In vivo RNAi screening identified MKK4 as a new therapeutic target for the improvement of liver regeneration

We established a unique system for performing direct in vivo RNAi screens to genetically dissect cellular signaling networks that regulate proliferation control of hepatocytes during liver damage and regeneration. By combining the well characterized FAH-/- mouse model with a transposon based vector system, we generated chimeric mouse livers. In these mouse livers each hepatocyte expresses FAH, a marker gene and a microRNA based shRNA. To such mouse livers any form of liver damaging protocol can be applied and the regenerative process can be studied. We conducted a first in vivo RNAi screen, using a focused shRNAmir library consisting of 631 constructs targeting 362 genes. Mouse livers were stably repopulated with the shRNAmir library and after repopulation mice were subjected to chronic CCl4 treatment to induce chronic liver damage. The representation of each shRNAmir in the pool, the chimeric mouse liver and after chronic liver damage was determined by deep sequencing analysis. ShRNAs targeting MKK4 showed strong enrichment during liver damage and regeneration. MKK4 belongs to the Map kinase family and is part of two stress kinase signaling pathways, the JNK as well as the p38 pathway. Functional in vivo validation experiments showed that stable knock down of MKK4 by different shRNAs can significantly increase the repopulation efficiency of mouse hepatocytes and also increases the regenerative capacity of chronically damaged mouse livers. In addition we could show that MKK4 suppression also confers an increased robustness of hepatocytes in a model of acute liver failure, resulting in better survival of these animals. Beyond this, primary hepatocytes with stable MKK4 knockdown showed prolonged survival in cell culture and these cells could even be passaged and used to repopulate FAH-/- mouse livers. Protein array and western blot analyses of MKK4 shRNAs versus control-shRNA expressing livers revealed a complex activation pattern of JNK- and p38-signaling downstream factors. Interestingly, the observed pattern does not merely represent an increased JNK over p38 signaling but our data rather suggest that complex cross signaling events between both pathways are involved. Also hepatocytes with stable MKK4 knock down already show higher levels of CyclinD1, suggesting a priming of the cells to leave the G0 state and entering the G1 state of the cell cycle. We developed a new screening platform for pinpointing new regulators of hepatocyte proliferation and liver regeneration. A first in vivo RNAi screen identified MKK4 as an interesting new therapeutic target structure for improving the regenerative capacity under acute and chronic liver damaging conditions.