Ismo Virtanen

Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor

Lymphatic vessels are essential for fluid homeostasis, immune surveillance and fat adsorption, and also serve as a major route for tumor metastasis in many types of cancer. We found that isolated human primary lymphatic and blood vascular endothelial cells (LECs and BECs, respectively) show interesting differences in gene expression relevant for their distinct functions in vivo. Although these phenotypes are stable in vitro and in vivo, overexpression of the homeobox transcription factor Prox‐1 in the BECs was capable of inducing LEC‐specific gene transcription in the BECs, and, surprisingly, Prox‐1 suppressed the expression of ∼40% of the BEC‐specific genes. Prox‐1 did not have global effects on the expression of LEC‐specific genes in other cell types, except that it up‐regulated cyclin E1 and E2 mRNAs and activated the cyclin e promoter in various cell types. These data suggest that Prox‐1 acts as a cell proliferation inducer and a fate determination factor for the LECs. Furthermore, the data provide insights into the phenotypic diversity of endothelial cells and into the possibility of transcriptional reprogramming of differentiated endothelial cells.

A SNAIL1-SMAD3/4 transcriptional repressor complex promotes TGF-β mediated epithelial-mesenchymal transition

Epithelial–mesenchymal transition (EMT) is essential for organogenesis and is triggered during carcinoma progression to an invasive state. Transforming growth factor-β (TGF-β) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT. The SNAIL1–SMAD3/4 complex was targeted to the gene promoters of CAR, a tight-junction protein, and E-cadherin during TGF-β-driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts of human breast cancer. We propose that activation of a SNAIL1–SMAD3/4 transcriptional complex represents a mechanism of gene repression during EMT.

Laminin isoforms in tumor invasion, angiogenesis and metastasis

Laminins are a growing family of alphabetagamma heterotrimeric proteins, commonly found in basement membranes (BMs). These large molecules promote cell adhesion and migration via integrins and other cell-surface receptors. Over 12 laminin isoforms are presently known. The various isoforms have a cell- and tissue-specific expression and are differentially recognized by integrins. Expression of laminin isoforms in tumors usually reflects expression in their normal counterparts. However, during tumor invasion, loss of the BM barrier occurs and a discontinuous pattern of laminin staining is observed. In carcinomas, tumor cells at the invading front strongly express intracellularly the gamma2 chain, a component of laminin-5. Remodeling of the vascular BM is observed during angiogenesis, and penetration of several BMs occurs during tumor dissemination and metastasis. Thus, disregulated cell-laminin interactions are major traits of malignant disorders.

Expression of Snail protein in tumor-stroma interface.

The product of Snail gene is a repressor of E-cadherin transcription and an inductor of the epithelial-to-mesenchymal transition in several epithelial tumor cell lines. In order to examine Snail expression in animal and human tissues, we have raised a monoclonal antibody (MAb) that reacts with the regulatory domain of this protein. Analysis of murine embryos shows that Snail is expressed in extraembryonic tissues and embryonic mesoderm, in mesenchymal cells of lungs and dermis as well as in cartilage. Little reactivity was detected in adult tissues as Snail was not constitutively expressed in most mesenchymal cells. However, Snail expression was observed in activated fibroblasts involved in wound healing in mice skin. Moreover, Snail was detected in pathological conditions causing hyperstimulation of fibroblasts, such as fibromatosis. Analysis of Snail expression in tumors revealed that it was highly expressed in sarcomas and fibrosarcomas. In epithelial tumors, it presented a more limited distribution, restricted to stromal cells placed in the vicinity of the tumor and to tumoral cells in the same areas. These results demonstrate that Snail is present in activated mesenchymal cells, indicate its relevance in the communication between tumor and stroma and suggest that it can promote the conversion of carcinoma cells to stromal cells.

Changes in the glycosylation pattern during embryonic development of mouse kidney as revealed with lectin conjugates.

Distribution of lectin-binding sites in adult and developing mouse kidney was studied with fluorochrome- and peroxidase-coupled lectins. Effects of fixation methods on lectin-binding patterns were also compared. Un-induced mesenchymal cells and ureter bud of the early metanephros reacted with Concanavalin A, Lens culinaris, Ricinus communis I, and wheat germ agglutinins, whereas binding sites for both soybean and peanut (PNA) agglutinins were seen only in ureter bud tissue. On induction, PNA positivity rapidly appeared in the induced, condensed areas of the metanephrogenic mesenchyme. Early glomeruli expressed heterogeneously terminal galactosyl and N-acetylgalactosaminyl moieties in the podocytes. Later, these sites disappeared and were apparently covered by sialic acids. Endothelia also displayed a comparable sialylation of terminal saccharide moieties during maturation. Binding sites for many of the above lectins were also found in the developing proximal and distal tubules. Terminal fucosyl residues, characteristic of mature proximal tubules, appeared during day 13 of development. Dolichos biflorus agglutinin reactivity, typically seen in the collecting ducts, appeared by day 13. Griffonia simplicifolia-I-B4 isolectin reactivity was exclusively localized to endothelial in adult kidney cortex, but in embryonic kidneys reactivity with collecting duct and podocytes was also seen. These results suggest that the compartmentalized expression of cell glycoconjugates in adult mouse kidney is acquired in a sequential manner during development. Such sequential appearance of the mature glycosylation pattern probably reflects functional maturation of the nephron.

Diagnostic Application of Monoclonal Antibodies to Intermediate Filamentsa

Most of the histopathologic practice in pathology laboratories is based on histochemical staining techniques that reveal the major components common to most tissues or cells, or on more specific staining reactions, that reveal structural components or products typical of certain tissues or cells. The assortment of the utilizable specific stains is, however, narrow and therefore the correct identification of the specific nature of a given lesion is critically dependent on the personal experience of the pathologist. In order to improve the accuracy and reliability of the histopathological diagnostics, much effort has been devoted to develop new and more specific techniques that could be used to reveal specific compositional features of cells and tissues. The most successful approach has been the use of antibodies-either conventional polyclonal or monoclonal antibodies raised against specific tissue components that, combined with immunohistochemical techniques, can be used to retrieve more specific information on the compositional features of a lesion, e.g. tumor, which may allow its identification. Such new reagents include, for instance, antibodies that enable us to identify specific cell types in tissue sections, such as endothelial cells, histiomonocytic cells, different types of lymphatic cells, epithelial cells, neuronal cells, and muscle-type of cells.’32

Fibronectin fibrillogenesis regulates three-dimensional neovessel formation

During vasculogenesis and angiogenesis, endothelial cell responses to growth factors are modulated by the compositional and mechanical properties of a surrounding three-dimensional (3D) extracellular matrix (ECM) that is dominated by either cross-linked fibrin or type I collagen. While 3D-embedded endothelial cells establish adhesive interactions with surrounding ligands to optimally respond to soluble or matrix-bound agonists, the manner in which a randomly ordered ECM with diverse physico-mechanical properties is remodeled to support blood vessel formation has remained undefined. Herein, we demonstrate that endothelial cells initiate neovascularization by unfolding soluble fibronectin (Fn) and depositing a pericellular network of fibrils that serve to support cytoskeletal organization, actomyosin-dependent tension, and the viscoelastic properties of the embedded cells in a 3D-specific fashion. These results advance a new model wherein Fn polymerization serves as a structural scaffolding that displays adhesive ligands on a mechanically ideal substratum for promoting neovessel development.

Transient coexpression of cytokeratin and vimentin in differentiating rat sertoli cells

The expression of cytokeratin and vimentin type intermediate filaments were studied in fetal, postnatal, and adult rat testes. Immunocytochemical observations were correlated with the light and electron microscopic analysis of the developing organs. The Sertoli cell precursors in 15-day-old fetal testes contained both cytokeratin and vimentin. A gradual reorganization of both filaments, accompanied by a decrease of cytokeratin-positivity, was observed toward the end of the fetal period. The simultaneous presence of cytokeratin and vimentin in the same cells was shown by double immunofluorescence of newborn testes and the primary culture of dissociated testicular cells. In postnatal Sertoli cells, cytokeratin-positivity continued to decrease and disappeared by the age of 14 days. The increase in vimentin content and the appearance of axially oriented vimentin filaments coincided with the acquisition of the columnar shape of the Sertoli cells. The presence of cytokeratin and vimentin in fetal and newborn testes, and only vimentin in the adult testes was confirmed by immunoblotting. The present results suggest that major qualitative changes in the expression of intermediate filament proteins can take place during the embryonic development. The expression of cytokeratin in developing Sertoli cells, although only transient, supports the epithelial origin of these cells and can be applied as a marker for embryonic and early postnatal Sertoli cells.

Mutation of the Cytoplasmic Domain of the Integrin Subunit DIFFERENTIAL EFFECTS ON CELL SPREADING, RECRUITMENT TO ADHESION PLAQUES, ENDOCYTOSIS, AND PHAGOCYTOSIS

The cytoplasmic domain of the β subunit of the αβ3integrin is required for cell spreading on fibrinogen. Here we report that deletion of six amino acids from the COOH terminus of the β3(ITYRGT) totally abolished cell spreading and formation of adhesion plaques, whereas retaining Ilepartially preserved these functions. We further found that substitution of Tyrwith Ala also abolished αβ3-mediated cell spreading. The effects of these and other mutations on additional functions of αβ3were also studied. Progressive truncations of β3, in which stop codons were inserted at amino acid positions 759-756, caused partial defects in the recruitment of αβ3to preestablished adhesion plaques and a gradual decrease in the ability of αβ3to mediate internalization of fibrinogen-coated particles. The Tyr Ala substitution mutant was almost totally inactive in both of these assays. Point mutations at Tyr, and at a conserved area close to the transmembrane domain of β3, decreased integrin recruitment to preestablished adhesion plaques but allowed αβ3-mediated formation of these structures and partial cell spreading. Deletion of the cytoplasmic domain of β3did not affect the constitutive endocytosis of αβ3.

Repression of PTEN Phosphatase by Snail1 Transcriptional Factor during Gamma Radiation-Induced Apoptosis

ABSTRACT The product of the Snail1 gene is a transcriptional repressor required for triggering the epithelial-to-mesenchymal transition. Furthermore, ectopic expression of Snail1 in epithelial cells promotes resistance to apoptosis. In this study, we demonstrate that this resistance to γ radiation-induced apoptosis caused by Snail1 is associated with the inhibition of PTEN phosphatase. In MDCK cells, mRNA levels of the p53 target gene PTEN are induced after γ radiation; the transfection of Snail1 prevents this up-regulation. Decreased mRNA levels of PTEN were also detected in RWP-1 cells after the ectopic expression of this transcriptional factor. Snail1 represses and associates to the PTEN promoter as detected both by the electrophoretic mobility shift assay and chromatin immunoprecipitation experiments performed with either endogenous or ectopic Snail1. The binding of Snail1 to the PTEN promoter increases after γ radiation, correlating with the stabilization of Snail1 protein, and prevents the association of p53 to the PTEN promoter. These results stress the critical role of Snail1 in the control of apoptosis and demonstrate the regulation of PTEN phosphatase by this transcriptional repressor.