61 results on '"Dada LA"'
Search Results
2. AMP-Activated Protein Kinase (AMPK) Regulates the Hypoxia-Induced Na,K-ATPase Endocytosis Via Direct Phosphorylation of PKCζ.
- Author
-
Dada, LA, primary, Gusarova, G, additional, Kelly, A, additional, Moazed, F, additional, Baker, M, additional, and Sznajder, JI, additional
- Published
- 2009
- Full Text
- View/download PDF
3. Hypoxia Leads to Ca2+/Calmodulin-Dependent Kinase Kinase β (CaMKKβ) Activation/AMPK Phosphorylation, Na,K-ATPase Endocytosis and Impaired Alveolar Epithelial Reabsorbtion.
- Author
-
Gusarova, GA, primary, Dada, LA, additional, Briva, A, additional, Trejo, H, additional, and Sznajder, JI, additional
- Published
- 2009
- Full Text
- View/download PDF
4. Effect of nitric oxide on rat adrenal zona fasciculata steroidogenesis
- Author
-
Cymeryng, CB, primary, Dada, LA, additional, and Podesta, EJ, additional
- Published
- 1998
- Full Text
- View/download PDF
5. Mitochondrial integrated stress response controls lung epithelial cell fate.
- Author
-
Han S, Lee M, Shin Y, Giovanni R, Chakrabarty RP, Herrerias MM, Dada LA, Flozak AS, Reyfman PA, Khuder B, Reczek CR, Gao L, Lopéz-Barneo J, Gottardi CJ, Budinger GRS, and Chandel NS
- Subjects
- Animals, Mice, NAD metabolism, NADH Dehydrogenase metabolism, Protons, RNA-Seq, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Single-Cell Gene Expression Analysis, Alveolar Epithelial Cells cytology, Alveolar Epithelial Cells metabolism, Alveolar Epithelial Cells pathology, Cell Differentiation, Cell Lineage, Lung cytology, Lung metabolism, Lung pathology, Mitochondria enzymology, Mitochondria metabolism, Stress, Physiological
- Abstract
Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia
1-6 . Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD+ and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD+ without proton pumping7,8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor9,10 or NAD+ precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD+ regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD+ regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction., (© 2023. The Author(s).)- Published
- 2023
- Full Text
- View/download PDF
6. Hypercapnia alters stroma-derived Wnt production to limit β-catenin signaling and proliferation in AT2 cells.
- Author
-
Dada LA, Welch LC, Magnani ND, Ren Z, Han H, Brazee PL, Celli D, Flozak AS, Weng A, Herrerias MM, Kryvenko V, Vadász I, Runyan CE, Abdala-Valencia H, Shigemura M, Casalino-Matsuda SM, Misharin AV, Budinger GRS, Gottardi CJ, and Sznajder JI
- Subjects
- Mice, beta Catenin metabolism, Cell Proliferation, COVID-19 complications, Animals, Hypercapnia metabolism, Wnt Signaling Pathway
- Abstract
Persistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with acute respiratory distress syndrome (ARDS) secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here - using a mouse model of hypercapnia exposure, cell lineage tracing, spatial transcriptomics, and 3D cultures - we show that hypercapnia limits β-catenin signaling in alveolar type II (AT2) cells, leading to their reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRα+ fibroblasts from those maintaining AT2 progenitor activity toward those that antagonize β-catenin signaling, thereby limiting progenitor function. Constitutive activation of β-catenin signaling in AT2 cells or treatment of organoid cultures with recombinant WNT3A protein bypasses the inhibitory effects of hypercapnia. Inhibition of AT2 proliferation in patients with hypercapnia may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier and increasing lung flooding, ventilator dependency, and mortality.
- Published
- 2023
- Full Text
- View/download PDF
7. Lung Injury Induces Alveolar Type 2 Cell Hypertrophy and Polyploidy with Implications for Repair and Regeneration.
- Author
-
Weng A, Maciel Herrerias M, Watanabe S, Welch LC, Flozak AS, Grant RA, Aillon RP, Dada LA, Han SH, Hinchcliff M, Misharin AV, Budinger GRS, and Gottardi CJ
- Subjects
- Alveolar Epithelial Cells metabolism, Cell Differentiation, Humans, Hypertrophy metabolism, Polyploidy, Lung Injury chemically induced, Lung Injury genetics, Lung Injury metabolism
- Abstract
Epithelial polyploidization after injury is a conserved phenomenon recently shown to improve barrier restoration during wound healing. Whether lung injury can induce alveolar epithelial polyploidy is not known. We show that bleomycin injury induces alveolar type 2 cell (AT2) hypertrophy and polyploidy. AT2 polyploidization is also seen in short term ex vivo cultures, where AT2-to-AT1 transdifferentiation is associated with substantial binucleation due to failed cytokinesis. Both hypertrophic and polyploid features of AT2 cells can be attenuated by inhibiting the integrated stress response using the small molecule ISRIB. These data suggest that AT2 hypertrophic growth and polyploidization may be a feature of alveolar epithelial injury. Because AT2 cells serve as facultative progenitors for the distal lung epithelium, a propensity for injury-induced binucleation has implications for AT2 self-renewal and regenerative potential upon reinjury, which may benefit from targeting the integrated stress response.
- Published
- 2022
- Full Text
- View/download PDF
8. Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease.
- Author
-
Kryvenko V, Vagin O, Dada LA, Sznajder JI, and Vadász I
- Subjects
- Cell Membrane metabolism, Ions metabolism, Protein Folding, Sodium-Potassium-Exchanging ATPase metabolism, Endoplasmic Reticulum metabolism
- Abstract
The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na
+ and K+ ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a β-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways. The maturation and trafficking of the Na,K-ATPase include co- and post-translational processing of the enzyme in the endoplasmic reticulum (ER) and the Golgi apparatus and subsequent delivery to the plasma membrane (PM). The ER folding of the enzyme is considered as the rate-limiting step in the membrane delivery of the protein. It has been demonstrated that only assembled Na,K-ATPase α:β-complexes may exit the organelle, whereas unassembled, misfolded or unfolded subunits are retained in the ER and are subsequently degraded. Loss of function of the Na,K-ATPase has been associated with lung, heart, kidney and neurological disorders. Recently, it has been shown that ER dysfunction, in particular, alterations in the homeostasis of the organelle, as well as impaired ER-resident chaperone activity may impede folding of Na,K-ATPase subunits, thus decreasing the abundance and function of the enzyme at the PM. Here, we summarize our current understanding on maturation and subsequent processing of the Na,K-ATPase in the ER under physiological and pathophysiological conditions., (© 2021. The Author(s).)- Published
- 2021
- Full Text
- View/download PDF
9. TRAF2 Is a Novel Ubiquitin E3 Ligase for the Na,K-ATPase β-Subunit That Drives Alveolar Epithelial Dysfunction in Hypercapnia.
- Author
-
Gabrielli NM, Mazzocchi LC, Kryvenko V, Tello K, Herold S, Morty RE, Grimminger F, Dada LA, Seeger W, Sznajder JI, and Vadász I
- Abstract
Several acute and chronic lung diseases are associated with alveolar hypoventilation leading to accumulation of CO
2 (hypercapnia). The β-subunit of the Na,K-ATPase plays a pivotal role in maintaining epithelial integrity by functioning as a cell adhesion molecule and regulating cell surface stability of the catalytic α-subunit of the transporter, thereby, maintaining optimal alveolar fluid balance. Here, we identified the E3 ubiquitin ligase for the Na,K-ATPase β-subunit, which promoted polyubiquitination, subsequent endocytosis and proteasomal degradation of the protein upon exposure of alveolar epithelial cells to elevated CO2 levels, thus impairing alveolar integrity. Ubiquitination of the Na,K-ATPase β-subunit required lysine 5 and 7 and mutating these residues (but not other lysines) prevented trafficking of Na,K-ATPase from the plasma membrane and stabilized the protein upon hypercapnia. Furthermore, ubiquitination of the Na,K-ATPase β-subunit was dependent on prior phosphorylation at serine 11 by protein kinase C (PKC)-ζ. Using a protein microarray, we identified the tumor necrosis factor receptor-associated factor 2 (TRAF2) as the E3 ligase driving ubiquitination of the Na,K-ATPase β-subunit upon hypercapnia. Of note, prevention of Na,K-ATPase β-subunit ubiquitination was necessary and sufficient to restore the formation of cell-cell junctions under hypercapnic conditions. These results suggest that a hypercapnic environment in the lung may lead to persistent epithelial dysfunction in affected patients. As such, the identification of the E3 ligase for the Na,K-ATPase may provide a novel therapeutic target, to be employed in patients with acute or chronic hypercapnic respiratory failure, aiming to restore alveolar epithelial integrity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gabrielli, Mazzocchi, Kryvenko, Tello, Herold, Morty, Grimminger, Dada, Seeger, Sznajder and Vadász.)- Published
- 2021
- Full Text
- View/download PDF
10. Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.
- Author
-
Dada LA, Vagin O, and Sznajder JI
- Subjects
- Epithelium, Humans, Ion Transport, Lung, COVID-19, SARS-CoV-2
- Published
- 2021
- Full Text
- View/download PDF
11. Linear ubiquitin assembly complex regulates lung epithelial-driven responses during influenza infection.
- Author
-
Brazee PL, Morales-Nebreda L, Magnani ND, Garcia JG, Misharin AV, Ridge KM, Budinger GRS, Iwai K, Dada LA, and Sznajder JI
- Subjects
- A549 Cells, Animals, Dogs, Humans, Influenza A Virus, H1N1 Subtype genetics, Interferon Regulatory Factor-1 genetics, Interferon Regulatory Factor-1 immunology, Lung pathology, Lung virology, Madin Darby Canine Kidney Cells, Mice, Mice, Knockout, Multiprotein Complexes genetics, Orthomyxoviridae Infections genetics, Orthomyxoviridae Infections pathology, Pneumonia, Viral genetics, Pneumonia, Viral pathology, Respiratory Mucosa pathology, Respiratory Mucosa virology, Ubiquitin-Protein Ligases deficiency, Ubiquitin-Protein Ligases immunology, Influenza A Virus, H1N1 Subtype immunology, Lung immunology, Multiprotein Complexes immunology, Orthomyxoviridae Infections immunology, Pneumonia, Viral immunology, Respiratory Mucosa immunology
- Abstract
Influenza A virus (IAV) is among the most common causes of pneumonia-related death worldwide. Pulmonary epithelial cells are the primary target for viral infection and replication and respond by releasing inflammatory mediators that recruit immune cells to mount the host response. Severe lung injury and death during IAV infection result from an exuberant host inflammatory response. The linear ubiquitin assembly complex (LUBAC), composed of SHARPIN, HOIL-1L, and HOIP, is a critical regulator of NF-κB-dependent inflammation. Using mice with lung epithelial-specific deletions of HOIL-1L or HOIP in a model of IAV infection, we provided evidence that, while a reduction in the inflammatory response was beneficial, ablation of the LUBAC-dependent lung epithelial-driven response worsened lung injury and increased mortality. Moreover, we described a mechanism for the upregulation of HOIL-1L in infected and noninfected cells triggered by the activation of type I IFN receptor and mediated by IRF1, which was maladaptive and contributed to hyperinflammation. Thus, we propose that lung epithelial LUBAC acts as a molecular rheostat that could be selectively targeted to modulate the immune response in patients with severe IAV-induced pneumonia.
- Published
- 2020
- Full Text
- View/download PDF
12. Hypercapnia Impairs Na,K-ATPase Function by Inducing Endoplasmic Reticulum Retention of the β-Subunit of the Enzyme in Alveolar Epithelial Cells.
- Author
-
Kryvenko V, Wessendorf M, Morty RE, Herold S, Seeger W, Vagin O, Dada LA, Sznajder JI, and Vadász I
- Subjects
- A549 Cells, Alveolar Epithelial Cells pathology, Animals, Endoplasmic Reticulum pathology, Humans, Hypercapnia pathology, Rats, Alveolar Epithelial Cells enzymology, Carbon Dioxide metabolism, Endoplasmic Reticulum enzymology, Hypercapnia enzymology, Protein Folding, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
Alveolar edema, impaired alveolar fluid clearance, and elevated CO
2 levels (hypercapnia) are hallmarks of the acute respiratory distress syndrome (ARDS). This study investigated how hypercapnia affects maturation of the Na,K-ATPase (NKA), a key membrane transporter, and a cell adhesion molecule involved in the resolution of alveolar edema in the endoplasmic reticulum (ER). Exposure of human alveolar epithelial cells to elevated CO2 concentrations caused a significant retention of NKA-β in the ER and, thus, decreased levels of the transporter in the Golgi apparatus. These effects were associated with a marked reduction of the plasma membrane (PM) abundance of the NKA-α/β complex as well as a decreased total and ouabain-sensitive ATPase activity. Furthermore, our study revealed that the ER-retained NKA-β subunits were only partially assembled with NKA α-subunits, which suggests that hypercapnia modifies the ER folding environment. Moreover, we observed that elevated CO2 levels decreased intracellular ATP production and increased ER protein and, particularly, NKA-β oxidation. Treatment with α-ketoglutaric acid (α-KG), which is a metabolite that has been shown to increase ATP levels and rescue mitochondrial function in hypercapnia-exposed cells, attenuated the deleterious effects of elevated CO2 concentrations and restored NKA PM abundance and function. Taken together, our findings provide new insights into the regulation of NKA in alveolar epithelial cells by elevated CO2 levels, which may lead to the development of new therapeutic approaches for patients with ARDS and hypercapnia.- Published
- 2020
- Full Text
- View/download PDF
13. Elevated CO 2 regulates the Wnt signaling pathway in mammals, Drosophila melanogaster and Caenorhabditis elegans.
- Author
-
Shigemura M, Lecuona E, Angulo M, Dada LA, Edwards MB, Welch LC, Casalino-Matsuda SM, Sporn PHS, Vadász I, Helenius IT, Nader GA, Gruenbaum Y, Sharabi K, Cummins E, Taylor C, Bharat A, Gottardi CJ, Beitel GJ, Kaminski N, Budinger GRS, Berdnikovs S, and Sznajder JI
- Subjects
- Animals, Bronchi cytology, Bronchi metabolism, Caenorhabditis elegans drug effects, Cell Line, Drosophila melanogaster drug effects, Gene Expression Profiling, Humans, Hypercapnia metabolism, Male, Mice, Mice, Inbred C57BL, Real-Time Polymerase Chain Reaction, Tissue Array Analysis, Caenorhabditis elegans metabolism, Carbon Dioxide pharmacology, Drosophila melanogaster metabolism, Wnt Signaling Pathway drug effects
- Abstract
Carbon dioxide (CO
2 ) is sensed by cells and can trigger signals to modify gene expression in different tissues leading to changes in organismal functions. Despite accumulating evidence that several pathways in various organisms are responsive to CO2 elevation (hypercapnia), it has yet to be elucidated how hypercapnia activates genes and signaling pathways, or whether they interact, are integrated, or are conserved across species. Here, we performed a large-scale transcriptomic study to explore the interaction/integration/conservation of hypercapnia-induced genomic responses in mammals (mice and humans) as well as invertebrates (Caenorhabditis elegans and Drosophila melanogaster). We found that hypercapnia activated genes that regulate Wnt signaling in mouse lungs and skeletal muscles in vivo and in several cell lines of different tissue origin. Hypercapnia-responsive Wnt pathway homologues were similarly observed in secondary analysis of available transcriptomic datasets of hypercapnia in a human bronchial cell line, flies and nematodes. Our data suggest the evolutionarily conserved role of high CO2 in regulating Wnt pathway genes.- Published
- 2019
- Full Text
- View/download PDF
14. Influenza A Virus Infection Induces Muscle Wasting via IL-6 Regulation of the E3 Ubiquitin Ligase Atrogin-1.
- Author
-
Radigan KA, Nicholson TT, Welch LC, Chi M, Amarelle L, Angulo M, Shigemura M, Shigemura A, Runyan CE, Morales-Nebreda L, Perlman H, Ceco E, Lecuona E, Dada LA, Misharin AV, Mutlu GM, Sznajder JI, and Budinger GRS
- Subjects
- Animals, Cells, Cultured, Disease Models, Animal, Forkhead Box Protein O3 metabolism, Humans, Interleukin-6 genetics, Janus Kinases metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Muscle Proteins genetics, SKP Cullin F-Box Protein Ligases genetics, STAT Transcription Factors metabolism, Signal Transduction, Influenza A virus physiology, Influenza, Human immunology, Interleukin-6 metabolism, Lung physiology, Muscle Proteins metabolism, Muscles pathology, Orthomyxoviridae Infections immunology, Pneumonia, Viral immunology, SKP Cullin F-Box Protein Ligases metabolism, Wasting Syndrome immunology
- Abstract
Muscle dysfunction is common in patients with adult respiratory distress syndrome and is associated with morbidity that can persist for years after discharge. In a mouse model of severe influenza A pneumonia, we found the proinflammatory cytokine IL-6 was necessary for the development of muscle dysfunction. Treatment with a Food and Drug Administration-approved Ab antagonist to the IL-6R (tocilizumab) attenuated the severity of influenza A-induced muscle dysfunction. In cultured myotubes, IL-6 promoted muscle degradation via JAK/STAT, FOXO3a, and atrogin-1 upregulation. Consistent with these findings, atrogin-1
+/- and atrogin-1-/- mice had attenuated muscle dysfunction following influenza infection. Our data suggest that inflammatory endocrine signals originating from the injured lung activate signaling pathways in the muscle that induce dysfunction. Inhibiting these pathways may limit morbidity in patients with influenza A pneumonia and adult respiratory distress syndrome., (Copyright © 2019 by The American Association of Immunologists, Inc.)- Published
- 2019
- Full Text
- View/download PDF
15. Ubiquitin-proteasome signaling in lung injury.
- Author
-
Magnani ND, Dada LA, and Sznajder JI
- Subjects
- Humans, Proteolysis, Lung Injury enzymology, Proteasome Endopeptidase Complex metabolism, Respiratory Distress Syndrome enzymology, Signal Transduction, Ubiquitin metabolism
- Abstract
Cell homeostasis requires precise coordination of cellular proteins function. Ubiquitination is a post-translational modification that modulates protein half-life and function and is tightly regulated by ubiquitin E3 ligases and deubiquitinating enzymes. Lung injury can progress to acute respiratory distress syndrome that is characterized by an inflammatory response and disruption of the alveolocapillary barrier resulting in alveolar edema accumulation and hypoxemia. Ubiquitination plays an important role in the pathobiology of acute lung injury as it regulates the proteins modulating the alveolocapillary barrier and the inflammatory response. Better understanding of the signaling pathways regulated by ubiquitination may lead to novel therapeutic approaches by targeting specific elements of the ubiquitination pathways., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
16. Splice Wars: The Role of MLCK Isoforms in Ventilation-induced Lung Injury.
- Author
-
Brazee PL and Dada LA
- Subjects
- Alternative Splicing, Calcium-Binding Proteins, Humans, Lung, Myosin-Light-Chain Kinase genetics, Protein Isoforms, RNA Splicing Factors, Lung Injury
- Published
- 2018
- Full Text
- View/download PDF
17. HIF and HOIL-1L-mediated PKCζ degradation stabilizes plasma membrane Na,K-ATPase to protect against hypoxia-induced lung injury.
- Author
-
Magnani ND, Dada LA, Queisser MA, Brazee PL, Welch LC, Anekalla KR, Zhou G, Vagin O, Misharin AV, Budinger GRS, Iwai K, Ciechanover AJ, and Sznajder JI
- Subjects
- A549 Cells, Animals, Apoptosis, COS Cells, Carrier Proteins genetics, Cell Hypoxia, Cell Membrane metabolism, Chlorocebus aethiops, Down-Regulation, Endocytosis, Epithelial Cells pathology, Humans, Hypoxia complications, Hypoxia-Inducible Factor 1, alpha Subunit genetics, Lung Injury etiology, Male, Mice, Mice, Inbred C57BL, Mice, Inbred ICR, Mice, Knockout, Mutation, Phosphorylation, Primary Cell Culture, Proteolysis, Pulmonary Alveoli cytology, Pulmonary Alveoli pathology, RNA Interference, RNA, Small Interfering metabolism, Rats, Rats, Sprague-Dawley, Sodium-Potassium-Exchanging ATPase genetics, Carrier Proteins metabolism, Hypoxia pathology, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Lung Injury pathology, Protein Kinase C metabolism, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
Organisms have evolved adaptive mechanisms in response to stress for cellular survival. During acute hypoxic stress, cells down-regulate energy-consuming enzymes such as Na,K-ATPase. Within minutes of alveolar epithelial cell (AEC) exposure to hypoxia, protein kinase C zeta (PKCζ) phosphorylates the α
1 -Na,K-ATPase subunit and triggers it for endocytosis, independently of the hypoxia-inducible factor (HIF). However, the Na,K-ATPase activity is essential for cell homeostasis. HIF induces the heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), which leads to PKCζ degradation. Here we report a mechanism of prosurvival adaptation of AECs to prolonged hypoxia where PKCζ degradation allows plasma membrane Na,K-ATPase stabilization at ∼50% of normoxic levels, preventing its excessive down-regulation and cell death. Mice lacking HOIL-1L in lung epithelial cells ( CreSPC /HOIL-1Lfl/fl ) were sensitized to hypoxia because they express higher levels of PKCζ and, consequently, lower plasma membrane Na,K-ATPase levels, which increased cell death and worsened lung injury. In AECs, expression of an α1 -Na,K-ATPase construct bearing an S18A (α1 -S18A) mutation, which precludes PKCζ phosphorylation, stabilized the Na,K-ATPase at the plasma membrane and prevented hypoxia-induced cell death even in the absence of HOIL-1L. Adenoviral overexpression of the α1 -S18A mutant Na,K-ATPase in vivo rescued the enhanced sensitivity of CreSPC/ HOIL-1Lfl/fl mice to hypoxic lung injury. These data suggest that stabilization of Na,K-ATPase during severe hypoxia is a HIF-dependent process involving PKCζ degradation. Accordingly, we provide evidence of an important adaptive mechanism to severe hypoxia, whereby halting the exaggerated down-regulation of plasma membrane Na,K-ATPase prevents cell death and lung injury., Competing Interests: The authors declare no conflict of interest.- Published
- 2017
- Full Text
- View/download PDF
18. Downregulation of PKCζ/Pard3/Pard6b is responsible for lung adenocarcinoma cell EMT and invasion.
- Author
-
Zhou Q, Dai J, Chen T, Dada LA, Zhang X, Zhang W, DeCamp MM, Winn RA, Sznajder JI, and Zhou G
- Subjects
- A549 Cells, Adenocarcinoma of Lung, Animals, Carboplatin pharmacology, Carcinoma, Non-Small-Cell Lung metabolism, Carcinoma, Non-Small-Cell Lung pathology, Cell Hypoxia drug effects, Cell Movement drug effects, Cell Polarity drug effects, Cisplatin pharmacology, Down-Regulation drug effects, Drug Resistance, Neoplasm drug effects, Fibronectins metabolism, Gene Silencing drug effects, Humans, Lung drug effects, Lung metabolism, Lung pathology, MAP Kinase Kinase Kinase 1 metabolism, Mice, Nude, Neoplasm Invasiveness, Adaptor Proteins, Signal Transducing metabolism, Adenocarcinoma metabolism, Adenocarcinoma pathology, Cell Cycle Proteins metabolism, Epithelial-Mesenchymal Transition drug effects, Lung Neoplasms metabolism, Lung Neoplasms pathology, Membrane Proteins metabolism, Protein Kinase C metabolism
- Abstract
Atypical protein kinase C ζ (PKCζ) forms an apico-basal polarity complex with Partitioning Defective (Pard) 3 and Pard6 to regulate normal epithelial cell apico-basolateral polarization. The dissociation of the PKCζ/Pard3/Pard6 complex is essential for the disassembly of the tight/adherens junction and epithelial-mesenchymal transition (EMT) that is critical for tumor spreading. Loss of cell polarity and epithelial organization is strongly correlated with malignancy and tumor progression in some other cancer types. However, it is unclear whether the PKCζ/Pard3/Pard6 complex plays a role in the progression of non-small-cell lung cancer (NSCLC). We found that hypoxia downregulated the PKCζ/Pard3/Pard6 complex, correlating with induction of lung cancer cell migration and invasion. Silencing of the PKCζ/Pard3/Pard6 polarity complex components induced lung cancer cell EMT, invasion, and colonization in vivo. Suppression of Pard3 was associated with altered expression of genes regulating wound healing, cell apoptosis/death and cell motility, and particularly upregulation of MAP3K1 and fibronectin which are known to contribute to lung cancer progression. Human lung adenocarcinoma tissues expressed less Pard6b and PKCζ than the adjacent normal tissues and in experimental mouse lung adenocarcinoma, the levels of Pard3 and PKCζ were also decreased. In addition, we showed that a methylation locus in the gene body of Pard3 is positively associated with the expression of Pard3 and that methylation of the Pard3 gene increased cellular sensitivity to carboplatin, a common chemotherapy drug. Suppression of Pard3 increased chemoresistance in lung cancer cells. Together, these results suggest that reduced expression of PKCζ/Pard3/Pard6 contributes to NSCLC EMT, invasion, and chemoresistance., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
19. FXYD5 Is an Essential Mediator of the Inflammatory Response during Lung Injury.
- Author
-
Brazee PL, Soni PN, Tokhtaeva E, Magnani N, Yemelyanov A, Perlman HR, Ridge KM, Sznajder JI, Vagin O, and Dada LA
- Abstract
The alveolar epithelium secretes cytokines and chemokines that recruit immune cells to the lungs, which is essential for fighting infections but in excess can promote lung injury. Overexpression of FXYD5, a tissue-specific regulator of the Na,K-ATPase, in mice, impairs the alveolo-epithelial barrier, and FXYD5 overexpression in renal cells increases C-C chemokine ligand-2 (CCL2) secretion in response to lipopolysaccharide (LPS). The aim of this study was to determine whether FXYD5 contributes to the lung inflammation and injury. Exposure of alveolar epithelial cells (AEC) to LPS increased FXYD5 levels at the plasma membrane, and FXYD5 silencing prevented both the activation of NF-κB and the secretion of cytokines in response to LPS. Intratracheal instillation of LPS into mice increased FXYD5 levels in the lung. FXYD5 overexpression increased the recruitment of interstitial macrophages and classical monocytes to the lung in response to LPS. FXYD5 silencing decreased CCL2 levels, number of cells, and protein concentration in bronchoalveolar lavage fluid (BALF) after LPS treatment, indicating that FXYD5 is required for the NF-κB-stimulated epithelial production of CCL2, the influx of immune cells, and the increase in alveolo-epithelial permeability in response to LPS. Silencing of FXYD5 also prevented the activation of NF-κB and cytokine secretion in response to interferon α and TNF-α, suggesting that pro-inflammatory effects of FXYD5 are not limited to the LPS-induced pathway. Furthermore, in the absence of other stimuli, FXYD5 overexpression in AEC activated NF-κB and increased cytokine production, while FXYD5 overexpression in mice increased cytokine levels in BALF, indicating that FXYD5 is sufficient to induce the NF-κB-stimulated cytokine secretion by the alveolar epithelium. The FXYD5 overexpression also increased cell counts in BALF, which was prevented by silencing the CCL2 receptor (CCR2), or by treating mice with a CCR2-blocking antibody, confirming that FXYD5-induced CCL2 production leads to the recruitment of monocytes to the lung. Taken together, the data demonstrate that FXYD5 is a key contributor to inflammatory lung injury.
- Published
- 2017
- Full Text
- View/download PDF
20. Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart: DISTINCT FUNCTIONAL PROPERTIES AND ISOFORM-SELECTIVE INHIBITORS.
- Author
-
Habeck M, Tokhtaeva E, Nadav Y, Ben Zeev E, Ferris SP, Kaufman RJ, Bab-Dinitz E, Kaplan JH, Dada LA, Farfel Z, Tal DM, Katz A, Sachs G, Vagin O, and Karlish SJ
- Subjects
- Adenosine Triphosphatases antagonists & inhibitors, Adenosine Triphosphatases chemistry, Animals, Cation Transport Proteins antagonists & inhibitors, Cation Transport Proteins chemistry, Cell Adhesion Molecules, Neuronal antagonists & inhibitors, Cell Adhesion Molecules, Neuronal chemistry, Dimerization, Enzyme Inhibitors metabolism, Humans, Isoenzymes antagonists & inhibitors, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes metabolism, Kinetics, Mice, Myocardium chemistry, Potassium chemistry, Potassium metabolism, Sodium chemistry, Sodium metabolism, Sodium-Potassium-Exchanging ATPase antagonists & inhibitors, Sodium-Potassium-Exchanging ATPase chemistry, Sodium-Potassium-Exchanging ATPase genetics, Adenosine Triphosphatases metabolism, Cation Transport Proteins metabolism, Cell Adhesion Molecules, Neuronal metabolism, Enzyme Inhibitors chemistry, Myocardium enzymology, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
The Na,K-ATPase α
2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+ , whereas α1 has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α2 in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α2 is preferentially expressed with β2 in T-tubules of cardiac myocytes, forming α2 β2 heterodimers. We have expressed human α1 β1 , α2 β1 , α2 β2 , and α2 β3 in Pichia pastoris, purified the complexes, and compared their functional properties. α2 β2 and α2 β3 differ significantly from both α2 β1 and α1 β1 in having a higher K0.5 K+ and lower K0.5 Na+ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K+ and shift of the E1 P-E2 P conformational equilibrium toward E1 P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α2 β2 and α2 β3 over α1 β1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K+ affinity of α2 β2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of α2 β2 for cardiac muscle contractility. The high sensitivity of α2 β2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α2 β2 -selective digoxin derivatives for reducing cardiotoxicity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2016
- Full Text
- View/download PDF
21. The O-glycosylated ectodomain of FXYD5 impairs adhesion by disrupting cell-cell trans-dimerization of Na,K-ATPase β1 subunits.
- Author
-
Tokhtaeva E, Sun H, Deiss-Yehiely N, Wen Y, Soni PN, Gabrielli NM, Marcus EA, Ridge KM, Sachs G, Vazquez-Levin M, Sznajder JI, Vagin O, and Dada LA
- Subjects
- A549 Cells, Amino Acids metabolism, Animals, Antibody Specificity, Cell Adhesion, Cell Line, Tumor, Cell Membrane metabolism, Dogs, Epithelial Cells metabolism, Gene Knockdown Techniques, Glycosylation, HEK293 Cells, Humans, Ion Channels, Madin Darby Canine Kidney Cells, Mice, Microfilament Proteins, Protein Binding, Protein Subunits chemistry, Rats, Sodium-Potassium-Exchanging ATPase chemistry, Membrane Glycoproteins metabolism, Neoplasm Proteins metabolism, Protein Multimerization, Protein Subunits metabolism, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
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., (© 2016. Published by The Company of Biologists Ltd.)
- Published
- 2016
- Full Text
- View/download PDF
22. Role of Linear Ubiquitination in Health and Disease.
- Author
-
Brazee P, Dada LA, and Sznajder JI
- Subjects
- Animals, Humans, Lung metabolism, Proteasome Endopeptidase Complex metabolism, Ubiquitin metabolism, Disease, Health, Ubiquitination
- Abstract
The covalent attachment of ubiquitin to target proteins is one of the most prevalent post-translational modifications, regulating a myriad of cellular processes including cell growth, survival, and metabolism. Recently, a novel RING E3 ligase complex was described, called linear ubiquitin assembly complex (LUBAC), which is capable of connecting ubiquitin molecules in a novel head-to-tail fashion via the N-terminal methionine residue. LUBAC is a heteromeric complex composed of heme-oxidized iron-responsive element-binding protein 2 ubiquitin ligase-1L (HOIL-1L), HOIL-1L-interacting protein, and shank-associated RH domain-interacting protein (SHARPIN). The essential role of LUBAC-generated linear chains for activation of nuclear factor-κB (NF-κB) signaling was first described in the activation of tumor necrosis factor-α receptor signaling complex. A decade of research has identified additional pathways that use LUBAC for downstream signaling, including CD40 ligand and the IL-1β receptor, as well as cytosolic pattern recognition receptors including nucleotide-binding oligomerization domain containing 2 (NOD2), retinoic acid-inducible gene 1 (RIG-1), and the NOD-like receptor family, pyrin domain containing 3 inflammasome (NLRP3). Even though the three components of the complex are required for full activation of NF-κB, the individual components of LUBAC regulate specific cell type- and stimuli-dependent effects. In humans, autosomal defects in LUBAC are associated with both autoinflammation and immunodeficiency, with additional disorders described in mice. Moreover, in the lung epithelium, HOIL-1L ubiquitinates target proteins independently of the other LUBAC components, adding another layer of complexity to the function and regulation of LUBAC. Although many advances have been made, the diverse functions of linear ubiquitin chains and the regulation of LUBAC are not yet completely understood. In this review, we discuss the various roles of linear ubiquitin chains and point to areas of study that would benefit from further investigation into LUBAC-mediated signaling pathways in lung pathophysiology.
- Published
- 2016
- Full Text
- View/download PDF
23. FXYD5 Protein Has a Pro-inflammatory Role in Epithelial Cells.
- Author
-
Lubarski-Gotliv I, Asher C, Dada LA, and Garty H
- Subjects
- Animals, Cell Line, Cell Line, Tumor, Chemokine CCL2 genetics, Chemokine CCL2 metabolism, Epithelial Cells metabolism, Gene Expression drug effects, Gene Knockdown Techniques, Humans, Inflammation Mediators metabolism, Ion Channels, Kinetics, Lipopolysaccharides pharmacology, Membrane Glycoproteins antagonists & inhibitors, Membrane Glycoproteins genetics, Membrane Proteins genetics, Mice, Microfilament Proteins, Neoplasm Proteins antagonists & inhibitors, Neoplasm Proteins genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Receptors, Tumor Necrosis Factor genetics, Receptors, Tumor Necrosis Factor metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism, Signal Transduction drug effects, Membrane Glycoproteins metabolism, Membrane Proteins metabolism, Neoplasm Proteins metabolism
- Abstract
The FXYD proteins are a family of small membrane proteins that share an invariant four amino acid signature motif F-X-Y-D and act as tissue-specific regulatory subunits of the Na,K-ATPase. FXYD5 (also termed dysadherin or RIC) is a structurally and functionally unique member of the FXYD family. As other FXYD proteins, FXYD5 specifically interacts with the Na,K-ATPase and alters its kinetics by increasing Vmax However, unlike other family members FXYD5 appears to have additional functions, which cannot be readily explained by modulation of transport kinetics. Knockdown of FXYD5 in MDA-MB-231 breast cancer cells largely decreases expression and secretion of the chemokine CCL2 (MCP-1). A related effect has also been observed in renal cell carcinoma cells. The current study aims to further characterize the relationship between the expression of FXYD5 and CCL2 secretion. We demonstrate that transfection of M1 epithelial cell line with FXYD5 largely increases lipopolysaccharide (LPS) stimulated CCL2 mRNA and secretion of the translated protein. We have completed a detailed analysis of the molecular events leading to the above response. Our key findings indicate that FXYD5 generates a late response by increasing the surface expression of the TNFα receptor, without affecting its total protein level, or mRNA transcription. LPS administration to mice demonstrates induced secretion of CCL2 and TNFα in FXYD5-expressing lung peripheral tissue, which suggests a possible role for FXYD5 in normal epithelia during inflammation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
- Full Text
- View/download PDF
24. High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation.
- Author
-
Dada LA, Trejo Bittar HE, Welch LC, Vagin O, Deiss-Yehiely N, Kelly AM, Baker MR, Capri J, Cohn W, Whitelegge JP, Vadász I, Gruenbaum Y, and Sznajder JI
- Subjects
- Amino Acid Sequence, Animals, Carbon Dioxide metabolism, Cell Line, Enzyme Activation, Homeodomain Proteins analysis, Homeodomain Proteins genetics, Humans, JNK Mitogen-Activated Protein Kinases analysis, Molecular Sequence Data, Mutation, Phosphorylation, Protein Interaction Maps, Rats, Sodium-Potassium-Exchanging ATPase analysis, Transcription Factors analysis, Transcription Factors genetics, Endocytosis, Homeodomain Proteins metabolism, Hypercapnia metabolism, JNK Mitogen-Activated Protein Kinases metabolism, Sodium-Potassium-Exchanging ATPase metabolism, Transcription Factors metabolism
- Abstract
The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis, and cell adhesion and cell migration by phosphorylating paxillin and β-catenin. JNK phosphorylation downstream of AMP-activated protein kinase (AMPK) activation is required for high CO2 (hypercapnia)-induced Na,K-ATPase endocytosis in alveolar epithelial cells. Here, we provide evidence that during hypercapnia, JNK promotes the phosphorylation of LMO7b, a scaffolding protein, in vitro and in intact cells. LMO7b phosphorylation was blocked by exposing the cells to the JNK inhibitor SP600125 and by infecting cells with dominant-negative JNK or AMPK adenovirus. The knockdown of the endogenous LMO7b or overexpression of mutated LMO7b with alanine substitutions of five potential JNK phosphorylation sites (LMO7b-5SA) or only Ser-1295 rescued both LMO7b phosphorylation and the hypercapnia-induced Na,K-ATPase endocytosis. Moreover, high CO2 promoted the colocalization and interaction of LMO7b and the Na,K-ATPase α1 subunit at the plasma membrane, which were prevented by SP600125 or by transfecting cells with LMO7b-5SA. Collectively, our data suggest that hypercapnia leads to JNK-induced LMO7b phosphorylation at Ser-1295, which facilitates the interaction of LMO7b with Na,K-ATPase at the plasma membrane promoting the endocytosis of Na,K-ATPase in alveolar epithelial cells., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
- Full Text
- View/download PDF
25. High CO2 levels cause skeletal muscle atrophy via AMP-activated kinase (AMPK), FoxO3a protein, and muscle-specific Ring finger protein 1 (MuRF1).
- Author
-
Jaitovich A, Angulo M, Lecuona E, Dada LA, Welch LC, Cheng Y, Gusarova G, Ceco E, Liu C, Shigemura M, Barreiro E, Patterson C, Nader GA, and Sznajder JI
- Subjects
- Animals, Base Sequence, Cell Line, DNA Primers, Forkhead Box Protein O3, Male, Mice, Mice, Inbred C57BL, Muscle, Skeletal metabolism, Real-Time Polymerase Chain Reaction, Tripartite Motif Proteins, Up-Regulation, Adenylate Kinase metabolism, Carbon Dioxide metabolism, Forkhead Transcription Factors metabolism, Muscle Proteins metabolism, Muscle, Skeletal pathology, Muscular Atrophy etiology, Ubiquitin-Protein Ligases metabolism
- Abstract
Patients with chronic obstructive pulmonary disease, acute lung injury, and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO2 had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO2 had reduced fiber diameter, protein/DNA ratios, and anabolic capacity. High CO2 induced the expression of MuRF1 in vivo and in vitro, whereas MuRF1(-/-) mice exposed to high CO2 did not develop muscle atrophy. AMP-activated kinase (AMPK), a metabolic sensor, was activated in myotubes exposed to high CO2, and loss-of-function studies showed that the AMPKα2 isoform is necessary for muscle-specific ring finger protein 1 (MuRF1) up-regulation and myofiber size reduction. High CO2 induced AMPKα2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO2 activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
- Full Text
- View/download PDF
26. Septin dynamics are essential for exocytosis.
- Author
-
Tokhtaeva E, Capri J, Marcus EA, Whitelegge JP, Khuzakhmetova V, Bukharaeva E, Deiss-Yehiely N, Dada LA, Sachs G, Fernandez-Salas E, and Vagin O
- Subjects
- Animals, Blotting, Western, Brain drug effects, Cell Line, Cell Line, Tumor, Dogs, Female, HEK293 Cells, Humans, Madin Darby Canine Kidney Cells, Male, Mice, Inbred BALB C, Microscopy, Confocal, PC12 Cells, Phenylurea Compounds pharmacology, Protein Binding drug effects, Protein Multimerization, Proteomics, Pyridines pharmacology, RNA Interference, Rats, Septins chemistry, Septins genetics, Synaptosomal-Associated Protein 25 metabolism, Brain metabolism, Exocytosis, Proteome metabolism, Septins metabolism
- Abstract
Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
- Full Text
- View/download PDF
27. Intratracheal administration of influenza virus is superior to intranasal administration as a model of acute lung injury.
- Author
-
Morales-Nebreda L, Chi M, Lecuona E, Chandel NS, Dada LA, Ridge K, Soberanes S, Nigdelioglu R, Sznajder JI, Mutlu GM, Budinger GR, and Radigan KA
- Subjects
- Animals, Body Weight, Brain virology, Histocytochemistry, Humans, Lung pathology, Lung virology, Male, Mice, Inbred C57BL, Nasal Cavity virology, Orthomyxoviridae Infections complications, Trachea virology, Viral Load, Acute Lung Injury pathology, Acute Lung Injury virology, Disease Models, Animal, Influenza A virus growth & development, Influenza A virus physiology, Orthomyxoviridae Infections pathology, Orthomyxoviridae Infections virology
- Abstract
Infection of mice with human or murine adapted influenza A viruses results in a severe pneumonia. However, the results of studies from different laboratories show surprising variability, even in genetically similar strains. Differences in inoculum size related to the route of viral delivery (intranasal vs. intratracheal) might explain some of this variability. To test this hypothesis, mice were infected intranasally or intratracheally with different doses of influenza A virus (A/WSN/33 [H1N1]). Daily weights, a requirement for euthanasia, viral load in the lungs and brains, inflammatory cytokines, wet-to-dry ratio, total protein and histopathology of the infected mice were examined. With all doses of influenza tested, intranasal delivery resulted in less severe lung injury, as well as smaller and more variable viral loads in the lungs when compared with intratracheal delivery. Virus was not detected in the brain following either method of delivery. It is concluded that compared to intranasal infection, intratracheal infection with influenza A virus is a more reliable method to deliver virus to the lungs., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
28. HOIL-1L functions as the PKCζ ubiquitin ligase to promote lung tumor growth.
- Author
-
Queisser MA, Dada LA, Deiss-Yehiely N, Angulo M, Zhou G, Kouri FM, Knab LM, Liu J, Stegh AH, DeCamp MM, Budinger GR, Chandel NS, Ciechanover A, Iwai K, and Sznajder JI
- Subjects
- Adenocarcinoma of Lung, Animals, Cell Hypoxia physiology, Cell Proliferation physiology, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Neoplastic, Humans, Mice, Protein Kinase C genetics, Transcription Factors, Ubiquitination physiology, Xenograft Model Antitumor Assays, Adenocarcinoma metabolism, Adenocarcinoma pathology, Cell Line, Tumor metabolism, Glioblastoma metabolism, Lung Neoplasms metabolism, Lung Neoplasms pathology, Protein Kinase C metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
Rationale: Protein kinase C zeta (PKCζ) has been reported to act as a tumor suppressor. Deletion of PKCζ in experimental cancer models has been shown to increase tumor growth. However, the mechanisms of PKCζ down-regulation in cancerous cells have not been previously described., Objectives: To determine the molecular mechanisms that lead to decreased PKCζ expression and thus increased survival in cancer cells and tumor growth., Methods: The levels of expression of heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), HOIL-1-interacting protein (HOIP), Shank-associated RH domain-interacting protein (SHARPIN), and PKCζ were analyzed by Western blot and/or quantitative real-time polymerase chain reaction in different cell lines. Coimmunoprecipitation experiments were used to demonstrate the interaction between HOIL-1L and PKCζ. Ubiquitination was measured in an in vitro ubiquitination assay and by Western blot with specific antibodies. The role of hypoxia-inducible factor (HIF) was determined by gain/loss-of-function experiments. The effect of HOIL-1L expression on cell death was investigated using RNA interference approaches in vitro and on tumor growth in mice models. Increased HOIL-1L and decreased PKCζ expression was assessed in lung adenocarcinoma and glioblastoma multiforme and documented in several other cancer types by oncogenomic analysis., Measurements and Main Results: Hypoxia is a hallmark of rapidly growing solid tumors. We found that during hypoxia, PKCζ is ubiquitinated and degraded via the ubiquitin ligase HOIL-1L, a component of the linear ubiquitin chain assembly complex (LUBAC). In vitro ubiquitination assays indicate that HOIL-1L ubiquitinates PKCζ at Lys-48, targeting it for proteasomal degradation. In a xenograft tumor model and lung cancer model, we found that silencing of HOIL-1L increased the abundance of PKCζ and decreased the size of tumors, suggesting that lower levels of HOIL-1L promote survival. Indeed, mRNA transcript levels of HOIL-1L were elevated in tumor of patients with lung adenocarcinoma, and in a lung adenocarcinoma tissue microarray the levels of HOIL-1L were associated with high-grade tumors. Moreover, we found that HOIL-1L expression was regulated by HIFs. Interestingly, the actions of HOIL-1L were independent of LUBAC., Conclusions: These data provide first evidence of a mechanism of cancer cell adaptation to hypoxia where HIFs regulate HOIL-1L, which targets PKCζ for degradation to promote tumor survival. We provided a proof of concept that silencing of HOIL-1L impairs lung tumor growth and that HOIL-1L expression predicts survival rate in cancer patients suggesting that HOIL-1L is an attractive target for cancer therapy.
- Published
- 2014
- Full Text
- View/download PDF
29. The Na-K-ATPase α₁β₁ heterodimer as a cell adhesion molecule in epithelia.
- Author
-
Vagin O, Dada LA, Tokhtaeva E, and Sachs G
- Subjects
- Animals, Cell Adhesion Molecules chemistry, Epithelial Cells chemistry, Humans, Intercellular Junctions chemistry, Intercellular Junctions metabolism, Protein Structure, Quaternary, Sodium-Potassium-Exchanging ATPase chemistry, Cell Adhesion Molecules metabolism, Epithelial Cells metabolism, Protein Multimerization, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
The ion gradients generated by the Na-K-ATPase play a critical role in epithelia by driving transepithelial transport of various solutes. The efficiency of this Na-K-ATPase-driven vectorial transport depends on the integrity of epithelial junctions that maintain polar distribution of membrane transporters, including the basolateral sodium pump, and restrict paracellular diffusion of solutes. The review summarizes the data showing that, in addition to pumping ions, the Na-K-ATPase located at the sites of cell-cell junction acts as a cell adhesion molecule by interacting with the Na-K-ATPase of the adjacent cell in the intercellular space accompanied by anchoring to the cytoskeleton in the cytoplasm. The review also discusses the experimental evidence on the importance of a specific amino acid region in the extracellular domain of the Na-K-ATPase β(1) subunit for the Na-K-ATPase trans-dimerization and intercellular adhesion. Furthermore, a possible role of N-glycans linked to the Na-K-ATPase β(1) subunit in regulation of epithelial junctions by modulating β(1)-β(1) interactions is discussed.
- Published
- 2012
- Full Text
- View/download PDF
30. Lord of the RING: ubiquitination and directed degradation of skeletal muscle in acute lung injury.
- Author
-
Vadász I, Dada LA, and Maltais F
- Subjects
- Animals, Male, Tripartite Motif Proteins, Acute Lung Injury genetics, Acute Lung Injury pathology, Muscle Proteins genetics, Muscular Atrophy genetics, Ubiquitin-Protein Ligases genetics
- Published
- 2012
- Full Text
- View/download PDF
31. Identification of the amino acid region involved in the intercellular interaction between the β1 subunits of Na+/K+ -ATPase.
- Author
-
Tokhtaeva E, Sachs G, Sun H, Dada LA, Sznajder JI, and Vagin O
- Subjects
- Amino Acid Sequence, Amino Acids chemistry, Amino Acids genetics, Amino Acids physiology, Animals, Cell Line, Dogs, Epithelial Cells cytology, Molecular Sequence Data, Protein Multimerization, Protein Structure, Tertiary genetics, Rats, Sodium-Potassium-Exchanging ATPase genetics, Sodium-Potassium-Exchanging ATPase metabolism, Cell Communication physiology, Epithelial Cells enzymology, Sodium-Potassium-Exchanging ATPase chemistry, Sodium-Potassium-Exchanging ATPase physiology
- Abstract
Epithelial junctions depend on intercellular interactions between β(1) subunits of the Na(+)/K(+)-ATPase molecules of neighboring cells. The interaction between dog and rat subunits is less effective than the interaction between two dog β(1) subunits, indicating the importance of species-specific regions for β(1)-β(1) binding. To identify these regions, the species-specific amino acid residues were mapped on a high-resolution structure of the Na(+)/K(+)-ATPase β(1) subunit to select those exposed towards the β(1) subunit of the neighboring cell. These exposed residues were mutated in both dog and rat YFP-linked β(1) subunits (YFP-β(1)) and also in the secreted extracellular domain of the dog β(1) subunit. Five rat-like mutations in the amino acid region spanning residues 198-207 of the dog YFP-β(1) expressed in Madin-Darby canine kidney (MDCK) cells decreased co-precipitation of the endogenous dog β(1) subunit with YFP-β(1) to the level observed between dog β(1) and rat YFP-β(1). In parallel, these mutations impaired the recognition of YFP-β(1) by the dog-specific antibody that inhibits cell adhesion between MDCK cells. Accordingly, dog-like mutations in rat YFP-β(1) increased both the (YFP-β(1))-β(1) interaction in MDCK cells and recognition by the antibody. Conversely, rat-like mutations in the secreted extracellular domain of the dog β(1) subunit increased its interaction with rat YFP-β(1) in vitro. In addition, these mutations resulted in a reduction of intercellular adhesion between rat lung epithelial cells following addition of the secreted extracellular domain of the dog β(1) subunit to a cell suspension. Therefore, the amino acid region 198-207 is crucial for both trans-dimerization of the Na(+)/K(+)-ATPase β(1) subunits and cell-cell adhesion.
- Published
- 2012
- Full Text
- View/download PDF
32. Evolutionary conserved role of c-Jun-N-terminal kinase in CO2-induced epithelial dysfunction.
- Author
-
Vadász I, Dada LA, Briva A, Helenius IT, Sharabi K, Welch LC, Kelly AM, Grzesik BA, Budinger GR, Liu J, Seeger W, Beitel GJ, Gruenbaum Y, and Sznajder JI
- Subjects
- Animals, Burkitt Lymphoma, Caenorhabditis elegans, Drosophila, Enzyme Activation drug effects, Epithelial Cells metabolism, Evolution, Molecular, Humans, JNK Mitogen-Activated Protein Kinases genetics, Phosphorylation drug effects, Protein Kinase C metabolism, Rats, Sodium-Potassium-Exchanging ATPase genetics, Sodium-Potassium-Exchanging ATPase metabolism, Carbon Dioxide toxicity, Epithelial Cells drug effects, Epithelial Cells enzymology, JNK Mitogen-Activated Protein Kinases metabolism, Pulmonary Alveoli cytology
- Abstract
Elevated CO(2) levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO(2) signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO(2) levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO(2) levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO(2)-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO(2) signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO(2) signaling in mammals, diptera and nematodes.
- Published
- 2012
- Full Text
- View/download PDF
33. Hypoxia leads to Na,K-ATPase downregulation via Ca(2+) release-activated Ca(2+) channels and AMPK activation.
- Author
-
Gusarova GA, Trejo HE, Dada LA, Briva A, Welch LC, Hamanaka RB, Mutlu GM, Chandel NS, Prakriya M, and Sznajder JI
- Subjects
- Animals, Blotting, Western, Calcium metabolism, Calcium Channels genetics, Calcium-Calmodulin-Dependent Protein Kinase Kinase genetics, Calcium-Calmodulin-Dependent Protein Kinase Kinase metabolism, Cell Hypoxia, Cell Line, Tumor, Cells, Cultured, Endoplasmic Reticulum metabolism, Enzyme Activation, HEK293 Cells, Humans, Hypoxia, In Vitro Techniques, Luminescent Proteins genetics, Luminescent Proteins metabolism, Lung metabolism, Male, Membrane Proteins genetics, Membrane Proteins metabolism, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, ORAI1 Protein, Pulmonary Alveoli cytology, Pulmonary Alveoli metabolism, RNA Interference, Rats, Rats, Sprague-Dawley, Reactive Oxygen Species metabolism, Sodium-Potassium-Exchanging ATPase genetics, Stromal Interaction Molecule 1, AMP-Activated Protein Kinases metabolism, Calcium Channels metabolism, Down-Regulation, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
To maintain cellular ATP levels, hypoxia leads to Na,K-ATPase inhibition in a process dependent on reactive oxygen species (ROS) and the activation of AMP-activated kinase α1 (AMPK-α1). We report here that during hypoxia AMPK activation does not require the liver kinase B1 (LKB1) but requires the release of Ca(2+) from the endoplasmic reticulum (ER) and redistribution of STIM1 to ER-plasma membrane junctions, leading to calcium entry via Ca(2+) release-activated Ca(2+) (CRAC) channels. This increase in intracellular Ca(2+) induces Ca(2+)/calmodulin-dependent kinase kinase β (CaMKKβ)-mediated AMPK activation and Na,K-ATPase downregulation. Also, in cells unable to generate mitochondrial ROS, hypoxia failed to increase intracellular Ca(2+) concentration while a STIM1 mutant rescued the AMPK activation, suggesting that ROS act upstream of Ca(2+) signaling. Furthermore, inhibition of CRAC channel function in rat lungs prevented the impairment of alveolar fluid reabsorption caused by hypoxia. These data suggest that during hypoxia, calcium entry via CRAC channels leads to AMPK activation, Na,K-ATPase downregulation, and alveolar epithelial dysfunction.
- Published
- 2011
- Full Text
- View/download PDF
34. Mitochondrial Ca²+ and ROS take center stage to orchestrate TNF-α-mediated inflammatory responses.
- Author
-
Dada LA and Sznajder JI
- Subjects
- ADAM Proteins metabolism, ADAM17 Protein, Animals, Calcium metabolism, Endothelial Cells cytology, Endotoxins metabolism, Lymphocytes metabolism, Macrophages metabolism, Mice, Signal Transduction, Calcium chemistry, Inflammation, Mitochondria metabolism, Reactive Oxygen Species, Tumor Necrosis Factor-alpha metabolism
- Abstract
Proinflammatory stimuli induce inflammation that may progress to sepsis or chronic inflammatory disease. The cytokine TNF-α is an important endotoxin-induced inflammatory glycoprotein produced predominantly by macrophages and lymphocytes. TNF-α plays a major role in initiating signaling pathways and pathophysiological responses after engaging TNF receptors. In this issue of JCI, Rowlands et al. demonstrate that in lung microvessels, soluble TNF-α (sTNF-α) promotes the shedding of the TNF-α receptor 1 ectodomain via increased mitochondrial Ca²+ that leads to release of mitochondrial ROS. Shedding mediated by TNF-α-converting enzyme (TACE) results in an unattached TNF receptor, which participates in the scavenging of sTNF-α, thus limiting the propagation of the inflammatory response. These findings suggest that mitochondrial Ca²+, ROS, and TACE might be therapeutically targeted for treating pulmonary endothelial inflammation.
- Published
- 2011
- Full Text
- View/download PDF
35. Extracellular signal-regulated kinase (ERK) participates in the hypercapnia-induced Na,K-ATPase downregulation.
- Author
-
Welch LC, Lecuona E, Briva A, Trejo HE, Dada LA, and Sznajder JI
- Subjects
- AMP-Activated Protein Kinases metabolism, Animals, Butadienes pharmacology, Down-Regulation, Endocytosis, Enzyme Inhibitors pharmacology, Mitogen-Activated Protein Kinase 1 antagonists & inhibitors, Mitogen-Activated Protein Kinase 3 antagonists & inhibitors, Nitriles pharmacology, Phosphorylation, Rats, Sodium-Potassium-Exchanging ATPase metabolism, Hypercapnia enzymology, Lung enzymology, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3 metabolism, Sodium-Potassium-Exchanging ATPase antagonists & inhibitors
- Abstract
Hypercapnia has been shown to impair alveolar fluid reabsorption (AFR) by decreasing Na,K-ATPase activity. Extracellular signal-regulated kinase pathway (ERK) is activated under conditions of cellular stress and has been known to regulate the Na,K-ATPase. Here, we show that hypercapnia leads to ERK activation in a time-dependent manner in alveolar epithelial cells (AEC). Inhibition of ERK by U0126 or siRNA prevented both the hypercapnia-induced Na,K-ATPase endocytosis and impairment of AFR. Moreover, ERK inhibition prevented AMPK activation, a known modulator of hypercapnia-induced Na,K-ATPase endocytosis. Accordingly, these data suggest that hypercapnia-induced Na,K-ATPase endocytosis is dependent on ERK activation in AEC and that ERK plays an important role in hypercapnia-induced impairment of AFR in rat lungs., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
36. E3 ubiquitin ligase Mule ubiquitinates Miz1 and is required for TNFalpha-induced JNK activation.
- Author
-
Yang Y, Do H, Tian X, Zhang C, Liu X, Dada LA, Sznajder JI, and Liu J
- Subjects
- Animals, Apoptosis drug effects, Cell Line, Enzyme Activation drug effects, Fibroblasts cytology, Fibroblasts drug effects, Fibroblasts metabolism, Humans, Immunoblotting, Immunoprecipitation, Mice, Mice, Knockout, Nuclear Proteins genetics, Protein Binding drug effects, Protein Inhibitors of Activated STAT genetics, RNA Interference, Transfection, Ubiquitin-Protein Ligases genetics, Ubiquitination drug effects, JNK Mitogen-Activated Protein Kinases metabolism, Nuclear Proteins metabolism, Protein Inhibitors of Activated STAT metabolism, Tumor Necrosis Factor-alpha pharmacology, Ubiquitin-Protein Ligases metabolism
- Abstract
The zinc finger transcription factor Miz1 is a negative regulator of TNFalpha-induced JNK activation and cell death through inhibition of TRAF2 K63-polyubiquitination in a transcription-independent manner. Upon TNFalpha stimulation, Miz1 undergoes K48-linked polyubiquitination and proteasomal degradation, thereby relieving its inhibition. However, the underling regulatory mechanism is not known. Here, we report that HECT-domain-containing Mule is the E3 ligase that catalyzes TNFalpha-induced Miz1 polyubiquitination. Mule is a Miz1-associated protein and catalyzes its K48-linked polyubiquitination. TNFalpha-induced polyubiquitination and degradation of Miz1 were inhibited by silencing of Mule and were promoted by ectopic expression of Mule. The interaction between Mule and Miz1 was promoted by TNFalpha independently of the pox virus and zinc finger domain of Miz1. Silencing of Mule stabilized Miz1, thereby suppressing TNFalpha-induced JNK activation and cell death. Thus, our study reveals a molecular mechanism by which Mule regulates TNFalpha-induced JNK activation and apoptosis by catalyzing the polyubiquitination of Miz1.
- Published
- 2010
- Full Text
- View/download PDF
37. Insulin regulates alveolar epithelial function by inducing Na+/K+-ATPase translocation to the plasma membrane in a process mediated by the action of Akt.
- Author
-
Comellas AP, Kelly AM, Trejo HE, Briva A, Lee J, Sznajder JI, and Dada LA
- Subjects
- Animals, Body Fluids drug effects, Body Fluids enzymology, Cattle, GTPase-Activating Proteins metabolism, Humans, Male, Phosphatidylinositol 3-Kinases metabolism, Protein Transport drug effects, Rats, Rats, Sprague-Dawley, Subcellular Fractions drug effects, Subcellular Fractions enzymology, rab GTP-Binding Proteins metabolism, Alveolar Epithelial Cells drug effects, Alveolar Epithelial Cells enzymology, Cell Membrane drug effects, Cell Membrane enzymology, Insulin pharmacology, Proto-Oncogene Proteins c-akt metabolism, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
Stimulation of Na(+)/K(+)-ATPase translocation to the cell surface increases active Na(+) transport, which is the driving force of alveolar fluid reabsorption, a process necessary to keep the lungs free of edema and to allow normal gas exchange. Here, we provide evidence that insulin increases alveolar fluid reabsorption and Na(+)/K(+)-ATPase activity by increasing its translocation to the plasma membrane in alveolar epithelial cells. Insulin-induced Akt activation is necessary and sufficient to promote Na(+)/K(+)-ATPase translocation to the plasma membrane. Phosphorylation of AS160 by Akt is also required in this process, whereas inactivation of the Rab GTPase-activating protein domain of AS160 promotes partial Na(+)/K(+)-ATPase translocation in the absence of insulin. We found that Rab10 functions as a downstream target of AS160 in insulin-induced Na(+)/K(+)-ATPase translocation. Collectively, these results suggest that Akt plays a major role in Na(+)/K(+)-ATPase intracellular translocation and thus in alveolar fluid reabsorption.
- Published
- 2010
- Full Text
- View/download PDF
38. Role of ubiquitination in Na,K-ATPase regulation during lung injury.
- Author
-
Helenius IT, Dada LA, and Sznajder JI
- Subjects
- Animals, Down-Regulation, Humans, Lysosomes enzymology, Phosphorylation, Pulmonary Alveoli enzymology, Pulmonary Edema pathology, Respiratory Distress Syndrome metabolism, Respiratory Mucosa pathology, Signal Transduction, Pulmonary Edema enzymology, Respiratory Distress Syndrome enzymology, Respiratory Mucosa enzymology, Sodium-Potassium-Exchanging ATPase metabolism, Ubiquitin metabolism, Ubiquitination physiology
- Abstract
During acute lung injury edema accumulates in the alveolar space, resulting in hypoxemia due to intrapulmonary shunt. The alveolar Na,K-ATPase, by effecting active Na(+) transport, is essential for removing edema from the alveolar spaces. However, during hypoxia it is endocytosed and degraded, which results in decreased Na,K-ATPase function and impaired lung edema clearance. Na,K-ATPase endocytosis and degradation require the phosphorylation and subsequent ubiquitination of the Na,K-ATPase. These events are the results of cross-talk between post-translational modifications, and how ubiquitination of a specific protein can result from injurious extracellular stimuli. Here, we review current knowledge on the regulation of Na,K-ATPase activity during lung injury, focusing on the role of Na,K-ATPase ubiquitination during hypoxia. A better understanding of these signaling pathways can be of relevance for the design of novel treatments to ameliorate the deleterious effects of acute lung injury.
- Published
- 2010
- Full Text
- View/download PDF
39. Hypoxia-induced alveolar epithelial-mesenchymal transition requires mitochondrial ROS and hypoxia-inducible factor 1.
- Author
-
Zhou G, Dada LA, Wu M, Kelly A, Trejo H, Zhou Q, Varga J, and Sznajder JI
- Subjects
- Animals, Cell Line, Transformed, Epithelial Cells metabolism, Gene Expression Regulation, Humans, Hypoxia metabolism, Mesoderm metabolism, Mice, Pulmonary Alveoli metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Transforming Growth Factor beta1 biosynthesis, Transforming Growth Factor beta1 genetics, Epithelial Cells pathology, Hypoxia pathology, Hypoxia-Inducible Factor 1 metabolism, Mesoderm pathology, Mitochondria metabolism, Pulmonary Alveoli pathology, Reactive Oxygen Species metabolism
- Abstract
Patients with acute lung injury develop hypoxia, which may lead to lung dysfunction and aberrant tissue repair. Recent studies have suggested that epithelial-mesenchymal transition (EMT) contributes to pulmonary fibrosis. We sought to determine whether hypoxia induces EMT in alveolar epithelial cells (AEC). We found that hypoxia induced the expression of alpha-smooth muscle actin (alpha-SMA) and vimentin and decreased the expression of E-cadherin in transformed and primary human, rat, and mouse AEC, suggesting that hypoxia induces EMT in AEC. Both severe hypoxia and moderate hypoxia induced EMT. The reactive oxygen species (ROS) scavenger Euk-134 prevented hypoxia-induced EMT. Moreover, hypoxia-induced expression of alpha-SMA and vimentin was prevented in mitochondria-deficient rho(0) cells, which are incapable of ROS production during hypoxia. CoCl(2) and dimethyloxaloylglycine, two compounds that stabilize hypoxia-inducible factor (HIF)-alpha under normoxia, failed to induce alpha-SMA expression in AEC. Furthermore, overexpression of constitutively active HIF-1alpha did not induce alpha-SMA. However, loss of HIF-1alpha or HIF-2alpha abolished induction of alpha-SMA mRNA during hypoxia. Hypoxia increased the levels of transforming growth factor (TGF)-beta1, and preincubation of AEC with SB431542, an inhibitor of the TGF-beta1 type I receptor kinase, prevented the hypoxia-induced EMT, suggesting that the process was TGF-beta1 dependent. Furthermore, both ROS and HIF-alpha were necessary for hypoxia-induced TGF-beta1 upregulation. Accordingly, we have provided evidence that hypoxia induces EMT of AEC through mitochondrial ROS, HIF, and endogenous TGF-beta1 signaling.
- Published
- 2009
- Full Text
- View/download PDF
40. Alpha1-AMP-activated protein kinase regulates hypoxia-induced Na,K-ATPase endocytosis via direct phosphorylation of protein kinase C zeta.
- Author
-
Gusarova GA, Dada LA, Kelly AM, Brodie C, Witters LA, Chandel NS, and Sznajder JI
- Subjects
- AMP-Activated Protein Kinases genetics, Animals, Enzyme Activation, Epithelial Cells cytology, Humans, Isoenzymes genetics, Isoenzymes metabolism, Mitochondria metabolism, Phosphorylation, Protein Kinase C genetics, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Rats, Rats, Sprague-Dawley, Reactive Oxygen Species metabolism, Signal Transduction physiology, Sodium-Potassium-Exchanging ATPase genetics, AMP-Activated Protein Kinases metabolism, Endocytosis physiology, Epithelial Cells metabolism, Hypoxia metabolism, Protein Kinase C metabolism, Pulmonary Alveoli cytology, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
Hypoxia promotes Na,K-ATPase endocytosis via protein kinase C zeta (PKC zeta)-mediated phosphorylation of the Na,K-ATPase alpha subunit. Here, we report that hypoxia leads to the phosphorylation of 5'-AMP-activated protein kinase (AMPK) at Thr172 in rat alveolar epithelial cells. The overexpression of a dominant-negative AMPK alpha subunit (AMPK-DN) construct prevented the hypoxia-induced endocytosis of Na,K-ATPase. The overexpression of the reactive oxygen species (ROS) scavenger catalase prevented hypoxia-induced AMPK activation. Moreover, hypoxia failed to activate AMPK in mitochondrion-deficient rho(0)-A549 cells, suggesting that mitochondrial ROS play an essential role in hypoxia-induced AMPK activation. Hypoxia-induced PKC zeta translocation to the plasma membrane and phosphorylation at Thr410 were prevented by the pharmacological inhibition of AMPK or by the overexpression of the AMPK-DN construct. We found that AMPK alpha phosphorylates PKC zeta on residue Thr410 within the PKC zeta activation loop. Importantly, the activation of AMPK alpha was necessary for hypoxia-induced AMPK-PKC zeta binding in alveolar epithelial cells. The overexpression of T410A mutant PKC zeta prevented hypoxia-induced Na,K-ATPase endocytosis, confirming that PKC zeta Thr410 phosphorylation is essential for this process. PKC zeta activation by AMPK is isoform specific, as small interfering RNA targeting the alpha1 but not the alpha2 catalytic subunit prevented PKC zeta activation. Accordingly, we provide the first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK alpha1 isoform, which binds and directly phosphorylates PKC zeta at Thr410, thereby promoting Na,K-ATPase endocytosis.
- Published
- 2009
- Full Text
- View/download PDF
41. Endothelin-1 impairs alveolar epithelial function via endothelial ETB receptor.
- Author
-
Comellas AP, Briva A, Dada LA, Butti ML, Trejo HE, Yshii C, Azzam ZS, Litvan J, Chen J, Lecuona E, Pesce LM, Yanagisawa M, and Sznajder JI
- Subjects
- Adenosine Triphosphatases metabolism, Animals, Cyclic GMP metabolism, Disease Models, Animal, Female, Humans, In Vitro Techniques, Lung Injury metabolism, Male, Rats, Rats, Transgenic, Receptor, Endothelin A metabolism, Respiratory Distress Syndrome metabolism, Endothelin-1 pharmacology, Endothelium, Vascular metabolism, Extravascular Lung Water metabolism, Nitric Oxide biosynthesis, Pulmonary Alveoli metabolism, Receptor, Endothelin B metabolism
- Abstract
Rationale: Endothelin-1 (ET-1) is increased in patients with high-altitude pulmonary edema and acute respiratory distress syndrome, and these patients have decreased alveolar fluid reabsorption (AFR)., Objectives: To determine whether ET-1 impairs AFR via activation of endothelial cells and nitric oxide (NO) generation., Methods: Isolated perfused rat lung, transgenic rats deficient in ETB receptors, coincubation of lung human microvascular endothelial cells (HMVEC-L) with rat alveolar epithelial type II cells or A549 cells, ouabain-sensitive 86Rb+ uptake., Measurements and Main Results: The ET-1-induced decrease in AFR was prevented by blocking the endothelin receptor ETB, but not ETA. Endothelial-epithelial cell interaction is required, as direct exposure of alveolar epithelial cells (AECs) to ET-1 did not affect Na,K-ATPase function or protein abundance at the plasma membrane, whereas coincubation of HMVEC-L and AECs with ET-1 decreased Na,K-ATPase activity and protein abundance at the plasma membrane. Exposing transgenic rats deficient in ETB receptors in the pulmonary vasculature (ET-B(-/-)) to ET-1 did not decrease AFR or Na,K-ATPase protein abundance at the plasma membrane of AECs. Exposing HMVEC-L to ET-1 led to increased NO, and the ET-1-induced down-regulation of Na,K-ATPase was prevented by the NO synthase inhibitor l-NAME, but not by a guanylate cyclase inhibitor., Conclusions: We provide the first evidence that ET-1, via an endothelial-epithelial interaction, leads to decreased AFR by a mechanism involving activation of endothelial ETB receptors and NO generation leading to alveolar epithelial Na,K-ATPase down-regulation in a cGMP-independent manner.
- Published
- 2009
- Full Text
- View/download PDF
42. Hypoxia-mediated Na-K-ATPase degradation requires von Hippel Lindau protein.
- Author
-
Zhou G, Dada LA, Chandel NS, Iwai K, Lecuona E, Ciechanover A, and Sznajder JI
- Subjects
- Animals, Basic Helix-Loop-Helix Transcription Factors physiology, COS Cells, Cell Membrane enzymology, Cells, Cultured, Chlorocebus aethiops, Deferoxamine pharmacology, Humans, Hypoxia-Inducible Factor 1, alpha Subunit physiology, Organometallic Compounds pharmacology, Salicylates pharmacology, Hypoxia metabolism, Sodium-Potassium-Exchanging ATPase metabolism, Von Hippel-Lindau Tumor Suppressor Protein physiology
- Abstract
Hypoxia inhibits Na-K-ATPase activity and leads to its degradation in mammalian cells. Von Hippel Lindau protein (pVHL) and hypoxia inducible factor (HIF) are key mediators in cellular adaptation to hypoxia; thus, we set out to investigate whether pVHL and HIF participate in the hypoxia-mediated degradation of plasma membrane Na-K-ATPase. We found that in the presence of pVHL hypoxia decreased Na-K-ATPase activity and promoted the degradation of plasma membrane Na-K-ATPase. In pVHL-deficient cells, hypoxia did not decrease the Na-K-ATPase activity and the degradation of plasma membrane Na-K-ATPase was prevented. pVHL-mediated degradation of Na-K-ATPase required the functional pVHL E3 ligase and Ubc5 since pVHL mutants and dominant-negative Ubc5 prevented Na-K-ATPase from degradation. The generation of reactive oxygen species was necessary for pVHL-mediated Na-K-ATPase degradation during hypoxia. Desferrioxamine, which stabilizes HIF1/2alpha, did not affect the half-life of plasma membrane Na-K-ATPase. In addition, stabilizing HIF1/2alpha by infecting mammalian cells with adenoviruses containing the oxygen-dependent degradation domain of HIF1alpha did not affect the plasma membrane Na-K-ATPase degradation. In cells with suppression of pVHL by short hairpin RNA, the Na-K-ATPase was not degraded during hypoxia, whereas cells with knockdown of HIF1/2alpha retained the ability to degrade plasma membrane Na-K-ATPase. These findings suggest that pVHL participates in the hypoxia-mediated degradation of plasma membrane Na-K-ATPase in a HIF-independent manner.
- Published
- 2008
- Full Text
- View/download PDF
43. Regulation of alveolar epithelial function by hypoxia.
- Author
-
Zhou G, Dada LA, and Sznajder JI
- Subjects
- Cell Membrane physiology, Epithelial Cells metabolism, Humans, Hypoxia complications, Hypoxia therapy, Intermediate Filaments metabolism, Pulmonary Alveoli cytology, Pulmonary Edema therapy, Respiratory Distress Syndrome therapy, Sodium-Potassium-Exchanging ATPase metabolism, Hypoxia physiopathology, Pulmonary Alveoli physiology, Pulmonary Edema physiopathology, Respiratory Distress Syndrome physiopathology
- Abstract
Patients with acute respiratory distress syndrome and high-altitude pulmonary oedema build up excess lung fluid, which leads to alveolar hypoxia. In patients with acute respiratory distress syndrome and hypoxia, there is a decrease in oedema fluid clearance, due in part to the downregulation of plasma membrane sodium-potassium adenosine triphosphatase (Na,K-ATPase). In alveolar epithelial cells, acute hypoxia promotes Na,K-ATPase endocytosis from the plasma membrane to intracellular compartments, resulting in inhibition of Na,K-ATPase activity. Exposure to prolonged hypoxia leads to degradation of plasma membrane Na,K-ATPase. The downregulation of plasma membrane Na,K-ATPase reduces adenosine triphosphate demand, as part of a survival mechanism of cellular adaptation to hypoxia. Hypoxia has also been shown to disassemble and degrade the keratin intermediate filament network, a fundamental component of the cell cytoskeleton, affecting epithelial barrier function. Accordingly, better understanding of the mechanisms regulating cellular adaptation to hypoxia may lead to the development of novel therapeutic strategies for acute respiratory distress syndrome and high-altitude pulmonary oedema patients.
- Published
- 2008
- Full Text
- View/download PDF
44. AMP-activated protein kinase regulates CO2-induced alveolar epithelial dysfunction in rats and human cells by promoting Na,K-ATPase endocytosis.
- Author
-
Vadász I, Dada LA, Briva A, Trejo HE, Welch LC, Chen J, Tóth PT, Lecuona E, Witters LA, Schumacker PT, Chandel NS, Seeger W, and Sznajder JI
- Subjects
- AMP-Activated Protein Kinases, Adrenergic beta-Agonists pharmacology, Animals, Calcium antagonists & inhibitors, Calcium metabolism, Calcium-Calmodulin-Dependent Protein Kinase Kinase antagonists & inhibitors, Calcium-Calmodulin-Dependent Protein Kinase Kinase genetics, Calcium-Calmodulin-Dependent Protein Kinase Kinase metabolism, Chelating Agents pharmacology, Cyclic AMP pharmacology, Extracellular Fluid metabolism, Humans, Isoproterenol pharmacology, Protein Kinase C metabolism, Pulmonary Alveoli enzymology, Rats, Rats, Sprague-Dawley, Respiratory Mucosa enzymology, Carbon Dioxide metabolism, Endocytosis drug effects, Endocytosis genetics, Hypercapnia enzymology, Multienzyme Complexes metabolism, Protein Serine-Threonine Kinases metabolism, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
Hypercapnia (elevated CO(2) levels) occurs as a consequence of poor alveolar ventilation and impairs alveolar fluid reabsorption (AFR) by promoting Na,K-ATPase endocytosis. We studied the mechanisms regulating CO(2)-induced Na,K-ATPase endocytosis in alveolar epithelial cells (AECs) and alveolar epithelial dysfunction in rats. Elevated CO(2) levels caused a rapid activation of AMP-activated protein kinase (AMPK) in AECs, a key regulator of metabolic homeostasis. Activation of AMPK was mediated by a CO(2)-triggered increase in intracellular Ca(2+) concentration and Ca(2+)/calmodulin-dependent kinase kinase-beta (CaMKK-beta). Chelating intracellular Ca(2+) or abrogating CaMKK-beta function by gene silencing or chemical inhibition prevented the CO(2)-induced AMPK activation in AECs. Activation of AMPK or overexpression of constitutively active AMPK was sufficient to activate PKC-zeta and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-alpha(1) prevented CO(2)-induced Na,K-ATPase endocytosis. The hypercapnia effects were independent of intracellular ROS. Exposure of rats to hypercapnia for up to 7 days caused a sustained decrease in AFR. Pretreatment with a beta-adrenergic agonist, isoproterenol, or a cAMP analog ameliorated the hypercapnia-induced impairment of AFR. Accordingly, we provide evidence that elevated CO(2) levels are sensed by AECs and that AMPK mediates CO(2)-induced Na,K-ATPase endocytosis and alveolar epithelial dysfunction, which can be prevented with beta-adrenergic agonists and cAMP.
- Published
- 2008
- Full Text
- View/download PDF
45. High CO2 levels impair alveolar epithelial function independently of pH.
- Author
-
Briva A, Vadász I, Lecuona E, Welch LC, Chen J, Dada LA, Trejo HE, Dumasius V, Azzam ZS, Myrianthefs PM, Batlle D, Gruenbaum Y, and Sznajder JI
- Subjects
- Animals, Body Fluids, Epithelial Cells cytology, Epithelial Cells enzymology, Male, Phosphorylation, Pulmonary Alveoli cytology, Pulmonary Alveoli enzymology, Rats, Rats, Sprague-Dawley, Sodium-Potassium-Exchanging ATPase chemistry, Sodium-Potassium-Exchanging ATPase metabolism, Carbon Monoxide metabolism, Hydrogen-Ion Concentration, Pulmonary Alveoli physiology
- Abstract
Background: In patients with acute respiratory failure, gas exchange is impaired due to the accumulation of fluid in the lung airspaces. This life-threatening syndrome is treated with mechanical ventilation, which is adjusted to maintain gas exchange, but can be associated with the accumulation of carbon dioxide in the lung. Carbon dioxide (CO2) is a by-product of cellular energy utilization and its elimination is affected via alveolar epithelial cells. Signaling pathways sensitive to changes in CO2 levels were described in plants and neuronal mammalian cells. However, it has not been fully elucidated whether non-neuronal cells sense and respond to CO2. The Na,K-ATPase consumes approximately 40% of the cellular metabolism to maintain cell homeostasis. Our study examines the effects of increased pCO2 on the epithelial Na,K-ATPase a major contributor to alveolar fluid reabsorption which is a marker of alveolar epithelial function., Principal Findings: We found that short-term increases in pCO2 impaired alveolar fluid reabsorption in rats. Also, we provide evidence that non-excitable, alveolar epithelial cells sense and respond to high levels of CO2, independently of extracellular and intracellular pH, by inhibiting Na,K-ATPase function, via activation of PKCzeta which phosphorylates the Na,K-ATPase, causing it to endocytose from the plasma membrane into intracellular pools., Conclusions: Our data suggest that alveolar epithelial cells, through which CO2 is eliminated in mammals, are highly sensitive to hypercapnia. Elevated CO2 levels impair alveolar epithelial function, independently of pH, which is relevant in patients with lung diseases and altered alveolar gas exchange.
- Published
- 2007
- Full Text
- View/download PDF
46. Phosphorylation and ubiquitination are necessary for Na,K-ATPase endocytosis during hypoxia.
- Author
-
Dada LA, Welch LC, Zhou G, Ben-Saadon R, Ciechanover A, and Sznajder JI
- Subjects
- Amino Acid Sequence, Animals, CHO Cells, Cell Hypoxia, Cell Line, Tumor, Cell Membrane enzymology, Cricetinae, Cricetulus, Humans, Lysine metabolism, Molecular Sequence Data, Mutation genetics, Phosphorylation, Phosphoserine metabolism, Protein Subunits metabolism, Rats, Sodium-Potassium-Exchanging ATPase chemistry, Endocytosis, Sodium-Potassium-Exchanging ATPase metabolism, Ubiquitin metabolism
- Abstract
As a cellular adaptative response, hypoxia decreases Na,K-ATPase activity by triggering the endocytosis of its alpha(1) subunit in alveolar epithelial cells. Here, we present evidence that the ubiquitin conjugating system is important in the Na,K-ATPase endocytosis during hypoxia and that ubiquitination of Na,K-ATPase alpha(1) subunit occurs at the basolateral membrane. Endocytosis and ubiquitination were prevented when the Ser 18 in the PKC phosphorylation motif of the Na,K-ATPase alpha(1) subunit was mutated to an alanine, suggesting that phosphorylation at Ser-18 is required for ubiquitination. Mutation of the four lysines surrounding Ser 18 to arginine prevented Na,K-ATPase ubiquitination and endocytosis during hypoxia; however, only one of them was sufficient to restore hypoxia-induced endocytosis. We provide evidence that ubiquitination plays an important role in cellular adaptation to hypoxia by regulating Na,K-ATPase alpha(1)-subunit endocytosis.
- Published
- 2007
- Full Text
- View/download PDF
47. Role of the small GTPase RhoA in the hypoxia-induced decrease of plasma membrane Na,K-ATPase in A549 cells.
- Author
-
Dada LA, Novoa E, Lecuona E, Sun H, and Sznajder JI
- Subjects
- Adenoviridae, Amides pharmacology, Bronchoalveolar Lavage Fluid, Cell Hypoxia, Cell Line, Cell Membrane genetics, Electron Transport Complex III metabolism, Enzyme Inhibitors pharmacology, Free Radical Scavengers metabolism, Genes, Dominant, Glutathione Peroxidase genetics, Glutathione Peroxidase metabolism, Humans, Hydrogen Peroxide metabolism, Iron-Sulfur Proteins metabolism, Mitochondria genetics, Mitochondria metabolism, Pyridines pharmacology, Sodium-Potassium-Exchanging ATPase genetics, Transduction, Genetic, rhoA GTP-Binding Protein antagonists & inhibitors, Cell Membrane enzymology, Endocytosis drug effects, Endocytosis genetics, Enzyme Activation drug effects, Enzyme Activation genetics, Respiratory Mucosa enzymology, Sodium-Potassium-Exchanging ATPase metabolism, rhoA GTP-Binding Protein metabolism
- Abstract
Hypoxia impairs alveolar fluid reabsorption by promoting Na,K-ATPase endocytosis, from the plasma membrane of alveolar epithelial cells. The present study was designed to determine whether hypoxia induces Na,K-ATPase endocytosis via reactive oxygen species (ROS)-mediated RhoA activation. In A549 cells, RhoA activation occurred within 15 minutes of cells exposure to hypoxia. This activation was inhibited in cells infected with adenovirus coding for gluthatione peroxidase (an H2O2 scavenger), in mitochondria depleted (rho0) cells or cells expressing decreased levels of the Rieske iron-sulfur protein (inhibitor of mitochondrial complex III), which suggests a role for mitochondrial ROS. Moreover, exogenous H2O2 treatment during normoxia mimicked the effects of hypoxia on RhoA, further supporting a role for ROS. Cells expressing dominant negative RhoA failed to endocytose the Na,K-ATPase during hypoxia or after H2O2 treatment. Na,K-ATPase endocytosis was also prevented in cells treated with Y-27632, a Rho-associated kinase (ROCK) inhibitor, and in cells expressing dominant negative ROCK. In summary, we provide evidence that in human alveolar epithelial cells exposed to hypoxia, RhoA/ROCK activation is necessary for Na,K-ATPase endocytosis via a mechanism that requires mitochondrial ROS.
- Published
- 2007
- Full Text
- View/download PDF
48. Hypoxic inhibition of alveolar fluid reabsorption.
- Author
-
Dada LA and Sznajder JI
- Subjects
- Humans, Hypoxia physiopathology, Pulmonary Alveoli pathology, Pulmonary Edema etiology, Sodium-Potassium-Exchanging ATPase metabolism, Sodium-Potassium-Exchanging ATPase physiology, Extracellular Fluid metabolism, Hypoxia pathology, Pulmonary Alveoli physiopathology, Water-Electrolyte Balance physiology
- Abstract
Alveolar hypoxia occurs during ascent to high altitude and is also observed in patients with ARDS and acute hypoxemic respiratory failure, in which alveolar flooding is associated with a decrease in edema fluid clearance and increased mortality. The mechanisms that lead to the impairment of alveolar fluid clearance are not completely understood. Alveolar fluid reabsorption is accomplished mostly by active Na+ transport across the alveolar epithelium which creates an osmotic gradient responsible for the clearance of lung edema from the alveolar spaces. In vivo and in vitro hypoxia inhibits both the epithelial sodium channels, responsible for the apical sodium entry, and the basolateral Na,K-ATPase, responsible for Na+ extrusion. We have shown that acute hypoxia inhibits Na,K-ATPase function by promoting its endocytosis from the plasma membrane to intracellular compartments. This process is mediated by the generation of mitochondrial reactive oxygen species (ROS) as shown by pharmacological and genetic approaches. Hypoxia and ROS promote the PKC-zeta dependent phosphorylation of the Na,K-ATPase alpha subunit triggering its endocytosis in a clathrin-AP2 dependent process. The phosphorylation occurs at the Ser-18 in the alpha subunit N-terminus, and mutation of this serine prevents both the decrease in function and the endocytosis. More prolonged hypoxia causes the ubiquitination and degradation of Na,K-ATPase. Thus, methods that counterbalance the inhibition of edema clearance during hypoxia and improve the lung's ability to clear pulmonary edema are needed. As such, a better understanding of the mechanisms that increase Na,K-ATPase function, (i.e., activation of dopaminergic or adrenergic receptors, gene transfer) may lead to the development of therapeutic approaches to upregulate the Na-K-ATPase function and increase edema clearance.
- Published
- 2007
- Full Text
- View/download PDF
49. Na,K-ATPase alpha1-subunit dephosphorylation by protein phosphatase 2A is necessary for its recruitment to the plasma membrane.
- Author
-
Lecuona E, Dada LA, Sun H, Butti ML, Zhou G, Chew TL, and Sznajder JI
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Epithelial Cells metabolism, Humans, Male, Protein Phosphatase 2, Protein Subunits, Protein Transport physiology, Rats, Rats, Sprague-Dawley, Receptors, G-Protein-Coupled agonists, Phosphoprotein Phosphatases metabolism, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
In alveolar epithelial cells, G-protein coupled-receptors agonists (GPCR) induce the recruitment of the Na,K-ATPase to the plasma membrane. Here we report that for the recruitment of the Na,K-ATPase to occur, dephosphorylation of its alpha1-subunit at serine 18 is necessary, as demonstrated by in vitro phosphorylation, mutation of the serine 18 to alanine, and use of a specific phospho-antibody. Several approaches strongly suggest dephosphorylation to be mediated by protein phosphatase 2A (PP2A): 1) Na,K-ATPase dephosphorylation and recruitment were prevented by okadaic acid (OA); 2) the Na,K-ATPase alpha1-subunit is an in vitro substrate for PP2A; and 3) glutathione S-transferase (GST)-fusion proteins binding assays demonstrate a direct interaction between the catalytic subunit of PP2A and the first 90 amino acids of the Na,K-ATPase alpha1-subunit. Finally, GPCR agonists induced a rapid translocation of PP2A from the cytosol to the membrane fraction, which corresponded with increased coimmunoprecipitation and colocalization of PP2A and the Na,K-ATPase. Accordingly, we provide evidence that GPCR agonists promote PP2A translocation to the membrane fraction, leading to the dephosphorylation of the Na,K-ATPase alpha1-subunit at the serine 18 residue and its recruitment to the cell plasma membrane, which is of biological and physiological importance.
- Published
- 2006
- Full Text
- View/download PDF
50. Hypoxia-mediated degradation of Na,K-ATPase via mitochondrial reactive oxygen species and the ubiquitin-conjugating system.
- Author
-
Comellas AP, Dada LA, Lecuona E, Pesce LM, Chandel NS, Quesada N, Budinger GR, Strous GJ, Ciechanover A, and Sznajder JI
- Subjects
- Animals, CHO Cells, Cell Membrane enzymology, Cells, Cultured, Cricetinae, Cricetulus, Humans, Hypoxia enzymology, Lysosomes metabolism, Male, Oxygen Consumption, Proteasome Endopeptidase Complex metabolism, Rats, Rats, Sprague-Dawley, Hypoxia metabolism, Mitochondria metabolism, Reactive Oxygen Species metabolism, Sodium-Potassium-Exchanging ATPase metabolism, Ubiquitin-Conjugating Enzymes metabolism
- Abstract
We set out to determine whether cellular hypoxia, via mitochondrial reactive oxygen species, promotes Na,K-ATPase degradation via the ubiquitin-conjugating system. Cells exposed to 1.5% O2 had a decrease in Na,K-ATPase activity and oxygen consumption. The total cell pool of alpha1 Na,K-ATPase protein decreased on exposure to 1.5% O2 for 30 hours, whereas the plasma membrane Na,K-ATPase was 50% degraded after 2 hours of hypoxia, which was prevented by lysosome and proteasome inhibitors. When Chinese hamster ovary cells that exhibit a temperature-sensitive defect in E1 ubiquitin conjugation enzyme were incubated at 40 degrees C and 1.5% O2, the degradation of the alpha1 Na,K-ATPase was prevented. Exogenous reactive oxygen species increased the plasma membrane Na,K-ATPase degradation, whereas, in mitochondrial DNA deficient rho(0) cells and in cells transfected with small interfering RNA against Rieske iron sulfur protein, the hypoxia-mediated Na,K-ATPase degradation was prevented. The catalase/superoxide dismutase (SOD) mimetic (EUK-134) and glutathione peroxidase overexpression prevented the hypoxia-mediated Na,K-ATPase degradation and overexpression of SOD1, but not SOD2, partially inhibited the Na+ pump degradation. Accordingly, we provide evidence that during hypoxia, mitochondrial reactive oxygen species are necessary to degrade the plasma membrane Na,K-ATPase via the ubiquitin-conjugating system.
- Published
- 2006
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.