Publications by authors named "Otmar Huber"

74 Publications

Post-translational modifications of tight junction transmembrane proteins and their direct effect on barrier function.

Biochim Biophys Acta Biomembr 2020 09 3;1862(9):183330. Epub 2020 May 3.

Institute of Biochemistry II, Jena University Hospital, Friedrich Schiller University Jena, Nonnenplan 2-4, 04473 Jena, Germany. Electronic address:

Post-translational modifications (PTMs) such as phosphorylation, ubiquitination or glycosylation are processes affecting the conformation, stability, localization and function of proteins. There is clear evidence that PTMs can act upon tight junction (TJ) proteins, thus modulating epithelial barrier function. Compared to transcriptional or translational regulation, PTMs are rapid and more dynamic processes so in the context of barrier maintenance they might be essential for coping with changing environmental or external impacts. The aim of this review is to extract literature deciphering PTMs in TJ proteins directly contributing to epithelial barrier changes in permeability to ions and macromolecules. It is not intended to cover the entire scope of PTMs in TJ proteins and should rather be understood as a digest of TJ protein modifications directly resulting in the tightening or opening of the epithelial barrier.
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http://dx.doi.org/10.1016/j.bbamem.2020.183330DOI Listing
September 2020

SIRT7: an influence factor in healthy aging and the development of age-dependent myeloid stem-cell disorders.

Leukemia 2020 08 25;34(8):2206-2216. Epub 2020 Mar 25.

Klinik für Innere Medizin II, Abteilung Hämatologie und Internistische Onkologie, Universitätsklinikum Jena, Jena, Germany.

Molecular alterations within the hematopoietic system influence cellular longevity and development of age-related myeloid stem-cell disorders like acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). A reduced SIRT7-expression in aged murine hematopoietic stem cells (HSC) resulted in reduced longevity and increased proliferation. In this study we investigated age-related changes of SIRT7-expression in healthy humans and relevant pathomechanisms in AML and CML. SIRT7-expression in leukocytes of healthy people decreased in an age-dependent manner. Low SIRT7 mRNA levels were also detected in AML and CML patients. With positive treatment response, SIRT7-expression increased, but showed reduction when patients progressed or relapsed. Pharmacologic inhibition of driver mutations in AML (FLT3-ITD) or CML (BCR-ABL) also restored SIRT7 levels in cell lines and patient samples. Furthermore, SIRT7-expression increased with time during PMA-mediated monocyte differentiation of THP-1 cells. SIRT7-overexpression in THP-1 cells resulted in increased expression of differentiation markers. BCR-ABL, FLT3-ITD, and differentiation-associated SIRT7-expression in general were positively regulated by C/EBPα, -β, and -ε binding to two different C/EBP-binding sites within the SIRT7 promoter. SIRT7 is important in human hematopoietic cell aging and longevity. It might act as tumor suppressor and could potentially serve as general biomarker for monitoring treatment response in myeloid stem-cell disorders.
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http://dx.doi.org/10.1038/s41375-020-0803-3DOI Listing
August 2020

Identification of PARP-1, Histone H1 and SIRT-1 as New Regulators of Breast Cancer-Related Aromatase Promoter I.3/II.

Cells 2020 02 12;9(2). Epub 2020 Feb 12.

Institute for Biochemistry II, Jena University Hospital, Friedrich Schiller University, 07743 Jena, Germany.

Paracrine interactions between malignant estrogen receptor positive (ER) breast cancer cells and breast adipose fibroblasts (BAFs) stimulate estrogen biosynthesis by aromatase in BAFs. In breast cancer, mainly the cAMP-responsive promoter I.3/II-region mediates excessive aromatase expression. A rare single nucleotide variant (SNV) in this promoter region, which caused 70% reduction in promoter activity, was utilized for the identification of novel regulators of aromatase expression. To this end, normal and mutant promoter activities were measured in luciferase reporter gene assays. DNA-binding proteins were captured by DNA-affinity and identified by mass spectrometry. The DNA binding of proteins was analyzed using electrophoretic mobility shift assays, immunoprecipitation-based in vitro binding assays and by chromatin immunoprecipitation in BAFs in vivo. Protein expression and parylation were analyzed by western blotting. Aromatase activities and RNA-expression were measured in BAFs. Functional consequences of poly (ADP-ribose) polymerase-1 (PARP-1) knock-out, rescue or overexpression, respectively, were analyzed in murine embryonic fibroblasts (MEFs) and the 3T3-L1 cell model. In summary, PARP-1 and histone H1 (H1) were identified as critical regulators of aromatase expression. PARP-1-binding to the SNV-region was crucial for aromatase promoter activation. PARP-1 parylated H1 and competed with H1 for DNA-binding, thereby inhibiting its gene silencing action. In MEFs (PARP-1 knock-out and wild-type) and BAFs, PARP-1-mediated induction of the aromatase promoter showed bi-phasic dose responses in overexpression and inhibitor experiments, respectively. The HDAC-inhibitors butyrate, panobinostat and selisistat enhanced promoter I.3/II-mediated gene expression dependent on PARP-1-activity. Forskolin stimulation of BAFs increased promoter I.3/II-occupancy by PARP-1, whereas SIRT-1 competed with PARP-1 for DNA binding but independently activated the promoter I.3/II. Consistently, the inhibition of both PARP-1 and SIRT-1 increased the NAD/NADH-ratio in BAFs. This suggests that cellular NAD/NADH ratios control the complex interactions of PARP-1, H1 and SIRT-1 and regulate the interplay of parylation and acetylation/de-acetylation events with low NAD/NADH ratios (reverse Warburg effect), promoting PARP-1 activation and estrogen synthesis in BAFs. Therefore, PARP-1 inhibitors could be useful in the treatment of estrogen-dependent breast cancers.
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http://dx.doi.org/10.3390/cells9020427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072628PMC
February 2020

Wnt Glycation Inhibits Canonical Signaling.

Cells 2019 10 25;8(11). Epub 2019 Oct 25.

Department of Biochemistry II, Jena University Hospital, Friedrich Schiller University Jena, 07743 Jena, Germany.

Glycation occurs as a non-enzymatic reaction between amino and thiol groups of proteins, lipids, and nucleotides with reducing sugars or α-dicarbonyl metabolites. The chemical reaction underlying is the Maillard reaction leading to the formation of a heterogeneous group of compounds named advanced glycation end products (AGEs). Deleterious effects have been observed to accompany glycation such as alterations of protein structure and function resulting in crosslinking and accumulation of insoluble protein aggregates. A substantial body of evidence associates glycation with aging. Wnt signaling plays a fundamental role in stem cell biology as well as in regeneration and repair mechanisms. Emerging evidence implicates that changes in Wnt/β-catenin pathway activity contribute to the aging process. Here, we investigated the effect of glycation of Wnt3a on its signaling activity.

Methods: Glycation was induced by treatment of Wnt3a-conditioned medium (CM) with glyoxal (GO). Effects on Wnt3a signaling activity were analyzed by Topflash/Fopflash reporter gene assay, co-immunoprecipitation, and quantitative RT-PCR.

Results: Our data show that GO-treatment results in glycation of Wnt3a. Glycated Wnt3a suppresses β-catenin transcriptional activity in reporter gene assays, reduced binding of β-catenin to T-cell factor 4 (TCF-4) and extenuated transcription of Wnt/β-catenin target genes.

Conclusions: GO-induced glycation impairs Wnt3a signaling function.
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http://dx.doi.org/10.3390/cells8111320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912562PMC
October 2019

Apoptotic Fragmentation of Tricellulin.

Int J Mol Sci 2019 Oct 1;20(19). Epub 2019 Oct 1.

Department of Biochemistry II, Jena University Hospital, Friedrich Schiller University Jena, 07743 Jena, Germany.

Apoptotic extrusion of cells from epithelial cell layers is of central importance for epithelial homeostasis. As a prerequisite cell-cell contacts between apoptotic cells and their neighbors have to be dissociated. Tricellular tight junctions (tTJs) represent specialized structures that seal polarized epithelial cells at sites where three cells meet and are characterized by the specific expression of tricellulin and angulins. Here, we specifically addressed the fate of tricellulin in apoptotic cells.

Methods: Apoptosis was induced by staurosporine or camptothecin in MDCKII and RT-112 cells. The fate of tricellulin was analyzed by Western blotting and immunofluorescence microscopy. Caspase activity was inhibited by Z-VAD-FMK or Z-DEVD-FMK.

Results: Induction of apoptosis induces the degradation of tricellulin with time. Aspartate residues 487 and 441 were identified as caspase cleavage-sites in the C-terminal coiled-coil domain of human tricellulin. Fragmentation of tricellulin was inhibited in the presence of caspase inhibitors or when Asp487 or Asp441 were mutated to asparagine. Deletion of the tricellulin C-terminal amino acids prevented binding to lipolysis-stimulated lipoprotein receptor (LSR)/angulin-1 and thus should impair specific localization of tricellulin to tTJs.

Conclusions: Tricellulin is a substrate of caspases and its cleavage in consequence contributes to the dissolution of tTJs during apoptosis.
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http://dx.doi.org/10.3390/ijms20194882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801678PMC
October 2019

A three-dimensional immunocompetent intestine-on-chip model as in vitro platform for functional and microbial interaction studies.

Biomaterials 2019 11 2;220:119396. Epub 2019 Aug 2.

Center for Sepsis Control and Care, Jena University Hospital, Friedrich-Schiller-University of Jena, Jena, Germany; Institute of Biochemistry II, Jena University Hospital, Jena, Germany. Electronic address:

Alterations of the microbial composition in the gut and the concomitant dysregulation of the mucosal immune response are associated with the pathogenesis of opportunistic infections, chronic inflammation, and inflammatory bowel disease. To create a platform for the investigation of the underlying mechanisms, we established a three-dimensional microphysiological model of the human intestine. This model resembles organotypic microanatomical structures and includes tissue resident innate immune cells exhibiting features of mucosal macrophages and dendritic cells. The model displays the physiological immune tolerance of the intestinal lumen to microbial-associated molecular patterns and can, therefore, be colonised with living microorganisms. Functional studies on microbial interaction between probiotic Lactobacillus rhamnosus and the opportunistic pathogen Candida albicans show that pre-colonization of the intestinal lumen of the model by L. rhamnosus reduces C. albicans-induced tissue damage, lowers its translocation, and limits fungal burden. We demonstrate that microbial interactions can be efficiently investigated using the in vitro model creating a more physiological and immunocompetent microenvironment. The intestinal model allows a detailed characterisation of the immune response, microbial pathogenicity mechanisms, and quantification of cellular dysfunction attributed to alterations in the microbial composition.
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http://dx.doi.org/10.1016/j.biomaterials.2019.119396DOI Listing
November 2019

CD147 (EMMPRIN) controls malignant properties of breast cancer cells by interdependent signaling of Wnt and JAK/STAT pathways.

Mol Cell Biochem 2019 Jan 18;451(1-2):197-209. Epub 2018 Jul 18.

Institute of Biochemistry II, Jena University Hospital, Nonnenplan 2-4, 07743, Jena, Germany.

EMMPRIN (extracellular matrix metalloproteinase inducer, EMN, CD147) is a member of the immunoglobulin superfamily expressed in numerous cell types both as a soluble and a membrane-spanning glycoprotein. It is involved in many physiological processes, as well as in cancer. This study addresses mechanisms of crosstalk between EMN-driven cancer-related cellular responses and the canonical Wnt-pathway in MCF-7 breast carcinoma cells. Genetic knockdown of EMN in MCF-7 resulted in characteristic changes in cellular shape, organization of the actin cytoskeleton and malignancy profile, indicating that EMN expression represses cell motility, but, in contrast, exerts a stimulatory effect on cell proliferation and invasive properties. Increased invasiveness coincided with elevated expression of Wnt-target genes and established invasion driver matrix metalloproteinase MMP14. Activation of the downstream Wnt-pathway by means of heterologous β-catenin and/or TCF-4 expression, through inhibition of GSK-3β by LiCl treatment, or by cell stimulation with insulin-like growth factor-1 (IGF-1) resulted in increased EMN expression. EMN over-expression raised the ratio of the two opposing Wnt pathway-driven transcription factors Sp1 and Sp5, leading to stimulation of the EMN promoter. Furthermore, the EMN promoter was activated by a feed-forward circuit involving an EMN-dependent drop in expression of the repressive signal transducer and activator of transcription 1 (STAT1). Taken together, we show that the influence of EMMPRIN on malignancy-related properties of breast cancer cells is functionally connected to both Wnt- and JAK/STAT pathways.
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http://dx.doi.org/10.1007/s11010-018-3406-9DOI Listing
January 2019

AAA+ ATPases Reptin and Pontin as potential diagnostic and prognostic biomarkers in salivary gland cancer - a short report.

Cell Oncol (Dordr) 2018 Aug 5;41(4):455-462. Epub 2018 Jun 5.

Gerhard-Domagk-Institute for Pathology, University Hospital of Münster, Albert Schweitzer Campus 1 D17, 48149, Münster, Germany.

Purpose: Salivary gland cancer (SGC) is a rare and heterogeneous disease with significant differences in recurrence and metastasis characteristics. As yet, little is known about the mechanisms underlying the initiation and/or progression of these diverse tumors. In recent years, the AAA+ ATPase family members Pontin (RuvBL1, Tip49a) and Reptin (RuvBL2, Tip49b) have been implicated in various processes, including transcription regulation, chromatin remodeling and DNA damage repair, that are frequently deregulated in cancer. The aim of this study was to assess the clinical and functional significance of Reptin and Pontin expression in SGC.

Methods: Immunohistochemical staining of Pontin, Reptin, β-catenin, Cyclin D1, TP53 and MIB-1 was performed on a collection of 94 SGC tumor samples comprising 13 different histological subtypes using tissue microarrays.

Results: We found that Reptin and Pontin were expressed in the majority of SGC samples across all histological subtypes. Patients with a high Reptin expression showed a significantly inferior 5-year overall survival rate compared to patients with a low Reptin expression (47.7% versus 78.3%; p = 0.033), whereas no such difference was observed for Pontin. A high Reptin expression strongly correlated with a high expression of the proliferation marker MIB-1 (p = 0.003), the cell cycle regulator Cyclin D1 (p = 0.006), accumulation of TP53 as a surrogate p53 mutation marker (p = 0.042) and cytoplasmic β-catenin expression (p = 0.002). Increased Pontin expression was found to significantly correlate with both cytoplasmic and nuclear β-catenin expression (p = 0.037 and p = 0.018, respectively), which is indicative for its oncogenic function.

Conclusions: Our results suggest a role of Reptin and Pontin in SGC tumor progression and/or patient survival. Therefore, SGC patients exhibiting a high Reptin expression may benefit from more aggressive therapeutic regimens. Future studies should clarify whether such patients may be considered for more radical surgery, extended adjuvant therapy and/or targeted therapy.
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http://dx.doi.org/10.1007/s13402-018-0382-8DOI Listing
August 2018

Underlying mechanism of subcortical brain protection during hypoxia and reoxygenation in a sheep model - Influence of α1-adrenergic signalling.

PLoS One 2018 29;13(5):e0196363. Epub 2018 May 29.

Institute for Biochemistry II, Jena University Hospital-Friedrich Schiller University, Jena, Germany.

While the cerebral autoregulation sufficiently protects subcortical brain regions during hypoxia or asphyxia, the cerebral cortex is not as adequately protected, which suggests that regulation of the cerebral blood flow (CBF) is area-specific. Hypoxia was induced by inhalation of 5% oxygen, for reoxygenation 100% oxygen was used. Cortical and subcortical CBF (by laser Doppler flowmetry), blood gases, mean arterial blood pressure (MABP), heart rate and renal blood flow were constantly monitored. Low dosed urapidil was used for α1A-adrenergic receptor blockade. Western blotting was used to determine adrenergic receptor signalling mediators in brain arterioles. During hypoxia cortical CBF decreased to 72 ± 11% (mean reduction 11 ± 3%, p < 0.001) of baseline, whereas subcortical CBF increased to 168±18% (mean increase 43 ± 5%, p < 0.001). Reoxygenation led to peak CBF of 194 ± 27% in the subcortex, and restored cortical CBF. α1A-Adrenergic blockade led to minor changes in cortical CBF, but massively reduced subcortical CBF during hypoxia and reoxygenation-almost aligning CBF in both brain regions. Correlation analyses revealed that α1A-adrenergic blockade renders all CBF-responses pressure-passive during hypoxia and reoxygenation, and confirmed the necessity of α1A-adrenergic signalling for coupling of CBF-responses to oxygen saturation. Expression levels and activation state of key signalling-mediators of α1-receptors (NOSs, CREB, ERK1/2) did not differ between cortex and subcortex. The dichotomy between subcortical and cortical CBF during hypoxia and reoxygenation critically depends on α1A-adrenergic receptors, but not on differential expression of signalling-mediators: signalling through the α1A-subtype is a prerequisite for cortical/subcortical redistribution of CBF.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196363PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973577PMC
August 2018

Redistribution of Cerebral Blood Flow during Severe Hypovolemia and Reperfusion in a Sheep Model: Critical Role of α1-Adrenergic Signaling.

Int J Mol Sci 2017 May 11;18(5). Epub 2017 May 11.

Institute for Biochemistry II, Jena University Hospital-Friedrich Schiller University, 07743 Jena, Germany.

Background: Maintenance of brain circulation during shock is sufficient to prevent subcortical injury but the cerebral cortex is not spared. This suggests area-specific regulation of cerebral blood flow (CBF) during hemorrhage.

Methods: Cortical and subcortical CBF were continuously measured during blood loss (≤50%) and subsequent reperfusion using laser Doppler flowmetry. Blood gases, mean arterial blood pressure (MABP), heart rate and renal blood flow were also monitored. Urapidil was used for α1A-adrenergic receptor blockade in dosages, which did not modify the MABP-response to blood loss. Western blot and quantitative reverse transcription polymerase chain reactions were used to determine adrenergic receptor expression in brain arterioles.

Results: During hypovolemia subcortical CBF was maintained at 81 ± 6% of baseline, whereas cortical CBF decreased to 40 ± 4% ( < 0.001). Reperfusion led to peak CBFs of about 70% above baseline in both brain regions. α1A-Adrenergic blockade massively reduced subcortical CBF during hemorrhage and reperfusion, and prevented hyperperfusion during reperfusion in the cortex. α1A-mRNA expression was significantly higher in the cortex, whereas α1D-mRNA expression was higher in the subcortex ( < 0.001).

Conclusions: α1-Adrenergic receptors are critical for perfusion redistribution: activity of the α1A-receptor subtype is a prerequisite for redistribution of CBF, whereas the α1D-receptor subtype may determine the magnitude of redistribution responses.
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http://dx.doi.org/10.3390/ijms18051031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454943PMC
May 2017

Tricellulin is a target of the ubiquitin ligase Itch.

Ann N Y Acad Sci 2017 06 24;1397(1):157-168. Epub 2017 Apr 24.

Department of Biochemistry II, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany.

Tricellulin, a member of the tight junction-associated MAGUK protein family, preferentially localizes to tricellular junctions in confluent polarized epithelial cell layers and is downregulated during the epithelial-mesenchymal transition. Posttranslational modifications are assumed to play critical roles in the process of downregulation of tricellulin at the protein level. Here, we report that the E3 ubiquitin ligase Itch forms a complex with tricellulin and thereby enhances its ubiquitination. Pull-down assays confirmed a direct interaction between tricellulin and Itch, which is mediated by the Itch WW domain and the N-terminus of tricellulin. Experiments in the presence of the proteasome inhibitor MG-132 did not show major changes in the levels of ubiquitinated tricellulin in epithelial cells, suggesting that ubiquitination is not primarily involved in proteasomal degradation of tricellulin, but it appears to be important for endocytosis or recycling. In contrast, in HEK-293 cells, MG-132 caused polyubiquitination. Moreover, we observed that well-differentiated RT-112 and de-differentiated Cal-29 bladder cancer cells show an inverse expression of tricellulin and Itch. We postulate that ubiquitination is an important posttranslational modification involved in the determination of the intracellular fate of tricellulin deserving of more detailed further investigations into the underlying molecular mechanisms and their regulation.
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http://dx.doi.org/10.1111/nyas.13349DOI Listing
June 2017

The expression of sialyltransferases is regulated by the bioavailability and biosynthesis of sialic acids.

Gene Expr Patterns 2017 01 27;23-24:52-58. Epub 2017 Mar 27.

Institute for Physiological Chemistry, Martin-Luther-University Halle-Wittenberg, Hollystraße 1, D-06114 Halle (Saale), Germany. Electronic address:

Glycosylation is the most frequent and important post-translational modification of proteins. It occurs on specific consensus sequences but the final structure of a particular glycan is not coded on the DNA, rather it depends on the expression of the required enzymes and the availability of substrates (activated monosaccharides). Sialic acid (Sia) is the terminal monosaccharide of most glycoproteins or glycolipids (= glycoconjugates) and involved in a variety of function on molecular (e.g. determination of protein stability and half-life) and cellular level (e.g. influenza infection). Sia are synthesized in the cytosol from UDP-GlcNAc by the Roseman-Warren pathway. The key enzyme of this pathway is the UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Sia are transferred on glycoconjugates by a family of Golgi-located enzymes, so called sialyltransferases (ST). There are 20 (human) ST known, which all transfer CMP-activated Sia to specific acceptor-sites on glycoconjugates. The regulation of the expression of ST is still not understood. Using a GNE-deficient embryonic stem cell line, which cannot synthesize Sia endogenously and by supplementation of soluble Sia precursors, we present data that the cellular availability of Sia strongly regulates the expression of ST on the level of transcription. In summary, we suggest that the concentration of the donor substrate of sialyltransferases, which can be regarded as a sensor for the environmental conditions of a cell, regulates not only total sialylation, but also the quality of sialylation. This allows a cell to response to altered environmental conditions.
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http://dx.doi.org/10.1016/j.gep.2017.03.003DOI Listing
January 2017

Trefoil factor 3 mediates resistance to apoptosis in colon carcinoma cells by a regulatory RNA axis.

Cell Death Dis 2017 03 9;8(3):e2660. Epub 2017 Mar 9.

Department of Veterinary Medicine, Institute of Veterinary Biochemistry, Freie Universität Berlin, Oertzenweg 19b, Berlin 14163, Germany.

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http://dx.doi.org/10.1038/cddis.2017.84DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386576PMC
March 2017

TFF3-dependent resistance of human colorectal adenocarcinoma cells HT-29/B6 to apoptosis is mediated by miR-491-5p regulation of lncRNA PRINS.

Cell Death Discov 2017 30;3:16106. Epub 2017 Jan 30.

Department of Veterinary Medicine, Institute of Veterinary Biochemistry, Freie Universität Berlin , Berlin, Germany.

Tumour necrosis factor- (TNF-) is a double-edged cytokine associated with pathogenesis of inflammatory-related cancers being also able to induce cancer cell death. In the process of tumour development or metastasis, cancer cells can become resistant to TNF-. In trefoil factor 3 (TFF3) overexpressing colorectal adenocarcinoma cells (HT-29/B6), we observed enhanced resistance against TNF-/interferon gamma-induced apoptosis. TFF3 is a secreted small peptide that supports intestinal tissue repair but is also involved in intestinal tumour progression and scattering. We hypothesised that TFF3 rescues intestinal epithelial cancer cells from TNF--induced apoptosis by involving regulatory RNA networks. -based expression analysis revealed TFF3-mediated regulation of selected microRNAs as well as long non-coding RNAs (lncRNAs), whereas miR-491-5p was identified to target the lncRNA 'psoriasis susceptibility-related RNA gene induced by stress' (PRINS). RNA interference-based gain- and loss-of-function experiments examined miR-491-PRINS axis to exert the TFF3-mediated phenotype. Chemical inhibition of selected pathways showed that phosphatidylinositol 3-kinase/AKT accounts for TFF3-mediated downregulation of miR-491-5p and accumulation of PRINS. Moreover, we showed that PRINS colocalises with PMAIP1 (NOXA) in nuclei of HT-29/B6 possessing inhibitory effects. Immunoprecipitation experiments proved molecular interaction of PMAIP1 with PRINS. Our study provides an insight into RNA regulatory networks that determine resistance of colorectal cancer cells to apoptosis.
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http://dx.doi.org/10.1038/cddiscovery.2016.106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5279457PMC
January 2017

SUMOylation regulates the intracellular fate of ZO-2.

Cell Mol Life Sci 2017 01 7;74(2):373-392. Epub 2016 Sep 7.

Institute of Biochemistry II, Jena University Hospital, Friedrich-Schiller-University Jena, Nonnenplan 2-4, 07743, Jena, Germany.

The zonula occludens (ZO)-2 protein links tight junctional transmembrane proteins to the actin cytoskeleton and associates with splicing and transcription factors in the nucleus. Multiple posttranslational modifications control the intracellular distribution of ZO-2. Here, we report that ZO-2 is a target of the SUMOylation machinery and provide evidence on how this modification may affect its cellular distribution and function. We show that ZO-2 associates with the E2 SUMO-conjugating enzyme Ubc9 and with SUMO-deconjugating proteases SENP1 and SENP3. In line with this, modification of ZO-2 by endogenous SUMO1 was detectable. Ubc9 fusion-directed SUMOylation confirmed SUMOylation of ZO-2 and was inhibited in the presence of SENP1 but not by an enzymatic-dead SENP1 protein. Moreover, lysine 730 in human ZO-2 was identified as a potential modification site. Mutation of this site to arginine resulted in prolonged nuclear localization of ZO-2 in nuclear recruitment assays. In contrast, a construct mimicking constitutive SUMOylation of ZO-2 (SUMO1ΔGG-ZO-2) was preferentially localized in the cytoplasm. Based on previous findings the differential localization of these ZO-2 constructs may affect glycogen-synthase-kinase-3β (GSK3β) activity and β-catenin/TCF-4-mediated transcription. In this context we observed that ZO-2 directly binds to GSK3β and SUMO1ΔGG-ZO-2 modulates its kinase activity. Moreover, we show that ZO-2 forms a complex with β-catenin. Wild-type ZO-2 and ZO-2-K730R inhibited transcriptional activity in reporter gene assays, whereas the cytosolic SUMO1ΔGG-ZO-2 did not. From these data we conclude that SUMOylation affects the intracellular localization of ZO-2 and its regulatory role on GSK3β and β-catenin signaling activity.
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http://dx.doi.org/10.1007/s00018-016-2352-5DOI Listing
January 2017

An integrative microfluidically supported in vitro model of an endothelial barrier combined with cortical spheroids simulates effects of neuroinflammation in neocortex development.

Biomicrofluidics 2016 Jul 5;10(4):044102. Epub 2016 Jul 5.

Institute of Biochemistry II, Jena University Hospital, Jena, Germany and Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany

The development of therapeutic substances to treat diseases of the central nervous system is hampered by the tightness and selectivity of the blood-brain barrier. Moreover, testing of potential drugs is time-consuming and cost-intensive. Here, we established a new microfluidically supported, biochip-based model of the brain endothelial barrier in combination with brain cortical spheroids suitable to detect effects of neuroinflammation upon disruption of the endothelial layer in response to inflammatory signals. Unilateral perfusion of the endothelial cell layer with a cytokine mix comprising tumor necrosis factor, IL-1β, IFNγ, and lipopolysaccharide resulted in a loss of endothelial von Willebrand factor and VE-cadherin expression accompanied with an increased leakage of the endothelial layer and diminished endothelial cell viability. In addition, cytokine treatment caused a loss of neocortex differentiation markers Tbr1, Tbr2, and Pax6 in the cortical spheroids concomitant with reduced cell viability and spheroid integrity. From these observations, we conclude that our endothelial barrier/cortex model is suitable to specifically reflect cytokine-induced effects on barrier integrity and to uncover damage and impairment of cortical tissue development and viability. With all its limitations, the model represents a novel tool to study cross-communication between the brain endothelial barrier and underlying cortical tissue that can be utilized for toxicity and drug screening studies focusing on inflammation and neocortex formation.
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http://dx.doi.org/10.1063/1.4955184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4947035PMC
July 2016

A novel role for the tumour suppressor Nitrilase1 modulating the Wnt/β-catenin signalling pathway.

Cell Discov 2016 5;2:15039. Epub 2016 Jan 5.

Institute of Biochemistry, Department of Biochemistry II, Jena University Hospital, Friedrich-Schiller-University Jena, Jena, Germany; Center for Sepsis Control and Care (CSCC), Jena University Hospital, Jena, Germany.

Nitrilase1 was classified as a tumour suppressor in association with the fragile histidine-triad protein Fhit. However, knowledge about nitrilase1 and its tumour suppressor function is still limited. Whereas nitrilase1 and Fhit are discrete proteins in mammals, they are merged in Drosophila melanogaster and Caenorhabditis elegans. According to the Rosetta-Stone hypothesis, proteins encoded as fusion proteins in one organism and as separate proteins in another organism may act in the same signalling pathway. Although a direct interaction of human nitrilase1 and Fhit has not been shown, our previous finding that Fhit interacts with β-catenin and represses its transcriptional activity in the canonical Wnt pathway suggested that human nitrilase1 also modulates Wnt signalling. In fact, human nitrilase1 forms a complex with β-catenin and LEF-1/TCF-4, represses β-catenin-mediated transcription and shows an additive effect together with Fhit. Knockdown of human nitrilase1 enhances Wnt target gene expression. Moreover, our experiments show that β-catenin competes away human nitrilase1 from LEF-1/TCF and thereby contributes to the activation of Wnt-target gene transcription. Inhibitory activity of human nitrilase1 on vertebrate Wnt signalling was confirmed by repression of Wnt-induced double axis formation in Xenopus embryogenesis. In line with this finding, the Drosophila fusion protein Drosophila NitFhit directly binds to Armadillo and represses the Wingless pathway in reporter gene assays. Genetic experiments confirmed the repressive activity of Drosophila NitFhit on Wingless signalling in the Drosophila wing imaginal disc. In addition, colorectal tumour microarray analysis revealed a significantly reduced expression of human nitrilase1 in poorly differentiated tumours. Taken together, repression of the canonical Wnt pathway represents a new mechanism for the human nitrilase1 tumour suppressor function.
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http://dx.doi.org/10.1038/celldisc.2015.39DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860965PMC
July 2016

Increase of cortical cerebral blood flow and further cerebral microcirculatory effects of Serelaxin in a sheep model.

Am J Physiol Heart Circ Physiol 2016 09 8;311(3):H613-20. Epub 2016 Jul 8.

Department of Neurology, Jena University Hospital, Friedrich Schiller University, Jena, Germany; and Orthopaedic Department, Jena University Hospital, Friedrich Schiller University, Jena, Germany

Serelaxin, recombinant human relaxin-2, modulates endothelial vasodilatory functionality and is under evaluation for treatment of acute heart failure. Little is known about acute effects on cerebral perfusion. We tested the hypothesis that Serelaxin might also have effects on the cerebral microcirculation in a sheep model, which resembles human brain structure quite well. We used laser Doppler flowmetry and sidestream dark-field (SDF) imaging techniques, which are reliable tools to continuously assess dynamic changes in cerebral perfusion. Laser Doppler flowmetry shows that bolus injection of 30 μg Serelaxin/kg body wt induces an increase (P = 0.006) to roughly 150% of cortical cerebral blood flow (CBF), whereas subcortical CBF remains unchanged (P = 0.688). The effects on area-dependent CBF were significantly different after the bolus injection (P = 0.042). Effects on cortical CBF were further confirmed by SDF imaging. The bolus injection of Serelaxin increased total vessel density to 127% (P = 0.00046), perfused vessel density to 145% (P = 0.024), and perfused capillary density to 153% (P = 0.024). Western blotting confirmed the expression of relaxin receptors RXFP1 and truncated RXFP2-variants in the respective brain regions, suggesting a possible contribution of RXFP1 on the effects of Serelaxin. In conclusion, the injection of a high dose of Serelaxin exerts quick effects on the cerebral microcirculation. Therefore, Serelaxin might be suitable to improve cortical microcirculation and exert neuroprotective effects in clinically relevant scenarios that involve cortical hypoperfusion. These findings need to be confirmed in relevant experimental settings involving cerebral cortical hypoperfusion and can possibly be translated into clinical practice.
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http://dx.doi.org/10.1152/ajpheart.00118.2016DOI Listing
September 2016

Real-time monitoring of hypertrophy in HL-1 cardiomyocytes by impedance measurements reveals different modes of growth.

Cytotechnology 2016 Oct 5;68(5):1897-907. Epub 2016 Jul 5.

Institute of Biochemistry II, Jena University Hospital, Friedrich-Schiller-University Jena, Nonnenplan 2-4, 07743, Jena, Germany.

Hypertrophic growth is a response of the heart to increased mechanical load or physiological stress. Thereby, cardiomyocytes grow in length and/or width to maintain cardiac pump function. Major signaling pathways involved in cardiomyocyte growth and remodeling have been identified during recent years including calcineurin-NFAT and PI3K-Akt signaling. Modulation of these pathways is of certain interest for therapeutic treatment of cardiac hypertrophy. However, quantification and characterization of hypertrophy in response to different stimuli or modulators is difficult. This study aims to test different read-out systems for detection and quantification of differences in hypertrophic growth in response to prohypertrophic stimuli. Real-time impedance measurements allowed the detection of distinct differences in hypertrophic growth in response to endothelin, norepinephrine, phenylephrine or BIO, which were not observable by other methods such as flow cytometry. Endothelin treatment induced a rapid and strong peak in the impedance signal concomitant with a massive reorientation of the actin cytoskeleton. Changes in expression of hypertrophy-associated genes were detected and stabilization of β-catenin was identified as a common response to all hypertrophic stimuli used in this study. Hypertrophic growth was blocked by the PI3K/mTOR inhibitor PI-103.
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http://dx.doi.org/10.1007/s10616-016-0001-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023564PMC
October 2016

Characterization of SKAP/kinastrin isoforms: the N-terminus defines tissue specificity and Pontin binding.

Hum Mol Genet 2016 07 11;25(13):2838-2852. Epub 2016 May 11.

Department of Biochemistry II, Jena University Hospital, 07743 Jena, Germany

Small Kinetochore-Associated Protein (SKAP)/Kinastrin is a multifunctional protein with proposed roles in mitosis, apoptosis and cell migration. Exact mechanisms underlying its activities in these cellular processes are not completely understood. SKAP is predicted to have different isoforms, however, previous studies did not differentiate between them. Since distinct molecular architectures of protein isoforms often influence their localization and functions, this study aimed to examine the expression profile and functional differences between SKAP isoforms in human and mouse. Analyses of various human tissues and cells of different origin by RT-PCR, and by Western blotting and immunocytochemistry applying newly generated anti-SKAP monoclonal antibodies revealed that human SKAP exists in two protein isoforms: ubiquitously expressed SKAP16 and testis/sperm-specific SKAP1. In mouse, SKAP1 expression is detectable in testis at 4 weeks postnatally, when the first wave of spermatogenesis in mice is complete and the elongated spermatids are present in the testes. Furthermore, we identified Pontin as a new SKAP1 interaction partner. SKAP1 and Pontin co-localized in the flagellar region of human sperm suggesting a functional relevance for SKAP1-Pontin interaction in sperm motility. Since most previous studies on SKAP were performed with the testis-specific isoform SKAP1, our findings provide a new basis for future studies on the role of SKAP in both human somatic cells and male germ cells, including studies on male fertility.
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http://dx.doi.org/10.1093/hmg/ddw140DOI Listing
July 2016

Recruitment of CD16(+) monocytes to endothelial cells in response to LPS-treatment and concomitant TNF release is regulated by CX3CR1 and interfered by soluble fractalkine.

Cytokine 2016 07 28;83:41-52. Epub 2016 Mar 28.

Center for Sepsis Control and Care, University Hospital Jena, Erlanger Allee 101, 07747 Jena, Germany; Institute of Biochemistry II, Jena University Hospital Jena, Nonnenplan 2-4, 07743 Jena, Germany. Electronic address:

Fractalkine (FKN, CX3CL1) is a regulator of leukocyte recruitment and adhesion, and controls leukocyte migration on endothelial cells (ECs). We show that FKN triggers different effects in CD16(+) and CD16(-) monocytes, the two major subsets of human monocytes. In the presence of ECs a lipopolysaccharide (LPS)-stimulus led to a significant increase in tumor necrosis factor (TNF)-secretion by CD16(+) monocytes, which depends on the interaction of CX3CR1 expressed on CD16(+) monocytes with endothelial FKN. Soluble FKN that was efficiently shed from the surface of LPS-activated ECs in response to binding of CD16(+) monocytes to ECs, diminished monocyte adhesion in down-regulating CX3CR1 expression on the surface of CD16(+) monocytes resulting in decreased TNF-secretion. In this process the TNF-converting enzyme (TACE) acts as a central player regulating FKN-shedding and TNFα-release through CD16(+) monocytes interacting with ECs. Thus, the release and local accumulation of sFKN represents a mechanism that limits the inflammatory potential of CD16(+) monocytes by impairing their interaction with ECs during the initial phase of an immune response to LPS. This regulatory process represents a potential target for therapeutic approaches to modulate the inflammatory response to bacterial components.
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http://dx.doi.org/10.1016/j.cyto.2016.03.017DOI Listing
July 2016

Monocyte-induced recovery of inflammation-associated hepatocellular dysfunction in a biochip-based human liver model.

Sci Rep 2016 Feb 23;6:21868. Epub 2016 Feb 23.

Institute of Biochemistry II, Jena University Hospital, 07743 Jena, Germany.

Liver dysfunction is an early event in sepsis-related multi-organ failure. We here report the establishment and characterization of a microfluidically supported in vitro organoid model of the human liver sinusoid. The liver organoid is composed of vascular and hepatocyte cell layers integrating non-parenchymal cells closely reflecting tissue architecture and enables physiological cross-communication in a bio-inspired fashion. Inflammation-associated liver dysfunction was mimicked by stimulation with various agonists of toll-like receptors. TLR-stimulation induced the release of pro- and anti-inflammatory cytokines and diminished expression of endothelial VE-cadherin, hepatic MRP-2 transporter and apolipoprotein B (ApoB), resulting in an inflammation-related endothelial barrier disruption and hepatocellular dysfunction in the liver organoid. However, interaction of the liver organoid with human monocytes attenuated inflammation-related cell responses and restored MRP-2 transporter activity, ApoB expression and albumin/urea production. The cellular events observed in the liver organoid closely resembled pathophysiological responses in the well-established sepsis model of peritoneal contamination and infection (PCI) in mice and clinical observations in human sepsis. We therefore conclude that this human liver organoid model is a valuable tool to investigate sepsis-related liver dysfunction and subsequent immune cell-related tissue repair/remodeling processes.
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http://dx.doi.org/10.1038/srep21868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4763209PMC
February 2016

A human macrophage-hepatocyte co-culture model for comparative studies of infection and replication of Francisella tularensis LVS strain and subspecies holarctica and mediasiatica.

BMC Microbiol 2016 Jan 6;16. Epub 2016 Jan 6.

Institute of Biochemistry II, Jena University Hospital, 07743, Jena, Germany.

Background: Francisella tularensis, a gram-negative bacterium replicates intracellularly within macrophages and efficiently evades the innate immune response. It is able to infect and replicate within Kupffer cells, specialized tissue macrophages of the liver, and to modulate the immune response upon infection to its own advantage. Studies on Francisella tularensis liver infection were mostly performed in animal models and difficult to extrapolate to the human situation, since human infections and clinical observations are rare.

Results: Using a human co-culture model of macrophages and hepatocytes we investigated the course of infection of three Francisella tularensis strains (subspecies holarctica--wildtype and live vaccine strain, and mediasiatica--wildtype) and analyzed the immune response triggered upon infection. We observed that hepatocytes support the intracellular replication of Franciscella species in macrophages accompanied by a specific immune response inducing TNFα, IL-1β, IL-6 and fractalkine (CX3CL1) secretion and the induction of apoptosis.

Conclusions: We could demonstrate that this human macrophage/hepatocyte co-culture model reflects strain-specific virulence of Francisella tularensis. We developed a suitable tool for more detailed in vitro studies on the immune response upon liver cell infection by F. tularensis.
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http://dx.doi.org/10.1186/s12866-015-0621-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4704405PMC
January 2016

A microfluidically perfused three dimensional human liver model.

Biomaterials 2015 Dec 25;71:119-131. Epub 2015 Aug 25.

Institute of Biochemistry II, Jena University Hospital, 07743 Jena, Germany; Center for Sepsis Control and Care, Jena University Hospital, 07747 Jena, Germany. Electronic address:

Within the liver, non-parenchymal cells (NPCs) are critically involved in the regulation of hepatocyte polarization and maintenance of metabolic function. We here report the establishment of a liver organoid that integrates NPCs in a vascular layer composed of endothelial cells and tissue macrophages and a hepatic layer comprising stellate cells co-cultured with hepatocytes. The three-dimensional liver organoid is embedded in a microfluidically perfused biochip that enables sufficient nutrition supply and resembles morphological aspects of the human liver sinusoid. It utilizes a suspended membrane as a cell substrate mimicking the space of Disse. Luminescence-based sensor spots were integrated into the chip to allow online measurement of cellular oxygen consumption. Application of microfluidic flow induces defined expression of ZO-1, transferrin, ASGPR-1 along with an increased expression of MRP-2 transporter protein within the liver organoids. Moreover, perfusion was accompanied by an increased hepatobiliary secretion of 5(6)-carboxy-2',7'-dichlorofluorescein and an enhanced formation of hepatocyte microvilli. From this we conclude that the perfused liver organoid shares relevant morphological and functional characteristics with the human liver and represents a new in vitro research tool to study human hepatocellular physiology at the cellular level under conditions close to the physiological situation.
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http://dx.doi.org/10.1016/j.biomaterials.2015.08.043DOI Listing
December 2015

Amyloid Precursor Protein (APP) Metabolites APP Intracellular Fragment (AICD), Aβ42, and Tau in Nuclear Roles.

J Biol Chem 2015 Sep 21;290(39):23515-22. Epub 2015 Aug 21.

the Jean Pierre Aubert Research Centre, Alzheimer & Tauopathies, INSERM, CHU-Lille, UMR-S 1172, University of Lille, F-59000 Lille, France.

Amyloid precursor protein (APP) metabolites (amyloid-β (Aβ) peptides) and Tau are the main components of senile plaques and neurofibrillary tangles, the two histopathological hallmarks of Alzheimer disease. Consequently, intense research has focused upon deciphering their physiological roles to understand their altered state in Alzheimer disease pathophysiology. Recently, the impact of APP metabolites (APP intracellular fragment (AICD) and Aβ) and Tau on the nucleus has emerged as an important, new topic. Here we discuss (i) how AICD, Aβ, and Tau reach the nucleus and how AICD and Aβ control protein expression at the transcriptional level, (ii) post-translational modifications of AICD, Aβ, and Tau, and (iii) what these three molecules have in common.
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http://dx.doi.org/10.1074/jbc.R115.677211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4583011PMC
September 2015

Diets rich in fructose, fat or fructose and fat alter intestinal barrier function and lead to the development of nonalcoholic fatty liver disease over time.

J Nutr Biochem 2015 Nov 16;26(11):1183-92. Epub 2015 Jun 16.

Institute of Nutritional Sciences, Model Systems of Molecular Nutrition, Friedrich-Schiller-University Jena, Jena, Germany. Electronic address:

General overnutrition but also a diet rich in certain macronutrients, age, insulin resistance and an impaired intestinal barrier function may be critical factors in the development of nonalcoholic fatty liver disease (NAFLD). Here the effect of chronic intake of diets rich in different macronutrients, i.e. fructose and/or fat on liver status in mice, was studied over time. C57BL/6J mice were fed plain water, 30% fructose solution, a high-fat diet or a combination of both for 8 and 16 weeks. Indices of liver damage, toll-like receptor 4 (TLR-4) signaling cascade, macrophage polarization and insulin resistance in the liver and intestinal barrier function were analyzed. Chronic exposure to a diet rich in fructose and/or fat was associated with the development of hepatic steatosis that progressed with time to steatohepatitis in mice fed a combination of macronutrients. The development of NAFLD was also associated with a marked reduction of the mRNA expression of insulin receptor, whereas hepatic expressions of TLR-4, myeloid differentiation primary response gene 88 and markers of M1 polarization of macrophages were induced in comparison to controls. Bacterial endotoxin levels in portal plasma were found to be increased while levels of the tight junction protein occludin and zonula occludens 1 were found to be significantly lower in the duodenum of all treated groups after 8 and 16 weeks. Our data suggest that chronic intake of fructose and/or fat may lead to the development of NAFLD over time and that this is associated with an increased translocation of bacterial endotoxin.
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http://dx.doi.org/10.1016/j.jnutbio.2015.05.011DOI Listing
November 2015

150th Anniversary Series: Desmosomes and the Hallmarks of Cancer.

Cell Commun Adhes 2015 2;22(1):15-28. Epub 2015 Jul 2.

c Institute of Pathology, Jena University Hospital, Friedrich-Schiller-University Jena , Ziegelmühlenweg 1, 07743 Jena , Germany.

Desmosomes represent adhesive, spot-like intercellular junctions that in association with intermediate filaments mechanically link neighboring cells and stabilize tissue architecture. In addition to this structural function, desmosomes also act as signaling platforms involved in the regulation of cell proliferation, differentiation, migration, morphogenesis, and apoptosis. Thus, deregulation of desmosomal proteins has to be considered to contribute to tumorigenesis. Proteolytic fragmentation and downregulation of desmosomal cadherins and plaque proteins by transcriptional or epigenetic mechanisms were observed in different cancer entities suggesting a tumor-suppressive role. However, discrepant data in the literature indicate that context-dependent differences based on alternative intracellular, signal transduction lead to altered outcome. Here, modulation of Wnt/β-catenin signaling by plakoglobin or desmoplakin and of epidermal growth factor receptor signaling appears to be of special relevance. This review summarizes current evidence on how desmosomal proteins participate in carcinogenesis, and depicts the molecular mechanisms involved.
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http://dx.doi.org/10.3109/15419061.2015.1039642DOI Listing
June 2016

Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions.

Biofabrication 2015 Mar 2;7(1):015013. Epub 2015 Mar 2.

Institute of Biochemistry II, Jena University Hospital, Nonnenplan 2-4, D-07743 Jena, Germany. Center for Sepsis Control and Care, Jena University Hospital, Erlanger Allee 101, D-07747 Jena, Germany.

Hemodynamic forces generated by the blood flow are of central importance for the function of endothelial cells (ECs), which form a biologically active cellular monolayer in blood vessels and serve as a selective barrier for macromolecular permeability. Mechanical stimulation of the endothelial monolayer induces morphological remodeling in its cytoskeleton. For in vitro studies on EC biology culture devices are desirable that simulate conditions of flow in blood vessels and allow flow-based adhesion/permeability assays under optimal perfusion conditions. With this aim we designed a biochip comprising a perfusable membrane that serves as cell culture platform multi-organ-tissue-flow (MOTiF biochip). This biochip allows an effective supply with nutrition medium, discharge of catabolic cell metabolites and defined application of shear stress to ECs under laminar flow conditions. To characterize EC layers cultured in the MOTiF biochip we investigated cell viability, expression of EC marker proteins and cell adhesion molecules of ECs dynamically cultured under low and high shear stress, and compared them with an endothelial culture in established two-dimensionally perfused flow chambers and under static conditions. We show that ECs cultured in the MOTiF biochip form a tight EC monolayer with increased cellular density, enhanced cell layer thickness, presumably as the result of a rapid and effective adaption to shear stress by remodeling of the cytoskeleton. Moreover, endothelial layers in the MOTiF biochip express higher amounts of EC marker proteins von-Willebrand-factor and PECAM-1. EC layers were highly responsive to stimulation with TNFα as detected at the level of ICAM-1, VCAM-1 and E-selectin expression and modulation of endothelial permeability in response to TNFα/IFNγ treatment under flow conditions. Compared to static and two-dimensionally perfused cell culture condition we consider MOTiF biochips as a valuable tool for studying EC biology in vitro under advanced culture conditions more closely resembling the in vivo situation.
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http://dx.doi.org/10.1088/1758-5090/7/1/015013DOI Listing
March 2015

Nuclear translocation uncovers the amyloid peptide Aβ42 as a regulator of gene transcription.

J Biol Chem 2014 Jul 30;289(29):20182-91. Epub 2014 May 30.

From the Institut fuer Chemie und Biochemie, Freie Universitaet Berlin, 14195 Berlin, Germany, the Department of Pharmacology and Therapeutics, Faculty of Medicine, McGill University, Montreal, Quebec H3G 0B1, Canada,

Although soluble species of the amyloid-β peptide Aβ42 correlate with disease symptoms in Alzheimer disease, little is known about the biological activities of amyloid-β (Aβ). Here, we show that Aβ peptides varying in lengths from 38 to 43 amino acids are internalized by cultured neuroblastoma cells and can be found in the nucleus. By three independent methods, we demonstrate direct detection of nuclear Aβ42 as follows: (i) biochemical analysis of nuclear fractions; (ii) detection of biotin-labeled Aβ in living cells by confocal laser scanning microscopy; and (iii) transmission electron microscopy of Aβ in cultured cells, as well as brain tissue of wild-type and transgenic APPPS1 mice (overexpression of amyloid precursor protein and presenilin 1 with Swedish and L166P mutations, respectively). Also, this study details a novel role for Aβ42 in nuclear signaling, distinct from the amyloid precursor protein intracellular domain. Chromatin immunoprecipitation showed that Aβ42 specifically interacts as a repressor of gene transcription with LRP1 and KAI1 promoters. By quantitative RT-PCR, we confirmed that mRNA levels of the examined candidate genes were exclusively decreased by the potentially neurotoxic Aβ42 wild-type peptide. Shorter peptides (Aβ38 or Aβ40) and other longer peptides (nontoxic Aβ42 G33A substitution or Aβ43) did not affect mRNA levels. Overall, our data indicate that the nuclear translocation of Aβ42 impacts gene regulation, and deleterious effects of Aβ42 in Alzheimer disease pathogenesis may be influenced by altering the expression profiles of disease-modifying genes.
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http://dx.doi.org/10.1074/jbc.M114.564690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106333PMC
July 2014