Inhibiting Sox9 as a tool to enhance LPC-mediated regeneration

Details

Supervisors

Leclercq, Isabelle

Degree label

Master [120] en sciences biomédicales, à finalité spécialisée: nutrition humaine

Abstract

Liver has the amazing capacity to regenerate. When 70% of its mass is resected, liver mass and function are rapidly restored through hepatocyte proliferation. However, when hepatocytes are injured such as in chronic liver diseases, the proliferation of hepatocytes is impaired. In such a condition, a ductular reaction (DR) emerges from the biliary cell compartment. Cells of the DR enclose a population of liver progenitor cells (LPCs) able to differentiate into hepatocytes, offering an alternative path for regeneration. Unfortunately, this alternative regeneration process is poorly efficient and DR-derived hepatocytes account for only a small percentage of all hepatocytes. The SRY-related HMG box transcription factor 9 (Sox9) is thought to inhibit LPC-to-hepatocyte differentiation. Downregulation of Sox9 through inhibition of EGFR signaling pathway has been reported to enhance LPC-mediated regeneration in zebrafish. The objective of this master’s thesis is to test the hypothesis that down regulation of Sox9 promotes LPC-to-hepatocyte differentiation and thereby, enhances LPC-driven regeneration in models of chronic liver injury in mice. We trace cells of the DR in the chronic intoxication to CCl4 model of hepatocellular damage using the OPN-iCreERT2/+:ROSAR26YFP/YFP transgenic mouse strain. With these mice, hepatocytes of biliary origin express the yellow fluorescent protein and are easily identifiable. In a first approach, we use the EGFR inhibitor Erlotinib to downregulate Sox9 expression. EGFR signaling induces the expression of Sox9. We found that chronic CCl4 treatment induced liver damage and important bridging fibrosis. We observed patches of DR-derived hepatocytes repopulating the parenchyma, accounting for 0,7% of lobular surface. Despite a 37% reduction in Sox9 expression, the total number of DR-derived hepatocytes and the number of differentiation events was similar in Erlotinib treated and control livers. Consistent with this, no improvement of liver damage or fibrosis was observed. We conclude that Erlotinib treatment does not improve LPC-to-hepatocyte differentiation. To obtain a more pronounced Sox9 deletion, we have used a transgenic approach. We generated a model allowing Sox9 deletion in an inducible fashion, and specific to cholangiocytes. We performed successive breedings to obtain the OPNCre/+:ROSAR26YFP/YFP:Sox9fl/fl and OPNCre/+:ROSAR26YFP/YFP:Sox9fl/+ triple transgenic mice. In Sox9fl/fl mice, Cre-mediated recombiantion completely cancels Sox9 expression in cholangiocytes. This promising mouse model of conditional Sox9 knockout in cholangiocyte will provide further insight to the role of Sox9 during the differentiation of LPCs. Finally, we characterize the DR in C57BL/6 mice fed a CDAA-HFD diet (choline deficient, L-amino acid defined, high-fat diet). CDAA diet induces steatosis, fibrosis and important invasion of biliary cells in the parenchyma. The use of this diet could be of real interest to study the behavior of DR in a fatty liver disease model.