Caracterización y estudio funcional de la α1-antiquimiotripsina. Papel en el daño hepático

Resumen

a1-antichymotrypsin (a1-ACT) or SERPINA3 is a plasmatic glycoprotein that inhibits serinproteases. It¿s an acute phase protein that plays and important role in inflammation and host defence. It has been implicated in several human disorders including liver diseases. In the present work we have studied the physiological role of a1-ACT in hepatic cells to evaluate its implication in liver damage. Our results show that Serpina3 expression is decreased in liver tissue from both alcoholic cirrhosis and HCC patients. Moreover, we developed mice models that revealed that Serpina3 expression was induced in LPS or turpentine-induced acute inflammation while its mRNA levels decreased in response to CCl4-induced acute liver injury and during the liver regeneration induced by partial hepatectomy. It has been confirmed the induction of Serpina3 in response to OSM, IL-6 and CT-1 in hepatic cells. In KO-AR, Serpina3 expression was similar than WT mice in the different experimental models but its mRNA levels was higher in liver tissue and also in isolated hepatocytes from KO-AR on respect the WT ones. The treatment with recombinant AR decreased Serpina3 expression in hepatic cells and also AR reduced Serpina3 induction by OSM. The molecular mechanisms involved in the interaction between AR and OSM revealed that MAPK (ERK or p38) could be, al least in part, responsible of the effect of AR. Finally, using reported gene assays, a transcriptional regulation of Serpina3 by AR and OSM was confirmed. We have confirmed the presence of an intracellular form of Serpina3 localized into the nucleus of hepatic cells and we observed that its levels increased in response to OSM. We developed Serpina3 expression vectors to study the effect of the intracellular Serpina3 overexpressed because the Serpina3 transfected also was found into the nucleus. Because of OSM exerts several actions on the apoptosis, proliferation or differentiation of hepatocytes, Serpina3 could also have any effect on this processes. We found that OSM has an antiapoptotic effect since it reduced the TGFbeta or okadaic acid-induced DNA fragmentation. However, the overexpression or the inhibition of Serpina3 hadn¿t any effect on the apoptosis because it didn¿t modified the levels of Bcl-XL, caspase 3 or DNA fragmentation with or without a proapoptotic agent. On the other hand, the overexpression of Serpina3 reduced cell proliferation and the inhibition of Serpina3 showed an increase in DNA synthesis and in the levels of molecules involved in the progression of cell cycle like PCNA, cyclin D1, CDK2 and p21. We confirmed that OSM presents an antiproliferative behaviour but the action of Serpina3 over cell growth was carried out also without OSM. We have demonstrated that the form of the protein that exerts this effect was the nuclear form of Serpina3 because the overexpression of a mutated a1-ACT that wasn¿t into the nucleus, presented a similar proliferation rate respect the control cells and higher than the cells that overexpressed the WT a1-ACT. Finally, we determined the involvement of Serpina3 in liver development since its expression level was very low in foetal liver and increase in the neonatal one. In addition, the effect of OSM in the differentiated state of hepatic cells was studied. We determined that this action required the induction of Serpina3 because it induced directly the expression of adult liver genes (MAT1A, HNF4a, albumin). The inhibition of Serpina3 expression blocked the induction of these genes by OSM. In conclusion, our data show that a nuclear form of Serpina3 exists and that it develops an intracellular role stimulating the differentiated state of hepatic cells and exerting an antiproliferative action. So, a1-ACT could have a therapeutic application potential to prevent the development of liver diseases such as alcoholic cirrhosis or HCC.