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  • 1
    Keywords: CELLS ; EXPRESSION ; PROTEIN ; PROTEINS ; EPITHELIA ; KERATINOCYTES ; IDENTIFICATION ; MERKEL CELLS ; MELANOMA-CELLS ; ADHERENS JUNCTIONS ; plakophilin-2 ; Asymmetric junctions ; CONTACTS ; CYTOKERATIN ; Heterotypic junctions ; HUMAN-FETAL SKIN
    Abstract: Merkel cells (MCs) are special neuroendocrine epithelial cells that occur as individual cells or as cell groups within the confinements of a major epithelium formed and dominated by other epithelial cells. In the epidermis and some of its appendages MCs are mostly located in the basal cell layer, occasionally also in suprabasal layers and generally occur in linear arrays in outer root sheath cell layers of hair follicles. As MCs are connected to the adjacent keratinocytes by a series of adhering junctions (AJs), of which the desmosomes are the most prominent, these junctions represent heterotypic cell-cell connections, i.e. a kind of structure not yet elucidated in molecular terms. Therefore, we have studied these AJs in order to examine the molecular composition of the desmosomal halves. Using light- and electron-microscopic immunolocalization and keratin 20 as the MC-specific cell type marker we show that the plaques of the MC half of the desmosomes specifically and constitutively contain plakophilin Pkp2. This protein, however, is absent in the keratinocyte half of such heterotypic desmosomes which instead contains Pkp1 and/or Pkp3. We discuss the developmental, tissue-architectonic and functional importance of such asymmetric junctions in normal physiology as well as in diseases, in particular in the formation of distant tumor cell metastasis.
    Type of Publication: Journal article published
    PubMed ID: 22006253
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  • 2
    Abstract: Occludin and several proteins of the claudin family have been identiried in simple epithelia and in endothelia as major and structure-determining transmembrane proteins clustered in the barrier-forming tight junctions (TJ), where they are associated with a variety of TJ plaque proteins, including protein ZO-1. To examine whether TJ also occur in the squamous stratified epithelium of the interfollicular human epidermis we have applied several microscopic and biochemical techniques. Using RT-PCR techniques, we have identiried mRNAs encoding protein ZO-1, occludin and claudins 1, 4, 7, 8, 11, 12, and 17 in both tissues, skin and cultured keratinocytes, whereas claudins i and 10 have only been detected in skin tissue. By immunocytochemistry we have localized claudin-1, occludin and protein ZO-1 in distinct plasma membrane structures representing cell-cell attachment zones. While claudin-1 occurs in plasma membranes of all living cell layers, protein ZO-1 is concentrated in or even restricted to the uppermost layers, and occludin is often detected only in the stratum granulosum. Using electron microscopy, typical TJ structures ("kissing points") as well as some other apparently related junctional structures have been detected in the stratum granulosum, interspersed between desmosomes. Modes and patterns of TJ formation have also been studied in experimental model systems, e.g., during wound healing and stratification as well as in keratinocyte cultures during Ca2+-induced stratification. We conclude that the epidermis contains in the stratum granulosum a continuous zonula occludens-equivalent structure with typical TJ morphology and molecular composition, characterized by colocalization of occludin, claudins and TJ plaque proteins. In addition, cell-cell contact structures and certain TJ proteins can also be detected in other epidermal cell layers in specific cell contacts. The pattern of formation and possible functions of epidermal TJ and related structures are discussed.
    Type of Publication: Journal article published
    PubMed ID: 12067061
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  • 3
    Keywords: ADHESION MOLECULE ; FREEZE-FRACTURE ; CORNEAL EPITHELIUM ; INTERCELLULAR-JUNCTIONS ; GAP-JUNCTIONS ; SEPTATE JUNCTIONS ; PARACELLULAR PERMEABILITY ; ZONULA OCCLUDENS ; ORAL EPITHELIUM ; PROTEIN ZO-1
    Abstract: The occurrence of extended tight junction (TJ) structures, including zonulae occludentes (ZO), and the spatial arrangement of TJ proteins in stratified mammalian epithelia has long been controversially discussed. Therefore, we have systematically examined the localization of TJ proteins in diverse stratified epithelial tissues (e.g., epidermis, heel pad, snout, gingiva, tongue, esophagus, exocervix, vagina, urothelium, cornea) of various species (human, bovine, rodents) as well as in human cell culture lines derived from stratified epithelia, by electron microscopy as well as by immunocytochemistry at both the light and the electron microscopic level, using antibodies to TJ proteins such as occludin, claudins 1 and 4, protein ZO-1, cingulin and symplekin. We have found an unexpected diversity of TJ-related structures of which only those showing colocalization with the most restricted transmembrane TJ marker protein, occludin, are presented here. While in epidermis and urothelium occludin is restricted to the uppermost living cell layer, TJ-related junctions are abundant in the upper third or even in the majority of the suprabasal cell layers in other stratified epithelia. Interfollicular epidermis contains, in the stratum granulosum, extended, probably continuous ZO-like structures which can also be traced at least through the Henle cell layer of hair follicles. Similar apical ZO-like structures have been seen in the upper living cell layers of all other stratified epithelia and cell cultures examined, but in most of them we have noticed, in addition, junctional regions showing relatively broad, ribbon-like membrane contacts which in cross-section often appear pentalaminar, with an electron-dense middle lamella ("lamellated TJs", coniunctiones laminosae). In suprabasal layers of several stratified epithelia we have further observed TJ protein-containing junctions of variable sizes which are characterized by a 10-30-nm dense lamina interposed between the two membranes ("sandwich junctions"; iuncturae structae). Moreover, we have often observed variously sized regions in which the intermembrane distance is rather regularly bridged by short rod-like elements ("cross-bridged cell walls"; parietes transtillati), often in close vicinity of TJ-related structures or desmosomes. The significance of these structures and their possible biological importance are discussed.
    Type of Publication: Journal article published
    PubMed ID: 12234014
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  • 4
    Keywords: CANCER ; ACTIVATION ; COMPARATIVE GENOMIC HYBRIDIZATION ; TUMOR-SUPPRESSOR GENE ; MALIGNANT-MELANOMA ; C-MYC ; LONG-TERM CULTURE ; RAS MUTATIONS ; SHORT ARM ; EPIDERMAL STEM-CELLS
    Abstract: Two developmentally highly divergent nonmelanoma skin cancers, the epidermal squamous cell carcinomas (SCC) and the neuroendocrine Merkel cell carcinomas (MCC), occur late in life at sun-exposed body sites. To determine whether these similarities may indicate common genetic alterations, we studied the genetic profile of 10 MCCs and analyzed 6 derived cell lines and 5 skin SCC lines by comparative genomic hybridization (CGH) and molecular genetic analyses. Although the MCCs were highly divergent-only 3 of the 10 tumors exhibited common gains and losses-they shared gain of 8q21-q22 and loss of 4p15-pter with the genetically much more homogeneous SCC lines. In addition, 2 of 5 SCC and 2 of 6 MCC lines exhibited UV-B-type-specific mutations in the p53 tumor-suppressor gene and a high frequency (9/11) of CC--〉TT double base changes in codon 27 of the Harvey (Ha)-ras gene. Since 45% of the tumor lines were homozygous for this nucleotide substitution compared to 14% of the controls and in 1 MCC patient the wild-type allele was lost in the tumor, this novel polymorphism may contribute to tumor development. On the other hand, loss of 3p, characteristic for SCCs, was rare in MCCs. Although in 2 of 3 SCC lines 3p loss was correlated with reduced expression of the FHIT (fragile histidine triad) gene, the potential tumor suppressor mapped to 3p14.2 and 2 MCC lines with normal 3p showed aberrant or no FHIT transcripts. Taken together, in addition to the common UV-B-specific mutations in the p53 and Ha-ras gene, MCCs and SCCs also share chromosomal imbalances that may point to a common environmental-derived (e.g., UV-A) oxidative damage.
    Type of Publication: Journal article published
    PubMed ID: 11992403
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  • 5
    Keywords: CANCER ; antibody ; METASTATIC MELANOMA ; MUTATIONS ; EPITOPE ; IMMUNOTHERAPY ; IMMUNE ESCAPE ; TUMOR-ANTIGENS ; EMBEDDED TISSUE-SECTIONS ; I ANTIGEN-EXPRESSION
    Abstract: Purpose: CD8(+) T lymphocytes can kill autologous melanoma cells, but their activity is impaired when poorly immunogenic tumor phenotypes evolve in the course of disease progression. Here, we analyzed three consecutive melanoma lesions obtained within one year of developing stage IV disease for their recognition by autologous T cells. Experimental Design: One skin (Ma-Mel-48a) and two lymph node (Ma-Mel-48b, Ma-Mel-48c) metastases were analyzed for T-cell infiltration. Melanoma cell lines established from the respective lesions were characterized, determining the T-cell-stimulatory capacity, expression of surface molecules involved in T-cell activation, and specific genetic alterations affecting the tumor-T-cell interaction. Results: Metastases Ma-Mel-48a and Ma-Mel-48b, in contrast with Ma-Mel-48c, were infiltrated by T cells. The T-cell-stimulatory capacity was found to be strong for Ma-Mel-48a, lower for Ma-Mel-48b, and completely abrogated for Ma-Mel-48c cells. The latter proved to be HLA class I-negative due to an inactivating mutation in one allele of the beta-2-microglobulin (B2M) gene and concomitant loss of the other allele by a deletion on chromosome 15q. The same deletion was already present in Ma-Mel-48a and Ma-Mel-48b cells, pointing to an early acquired genetic event predisposing to development of beta 2m deficiency. Notably, the same chronology of genetic alterations was also observed in a second beta 2m-deficient melanoma model. Conclusion: Our study reveals a progressive loss in melanoma immunogenicity during the course of metastatic disease. The genetic evolvement of T-cell resistance suggests screening tumors for genetic alterations affecting immunogenicity could be clinically relevant in terms of predicting patient responses to T-cell-based immunotherapy.
    Type of Publication: Journal article published
    PubMed ID: 25294904
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  • 6
    Keywords: EXPRESSION ; IN-VIVO ; LIGAND ; FEATURES ; BLOOD-BRAIN-BARRIER ; NOTCH ; CCM1 ; INHIBITS SPROUTING ANGIOGENESIS ; MUSCLE-CELL FORMATION ; VASCULAR INTEGRITY
    Abstract: Background and Purpose-Cerebral cavernous malformation (CCM) is a neurovascular dysplasia characterized by conglomerates of enlarged endothelial channels in the central nervous system, which are almost devoid of pericytes or smooth muscle cells. This disease is caused by loss-of-function mutations in CCM1, CCM2, or CCM3 genes in endothelial cells, making blood vessels highly susceptible to angiogenic stimuli. CCM1- and CCM3-silenced endothelial cells have a reduced expression of the Notch ligand Delta-like 4 (DLL4) resulting in impaired Notch signaling and irregular sprouting angiogenesis. This study aimed to address if DLL4, which is exclusively expressed on endothelial cells, may influence interactions of endothelial cells with pericytes, which express Notch3 as the predominant Notch receptor. Methods-Genetic manipulation of primary human endothelial cells and brain pericytes. Transgenic mouse models were also used. Results-Endothelial cell-specific ablation of Ccm1 and Ccm2 in different mouse models led to the formation of CCM-like lesions, which were poorly covered by periendothelial cells. CCM1 silencing in endothelial cells caused decreased Notch3 activity in cocultured pericytes. DLL4 proteins stimulated Notch3 receptors on human brain pericytes. Active Notch3 induced expression of PDGFRB2, N-Cadherin, HBEGF, TGFB1, NG2, and S1P genes. Notch3 signaling in pericytes enhanced the adhesion strength of pericytes to endothelial cells, limited their migratory and invasive behavior, and enhanced their antiangiogenic function. Pericytes silenced for Notch3 expression were more motile and could not efficiently repress angiogenesis. Conclusions-The data suggest that Notch signaling in pericytes is important to maintain the quiescent vascular phenotype. Deregulated Notch signaling may, therefore, contribute to the pathogenesis of CCM.
    Type of Publication: Journal article published
    PubMed ID: 25791711
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  • 7
    Keywords: ENDOTHELIAL-CELLS ; IN-VITRO ; BINDING ; TUMOR-GROWTH ; DLL4 ; INHIBITS SPROUTING ANGIOGENESIS ; DELTA-LIKE 1 ; DSL DOMAIN ; TIP ; JAGGED1
    Abstract: Aims Notch signalling is essential for blood vessel formation. During angiogenesis, the Notch ligand DLL4 on the leading tip cell activates Notch receptors on the adjacent stalk cells. DLL4-Notch signalling is impaired by the Notch ligand JAG1 in endothelial cells. The Delta/Serrate/Lag2 (DSL) domain of the Notch ligands binds to the EGF-like repeats 11-13 of the Notch receptor. This study aimed to elucidate how soluble proteins containing these short domains interfere with Notch signalling during angiogenesis. Methods and results Adenoviral vectors were generated to express the DSL domains of DLL1, DLL4, JAG1, and the Notch1 EGF-like repeats 11-13 fused to immunoglobulin-G heavy chain. These soluble ligand peptides inhibited Notch signalling in endothelial cells and this caused hyperbranching in cellular angiogenesis assays and in the neonatal mouse retina. The soluble Notch receptor peptides bound stronger to JAG1 than DLL4 ligands, resulting in increased signalling activity. This led to impaired tip cell formation and less vessel sprouting in the retina. Conclusion The minimal binding domains of Notch ligands are sufficient to interfere with Notch signalling. The corresponding soluble Notch1 EGF11-13 peptide binds stronger to inhibitory Notch ligands and thereby promotes Notch signalling in endothelial cells.
    Type of Publication: Journal article published
    PubMed ID: 25975260
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  • 8
  • 9
    Keywords: CELLS ; EXPRESSION ; tumor ; carcinoma ; CELL ; Germany ; human ; MICROSCOPY ; PROTEIN ; PROTEINS ; EPITHELIA ; TISSUE ; TISSUES ; PRIMARY CULTURES ; immunohistochemistry ; metastases ; REGION ; REGIONS ; SURFACE ; MONOCLONAL-ANTIBODIES ; CARCINOMAS ; squamous cell carcinoma ; pathology ; epidermis ; PERMEABILITY BARRIER ; TIGHT JUNCTIONS ; LAYER ; CYTOKERATINS ; MAMMALIAN-TISSUES ; electron microscopy ; FREEZE-FRACTURE ; HASSALLS CORPUSCLES ; HUMAN EPIDERMIS ; NORMAL HUMAN THYMUS ; stratified epithelia,tight junctions,occludin,claudins,squamous cell carcinoma,thymus,Hassall's corp
    Abstract: Tight junctions (TJs), hallmark structures of one-layered epithelia and of endothelia, are of central biological importance as intramembranous "fences" and as hydrophobic "barriers" between lumina represented by liquid- or gas-filled spaces on the one hand and the mesenchymal space on the other. They have long been thought to be absent from stratified epithelia. Recently, however, constitutive TJ proteins and TJ-related structures have also been identified in squamous stratified epithelia, including the epidermis, where they occur in special positions, most prominently in the uppermost living epidermal cell layer, the stratum granulosum. Much to our surprise, however, we have now also discovered several major TJ proteins (claudins 1 and 4, occludin, cingulin, symplekin, protein ZO-1) and TJ-related structures in specific positions of formations of epithelium-derived tissues that lack any lumen and do not border on luminal or body surfaces. Using immunohistochemistry and electron microscopy we have localized TJ proteins and structures in peripheral cells of the Hassall's corpuscles of human and bovine thymi as well as in specific central formations of tumor nests in squamous cell carcinomas, including the so-called "horn pearls". Such structures have even been found in carcinoma metastases. In carcinomas, they often seem to separate certain tumor regions from others or from stroma. The structural significance and the possible functional relevance of the locally restricted synthesis of TJ proteins and of the formations of TJ-related structures are discussed. It is proposed to include the determination of the presence or absence of such proteins and structures in the diagnostic program of tumor pathology
    Type of Publication: Journal article published
    PubMed ID: 14533737
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  • 10
    Keywords: ADHERENS JUNCTIONS, BARRIER, BIOLOGY, CELL, CELLS, culture, CULTURED KERATINOCYTES, CYTOKERATIN EXPR
    Abstract: In stratified squamous epithelia constituent proteins of tight junctions (TJs) are not restricted to the zonula occludens-related structures of the uppermost living cell layer such as the stratum granulosum of the epidermis but TJ membrane proteins such as occludin and certain members of the claudin family as well as TJ plaque proteins, notably cingulin and protein ZO-1, have also been identified by immunofluorescence and immunoelectron microscopy in more basal layers where they form special cell-cell-connecting structures such as the "lamellated" and the "sandwich" junctions. In the present study, we describe another TJ protein-containing structure, the very small puncta occludentia ("stud junctions"), as the smallest identifiable TJ-Iike unit that occurs in most, perhaps all strata. We have also determined the specific distributions of TJ proteins in the cell layers of squamous cell metaplasias of the human bronchial tract. Moreover, we show that the occl ud in- related tetraspanin protein tricellulin-alpha connects and seals the membranes of adjacent "three corner" cell structures of the uppermost layer in keratinocytes growing in culture. We hypothesize the possible occurrence of tricellulin-beta in more basal cell layers of keratinocyte cultures and the general occurrence of different tricellulin splice forms in stratified epithelia in situ, and discuss the possible functions of TJ proteins in stratified epithelia and tumors derived therefrom. (c) 2007 Elsevier GmbH. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17291627
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