Mechanism of MEK Kinase 1 in Mouse Eyelid Development

描述（由申请人提供）：拟议研究的长期目标是阐明信号转导途径调节眼发育和眼表形态发生的分子机制。胚胎眼睑闭合对于正常脊椎动物眼表形态发生是必需的;其在小鼠中的缺陷导致出生时睁眼（EOB）和各种眼部病理。许多人类先天性疾病，如结膜-角膜营养不良、上睑下垂和小眼症，表现出与患有EOB的小鼠中发现的病理相似，并且可能与眼睑发育失败有关。了解眼睑闭合中的分子事件可能会揭示先天性眼病的原因，帮助开发诊断工具并确定可能的药物干预目标。在小鼠中切除编码细胞质蛋白激酶的Mekkl基因导致EOB表型。MEKK 1消融导致发育中眼睑上皮中F-肌动蛋白形成的缺陷和角质形成细胞迁移的损伤。MEKK 1介导的Jun N-末端激酶（JNK）级联导致转录因子cJun的磷酸化和基因表达模式的伴随变化，促进细胞运动和增殖，但阻止上皮末端分化。这些发现导致了这样的假设，即在发育中的眼睑中MEKK 1通路的诱导导致AP-1转录复合物的成员的磷酸化和活化，负责基因表达、上皮细胞迁移和胚胎眼睑闭合的变化。本申请中提出的研究将利用我们实验室中可获得的野生型和Mekk 1-/-小鼠的发育中的眼睑组织和培养的角质形成细胞来研究参与MEKK 1活性诱导和AP-1功能调节的分子途径。我建议：（1）阐明MEKK 1信号传导的分子过程，导致角质形成细胞迁移和介导眼睑闭合;（2）确定MEKK 1依赖的AP-1磷酸化对角质形成细胞迁移至关重要;（3）确定MEKK 1在调节AP-1转录功能中的作用，AP-1转录功能与眼睑闭合过程中基因表达的变化有关。这项工作的结果将扩展我们对眼睑发育的分子调控的理解，并推进我们对上皮细胞迁移和眼睑闭合的关键信号网络和分子事件的了解。

DESCRIPTION (provided by applicant): The long-term goal of the proposed study is to elucidate the molecular mechanisms by which signal transduction pathways regulate eye development and ocular surface morphogenesis. Embryonic eyelid closure is essential for normal vertebrate ocular surface morphogenesis; its deficiency in mouse results in eyes open at birth (EOB) and various eye pathologies. Many human congenital diseases, such as conjunctiva-corneal dystrophy, ptosis and microophthalmia, exhibit similarities to pathologies found in mice with EOB and are possibly linked to failure in eyelid development. Understanding the molecular events in eyelid closure may uncover the causes of congenital eye diseases, help develop diagnostic tools and identify targets for possible pharmaceutical intervention. Ablation in mice of the Mekkl gene, encoding a cytoplasmic protein kinase, causes EOB phenotype. MEKK1 ablation results in defects in F-actin formation in developing eyelid epithelium and impairments in keratinocyte cell migration. MEKK1-mediated Jun N-terminal kinase (JNK) cascade leads to phosphorylation of the transcription factor cJun and a concomitant change in gene expression pattern that promotes cell movement and proliferation, but prevents epithelium terminal differentiation. These findings lead to the hypothesis that induction of the MEKK1 pathway in the developing eyelid leads to phosphorylation and activation of members of the AP-1 transcription complex, responsible for changes in gene expression, epithelial cell migration and embryonic eyelid closure. The studies proposed in this application will utilize developing eyelid tissues and cultured keratinoctyes from wild type and Mekk1-/-mice available in our laboratory to investigate the molecular pathways involved in the induction of MEKK1 activity and the regulation of AP-1 function. I propose (1) to elucidate the molecular processes of MEKK1 signaling that lead to keratinocyte migration and mediate eyelid closure; (2) to identify the MEKK1-dependent phosphorylation of AP-1 critical for keratinocyte migration; and (3) to define the role of MEKK1 in the regulation of AP-1 transcription functions pertinent to changes in gene expression during eyelid closure. Results from this work will extend our understanding of the molecular regulation of eyelid development and advance our knowledge on the signaling networks and molecular events critical for epithelial cell migration and eyelid closure.