancer treatment. Here, we have investigated the potential of targeting the axis of the insulin-like growth factor-1 receptor (IGF-1R) and PI3K signaling in two common cancers of childhood: neuroblastoma, the most common extracranial tumor in children and medulloblastoma, the most frequent malignant childhood brain tumor. By treating neuroblastoma and medulloblastoma cells with R1507, a specific humanized monoclonal antibody against the IGF-1R, we could observe cell line-specific responses and in some cases a strong decrease in cell proliferation. In contrast, targeting the PI3K p110α with the specific inhibitor PIK75 resulted in broad anti-proliferative effects in a panel of neuro- and medulloblastoma cell lines. Additionally, sensitization to commonly used chemotherapeutic agents occurred in neuroblastoma cells upon treatment with R1507 or PIK75. Furthermore, by studying the expression and phosphorylation state of IGF-1R/PI3K downstream signaling targets we found down-regulated signaling pathway activation. In addition, apoptosis occurred in embryonal tumor cells after treatment with PIK75 or R1507. Together, our studies demonstrate the potential of targeting the IGF-1R/PI3K signaling axis in embryonal tumors. Hopefully, this knowledge will contribute to the development of urgently required new targeted therapies for embryonal tumors.

Androgenetic alopecia: identification of four genetic risk loci and evidence for the contribution of WNT signaling to its etiology.

The pathogenesis of androgenetic alopecia (AGA, male-pattern baldness) is driven by androgens, and genetic predisposition is the major prerequisite. Candidate gene and genome-wide association studies have reported that single-nucleotide polymorphisms (SNPs) at eight different genomic loci are associated with AGA development. However, a significant fraction of the overall heritable risk still awaits identification. Furthermore, the understanding of the pathophysiology of AGA is incomplete, and each newly associated locus may provide novel insights into contributing biological pathways. The aim of this study was to identify unknown AGA risk loci by replicating SNPs at the 12 genomic loci that showed suggestive association (5 × 10(-8)<P<10(-5)) with AGA in a recent meta-analysis. We analyzed a replication set comprising 2,759 cases and 2,661 controls of European descent to confirm the association with AGA at these loci. Combined analysis of the replication and the meta-analysis data identified four genome-wide significant risk loci for AGA on chromosomes 2q35, 3q25.1, 5q33.3, and 12p12.1. The strongest association signal was obtained for rs7349332 (P=3.55 × 10(-15)) on chr2q35, which is located intronically in WNT10A. Expression studies in human hair follicle tissue suggest that WNT10A has a functional role in AGA etiology. Thus, our study provides genetic evidence supporting an involvement of WNT signaling in AGA development.

Experimental cerebral malaria develops independently of caspase recruitment domain-containing protein 9 signaling.

The outcome of infection depends on multiple layers of immune regulation, with innate immunity playing a decisive role in shaping protection or pathogenic sequelae of acquired immunity. The contribution of pattern recognition receptors and adaptor molecules in immunity to malaria remains poorly understood. Here, we interrogate the role of the caspase recruitment domain-containing protein 9 (CARD9) signaling pathway in the development of experimental cerebral malaria (ECM) using the murine Plasmodium berghei ANKA infection model. CARD9 expression was upregulated in the brains of infected wild-type (WT) mice, suggesting a potential role for this pathway in ECM pathogenesis. However, P. berghei ANKA-infected Card9(-/-) mice succumbed to neurological signs and presented with disrupted blood-brain barriers similar to WT mice. Furthermore, consistent with the immunological features associated with ECM in WT mice, Card9(-/-) mice revealed (i) elevated levels of proinflammatory responses, (ii) high frequencies of activated T cells, and (iii) CD8(+) T cell arrest in the cerebral microvasculature. We conclude that ECM develops independently of the CARD9 signaling pathway.

Merlin, a "Magic" Linker between Extracellular Cues and Intracellular Signaling Pathways that Regulate Cell Motility, Proliferation, and Survival.

Genetic alterations of neurofibromatosis type 2 (NF2) gene lead to the development of schwannomas, meningiomas, and ependymomas. Mutations of NF2 gene were also found in thyroid cancer, mesothelioma, and melanoma, suggesting that it functions as a tumor suppressor in a wide spectrum of cells. The product of NF2 gene is merlin (moesin-ezrin-radixin-like protein), a member of the Band 4.1 superfamily proteins. Merlin shares significant sequence homology with the ERM (Ezrin-Radixin-Moesin) family proteins and serves as a linker between transmembrane proteins and the actin-cytoskeleton. Merlin is a multifunctional protein and involved in integrating and regulating the extracellular cues and intracellular signaling pathways that control cell fate, shape, proliferation, survival, and motility. Recent studies showed that merlin regulates the cell-cell and cell-matrix adhesions and functions of the cell surface adhesion/extracellular matrix receptors including CD44 and that merlin and CD44 antagonize each other's function and work upstream of the mammalian Hippo signaling pathway. Furthermore, merlin plays important roles in stabilizing the contact inhibition of proliferation and in regulating activities of several receptor tyrosine kinases. Accumulating data also suggested an emerging role of merlin as a negative regulator of growth and progression of several non-NF2 associated cancer types. Together, these recent advances have improved our basic understanding about merlin function, its regulation, and the major signaling pathways regulated by merlin and provided the foundation for future translation of these findings into the clinic for patients bearing the cancers in which merlin function and/or its downstream signaling pathways are impaired or altered.

The puzzles of the prokineticin 2 pathway in human reproduction.

Prokineticin, 1 (PROK1) and prokineticin 2 (PROK2), are two closely related proteins that were identified as the mammalian homologs of their two amphibian homologs, mamba intestinal toxin (MIT-1) and Bv8. MIT-1 was initially identified as a non-toxic constituent in the venom of the black mamba snake (Dendroaspis polylepis) (Joubert and Strydom, 1980) while Bv8 was identified in the skin secretion of the toad, Bombina variegate (Mollay et al., 1999). All three homologs stimulate gastrointestinal motility thus accounting for their family name "prokineticins" (Schweitz et al., 1990, 1999). However, since its initial description, both PROK1 and PROK2 have been found to regulate a dazzling array of biological functions throughout the body. In particular, PROK1 acts as a potent angiogenic mitogen on endocrine vascular epithelium, thus earning its other name, Endocrine gland-vascular endothelial factor (EG-VEGF) (LeCouter et al., 2002). In contrast, the PROK2 signaling pathway is a critical regulator of olfactory bulb morphogenesis and sexual maturation in mammals and this function is the focus of this review.

Yellow submarine of the Wnt/Frizzled signaling: submerging from the G protein harbor to the targets.

The Wnt/Frizzled signaling pathway plays multiple functions in animal development and, when deregulated, in human disease. The G-protein coupled receptor (GPCR) Frizzled and its cognate heterotrimeric Gi/o proteins initiate the intracellular signaling cascades resulting in cell fate determination and polarization. In this review, we summarize the knowledge on the ligand recognition, biochemistry, modifications and interacting partners of the Frizzled proteins viewed as GPCRs. We also discuss the effectors of the heterotrimeric Go protein in Frizzled signaling. One group of these effectors is represented by small GTPases of the Rab family, which amplify the initial Wnt/Frizzled signal. Another effector is the negative regulator of Wnt signaling Axin, which becomes deactivated in response to Go action. The discovery of the GPCR properties of Frizzled receptors not only provides mechanistic understanding to their signaling pathways, but also paves new avenues for the drug discovery efforts.

Regulation of epithelial-mesenchymal IL-1 signaling by PPARbeta/delta is essential for skin homeostasis and wound healing.

Skin morphogenesis, maintenance, and healing after wounding require complex epithelial-mesenchymal interactions. In this study, we show that for skin homeostasis, interleukin-1 (IL-1) produced by keratinocytes activates peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) expression in underlying fibroblasts, which in turn inhibits the mitotic activity of keratinocytes via inhibition of the IL-1 signaling pathway. In fact, PPARbeta/delta stimulates production of the secreted IL-1 receptor antagonist, which leads to an autocrine decrease in IL-1 signaling pathways and consequently decreases production of secreted mitogenic factors by the fibroblasts. This fibroblast PPARbeta/delta regulation of the IL-1 signaling is required for proper wound healing and can regulate tumor as well as normal human keratinocyte cell proliferation. Together, these findings provide evidence for a novel homeostatic control of keratinocyte proliferation and differentiation mediated via PPARbeta/delta regulation in dermal fibroblasts of IL-1 signaling. Given the ubiquitous expression of PPARbeta/delta, other epithelial-mesenchymal interactions may also be regulated in a similar manner.

ER stress activates the NLRP3 inflammasome via an UPR-independent pathway.

Uncontrolled endoplasmic reticulum (ER) stress responses are proposed to contribute to the pathology of chronic inflammatory diseases such as type 2 diabetes or atherosclerosis. However, the connection between ER stress and inflammation remains largely unexplored. Here, we show that ER stress causes activation of the NLRP3 inflammasome, with subsequent release of the pro-inflammatory cytokine interleukin-1β. This ER-triggered proinflammatory signal shares the same requirement for reactive oxygen species production and potassium efflux compared with other known NLRP3 inflammasome activators, but is independent of the classical unfolded protein response (UPR). We thus propose that the NLRP3 inflammasome senses and responds to ER stress downstream of a previously uncharacterized ER stress response signaling pathway distinct from the UPR, thus providing mechanistic insight to the link between ER stress and chronic inflammatory diseases.

Atopic dermatitis-like disease and associated lethal myeloproliferative disorder arise from loss of Notch signaling in the murine skin.

BACKGROUND: The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. Moreover, skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. However, much less is known about the consequences of loss of Notch signaling after birth. METHODOLOGY AND PRINCIPAL FINDINGS: To study the function of Notch signaling in the skin of adult mice, we made use of a series of conditional gene targeted mice that allow inactivation of several components of the Notch signaling pathway specifically in the skin. We demonstrate that skin-specific inactivation of Notch1 and Notch2 simultaneously, or RBP-J, induces the development of a severe form of atopic dermatitis (AD), characterized by acanthosis, spongiosis and hyperkeratosis, as well as a massive dermal infiltration of eosinophils and mast cells. Likewise, patients suffering from AD, but not psoriasis or lichen planus, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is cell non-autonomous and caused by dramatic microenvironmental alterations. Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia. SIGNIFICANCE: Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.

Characterization of cytoplasmic antiviral signaling pathways

Summary Multicellular organisms have evolved the immune system to protect from pathogen such as viruses, bacteria, fungi or parasites. Detection of invading pathogens by the host innate immune system is crucial for mounting protective responses and depends on the recognition of microbial components by specific receptors. The results presented in this manuscript focus on the signaling pathways involved in the detection of viral infection by the sensing of viral nucleic acids. First, we describe a new regulatory mechanism controlling RNA-sensing antiviral pathways. Our results indicate that TRIF and Cardif, the crucial adaptor proteins for endosomal and cytoplasmic RNA detection signaling pathway, are processed and inactivated by caspases. The second aspect investigated here involves a signaling pathway triggered upon cytosolic DNA sensing. The interferon inducible protein DAI was recently described as a DNA sensor able to induce the activation of IRFs and NF-κΒ transcription factors leading to type I interferon production. Here we identify two RIP homotypic interaction motifs (RHIMs) in DAI and demonstrate that they mediate the recruitment of RIP1 and RIP3 and the subsequent NF-κΒ activation. Moreover, we observed that the mouse cytomegalovirus RHIM- containing protein M45 has the potential to block this signaling cascade by interfering with the formation of the DAI-RIP1/3 signaling complex. Finally, we report the generation and the initial characterization of NLRX1-deficient mice. NLRX1 is a member of the NOD-like receptor family localized to the mitochondria. The function of NLRX1 is still controversial: one study proposed that NLRX1 acts as an inhibitor of the RIG-like receptor (RLR) antiviral pathway by binding the adaptor protein Cardif, whereas another report implicated NLRX1 in the generation of reactive oxygen species (ROS) and the amplification of NF-κΒ and JNK triggered by TNF-α, poly(I:C) or Shigella infection. Collectively, our results indicate that NLRX1-deficiency does not affect RLR signaling nor TNF-α induced responses. Proteomics analysis identified UQCRC2, a subunit of the complex III of the mitochondrial respiratory chain, as a NLRX1 binding partner. This observation might reveal a possible functional link between NLRX1 and mitochondrial respiration and/or ROS generation. Résumé Au cours de l'évolution, les organismes multicellulaires ont développé le système immunitaire afin de se protéger contre les pathogènes. Une étape cruciale pour le déclenchement des réponses protectrices est la reconnaissance par les cellules du système immunitaire de molécules propres aux microbes grâce à des récepteurs spécifiques. Les résultats présentés dans cette thèse décrivent des nouveaux aspects concernant les voies de signalisation impliquées dans la détection des virus. Le premier projet décrit un mécanisme de régulation des voies activées par la détection d'ARN virale. Nos résultats montrent que TRIF et Cardif, des protéines adaptatrices des voies déclenchées par la reconnaissance de ces acides nucléiques au niveau des endosomes et du cytoplasme, sont clivés et inactivés par les caspases. Le projet suivant de notre recherche concerne une voie de signalisation activée par la détection d'ADN au niveau du cytoplasme. La protéine DAI a été récemment décrite comme un senseur pour cet ADN capable d'activer les facteurs de transcription IRF et NF-κΒ et d'induire ainsi la production des interférons de type I. Ici on démontre que DAI interagit avec RIP1 et RIP3 par le biais de domaines appelés RHIM et que ce complexe est responsable de l'activation de NF-κΒ. On a aussi identifié une protéine du cytomégalovirus de la souris, M45, qui contient ce même domaine et on a pu démontrer qu'elle a la capacité d'interférer avec la formation du complexe entre DAI et RIP1/RIP3 bloquant ainsi l'activation de NF-κΒ. Enfin on décrit ici la génération de souris déficientes pour le gène qui code pour la protéine NLRX1. Cette protéine fait partie de la famille des récepteurs NOD et est localisée dans la mitochondrie. Une étude a suggéré que NLRX1 agit comme un inhibiteur des voies antivirales activées par les récepteurs du type RIG-I (RLR) en interagissant avec la protéine adaptatrice Cardif. Une autre étude propose par contre que NLRX1 participe à la production des dérivés réactifs de l'oxygène et contribue ainsi à augmenter l'activation de NF- κΒ et JNK induite par le TNF-α ou le poly(I:C). Nos résultats montrent que l'absence de NLRX1 ne modifie ni la voie de signalisation RLR ni les réponses induites par le TNF-α. Des analyses ultérieures ont permis d'identifier comme partenaire d'interaction de NLRX1 la protéine UQCRC2, une des sous-unités qui composent le complexe III de la chaîne respiratoire mitochondriale. Cette observation pourrait indiquer un lien fonctionnel entre NLRX1 et la respiration mitochondriale ou la production des dérivés réactifs de l'oxygène au niveau de cette organelle.

BCL9 is an essential component of canonical Wnt signaling that mediates the differentiation of myogenic progenitors during muscle regeneration.

Muscle stem cells and their progeny play a fundamental role in the regeneration of adult skeletal muscle. We have previously shown that activation of the canonical Wnt/beta-catenin signaling pathway in adult myogenic progenitors is required for their transition from rapidly dividing transient amplifying cells to more differentiated progenitors. Whereas Wnt signaling in Drosophila is dependent on the presence of the co-regulator Legless, previous studies of the mammalian ortholog of Legless, BCL9 (and its homolog, BCL9-2), have not revealed an essential role of these proteins in Wnt signaling in specific tissues during development. Using Cre-lox technology to delete BCL9 and BCL9-2 in the myogenic lineage in vivo and RNAi technology to knockdown the protein levels in vitro, we show that BCL9 is required for activation of the Wnt/beta-catenin cascade in adult mammalian myogenic progenitors. We observed that the nuclear localization of beta-catenin and downstream TCF/LEF-mediated transcription, which are normally observed in myogenic progenitors upon addition of exogenous Wnt and during muscle regeneration, were abrogated when BCL9/9-2 levels were reduced. Furthermore, reductions of BCL9/9-2 inhibited the promotion of myogenic differentiation by Wnt and the normal regenerative response of skeletal muscle. These results suggest a critical role of BCL9/9-2 in the Wnt-mediated regulation of adult, as opposed to embryonic, myogenic progenitors.

A fresh look at interferon-alpha signaling and treatment outcomes in chronic hepatitis C.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The current standard therapy for chronic hepatitis C (CHC) consists of a combination of pegylated IFN alpha (pegIFNalpha) and ribavirin. It achieves a sustained viral clearance in only 50-60% of patients. To learn more about molecular mechanisms underlying treatment failure, we investigated IFN-induced signaling in paired liver biopsies collected from CHC patients before and after administration of pegIFNalpha. In patients with a rapid virological response to treatment, pegIFNalpha induced a strong up-regulation of IFN-stimulated genes (ISGs). As shown previously, nonresponders had high expression levels of ISGs before therapy. Analysis of posttreatment biopsies of these patients revealed that pegIFNalpha did not induce expression of ISGs above the pretreatment levels. In accordance with ISG expression data, phosphorylation, DNA binding, and nuclear localization of STAT1 indicated that the IFN signaling pathway in nonresponsive patients is preactivated and refractory to further stimulation. Some features characteristic of nonresponders were more accentuated in patients infected with HCV genotypes 1 and 4 compared with genotypes 2 and 3, providing a possible explanation for the poor response of the former group to therapy. Taken together with previous findings, our data support the concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection but also may impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway.

Contribution of S6K1/MAPK signaling pathways in the response to oxidative stress: activation of RSK and MSK by hydrogen peroxide

Cells respond to different kind of stress through the coordinated activation of signaling pathways such as MAPK or p53. To find which molecular mechanisms are involved, we need to understand their cell adaptation. The ribosomal protein, S6 kinase 1 (S6K1), is a common downstream target of signaling by hormonal or nutritional stress. Here, we investigated the initial contribution of S6K1/MAPK signaling pathways in the cell response to oxidative stress produced by hydrogen peroxide (H2O2). To analyze S6K1 activation, we used the commercial anti-phospho-Thr389-S6K1 antibody most frequently mentioned in the bibliography. We found that this antibody detected an 80-90 kDa protein that was rapidly phosphorylated in response to H2O2 in several human cells. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI3K inhibitors, and knock-down experiments showed that this protein was not S6K1. RSK and MSK proteins were candidate targets of this phosphorylation. We demonstrated that H2O2 stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. This phosphorylation required the activity of either p38 or ERK MAP kinases. Kinase assays showed activation of RSK and MSK by H2O2. Experiments with mouse embryonic fibroblasts from p38 animals" knockout confirmed these observations. Altogether, these findings show that the S6K1 signaling pathway is not activated under these conditions, clarify previous observations probably misinterpreted by non-specific detection of proteins RSK and MSK by the anti-phospho-Thr389-S6K1 antibody, and demonstrate the specific activation of MAPK signaling pathways through ERK/p38/RSK/MSK by H2O2.

Notch signaling in the immune system.

The Notch signaling pathway regulates many aspects of embryonic development, as well as differentiation processes and tissue homeostasis in multiple adult organ systems. Disregulation of Notch signaling is associated with several human disorders, including cancer. In the last decade, it became evident that Notch signaling plays important roles within the hematopoietic and immune systems. Notch plays an essential role in the development of embryonic hematopoietic stem cells and influences multiple lineage decisions of developing lymphoid and myeloid cells. Moreover, recent evidence suggests that Notch is an important modulator of T cell-mediated immune responses. In this review, we discuss Notch signaling in hematopoiesis, lymphocyte development, and function as well as in T cell acute lymphoblastic leukemia.