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Collagen is one the most abundant proteins and the main cargo of the secretory pathway, contributing to hepatic fibrosis and cirrhosis due to excessive deposition of extracellular matrix. Here we investigated the possible contribution of the unfolded protein response, the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 reduced liver damage and diminished collagen deposition in models of liver fibrosis triggered by carbon tetrachloride (CCl4) administration or by high fat diet. Proteomic and transcriptomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1), which is known to be critical for collagen maturation, as a major IRE1-induced gene. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER and altered secretion, a phenotype rescued by P4HB overexpression. Taken together, our results collectively establish a role of the IRE1/P4HB axis in the regulation of collagen production and its significance in the pathogenesis of various disease states.

The endoplasmic reticulum (ER) stress sensor IRE1 is a a major player of the unfolded protein response (UPR), the main pathway driving adaptation processes to restore proteostasis. In addition, overactivation of IRE1 signaling contributes to a variety of pathologies including diabetes, neurodegenerative diseases, and cancer. Under ER stress, IRE1 auto-transphosphorylates and oligomerizes, triggering the activation of its endoribonuclease domain located in the cytosolic region. Active IRE1 catalyzes the splicing of the mRNA encoding for the XBP1 transcription factor, in addition to degrade several RNAs through a process known as regulated IRE1-dependent decay of mRNA (RIDD). Besides its role as an UPR transducer, several posttranslational modifications and protein-protein interactions can regulate IRE1 activity and modulate its signaling in the absence of stress. Thus, investigating the function of IRE1 in physiology and disease requires the use of complementary approaches. Here, we provide detailed protocols to perform four different assays to study IRE1 activation and signaling: (i) Phos-tag gels to evaluate the phosphorylation status of IRE1, (ii) microscopy using TREX-IRE1-GFP cells to measure IRE1 oligomerization, (iii) conventional RT-PCR to assess XBP1 mRNA processing, and (iv) quantitative PCR to determine the levels of canonical UPR target genes and the degradation of several mRNAs that are target of RIDD. We propose to use these experimental strategies as "gold standards" to study IRE1 signaling.

Recessive gene mutations underlie many developmental disorders and often lead to disabling neurological problems. Here, we report identification of a homozygous c.170G>A (p.Cys57Tyr or C57Y) mutation in the gene coding for protein disulfide isomerase A3 (PDIA3, also known as ERp57), an enzyme that catalyzes formation of disulfide bonds in the endoplasmic reticulum, to be associated with syndromic intellectual disability. Experiments in zebrafish embryos show that PDIA3C57Y expression is pathogenic and causes developmental defects such as axonal disorganization as well as skeletal abnormalities. Expression of PDIA3C57Y in the mouse hippocampus results in impaired synaptic plasticity and memory consolidation. Proteomic and functional analyses reveal that PDIA3C57Y expression leads to dysregulation of cell adhesion and actin cytoskeleton dynamics, associated with altered integrin biogenesis and reduced neuritogenesis. Biochemical studies show that PDIA3C57Y has decreased catalytic activity and forms disulfide-crosslinked aggregates that abnormally interact with chaperones in the endoplasmic reticulum. Thus, rare disease gene variant can provide insight into how perturbations of neuronal proteostasis can affect the function of the nervous system.

Description, RECS1, a member of the TMBIM family, triggers lysosomal membrane permeabilization in cells undergoing lysosomal stress., Programmed cell death is regulated by the balance between activating and inhibitory signals. Here, we have identified RECS1 (responsive to centrifugal force and shear stress 1) [also known as TMBIM1 (transmembrane BAX inhibitor motif containing 1)] as a proapoptotic member of the TMBIM family. In contrast to other proteins of the TMBIM family, RECS1 expression induces cell death through the canonical mitochondrial apoptosis pathway. Unbiased screening indicated that RECS1 sensitizes cells to lysosomal perturbations. RECS1 localizes to lysosomes, where it regulates their acidification and calcium content, triggering lysosomal membrane permeabilization. Structural modeling and electrophysiological studies indicated that RECS1 is a pH-regulated calcium channel, an activity that is essential to trigger cell death. RECS1 also sensitizes whole animals to stress in vivo in Drosophila melanogaster and zebrafish models. Our results unveil an unanticipated function for RECS1 as a proapoptotic component of the TMBIM family that ignites cell death programs at lysosomes.

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1α (IRE1α), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP-1 (X-box-binding protein-1). Through a global proteomic approach we identified the BCL-2 family member PUMA as a novel IRE1α interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1α with BIM, another BH3-only protein. BIM and PUMA double-knockout cells failed to maintain sustained XBP-1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1α, inhibiting its effects on XBP-1 mRNA splicing. Unexpectedly, this regulation required BCL-2 and was antagonized by BAD or the BH3 domain mimetic ABT-737. The modulation of IRE1α RNAse activity by BH3-only proteins was recapitulated in a cell-free system suggesting a direct regulation. Moreover, BH3-only proteins controlled XBP-1 mRNA splicing in vivo and affected the ER stress-regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL-2 family members participates in a new UPR-regulatory network, thus assuming apoptosis-unrelated functions.

In the version of this Article originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Article.

The development of the central nervous system requires a series of morphogenetic events that shape brain and spinal cord structures. Several brain regions and neural circuits are formed by differential gene expression patterns and cell migration events involving neurons. During neurogenesis and neuritogenesis, increased demand for protein synthesis occurs to express key neuronal proteins to generate axons, dendrites, and synapsis. The endoplasmic reticulum (ER) is a central hub controlling protein homeostasis (proteostasis), impacting a wide range of cellular processes required for brain function. Although most of the field has focused on studying the role of ER stress in neurodegenerative diseases marked by abnormal protein aggregation, accumulating evidence indicates that ER proteostasis contributes to brain development and may impact neurodevelopmental processes such as neuronal migration, differentiation, and function. Here, we review emerging evidence linking neurodevelopment with ER proteostasis and its relevance to human disorders.

Caveolin-1 (CAV1), is a broadly expressed, membrane-associated scaffolding protein that acts both, as a tumor suppressor and a promoter of metastasis, depending on the type of cancer and stage. CAV1 is downregulated in human tumors, tumor cell lines and oncogene-transformed cells. The tumor suppressor activity of CAV1 is generally associated with its presence at the plasma membrane, where it participates, together with cavins, in the formation of caveolae and also has been suggested to interact with and inhibit a wide variety of proteins through interactions mediated by the scaffolding domain. However, a pool of CAV1 is also located at the endoplasmic reticulum (ER), modulating the secretory pathway in a manner dependent on serine-80 (S80) phosphorylation. In melanoma cells, CAV1 expression suppresses tumor formation, but the protein is largely absent from the plasma membrane and does not form caveolae. Perturbations to the function of the ER are emerging as a central driver of cancer, highlighting the activation of the unfolded protein response (UPR), a central pathway involved in stress mitigation. Here we provide evidence indicating that the expression of CAV1 represses the activation of the UPR in vitro and in solid tumors, reflected in the attenuation of PERK and IRE1α signaling. These effects correlated with increased susceptibility of cells to ER stress and hypoxia. Interestingly, the tumor suppressor activity of CAV1 was abrogated by site-directed mutagenesis of S80, correlating with a reduced ability to repress the UPR. We conclude that the tumor suppression by CAV1 involves the attenuation of the UPR, and identified S80 as essential in this context. This suggests that intracellular CAV1 regulates cancer through alternative signaling outputs.

Different perturbations alter the function of the endoplasmic reticulum (ER), resulting in the accumulation of misfolded proteins in its lumen, a condition termed ER stress. To restore ER proteostasis, a highly conserved pathway is engaged, known as the unfolded protein response (UPR), triggering adaptive programs or apoptosis of terminally damaged cells. IRE1α (also known as ERN1), the most conserved UPR sensor, mediates the activation of responses to determine cell fate under ER stress. The complexity of IRE1α regulation and its signaling outputs is mediated in part by the assembly of a dynamic multi-protein complex, named the UPRosome, that regulates IRE1α activity and the crosstalk with other pathways. We discuss several studies identifying components of the UPRosome that have illuminated novel functions in cell death, autophagy, DNA damage, energy metabolism and cytoskeleton dynamics. Here, we provide a theoretical analysis to assess the biological significance of the UPRosome and present the results of a systematic bioinformatics analysis of the available IRE1α interactome data sets followed by functional enrichment clustering. This in silico approach decoded that IRE1α also interacts with proteins involved in the cell cycle, transport, differentiation, response to viral infection and immune response. Thus, defining the spectrum of IRE1α-binding partners will reveal novel signaling outputs and the relevance of the pathway to human diseases.

Cyclosporine, a widely used immunosuppressant in organ transplantation and in treatment of various autoimmune diseases, activates the unfolded protein response (UPR), an ER stress coping response. In this study we discovered a new and unanticipated cyclosporine-dependent signaling pathway, with cyclosporine triggering direct activation of the UPR. COX-2 binds to and activates IRE1α, leading to IRE1α splicing of XBP1 mRNA. Molecular interaction and modeling analyses identified a novel interaction site for cyclosporine with COX-2 which caused enhancement of COX-2 enzymatic activity required for activation of the IRE1α branch of the UPR. Cyclosporine-dependent activation of COX-2 and IRE1α in mice indicated that cyclosporine-COX-2-IRE1α signaling pathway was functional in vivo. These findings identify COX-2 as a new IRE1α binding partner and regulator of the IRE1α branch of the UPR pathway, and establishes the mechanism underlying cytotoxicity associated with chronic cyclosporine exposure.

Cancer cells are exposed to adverse conditions within the tumor microenvironment that challenge cells to adapt and survive. Several of these homeostatic perturbations insults alter the normal function of the endoplasmic reticulum (ER), resulting in the accumulation of misfolded proteins. ER stress triggers a conserved signaling pathway known as the unfolded protein response (UPR) to cope with the stress or trigger apoptosis of damaged cells. The UPR has been described as a major driver in the acquisition of malignant characteristics that ultimately lead to cancer progression. Although, several reports describe the relevance of the UPR in tumor growth, the possible crosstalk with other cancer-related pathways is starting to be elucidated. The Forkhead Box O (FoxO) subfamily of proteins has a major role in cancer progression, where chromosomal translocations and deregulated signaling lead to loss-of-function of FoxO proteins, contributing to tumor progression. Here we discuss the homeostatic connection between the UPR and FoxO proteins and its possible implications to tumor progression and the acquisition of several hallmarks of cancer. In addition, studies linking a crosstalk between the UPR and FoxO proteins in other diseases, including neurodegeneration and metabolic disorders is provided.

Mitochondria-associated membranes (MAMs) are central microdomains that fine-tune bioenergetics by the local transfer of calcium from the endoplasmic reticulum to the mitochondrial matrix. Here, we report an unexpected function of the endoplasmic reticulum stress transducer IRE1α as a structural determinant of MAMs that controls mitochondrial calcium uptake. IRE1α deficiency resulted in marked alterations in mitochondrial physiology and energy metabolism under resting conditions. IRE1α determined the distribution of inositol-1,4,5-trisphosphate receptors at MAMs by operating as a scaffold. Using mutagenesis analysis, we separated the housekeeping activity of IRE1α at MAMs from its canonical role in the unfolded protein response. These observations were validated in vivo in the liver of IRE1α conditional knockout mice, revealing broad implications for cellular metabolism. Our results support an alternative function of IRE1α in orchestrating the communication between the endoplasmic reticulum and mitochondria to sustain bioenergetics. This work was funded by FONDECYT 1140549, FONDAP program 15150012, the Millennium Institute P09-015-F and the European Commission R&D MSCA-RISE 734749 (to C.H.); the Michael J. Fox Foundation for Parkinson’s Research target validation grant number 9277, FONDEF ID16I10223, FONDEF D11E1007, US Office of Naval Research-Global N62909-16-1-2003, US Air Force Office of Scientific Research FA9550-16-1-0384, ALSRP Therapeutic Idea Award AL150111, Muscular Dystrophy Association 382453, Seed grant Leading House for the Latin American Region, Switzerland and CONICYT-Brazil 441921/2016-7 (to C.H.), FONDECYT 1160332 and FONDAP15150012 (to J.C.C.); the Spanish Ministry of Economy and Competitiveness SAF2014-52228-R, Unidad de Excelencia María de Maeztu, funded by the MINECO (ref: MDM-2014-0370) and Fundació la Marató de TV3 20134030 (to R.V.); NIH NS095892 (to R.L.W.); FONDECYT 11180825 (to H.U.); FONDECYT 3150113 and EMBO ASTF 385-2016 (to A.C.-S.); FONDECYT 3140355 (to E.R.-F.); FONDECYT 3140458 and 11170291 (to F.J.); FONDECYT 3180427 (to Y.H.); FONDECYT 3190738 (to A.S.-C.); FONDECYT 11170546 and CONICYT PAI 77170091 (to C.T.-R.); R01DK113171, R01CA198103 and R01DK103185 (to R.J.K.); FONDECYT 1150766 (to F.A.C.); the Research Council KU Leuven grant OT14/101 (to G.B.); the Research Foundation – Flanders (FWO) G.0C91.14N, G.0A34.16N and the FWO Scientific Research Community “Ca2+ signaling in health, disease and therapy” W0.019.17 (to G.B.); FWO (G049817N, G076617N) and KU Leuven (C16/15/073) (to P.A.); a FWO doctorate fellowship (to M.K.); the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant 675448 (to M.L.S.); CONICYT fellowship PCHA/Doctorado Nacional/2016-21160232 (to M.G.-Q.); the George E. Hewitt Foundation for a postdoctoral fellowship (to D.E.M.); Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche – Projets blancs; under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; Association pour la recherche sur le cancer; Cancéropôle Ile-de-France; Chancelerie des universités de Paris (Legs Poix), Fondation pour la Recherche Médicale; a donation from Elior; the European Research Area Network on Cardiovascular Diseases (MINOTAUR), Gustave Roussy Odyssea, the European Union Horizon 2020 Project Oncobiome; Fondation Carrefour; High-end Foreign Expert Program in China (GDW20171100085); Institut National du Cancer; Inserm (HTE); Institut Universitaire de France; LeDucq Foundation; the LabEx Immuno-Oncology; the RHU Torino Lumière; the Seerave Foundation; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination; and the SIRIC Cancer Research and Personalized Medicine (to G.K.).

International audience; In the version of this Article originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Article.

International audience; Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR.

Endoplasmic reticulum (ER) stress is generated by various physiological and pathological conditions that induce an accumulation of misfolded proteins in its lumen. ER stress activates the unfolded protein response (UPR), an adaptive reaction to cope with protein misfolding to and restore proteostasis. However, chronic ER stress results in apoptosis. In solid tumors, the UPR mediates adaptation to various environmental stressors, including hypoxia, low in pH and low nutrients availability, driving positive selection. Recent findings support the concept that UPR signaling also contributes to other relevant cancer-related event that may not be related to ER stress, including angiogenesis, genomic instability, metastasis and immunomodulation. In this article, we overview novel discoveries highlighting the impact of the UPR to different aspects of cancer biology beyond its known role as a survival factor to the hypoxic environment observed in solid tumors.

International audience; Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a signalling network known as the unfolded protein response (UPR). Here, we identified filamin A as a major binding partner of the ER stress transducer IRE1 alpha. Filamin A is an actin crosslinking factor involved in cytoskeleton remodelling. We show that IRE1 alpha controls actin cytoskeleton dynamics and affects cell migration upstream of filamin A. The regulation of cytoskeleton dynamics by IRE1 alpha is independent of its canonical role as a UPR mediator, serving instead as a scaffold that recruits and regulates filamin A. Targeting IRE1 alpha expression in mice affected normal brain development, generating a phenotype resembling periventricular heterotopia, a disease linked to the loss of function of filamin A. IRE1 alpha also modulated cell movement and cytoskeleton dynamics in fly and zebrafish models. This study unveils an unanticipated biological function of IRE1 alpha in cell migration, whereby filamin A operates as an interphase between the UPR and the actin cytoskeleton.

Mitochondria-associated membranes (MAMs) are central microdomains that fine-tune bioenergetics by the local transfer of calcium from the endoplasmic reticulum to the mitochondrial matrix. Here, we report an unexpected function of the endoplasmic reticulum stress transducer IRE1α as a structural determinant of MAMs that controls mitochondrial calcium uptake. IRE1α deficiency resulted in marked alterations in mitochondrial physiology and energy metabolism under resting conditions. IRE1α determined the distribution of inositol-1,4,5-trisphosphate receptors at MAMs by operating as a scaffold. Using mutagenesis analysis, we separated the housekeeping activity of IRE1α at MAMs from its canonical role in the unfolded protein response. These observations were validated in vivo in the liver of IRE1α conditional knockout mice, revealing broad implications for cellular metabolism. Our results support an alternative function of IRE1α in orchestrating the communication between the endoplasmic reticulum and mitochondria to sustain bioenergetics.

Endoplasmic reticulum (ER) stress is a common feature of several physiological and pathological conditions affecting the function of the secretory pathway. To restore ER homeostasis, an orchestrated signaling pathway is engaged that is known as the unfolded protein response (UPR). The UPR has a primary function in stress adaptation and cell survival; however, under irreversible ER stress a switch to pro-apoptotic signaling events induces apoptosis of damaged cells. The mechanisms that initiate ER stress-dependent apoptosis are not fully understood. Several pathways have been described where we highlight the participation of the BCL-2 family of proteins and ER calcium release. In addition, recent findings also suggest that microRNAs and oxidative stress are relevant players on the transition from adaptive to cell death programs. Here we provide a global and integrated overview of the signaling networks that may determine the elimination of a cell under chronic ER stress. This article is part of a Special Section entitled: Cell Death Pathways. Guest Editors: Frank Madeo and Slaven Stekovic. (C) 2013 Elsevier B.V. All rights reserved.

International audience; Tumor cells are often exposed to intrinsic and external factors that alter protein homeostasis, thus producing endoplasmic reticulum (ER) stress. To cope with this, cells evoke an adaptive mechanism to restore ER proteostasis known as the unfolded protein response (UPR). The three main UPR signaling branches initiated by IRE1α, PERK, and ATF6 are crucial for tumor growth and aggressiveness as well as for microenvironment remodeling or resistance to treatment. We provide a comprehensive overview of the contribution of the UPR to cancer biology and the acquisition of malignant characteristics, thus highlighting novel aspects including inflammation, invasion and metastasis, genome instability, resistance to chemo/radiotherapy, and angiogenesis. The therapeutic potential of targeting ER stress signaling in cancer is also discussed.

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1α (IRE1α), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP-1 (X-box-binding protein-1). Through a global proteomic approach we identified the BCL-2 family member PUMA as a novel IRE1α interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1α with BIM, another BH3-only protein. BIM and PUMA double-knockout cells failed to maintain sustained XBP-1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1α, inhibiting its effects on XBP-1 mRNA splicing. Unexpectedly, this regulation required BCL-2 and was antagonized by BAD or the BH3 domain mimetic ABT-737. The modulation of IRE1α RNAse activity by BH3-only proteins was recapitulated in a cell-free system suggesting a direct regulation. Moreover, BH3-only proteins controlled XBP-1 mRNA splicing in vivo and affected the ER stress-regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL-2 family members participates in a new UPR-regulatory network, thus assuming apoptosis-unrelated functions.

PERK is a major sensor of the unfolded protein response controlling cell fate under endoplasmic reticulum (ER) stress. A new study reveals an additional step for optimal PERK signaling, involving the binding of CNPY2 to PERK's luminal domain. The PERK–CNPY2 axis was shown to enhance cell death under ER stress in vivo influence liver disease.

The endoplasmic reticulum (ER) is a fundamental cellular organelle responsible for a variety of physiological processes, including the synthesis, folding and post-translational modification of the majority of membrane and secreted proteins, calcium storage, and lipid synthesis among other important functions. Besides, many pathological conditions are associated with the occurrence of uncontrolled ER stress, including diabetes, neurodegeneration, and cancer.1

Tumors rely on the unfolded protein response (UPR) and angiogenesis to survive the metabolic stress of hypoxia. Karali et al. (2014) revealed that VEGF signaling engages UPR sensors in an unconventional manner that is independent of endoplasmic reticulum (ER) stress, mediated by mTOR signaling to promote endothelial cell survival and angiogenesis.

The disruption of the energy or nutrient balance triggers endoplasmic reticulum (ER) stress, a process that mobilizes various strategies, collectively called the unfolded protein response (UPR), which reestablish homeostasis of the ER and cell. Activation of the UPR stress sensor IRE1 alpha (inositol-requiring enzyme 1 alpha) stimulates its endoribonuclease activity, leading to the generation of the mRNA encoding the transcription factor XBP1 (X-box binding protein 1), which regulates the transcription of genes encoding factors involved in controlling the quality and folding of proteins. We found that the activity of IRE1 alpha was regulated by the ER oxidoreductase PDIA6 (protein disulfide isomerase A6) and the microRNA miR-322 in response to disruption of ER Ca2+ homeostasis. PDIA6 interacted with IRE1 alpha and enhanced IRE1 alpha activity as monitored by phosphorylation of IRE1 alpha and XBP1 mRNA splicing, but PDIA6 did not substantially affect the activity of other pathways that mediate responses to ER stress. ER Ca2+ depletion and activation of store-operated Ca2+ entry reduced the abundance of the microRNA miR-322, which increased PDIA6 mRNA stability and, consequently, IRE1 alpha activity during the ER stress response. In vivo experiments with mice and worms showed that the induction of ER stress correlated with decreased miR-322 abundance, increased PDIA6 mRNA abundance, or both. Together, these findings demonstrated that ER Ca2+, PDIA6, IRE1 alpha, and miR-322 function in a dynamic feedback loop modulating the UPR under conditions of disrupted ER Ca2+ homeostasis.

The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10 (E-cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10 (cav-1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity, and cell migration observed with B16F10 (cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells.

Helicobacter pylori infects the human stomach and modifies signaling pathways that affect gastric epithelial cell proliferation and viability. Chronic exposure to this pathogen contributes to the onset of gastric atrophy, an early event in the genesis of gastric cancer associated with H. pylori infection. Susceptibility to H. pylori-induced cell death ultimately depends on the presence of protective host cell factors. Although expression of the inhibitor-of-apoptosis protein survivin in adults is frequently linked to the development of cancer, evidence indicating that the protein is present in normal gastric mucosa is also available. Thus, we investigated in human gastric tissue samples and cell lines whether H. pylori infection is linked to loss of survivin and increased cell death. Our results show that infection with H. pylori decreased survivin protein levels in the mucosa of patients with gastritis. Furthermore, survivin down-regulation correlated with apoptosis and loss of cell viability in gastrointestinal cells cocultured with different H. pylori strains. Finally, overexpression of survivin in human gastric cells was sufficient to reduce cell death after infection. Taken together, these findings implicate survivin as an important survival factor in the gastric mucosa of humans.

Background Labial salivary glands (LSG) from Sjogren9s syndrome (SS) patients show impaired cell-polarity, alterations of SNARE proteins localization and presence of salivary mucins in the extracellular matrix. Exocytosis and Ca + signaling are polarized processes in acinar-cells. Synaptotagmin-I (Syt-I) is a Ca + -sensor protein localized in the membrane of secretory granules and is a linker between Ca + stimulus and the exocytic machinery. A significant upregulation of SytI observed in a microarray assay and a Ca + response less sensitive to acetylcholine stimulation have been observed in LSG from SS patients. These data suggest that impaired cell-polarity observed in SS-patients could affect the behavior of Ca + signaling and alter the expression of Syt-I and the exocytic process. Objectives To determine the expression and localization of Syt-I in LSG from SS patients and controls. To evaluate Ca + signaling and exocytic events in normal and polarity-altered three dimensional (3D) HSG acini. Methods LSG were obtained from 22 healthy controls and 28 SS patients selected according to the American/European classification criteria (2002). Syt-I mRNA and protein levels were determined in protein extracts of LSG by qPCR and Western blotting, respectively. Syt-I localization was addressed by immunofluorescence and confocal microscopy. To evaluate the formation of SDS-resistant SNARE complexes, some aliquots of proteins were maintained at 25°C and other aliquots were boiled for 3 min at 100°C to disassemble SNARE complexes. Ca + signaling was measured in 3D HSG acini obtained by culturing HSG cells on basal lamina extract. 3D acini like structures were treated with the a6 integrin blocking antibody (GoH3) that alters cell polarity. Ca + measurements were performed with fluorescent dyes sensitive to changes of Ca + concentration upon carbachol and isoproterenol stimulation. Exocytosis events were measured by FM 1-43 fluorescence and carbon fiber amperometry. Results LSG of SS-patients showed a significant increase of mRNA and protein levels of Syt-I. An increased Syt-I staining intensity co-localizing with STX4 mainly in the basal pole was also observed in LSG of SS-patients. An increased amount of Syt-I was observed in pre-formed SNARE complexes of LSG extracts from SS-patients. Ca + signaling and exocytic events were dependent on extracellular Ca + and acinar cell-polarity in 3D HSG acini. Conclusions Altered distribution of SNARE complexes and increased Syt-I levels in LSG from SS-patients might indicate Ca + signaling alterations. These alterations could explain altered polarized exocytosis and the presence of salivary mucins in the extracellular matrix of LSG from SS-patients. We demonstrate that 3D HSG acini are useful to evaluate the effects of disrupting Ca + signaling and Syt-I over-expression on exocytosis, as a model to explain observations in LSG from SS-patients. Acknowledgements Fondecyt-Chile 1120062 (MJGB, SA, CM, SG), CONICYT-Chile PhD fellowship (MJB, JC and HU). Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.2393

Caveolin-1 is known to promote cell migration, and increased caveolin-1 expression is associated with tumor progression and metastasis. In fibroblasts, caveolin-1 polarization and phosphorylation of tyrosine-14 are essential to promote migration. However, the role of caveolin-1 in migration of metastatic cells remains poorly defined. Here, caveolin-1 participation in metastatic cell migration was evaluated by shRNA targeting of endogenous caveolin-1 in MDA-MB-231 human breast cancer cells and ectopic expression in B16-F10 mouse melanoma cells. Depletion of caveolin-1 in MDA-MB-231 cells reduced, while expression in B16-F10 cells promoted migration, polarization and focal adhesion turnover in a sequence of events that involved phosphorylation of tyrosine-14 and Rac-1 activation. In B16-F10 cells, expression of a non-phosphorylatable tyrosine-14 to phenylalanine mutant failed to recapitulate the effects observed with wild-type caveolin-1. Alternatively, treatment of MDA-MB-231 cells with the Src family kinase inhibitor PP2 reduced caveolin-1 phosphorylation on tyrosine-14 and cell migration. Surprisingly, unlike for fibroblasts, caveolin-1 polarization and re-localization to the trailing edge were not observed in migrating metastatic cells. Thus, expression and phosphorylation, but not polarization of caveolin-1 favor the highly mobile phenotype of metastatic cells.