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，是皮肤最外层的一层，提供了抵御各种环境攻击的屏障。在一个持续不断的自我更新过程中，分裂的角质形成细胞离开基底层，随着它们迁移到死亡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，部分是由控制角质形成细胞中基因表达的转录因子来调节的。AP-2家族转录因子在哺乳动物表皮中高水平表达，调节多种表皮启动子，可能在皮肤表皮发育中发挥重要作用。我们的初步研究揭示了AP-2α和AP-2γ之间的功能相互作用，以及一种新的角质形成细胞特异性核因子Karp。我们发现，Karp可以将AP-2蛋白招募到存在于表皮特异性增强剂中的调节DNA序列中。我们推测AP-2α在胚胎皮肤中的分化标志物的表达变化表明，AP-2α在表皮分化中起作用。因此，我们将建立AP-2α缺失胚胎皮肤的原代角质形成细胞培养体系，这将为实验操作提供一个有用的系统。我们发现，AP-2蛋白的功能可以被靶向显性负值消除，它可以与多个家族成员同源和异源二聚体，从而使它们不能与DNA结合。这种显性负性将被用来阻断角质形成细胞中表达的所有AP-2蛋白的活性。 在目前的方案中，我们试图纯化和鉴定一种新的与AP-2相互作用的角质形成细胞特异性因子Karp蛋白(目标1)，利用AP-2α缺失的角质形成细胞作为手段来证实AP-2α是AP-2α-Karp复合体的重要组成部分，并证明AP-2αq单独或与Karp联合调节角质形成细胞的增殖和分化(目标2)，并利用AP-2的主要阴性形式在活体模型系统中评估AP-2在表皮发育和分化中的生物学作用(目标3)。我们的研究将为研究AP-2及其相互作用的伙伴如Karp控制上皮生长和分化的分子机制提供工具，并将确定这些过程的关键调节因子。这些研究的长期目标是确定控制表皮特异性和分化特异性基因表达的转录调控机制。这些知识将提供对表皮生物学的基本见解，并可能直接影响到为一系列困扰人类皮肤的疾病开发新的治疗方法。

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.