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: 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
• 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

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）：胚胎干（ES）和癌（EC）细胞在用视黄酸处理时在培养物中发生分化。大多数ES细胞分化成具有类似于早期胚胎的原始内胚层的特性的细胞。视黄酸诱导的原始内胚层分化导致细胞生长抑制，我们发现这是由于来自细胞核的活性MAPK的限制，并且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，在大多数培养的细胞中，血清有丝分裂原刺激细胞，导致活化的MAPK容易进入细胞核。活化的MAPK的定位与细胞核和细胞质MAPK底物的磷酸化程度相关，例如分别为Elk 1和cPLA 2。目前的建议是调查一个潜在的机制，占在分化的ES细胞活化MAPK的细胞质滞留。 在初步研究中，我们发现ES细胞的内胚层分化显著增加核膜蛋白Nesprin-1的表达。Nesprins是连接核膜和骨架与细胞骨架的LINC复合物的蛋白质家族，被认为影响核结构和位置以及细胞骨架稳定性和细胞移动性。已报道Nesprin-2结合平滑肌细胞中的MAPK。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。Nesprin-1在未分化的ES细胞中的有限表达将允许MAPK进入核，并因此增强增殖。我们将在以下三个实验目标中验证这一假设：（1）以生物化学方式表征Nesprin- 1/MAPK缔合;（2）分析Nesprin-1和MAPK在ES细胞分化中的缔合;以及（3）确定Nesprin-1抑制对MAPK核进入和信号传导以及细胞分化和增殖的影响。 细胞生长和增殖的控制是在发育中的胚胎和成体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细胞质定位和核进入。如果实验表明这一假设是有效的，我们可能会发现一个机制，调节MAPK的定位和细胞增殖分化的细胞。我们推测，在体内激活的MAPK的核进入的调节是普遍的，并且在大多数培养的细胞中克服了这个调节步骤。