Dynamin GTPase activity mediates dedifferentiation of cultured hepatocytes via transcriptional changes

I Ilkavets; S Lukowski; A Bachmann; C Sticht; C Meyer; N Gretz; S Dooley

doi:10.1055/s-0029-1246412

Zeitschrift für Gastroenterologie, Inhaltsverzeichnis

Dynamin GTPase activity mediates dedifferentiation of cultured hepatocytes via transcriptional changes

I Ilkavets ¹, S Lukowski ¹, A Bachmann ², C Sticht ³, C Meyer ⁴, N Gretz ³, S Dooley ²

¹Molecular Hepatology - Alcohol Dependent Diseases; II. Medical Clinic; Faculty of Medicine Mannheim; University of Heidelberg, Mannheim
²II. Med. Universitätsklinik (Gastroenterologie, Hepatologie & Infektionskrankheiten), Mannheim
³Medical Research Center, Medical Faculty of Medicine at Mannheim, University of Heidelberg, Mannheim
⁴Molecular Alcohol Research in Gastroenterology, Department of Medicine II, Faculty of Medicine at Mannheim, University of Heidelberg, Mannheim

Abstract

Drug and metabolite endocytosis, viral and bacterial infections, and transcitosis are facilitated by large GTPase dynamin. However, the underlying molecular mechanisms of dynamin action on homeostasis in hepatocytes without additional external signals (like growth factors, transferrin or drugs) are poorly understood. Therefore, we depleted hepatocytes of functional dynamin by adenoviral overexpression of a GTPase deficient dynamin mutant (DN-dynamin) for 48h and compared genome wide oligonucleotide microarrays, morphology and biochemical functions with AdLacZ infected controls. Blunting dynamin function leads to decreased levels of early endosomal protein EEA1 and subsequent reduction in cell size and spreading. Moreover, hepatocyte arrangement in sheets is established and stabilized, representing the typical hepatocyte organization in the liver sinusoid. DN-dynamin decreases levels of FAK, pSrc, pAkt and pERK1/2, thus sensitizing hepatocytes to TGF-beta mediated apoptosis. These physiological changes are accompanied by differential expression of 504 genes (fold change greater than 1.3) related to several functional groups, including GTPase regulator activity, endocytosis, endoplasmic reticulum, Golgi apparatus, mitochondria, transcription factors, transporters, cytoskeleton, apical membrane and intercellular junctions. Our data suggest that membrane vesicle fission mediated via dynamin GTPase activity is required for fibroblastoid spreading and dedifferentiation of cultured hepatocytes. Abrogating dynamin function in hepatocyte culture decreases survival, sensitizes to cell death and stabilizes hepatocyte morphology and physiology.

GTPase - apoptosis - differentiation - dynamin - hepatocytes - microarray - morphology