Relevanz von p21 für die Progression der akuten und chronischen Lebererkrankung im NEMOdeltahepa-Modell

Every year 500 000 people die worldwide due to the consequences of a hepatocellular carcinoma (HCC). This makes the disease an enormous health problem (Severi et al. 2010). One major reason for the high lethality is the late diagnosis of the disease and insufficient therapeutic options to treat the tumor. The NF-kappaB signaling pathway plays an important role in HCC pathogenesis. It was shown by Luedde et al. (2007), that the hepatocyte specific deletion of NEMO (NEMOdeltahepa) and the resulting inactivation of the transcription factor NF-kappaB leads to steatohepatitis and as a consequence to the development of cirrhosis and HCCs. The phenotype of the NEMOdeltahepa liver is associated with an overexpression of the cell cycle inhibitor p21 (Malato & Ehedego et al. 2012). p21 is known to inhibit cell proliferation, by binding to cyclin/cdk2 complexes (Harper et al. 1993). p21 is furthermore a key mediator of the p53-dependent cell cycle arrest after DNA-damage (Gartel et al. 1996 and 1998). So far, the function of p21 in tumorgenesis is not fully understood. There are studies describing oncogenic as well as tumor suppressor function for p21. Therefore, in the present study the relevance and function of the p21 overexpression for the progression of the chronic and acute liver disease was investigated in the NEMOdeltahepa model. The loss of p21 in NEMOdeltahepa livers led to an overexpression of the cell cycle mediators cyclin A2, cyclin E2 and E2F1. Overexpression of the G1- and S-phase cyclins had no influence on hepatocyte proliferation, this was shown by immunological staining using the proliferation markers Ki67 and BrdU in NEMOdeltahepa compared to NEMOdeltahepap21-/- livers. The missing increase of cdk2 activity was an additional sign that proliferation in NEMOdeltahepa liver was not affected after loss of p21 expression. Tane & Chibazakura (2009) showed for the first time that ectopic overexpression of cyclin A or E in mouse embryonic fibroblast (MEFs) leads to an increase in DNA double strand breakage. In this actual work it could be shown, that there is a correlation between the increase of DNA-damage and the overexpression of cyclin A or cyclin E2 in NEMOdeltahepap21-/- livers. This was quantified by immunofluorescence staining with a pH2AX antibody. This observation explains the increase of phosphorylated p53 in NEMOdeltahepap21-/- livers, which accumulates in response to DNA-damage in the cells. As a consequence of the DNA-damage the expression of important mediators (BRCA1 and FoxM1) for DNA repair are enhanced in NEMOhepap21-/- livers. This observation is normally a disadvantage for the treatment of HCCs, because overexpression of DNA repair proteins lead to a increased resistance to chemotherapy and a more aggressive progression of the HCCs. Thus aging experiments with 52 week old animals were performed. Loss of p21 in NEMOdeltahepa livers enhanced hepatocarcinogenesis and in addition a strong cholestasis phenotype was found. Immunological studies in NEMOdeltahepa and NEMOdeltahepap21-/- HCCs showed an increase in CK19 positive cells in the double knockout HCCs. This was an additional sign, that the p21-deficient HCCs develop a more malignant HCC phenotype. To further characterise the protective function of p21 overexpression in NEMOdeltahepa livers, experiments were performed in the acute LPS-model. The loss of p21 caused much stronger liver injury and DNA-damage in the NEMOdeltahepa animals, which resulted in increased lethality of the animals. The enhanced sensitivity against LPS could be reversed in the NEMOdeltahepap21-/- mice by deleting TNFR1. Deletion of TNFR1 also ameliorated the spontaneous phenotype of the NEMOdeltahepap21-/- animals, so that this animals in aging experiments revealed a similar phenotype as NEMOdeltahepa animals. In summary this study shows, that p21 overexpression in NEMOdeltahepa livers inhibits the progression of chronic liver disease. Investigations after acute LPS-stress and in chronic injury prove, that p21 facilitates it’s protective influence not by changing the cell cycle progression, but the cell cycle inhibitor is protecting from DNA-damage. This study describes a new, until now an unknown function of p21, which could be an initial point for the development of new biomarkers and therapeutic strategies