EXPRESSION, STRUCTURE AND FUNCTION OF FILAGGRIN

侧聚蛋白是表达于表皮颗粒层的主要结构蛋白。它是一种多蛋白前体，由10、11或12个聚丝蛋白重复序列组成，并由短连接序列连接。在终末分化过程中，这个前体被分裂成单独的聚丝蛋白分子，根据体外实验，它被认为参与了表皮完全分化、死亡、角化细胞层中角蛋白中间丝的聚集和特异性排列。在早期的研究中，我们已经探索了侧聚蛋白基因的表达调控，并表征了其近端启动子。AP1位点及其同源结合的c-fos和c-jun蛋白赋予该基因与邻近的Sp1、ets-like和NF-KB元件协同的角化细胞特异性表达。我们发现了一种新的ets转录蛋白，它似乎在调节表皮中几种晚期分化基因的表达中起作用，包括侧聚蛋白。我们现在正在研究一系列新的Oct转录因子在侧聚蛋白基因表达中的作用。Morasso及其合作者的研究已经确定了一种同源结构域转录因子，称为远端- 3 (Dlx3)，它在激活结构蛋白的表达方面起着核心作用，而结构蛋白是形成锥体层所必需的。在表皮发育过程中，Dlx3的表达仅限于小鼠层状表皮的分化（基底上）细胞，主要是颗粒细胞。通过使用基底特异性角蛋白5启动子异位驱动转基因小鼠基底细胞中Dlx3的表达，功能获得实验导致表皮表型严重异常，导致围产期死亡，因为无法形成功能性角化层，构成皮肤的水屏障并防止脱水。重要的是，转基因表皮中的基底细胞停止增殖，并表达晚期分化表皮标志物，如loricrin和聚丝蛋白。为了确定Dlx3在表皮中激活的信号通路，我们研究了体外培养的小鼠角质形成细胞。在模拟皮肤中存在的内源性Ca++梯度的情况下，通过将培养基中的Ca++浓度从0.05 mM增加到0.12 mM，可以诱导基底角化细胞在体外分化。Ca信号分化途径与蛋白激酶C （PKC）的激活有关。最近的数据表明PKC信号在表皮分化后期的作用，表明PKC的激活是晚期分化标志物loricrin和profilaggrin表达的必要条件。Dlx3启动子也会响应ca2 +的转移，这表明该区域包含响应ca2 +分化信号通路所需的调控元件。为了确定诱导Dlx3基因表达的因素，我们克隆了1.2 kb的小鼠基因近端区域，并分析了其顺式调控元件和潜在的反式作用因子。Dlx3基因近端具有典型的TATA盒和CCAAT盒，转录起始位点位于翻译起始位点上游205个碱基对处。序列缺失分析显示？84和？34在未分化和分化的原代小鼠角质形成细胞中均具有最大的启动子活性。凝胶阻滞试验和突变分析表明，转录调节因子NF-Y（也称为CBF）与该区域内的CCAAT盒基元结合，并负责大部分Dlx3启动子活性。此外，Sp1结合位点位于转录起始位点的上游，该位点作为Dlx3启动子的正调控元件，独立于CCAAT盒基序。重要的是，在角化细胞分化过程中，位于Dlx3基因+30至+60之间的元件负责Ca++依赖性诱导Dlx3。这些体外实验应该有助于阐明一系列事件，这些事件始于Ca信号，最终以与角化细胞分化过程相关的特定结构表皮基因的激活而结束。Dlx3在发育和分化过程中起转录激活剂的作用。转录因子的功能需要细胞核定位。在钙（Ca++）诱导分化的角质形成细胞中，检测了与Dlx3融合的绿色荧光蛋白（GFP）的细胞内定位。在基底和分化的角质形成细胞中，GFP-Dlx3定位于细胞核。在同源结构域的氨基端鉴定出一个二部核定位信号（NLS）。Dlx3二部NLS序列的插入将细胞质融合蛋白gfp -角蛋白14 （K14）定位到细胞核。这是首次在脊椎动物同源结构域蛋白中发现的两部分NLS之一，该蛋白被证明是核定位所必需和充分的。发现Dlx3 NLS序列不仅对核定位至关重要，而且对Dlx3的反激活电位、特异性DNA结合以及与Msx1蛋白的相互作用至关重要。在了解Dlx3的功能作用的平行方法中，对该基因进行了靶向删除，并对表型进行了分析。Dlx3的靶向缺失导致胚胎发育在9.5-10天左右停止，与胎盘发生适当形态发生的严重失败有关。由于胚胎死亡明显早于表皮分层发生，因此无法评估Dlx3功能丧失对表皮分化的影响（E15.5）。在大量导致胚胎发生早期死亡的基因靶向突变中，一些由于胎盘发育缺陷而导致的基因突变已经通过四倍体聚集实验（即Mash-2， Ets-2）得以挽救。通过四倍体聚集实验，修复了Dlx3 -/-胚胎的滋养细胞缺陷，并将零胚的存活时间延长至发育的第13.5天。这些结果促使我们开始了一个条件靶向Dlx3的项目，以便能够专门评估这种同源结构域转录因子在表皮分层中的作用。

Profilaggrin is a major structural protein expressed in the granular layer of the epidermis. It is a polyprotein precursor consisting 10, 11 or 12 filaggrin repeats which are adjoined by a short linker sequences. During terminal differentiation, this precursor is cleaved into individual filaggrin molecules, which based on in vitro experiments, are thought to be involved in the aggregation and specific alignment of the keratin intermediate filaments in the fully differentiated, dead, cornified cell layers of the epidermis. In earlier studies, we have explored the regulation of expression of the profilaggrin gene and have characterized its proximal promoter. An AP1 site and its cognate binding c-fos and c-jun proteins confer keratinocyte-specific expression to the gene, in concert with neighboring Sp1, ets-like, and NF-KB elements. We have discovered a novel ets transcription protein that seems to function in the regulation of expression of several late differentiation genes in the epidermis, including profilaggrin. We are now studying the role of a novel series of Oct transcription factors on the expression of the profilaggrin gene. Studies by Morasso and collaborators have identified a homeodomain transcription factor, known as Distal-less 3 (Dlx3), that plays a central role in activating the expression of structural proteins that are necessary for the formation of the cornified layer. During epidermal development, the expression of Dlx3 is restricted to the differentiated (suprabasal) cells, predominantly the granular cells, of the mouse stratified epidermis. Gain-of-function experiments by ectopically driving the expression of Dlx3 in the basal cells of transgenic mice using the basal-specific keratin 5 promoter, resulted in a severely abnormal epidermal phenotype leading to perinatal lethality because of the inability to form a functional cornified layer, that constitutes the water-barrier of the skin and prevents dehydration. Importantly, the basal cells in the transgenic epidermis ceased to proliferate and expressed late differentiation epidermal markers such as loricrin and filaggrin. In order to determine the signaling pathway by which Dlx3 is activated in the epidermis, we have studied mouse keratinocytes cultivated in vitro. Basal keratinocytes can be induced to differentiate in vitro by increasing the Ca++ concentration from 0.05 mM to 0.12 mM in the culture medium, in a situation that mimics the endogenous Ca++ gradient present in the skin. The Ca signaling differentiation pathway is associated with activation of protein kinase C (PKC). Recent data demonstrate a role of PKC signaling in the late stages of epidermal differentiation, showing that activation of PKC is necessary for expression of the late differentiation markers loricrin and profilaggrin. The Dlx3 promoter is also responsive to the Ca++ shift, suggesting that this region contains the regulatory elements required to respond to the Ca++ signaling pathway of differentiation. To determine the factors that induce Dlx3 gene expression, we have cloned the 1.2-kb proximal region of murine gene and analyzed its cis-regulatory elements and potential trans-acting factors. The proximal region of the Dlx3 gene has a canonical TATA box and CCAAT box, and the transcription start site was located 205 base pairs upstream from the initiation of translation site. Serial deletion analysis showed that the region between ?84 and ?34 confers the maximal promoter activity both in undifferentiated and differentiated primary mouse keratinocytes. Gel retardation assays and mutational analysis demonstrated that the transcriptional regulator NF-Y (also referred to as CBF) binds to a CCAAT box motif within this region and is responsible for the majority of the Dlx3 promoter activity. In addition, an Sp1 binding site was located immediately upstream of transcription start site that acts as a positive regulatory element of the Dlx3 promoter, independent of the CCAAT box motif. Importantly, elements residing between +30 to +60 of the Dlx3 gene are responsible for the Ca++-dependent induction of Dlx3 during keratinocyte differentiation. These in vitro assays should help elucidate the cascade of events that begin with the Ca signal and culminate with the activation of specific structural epidermal genes in events that are linked with the keratinocyte differentiation process. Dlx3 acts as a transcriptional activator during development and differentiation. Transcription factor function requires nuclear localization. The intracellular localization of the green fluorescent protein (GFP) fused to Dlx3 was examined in keratinocytes induced to differentiate by calcium (Ca++). In both basal and differentiated keratinocytes, GFP-Dlx3 localized to the nucleus. A bipartite nuclear localization signal (NLS) was identified at the amino terminus of the homeodomain. The insertion of the Dlx3 bipartite NLS sequence, localized the cytoplasmic fusion protein, GFP-keratin 14 (K14), to the nucleus. This is one of the first bipartite NLS identified in a vertebrate homeodomain protein shown to be necessary and sufficient for nuclear localization. Dlx3 NLS sequences were found to be critical not only for nuclear localization, but also were essential for Dlx3 transactivation potential, specific DNA binding, and interaction with Msx1 protein. In a parallel approach to understand the functional role of Dlx3, a targeted deletion of the gene was performed and the analysis of the phenotype has been submitted for publication. Targeted deletion of Dlx3 results in embryonic developmental arrest around day 9.5-10, associated with a gross failure of the placenta to undergo proper morphogenesis. It was not possible to assess the effects of Dlx3 loss of function on epidermal differentiation, since embryonic death occurs significantly earlier than the onset of epidermal stratification (E15.5). Of the large number of targeted mutagenesis of genes that lead to early death in embryogenesis, several of those due to defects in placenta development have been rescued by tetraploid aggregation experiments (i.e. Mash-2, Ets-2). Rescue of the trophoblast defect in the Dlx3 -/- embryos by tetraploid aggregation experiments has been performed, and extended the survival of the null embryos only to day 13.5 of development. These results have led us to commence a project for conditional targeting of Dlx3 to be able to specifically assess the role of this homeodomain transcription factor in epidermal stratification.