Transcriptional control of epidermal differentiation

描述（由申请人提供）： 角质形成细胞是表皮的主要细胞类型，表皮是皮肤的最外层，为各种环境攻击提供屏障。在一个持续的自我更新过程中，分裂的角质形成细胞离开基底层，并随着它们迁移到死鳞片的最外层而逐渐分化。表皮中的这些过程受到很好的调节，部分是通过控制角质形成细胞中基因表达的转录因子。AP-2家族转录因子在哺乳动物表皮中高水平表达，调节许多表皮启动子，因此可能在皮肤表皮发育中起重要作用。我们的初步研究发现了AP-2 α和AP-2 γ与一种新的角质形成细胞特异性核因子KARP之间的功能性相互作用。我们表明，KARP可以招募AP-2蛋白的监管DNA序列存在于表皮特异性增强子。我们假设AP-2 α在表皮分化中起作用，如AP-2 α胚胎皮肤中分化标志物表达改变所示。因此，我们将建立一个原代角质形成细胞培养系统从AP-2 α空胚胎皮肤，这将提供一个有用的系统，是经得起实验操作。我们表明，AP-2蛋白的功能可以被废除与有针对性的显性负，它可以同源和异源二聚体与多个家庭成员，从而使他们无法DNA结合。该显性阴性将用于阻断角质形成细胞中表达的所有AP-2蛋白的活性。 在本发明中，我们试图纯化和鉴定KARP蛋白，一种与AP-2相互作用的新的角质形成细胞特异性因子（目的1），利用AP-2 α无效的角质形成细胞作为手段来证实AP-2 α是AP-2 α-KARP复合物的重要组分，并显示AP-2 α q单独或与KARP联合调节角质形成细胞的增殖和分化（目的2），并采用AP-2的显性阴性形式来评估AP-2在体内模型系统中表皮发育和分化过程中的生物学作用（目的3）。我们的研究将提供工具来研究AP-2及其相互作用伙伴（如KARP）控制上皮生长和分化的分子机制，并将确定这些过程的关键调节剂。这些研究的长期目标是确定调控表皮特异性和分化特异性基因表达的转录控制机制。这些知识将为表皮生物学提供基本的见解，并可能对开发用于折磨人类皮肤的各种疾病的新疗法产生直接影响。

DESCRIPTION (provided by applicant): Keratinocytes are the principal cell-type of the epidermis, the outermost layer of the skin that provides a barrier against various environmental assaults. In a continual process of self-renewal, the dividing keratinocytes leave the basal layer and progressively differentiate as they migrate to the outermost layer of dead squames. These processes in the epidermis are well regulated, in part by transcription factors that control gene expression in the keratinocytes. The AP-2 family of transcription factors are expressed at high levels in mammalian epidermis, regulate many epidermal promoters and hence, may play an important role in skin epidermal development. Our preliminary studies have uncovered a functional interaction between AP-2alpha and AP-2gamma and a novel keratinocyte-specific nuclear factor KARP. We show that KARP can recruit AP-2 proteins to regulatory DNA sequences present in epidermal-specific enhancers. We hypothesize that AP-2alpha plays a role in the epidermal differentiation as shown by altered expression of differentiation markers in AP-2alpha embryonic skin. Hence, we will establish a primary keratinocyte cell culture system from AP-2alpha null embryonic skin, which will provide a useful system that is amenable to experimental manipulation. We show that the function of AP-2 proteins can be abrogated with a targeted dominant negative, which can homo-and-heterodimerize with multiple family members, thus rendering them incapable of DNA-binding. This dominant negative will be utilized to block the activity of all AP-2 proteins expressed in keratinocytes. In the present proposal we seek to purify and identify the KARP protein, a novel keratinocyte specific factor that interacts with AP-2 (Aim 1), utilize AP-2alpha null keratinocytes as means to confirm that AP-2alpha is an important component of the AP-2alpha-KARP complex and show that AP-2alphaq alone or in association with KARP regulates the proliferation and differentiation of keratinocytes (Aim 2), and employ a dominant negative form of AP-2 to assess the biological role of AP-2 during epidermal development and differentiation in an in vivo model system (Aim 3). Our studies will provide the tools to investigate the molecular mechanisms by which AP-2 and its interacting partners such as KARP control epithelial growth and differentiation and will identify key modulators of these processes. The long-term goal of these studies is to define the transcriptional control mechanisms that govern epidermal-specific and differentiation-specific gene expression. Such knowledge will provide fundamental insights into epidermal biology and is likely to have direct bearing on developing new therapies for a wide array of diseases that afflict the human skin.