| 1. |
- Dabral, Swati, et al.
(författare)
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A RASSF1A-HIF1 alpha loop drives Warburg effect in cancer and pulmonary hypertension
- 2019
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Hypoxia signaling plays a major role in non-malignant and malignant hyperproliferative diseases. Pulmonary hypertension (PH), a hypoxia-driven vascular disease, is characterized by a glycolytic switch similar to the Warburg effect in cancer. Ras association domain family 1A (RASSF1A) is a scaffold protein that acts as a tumour suppressor. Here we show that hypoxia promotes stabilization of RASSF1A through NOX-1- and protein kinase C- dependent phosphorylation. In parallel, hypoxia inducible factor-1 alpha (HIF-1 alpha) activates RASSF1A transcription via HIF-binding sites in the RASSF1A promoter region. Vice versa, RASSF1A binds to HIF-1 alpha, blocks its prolyl-hydroxylation and proteasomal degradation, and thus enhances the activation of the glycolytic switch. We find that this mechanism operates in experimental hypoxia-induced PH, which is blocked in RASSF1A knockout mice, in human primary PH vascular cells, and in a subset of human lung cancer cells. We conclude that RASSF1A-HIF-1 alpha forms a feedforward loop driving hypoxia signaling in PH and cancer.
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| 2. |
- Alberro-Brage, Andres, et al.
(författare)
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Influenza virus decreases albumin uptake and megalin expression in alveolar epithelial cells
- 2023
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Ingår i: Frontiers in Immunology. - 1664-3224. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- IntroductionAcute respiratory distress syndrome (ARDS) is a common complication of influenza virus (IV) infection. During ARDS, alveolar protein concentrations often reach 40-90% of plasma levels, causing severe impairment of gas exchange and promoting deleterious alveolar remodeling. Protein clearance from the alveolar space is at least in part facilitated by the multi-ligand receptor megalin through clathrin-mediated endocytosis.MethodsTo investigate whether IV infection impairs alveolar protein clearance, we examined albumin uptake and megalin expression in MLE-12 cells and alveolar epithelial cells (AEC) from murine precision-cut lung slices (PCLS) and in vivo, under IV infection conditions by flow cytometry and western blot. Transcriptional levels from AEC and broncho-alveolar lavage (BAL) cells were analyzed in an in-vivo mouse model by RNAseq.ResultsIV significantly downregulated albumin uptake, independently of activation of the TGF- β1/GSK3β axis that has been previously implicated in the regulation of megalin function. Decreased plasma membrane abundance, total protein levels, and mRNA expression of megalin were associated with this phenotype. In IV-infected mice, we identified a significant upregulation of matrix metalloproteinase (MMP)-14 in BAL fluid cells. Furthermore, the inhibition of this protease partially recovered total megalin levels and albumin uptake.DiscussionOur results suggest that the previously described MMP-driven shedding mechanisms are potentially involved in downregulation of megalin cell surface abundance and clearance of excess alveolar protein. As lower alveolar edema protein concentrations are associated with better outcomes in respiratory failure, our findings highlight the therapeutic potential of a timely MMP inhibition in the treatment of IV-induced ARDS.
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| 3. |
- Giordano, Luca, et al.
(författare)
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Essential Role of Mitochondrial Cytochrome c Oxidase Subunit 4 Isoform 2 (Cox4i2) for Acute Pulmonary Oxygen Sensing
- 2022
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier. - 0005-2728 .- 1879-2650. ; 1863:Supplement
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondrial Cytochrome c Oxidase Subunit 4 Isoform 2 (Cox4i2) is essential for acute oxygen sensing and signaling in pulmonary arterial smooth muscle cells (PASMCs) by triggering the production of superoxide during acute hypoxia [1]. However, the molecular mechanism underlying Cox4i2-dependent oxygen sensing remains elusive. We analysed oxygen-dependent respiration by high resolution respirometry, redox changes of the electron transport chain (ETC) by RAMAN spectroscopy, and supercomplex formation by blue native gel analysis of PASMCs isolated from wild type (WT) and Cox4i2-/- mice. To understand the role of Cox4i2-specific cysteine residues we determined hypoxia-induced superoxide production and oxygen affinity in a mouse epithelial cell line (CMT167 cells) overexpressing either Cox4i1, or WT Cox4i2, or Cox4i2 mutants (C41S, C55A, C109S). Respiration and supercomplex formation were similar in WT and Cox4i2-/- PASMCs. Interestingly, hypoxia-induced reduction of ETC components (NADH, ubiquinol, and reduced cytochrome c) was prevented in Cox4i2-/- PASMCs. CMT167 cells expressing either Cox4i1, or Cox4i2 mutants lacked hypoxia-induced superoxide release, which was detected only in cells expressing WT Cox4i2. In contrast, overexpression of Cox4i1, or Cox4i2, or Cox4i2 mutants did not affect oxygen affinity. Our findings suggest that Cox4i2 does not alter superoxide production by rearrangement of supercomplexes, whereas its specific cysteines are needed for the superoxide release. In conclusion, Cox4i2 plays a major role in the hypoxia-induced reduction of ETC components, likely mediated through its redox-active cysteine residues.
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| 4. |
- Klymenko, Oleksiy, et al.
(författare)
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Regulation and role of the ER stress transcription factor CHOP in alveolar epithelial type-II cells
- 2019
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Ingår i: Journal of Molecular Medicine. - : Springer Science and Business Media LLC. - 0946-2716 .- 1432-1440. ; 97:7, s. 973-990
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Tidskriftsartikel (refereegranskat)abstract
- Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by type-II alveolar epithelial cell (AECII) injury and fibroblast hyperproliferation. Severe AECII endoplasmic reticulum (ER) stress is thought to underlie IPF, but is yet incompletely understood. We studied the regulation of C/EBP homologous protein (CHOP), a proapoptotic ER-stress-related transcription factor (TF) in AECII-like cells. Interestingly, single or combined overexpression of the active ER stress transducers activating transcription factor-4 (Atf4) and activating transcription factor-6 (p50Atf6) or spliced x-box-binding protein-1 (sXbp1) in MLE12 cells did not result in a substantial Chop induction, as compared to the ER stress inducer thapsigargin. Employing reporter gene assays of distinct CHOP promoter fragments, we could identify that, next to the conventional amino acid (AARE) and ER stress response elements (ERSE) within the CHOP promoter, activator protein-1 (AP-1) and c-Ets-1 TF binding sites are necessary for CHOP induction. Serial deletion and mutation analyses revealed that both AP-1 and c-Ets-1 motifs act in concert to induce CHOP expression. In agreement, CHOP promoter activity was greatly enhanced upon combined versus single overexpression of AP-1 and c-Ets-1. Moreover, combined overexpression of AP-1 and c-Ets-1 in MLE12 cells alone in the absence of any other ER stress inducer was sufficient to induce Chop protein expression. Further, AP-1 and c-Ets-1 were upregulated in AECII under ER stress conditions and in human IPF. Finally, Chop overexpression in vitro resulted in AECII apoptosis, lung fibroblast proliferation, and collagen-I production. We propose that CHOP activation by AP-1 and c-Ets-1 plays a key role in AECII maladaptive ER stress responses and consecutive fibrosis, offering new therapeutic prospects in IPF.Key messagesOverexpression of active ER stress sensors Atf4, Atf6, and Xbp1 does not induce Chop.AP-1 and c-Ets-1 TFs are necessary for induction of the ER stress factor Chop.AP-1 and c-Ets-1 alone induce Chop expression in the absence of any ER stress inducers.AP-1 and c-Ets-1 are induced in AECII under ER stress conditions and in human IPF.Chop expression alone triggers AECII apoptosis and consecutive profibrotic responses.
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| 5. |
- Kryvenko, Vitalii, et al.
(författare)
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Clathrin-Mediated Albumin Clearance in Alveolar Epithelial Cells of Murine Precision-Cut Lung Slices
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 24:3
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Tidskriftsartikel (refereegranskat)abstract
- A hallmark of acute respiratory distress syndrome (ARDS) is an accumulation of protein-rich alveolar edema that impairs gas exchange and leads to worse outcomes. Thus, understanding the mechanisms of alveolar albumin clearance is of high clinical relevance. Here, we investigated the mechanisms of the cellular albumin uptake in a three-dimensional culture of precision-cut lung slices (PCLS). We found that up to 60% of PCLS cells incorporated labeled albumin in a time- and concentration-dependent manner, whereas virtually no uptake of labeled dextran was observed. Of note, at a low temperature (4 °C), saturating albumin receptors with unlabeled albumin and an inhibition of clathrin-mediated endocytosis markedly decreased the endocytic uptake of the labeled protein, implicating a receptor-driven internalization process. Importantly, uptake rates of albumin were comparable in alveolar epithelial type I (ATI) and type II (ATII) cells, as assessed in PCLS from a SftpcCreERT2/+: tdTomatoflox/flox mouse strain (defined as EpCAM+CD31−CD45−tdTomatoSPC−T1α+ for ATI and EpCAM+CD31−CD45−tdTomatoSPC+T1α− for ATII cells). Once internalized, albumin was found in the early and recycling endosomes of the alveolar epithelium as well as in endothelial, mesenchymal, and hematopoietic cell populations, which might indicate transcytosis of the protein. In summary, we characterize albumin uptake in alveolar epithelial cells in the complex setting of PCLS. These findings may open new possibilities for pulmonary drug delivery that may improve the outcomes for patients with respiratory failure.
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| 6. |
- Liontos, Andreas, et al.
(författare)
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Secretory cell distribution in the adult airways follows graded proximodistal differentiation programs controlled by postnatal FGFR signaling
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Respiratory organs are composed of branched tubular epithelial networks that optimize gas flow and shield against airborne microbes and noxious chemicals. A systematic account of lung epithelial heterogeneity is expected to elucidate epithelial cell functions and facilitate new repair strategies upon infection. Here, we combined mouse genetics, single-cell mRNA sequencing and multiplex in situ mapping to generate a topological lineage map of adult airway epithelial cells. Apart from ciliated cells, we identified and focused on 4 major adult secretory cell states with distinct localizations along the proximo-distal axis of intrapulmonary airways. Positional differences in cell type occurrence reveal a gradient of epithelial heterogeneity, where proximal and distal epithelial cells highly express characteristic gene programs, whereas cells in intermediate positions of the airway stalks are characterized by low levels of both proximal and distal gene programs. Lineage-tracing experiment combined with single cell sequencing and spatial analysis suggest the hierarchical activation of genetic programs establishing mature secretory and ciliated epithelial cell types from embryonic progenitors. We tested if the distinct transcriptomic states of secretory cells reflect functional differences in vitro, by comparing differentiation potentials of secretory cells isolated from distinct locations. Distally located secretory cells showed an increased potency compared to proximal cells and Scgb1a1-Sftpc double positive cells (DPs). To identify distal determinant signals that generate this diversity, we investigated the significance of postnatal upregulation of FGF signaling components in secretory cells. Conditional inactivation of Fgfr2 suggests that FGF signaling selectively controls the identity of distal secretory cells by upregulating surfactant and lipid metabolism genes and repressing alveolar type I genes, without affecting the expression of proximal-distal graded programs. Our work elucidates the spatial heterogeneity of the airway secretory epithelium and reveals its developmental origin.
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| 7. |
- Menkveld, Albert J., et al.
(författare)
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Nonstandard Errors
- 2024
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Ingår i: JOURNAL OF FINANCE. - : Wiley-Blackwell. - 0022-1082 .- 1540-6261. ; 79:3, s. 2339-2390
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Tidskriftsartikel (refereegranskat)abstract
- In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty-nonstandard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for more reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants.
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| 8. |
- Seeger, Christian, et al.
(författare)
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Histaminergic pharmacology of homo-oligomeric beta 3 gamma-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology
- 2012
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Ingår i: Biochemical Pharmacology. - : Elsevier BV. - 0006-2952 .- 1356-1839. ; 84:3, s. 341-351
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Tidskriftsartikel (refereegranskat)abstract
- A surface plasmon resonance biosensor assay was established for studying the interactions of 51 histaminergic and 15 GABAergic ligands with homo-oligomeric beta 3 GABA(A) receptors. Detergent solubilized receptors were successfully immobilized via affinity-capture on biosensor surfaces. The interaction kinetics of both histaminergic and GABAergic ligands were very rapid but affinities could be determined by steady-state analysis. Binding of several GABAergic ligands was observed, in agreement with previous data. Histamine and 16 histaminergic ligands were detected to directly bind to beta 3 GABA(A) receptors with micromolar affinity (K-D <300 mu M), thus extending previous evidence that beta 3 GABA(A) receptors can interact with histaminergic ligands. Histamine exhibited an affinity for these receptors comparable to that for human histamine type 1 (H1) or type 2 (H2) receptors. Furthermore, 13 of these histaminergic ligands appeared to compete with histamine. The discovery that H2, H3 and H4 receptor ligands interact with beta 3 receptors indicates a unique histaminergic pharmacology of these receptors. Due to their low affinity for the homo-pentameric beta 3 receptors these histaminergic drugs are not expected to modulate these receptors at clinically relevant concentrations. The results support the use of the new biosensor assay for the identification of drugs interacting with full length receptors and for fragment-based drug discovery of high affinity ligands for beta 3 receptors. Drugs with high affinity and selectivity for these receptors can be used to clarify the question whether beta 3 receptors do exist in the brain, and provide new avenues for the development of therapeutically active compounds targeting this novel histamine binding site.
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| 9. |
- Sommer, Natascha, et al.
(författare)
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Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensing
- 2020
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Ingår i: Science Advances. - : American Association for the Advancement of Science. - 2375-2548. ; 6:16
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondria play an important role in sensing both acute and chronic hypoxia in the pulmonary vasculature, but their primary oxygen-sensing mechanism and contribution to stabilization of the hypoxia-inducible factor (HIF) remains elusive. Alteration of the mitochondrial electron flux and increased superoxide release from complex III has been proposed as an essential trigger for hypoxic pulmonary vasoconstriction (HPV). We used mice expressing a tunicate alternative oxidase, AOX, which maintains electron flux when respiratory complexes III and/or IV are inhibited. Respiratory restoration by AOX prevented acute HPV and hypoxic responses of pulmonary arterial smooth muscle cells (PASMC), acute hypoxia-induced redox changes of NADH and cytochrome c, and superoxide production. In contrast, AOX did not affect the development of chronic hypoxia-induced pulmonary hypertension and HIF-1α stabilization. These results indicate that distal inhibition of the mitochondrial electron transport chain in PASMC is an essential initial step for acute but not chronic oxygen sensing.
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| 10. |
- Sountoulidis, Alexandros, et al.
(författare)
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SCRINSHOT enables spatial mapping of cell states in tissue sections with single-cell resolution
- 2020
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Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 18:11
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Tidskriftsartikel (refereegranskat)abstract
- Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.
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