MAP kinases regulate involucrin gene expression

描述(申请人提供)：Involucrin是角质形成细胞角化包膜的重要前体。在表皮中，总蛋白和mRNA的表达是角质形成细胞特异性的，并且依赖于分化(即，表达局限于上基底层)。我们的转基因小鼠和细胞培养研究表明，人类总蛋白(HINV)基因上游调控区足以进行这种表达，该区域的特定DNA元件结合API、C/EBP和SPL转录因子来驱动表达。在过去的资金支持期间，我们发现了一个新的PKC、RAS、MEKK1、MEK3和p388-Erk1/2信号级联，它调节API、SPL和C/EBP转录因子的活性和水平，以增加总蛋白基因的表达。尽管识别这一级联反应代表着我们在理解与分化相关的基因调控方面向前迈进了一大步，但我们对这一信号转导级联反应的理解还远未完成。在确定了这一途径的重要性后，我们现在提出旨在扩大这些发现的实验。我们的研究表明，在正常角质形成细胞中，包括PKC5在内的新的PKC亚型是hINV基因表达的主要激活剂，分化因子可以触发PKC8的酪氨酸磷酸化来调节基因的表达。然而，PKC8酪氨酸磷酸化在角质形成细胞中的重要性是复杂的，有争议的，并且还没有得到彻底的研究。我们的第一个主要目标是确定PKC8的酪氨酸磷酸化对PKC8的活性、亚细胞定位和调节下游信号事件的能力的作用。一个重要的观察结果是，正常人角质形成细胞的信号转导在p388上的MAPK水平汇聚。我们的初步研究表明，MEK3和MEK6与P38S的不同相互作用可能调节角质形成细胞分化和凋亡之间的平衡。这项研究的第二个主要目标是确定这些激酶在调节分化和凋亡之间的平衡中所起的作用。特别是，我们将重点介绍PKC8、MEK3和p388在调节角质形成细胞分化中的作用。这项建议的总体目标是扩大我们对驱动角质形成细胞分化的分子机制的了解。这项工作尤其重要，因为这个丝裂原活化蛋白激酶(MAPK)信号级联被认为参与调节角质形成细胞的生存、分化、死亡和转化。人们希望，更好地了解这一级联反应将导致新的有效治疗表皮疾病的方法。

DESCRIPTION (provided by applicant): Involucrin is an important precursor of the keratinocyte cornified envelope. In epidermis, expression of involucrin protein and mRNA is keratinocyte-specific and differentiation-dependent (i.e., expression is confined to the suprabasal layers). Our transgenic mouse and cell culture studies show that the human involucrin (hINV) gene upstream regulatory region is sufficient for this expression and that specific DNA elements in this region bind API, C/EBP and Spl transcription factors to drive expression. During the past funding period we identified a novel PKC, Ras, MEKK1, MEK3 and p388-ERKl/2 signaling cascade that regulates the activity and level of API, Spl and C/EBP transcription factors to increase involucrin gene expression. Although identifying this cascade represents a major step forward in our understanding of differentiation associated gene regulation, our understanding of this signal transduction cascade is far from complete. Having established the importance of this pathway, we now propose experiments designed to extend these findings. Our studies show that novel PKC isoforms, including PKC5, are the major activators of hINV gene expression in normal keratinocytes and that differentiation agents trigger tyrosine phosphorylation of PKC8 to regulate gene expression. However, the importance of PKC8 tyrosine phosphorylation in keratinocytes is complicated, controversial, and has not been thoroughly studied. Our first major goal is to identify the role of tyrosine phosphorylation of PKC8 on PKC8 activity, subcellular localization, and ability to regulate downstream signaling events. An important observation is that signal transduction in normal human keratinocytes converges at the MAPK level on p388. Our preliminary studies suggest that differential interaction of MEK3 and MEK6 with p38S may function to regulate the balance between keratinocyte differentiation and apoptosis. The second major goal of this study is to characterize the role of these kinases in regulating the balance between differentiation and apoptosis. In particular, we will focus on the role of PKC8, MEK3 and p388 in regulating keratinocyte differentiation. The overall goal of this proposal is to expand our knowledge regarding the molecular mechanisms that drive differentiation in keratinocytes. This work is particularly important, as this mitogen-activated protein kinase (MAPK) signaling cascade has been implicated in regulating keratinocyte survival, differentiation, death, and transformation. It is hoped that a better understand of this cascade will lead to new and effective therapies for epidermal disease.