MAP kinases regulate involucrin gene expression

外膜蛋白是角质形成细胞外膜的重要前体。在表皮中，总苞蛋白的表达 蛋白质和mRNA是角质形成细胞特异性的和分化依赖性的（即，表达式仅限于 基底上层）。我们的转基因小鼠和细胞培养研究表明，人类外皮蛋白（hINV）基因 上游调控区足以用于该表达，且该区域中的特异性DNA元件结合API， C/EBP和Spl转录因子来驱动表达。在过去的资助期间，我们确定了一个新的PKC， Ras、MEKK 1、MEK 3和p388-ERK 1/2信号级联，其调节API、Sp1和 C/EBP转录因子增加外皮蛋白基因表达。虽然识别这种级联代表了一种 我们对分化相关基因调控的理解向前迈出了一大步，我们对这种信号的理解 转导级联还远未完成。在确定了这一途径的重要性之后，我们现在提出 旨在扩展这些发现的实验。我们的研究表明，新的PKC亚型，包括PKC 5，是 正常角质形成细胞中hINV基因表达的主要激活剂和分化剂触发酪氨酸 PKC 8磷酸化以调节基因表达。然而，PKC 8酪氨酸磷酸化的重要性， 角质形成细胞是复杂的、有争议的，并且尚未被彻底研究。我们的第一个主要目标是 PKC 8酪氨酸磷酸化对PKC 8活性、亚细胞定位和调控能力作用 下游信号事件。一个重要的观察是正常人角质形成细胞中的信号转导 在p388的MAPK水平上聚合。我们的初步研究表明，不同的相互作用MEK 3和 MEK 6与p38 S可能起调节角质形成细胞分化与凋亡之间的平衡的作用。的 本研究的第二个主要目标是描述这些激酶在调节 分化和凋亡。特别是，我们将重点关注PKC 8，MEK 3和p388在调节细胞凋亡中的作用。 角质形成细胞分化这项提案的总体目标是扩大我们对分子生物学的认识， 驱动角质形成细胞分化的机制。这项工作特别重要，因为这种丝裂原激活 蛋白激酶（MAPK）信号级联与调节角质形成细胞存活，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和有效的治疗方法 治疗表皮疾病

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 bindAPI, C/EBP and Spl transcription factors to drive expression. During the past funding period we identified a novelPKC, 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.