MAP KINASES REGULATE INVOLUCRIN GENE EXPRESSION

分化依赖基因表达是生物系统/细胞类型经历分化的标志。因此，了解正常和异常角质细胞分化，需要了解调节基因表达的机制。这包括识别启动调控的配体，了解将调控从细胞表面转移到细胞核的事件，以及描述调控靶基因的核机制。我们已经开发了人类花苞蛋白（hINV）基因作为研究分化依赖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），这些转录因子通过结合艾滋病毒基因上游调控区域内特定的DNA结合位点来调节艾滋病毒基因的表达。对这些核事件的了解使得开始研究将基因调控信号从细胞表面转移到细胞核中这些转录因子的信号转导级联成为可能（Effimova et al.， J. Biol）。化学。273:24387,1998)。我们之所以启动这项研究，是因为尽管这些信号级联在其他系统中很重要，但我们对分化过程中角质形成细胞基因向细胞核传递调控信息的步骤知之甚少。这项发表的研究为理解分化调控基因的信号转导提供了全面的努力，并为本提案中概述的实验提供了基本原理。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、MEKK1、MEK3/MEK6、p38和AP1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hiv基因表达。这些研究也是创新的，因为p38 MAPKs在分化过程中没有被认为是基因表达的调节剂。然而，还有很多东西需要学习，在本提案中概述的研究中，我们将重点关注我们所知有限的这个级联中的关键点。我们将(i)确定参与这种调节的PKC亚型，（ii）研究MEK3和MEK6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AP1转录因子分布、水平和激活状态的研究，以及（iv）评估这些激酶的作用，在体内，使用DNA弹道学（基因枪）递送到小鼠表皮。这些研究旨在为表皮分化依赖基因表达的调控提供新的见解。由于天花素是分化过程中受调控的众多基因之一，我们希望并期望这些研究提供的理解将对其他研究者有用。

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.