Tenascin expression in normal and pathological conditions of the musculoskeletal system

Tenascin is an extracellular matrix protein with highly regulated expression and uncertain functions. It is prominently expressed during musculoskeletal embryogenesis. The pattern of distribution of tenascin in healthy adult musculoskeletal tissues is spatially and temporally restricted. It can be only detected in a small amount in the muscle-tendon junctions, tendons, perichondrium, periosteum, endosteum, the superficial layer of articular cartilage and the subintimal connective tissue of synovium. Elevated tenascin expression is found in inflammatory, degenerative and neoplastic lesions of the musculoskeletal system. The peculiar pattern of tenascin expression suggests it may play a role in the regulation of cell behavior at the interfaces between different elements of the musculoskeletal system and in various pathological processes, in particular those involving attachment and or detachment of cells from the extracellular matrix and their proliferation and collagenase secretion.Biomedical Reviews 1996; 6: 83-94

Association of invasion-promoting tenascin-C additional domains with breast cancers in young women

Introduction: Tenascin-C (TNC) is a large extracellular matrix glycoprotein that shows prominent stromal expression in many solid tumours. The profile of isoforms expressed differs between cancers and normal breast, with the two additional domains AD1 and AD2 considered to be tumour associated. The aim of the present study was to investigate expression of AD1 and AD2 in normal, benign and malignant breast tissue to determine their relationship with tumour characteristics and to perform in vitro functional assays to investigate the role of AD1 in tumour cell invasion and growth. Methods: Expression of AD1 and AD2 was related to hypoxanthine phosphoribosyltransferase 1 as a housekeeping gene in breast tissue using quantitative RT-PCR, and the results were related to clinicopathological features of the tumours. Constructs overexpressing an AD1-containing isoform (TNC-14/AD1/16) were transiently transfected into breast carcinoma cell lines (MCF-7, T-47 D, ZR-75-1, MDA-MB-231 and GI-101) to assess the effect in vitro on invasion and growth. Statistical analysis was performed using a nonparametric Mann-Whitney test for comparison of clinicopathological features with levels of TNC expression and using Jonckheere-Terpstra trend analysis for association of expression with tumour grade. Results: Quantitative RT-PCR detected AD1 and AD2 mRNA expression in 34.9% and 23.1% of 134 invasive breast carcinomas, respectively. AD1 mRNA was localised by in situ hybridisation to tumour epithelial cells, and more predominantly to myoepithelium around associated normal breast ducts. Although not tumour specific, AD1 and AD2 expression was significantly more frequent in carcinomas in younger women (age ≤40 years; P < 0.001) and AD1 expression was also associated with oestrogen receptor-negative and grade 3 tumours (P < 0.05). AD1 was found to be incorporated into a tumour-specific isoform, not detected in normal tissues. Overexpression of the TNC-14/AD1/16 isoform significantly enhanced tumour cell invasion (P < 0.01) and growth (P < 0.01) over base levels. Conclusions: Together these data suggest a highly significant association between AD-containing TNC isoforms and breast cancers in younger women (age ≤40 years), which may have important functional significance in vivo

The role of tenascin-C in tissue injury and tumorigenesis

The extracellular matrix molecule tenascin-C is highly expressed during embryonic development, tissue repair and in pathological situations such as chronic inflammation and cancer. Tenascin-C interacts with several other extracellular matrix molecules and cell-surface receptors, thus affecting tissue architecture, tissue resilience and cell responses. Tenascin-C modulates cell migration, proliferation and cellular signaling through induction of pro-inflammatory cytokines and oncogenic signaling molecules amongst other mechanisms. Given the causal role of inflammation in cancer progression, common mechanisms might be controlled by tenascin-C during both events. Drugs targeting the expression or function of tenascin-C or the tenascin-C protein itself are currently being developed and some drugs have already reached advanced clinical trials. This generates hope that increased knowledge about tenascin-C will further improve management of diseases with high tenascin-C expression such as chronic inflammation, heart failure, artheriosclerosis and cancer

The expression and role of the extracellular matrix protein tenascin in ovarian cancer

Tenascin (TN) is a large hexameric glycoprotein which is expressed transiently in the extracellular matrix at times of tissue development, proliferation and reorganisation. It is overexpressed in the stroma of several types of malignant tumours. The expression of TN in ovarian cancer has not previously been investigated. The aims of the project were therefore to (i) define the expression of TN in ovarian cancer, (ii) examine factors controlling its production and (iii) explore the functionality of the protein.Initial studies characterised the expression of TN in a series of malignant, benign and borderline ovarian tumours. TN protein was examined by immunohistochemical staining of frozen sections of tumours. Malignant and borderline tumours showed a significantly greater incidence and intensity of stromal staining as compared to benign tumours. Omental metastases showed a pattern of protein distribution strikingly similar to their primary counterpart. These data suggest that TN is overexpressed in malignant ovarian tumours.TN exists in a number of different isoforms, which are produced by alternative splicing of the RNA transcript. The pattern of different isoforms expressed was investigated using RT-PCR and hybridisation to specific oligonucleotides. The smallest TN splice variant was found in all tumours examined while the appearance of larger molecular weight transcripts was predominantly limited to malignant tumoursIn order to determine which cell types were capable of producing TN, in vitro, fibroblast cells, cultured from ascitic fluids of ovarian cancer patients, and established ovarian carcinoma cell lines were studied. TN levels in conditioned media were measured by ELISA. "Basal" levels of TN secretion were determined by culturing cells in serum-free media; under these conditions the ovarian fibroblasts secreted levels of TN over 100 fold greater than the epithelial cells. RT-PCR data showed that epithelial and fibroblast cell lines express TN RNA and display multiple RNA splice forms.To examine whether a paracrine interaction between the fibroblast and epithelial cells can influence TN production, the cells were co-cultured in compartments separated by a filter, which allowed diffusion of soluble factors. The co-cultured populations of cells produced significantly more TN than either cell type alone. The effects of a number of potential modulating factors, on secretion of TN, have been investigated. Several factors (IGF II, TGFB, progesterone and EGF) stimulate TN secretion by fibroblasts while other factors (gonadotrophins and interferon) inhibit TN secretion in the same cell type. Of the factors studied TGF-B provided the greatest induction of TN in fibroblasts. None of these factors induced the PE01 epithelial cell line to produce measurable levels of TN.Adhesion and migration assays were used to examine how ovarian carcinoma cell lines interacted with TN, as compared with the ECM proteins fibronectin and collagen IV. The assays demonstrated that TN promoted cell adhesion, spreading and migration in the SKOV-3, 59M, PE01 and PE04 ovarian carcinoma cell lines, however, fibronectin and collagen IV appeared to be preferable substrates. Immuno-staining and analysis by flow cytometry of these cell lines demonstrated that all expressed the integrin a2B1which can bind TN, the SKOV-3 cell line also expressed the integrin aVB3.These studies have demonstrated that TN is overexpressed in malignant ovarian tumours, and paracrine growth factors, such as TGFB, can induce the synthesis of TN in ovarian fibroblasts. Tumour cells can adhere to, and migrate on TN. These data would be consistent with TN playing a role in the invasion and metastasis of ovarian cancer

Analysis of tenascin-C expression pattern in post myocardial infarction remodelling

Das Protein Tenascin-C (TN-C) ist Bestandteil der extrazellulären Matrix (EZM) und gehört zur Familie der matrizellulären Proteine. Matrizelluläre Proteine beeinflussen wichtige Zellfunktionen und den Aufbau der EZM. Sie binden an zelluläre Rezeptoren, modulieren die Genexpression und regulieren die Aktivität von verschiedenen Wachstumsfaktoren, Zytokinen und Proteinasen. Die EZM stellt somit ein sehr dynamisches Netzwerk dar. Während der Embryonalentwicklung spielt TN-C in vielen Geweben, wie zum Beispiel dem Herzen, eine wichtige Rolle. Im adulten, gesunden Organismus wird TN-C nur sehr lokal, wie etwa in den myotendinous junctions, dem Darm, der Haut und lymphatischen Organen exprimiert. Unter pathologischen Bedingungen wie z.B. bei Tumorentstehung, Wundheilung, Infektion, Inflammation, Atherosklerose, mechanischem Stress und dem Remodelling Prozess kommt es erneut zu einer Hochregulation von TN-C. Während im adulten, gesunden Herzen das Protein nicht exprimiert wird, kommt es unter pathologischen Umständen, wie Myokardinfarkt (MI), Myokarditis oder dilatativer Kardiomyopathie, erneut zur Proteinexpression. Nach einem Myokardinfarkt ist TN-C in der Randzone zwischen infarziertem und nicht infarziertem Gewebe innerhalb von 24 Stunden detektierbar, wobei die Hauptquelle der TN-C Produktion die Fibroblasten darstellen. TN-C spielt eine duale Rolle im kardialen Wundheilungsprozess: Auf der einen Seite kommt es durch Deadhäsion der Kardiomyozyten zu einer Umstrukturierung des infarzierten Gewebes. Myofibroblasten können leicht in das betroffene Areal einwandern und Matrixkomponenten für die Stärkung der EZM produzieren. Zudem wirkt TN-C mit seiner Elastizität der enormen mechanischen Last, die auf die Randzone des Infarktareals wirkt, entgegen. Auf der anderen Seite können Kardiomyozyten von der EZM abrutschen und inflammatorische Zellen einwandern. Matrixmetalloproteinasen (MMPs) werden verstärkt exprimiert, was zur Degradation der EZM führt. Dadurch kann es zu einer Ausdünnung des linken Ventrikels, einer Dilatation und im schlimmsten Fall zur Ruptur des Ventrikels kommen. Im Rahmen der vorliegenden Arbeit liegt das Hauptinteresse auf dem zeitlichen und räumlichen TN-C Expressionsmuster nach einem Myokardinfarkt, welches am Rattenmodell untersucht wurde. Insgesamt wurden acht verschiedene Zeitpunkte (0.5, 1, 2, 3, 5, 7, 10 und 14 Tage) nach Infarkt untersucht, und mit einer sham operierten Kontrollgruppe verglichen. Zu jedem Zeitpunkt wurden Proben für histologische und molekularbiologische Analysen (Real time-PCR, PCR, Western Blot und Sequenzierungen) entnommen. Zuerst wurde die gesamte, bisher unbekannte, TN-C cDNA Ratten-Sequenz mit 6.057bp (GenBank, EU596506) sequenziert. Die Untersuchungen zeigten, dass verschiedene TN-C Isoformen zu unterschiedlichen Zeitpunkten post MI vorhanden sind. Diese Isoformen kommen durch alternatives Splicing der Fibronektin Typ-III Domänen (FN-III) zustande. Es wurde ein zeitlicher Shift der Isoformen nachgewiesen und insgesamt 4 verschiedene Isoformen, mit keiner, 1, 2 und 5 FN-III Domänen identifiziert. Es ist bekannt, dass Isoformen mit wenigen bzw. keinen FN-III in der alternativ gesplicten Region mit einer gesunden und „normalen“ EZM korrelieren, während größere Varianten mit vielen FN-III Domänen, mit pathologischen Veränderungen im Zusammenhang stehen. Aus diesen Fakten kann man schließen, dass die Funktion der unterschiedlich gesplicten TN-C Isoformen durch einen zeitlichen Shift nach einem MI reguliert wird.Tenascin-C (TN-C) is a component of the extracellular matrix (ECM) and member of the matricellular protein family. Matricellular proteins have a special role to influence cellular function and matrix composition. Those proteins bind to cellular receptors, modulate gene expression and regulate activity of various growth factors, cytokines, and proteinases. Therefore, composition of the ECM is a very dynamic network. TN-C plays an important role during embryonic development in numerous organs, e.g. in the heart. In the adult, healthy organism TN-C expression is highly restricted to a few regions, such as myotendinous junctions, gut, skin and lymphopoietic organs. Under pathological conditions, such as tumour formation, wound healing, infection, inflammation, atherosclerosis, mechanical stress, and during tissue remodelling TN-C expression is up-regulated once again. In healthy, adult myocardial tissue TN-C is not expressed but up-regulation starts under pathological conditions, such as myocardial infarction (MI), myocarditis, and dilated cardiomyopathy. After MI TN-C is mainly expressed by fibroblasts and up-regulated in the border zone between infarcted and non-infarcted region of the left ventricle within 24 hours. TN-C plays a dual role in cardiac healing post MI: On one hand, TN-C is important in loosen strong adhesion of cardiomyocytes and helps to rearrange surviving cells in the infarcted region. Myofibroblasts were recruited and production of matrix components is stimulated to strengthening the cardiac matrix. The elastic properties of TN-C may also help to resist the increased mechanical loading to which the border zone of the infarct is subjected. On the other hand, TN-C may loosen cardiomyocytes from the ECM, causing slippage of cardiomyocytes and supports invasion of inflammatory cells. Additionally TN-C up-regulates matrix metalloproteinases (MMPs), promotes ECM degradation and loosen cardiac tissue, which leads to an increasing risk of wall thinning, cardiac dilatation and left ventricle rupture. The aim of this thesis was to investigate TN-C regulation post MI and to study the temporal and spatial expression pattern in an experimental MI animal model. Eight time points during the early phase post infarction (0.5, 1, 2, 3, 5, 7, 10, and 14 days post MI) were analysed and compared to a sham operated control group. At each time point samples were collected for histological and molecular biological analysis (real time-PCR, PCR, western blot and sequencing). First, the complete, until now unknown, rat cDNA TN-C sequence (GenBank, EU596506) encompassing an open reading frame of 6.057bp was sequenced. The experiments showed that different TN-C isoforms were up-regulated at different time points post MI. Those isoforms differ in size, because of alternative splicing processes within the fibronectin type III (FN-III) domain. Overall a time dependent isoform shift could be shown and 4 different isoforms, with none, 1, 2, and 5 FN-III domains, which are up-regulated post MI were identified. Small TN-C (TN-CS) isoforms, which exhibit none or only a few FN-III domains in the alternative spliced side, are present within `normal´ and healthy ECM. In contrast, large TN-C (TN-CL) isoforms are up-regulated under pathologic conditions. Those facts suggest that the function of different spliced TN-C isoforms can be regulated through a time dependent shift during tissue remodelling processes post MI

Targeted splicing therapy: new strategies for colorectal cancer

RNA splicing is the process of forming mature mRNA, which is an essential phase necessary for gene expression and controls many aspects of cell proliferation, survival, and differentiation. Abnormal gene-splicing events are closely related to the development of tumors, and the generation of oncogenic isoform in splicing can promote tumor progression. As a main process of tumor-specific splicing variants, alternative splicing (AS) can promote tumor progression by increasing the production of oncogenic splicing isoforms and/or reducing the production of normal splicing isoforms. This is the focus of current research on the regulation of aberrant tumor splicing. So far, AS has been found to be associated with various aspects of tumor biology, including cell proliferation and invasion, resistance to apoptosis, and sensitivity to different chemotherapeutic drugs. This article will review the abnormal splicing events in colorectal cancer (CRC), especially the tumor-associated splicing variants arising from AS, aiming to offer an insight into CRC-targeted splicing therapy

Monoklonale Antikörper für die Analyse der Genexpression in neuronalen Geweben

Monoklonale Antikörper sind unverzichtbare Hilfsmittel, um Proteinkomplexe aus Zellen zu isolieren oder Proteine in Gewebeschnitten zu lokalisieren. Sie dienen auch dazu, Entwicklungsvorgänge aufzuklären. Dabei wird als Modellorganismus für Vertebraten oft der Zebrafisch gewählt, da er sich asaisonal vermehrt, eine zahlreiche Nachkommenschaft hat und sowohl die Befruchtung als auch die Entwicklung außerhalb des Mutterleibs erfolgt. Im Rahmen dieser Arbeit wurden monoklonale Antikörper generiert, die spezifisch mit neuronalen Geweben und Organen des Zebrafisches reagieren. Zur Immunisierung wurde Gehirngewebe des Zebrafisches verwendet. Immunisiert wurden Ratten. Antikörperbildende B-Zellen aus der Ratte wurden mit einer Mausmyelom-Zelllinie fusioniert. Proteine von Interesse wurden mit Hilfe der Antikörper aus Zelllysaten des Zebrafisch-Gehirns immunpräzipitiert und durch Elektrophorese in Polyacrylamidgelen aufgetrennt. Die durch Antikörper detektierbaren Banden wurden ausgeschnitten und die enthaltenen Proteine mit massenspektrometrischen Techniken identifiziert. In einem weiteren Ansatz diente eine in λ-Phagen einklonierte Genbank der Expression der Proteine. Die Proteine wurden ebenfalls mit monoklonalen Antikörpern identifiziert. Die Phagen, die diese Proteine produzierten, wurden vermehrt und die für das Protein kodierende DNA sequenziert. Wir haben unsere Anstrengungen vor allem auf Proteine neuronalen Ursprungs konzentriert, weil diese Strukturen in den Fischen besonders deutlich markiert wurden. Histologische Untersuchungen an anderen Spezies ergaben, dass die Antikörper mit neuronalen Strukturen vieler Spezies reagierten, was auf eine hohe Konservierung der Proteine in der Phylogenese schließen lässt. Aus drei Fusionen mit Milzzellen von immunisierten Ratten wurden 2400 Zellüberstände erzeugt, die auf ihre Immunglobulin-Subklasse getestet wurden. IgG-positive Überstände wurden auf histologischen Schnitten untersucht. Schließlich wurden 17 Klone etabliert, die mit Nervengewebe des Zebrafisches reagierten, und weitere 9 Klone, die sowohl mit neuronalen Zellen des Zebrafisches als auch mit neuronalem Gewebe anderer Spezies reagierten. Die von den einzelnen Antikörpern erkannten Proteine wurden entweder massenspektrometrisch oder mittels einer Expressionsgenbank, die aus drei Tage alten Zebrafischlarven hergestellt wurde, identifiziert. Es wurden Antikörper gegen folgende Proteine gefunden: 1. Tenascin R 2. Plasticin 3. TOPAP 4. VAT-1 Es wurden 16 monoklonale Antikörper, die gegen fünf verschiedene humane Antigene hergestellt worden waren, auf Kreuzreaktivität mit Zebrafischgehirn getestet. Die Antikörper reagierten sowohl mit dem Hirn des Zebrafisches als auch mit dem Hirn acht verschiedener Säugerspezies. Im zweiten Teil der Arbeit wurde der Versuch unternommen, gezielt gegen ein Fusionskonstrukt, das Teile des humanen Parkins enthielt, monoklonale Antikörper herzustellen. Aus vier Fusionen wurden nur drei spezifisch mit dem Antigen reagierende Antikörper selektiert, die auch im Western-Blot mit Parkin reagierten. In vivo wurde das Antigen in histologischen Schnitten jedoch nicht erkannt