Publications by authors named "Uilke Brouwer"

10 Publications

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Autophagy induction during stem cell activation plays a key role in salivary gland self-renewal.

Autophagy 2021 May 19:1-16. Epub 2021 May 19.

Department of Biomedical Sciences of Cells and Systems, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Relatively quiescent tissues like salivary glands (SGs) respond to stimuli such as injury to expand, replace and regenerate. Resident stem/progenitor cells are key in this process because, upon activation, they possess the ability to self-renew. Macroautophagy/autophagy contributes to and regulates differentiation in adult tissues, but an important question is whether this pathway promotes stem cell self-renewal in tissues. We took advantage of a 3D organoid system that allows assessing the self-renewal of mouse SGs stem cells (SGSCs). We found that autophagy in dormant SGSCs has slower flux than self-renewing SGSCs. Importantly, autophagy enhancement upon SGSCs activation is a self-renewal feature in 3D organoid cultures and SGs regenerating . Accordingly, autophagy ablation in SGSCs inhibits self-renewal whereas pharmacological stimulation promotes self-renewal of mouse and human SGSCs. Thus, autophagy is a key pathway for self-renewal activation in low proliferative adult tissues, and its pharmacological manipulation has the potential to promote tissue regeneration.
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http://dx.doi.org/10.1080/15548627.2021.1924036DOI Listing
May 2021

Cellular senescence contributes to radiation-induced hyposalivation by affecting the stem/progenitor cell niche.

Cell Death Dis 2020 10 14;11(10):854. Epub 2020 Oct 14.

Department of Biomedical Sciences of Cells & Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Radiotherapy for head and neck cancer is associated with impairment of salivary gland function and consequent xerostomia, which has a devastating effect on the quality of life of the patients. The mechanism of radiation-induced salivary gland damage is not completely understood. Cellular senescence is a permanent state of cell cycle arrest accompanied by a secretory phenotype which contributes to inflammation and tissue deterioration. Genotoxic stresses, including radiation-induced DNA damage, are known to induce a senescence response. Here, we show that radiation induces cellular senescence preferentially in the salivary gland stem/progenitor cell niche of mouse models and patients. Similarly, salivary gland-derived organoids show increased expression of senescence markers and pro-inflammatory senescence-associated secretory phenotype (SASP) factors after radiation exposure. Clearance of senescent cells by selective removal of p16Ink4a-positive cells by the drug ganciclovir or the senolytic drug ABT263 lead to increased stem cell self-renewal capacity as measured by organoid formation efficiency. Additionally, pharmacological treatment with ABT263 in mice irradiated to the salivary glands mitigates tissue degeneration, thus preserving salivation. Our data suggest that senescence in the salivary gland stem/progenitor cell niche contributes to radiation-induced hyposalivation. Pharmacological targeting of senescent cells may represent a therapeutic strategy to prevent radiotherapy-induced xerostomia.
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http://dx.doi.org/10.1038/s41419-020-03074-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566836PMC
October 2020

KRAB-Induced Heterochromatin Effectively Silences Gene Expression in Somatic Cells and is Resilient to TGFβ1 Activation.

Int J Mol Sci 2020 May 21;21(10). Epub 2020 May 21.

Epigenetic Editing Laboratory, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Hanzeplein 1 EA11, 9713 GZ Groningen, The Netherlands.

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete silencing. Interestingly, both long-term TGFβ1-induced, as well as unstimulated expression, was completely repressed by KRAB, while M.SssI only prevented the TGFβ1-induced expression. Targeting transiently expressed dCas9-KRAB resulted in sustained repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFβ1-induced and uninduced expression.
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http://dx.doi.org/10.3390/ijms21103634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279273PMC
May 2020

Protocadherin-1 Localization and Cell-Adhesion Function in Airway Epithelial Cells in Asthma.

PLoS One 2016 4;11(10):e0163967. Epub 2016 Oct 4.

Department of Pediatric Pulmonology and Pediatric Allergology, Beatrix Children's Hospital, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Background: The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma.

Methods: We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance.

Results: PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding.

Conclusions: In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163967PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5049773PMC
June 2017

Protocadherin-1 binds to SMAD3 and suppresses TGF-β1-induced gene transcription.

Am J Physiol Lung Cell Mol Physiol 2015 Oct 24;309(7):L725-35. Epub 2015 Jul 24.

University of Groningen, University Medical Center Groningen, Experimental Pulmonology and Inflammation Research (EXPIRE), Department of Pathology & Medical Biology, Groningen, The Netherlands; GRIAC Research Institute, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands;

Genetic studies have identified Protocadherin-1 (PCDH1) and Mothers against decapentaplegic homolog-3 (SMAD3) as susceptibility genes for asthma. PCDH1 is expressed in bronchial epithelial cells and has been found to interact with SMAD3 in yeast two-hybrid (Y2H) overexpression assays. Here, we test whether PCDH1 and SMAD3 interact at endogenous protein levels in bronchial epithelial cells and evaluate the consequences thereof for transforming growth factor-β1 (TGF-β1)-induced gene transcription. We performed Y2H screens and coimmunoprecipitation (co-IP) experiments of PCDH1 and SMAD3 in HEK293T and 16HBE14o(-) (16HBE) cell lines. Activity of a SMAD3-driven luciferase reporter gene in response to TGF-β1 was measured in BEAS-2B cells transfected with PCDH1 and in 16HBE cells transfected with PCDH1-small-interfering RNA (siRNA). TGF-β1-induced gene expression was quantified in BEAS-2B clones overexpressing PCDH1 and in human primary bronchial epithelial cells (PBECs) transfected with PCDH1-siRNA. We confirm PCDH1 and SMAD3 interactions by Y2H and by co-IP in HEK293T cells overexpressing both proteins, and at endogenous protein levels in 16HBE cells. TGF-β-induced activation of a SMAD3-driven reporter was reduced by exogenous PCDH1 in BEAS2B cells, whereas it was increased by siRNA-mediated knockdown of endogenous PCDH1 in 16HBE cells. Overexpression of PCDH1 suppressed expression of TGF-β target genes in BEAS-2B cells, whereas knockdown of PCDH1 in human PBECs increased TGF-β-induced gene expression. In conclusion, we demonstrate that PCDH1 binds to SMAD3 and regulates its activation by TGF-β signaling in bronchial epithelial cells. We propose that PCDH1 and SMAD3 act in a single pathway in asthma susceptibility that affects sensitivity of the airway epithelium to TGF-β.
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http://dx.doi.org/10.1152/ajplung.00346.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4593836PMC
October 2015

Genetic variation associates with susceptibility for cigarette smoke-induced neutrophilia in mice.

Am J Physiol Lung Cell Mol Physiol 2015 Apr 30;308(7):L693-709. Epub 2015 Jan 30.

University of Groningen, University Medical Center Groningen, Department of Pathology and Medical Biology, Experimental Pulmonology and Inflammation Research, Groningen, The Netherlands; GRIAC Research Institute, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands;

Neutrophilic airway inflammation is one of the major hallmarks of chronic obstructive pulmonary disease and is also seen in steroid resistant asthma. Neutrophilic airway inflammation can be induced by different stimuli including cigarette smoke (CS). Short-term exposure to CS induces neutrophilic airway inflammation in both mice and humans. Since not all individuals develop extensive neutrophilic airway inflammation upon smoking, we hypothesized that this CS-induced innate inflammation has a genetic component. This hypothesis was addressed by exposing 30 different inbred mouse strains to CS or control air for 5 consecutive days, followed by analysis of neutrophilic lung inflammation. By genomewide haplotype association mapping, we identified four susceptibility genes with a significant association to lung tissue levels of the neutrophil marker myeloperoxidase under basal conditions and an additional five genes specifically associated with CS-induced tissue MPO levels. Analysis of the expression levels of the susceptibility genes by quantitative RT-PCR revealed that three of the four genes associated with CS-induced tissue MPO levels had CS-induced changes in gene expression levels that correlate with CS-induced airway inflammation. Most notably, CS exposure induces an increased expression of the coiled-coil domain containing gene, Ccdc93, in mouse strains susceptible for CS-induced airway inflammation whereas Ccdc93 expression was decreased upon CS exposure in nonsusceptible mouse strains. In conclusion, this study shows that CS-induced neutrophilic airway inflammation has a genetic component and that several genes contribute to the susceptibility for this response.
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http://dx.doi.org/10.1152/ajplung.00118.2014DOI Listing
April 2015

Mouse protocadherin-1 gene expression is regulated by cigarette smoke exposure in vivo.

PLoS One 2014 3;9(7):e98197. Epub 2014 Jul 3.

Laboratory of Allergology and Pulmonary Diseases, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen (UMCG), Groningen, the Netherlands; GRIAC research institute, University of Groningen, University Medical Center Groningen (UMCG), Groningen, the Netherlands.

Protocadherin-1 (PCDH1) is a novel susceptibility gene for airway hyperresponsiveness, first identified in families exposed to cigarette smoke and is expressed in bronchial epithelial cells. Here, we asked how mouse Pcdh1 expression is regulated in lung structural cells in vivo under physiological conditions, and in both short-term cigarette smoke exposure models characterized by airway inflammation and hyperresponsiveness and chronic cigarette smoke exposure models. Pcdh1 gene-structure was investigated by Rapid Amplification of cDNA Ends. Pcdh1 mRNA and protein expression was investigated by qRT-PCR, western blotting using isoform-specific antibodies. We observed 87% conservation of the Pcdh1 nucleotide sequence, and 96% conservation of the Pcdh1 protein sequence between men and mice. We identified a novel Pcdh1 isoform encoding only the intracellular signalling motifs. Cigarette smoke exposure for 4 consecutive days markedly reduced Pcdh1 mRNA expression in lung tissue (3 to 4-fold), while neutrophilia and airway hyperresponsiveness was induced. Moreover, Pcdh1 mRNA expression in lung tissue was reduced already 6 hours after an acute cigarette-smoke exposure in mice. Chronic exposure to cigarette smoke induced loss of Pcdh1 protein in lung tissue after 2 months, while Pcdh1 protein levels were no longer reduced after 9 months of cigarette smoke exposure. We conclude that Pcdh1 is highly homologous to human PCDH1, encodes two transmembrane proteins and one intracellular protein, and is regulated by cigarette smoke exposure in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098197PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4081120PMC
November 2015

Susceptibility to chronic mucus hypersecretion, a genome wide association study.

PLoS One 2014 8;9(4):e91621. Epub 2014 Apr 8.

Channing Division of Network Medicine, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America; Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America; Harvard Medical School, Boston, Massachusetts, United States of America.

Background: Chronic mucus hypersecretion (CMH) is associated with an increased frequency of respiratory infections, excess lung function decline, and increased hospitalisation and mortality rates in the general population. It is associated with smoking, but it is unknown why only a minority of smokers develops CMH. A plausible explanation for this phenomenon is a predisposing genetic constitution. Therefore, we performed a genome wide association (GWA) study of CMH in Caucasian populations.

Methods: GWA analysis was performed in the NELSON-study using the Illumina 610 array, followed by replication and meta-analysis in 11 additional cohorts. In total 2,704 subjects with, and 7,624 subjects without CMH were included, all current or former heavy smokers (≥20 pack-years). Additional studies were performed to test the functional relevance of the most significant single nucleotide polymorphism (SNP).

Results: A strong association with CMH, consistent across all cohorts, was observed with rs6577641 (p = 4.25×10(-6), OR = 1.17), located in intron 9 of the special AT-rich sequence-binding protein 1 locus (SATB1) on chromosome 3. The risk allele (G) was associated with higher mRNA expression of SATB1 (4.3×10(-9)) in lung tissue. Presence of CMH was associated with increased SATB1 mRNA expression in bronchial biopsies from COPD patients. SATB1 expression was induced during differentiation of primary human bronchial epithelial cells in culture.

Conclusions: Our findings, that SNP rs6577641 is associated with CMH in multiple cohorts and is a cis-eQTL for SATB1, together with our additional observation that SATB1 expression increases during epithelial differentiation provide suggestive evidence that SATB1 is a gene that affects CMH.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091621PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979657PMC
November 2014
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