Hepatocyte Nuclear Factors in Regenerating Liver

描述（由申请人提供）：哺乳动物肝脏是少数能够对损伤或三分之二部分肝切除术（其中诱导终末分化的肝细胞重新进入细胞周期并进行DNA复制和有丝分裂）做出完全自我再生反应的成年器官之一。分化的肝细胞的增殖反应由刺激立即早期转录因子的表达和核转位的生长因子和细胞因子的释放启动。然而，介导肝细胞进入S期和有丝分裂的转录机制还不完全清楚。在上一个资助期，我们开发了转基因小鼠，其中甲状腺素运载蛋白启动子的功能是过早表达叉头盒（福克斯）M1 B（以前称为HFH-11B）转录因子。这些TG小鼠的肝再生研究表明，过早的FoxM 1B表达通过早期诱导细胞周期调控基因加速肝细胞增殖的发生。此外，白蛋白增强子/启动子驱动的Cre重组酶（AIb-Cre）介导的出生后肝细胞中FoxM 1b fl/fl（LoxP靶向）等位基因缺失导致再生肝细胞DNA复制和有丝分裂显著减少。这与细胞周期蛋白依赖性激酶（Cdk）抑制剂p21 Cip 1蛋白水平升高和Cdc 25 B磷酸酶（Cdk 1活性和进入有丝分裂所需）表达检测不到有关。我们广泛的长期目标是确定Foxm 1b在介导再生肝细胞增殖和肝脏形态发生中的作用，并检查Foxm 1b是否是肝细胞癌发展所必需的。我们提出了以下三个具体目的：（1）验证缺失cdk抑制剂p21 Cip 1基因并恢复Cdc 25 B表达将增加再生AIb-Cre Foxm 1b-/-肝中肝细胞增殖的假设。我们已经证明，胚胎肝脏中Foxm 1b fl/fl等位基因的缺失会导致肝脏发育异常和胚胎死亡。(2)进一步表征Foxm 1b胚胎肝缺陷，并使用诱导型肝Cre重组酶在肝脏发育期间的不同时间删除Foxm 1b fl/fl等位基因，以确定需要Foxm 1b功能的肝脏发育阶段。(3)由于Foxm 1b对于再生肝细胞增殖是必不可少的，因此我们将检验AIb-Cre Foxm 1b-/-肝细胞对二乙基亚硝胺/苯巴比妥肝肿瘤诱导方案的反应难以发展为肝细胞癌的假设。

DESCRIPTION (provided by applicant): The mammalian liver is one of the few adult organs capable of completely regenerating itself in response to injury or two-thirds partial hepatectomy in which terminally differentiated hepatocytes are induced to reenter the cell cycle and undergo DNA replication and mitosis. The proliferative response of differentiated hepatocytes is initiated by the release of growth factors and cytokines that stimulate expression and nuclear translocation of immediate early transcription factors. However, the transcriptional mechanisms mediating hepatocyte entry into S-phase and mitosis are not completely understood. In the previous funding period, we developed transgenic mice in which the Transthyretin promoter functioned to prematurely express the Forkhead Box (Fox) M1B (previously called HFH-11B) transcription factor. Liver regeneration studies with these TG mice demonstrated that premature FoxM1B expression accelerates the onset of hepatocyte proliferation through earlier induction of cell cycle regulatory genes. Furthermore, albumin enhancer/promoter driven Cre recombinase (AIb-Cre) mediated deletion of the FoxM1b fl/fl (LoxP targeted) allele in postnatal hepatocytes resulted in significant reduction in regenerating hepatocyte DNA replication and mitosis. This was associated with increased levels of the cyclin dependent kinase (Cdk) inhibitor p21Cip1 protein and undetectable expression of Cdc25B phosphatase, which is required for Cdk1 activity and entry into mitosis. Our broad, long-term goals are to determine the role of Foxm1b in mediating regenerating hepatocyte proliferation and liver morphogenesis and examine whether Foxm1b is required for development of hepatocellular carcinoma. We propose the following three Specific Aims: (1) To test the hypothesis that deleting the cdk inhibitor p21Cip1 gene and restoring Cdc25B expression will increase hepatocyte proliferation in regenerating AIb-Cre Foxm1b -/- liver. We have shown that deletion of the Foxm1b fl/fl allele in the embryonic liver causes abnormal liver development and embryonic lethality. (2) To further characterize the Foxm1b embryonic liver defect and use of inducible hepatic Cre recombinase to delete the Foxm1b fl/fl allele at various times during liver development to determine the stages of liver development at which Foxm1b function is required. (3) Because Foxm1b is essential for regenerating hepatocyte proliferation, we will test the hypothesis that AIb-Cre Foxm1b -/- hepatocytes are refractory to developing hepatocellular carcinoma in response to a Diethylnitrosamine/Phenobarbital liver tumor induction protocol.