Nrf2 Deficiency Causes Transient but Massive Hepatocyte Dedifferentiation during Liver Regeneration
Yuhong Zou, Joonyong Lee, Shashank M. Nambiar, Min Hu, Qi Bao, Guoli Dai
Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN
Nuclear factor erythroid 2-related factor 2 (Nrf2), a basic leucine zipper transcription factor, is a central regulator of cellular defense responses against xenobiotic insults, oxidative stress, and inflammation. Several studies indicate important roles for Nrf2 in modulating liver injury and repair. To gain insights into how Nrf2 participates in regulating liver regeneration, wild-type and Nrf2-null mice were subjected to 2/3 partial hepatectomy (PH) and were sacrificed at multiple time points after surgery for various analyses. Nrf2-null mice exhibited sluggish liver regrowth, although the lost liver mass was eventually restored 7 days after PH. Nrf2 deficiency did not affect the number of hepatocytes entering the cell cycle, but delayed hepatocyte mitosis due to excessive accumulation of Cyclin A2 protein and reduced activity of Wee1/Cdc2/Cyclin B1 pathway in regenerating livers. Both strains of mice showed an overall trend of progressively increased hepatocyte hypertrophy during the entire course of liver regeneration. However, at 60 h after PH, hepatocytes lacking Nrf2 had markedly reduced cell sizes and condensed nuclei, accompanied by severe liver regrowth retardation. Moreover, DNA microarray analysis revealed three hepatic progenitor marker genes which were dramatically upregulated at the time point due to Nrf2 absence. They are CD133, tumor necrosis factor-like weak inducer of apoptosis (TWEAK) receptor (Fn14), and trefoil factor family 3 (TFF3). Verification at protein level by western blotting showed that hepatic CD133, Fn14, and TFF3 proteins were drastically activated at 60 h after PH in Nrf2-null, but not wild-type, mice during the entire course of liver regeneration. Immunohistochemically, almost all hepatocytes lacking Nrf2 expressed CD133 evenly in their cytosol, Fn14 on their surface, and TFF3 cytosolically concentrated as aggregates. Thus, the vast majority of hepatocytes deficient in Nrf2 exhibited a phenotype of CD133+/Fn14+/TFF3+. Reduction in cell size and activation of the three hepatic progenitor markers indicate that Nrf2-null hepatocytes were undergoing transient but massive dedifferentiation. Taken together, we demonstrate that Nrf2 is required for timely M phase entry of replicating hepatocytes by ensuring proper regulation of Cyclin A2 and Wee1/Cdc2/Cyclin B1 pathway. Most remarkably, we, for the first time, discovered a phenomenon of massive hepatocyte dedifferentiation in vivo in a pathological condition (Nrf2 absence and liver injury). The finding indicates a role for Nrf2 in maintaining hepatocyte identity during liver regeneration.
The following people have nothing to disclose: Yuhong Zou, Joonyong Lee, Shashank M. Nambiar, Min Hu, Qi Bao, Guoli Dai