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

• 批准号: 8228742
• 负责人: ELIZABETH R SMITH
• 金额: $ 7.65万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-05 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8228742
• 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

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. PUBLIC HEALTH RELEVANCE: In this proposal we plan to investigate a possible mechanism for the restriction of nuclear entry of activated MAPK in differentiated ES cells. We suggest that this restriction is a prevalent, important regulatory step in vivo, which is required to maintain the differentiated phenotype. Moreover, it may be lost upon adaptation of cells in culture. Thus, this study may have the potential to uncover a regulatory mechanism for the Ras/MAPK signaling pathway that is highly critical in the regulation of cell growth, differentiation, and function.

描述(申请人提供)：胚胎干细胞(ES)和癌细胞(EC)在培养过程中用维甲酸处理时会发生分化。大多数ES细胞分化为具有类似于早期胚胎原始内胚层的特性的细胞。维甲酸诱导的原始内胚层分化导致细胞生长抑制，我们发现这是由于限制了细胞核中活性的MAPK，并需要完整的细胞骨架。在以前的研究中，我们已经发现，在分化的细胞中，活化的磷酸化MAPK主要保持在细胞质中，而不是进入细胞核，这与大多数培养细胞不同，在血清有丝分裂原刺激细胞时，激活的MAPK很容易进入细胞核。活化的MAPK的定位分别与核底物ELK1和细胞质MAPK底物cPLA2的磷酸化程度有关。目前的建议是研究一种潜在的机制，解释激活的MAPK在分化的ES细胞中的胞浆滞留。在初步研究中，我们发现ES细胞的内胚层分化显著增加了核膜蛋白Nesprin-1的表达。Nesprs是LINC复合体的一个蛋白质家族，将核膜和骨架与细胞骨架联系起来，被认为影响核结构和位置以及细胞骨架的稳定性和细胞移动性。Nesprin-2已被报道与平滑肌细胞中的MAPK结合。因此，在分化为原始内胚层细胞的ES细胞中Nesprin-1的表达增加可能起到限制MAPK核进入的作用。在未分化的ES细胞中，Nesprin-1的有限表达将允许MAPK核进入，从而促进增殖。我们将在以下三个实验目标中验证这一假说：(1)生化表征Nesprin-1/MAPK的关联；(2)分析ES细胞分化中Nesprin-1与MAPK的关联；以及(3)确定Nesprin-1抑制对MAPK核进入和信号转导以及细胞分化和增殖的影响。控制细胞的生长和增殖是在发育中的胚胎和成年生物体中获得和维持分化细胞的表型和功能的必要步骤，并且需要调节MAPK的细胞质定位和核进入。如果实验表明这一假说是有效的，我们可能会发现一种调节分化细胞中MAPK定位和细胞增殖的机制。我们推测，激活的MAPK的核进入调控在体内是普遍存在的，而这一调控步骤在大多数培养细胞中都被克服了。 公共卫生相关性：在这项建议中，我们计划研究一种可能的机制，以限制活化的MAPK在分化的ES细胞中的核进入。我们认为，这种限制是体内普遍的、重要的调节步骤，这是维持分化表型所必需的。此外，当细胞在培养中适应时，它可能会丢失。因此，这项研究可能有可能揭示RAS/MAPK信号通路的调节机制，该通路在细胞生长、分化和功能调节中起着至关重要的作用。