The molecular and cell biological characterization of cell-cell junctions in mesenchymally derived cells and tissues

Nach dem herrschenden Lehrbuch Dogma exestieren zwei – und nur zwei – verschiedene Typen von zell-zell-verbindenden, Calcium-abhängigen Zellkontakten ("Adhering Junctions", AJs), die durch dicht-gepackte zytoplasmatische Plaques gekennzeichnet sind und so regelmäßig Filamente des Zytoskeletts verankern. AJs sind somit architektonisch und funktionell wichtige Strukturen, die bisher insbesondere in Epithelien und davon abgeleiteten Tumoren, vor allem den Karzinomen, untersucht worden sind. Hier unterscheided man vor allem zwischen den Intermediärfilamente verankernden Desmosomen (Maculae adhaerentes) und den Mikrofilament-Bündel verankernden Zellkontakten der Adherenz-Kategorie, die in verschiedenen Größen und Morphotypen vorkommen (Puncta, Fasziae, Zonulae). Die molekulare Zusammensetzung dieser AJ-Arten ist weitgehend bekannt, und spezifische molekular-diagnostische Antikörper werden routinemäßig in der Zell- und Entwicklungsbiologie sowie der diagnostischen Pathologie eingesetzt. Im Vergleich dazu ist sehr wenig über die molekulare Zusammensetzung der AJs, die nicht-epitheliale Zellen verbinden, bekannt, insbesondere die der mesenchymalen Gewebe und der mesenchymal-abgeleiteten Tumore. Aus diesem Grund und wegen des allgemeinen Bedarfs an verbesserten immunzytochemisch-diagnostischen Reagenzien und molekular-therapeutischen Ansätzen war es das Ziel, eine zellbiologische und molekular-analytische Grundlage für die diagnostische Identifizierung und Charakterisierung solcher AJs von mesenchymal-abgeleiteten Tumoren zu liefern. Der erste neuartige und weitverbreitete AJ-Typ ist einer, der ein grundlegendes "mesenchymales" Ensemble von N-Cadherin und/oder Cadherin-11 als transmembrane Glykoproteine aufweist, die in einem subplasmalemmalen Plaque verankert sind, der von typischen armadillo Proteinen wie alpha and beta-Catenin, Plakoglobin, den Proteinen p120, p0071 und/oder ARVCF sowie den Aktin-Mikrofilamente-bindenden Proteinen alpha and beta-Catenin und alpha-Actinin gebildet wird, hier aber zusätzlich das bedeutende Plaque-Protein Plakophilin-2 (Pkp2), oft zusammen mit Pkp3, enthält. Ich habe das Vorkommen solcher Pkp2-haltigen AJs – mit oder ohne Pkp3 – (Coniunctiones adhaerentes) in verschiedenen Säugetier-Zellkulturlinien gezeigt. Des Weiteren wurden diese AJs in einigen Tumoren in situ nachgewiesen, einschließlich bestimmter Tumore, bei denen dieses Protein durchweg ein diagnostisch relevanter molekularer Marker zu sein scheint, wie zum Beispiel den kardialen Myxomen. Basierend auf ersten Experimenten mit einer siRNA-vermittelten Genprodukt-Reduktion und ausgehend von jüngsten Erkenntnissen über eine allgemeine Rolle von Pkp2 als stabilisierendes Zell-Zell-Verbindungs-Protein wie z.B. im Säugetier-Herz, diskutiere ich die Möglichkeit dass eine Integration von Pkp-Molekülen in AJs von mesenchymal-abgeleiteten Zellen wesentlich zur strukturellen Stabilisierung und Festigkeit beiträgt und vor allem einen Vorteil beim Zusammenhalten proliferierender Zellen darstellt. Als dritte und strukturell völlig verschiedene Cadherin-Anordnung habe ich in einigen Subtypen von in Zellkultur-gewachsenen menschlichen Melanomen und Melanozyten das weit verbreitete in die Plasmamembran integrierte Glykoprotein Desmoglein Dsg2 entdeckt, dass dort sich offenbar regelmäßig in einzelnen Molekülen über große Areale der Zelloberfläche hin erstreckt und in Spiegelbild-symmetrischer Anordnung Zellen verbindet. Letztlich, habe ich die spontane, nicht induzierte, oft kumulative Synthese von Zellverbindungsmolekülen in bereits seit langem etablierten hämatopoetischen Zellkulturlinien (K562, RPMI 8226) und die Ansammlung solcher Moleküle zu einem Spektrum von Zell-Zell AJ-Strukturen untersucht. Ich habe verschiedene Arten von unterschiedlich großen, meist punktförmigen Zell-Zell-Verbindungs-Strukturen der AJ-Kategorie identifiziert, von denen die große Mehrheit auf distinkte Dsg2-Plasmamembran-Anhäufungen basiert. Am häufigsten habe ich AJ-Plaque-ähnliche Zusammensetzungen mit Pkp2 – mit oder ohne Pkp3 – mit Plakoglobin bzw. eher selten mit anderen armadillo Proteinen nachgewiesen. Es ist offensichtlich, dass solch unerwartete Analyseergebnisse, vor allem etwa die zuletzt geschilderte Anhäufung von Karzinom-charakteristischen Molekülen und Strukturen in mesenchymalen Tumoren erst recht in unizellulären Bluttumorzellen, in der Tumordiagnostik und für die therapeutische Behandlung, Beunruhigungen hervorrufen. Diese spontanen Veränderungen der Zell-Differenzierungs-Eigenschaften sowie des Zell-Charakters und -Verhaltens werden in Bezug auf entsprechende vereinzelte Angaben in der Literatur diskutiert. Die Feststellung solcher sehr unterschiedlicher AJ-Strukturen, in nicht-epithelialen Tumorzellen gezeigt, dass die Lehrbuchkapitel über Zell-Zell-Kontakte in der Zell- und Tumorbiologie wiedereröffnet und mit Fakten der ultrastrukturellen und molekularen Analyse erneut und verbessert dargestellt werden sollten

Desmosomal Molecules In and Out of Adhering Junctions: Normal and Diseased States of Epidermal, Cardiac and Mesenchymally Derived Cells

Current cell biology textbooks mention only two kinds of cell-to-cell adhering junctions coated with the cytoplasmic plaques: the desmosomes (maculae adhaerentes), anchoring intermediate-sized filaments (IFs), and the actin microfilament-anchoring adherens junctions (AJs), including both punctate (puncta adhaerentia) and elongate (fasciae adhaerentes) structures. In addition, however, a series of other junction types has been identified and characterized which contain desmosomal molecules but do not fit the definition of desmosomes. Of these special cell-cell junctions containing desmosomal glycoproteins or proteins we review the composite junctions (areae compositae) connecting the cardiomyocytes of mature mammalian hearts and their importance in relation to human arrhythmogenic cardiomyopathies. We also emphasize the various plakophilin-2-positive plaques in AJs (coniunctiones adhaerentes) connecting proliferatively active mesenchymally-derived cells, including interstitial cells of the heart and several soft tissue tumor cell types. Moreover, desmoplakin has also been recognized as a constituent of the plaques of the complexus adhaerentes connecting certain lymphatic endothelial cells. Finally, we emphasize the occurrence of the desmosomal transmembrane glycoprotein, desmoglein Dsg2, out of the context of any junction as dispersed cell surface molecules in certain types of melanoma cells and melanocytes. This broadening of our knowledge on the diversity of AJ structures indicates that it may still be too premature to close the textbook chapters on cell-cell junctions

Homo- and Heterotypic Cell Contacts in Malignant Melanoma Cells and Desmoglein 2 as a Novel Solitary Surface Glycoprotein

During progression of melanomas, a crucial role has been attributed to alterations of cell–cell adhesions, specifically, to a “cadherin switch” from E- to N-cadherin (cad). We have examined the adhesion of melanoma cells to each other and to keratinocytes. When different human melanoma cell lines were studied by protein analysis and immunofluorescence microscopy, six of eight lines contained N-cad, three E-cad, and five P-cad, and some lines had more than one cad. Surprisingly, two N-cad-positive lines, MeWo and C32, also contained desmoglein 2 (Dsg2), a desmosomal cad previously not reported for melanomas, whereas other desmosome-specific proteins were absent. This finding was confirmed by reverse transcriptase–PCR, immunoprecipitation, and matrix-assisted laser desorption ionization–time of flight analyses. Double-label confocal and immunoelectron microscopy showed N-cad, α- and β-catenin in plaque-bearing puncta adhaerentia, whereas Dsg2 was distributed rather diffusely over the cell surface. In cocultures with HaCaT keratinocytes Dsg2 was found in heterotypic cell contact regions. Correspondingly, immunohistochemistry revealed Dsg2 in five of 10 melanoma metastases. Together, we show that melanoma cell adhesions are more heterogeneous than expected and that certain cells devoid of desmosomes contain Dsg2 in a non-junction-restricted form. Future studies will have to clarify the diagnostic and prognostic significance of these different adhesion protein subtypes

Striatins as plaque molecules of zonulae adhaerentes in simple epithelia, of tessellate junctions in stratified epithelia, of cardiac composite junctions and of various size classes of lateral adherens junctions in cultures of epithelia- and carcinoma-derived cells

Proteins of the striatin family (striatins 1–4; sizes ranging from 90 to 110 kDa on SDS-polyacrylamide gel electrophoresis) are highly homologous in their amino acid sequences but can differ in their cell-type-specific gene expression patterns and biological functions. In various cell types, we have found one, two or three polypeptides of this evolutionarily old and nearly ubiquitous family of proteins known to serve as scaffold proteins for diverse protein complexes. Light and electron microscopic immunolocalization methods have revealed striatins in mammalian cell-cell adherens junctions (AJs). In simple epithelia, we have localized striatins as constitutive components of the plaques of the subapical zonulae adhaerentes of cells, including intestinal, glandular, ductal and urothelial cells and hepatocytes. Striatins colocalize with E-cadherin or E–N-cadherin heterodimers and with the plaque proteins α- and β-catenin, p120 and p0071. In some epithelia and carcinomas and in cultured cells derived therefrom, striatins are also seen in lateral AJs. In stratified epithelia and in corresponding squamous cell carcinomas, striatins can be found in plaques of some forms of tessellate junctions. Moreover, striatins are major plaque proteins of composite junctions (CJs; areae compositae) in the intercalated disks connecting cardiomyocytes, colocalizing with other CJ molecules, including plectin and ankyrin-G. We discuss the “multimodulator” scaffold roles of striatins in the initiation and regulation of the formation of various complex particles and structures. We propose that striatins are included in the diagnostic candidate list of proteins that, in the CJs of human hearts, can occur in mutated forms in the pathogeneses of hereditary cardiomyopathies, as seen in some types of genetically determined heart damage in boxer dogs.German-Israeli Foundation for Scientific Research and Development (GIF grant I-1098-43.11/2010

E–N-cadherin heterodimers define novel adherens junctions connecting endoderm-derived cells

Contradicting the “cadherin switch” model, mixed E-cadherin–N-cadherin heterodimeric adherens junctions are prevalent in a variety of endodermal cells and endoderm-derived tumors

The cell-cell junctions of mammalian testes: I. The adhering junctions of the seminiferous epithelium represent special differentiation structures

The seminiferous tubules and the excurrent ducts of the mammalian testis are physiologically separated from the mesenchymal tissues and the blood and lymph system by a special structural barrier to paracellular translocations of molecules and particles: the “blood–testis barrier”, formed by junctions connecting Sertoli cells with each other and with spermatogonial cells. In combined biochemical as well as light and electron microscopical studies we systematically determine the molecules located in the adhering junctions of adult mammalian (human, bovine, porcine, murine, i.e., rat and mouse) testis. We show that the seminiferous epithelium does not contain desmosomes, or “desmosome-like” junctions, nor any of the desmosome-specific marker molecules and that the adhering junctions of tubules and ductules are fundamentally different. While the ductules contain classical epithelial cell layers with E-cadherin-based adherens junctions (AJs) and typical desmosomes, the Sertoli cells of the tubules lack desmosomes and “desmosome-like” junctions but are connected by morphologically different forms of AJs. These junctions are based on N-cadherin anchored in cytoplasmic plaques, which in some subforms appear thick and dense but in other subforms contain only scarce and loosely arranged plaque structures formed by α- and β-catenin, proteins p120, p0071 and plakoglobin, together with a member of the striatin family and also, in rodents, the proteins ZO-1 and myozap. These N-cadherin-based AJs also include two novel types of junctions: the “areae adhaerentes”, i.e., variously-sized, often very large cell-cell contacts and small sieve-plate-like AJs perforated by cytoplasm-to-cytoplasm channels of 5–7 nm internal diameter (“cribelliform junctions”). We emphasize the unique character of this epithelium that totally lacks major epithelial marker molecules and structures such as keratin filaments and desmosomal elements as well as EpCAM- and PERP-containing junctions. We also discuss the nature, development and possible functions of these junctions.German-Israeli Foundation for Scientific Research and Development (GIF grant I-1098-43.11/2010

Transmembrane protein PERP is a component of tessellate junctions and of other junctional and non-junctional plasma membrane regions in diverse epithelial and epithelium-derived cells

Protein PERP (p53 apoptosis effector related to PMP-22) is a small (21.4 kDa) transmembrane polypeptide with an amino acid sequence indicative of a tetraspanin character. It is enriched in the plasma membrane and apparently contributes to cell-cell contacts. Hitherto, it has been reported to be exclusively a component of desmosomes of some stratified epithelia. However, by using a series of newly generated mono- and polyclonal antibodies, we show that protein PERP is not only present in all kinds of stratified epithelia but also occurs in simple, columnar, complex and transitional epithelia, in various types of squamous metaplasia and epithelium-derived tumors, in diverse epithelium-derived cell cultures and in myocardial tissue. Immunofluorescence and immunoelectron microscopy allow us to localize PERP predominantly in small intradesmosomal locations and in variously sized, junction-like peri- and interdesmosomal regions (“tessellate junctions”), mostly in mosaic or amalgamated combinations with other molecules believed, to date, to be exclusive components of tight and adherens junctions. In the heart, PERP is a major component of the composite junctions of the intercalated disks connecting cardiomyocytes. Finally, protein PERP is a cobblestone-like general component of special plasma membrane regions such as the bile canaliculi of liver and subapical-to-lateral zones of diverse columnar epithelia and upper urothelial cell layers. We discuss possible organizational and architectonic functions of protein PERP and its potential value as an immunohistochemical diagnostic marker

Immunogenic Chemotherapy Sensitizes Tumors to Checkpoint Blockade Therapy

Checkpoint blockade immunotherapies can be extraordinarily effective, but might benefit only the minority of patients whose tumors are pre-infiltrated by T cells. Here, using lung adenocarcinoma mouse models, including genetic models, we show that autochthonous tumors that lacked T cell infiltration and resisted current treatment options could be successfully sensitized to host antitumor T cell immunity when appropriately selected immunogenic drugs (e.g., oxaliplatin combined with cyclophosphamide for treatment against tumors expressing oncogenic Kras and lacking Trp53) were used. The antitumor response was triggered by direct drug actions on tumor cells, relied on innate immune sensing through toll-like receptor 4 signaling, and ultimately depended on CD8 + T cell antitumor immunity. Furthermore, instigating tumor infiltration by T cells sensitized tumors to checkpoint inhibition and controlled cancer durably. These findings indicate that the proportion of cancers responding to checkpoint therapy can be feasibly and substantially expanded by combining checkpoint blockade with immunogenic drugs

Plakophilin-2: a cell-cell adhesion plaque molecule of selective and fundamental importance in cardiac functions and tumor cell growth

Within the characteristic ensemble of desmosomal plaque proteins, the armadillo protein plakophilin-2 (Pkp2) is known as a particularly important regulatory component in the cytoplasmic plaques of various other cell–cell junctions, such as the composite junctions (areae compositae) of the myocardiac intercalated disks and in the variously-sized and -shaped complex junctions of permanent cell culture lines derived therefrom. In addition, Pkp2 has been detected in certain protein complexes in the nucleoplasm of diverse kinds of cells. Using a novel set of highly sensitive and specific antibodies, both kinds of Pkp2, the junctional plaque-bound and the nuclear ones, can also be localized to the cytoplasmic plaques of diverse non-desmosomal cell–cell junction structures. These are not only the puncta adhaerentia and the fasciae adhaerentes connecting various types of highly proliferative non-epithelial cells growing in culture but also some very proliferative states of cardiac interstitial cells and cardiac myxomata, including tumors growing in situ as well as fetal stages of heart development and cultures of valvular interstitial cells. Possible functions and assembly mechanisms of such Pkp2-positive cell–cell junctions as well as medical consequences are discussed