Embryonic lethality and fetal liver apoptosis in mice lacking all three small Maf proteins

Embryogenesis is a period during which cells are exposed to dynamic changes of various intracellular and extracellular stresses. Oxidative stress response genes are regulated by heterodimers composed of Cap'n'Collar (CNC) and small Maf proteins (small Mafs) that bind to antioxidant response elements (ARE). Whereas CNC factors have been shown to contribute to the expression of ARE-dependent cytoprotective genes during embryogenesis, the specific contribution of small Maf proteins to such gene regulation remains to be fully examined. To delineate the small Maf function in vivo, in this study we examined mice lacking all three small Mafs (MafF, MafG, and MafK). The small Maf triple-knockout mice developed normally until embryonic day 9.5 (E9.5). Thereafter, however, the triple-knockout embryos showed severe growth retardation and liver hypoplasia, and the embryos died around E13.5. ARE-dependent cytoprotective genes were expressed normally in E10.5 triple-knockout embryos, but the expression was significantly reduced in the livers of E13.5 mutant embryos. Importantly, the embryonic lethality could be completely rescued by transgenic expression of exogenous MafG under MafG gene regulatory control. These results thus demonstrate that small Maf proteins are indispensable for embryonic development after E9.5, especially for liver development, but early embryonic development does not require small Mafs.     

MafT, a new member of the small Maf protein family in zebrafish

Small Maf proteins play critical roles on morphogenesis and homeostasis through associating with CNC proteins. To date, three small Maf proteins, MafF, MafG, and MafK, have been reported in vertebrates, which share redundant functions. In this study, we tried to identify and characterize small Maf proteins in zebrafish to elucidate their conservation and diversity in the fish kingdom. We identified homolog genes of MafG and MafK but not MafF in zebrafish, indicating the former two are conserved among vertebrates. In addition, a novel type of small Maf protein MafT was identified. MafT protein bound MARE sequence as a homodimer or heterodimers with zebrafish Nrf2 or p45 Nfe2. Co-overexpression of MafT and Nrf2 synergistically activated MARE-mediated gene expression in zebrafish embryos. These results indicated that MafT is a new member of small Maf proteins and involved in the Nrf2-dependent gene regulation in cellular defense system.     

Nrf2 Enhances Cholangiocyte Expansion in Pten-Deficient Livers

Keap1-Nrf2 system plays a central role in the stress response. While Keap1 ubiquitinates Nrf2 for degradation under unstressed conditions, this Keap1 activity is abrogated in response to oxidative or electrophilic stresses, leading to Nrf2 stabilization and coordinated activation of cytoprotective genes. We recently found that nuclear accumulation of Nrf2 is significantly increased by simultaneous deletion of Pten and Keap1, resulting in the stronger activation of Nrf2 target genes. To clarify the impact of the cross talk between the Keap1-Nrf2 and Pten–phosphatidylinositide 3-kinase–Akt pathways on the liver pathophysiology, in this study we have conducted closer analysis of liver-specific Pten::Keap1 double-mutant mice (Pten::Keap1-Alb mice). The Pten::Keap1-Alb mice were lethal by 1 month after birth and displayed severe hepatomegaly with abnormal expansion of ductal structures comprising cholangiocytes in a Nrf2-dependent manner. Long-term observation of Pten::Keap1-Alb::Nrf2^+/− mice revealed that the Nrf2-heterozygous mice survived beyond 1 month but developed polycystic liver fibrosis by 6 months. Gsk3 directing the Keap1-independent degradation of Nrf2 was heavily phosphorylated and consequently inactivated by the double deletion of Pten and Keap1 genes. Thus, liver-specific disruption of Keap1 and Pten augments Nrf2 activity through inactivation of Keap1-dependent and -independent degradation of Nrf2 and establishes the Nrf2-dependent molecular network promoting the hepatomegaly and cholangiocyte expansion.