Elucidation of the role of connexin proteins in the control of hepatocellular homeostasis: development of a hepatocyte-based in vitro model for pre-clinical pharmaco-toxicological research

1) Objectives and motivation Preclinical pharmaco-toxicological research is frequently carried out on experimental animals. However, from a scientific, ethical and economic point of view this seems not longer acceptable. Therefore, the need for the development of appropriate in vitro models is urgently growing. Primary hepatocyte cultures are promising in vitro systems, since they provide a good reflection of the in vivo situation. However, their use is currently limited to short-term purposes by the progressive occurrence of dedifferentiation [1]. Our group and others have shown that dedifferentiation can be partly reduced by establishing organotypical cultivation condition. As a result, our group has built up great expertise in the field of cultivation of primary hepatocytes [2-19] (for an overview of our publications, see http://minf.ac.be/~fafy). Since 1999, our laboratory explores an innovative anti-dedifferentiation strategy i.e. the use of histone deacetylase inhibitors as culture medium additives. Thusfar, we have shown that these molecules prevent proliferation and apoptosis of primary hepatocytes, whereas the maintenance of their differentiated phenotype is strongly promoted [20-25]. In particular, the doctoral research of the applicant concerned the study of the effects of histone deacetylase inhibitors on gap junctional intercellular communication in primary hepatocyte cultures. This doctoral research could show that histone deacetylase inhibitors greatly enhance gap junctional intercellular communication. This is associated with differential effects on the expression of connexins, the structural units of gap junctions [26,27]. This research also demonstrated that connexins behave in distinct ways upon hepatocyte proliferation [28]. Based on these findings, the hypothesis is raised that connexins perform specific functions in the control of hepatocellular homeostasis. In a first part of this post-doc research, it will be studied whether this hypothesis holds. Based on the results obtained, primary hepatocytes will then be transfected with selected connexin genes in order stabilise the differentiated hepatocyte phenotype in vitro. 2) Aims (i) Elucidation of the specific role of Cx26 and Cx32 in the control of hepatocellular homeostasis, particularly in apoptosis, differentiation and proliferation. (ii) The development of primary hepatocyte cultures in which the differentiated phenotype can be kept at long-term, by transfecting primary hepatocytes with individual connexin genes.