Mechanism of MAPK Cytoplasmic Retention in Differentiation of ES Cells

描述（由申请人提供）：胚胎干细胞（ES）和癌细胞（EC）在维甲酸处理后在培养中分化。大多数胚胎干细胞分化为具有类似早期胚胎原始内胚层特性的细胞。维甲酸诱导的原始内胚层分化导致细胞生长受到抑制，我们发现这是由于细胞核中活性MAPK受到限制，并且需要完整的细胞骨架。在之前的研究中，我们发现，在分化的细胞中，活化的磷酸化MAPK主要停留在细胞质中，而不是进入细胞核，这与大多数培养细胞形成了对比，在大多数培养细胞中，血清有丝分裂原的刺激导致活化的MAPK很容易进入细胞核。活化MAPK的定位与细胞核和细胞质MAPK底物（如Elk1和cPLA2）的磷酸化程度相关。目前的建议是研究在分化的胚胎干细胞中激活的MAPK的细胞质保留的潜在机制。在初步研究中，我们发现胚胎干细胞的内胚层分化显著增加了核膜蛋白nesprin1的表达。Nesprins是连接核膜和骨架与细胞骨架的LINC复合物的一个蛋白质家族，被认为影响核结构和位置，以及细胞骨架的稳定性和细胞的流动性。据报道，nesprin2在平滑肌细胞中与MAPK结合。因此，在分化为原始内胚层细胞的ES细胞中，nesprin1的表达增加可能具有限制MAPK进入核的功能。nesprin1在未分化的胚胎干细胞中的有限表达将允许MAPK进入细胞核，从而增强增殖。我们将通过以下三个实验目的来验证这一假设：(1)生物化学表征nesprin1 /MAPK关联；(2)分析nesprin1与MAPK在ES细胞分化中的关联；(3)确定nesprin1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发育中的胚胎和成年生物体中分化细胞获得和维持表型和功能的必要步骤，需要调节MAPK的细胞质定位和核进入。如果实验表明这一假设是有效的，我们可能会发现在分化细胞中调节MAPK定位和细胞增殖的机制。我们推测活化的MAPK进入细胞核的调控在体内是普遍存在的，并且在大多数培养细胞中克服了这一调控步骤。

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.