A differentiation protocol consisting of a sequential treatment with a demethylating agent 5-aza-2'-deoxycytidine followed by a combination of FGF+OSM+HGF in the continuos presence of a synthetic glucocorticoid dexamethasone (Dex) was developed and applied to stem-like rat liver epithelial (RLE) cell lines derived from adult rat. This treatment resulted in a remarkable enlargement in cell size and organelle complexity as shown by FACS and confocal microscopy. Morphological maturation of RLE was paralleled by a decrease in cell proliferation. More significantly, RT-PCR analysis revealed an induction of hepatocyte specific markers AFP, TTR, TAT and connexin 32. In addition, mRNAs encoding liver enriched transcription factors HNF 1a/b, HNF 3a/b, HNF 4, and C/EBPb as well as Hex and GATA 4/5 were markedly induced along with hepatocytic differentiation. The RT-PCR results were supported by a cDNA microarray analysis. Comparison of the gene expression profiles following the treatment protocol reflected an activation of the hepatocytic differentiation program as judged by increased expression of a differentiation-associated gene set and decreased expression of a cell cycle regulated gene set. Among the expressed genes related to hepatic specification/differentiation were BMP4, BMP6, Enothelin 1, as well as genes involved in heme and xenobiotic metabolism. In addition, alteration in expression of structural genes critical for extracellular matrix remodeling and organization of cytoskeleton including MMP1, TIMP-1, collagens, laminin, a-actinin was observed. Of importance, expression of b-catenin, Hdac1 as well as c-myc was increased along with hepatocytic differentiation. In contrast, down-regulation of Wnt/b-catenin pathway was reported in vitro system of fetal hepatic stem cell differentiation. Hence, it is possible that different regulatory pathways are responsible for the control of adult vs. embryonic stem cells differentiation. Furthermore, we found induction of genes implicated in biliary epithelial as well as pancreatic differentiation reflecting a broad developmental capacity of the RLE cells. Analysis of gene expression profiles induced by individual differentiating factors and their combinations, with genes commonly and preferentially expressed in purified hepatocytes, oval cells or biliary epithelial cells is underway to identify genes expression modules characteristic for the differentiation pathways of adult hepatic stem cells. Characterization of molecular and cellular events accompanying the expansion and differentiation of the stem cell compartment is of fundamental importance for understanding the biology of the liver stem cells and facilitating its clinical application. TNFa, an inflammatory cytokine that induces both hepatocyte proliferation and apoptosis, has been involved in different types of liver damage. In this work, we examined whether the differentiation of a rat liver epithelial cell line (RLE13) promoted the acquisition of sensitivity to TNFa-mediated apoptosis. To induce differentiation towards the hepatocyte lineage we treated RLE cells with a combination of 5-Aza-2'-deoxycytidine+Dexamethasone+Oncostatin M. While control RLE were resistant to TNFa, a massive apoptosis was observed in differentiated RLE. To clarify the mechanism of apoptosis induced by TNFa, cascade of caspase activation and mitochondrial functions were investigated. In both control and differentiated RLE, TNFa induced all early events required for apoptosis, including activation of caspase 8, loss of mitochondria membrane potential, release of cytochrome c and cleavage of caspases 9. However, only in differentiated RLE did TNFa treatment culminated in activation of the effector caspase 3. Western blot analysis of the phosphorylation and degradation of IkBa as well as gel shift analysis of NFkB DNA-binding activity showed a similar activation of this pathway in both control and differentiated RLE. Therefore, a deregulation of the NFkB survival pathway was excluded as a potential explanation for the differential response elicited by TNFa in both cell types. Interestingly, hepatocytic differentiation was associated with changes in the expression pattern of XIAP, a member of the inhibitor of apoptosis protein family (IAPs). While control RLE showed abundant presence of XIAP in soluble cytosolic fractions, this protein was hardly detectable in differentiated RLE. Since western blot analysis in whole cell lysates showed a comparable global expression of XIAP in both cell types, we are testing the hypothesis that changes in intracellular distribution, conformation or binding complexes involving this protein could account for the inability of XIAP to inhibit apoptosis in differentiated RLE.
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