MAP KINASES REGULATE INVOLUCRIN GENE EXPRESSION

分化依赖性基因表达是经历分化的生物系统/细胞类型的标志。因此，了解正常和异常的角质形成细胞分化，需要了解调节基因表达的机制。这包括识别启动调节的配体，理解将调节从细胞表面转移到细胞核的事件，并描述调节靶基因的核机制。我们已经开发了人类involucrin（hINV）基因作为研究分化依赖性基因表达的先进模型。作为这项工作的一部分，我们已经确定了转录因子（junB，junD，Fra-1），通过结合到hINV基因上游调控区内的特异性DNA结合位点来调节hINV基因的表达。这些核事件的知识使得有可能开始检查将基因调节信号从细胞表面转移到核中的这些转录因子的信号转导级联（Effimova et al.，J.Biol.Chem.273：24387，1998）。我们开始这项研究，因为尽管这些信号级联在其他系统中的重要性，很少有人知道有关的步骤，转移调节信息到细胞核的角质形成细胞的基因，在分化过程中受到调节。这项已发表的研究启动了一项全面的努力，以了解分化调节基因的信号转导，并提供了本提案中概述的实验的基本原理。我们的研究涉及一个丝裂原活化蛋白激酶（MAPK）信号级联，涉及蛋白激酶-c、Ras、MEKK 1、MEK 3/MEK 6、p38和AP 1家族成员（junB、junD、Fra-1）。这种级联反应显然很重要，因为抑制这些激酶会关闭hINV基因表达。这些研究也是创新性的，因为p38 MAPK不被认为在分化过程中起基因表达调节剂的作用。然而，需要学习的东西很多，在本建议概述的研究中，我们将侧重于我们知识有限的这一级联中的关键点。我们将（i）鉴定参与这种调节的PKC亚型，（ii）研究MEK 3和MEK 6作为p38活性调节剂的作用，并确定哪些p38亚型参与这种调节，（iii）进行旨在确定p38如何调节AP 1转录因子分布、水平和激活状态的研究，（iv）评估这些激酶在体内的作用，使用DNA弹道（基因枪）递送到小鼠表皮。这些研究旨在为表皮中分化依赖性基因表达的调控提供新的见解。由于外皮蛋白是分化过程中调控的许多基因之一，我们希望并期望这些研究提供的理解将有助于其他研究人员。

Differentiation-dependent gene expression is a hallmark of biological systems/cell types that undergo differentiation. Thus, understanding normal and abnormal keratinocyte differentiation, requires an understanding of the mechanisms that regulate gene expression. This includes identifying ligands that initiate the regulation, understanding events that transfer the regulation from the cell surface to the nucleus, and describe nuclear mechanisms that regulate target genes. We have developed the human involucrin (hINV) gene as an advanced model for the study of differentiation-dependent gene expression. As part of this effort, we have identified transcription factors (junB, junD, Fra-1) that regulate hINV gene expression by binding to specific DNA binding sites within the hINV gene upstream regulatory region. Knowledge of these nuclear events has made it possible to begin examining the signal transduction cascade that transfers the gene regulatory signal from the cell surface to these transcription factors in the nucleus (Effimova et al., J. Biol. Chem. 273:24387, 1998). We initiated this study because in spite of the importance of these signaling cascades in other systems, little is known regarding the steps that transfer regulatory information to the nucleus for keratinocyte genes that are regulated during differentiation. This published study initiates a comprehensive effort to understand signal transduction for a differentiation-regulated gene, and provides the rationale for the experiments outlined in this proposal. Our studies implicate a mitogen-activated protein kinase (MAPK) signaling cascade that involves protein kinase-c, Ras, MEKK1, MEK3/MEK6, p38, and AP1 family members (junB, junD, Fra-1). This cascade is clearly important, as inhibition of these kinases turn off hINV gene expression. These studies are also innovative, as the p38 MAPKs have not been thought to function as regulators of gene expression during differentiation. However, much needs to be learned, and in the studies outlined in this proposal, we will focus on key points in this cascade for which we have limited knowledge. We will (i) identify the PKC isoforms involved in this regulation, (ii) study the role of MEK3 and MEK6 as regulators of p38 activity and determine which p38 isoforms are involved in this regulation, (iii) perform studies designed to determine how p38 regulates AP1 transcription factor distribution, level and activation state, and (iv) evaluate the role of these kinases, in vivo, using DNA ballistics (gene gun) delivery to mouse epidermis. These studies are designed to provide new insights regarding regulation of differentiation-dependent gene expression in epidermis. As involucrin is one of many genes that are regulated during differentiation, we hope and expect that understanding provided by these studies will be useful to other investigators.