A defining feature of the mammalian liver is polyploidy, a numerical change in the entire complement of chromosomes. The first step of polyploidization involves cell division with failed cytokinesis. Although polyploidy is common, affecting ~90% of hepatocytes in mice and 50% in humans, the specialized role played by polyploid cells in liver homeostasis and disease remains poorly understood. The goal of this study was to identify novel signals that regulate polyploidization, and we focused on microRNAs (miRNAs). First, to test whether miRNAs could regulate hepatic polyploidy we examined livers from Dicer1 liver-specific knockout mice, which are devoid of mature miRNAs. Loss of miRNAs resulted in a 3-fold reduction in binucleate hepatocytes, indicating that miRNAs regulate polyploidization. Secondly, we surveyed age-dependent expression of miRNAs in wild-type mice and identified a subset of miRNAs, including miR-122, that is differentially expressed at 2–3 weeks, a period when extensive polyploidization occurs. Next, we examined Mir122 knockout mice and observed profound, life-long depletion of polyploid hepatocytes, proving that miR-122 is required for complete hepatic polyploidization. Moreover, the polyploidy defect in Mir122 knockout mice was ameliorated by adenovirus-mediated over-expression of miR-122, underscoring the critical role miR-122 plays in polyploidization. Finally, we identified direct targets of miR-122 (Cux1, Rhoa, Iqgap1, Mapre1, Nedd4l and Slc25a34) that regulate cytokinesis. Inhibition of each target induced cytokinesis failure and promoted hepatic binucleation. Conclusion Our data demonstrate that miR-122 is both necessary and sufficient in liver polyploidization. Among the different signals that have been associated with hepatic polyploidy, miR-122 is the first liver-specific signal identified. These studies will serve as the foundation for future work investigating miR-122 in liver maturation, homeostasis and disease.

Activation of Wnt/b-catenin signaling during liver regeneration (LR) after partial hepatectomy (PH) is observed in several species. However, how this pathway is turned off when hepatocyte proliferation is no longer required is unknown. We assessed LR in liver-specific knockouts of Wntless (Wls-LKO), a protein required for Wnt secretion from a cell. When subjected to PH, Wls-LKO showed prolongation of hepatocyte proliferation for up to 4 days compared with littermate controls. This coincided with increased b-catenineT-cell factor 4 interaction and cyclin-D1 expression. Wls-LKO showed decreased expression and secretion of inhibitory Wnt5a during LR. Wnt5a expression increased between 24 and 48 hours, and Frizzled-2 between 24 and 72 hours, after PH in normal mice. Treatment of primary mouse hepatocytes and liver tumor cells with Wnt5a led to a notable decrease in b-catenineT-cell factor activity, cyclin-D1 expression, and cell proliferation. Intriguingly, Wnt5a-LKO did not display any prolongation of LR because of compensation by other cells. In addition, Wnt5a-LKO hepatocytes failed to respond to exogenous Wnt5a treatment in culture because of a compensatory decrease in Frizzled-2 expression.In conclusion, we demonstrate Wnt5a to be, by default, a negative regulator of b-catenin signaling and hepatocyte proliferation, both in vitro and in vivo. We also provide evidence that the Wnt5a/Frizzled-2 axis suppresses b-catenin signaling in hepatocytes in an autocrine manner, thereby contributing to timely conclusion of the LR process. (Am J Pathol 2015, 185: 2194e2205; http:// dx

AlanGrant, Baee, Corofin, OrangeOswald, and Vincenzo are newly isolated phages of Mycobacterium smegmatis mc2155 discovered in Pittsburgh, Pennsylvania, USA. All five phages share nucleotide similarity with cluster B mycobacteriophages but span considerable diversity with Corofin and OrangeOswald in subcluster B3, AlanGrant and Vincenzo in subcluster B4, and Baee in subcluster B5.

Liver regeneration (LR) occurs when part of the liver is damaged or surgically removed by partial hepatectomy (PH). The organ responds by cell division to restore the lost mass. Determining the mechanism by which hepatocytes stop proliferating once LR is complete may provide insight into termination of LR. Several pathways can induce hepatocyte proliferation after PH, including β‐Catenin, which stimulates cyclin‐D1 expression. However, it is unclear how β‐catenin is inhibited once LR is accomplished. Using LR samples from different times after PH in mice, we identified Wnt‐5a increase at 24–72h post‐PH, only subsequent to β‐catenin activation. To determine its role, primary mouse hepatocytes were cultured in the presence of Wnt‐5a. A significant decrease in thymidine incorporation was observed with Wnt‐5a. Western blots using lysates from Wnt‐5a treated hepatocytes revealed a decrease in canonical Wnt pathway components and targets such as β‐catenin, cyclin D‐1, ser9‐GSK3β, and Frizzled‐4 levels, while an increase was detected in Ror2, an inhibitory Wnt receptor. Additionally, Wnt‐5a treated hepatoma cells showed decreased β‐catenin reporter activity. In conclusion, we have identified Wnt‐5a to inhibit β‐catenin activity in liver cells by inducing its degradation and may be terminating β‐catenin signaling following adequate hepatocyte proliferation during LR.