Mechanism of MAPK Cytoplasmic Retention in Differentiation of ES Cells

MAPK胞质保留在ES细胞分化中的机制

基本信息

批准号：
8436213
负责人：
ELIZABETH R SMITH
金额：
$ 7.26万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-05 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8436213
关键词：
Accounting Actins Adult Architecture Binding C-terminal Cell Differentiation process Cell Maintenance Cell Mobility Cell Nucleus Cell Proliferation Cells Chimeric Proteins Co-Immunoprecipitations Complex Cultured Cells Cytoplasm Cytoskeleton Down-Regulation Dystrophin Embryo Endoderm Endoderm Cell Epithelial Cells Exclusion FOS gene Future Goals Growth Immunofluorescence Immunologic Link MAPK3 gene Malignant Epithelial Cell Mitogen Activated Protein Kinase 1 Mitogen-Activated Protein Kinases Mitogens N-terminal Nuclear Nuclear Envelope Nuclear Translocation Organism Phenotype Phosphorylation Positioning Attribute Prevalence Property Protein Binding Protein Family Proteins Regulation Reporting Serum Signal Pathway Signal Transduction Site Skeleton Small Interfering RNA Smooth Muscle Myocytes Spectrin Testing Tretinoin Undifferentiated Utrophin base biological systems blastomere structure calponin cell growth cell growth regulation cell type design embryonic stem cell env Gene Products established cell line in vivo prevent research study retinal rods small hairpin RNA stem stem cell differentiation

项目摘要

DESCRIPTION (provided by applicant): Embryonic stem (ES) and carcinoma (EC) cells undergo differentiation in culture when treated with retinoic acid. The majority of the ES cells differentiate into cells with properties that resemble the primitive endoderm of early embryos. Retinoic acid-induced primitive endoderm differentiation leads to suppression of cell growth, which we found to result from restriction of active MAPK from the nucleus and to require an intact cytoskeleton. In previous studies, we have found that in differentiated cells, activated phospho-MAPK remains principally cytoplasmic rather than entering the nucleus, in contrast to most cultured cells, in which stimulation of cells by serum mitogens results in activated MAPK readily entering the nucleus. The localization of activated MAPK correlates with the degree of phosphorylation of nuclear and cytoplasmic MAPK substrates, such as Elk1 and cPLA2, respectively. The current proposal is to investigate a potential mechanism that accounts for the cytoplasmic retention of activated MAPK in differentiated ES cells. In preliminary studies, we found that endoderm differentiation of ES cells dramatically increases expression of the nuclear envelope protein Nesprin-1. Nesprins, a family of proteins of the LINC complex that links the nuclear envelope and skeleton to the cytoskeleton, are believed to influence nuclear architecture and position as well as cytoskeletal stability and cell mobility. Nesprin-2 has been reported to bind MAPK in smooth muscle cells. The increased expression of Nesprin-1 in ES cells differentiated to primitive endoderm cells may therefore function to restrict MAPK nuclear entry. The limited expression of Nesprin-1 in undifferentiated ES cells would be permissive for MAPK nuclear entry and, as a consequence, enhance proliferation. We will test this hypothesis in the following three experimental aims: (1) Characterize Nesprin- 1/MAPK association biochemically; (2) Analyze Nesprin-1 and MAPK association in ES cell differentiation; and (3) Determine the impact of Nesprin-1 suppression on MAPK nuclear entry and signaling and cellular differentiation and proliferation. Control of cell growth and proliferation is an obligate step to attain and maintain the phenotype and function of differentiated cells in the developing embryo and in the adult organism, and requires regulation of MAPK cytoplasmic localization and nuclear entry. If the experiments suggest the hypothesis is valid, we may uncover a mechanism regulating MAPK localization and cell proliferation in differentiated cells. We speculate that regulation of nuclear entry of activated MAPK is prevalent in vivo, and this regulatory step is overcome in most cultured cells.

描述（由申请人提供）：用视黄酸治疗时胚胎（ES）和癌（EC）细胞在培养中经历分化。大多数ES细胞将具有类似于早期胚胎的原始内胚层的特性分化为细胞。维甲酸诱导的原始内胚层分化导致细胞生长的抑制，我们发现这是由于对核的活性MAPK的限制造成的，需要完整的细胞骨架。在先前的研究中，我们发现在分化的细胞中，激活的磷酸化含量仍然是细胞质的，而不是与大多数培养的细胞相反，与大多数培养的细胞相反，在这种细胞中，血清有丝分裂剂刺激细胞会导致活化的MAPK很容易进入细胞核。活化MAPK的定位分别与核和细胞质MAPK底物（例如ELK1和CPLA2）的磷酸化程度相关。当前的建议是研究一种潜在的机制，该机制解释了分化ES细胞中激活的MAPK的细胞质保留率。在初步研究中，我们发现ES细胞的内胚层分化显着增加了核包膜蛋白Nesprin-1的表达。 Nesprins是将核包膜和骨骼与细胞骨架联系起来的LINC复合物的蛋白质家族，它被认为会影响核结构和位置以及细胞骨架稳定性和细胞迁移率。据报道，Nesprin-2在平滑肌细胞中结合MAPK。因此，NESPRIN-1在与原始内胚细胞区分开的ES细胞中的表达增加可能起作用可限制MAPK核进入。 NESPRIN-1在未分化的ES细胞中的表达有限，将允许MAPK核进入，并因此增强增殖。我们将在以下三个实验目的中检验这一假设：（1）在生化上表征Nesprin-1/MAPK关联；（2）分析ES细胞分化中的Nesprin-1和MAPK关联；（3）确定Nesprin-1抑制对MAPK核进入以及信号传导以及细胞分化和增殖的影响。控制细胞生长和增殖是在发育中的胚胎和成人生物体中分化细胞的表型和功能的强大步骤，并且需要调节MAPK细胞质定位和核进入。如果实验表明该假设有效，我们可能会发现调节分化细胞中MAPK定位和细胞增殖的机制。我们推测，活化MAPK的核进入的调节在体内很普遍，并且在大多数培养的细胞中都可以克服这种调节步骤。