Mechanism of MEK Kinase 1 in Mouse Eyelid Development

描述（由申请人提供）：本研究的长期目标是阐明信号转导通路调节眼睛发育和眼表形态发生的分子机制。胚胎眼睑闭合是正常脊椎动物眼表形态发生的必要条件；小鼠体内缺乏这种物质会导致出生时睁眼（EOB）和各种眼部病变。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，与EOB小鼠的病理相似，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揭示先天性眼病的原因，有助于开发诊断工具并确定可能的药物干预目标。编码细胞质蛋白激酶的Mekkl基因在小鼠中被切除，导致EOB表型。MEKK1消融导致发育中的眼睑上皮中f -肌动蛋白形成缺陷和角化细胞细胞迁移障碍。mekk1介导的Jun n -末端激酶（JNK）级联可导致转录因子cJun的磷酸化，并伴随基因表达模式的改变，从而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一种假设：在眼睑发育过程中，MEKK1通路的诱导导致AP-1转录复合体成员的磷酸化和激活，而AP-1转录复合体负责基因表达、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提供的野生型和Mekk1-/-小鼠的发育中的眼睑组织和培养的角质形成细胞来研究诱导Mekk1活性和调节AP-1功能的分子途径。我建议(1)阐明导致角质细胞迁移和介导眼睑闭合的MEKK1信号传导的分子过程；(2)鉴定对角质形成细胞迁移至关重要的mekk1依赖性AP-1磷酸化；(3)明确MEKK1在闭眼过程中与AP-1基因表达变化相关的转录功能调控中的作用。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，并提高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认识。

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.