The Keap1/Nrf2Signaling Pathway: Regulation Of Hepatocyte Cell Cycle Duirng Liver Regeneration

Introduction: Actin-binding Kelch-like ECH-associated protein1(Keap1) and aleucine zipper motif-containing transcription factor nuclear factor erythroid2-relatedfactor2(Nrf2) form a pathway combating a variety of xenobiotic and endobioticinsults. In the quiescent state, cytosolic Keap1, as an adaptor, tethers Nrf2for ubiquitinconjugation by a Cul3-containing E3ubiquitin ligase, targeting Nrf2for proteasomaldegradation. When oxidative stress occurs, Keap1, as a redox sensor, undergoes a con-formational change because of the modification of its ultrasensitive cysteine residues byoxidative stress-causing highly active molecules such as electrophiles. As a result, Nrf2escapes from ubiqui tin conjugation, translocates into the nucleus, and, in turn, trans-criptionally activates a group of genes encoding cytoprotective molecules, includingdetoxifying enzymes, GSH synthesis enzymes, and antioxidant proteins. These mole-cules coordinately combat oxidative stress, thus restoring cellular homeostasis.Subsequently, Keap1translocates into the nucleus and escorts nuclear export of Nrf2forcontinuous proteasomal degradation in the cytoplasm, thus terminating Nrf2activation.Moreover, Keap1can interact with other proteins and Nrf2activity can be regulated attran-scriptional level or by phosphorylation, suggesting their independent regulationand roles in different biological or pathological processes.It has been shown that Nrf2deficiency causes impairment of liver regenerationdue to oxidative stress-mediated insulin/insulin-like growth factor resistance and dimi-nished Notch1signaling. Those few studies indicate that Nrf2participates in regulatinghepatocyte proliferation during liver regeneration. However, how the Keap1/Nrf2signaling pathway controls hepatocyte replication has never been precisely and sys-tematically investigated. Object: The aim of our study was to gain further insights into the roles of theKeap1/Nrf2signaling pathway in regulating liver regeneration, with a particular focuson determining whether and how Keap1and Nrf2modulate the cell cycle of rege-nerating hepatocytes.Methods: Wild-type and Keap1knockdown mice and wild-type and Nrf2-null micewere subjected to2/3partial hepatectomy (PH), and Various experimental endpointswill be analyzed including hepatocyte cell cycle progression, the expression of the cellcycle components, and the functional states of mitogenic signaling molecules.Results: We found that Keap1knockdown did not affect the overall hepatic regrowth inresponse to liver resection but resulted in a delay in S phase entry, disruption of S phaseprogression, and loss of mitotic rhythm of replicating hepatocytes. These defects causedby Keap1knockdown are associated with decreased phosphorylation of c-Met andEGFR, aberrant regulation of Akt1and p70SP6K activities, and abnormal expression ofCyclin s A2and B1in regenerating livers. Astonishingly, we found that wild-typeregenerating livers exhibited a surprisingly strong wave of redox fluctuation at the G1/Sboundary, followed by two additional progressively reduced waves in the S phase,without Nrf2activation, during the first round of hepatocyte replication following PH.Keap1knockdown caused severely disrupted hepatic redox cycle, which was closelycorrelated with dysregulated hepatocyte cell cycle, in regenerating livers.Nrf2-null mice exhibited sluggish liver regrowth, although the lost liver mass waseventually restored seven days after PH. During the first wave of hepatocyte replicationfollowing PH, Nrf2deficiency caused a delay in hepatocyte mitosis due to excessiveaccumulation of Cyclin A2protein and dysregulation of the Wee1/Cdc2/Cyclin B1pathway in regenerating livers. Most strikingly, at60h post-PH, the vast majority ofhepatocytes lacking Nrf2reduced their sizes, activated hepatic progenitor markers(CD133, TWEAK receptor, and trefoil factor family3), and downregulated the expression of a group of genes critical for their functions, which was accompanied byp70S6K inactivation. Thus, Nrf2-null hepatocytes were undergoing transient butmassive dedifferentiation in response to liver mass loss.Conclusion: We demonstrated that the Keap1/Nrf2signaling pathway is very importantin regulating liver regeneration. The proliferating hepatocytes require preciselyregulated Keap1expression to enter and progress through the S phase and exhibitmitotic rhythm during liver regeneration. Nrf2is required for timely M phase entry ofreplicating hepatocytes and for the maintenance of newly regenerated hepatocytes in afully differentiated state.