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FXYD5 (also known as dysadherin), a regulatory subunit of the Na,K-ATPase, impairs intercellular adhesion by a poorly understood mechanism. Here, we determined whether FXYD5 disrupts the trans-dimerization of Na,K-ATPase molecules located in neighboring cells. Mutagenesis of the Na,K-ATPase β1 subunit identified four conserved residues, including Y199, that are crucial for the intercellular Na,K-ATPase trans-dimerization and adhesion. Modulation of expression of FXYD5 or of the β1 subunit with intact or mutated β1?β1 binding sites demonstrated that the anti-adhesive effect of FXYD5 depends on the presence of Y199 in the β1 subunit. Immunodetection of the plasma membrane FXYD5 was prevented by the presence of O-glycans. Partial FXYD5 deglycosylation enabled antibody binding and showed that the protein level and the degree of O-glycosylation were greater in cancer than in normal cells. FXYD5-induced impairment of adhesion was abolished by both genetic and pharmacological inhibition of FXYD5 O-glycosylation. Therefore, the extracellular O-glycosylated domain of FXYD5 impairs adhesion by interfering with intercellular β1?β1 interactions, suggesting that the ratio between FXYD5 and α1?β1 heterodimer determines whether the Na,K-ATPase acts as a positive or negative regulator of intercellular adhesion. Fil: Tokhtaeva, Elmira. University of California at Los Angeles; Estados Unidos Fil: Sun, Haying. Northwestern University; Estados Unidos Fil: Deiss Yehiely, Nimrod. Northwestern University; Estados Unidos Fil: Wen, Yi. University of California at Los Angeles; Estados Unidos Fil: Soni, Pritin N.. Northwestern University; Estados Unidos Fil: Gabrielli, Nieves María. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Northwestern University; Estados Unidos Fil: Marcus, Elizabeth A.. University of California at Los Angeles; Estados Unidos Fil: Ridge, Karen M.. Northwestern University; Estados Unidos Fil: Sachs, George. University of California at Los Angeles; Estados Unidos Fil: Vazquez, Monica Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Sznajder, Jacob I.. Northwestern University; Estados Unidos Fil: Vagin, Olga. University of California at Los Angeles; Estados Unidos Fil: Dada, Laura Andrea. Northwestern University; Estados Unidos

INTRODUCTION: In patients with acute respiratory distress syndrome (ARDS) disruption of the epithelial barrier results in accumulation of edema fluid in the airspaces, impairing gas exchange and leading to elevated CO2 levels (hypercapnia). Moreover, lung-protective mechanical ventilation in patients often results in further hypercapnia. The formation of new cell-cell contacts after ARDS is essential for the re-establishment of an intact and functional alveolar epithelium, capable of clearing lung edema and performing gas exchange. The Na,K-ATPase beta-subunit is a cell-adhesion molecule with a key role in the formation and stability of cell junctions and therefore might be important in the repair of the alveolar epithelium. However, the effects of hypercapnia on the stability of the Na,K-ATPase beta-subunit and on the re-organization of adherens junctions have not been studied before. In the present work we tested the hypothesis that hypercapnia inhibits cell-cell contact formation by promoting the down-regulation of the Na,K-ATPase beta-subunit. We aimed to elucidate the molecular mechanism underlying the (dis)regulation of adherens junction formation by hypercapnia.METHODS AND RESULTS: Exposing alveolar epithelial cells to elevated CO2 at constant extracellular pH of 7.4 led to the ubiquitination of the Na,K-ATPase beta-subunit at the plasma membrane which resulted in a significant reduction of the protein abundance at the cell surface, as determined by cell-surface biotinylation and confocal microscopy. Internalization of the Na,K-ATPase beta-subunit was followed by proteasome-dependent degradation of the protein, as assessed by pulse-chase experiments with impermeable biotin. The simultaneous mutations of lysines 5 and 7 of the Na,K-ATPase beta-subunit to arginine prevented hypercapnia-induced ubiquitination and endocytosis of the protein, demonstrating that ubiquitin chains covalently-attached to the Na,K-ATPase beta-subunit during hypercapnia act as an endocytosis signal. Mutation of serine 11 of the Na,K-ATPase beta-subunit to alanine prevented hypercapnia-induced ubiquitination and degradation of the protein. In contrast, mutation of serine 11 to aspartate which mimics phosphorylation did not prevent hypercapnia-induced effects, suggesting that phosphorylation of the Na,K-ATPase beta-subunit at serine 11 is a prerequisite for the ubiquitination of the protein. Coimmunoprecipitation and in vitro interaction studies showed that the serine/threonine kinase protein kinase C-zeta; (PKC-zeta), known to be activated by hypercapnia, interacted with the Na,K-ATPase beta-subunit and this interaction was dependent on the serine 11 of the Na,K-ATPase beta-subunit. Moreover, PKC-zeta activity was required for the hypercapnia-mediated effects, since the chemical inhibition or knock-down of the kinase prevented the endocytosis of the Na,K-ATPase beta-subunit under hypercapnic conditions. By the use of a protein-interaction microarray we identified the E3 ligase TRAF2 as an interactive partner for Na,K-ATPase beta-subunit. This interaction was further confirmed by coimmunoprecipitation and in vitro interaction studies. TRAF2 led to ubiquitination of the Na,K-ATPase beta-subunit in vitro and in vivo. Moreover, knock-down of TRAF2 prevented the hypercapnia-induced endocytosis of the Na,K-ATPase beta-subunit, demonstrating that TRAF2 is the E3 ligase that mediates the Na,K-ATPase beta-subunit ubiquitination in hypercapnia. Most importantly, by cell aggregation assays we demonstrated that hypercapnia led to impaired cell junction formation, effect that was prevented by the simultaneous mutations of lysines 5 and 7 of the Na,K-ATPase beta-subunit, demonstrating that hypercapnia-mediated down-regulation of the Na,K-ATPase beta-subunit at the plasma membrane is the underlying mechanism by which hypercapnia inhibits cell-cell adhesion.CONCLUSIONS: Here we report a novel mechanism by which hypercapnia affects the function of the alveolar epithelium. We provide evidence that hypercapnia (independently of pH) promotes ubiquitination of the Na,K-ATPase beta-subunit at the plasma membrane leading to the endocytosis of the protein, which results in reduced ability of alveolar cells to form intercellular junctions. We demonstrate that ubiquitination depends on PKC-zeta activity and we identify TRAF2 as the E3 ligase that mediates the hypercapnia-induced ubiquitination of the Na,K-ATPase beta-subunit. Thus, hypercapnia may impair restoration of the alveolo-capillary barrier in patients with ARDS upon hypercapnia by the down-regulation of the Na,K-ATPase beta-subunit., EINLEITUNG: Die Schädigung der epithelialen Schranke in Patienten mit akutem Lungenversagen (engl. acute respiratory distress syndrome, ARDS) führt zur Ausbildung alveolärer Ödeme und folglich zu erhöhten CO2-Leveln (Hyperkapnie). Zudem kann die lungenprotektive Beatmung mit geringen Tidalvolumen Hyperkapnie induzieren. Zur Auflösung von ARDS ist daher die Ausbildung neuer Zell-Zell-Kontakte für die Wiederherstellung eines intakten und funktionalen alveolären Epitheliums, das zur Resorption der Ödemflüssigkeit und Aufnahme des normalen Gasaustausches in der Lage ist, essenziell. Die beta-Untereinheit der Na,K-ATPase ist ein Zelladhäsionsmolekül und spielt eine Schlüsselrolle in Ausbildung und Aufrechterhaltung von Zell-Zell-Kontakten und kann daher auch als wichtige Komponente für die Wiederherstellung der alveolo-kapillaren Schranke von Interesse sein. Allerdings ist bisher nicht bekannt, inwieweit erhöhte CO2-Level einen Einfluss auf die Stabilität der Na,K-ATPase beta-Untereinheit und die Reorganisation von Adherens Junctions haben. In der vorliegenden Arbeit sollte daher untersucht werden, ob und über welche molekularen Mechanismen Hyperkapnie die Regulation von Adherens Junctions reguliert und ob die Ausbildung von Zell-Zell-Kontakten durch eine Herunterregulation der Na,K-ATPase beta-Untereinheit inhibiert wird.METHODEN AND ERGEBNISSE: Oberflächen-Biotinylierung und konfokale Mikroskopie alveolarer Epithelzellen zeigen, dass die Behandlung mit hohen CO2-Konzentrationen zu einer Ubiquitinierung der an der Plasmamembran befindlichen Na,K-ATPase beta-Untereinheit, und in Folge dessen zu einer signifikanten Reduktion der Häufigkeit dieser Untereinheit an der Plasmamembran führt. Pulse-Chase Experimente mit nicht-zellpermeablem Biotin demonstrieren, dass nach der Hyperkapnie-induzierten Internalisierung der Na,K-ATPase beta-Untereinheit eine proteosomale Degradation des Proteins folgt. Die gleichzeitige Mutation von Lysin 5 und 7 der Na,K-ATPase beta-Untereinheit verhindert die Hyperkapnie-induzierte Ubiquitinierung und Endozytose des Proteins. Die Mutation von Serin 11 der Na,K-ATPase beta-Untereinheit zu Alanin verhindert die Hyperkapnie-induzierte Ubiquitinierung und Degradierung, wohingegen die Mutation von Serin 11 zu Aspartat, was eine Phosphorylierung imitiert, die Hyperkapnie-induzierten Effekte nicht verhindert. Koimmunopräzipitation und in vitro Experimente zur Interaktion der Protein belegen, dass die PKC-zeta Serin/Threonin-Kinase, die bekannterweise während der Hyperkapnie aktiviert wird, mit der Na,K-ATPase beta-Untereinheit interagiert und diese Interaktion abhängig ist von Serin 11 der Na,K-ATPase beta-Untereinheit. Weiterhin verhindert die chemische Inhibition oder der knock-down von PKC-zeta die Hyperkapnie-induzierte Endozytose der Na,K-ATPase beta-Untereinheit. In einem Protein-Microarray zur Identifikation von Bindungspartnern der Na,K-ATPase beta-Untereinheit wurde die E3 Ligase TRAF2 gefunden, die auch in Koimmunopräzipitationsstudien und in in vitro Interaktionsstudien die Na,K-ATPase beta-Untereinheit bindet. TRAF2 induziert die Ubiquitinierung der Na,K-ATPase beta-Untereinheit in vitro und in vivo. Außerdem verhindert der Knock-down von TRAF2 die Hyperkapnie-induzierte Endocytose der Na,K-ATPase beta-Untereinheit. Durch Zellaggregationsassays konnten wir zeigen, dass Hyperkapnie zu gestörten Zell-Zell-Adhäsionen führt und dieser Effekt durch Mutation der Lysine 5 und 7 der Na,K-ATPase beta-Untereinheit verhindert werden konnte.ERGEBNIS: In dieser Studie zeigen wir einen neuen Mechanismus auf, über den Hyperkapnie Einfluss auf die Funktion des alveolären Epitheliums nimmt. Wir belegen, dass Hyperkapnie unabhängig vom pH die Ubiquitinierung und infolgedessen die Endozytose der Na,K-ATPase beta-Untereinheit induziert, wodurch die Fähigkeit alveolarer Zellen zur Ausbildung von Zell-Zell-Kontakten reduziert wird. Weiterhin demonstrieren wir, dass die Ubiquitinierung der Na,K-ATPase beta-Untereinheit von der Aktivität von PKC-zeta abhängt, und dass TRAF2 die E3 Ligase ist, die die Hyperkapnie-induzierte Ubiquitinierung vermittelt. Demzufolge könnte Hyperkapnie die Wiederherstellung der alveolo-kapillaren Schranke in Patienten mit ARDS durch eine Herunterregulation der Na,K-ATPase beta-Untereinheit behindern.

La interacción entre las células somáticas y entre las gametas involucra una serie de eventos moleculares que no han sido dilucidados totalmente. Nuestro grupo de investigación ha desarrollado proyectos dirigidos a profundizar el conocimiento de dichos eventos. Los estudios han comprendido el análisis de moduladores de la funcionalidad espermática (ej. efecto de la temperatura de incubación, las concentraciones del ión calcio, los anticuerpos antiespermáticos de fluidos biológicos en la motilidad, la capacitación y la exocitosis acrosomal). Asimismo, hemos caracterizado componentes del espermatozoide (ej. CaM Kinasa IV, proacrosina/acrosina) y de secreciones del tracto femenino (ej. Grp78/BiP), evaluado su rol en el desarrollo de capacidad fecundante y, en algunos casos, investigado su relación con la infertilidad. En años recientes, nuestros proyectos se han extendido al estudio de las cadherinas en eventos de adhesión celular durante la fecundación; hemos caracterizado la expresión de cadherina epitelial y neural en tejidos reproductivos y gametas y evaluado su participación en la fecundación. Dada su reconocida relevancia en el cáncer, hemos abordado estudios en diversos modelos tumorales. Nuestras investigaciones han contribuido a la comprensión de los eventos de interacción de las gametas durante la fecundación así como entre las células somáticas durante la progresión tumoral. Cell-cell interaction between somatic cells as well as gametes involves molecular events that have not been completely elucidated. Our research group has developed projects aimed at studying proteins and mechanisms participating in these interactions. Several modulators of sperm functions have been analyzed (i.e. incubation temperature, calcium ion concentration, and antisperm antibodies present in biological fluids upon sperm motility, capacitation and acrosomal exocytosis). In addition, proteins from spermatozoa (i.e. CaM Kinase IV, proacrosin/acrosin) and from secretions of the female tract (Grp78/BiP) have been characterized, and their role in the development of sperm fertilizing ability assessed. In some cases, their relationship with infertility was evaluated. In recent years, our projects have been extended to study members of the cadherin superfamily and related proteins; in particular, the expression of epithelial and neural cadherin in reproductive tissues and gametes was characterized and evidence of their participation in fertilization-related cell-cell adhesion events shown. Based on the vast evidence of the role of these proteins in tumor progression, our current research also involves studies of cancer models. Our projects have contributed to the understanding of the molecular basis of cell-cell interaction during fertilization as well as during tumor progression. A interação entre as células somáticas e entre os gametas envolve uma série de eventos moleculares que não têm sido elucidados totalmente. Nosso grupo de pesquisa tem desenvolvido projetos encaminhados a aprofundar o conhecimento de tais eventos. Os estudos têm compreendido a análise de moduladores da funcionalidade espermática (ex. efeito da temperatura de incubação, as concentrações do íon cálcio, os anticorpos antiespermáticos de fluidos biológicos na motilidade, a capacitação e a exocitose acrossomal). Do mesmo modo, caracterizamos componentes do espermatozoide (ex. CaM Kinase IV, proacrosina /acrosina) e de secreções do trato feminino (ex. Grp78/BiP), avaliamos seu papel no desenvolvimento de capacidade fecundante e, em alguns casos, investigamos sua relação com a infertilidade. Em anos recentes, nossos projetos se têm estendido ao estudo das caderinas em eventos de adesão celular durante a fecundação; temos caracterizado a expressão de caderina epitelial e neural em tecidos reprodutivos e gametas e avaliamos sua participação na fecundação. Dada sua reconhecida relevância no câncer, temos abordado estudos em diversos modelos tumorais. Nossas pesquisas têm contribuído à compreensão dos eventos de interação dos gametas durante a fecundação bem como entre as células somáticas durante a progressão tumoral Fil: Vazquez, Monica Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Furlong, Laura I.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Marin Briggiler, Clara Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Veaute, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Veiga, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Matos, María L.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Lapyckyj, Lara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Gabrielli, Nieves María. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Rosso, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Arzondo, María M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Edelsztein, Nadia Yasmín. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Besso, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina