Aging Features in Mice with Conditional Expression of p16Ink4a

描述(申请人提供)：衰老是一个复杂的过程，伴随着大分子损伤的逐渐积累和组织再生的丧失。很难梳理出这些因素的相对贡献。P16INK4a(P16)是一种特异性的细胞周期抑制因子和肿瘤抑制因子，随着年龄的增长而积聚，已被认为是衰老的生物标志物。在组织培养中，p16与许多正常细胞类型的复制衰老有关，我们已经证明，p16的诱导足以在培养的肿瘤细胞系中施加衰老。对p16缺失小鼠的遗传分析表明，p16限制了老年小鼠几个组织中祖细胞的增殖。然而，无论p16 表达是否足以导致体内正常细胞停滞或衰老或施加衰老特征尚不清楚。令人惊讶的是，p16‘S靶向细胞周期蛋白依赖性激酶(CDK)在小鼠体内都不是必需的。为了解决这些问题，我们产生了转基因小鼠，在其中p16的条件表达通过抗生素多西环素(Dox)的处理实现。这个简单的系统可以对细胞增殖进行前所未有的控制。诱导的p16与CDK4结合，抑制包括Lgr5+肠干细胞在内的肠上皮细胞的增殖。对幼鼠的广泛诱导会造成早期衰老表型，特征是毛发色素减少和脱落、体重减轻、后凸和贫血。在目标1中，我们建议研究选择性p16表达对三种不同组织--胰岛、皮肤和肠道--的衰老参数和细胞增殖的影响。这些组织被选为与AG相关的表型、内源性p16在年龄相关功能障碍中作用的证据、已建立的用于Dox敏感转基因诱导的反式激活物系以及经遗传验证的干细胞组织学标记。在目标2中，我们将测试p16强加的衰老特征和细胞周期停滞是否可逆。这些研究将证实，细胞增殖丧失S足以导致衰老表型，挑战需要大分子损伤的范式。此外，这项工作将探索这种衰老特征的基本可逆性，同时定义它们与干细胞/祖细胞停滞和衰老的关系。 公共卫生相关性：这项建议在小鼠模型中检验了肿瘤抑制因子和细胞周期抑制因子p16INK4a是衰老和与年龄相关的器官功能障碍的效应因子的假设。我们建议测试p16介导的细胞周期抑制是否足以强加衰老的特征特征，是否涉及组织干细胞增殖的缺陷，以及这些特征是否从根本上是可逆的。

DESCRIPTION (provided by applicant): Aging is a complex process associated with gradual accumulation of macromolecular damage and loss of tissue regeneration. It has been difficult to tease out the relative contributions of these factors. p16Ink4a (p16) is a specific cell cycle inhibitor and tumor suppressor that accumulates with age and has been proposed as a biomarker of aging. p16 has been implicated in replicative senescence of many normal cell types in tissue culture, and we have shown that induction of p16 is sufficient to impose senescence in a cultured tumor cell line. Genetic analyses using p16-null mice revealed that p16 limits proliferation of progenitor cells in several tissues of older mice. However, whether p16 expression is sufficient to cause normal cells in vivo to arrest or senesce or to impose aging features is unknown. Surprisingly, none of p16's target Cyclin dependent kinases (Cdks) is essential in the mouse. To address these issues, we generated transgenic mice in which facile conditional expression of p16 is achieved by treatment with the antibiotic doxycycline (Dox). This simple system allows unprecedented control over cell proliferation. Induced p16 binds Cdk4 and inhibits proliferation of intestinal epithelial cells, including Lgr5+ intestinal stem cels. Broad induction in young mice imposes early aging phenotypes characterized by hair hypopigmentation and loss, weight loss, kyphosis, and anemia. We propose in Aim 1 to examine effects of selective p16 expression on aging parameters and cell proliferation in three different tissues--pancreatic islets, skin, and intestine. These tissues were selected for their ag-related phenotypes, evidence for roles for endogenous p16 in age-related dysfunction, established transactivator lines for Dox-sensitive transgene induction, and genetically validated histological markers of stem cells. In Aim 2, we will test whether the p16-imposed aging features and cell cycle arrest are reversible. These studies will establish that loss of cell proliferation s sufficient to confer aging phenotypes, challenging the paradigm that macromolecular damage is needed. Further, this work will explore the fundamental reversibility of such aging features while defining their relationship to stem/progenitor cell arrest and senescence. PUBLIC HEALTH RELEVANCE: This proposal examines in mouse models the hypothesis that the tumor suppressor and cell cycle inhibitor p16Ink4a is an effector of aging and age-related organ dysfunction. We propose to test whether p16-mediated cell cycle inhibition is sufficient to impose signature features of aging, whether defects in tissue stem cell proliferation are involved, and whether these features are fundamentally reversible.