Studying the role of fascin-1 in mechanically stressed podocytes

Glomerular hypertension causes glomerulosclerosis via the loss of podocytes, which are challenged by increased mechanical load. We have demonstrated that podocytes are mechanosensitive. However, the response of podocytes to mechanical stretching remains incompletely understood. Here we demonstrate that the actin-bundling protein fascin-1 plays an important role in podocytes that are exposed to mechanical stress. Immunofluorescence staining revealed colocalization of fascin-1 and nephrin in mouse kidney sections. In cultured mouse podocytes fascin-1 was localized along actin fibers and filopodia in stretched and unstretched podocytes. The mRNA and protein levels of fascin-1 were not affected by mechanical stress. By Western blot and 2D-gelelectrophoresis we observed that phospho-fascin-1 was significantly downregulated after mechanical stretching. It is known that phosphorylation at serine 39 (S39) regulates the bundling activity of fascin-1, e.g. required for filopodia formation. Podocytes expressing wild type GFP-fascin-1 and non-phosphorylatable GFP-fascin-1-S39A showed marked filopodia formation, being absent in podocytes expressing phosphomimetic GFP-fascin-1-S39D. Finally, the immunofluorescence signal of phosphorylated fascin-1 was strongly reduced in glomeruli of patients with diabetic nephropathy compared to healthy controls. In summary, mechanical stress dephosphorylates fascin-1 in podocytes in vitro and in vivo thereby fascin-1 may play an important role in the adaptation of podocytes to mechanical forces

Interaktion des Repressors Opi1 der Phospholipid-Biosynthese mit Regulatoren des Phosphat-Stoffwechsels und pleiotropen Corepressoren in der Hefe Saccharomyces cerevisiae

In der Hefe Saccharomyces cerevisiae werden die Strukturgene der Phospholipid-Biosynthese auf Transkriptionsebene in Abhängigkeit der Verfügbarkeit der Phospholipidvorstufen Inositol und Cholin (IC) über ein in der Promotorregion befindliches UAS-Element, genannt ICRE („inositol/choline-responsive element“), reguliert. Bei Mangel an IC kommt es zu einer Anhäufung des Intermediats Phosphatidsäure, wodurch der Repressor Opi1 außerhalb des Zellkerns am endoplasmatischen Reticulum verankert wird. Dadurch kann ein Heterodimer, bestehend aus den bHLH-Proteinen Ino2 und Ino4, an das ICRE-Motiv binden und die transkriptionelle Aktivierung vermitteln. Ist ausreichend IC vorhanden, gelangt der Repressor Opi1 in den Zellkern und bindet an Ino2. Dadurch ist eine Aktivierung nicht mehr möglich. Ferner kontaktiert Opi1 über seine Opi1-Sin3-Interaktionsdomäne (OSID) die Corepressor-Komplexe Sin3 und Cyc8/Tup1, die durch Rekrutierung von Histondeacetylasen (HDACs) zur Chromatinverdichtung und damit zur Genrepression führen. In einer früheren Arbeit wurde beobachtet, dass die regulierte Expression von Genen der Phospholipid-Biosynthese auch durch die Phosphatkonzentration beeinflusst wird. Es konnte festgestellt werden, dass bei Phosphatmangelbedingungen die Expression ICRE-abhängiger Gene auf 10 % reduziert ist. Eine Δopi1-Mutante zeigte dieses Expressionsmuster jedoch nicht mehr. Dieser Befund wies darauf hin, dass Opi1 seine Repressorfunktion sowohl bei IC-Überschuss als auch bei Phosphatmangel ausführt. Ein Protein, welches die Phosphatverfügbarkeit an Opi1 möglicherweise über eine Phosphorylierung vermitteln könnte, ist die cyclinabhängige Proteinkinase Pho85, für die eine in vitro Interaktion mit Opi1 gezeigt wurde. Um diese Hypothese zu überprüfen, wurden mittels gerichteter Mutagenese Aminosäurereste mutmaßlicher Pho85-Phosphorylierungsstellen im Opi1-Protein (S321, T51) gegen das nicht mehr phosphorylierbare Alanin ausgetauscht. Hefestämme, die solche Opi1-Protein-varianten (S321A, T51A) synthetisierten, zeigten jedoch weiterhin einen klaren Einfluss des Phosphatmangels auf die Expression eines ICRE-regulierten Reportergens. Dies lässt darauf schließen, dass die Repression unter Phosphatmangelbedingungen nicht über eine Phosphorylierung von Opi1 durch Pho85 zu Stande kommt. Parallel durchgeführte in vitro-Interaktionsstudien zeigten, dass die Bindung von Pho85 an Opi1 über zwei unabhängig voneinander funktionierende Interaktionsdomänen im Opi1-Protein (aa 30-70 und aa 321-350) erfolgt. Mit Hilfe des „Two-Hybrid“-Systems wurde festgestellt, dass die Opi1-Pho85 Wechselwirkung in vivo phosphatabhängig stattfindet. Die Befunde erlauben die Hypothese, dass Pho85 bei Phosphatüberschuss u. a. die OSID im Opi1 abdeckt, dadurch die Wechsel-wirkung mit Sin3/Cyc8 verhindert und eine gesteigerte Genexpression zulässt. Mittels Chromatin-Immunopräzipitation (ChIP) konnte gezeigt werden, dass Opi1, Co-Repressoren wie Sin3 und Cyc8 als auch die HDACs Hda1 und Hos1 an Promotoren ICRE-regulierter Gene Ino2-abhängig anwesend sind. Des Weiteren wurde festgestellt, dass sich Sin3 unabhängig von Opi1 an ICRE-haltigen Promotoren befindet. Dieses Ergebnis wider-sprach einer früheren Arbeitshypothese, konnte aber durch weitere Versuche, die eine direkte in vitro Interaktion von Sin3 mit dem Ino2-Aktivator zeigten, plausibel in ein neues Rekrutierungsmodell eingefügt werden. Abschließend wurden die am Beispiel von Opi1 gewonnenen Erkenntnisse durch in vitro Interaktionsanalysen diverser spezifischer Repressoren mit den pleiotropen Co-Repressoren Sin3 und Cyc8/Tup1 erweitert. Für zahlreiche Repressoren wurde gefunden, dass sie parallel mit Sin3 und Cyc8 interagieren (u. a. Rox1, Yox1, Dal80 und Mot3). Durch Kartierungsexperimente konnten minimale Repressordomänen charakterisiert werden, die die Interaktion zu Sin3 bzw. Cyc8 vermitteln, und sequenzhomologe Domänenstrukturen analysiert werden. Des Weiteren zeigte sich, dass alle Repressoren, die mit Sin3 wechselwirken, dessen Domänen PAH1 oder PAH2 („paired amphipathic helix“) kontaktieren.In the yeast Saccharomyces cerevisiae, structural genes of phospholipid biosynthesis are regulated at the transcriptional level by the availability of phospholipid precursors inositol and choline (IC). Regulation is mediated by a UAS element in promoter regions, designated ICRE (“inositol/choline-responsive element"). A heterodimer consisting of bHLH proteins Ino2 and Ino4 binds the ICRE motif in the absence of IC and triggers transcriptional activation. With IC in excess, repressor Opi1 is imported into the nucleus and binds to Ino2, thereby counteracting activation. Using its Opi1-Sin3-interaction domain (OSID), Opi1 also contacts pleiotropic co-repressor complexes Sin3 and Cyc8/Tup1 which subsequently recruit histone deacetylases (HDACs), resulting in chromatin silencing and thus gene repression. Previous work has shown that phospholipid biosynthetic genes are also regulated by the concentration of free phosphate. In a yeast wild-type strain grown under phosphate limitation, expression of ICRE-dependent genes decreases to 10%, compared with high phosphate. This expression pattern was no longer observed in an opi1 mutant. Thus, repressor Opi1 counteracts Ino2 in the presence of high IC as well as under conditions of phosphate limitation. It could be also shown that Opi1 interacts with the cyclin-dependent protein kinase Pho85 which is active in the presence of high phosphate. Possibly, interaction of Opi1 with Sin3 and Ino2 is affected by Pho85-dependent phosphorylation. To test this hypothesis, selected amino acid residues in the Opi1 protein which match Pho85 phosphorylation sites (SP, TP) have been exchanged by site-directed mutagenesis for amino acids that can no longer be phosphorylated (mutations S321A and T51A). Similarly, variants carrying a permanent negative charge were also constructed. However, even with these Opi1 variants, expression of an ICRE-dependent reporter gene was still clearly influenced by phosphate deficiency. The phosphomimetic OPI1 variant S321E did not show a permanent expression of the reporter gene. These findings suggest that gene repression under phosphate limitation is not mediated by the degree of phosphorylation of the tested Opi1 amino acid positions. In vitro interaction studies could show that binding of Opi1 to Pho85 maps to two distinct domains in the region of amino acids 30-70 and 321-350. Interestingly, these domains are also responsible for Opi1 interaction with Sin3 and Ino2, respectively. Use of the “yeast two-hybrid” system demonstrated that in vivo interaction between Pho85 and Opi1 is regulated by the availability of phosphate. A working hypothesis postulates that Opi1 interactions with Sin3 and Ino2 are prevented by Pho85 in the presence of high phosphate. Consequently, Opi1 and Sin3/Cyc8 are no longer recruited to ICRE-containing promoters, resulting in an increased gene expression. Chromatin immunoprecipitation (ChIP) analyses demonstrated Ino2-dependent recruitment of negative regulators Opi1, Sin3 and Cyc8 as well as HDACs Hda1 and Hos1 to promoters of ICRE-regulated genes. In contrast to a previous hypothesis it was shown that Sin3 binds to ICRE-containing promoters independently of Opi1. This surprising result could be explained by the finding of a direct in vitro interaction between activator Ino2 and Sin3, leading to a modified recruitment model. Finally, results obtained on Opi1-dependent co-repressor interaction were also tested for additional pathway-specific repressor proteins. In vitro interaction studies showed that various repressor proteins (such as Rox1, Yox1, Dal80 and Mot3) are indeed able to bind pleiotropic co-repressors Sin3 and Cyc8/Tup1. All repressors interacting with Sin3 contact domains PAH1 or PAH2 ("paired amphipathic helix"). Mapping experiments allowed the characterization of minimum repressor domains and to derive a sequence pattern which may be important for repressor interaction with Cyc8 or Sin3

The transcription factor Dach1 is essential for podocyte function

Dedifferentiation and loss of podocytes are the major cause of chronic kidney disease. Dach1, a transcription factor that is essential for cell fate, was found in genome-wide association studies to be associated with the glomerular filtration rate. We found that podocytes express high levels of Dach1 in\ua0vivo and to a much lower extent in\ua0vitro. Parietal epithelial cells (PECs) that are still under debate to be a type of progenitor cell for podocytes expressed Dach1 only at low levels. The transfection of PECs with a plasmid encoding for Dach1 induced the expression of synaptopodin, a podocyte-specific protein, demonstrated by immunocytochemistry and Western blot. Furthermore, synaptopodin was located along actin fibres in a punctate pattern in Dach1-expressing PECs comparable with differentiated podocytes. Moreover, dedifferentiating podocytes of isolated glomeruli showed a significant reduction in the expression of Dach1 together with synaptopodin after 9\ua0days in cell culture. To study the role of Dach1 in\ua0vivo, we used the zebrafish larva as an animal model. Knockdown of the zebrafish ortholog Dachd by morpholino injection into fertilized eggs resulted in a severe renal phenotype. The glomeruli of the zebrafish larvae showed morphological changes of the glomerulus accompanied by down-regulation of nephrin and leakage of the filtration barrier. Interestingly, glomeruli of biopsies from patients suffering from diabetic nephropathy showed also a significant reduction of Dach1 and synaptopodin in contrast to control biopsies. Taken together, Dach1 is a transcription factor that is important for podocyte differentiation and proper kidney function