IKKbeta:Bi-Functional Regulator of Hepatocyte Proliferation

描述（由申请人提供）：本提案的长期目标是旨在了解肝脏再生和肝癌发生的机制。原癌基因周期蛋白D1是一种主要的细胞周期蛋白，是正常成人肝细胞增殖所必需的。在肝细胞中，已经发现了许多调节cyclin D1表达的细胞内趋同通路，但没有IKK¿是其中之一的报道。IKK激酶是激活转录因子NF-KB和预防肝细胞凋亡所必需的，肝细胞凋亡是由细胞结合而不是由循环TNFa介导的。新的研究表明，在70%的部分肝切除术后，小鼠肝脏再生开始更快，并且在携带靶向肝细胞特异性IKK¿缺失的小鼠中，二乙基亚硝胺诱导的肝细胞癌（HCC）发生率更高。相比之下，成年小鼠的肝脏再生变得迟钝，在整个肝脏（包括其造血细胞成分）携带细胞IKKB缺失的小鼠中，HCC的发生率降低。现有证据进一步表明，在正常肝再生（对70%部分肝切除术的反应）和化学肝癌发生的小鼠模型中，正常肝细胞-而不是肝干细胞-是代偿性增殖和HCC的前体。因此，生长调节系统的变化增加了肝细胞增殖的可能性，很可能使肝细胞对生长因子和致癌转化更敏感。因此，由于它们与化学性肝癌发生的关系，本提案将重点研究正常肝细胞中与IKK¿相关的肝细胞生长改变。具体来说，观察结果表明IKK¿是成人肝细胞增殖的双功能调节剂：直接在肝细胞内；间接地在非实质肝细胞内。这些观察结果暗示了肝细胞和非实质肝细胞IKK¿的肿瘤抑制和肿瘤促进作用。细胞和分子生物学的标准程序，小鼠遗传学，以及来自正常小鼠和基因敲除小鼠的特定菌株的组织和细胞的分析，将用于在成年肝细胞中研究这些预测。因此，三个具体目标将测试以下假设：1]IKK¿是成人肝细胞增殖的“被动”负调节因子；2] IKK¿在非实质肝细胞中是必需的，以提供刺激成人肝细胞增殖的生长因子；[3]肝细胞IKKI2，缺失通过增加JNK1和/或降低JNK2的表达，延长c-JUN的表达，增强AP-1的激活，刺激cyclin D1的早发性表达，促进生长刺激的肝细胞提前进入s期。减弱肝细胞IKK¿和JNK2的共调控表达可能促进肝细胞增殖并增加肝细胞对癌变的易感性。

DESCRIPTION (provided by applicant): The long-term goals of this proposal are directed towards understanding mechanisms of liver regeneration and hepatocarcinogenesis. Proto-oncogene cyclin D1 is a major cell-cycle protein that is necessary for normal adult hepatocyte proliferation. Many convergent intracellular pathways have been identified that regulate cyclin D1 expression in hepatocytes, but there are no reports that IKK¿ is among them. IKK¿ kinase is required for activation of transcription factor NF-KB and for the prevention of hepatocyte apoptosis mediated by cell-bound but not by circulating TNFa. New investigations have revealed that following 70% partial hepatectomy, mouse liver regeneration starts faster and the diethylnitrosamine- induced incidence of hepatocellular carcinoma (HCC) is higher in mice carrying targeted hepatocyte-specific IKK¿ deletions. In contrast, adult mouse liver regeneration is blunted and the incidence of HCC is attenuated in mice carrying cellular IKKB deletions throughout the liver including its hematopoietic cellular components. Available evidence further suggests that, in this mouse model of normal liver regeneration (in response to 70% partial hepatectomy) and chemical hepatocarcinogenesis, normal hepatocytes - not liver stem cells -- are precursors of compensatory proliferation and HCC. Thus, changes in growth regulatory systems that increase the probabilities of hepatocyte proliferation may well make such hepatocytes more sensitive to growth factors and to carcinogenic transformation. Therefore, owing to their relationship to chemical hepatocarcinogenesis, this proposal will focus on hepatocyte growth alterations related to IKK¿ in normal hepatocytes. Specifically, the observations suggest that IKK¿ is a bi-functional regulator of adult hepatocyte proliferation: directly inside hepatocytes; and, indirectly inside non-parenchymal liver cells. Such observations implicate tumor suppressor and tumor promoter roles for hepatocyte and non-parenchymal liver cell IKK¿. Standard procedures of cellular and molecular biology, mouse genetics, and analyses of tissues and cells derived from specific strains of normal and knockout mice, will be used to investigate these predictions in adult liver cells. Accordingly, three specific aims will test the following hypotheses: 1] IKK¿ is a 'passive' negative regulator of adult hepatocyte proliferation; 2] IKK¿ is required in nonparenchymal liver cells for provision of growth factors that stimulate adult hepatocyte proliferation; and, 3] Hepatocyte IKKI2, deletion facilitates precocious S-phase entry in growth-stimulated hepatocytes by increasing JNK1 and/or reducing JNK2 expression which prolong c-JUN expression, enhance AP-1 activation and stimulate early onset expression of cyclin D1. Attenuated co-regulated expression of hepatocyte IKK¿ and JNK2 may both facilitate hepatocyte proliferation and increase the susceptibility of hepatocytes to carcinogenesis.