Based on a recent description of an apoptosis stimulating property for hepatocyte derived isoferritins, this investigation demonstrates that ferritin, released in vitro from hepatocytes substantially contributes to density dependent apoptosis in primary hepatocytes and is significantly (P < or = 0.05) inhibited by anti-H-ferritin antibody rH02. Furthermore, total protein release and albumin secretion rapidly decline in a time and density dependent mode under serum-free conditions, whereas ferritin secretion, which is upregulated at initial stages of primary culture is not affected by cell density. Supplementation with dexamethasone (DEX) or proliferative stimulation by epidermal growth factor (EGF) and insulin strongly suppresses density dependent apoptosis. Both regimens have previously been shown to inhibit isoferritin mediated apoptosis in hepatocytes, most likely by interrupting proapotitc mitochondrial signalling. Finally, FasL/Fas also participates in density dependent apoptosis, since apoptosis is significantly (P < or = 0.005) reduced in high density cultures supplemented with an anti-FasL antibody. This antibody has also been shown to neutralise ferritin mediated apoptosis in primary hepatocytes, suggesting a linkage of ferritin and Fas in density dependent apoptosis. In conclusion, ferritin contributes to apoptosis in primary hepatocytes in an autocrine, density dependent mode, involving Fas stimulation and proapoptotic mitochondrial signalling. With respect to liver physiology, these findings may indicate that ferritin plays a yet unrecognised role as an acute phase signalling molecule in early stages of tissue repair and liver regeneration, and may also be responsible for the limited ability to propagate human hepatocytes in culture and the limited expansion of donor cells in the recipient liver upon cell transplantation.
Previously we have demonstrated an apoptosis inducing activity for a rat hepatocyte conditioned medium (CM) presumably mediated by acidic isoferritins. Here, we present support for this assumption since isoferritins purified from different rat hepatocyte CM significantly enhanced the frequency of apoptotic cells in primary rat hepatocytes, an effect completely inhibited by a neutralizing anti-H-ferritin antibody. The apoptosis induction appears to be related to a 43 kDa ferritin subunit contained in the isoferritins released from primary hepatocytes, presumably representing a ferritin heavy/light chain heterodimer. In addition, these isoferritins immunologically crossreact with antibodies raised against placental isoferritin p43-PLF (which also contains a 43 kDa ferritin subunit) and melanoma-derived H-chain ferritin, representing ferritin isoforms which reveal immunomodulatory properties. Furthermore, p53 and FasL are upregulated upon isoferritin treatment in a time dependent mode, and apoptosis induction can be suppressed by neutralizing anti-FasL antibodies. Proapoptotic Bid is upregulated too and translocated into mitochondria in primary hepatocytes exposed to the isoferritins purified from the CM. Finally, epidermal growth factor (EGF) and dexamethasone (DEX), which counteract proapoptotic mitochondrial signalling, almost completely abolished the proapoptotic effect of the hepatocyte derived isoferritins. In conclusion, our findings demonstrate that acidic isoferritins with homology to immunomodulatory ferritin isoforms (p43-PLF, melanoma-derived-H-chain ferritin) are released from hepatocytes in vitro, and are able to stimulate upregulation of p53 and mediate apoptosis involving Fas (CD95) signalling as well as addressing the intrinsic mitochondrial proapoptotic pathway.
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