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Pubblicazioni Aperte DIgitali Sapienza > Biotecnologie cellulari ed ematologia > BIOLOGIA UMANA E GENETICA >

Please use this identifier to cite or link to this item: http://hdl.handle.net/10805/2308

Title: Identification of the mechanism for physiological hepatocyte polyploidization
Authors: DE SANTIS PUZZONIA, MARCO
Tutor: AMICONE, LAURA
Keywords: liver
polyploidy
TGF beta
EMT
MET
cytokinesis failure
binucleated cell
Issue Date: 5-Feb-2014
Abstract: Polyploidy is the condition in which a cell possesses more than two sets of homologous chromosomes. Although polyploidy is not common in mammalians, some specific tissues including heart, muscle cells, megakaryocytes, trophoblast giant cells and parenchyma liver cells are physiologically polyploid. In liver an extensive polyploidization occurs during the weaning period between the 15th and 40th day after birth, leading to the formation of tetraploid and octapoloid cells with one or two nuclei. Polyploid hepatocytes retain an highly proliferative potentiality; they can undergo multipolar mitosis due to extra centrosomes generating 3 or 4 aneuploid daughter cells. Hepatic aneuploidy was proposed to be a substrate for selection of more resistant clones during liver injury. In fact it has been demonstrated that specific aneuploid injury resistant nodules emerged after chronic liver damage. However, how polyploidization is triggered, established and regulated remains an open question. Several data suggest that microenvironmental elements (mainly soluble factors), rather than cell autonomous mechanisms, play a direct role in the appearance of polyploidy. For example, insulin and T3 hormone were identified as positive regulators and TGFα as a negative regulator of the binucleation. We screened the effects of a set of soluble factors involved in liver development and/or mass liver control on a murine hepatocyte cell line. Within the screening, we identified the cytokine TGFβ as a strong inducer of hepatocyte binucleation. Time lapse video microscopy highlighted that binucleated cells originated from a mechanism of cytokinesis failure without affecting the kinetics of the mitosis; this suggests that binucleation is a physiological alternative program of hepatocyte division. Very interestingly, in vivo experiments performed with the oral administration of a TGFβ receptor chemical inhibitor, during days 18-32 post-birth in mice, produced a significant decrease of binucleated hepatocytes, confirming the crucial role played by this cytokine in the polyploidization of the liver cells. In-vivo data showed that the hepatocytes cellular model recapitulated the physiology of the whole organism. Analysis of TGFβ downstream elements, known to be involved in cytoskeleton rearrangement, showed that Src kinase activity has a role in the polyploidization process of hepatocytes, most probably through the control of the GTPase protein RhoA, an actin cytoskeleton regulator, crucial for the cytokinesis. In TGFβ treated cells, in fact, together with the activation of Src, we observed a delocalization from the mid-body structure of the active form of RhoA. TGFβ is well known to trigger Epithelial to Mesenchymal Transition (EMT) in hepatocytes. The TGFβ- induced binucleated cells, in fact, showed a fibroblastoid morphology with upregulation of mesenchymal markers and downregulation of epithelial ones. TGFβ withdrawal was also performed and it produced an increase in ploidy levels with the appearance of an octaploid population and giant nuclei. Together with a ploidy increase, TGFβ withdrawal triggered the Mesenchymal to Epithelial Transition (MET) characterized by upregulation of ephitelial markers and downregulation of mesenchymal markers and generating a full epithelial polyploid population. In conclusion, TGFβ seems to be one of the major stimuli for hepatocyte binucleation; it acts through Src kinases and involves the activity of RhoA during cytokinesis. Moreover, intriguing is the relationship between EMT/MET and polyploidization that might reveal a new biological process in which these transdifferentiation mechanisms are involved and thus, requires further study.
URI: http://hdl.handle.net/10805/2308
Appears in PhD:BIOLOGIA UMANA E GENETICA

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