Publications by authors named "Laura A Dada"

45 Publications

TRAF2 Is a Novel Ubiquitin E3 Ligase for the Na,K-ATPase β-Subunit That Drives Alveolar Epithelial Dysfunction in Hypercapnia.

Front Cell Dev Biol 2021 2;9:689983. Epub 2021 Jul 2.

Member of the German Center for Lung Research (DZL), Department of Internal Medicine, Justus Liebig University Giessen, Universities of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany.

Several acute and chronic lung diseases are associated with alveolar hypoventilation leading to accumulation of CO (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 CO 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.
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http://dx.doi.org/10.3389/fcell.2021.689983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8283768PMC
July 2021

Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease.

J Membr Biol 2021 Jun 10. Epub 2021 Jun 10.

Department of Internal Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Justus Liebig University, Klinikstrasse 33, 35392, Giessen, Germany.

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.
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http://dx.doi.org/10.1007/s00232-021-00184-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192048PMC
June 2021

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Am J Physiol Lung Cell Mol Physiol 2021 06 21;320(6):L1183-L1185. Epub 2021 Apr 21.

Northwestern University Feinberg School of Medicine, Chicago, Illinois.

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http://dx.doi.org/10.1152/ajplung.00170.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238444PMC
June 2021

Hypercapnia Impairs Na,K-ATPase Function by Inducing Endoplasmic Reticulum Retention of the β-Subunit of the Enzyme in Alveolar Epithelial Cells.

Int J Mol Sci 2020 Feb 21;21(4). Epub 2020 Feb 21.

Department of Internal Medicine, Justus Liebig University, Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), 35392 Giessen, Germany.

Alveolar edema, impaired alveolar fluid clearance, and elevated CO 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 CO 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 CO 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 CO 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 CO levels, which may lead to the development of new therapeutic approaches for patients with ARDS and hypercapnia.
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http://dx.doi.org/10.3390/ijms21041467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073107PMC
February 2020

Elevated CO regulates the Wnt signaling pathway in mammals, Drosophila melanogaster and Caenorhabditis elegans.

Sci Rep 2019 12 3;9(1):18251. Epub 2019 Dec 3.

Division of Pulmonary and Critical Care, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States of America.

Carbon dioxide (CO) 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 CO 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 CO in regulating Wnt pathway genes.
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http://dx.doi.org/10.1038/s41598-019-54683-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890671PMC
December 2019

Linear ubiquitin assembly complex regulates lung epithelial-driven responses during influenza infection.

J Clin Invest 2020 03;130(3):1301-1314

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Northwestern University, Chicago, Illinois, USA.

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.
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http://dx.doi.org/10.1172/JCI128368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269564PMC
March 2020

Influenza A Virus Infection Induces Muscle Wasting via IL-6 Regulation of the E3 Ubiquitin Ligase Atrogin-1.

J Immunol 2019 01 7;202(2):484-493. Epub 2018 Dec 7.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611;

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, and 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.
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http://dx.doi.org/10.4049/jimmunol.1701433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324970PMC
January 2019

Ubiquitin-proteasome signaling in lung injury.

Transl Res 2018 08 23;198:29-39. Epub 2018 Apr 23.

Pulmonary and Critical Care Division, Northwestern Feinberg School of Medicine, Chicago, Illinois. Electronic address:

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.
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http://dx.doi.org/10.1016/j.trsl.2018.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986356PMC
August 2018

Splice Wars: The Role of MLCK Isoforms in Ventilation-induced Lung Injury.

Am J Respir Cell Mol Biol 2018 05;58(5):549-550

1 Division of Pulmonary and Critical Care Northwestern University Chicago, Illinois.

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http://dx.doi.org/10.1165/rcmb.2017-0385EDDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946334PMC
May 2018

HIF and HOIL-1L-mediated PKCζ degradation stabilizes plasma membrane Na,K-ATPase to protect against hypoxia-induced lung injury.

Proc Natl Acad Sci U S A 2017 11 6;114(47):E10178-E10186. Epub 2017 Nov 6.

Pulmonary and Critical Care Division, Northwestern Feinberg School of Medicine, Chicago, IL, 60611;

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-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 ( ) 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 α-Na,K-ATPase construct bearing an S18A (α-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 α-S18A mutant Na,K-ATPase in vivo rescued the enhanced sensitivity of 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.
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http://dx.doi.org/10.1073/pnas.1713563114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703319PMC
November 2017

Downregulation of PKCζ/Pard3/Pard6b is responsible for lung adenocarcinoma cell EMT and invasion.

Cell Signal 2017 10 24;38:49-59. Epub 2017 Jun 24.

Department of Pediatrics, University of Illinois at Chicago, Chicago, IL, USA; Cancer Center, University of Illinois at Chicago, Chicago, IL, USA; State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Diseases, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cellsig.2017.06.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555371PMC
October 2017

FXYD5 Is an Essential Mediator of the Inflammatory Response during Lung Injury.

Front Immunol 2017 1;8:623. Epub 2017 Jun 1.

Pulmonary and Critical Care Division, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States.

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.
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http://dx.doi.org/10.3389/fimmu.2017.00623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451504PMC
June 2017

Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart: DISTINCT FUNCTIONAL PROPERTIES AND ISOFORM-SELECTIVE INHIBITORS.

J Biol Chem 2016 10 13;291(44):23159-23174. Epub 2016 Sep 13.

From the Department of Biomolecular Sciences and

The Na,K-ATPase α subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca, whereas α 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 α 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 α is preferentially expressed with β in T-tubules of cardiac myocytes, forming αβ heterodimers. We have expressed human αβ, αβ, αβ, and αβ in Pichia pastoris, purified the complexes, and compared their functional properties. αβ and αβ differ significantly from both αβ and αβ in having a higher KK and lower KNa for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K and shift of the EP-EP conformational equilibrium toward EP. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of αβ and αβ over αβ (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 αβ could allow an acute response to raised ambient K concentrations in physiological conditions and explain the importance of αβ for cardiac muscle contractility. The high sensitivity of αβ to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of αβ-selective digoxin derivatives for reducing cardiotoxicity.
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http://dx.doi.org/10.1074/jbc.M116.751735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087734PMC
October 2016

The O-glycosylated ectodomain of FXYD5 impairs adhesion by disrupting cell-cell trans-dimerization of Na,K-ATPase β1 subunits.

J Cell Sci 2016 06 3;129(12):2394-406. Epub 2016 May 3.

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA

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.
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http://dx.doi.org/10.1242/jcs.186148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920254PMC
June 2016

FXYD5 Protein Has a Pro-inflammatory Role in Epithelial Cells.

J Biol Chem 2016 May 22;291(21):11072-82. Epub 2016 Mar 22.

From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 7610001, Israel and.

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.
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http://dx.doi.org/10.1074/jbc.M115.699041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900257PMC
May 2016

Role of Linear Ubiquitination in Health and Disease.

Am J Respir Cell Mol Biol 2016 06;54(6):761-8

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Northwestern University, Chicago, Illinois.

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.
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http://dx.doi.org/10.1165/rcmb.2016-0014TRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4942223PMC
June 2016

High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation.

Mol Cell Biol 2015 Dec 14;35(23):3962-73. Epub 2015 Sep 14.

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

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.
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http://dx.doi.org/10.1128/MCB.00813-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628060PMC
December 2015

High CO2 levels cause skeletal muscle atrophy via AMP-activated kinase (AMPK), FoxO3a protein, and muscle-specific Ring finger protein 1 (MuRF1).

J Biol Chem 2015 Apr 17;290(14):9183-94. Epub 2015 Feb 17.

From the Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, Illinois 60611,

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.
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http://dx.doi.org/10.1074/jbc.M114.625715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4423704PMC
April 2015

Septin dynamics are essential for exocytosis.

J Biol Chem 2015 Feb 9;290(9):5280-97. Epub 2015 Jan 9.

From the Departments of Physiology and Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073,

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.
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http://dx.doi.org/10.1074/jbc.M114.616201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342448PMC
February 2015

Intratracheal administration of influenza virus is superior to intranasal administration as a model of acute lung injury.

J Virol Methods 2014 Dec 17;209:116-20. Epub 2014 Sep 17.

Division of Pulmonary and Critical Care Medicine, Northwestern University, Feinberg School of Medicine, Chicago, IL, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jviromet.2014.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4315182PMC
December 2014

HOIL-1L functions as the PKCζ ubiquitin ligase to promote lung tumor growth.

Am J Respir Crit Care Med 2014 Sep;190(6):688-98

1 Division of Pulmonary and Critical Care Medicine.

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.
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http://dx.doi.org/10.1164/rccm.201403-0463OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4214108PMC
September 2014

Evolutionary conserved role of c-Jun-N-terminal kinase in CO2-induced epithelial dysfunction.

PLoS One 2012 8;7(10):e46696. Epub 2012 Oct 8.

Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, Illinois, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046696PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3466313PMC
May 2013

Identification of the amino acid region involved in the intercellular interaction between the β1 subunits of Na+/K+ -ATPase.

J Cell Sci 2012 Mar 10;125(Pt 6):1605-16. Epub 2012 Feb 10.

Department of Physiology, School of Medicine, UCLA and Veterans Administration Greater Los Angeles Health Care System, Los Angeles, VAGLAHS/West LA, Building 113, Room 324, 11301 Wilshire Boulevard, Los Angeles, CA 90073, USA.

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.
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http://dx.doi.org/10.1242/jcs.100149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3336383PMC
March 2012

The Na-K-ATPase α₁β₁ heterodimer as a cell adhesion molecule in epithelia.

Am J Physiol Cell Physiol 2012 May 25;302(9):C1271-81. Epub 2012 Jan 25.

Department of Physiology, School of Medicine, University of California Los Angeles and Veterans Administration Greater Los Angeles Health Care System, Los Angeles, California, USA.

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.
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http://dx.doi.org/10.1152/ajpcell.00456.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361946PMC
May 2012

Hypoxia leads to Na,K-ATPase downregulation via Ca(2+) release-activated Ca(2+) channels and AMPK activation.

Mol Cell Biol 2011 Sep 5;31(17):3546-56. Epub 2011 Jul 5.

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

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.
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http://dx.doi.org/10.1128/MCB.05114-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165547PMC
September 2011

Mitochondrial Ca²+ and ROS take center stage to orchestrate TNF-α-mediated inflammatory responses.

J Clin Invest 2011 May 25;121(5):1683-5. Epub 2011 Apr 25.

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

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.
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http://dx.doi.org/10.1172/JCI57748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083786PMC
May 2011

Extracellular signal-regulated kinase (ERK) participates in the hypercapnia-induced Na,K-ATPase downregulation.

FEBS Lett 2010 Sep 6;584(18):3985-9. Epub 2010 Aug 6.

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

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.
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http://dx.doi.org/10.1016/j.febslet.2010.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2966388PMC
September 2010

E3 ubiquitin ligase Mule ubiquitinates Miz1 and is required for TNFalpha-induced JNK activation.

Proc Natl Acad Sci U S A 2010 Jul 12;107(30):13444-9. Epub 2010 Jul 12.

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

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.
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http://dx.doi.org/10.1073/pnas.0913690107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2922175PMC
July 2010

Insulin regulates alveolar epithelial function by inducing Na+/K+-ATPase translocation to the plasma membrane in a process mediated by the action of Akt.

J Cell Sci 2010 Apr 23;123(Pt 8):1343-51. Epub 2010 Mar 23.

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

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.
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http://dx.doi.org/10.1242/jcs.066464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2848117PMC
April 2010
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