Jukka Tienari

Neuron-like derivatives of cultured F9 embryonal carcinoma cells express characteristics of parietal endoderm cells

Murine F9 embryonal carcinoma cells exposed to retinoic acid and dibutyryl cyclic AMP gradually arborize and acquire a neuron-like morphology in monolayer culture. Whether F9 cells can be induced to differentiate into cells with features specific to neural cells is controversial. We analyzed the intermediate filament content and pericellular matrix proteins of F9 cells after exposing them to retinoic acid, dibutyryl cyclic AMP, and nerve growth factor. In long-term cultures, a great majority of the cells appeared neuron-like, but showed intra- and pericellular laminin and type IV collagen, and frequently cytokeratin filaments as well. Several monoclonal antibodies to neurofilaments did not react with these cells in immunofluorescence or immunoblotting, though they recognize either all or individual mouse neurofilament triplet proteins. Polyclonal antibodies to neurofilament proteins gave a diffuse, nonfibrillar, vinblastine-resistant fluorescence in the morphologically neuron-like cells, but in immunoblotting failed to reveal polypeptides compatible with neurofilament triplet proteins. In long-term cultures, most of the cells appeared to have partially or totally lost the intermediate filaments. This was confirmed with anti-IFA antibodies which normally react with all intermediate filament proteins. The F9-derived cells did not respond to nerve growth factor in any detectable way. We conclude that the morphologically neuron-like derivatives of F9 cells display characteristics of modified parietal endoderm-like cells and do not show unequivocal features of neural cells.

Embryonal carcinoma cells adhere preferentially to fibronectin and laminin but their endodermal differentiation leads to a reduced adherence to laminin

F9 and PC13 embryonal carcinoma (EC) cells adhered rapidly to growth substrata coated with fibronectin or laminin. When F9 cells were induced to differentiate into visceral or parietal endoderm-like cells, their ability to adhere to laminin diminished, but their adherence to fibronectin remained unchanged. Correspondingly, permanently differentiated teratocarcinoma-derived endoderm cells (PYS-2 and PSA-5e) adhered markedly less efficiently to laminin than to fibronectin. F9 cells adhered to proteolytic fibronectin fragments containing the cell-binding site but not to fragments containing gelatin- or heparin-binding sites. They also adhered slowly to gelatin, but this adhesion was completely blocked by cycloheximide. The results show that the teratocarcinoma stem cells may have specific mechanisms mediating adhesion to fibronectin and laminin and that endodermal differentiation leads to a reduction in their capacity to adhere to laminin but not to fibronectin.

Implanted solid human tumours grow under the renal capsule of cyclosporin-immunosuppressed rats.

Cyclosporin (CsA) is a potent immunosuppressive drug widely used in organ transplantation. We transplanted fresh surgical samples from human solid malignant tumours into 45 CsA‐immunosuppressed rats. Eight out of nine tumour types grew and remained viable for 5 weeks or more in at least two of the transplanted rats. In 29 rats (64%) a distinct growth of primary human tumours was recorded.

Regulation of nephrin gene by the Ets transcription factor, GA-binding protein

BACKGROUND Transcription factor GA-binding protein (GABP) is suggested to be involved in the formation of the neuromuscular junctions by regulating the transcription of synapse genes. Interestingly, neurons and podocytes share molecular and functional similarities that led us to investigate the expression and function of GABP in podocytes and its role in transcriptional regulation of nephrin, the key molecule of the podocyte slit diaphragm that is essential for normal glomerular ultrafiltration. METHODS The expression and localization of GABP in the rat and human kidney as well as in human embryonic kidney A293 cells and undifferentiated and differentiated human podocytes were analysed by immunoblotting and immunostaining. The role of GABP in activating the nephrin promoter was investigated by reporter gene assay and site-directed mutagenesis of the GABP-binding elements, and the interaction of GABP with the nephrin promoter was analysed by chromatin immunoprecipitation. The function of GABP in podocytes was studied by knocking down GABPα in differentiated human podocytes using lentiviral shRNA targeting GABPα. RESULTS GABP is expressed in the nuclei in rat and human glomeruli. In addition, in A293 cells and undifferentiated and differentiated human podocytes, GABP highly enriches in the nucleus. GABP activates and binds nephrin proximal promoter and Ets sites are essential for this activity. Knock-down of GABPα stimulates apoptosis in cultured podocytes. CONCLUSIONS The results show that GABP is expressed in podocytes and is involved in the regulation of nephrin gene expression. Furthermore, GABP may be important in the maintenance of podocyte function by regulating apoptosis.

PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease

Nephrin is a core component of podocyte (glomerular epithelial cell) slit diaphragm and is required for kidney ultrafiltration. Down‐regulation or mislocalization of nephrin has been observed in diabetic kidney disease (DKD), characterized by albuminuria. Here, we investigate the role of protein kinase C and casein kinase 2 substrate in neurons 2 (PACSIN2), a regulator of endocytosis and recycling, in the trafficking of nephrin and development of DKD. We observe that PACSIN2 is up‐regulated and nephrin mislocalized in podocytes of obese Zucker diabetic fatty (ZDF) rats that have altered renal function. In cultured podocytes, PACSIN2 and nephrin colocalize and interact. We show that nephrin is endocytosed in PACSIN2‐positive membrane regions and that PACSIN2 overexpression increases both nephrin endocytosis and recycling. We identify rabenosyn‐5, which is involved in early endosome maturation and endosomal sorting, as a novel interaction partner of PACSIN2. Interestingly, rabenosyn‐5 expression is increased in podocytes in obese ZDF rats, and, in vitro, its overexpression enhances the association of PACSIN2 and nephrin. We also show that palmitate, which is elevated in diabetes, enhances this association. Collectively, PACSIN2 is up‐regulated and nephrin is abnormally localized in podocytes of diabetic ZDF rats. In vitro, PACSIN2 enhances nephrin turnover apparently via a mechanism involving rabenosyn‐5. The data suggest that elevated PACSIN2 expression accelerates nephrin trafficking and associates with albuminuria.—Dumont, V., Tolvanen, T. A., Kuusela, S., Wang, H., Nyman, T. A., Lindfors, S., Tienari, J., Nisen, H., Suetsugu, S., Plomann, M., Kawachi, H., Lehtonen, S. PACSIN2 accelerates nephrin trafficking and is up‐regulated in diabetic kidney disease. FASEB J. 31, 3978–3990 (2017). www.fasebj.org—Dumont, Vincent, Tolvanen, Tuomas A., Kuusela, Sara, Wang, Hong, Nyman, Tuula A., Lindfors, Sonja, Tienari, Jukka, Nisen, Harry, Suetsugu, Shiro, Plomann, Markus, Kawachi, Hiroshi, Lehtonen, Sanna PACSIN2 accelerates nephrin trafficking and is up‐regulated in diabetic kidney disease. FASEB J. 31, 3978–3990 (2017)

A cloned human germ cell tumor‐derived cell line differentiating in culture

We have derived a clonal cell line (HGCT‐1) from a lymph node metastasis of a primary testicular germ cell tumor (GCT). The tumor was negative for the embryonal carcinoma (EC) cell marker BerH2 but positive for vimentin, cytokeratin (CK) and desmin. Comparative genomic hybridization (CGH) revealed a high‐level amplification at 12p that was observed in both the metastatic tumor and in the cultured HGCT‐1 cells. In vitro, the phenotype of HGCT‐1 cells was modulated by the culture conditions. In the presence of 10% fetal calf serum (FCS), the majority of HGCT‐1 cells lacked CK and desmin. If cultured in 0.5% FCS, HGCT‐1 cells acquired a uniform co‐expression of vimentin, CK and desmin. Upon treatment with retinoic acid (RA), HGCT‐1 cells lost the expression of desmin, but exhibited abundant CK filaments. Simultaneously, they started to express desmoplakin, form desmosomes and flatten on the culture substratum. The RA‐induced changes were irreversible, whereas those following the culture in 0.5% FCS were at least partially reversible. When xenografted into an immunosuppressed rat, HGCT‐1 cells formed a tumor consisting of epithelial‐ and mesenchymal‐like structures. HGCT‐1 cells thus represent a pluripotential cell system with a capacity for reversible phenotypic modulation and for irreversible differentiation into epithelial‐type cells. The behavior of this novel cell line, distinct from established EC cell models, suggests a complex regulation of GCT cell differentiation. Int. J. Cancer 77:710–719, 1998.

Actin cytoskeleton of extraembryonic endoderm and teratocarcinoma-derived endoderm cells☆

Distinct F-actin- and myosin-containing stress fibers were observed in situ in many endoderm cells of parietal yolk sacs from 11-day mouse embryos. In visceral endoderm (VE) such fibers were not seen, and F-actin was concentrated in the cell periphery. Correspondingly, in electron microscopy ventral cell membrane-associated bundles of microfilaments were revealed in the periphery of parietal endoderm (PE) cells but not in VE cells. Both PE and VE cells formed stress fibers in primary cultures. Undifferentiated F9 embryonal carcinoma cells formed only short actin spikes and fibrils irrespective of growth substratum. In PE-like derivatives of F9 cells, on the other hand, distribution of F-actin was markedly affected by the growth substratum: They formed distinct stress fibers when plated on fibronectin but did not when plated on gelatin. Similarly, in teratocarcinoma-derived PE cells (PYS-2) adhesion to fibronectin induced the formation of distinct bundles of F-actin and plaques of vinculin. The results suggest that the susceptibility of teratocarcinoma cell actin cytoskeleton to the influence of molecular composition of surrounding matrix is developmentally regulated. On the other hand, the reason for the presence of stress fibers in PE cells and for their absence in VE cells is unclear.