Introduction During chronic liver disease the liver can be regenerated from a population of endogenous hepatic progenitor cells (HPCs). These cells are known to be bipotent and can regenerate both hepatocytes and cholangiocytes, however the mechanisms by which these lineages are delineated from HPCs is currently unknown. Here we describe how the Notch and Wnt signalling pathways are differentially regulated and interact via NUMB to define cholangiocytes and hepatocytes from HPCs.
Results We have found that during biliary regeneration in primary sclerosing cholangitis and primary biliary cirrhosis, as well as mouse models of such, the Notch pathway is activated as demonstrated through nuclear Notch-1 in HPCs. This Notch mechanism is mediated through interaction of Jagged-1 supplied by a transient myofibroblast niche in mouse, or autonomous cell signalling in established human disease. In vitro when murine HPCs are co-cultured with myofibroblasts y-secretase inhibition significantly reduced expression of the Notch effectors HeyL (p<0.001 3.133±0.32 vs 0.98±0.09) and Hey1 (p<0.001 1.67±0.34 vs 0.08±0.01); associated with this reduction we found a significant reduction in expression of biliary genes HNF6 (p<0.001 77.94±12.52 vs 14.97±4.57) and GGT (p<0.01 6082±757.70 vs 3169±449.0). In vivo administration of DAPT during murine biliary regeneration demonstrates a significant reduction of cholangiocyte numbers (p<0.05 957.7±114.2 vs 1787±266) and also a reduction in pro-biliary genes including HNF6 and HNF1β. NUMB, a negative regulator of Notch signalling is lost during murine biliary regeneration (p<0.01; 1.19±0.07 vs 0.10±0.02) as well as during PSC/PBC (p<0.01; 8748±3090 vs 1814±277). During regeneration of hepatocytes from HPCs in HCV and in murine models NUMB levels remain high and Notch signalling is suppressed. During murine hepatocyte regeneration NUMB is maintained via macrophage derived Wnt. In vitro activation of the Wnt pathway in HPCs resulted in a twofold induction of NUMB and 35-fold induction of HNF4, without significantly affecting HNF1β. We ablated macrophages using liposomal clodronate this resulted in a conversion of hepatocyte HPCs into biliary HPCs which formed luminal structures with membranous b-catenin, lost expression of Wnt pathway target Axin2, NUMB and HNF4 as well as inducing expression of Hes-1, HNF1β and HNF6.
Conclusion During regeneration of the adult liver, Notch signalling is required for biliary specification however is actively sequestered via NUMB during regeneration of hepatocytes. This hepatocyte phenotype is induced through signalling via the Wnt pathway, removal of which results in a biliary phenotype during hepatocyte regeneration. We also describe how this mechanism is conserved from mouse models to human disease, where during biliary diseases such as PSC and PBC the Notch pathway is activated, however is actively restricted during regeneration of in hepatocytes during HCV.
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