Browsing by Author "Soza, Andrea"

Now showing 1 - 6 of 6
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    Antibody to AP1B adaptor blocks biosynthetic and recycling routes of basolateral proteins at recycling endosomes
    (AMER SOC CELL BIOLOGY, 2007) Cancino, Jorge; Torrealba, Carolina; Soza, Andrea; Yuseff, Maria Isabel; Gravotta, Diego; Henklein, Peter; Rodriguez Boulan, Enrique; Gonzalez, Alfonso
    The epithelial-specific adaptor AP1B sorts basolateral plasma membrane (PM) proteins in both biosynthetic and recycling routes, but the site where it carries out this function remains incompletely defined. Here, we have investigated this topic in Fischer rat thyroid (FRT) epithelial cells using an antibody against the medium subunit mu 1B. This antibody was suitable for immunofluorescence and blocked the function of AP1B in these cells. The antibody blocked the basolateral recycling of two basolateral PM markers, Transferrin receptor (TfR) and LDL receptor (LDLR), in a perinuclear compartment with marker and functional characteristics of recycling endosomes (RE). Live imaging experiments demonstrated that in the presence of the antibody two newly synthesized GFP-tagged basolateral proteins (vesicular stomatitis virus G [VSVG] protein and TfR) exited the trans-Golgi network (TGN) normally but became blocked at the RE within 3-5 min. By contrast, the antibody did not block trafficking of green fluorescent protein (GFP)-LDLR from the TGN to the PM but stopped its recycling after internalization into RE in similar to 45 min. Our experiments conclusively demonstrate that 1) AP1B functions exclusively at RE; 2) TGN-to-RE transport is very fast and selective and is mediated by adaptors different from AP1B; and 3) the TGN and AP1B-containing RE cooperate in biosynthetic basolateral sorting.
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    AP1B sorts basolateral proteins in recycling and biosynthetic routes of MDCK cells
    (NATL ACAD SCIENCES, 2007) Gravotta, Diego; Deora, Ami; Perret, Emilie; Oyanadel, Claudia; Soza, Andrea; Schreiner, Ryan; Rodriguez Boulan, Enrique
    The epithelial-specific adaptor AP1B sorts basolateral proteins, but the trafficking routes where it performs its sorting role remain controversial. Here, we used an RNAi approach to knock down the medium subunit of AP1B (mu 1B) in the prototype epithelial cell line Madin-Darby canine kidney (MDCK). mu 1B-knocked down MDCK cells displayed loss of polarity of several endogenous and exogenous basolateral markers transduced via adenovirus vectors, but exhibited normal polarity of apical markers. We chose two well characterized basolateral protein markers, the transferrin receptor (TfR) and the vesicular stomatitis virus G protein, to study the sorting role of AP1B. A surface-capture assay introduced here showed that mu 1B-knocked down MDCK cells plated on filters at confluency and cultured for 4.5 d, sorted TfR correctly in the biosynthetic route but incorrectly in the recycling route. In contrast, these same cells missorted vesicular stomatitis virus G apically in the biosynthetic route. Strikingly, recently confluent MDCK cells (1-3 d) displayed AP1B-dependence in the biosynthetic route of TfR, which decreased with additional days in culture. Sucrose density gradient analysis detected AP1B predominantly in TfR-rich endosomal fractions in MDCK cells confluent for 1 and 4 d. Our results are consistent with the following model: AP1B sorts basolateral proteins in both biosynthetic and recycling routes of MDCK cells, as a result of its predominant functional localization in recycling endosomes, which constitute a post-Golgi station in the biosynthetic route of some plasma membrane proteins. TfR utilizes a direct route from Golgi to basolateral membrane that is established as the epithelial monolayer matures.
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    Galectin-8 as an immunosuppressor in experimental autoimmune encephalomyelitis and a target of human early prognostic antibodies in multiple sclerosis
    (2017) Pardo, Evelyn; Cárcamo, Claudia; Uribe-San Martín, Reinaldo; Ciampi, Ethel; Segovia-Miranda, Fabián; Curkovic-Peña, Cristobal; Montecino, Fabián; Holmes, Christopher; Tichauer, Juan Enrique; Acuña, Eric; Osorio-Barrios, Francisco; Castro, Marjorie; Cortes, Priscilla; Oyanadel, Claudia; Valenzuela, David M.; Pacheco, Rodrigo; Naves, Rodrigo; Soza, Andrea; González, Alfonso
    Galectin-8 (Gal-8) is a member of a glycan-binding protein family that regulates the immune system, among other functions, and is a target of antibodies in autoimmune disorders. However, its role in multiple sclerosis (MS), an autoimmune inflammatory disease of the central nervous system (CNS), remains unknown. We study the consequences of Gal-8 silencing on lymphocyte subpopulations and the development of experimental autoimmune encephalitis (EAE), to then assess the presence and clinical meaning of anti-Gal-8 antibodies in MS patients. Lgals8/Lac-Z knock-in mice lacking Gal-8 expression have higher polarization toward Th17 cells accompanied with decreased CCR6+ and higher CXCR3+ regulatory T cells (Tregs) frequency. These conditions result in exacerbated MOG35-55 peptide-induced EAE. Gal-8 eliminates activated Th17 but not Th1 cells by apoptosis and ameliorates EAE in C57BL/6 wild-type mice. β-gal histochemistry reflecting the activity of the Gal-8 promoter revealed Gal-8 expression in a wide range of CNS regions, including high expression in the choroid-plexus. Accordingly, we detected Gal-8 in human cerebrospinal fluid, suggesting a role in the CNS immune-surveillance circuit. In addition, we show that MS patients generate function-blocking anti-Gal-8 antibodies with pathogenic potential. Such antibodies block cell adhesion and Gal-8-induced Th17 apoptosis. Furthermore, circulating anti-Gal-8 antibodies associate with relapsing-remitting MS (RRMS), and not with progressive MS phenotypes, predicting clinical disability at diagnosis within the first year of follow-up. Our results reveal that Gal-8 has an immunosuppressive protective role against autoimmune CNS inflammation, modulating the balance of Th17 and Th1 polarization and their respective Tregs. Such a role can be counteracted during RRMS by anti-Gal-8 antibodies, worsening disease prognosis. Even though anti-Gal-8 antibodies are not specific for MS, our results suggest that they could be a potential early severity biomarker in RRMS.
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    Galectin-8 Induces Apoptosis in Jurkat T Cells by Phosphatidic Acid-mediated ERK1/2 Activation Supported by Protein Kinase A Down-regulation
    (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2009) Norambuena, Andres; Metz, Claudia; Vicuna, Lucas; Silva, Antonia; Pardo, Evelyn; Oyanadel, Claudia; Massardo, Loreto; Gonzalez, Alfonso; Soza, Andrea
    Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by function-blocking autoantibodies in autoimmunity.
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    Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling
    (PUBLIC LIBRARY SCIENCE, 2012) Diskin, Shiri; Chen, Wei Sheng; Cao, Zhiyi; Gyawali, Smita; Gong, Haiyan; Soza, Andrea; Gonzalez, Alfonso; Panjwani, Noorjahan
    Purpose: The trabecular meshwork (TM) cell-matrix interactions and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. The current study was designed to evaluate the role of a carbohydrate-binding protein, galectin-8 (Gal8), in TM cell adhesion and Rho signaling.
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    Phosphatidic Acid Induces Ligand-independent Epidermal Growth Factor Receptor Endocytic Traffic through PDE4 Activation
    (AMER SOC CELL BIOLOGY, 2010) Norambuena, Andres; Metz, Claudia; Jung, Juan E.; Silva, Antonia; Otero, Carolina; Cancino, Jorge; Retamal, Claudio; Valenzuela, Juan C.; Soza, Andrea; Gonzalez, Alfonso
    Endocytosis modulates EGFR function by compartmentalizing and attenuating or enhancing its ligand-induced signaling. Here we show that it can also control the cell surface versus intracellular distribution of empty/inactive EGFR. Our previous observation that PKA inhibitors induce EGFR internalization prompted us to test phosphatidic acid (PA) generated by phospholipase D (PLD) as an endogenous down-regulator of PKA activity, which activates rolipram-sensitive type 4 phosphodiesterases (PDE4) that degrade cAMP. We found that inhibition of PA hydrolysis by propranolol, in the absence of ligand, provokes internalization of inactive (neither tyrosine-phosphorylated nor ubiquitinated) EGFR, accompanied by a transient increase in PA levels and PDE4s activity. This EGFR internalization is mimicked by PA micelles and is strongly counteracted by PLD2 silencing, rolipram or forskolin treatment, and PKA overexpression. Accelerated EGFR endocytosis seems to be mediated by clathrin-dependent and -independent pathways, leading to receptor accumulation in juxtanuclear recycling endosomes, also due to a decreased recycling. Internalized EGFR can remain intracellular without degradation for several hours or return rapidly to the cell surface upon discontinuation of the stimulus. This novel regulatory mechanism of EGFR, also novel function of signaling PA, can transmodulate receptor accessibility in response to heterologous stimuli.