Role of force regulated nuclear structure in expression of osteogenesis

翻译后摘要：力调节的核结构在成骨表达的作用 骨髓间充质干细胞（MSC）以多潜能状态存在， 并且脂肪形成基因组在内核小叶的异染色质中沉默。激活 成骨分化程序涉及多个调节因子，包括物理因子， 力，这是在动态运动过程中产生的骨髓空间。MSC经验 机械力通过它们的细胞骨架附着到基质上，诱导信号传导， 改变基因表达我们发现，核内肌动蛋白结构的变化， 细胞质细胞骨架，和直接的变化，核内肌动蛋白由于敲低 核限制性mDia 2（阻止核内肌动蛋白聚合）发挥深刻的调节作用， 控制基因表达。此外，尽管动态和静态机械力都激活了 RhoA控制肌动蛋白细胞骨架的形成，这些力量的性质似乎具有 对基因表达的广泛变化的影响-动力抑制脂肪形成并促进 多潜能性，而静态力与骨生成相关。动态与静态 应用可以通过在细胞核上产生不同的力来影响基因表达， 影响机械应答者、雅普和β-连环蛋白的核结构和核通路。 我们在这里假设，核结构，修改武力激活肌动蛋白聚合， 有助于选择性MSC分化和命运。为了解决这个问题，我们建议 定义动态或静态机械力如何导致细胞肌动蛋白结构的差异 调节核结构和基因表达。在SA 1中，我们将发现动态和静态应变 差异修饰核结构（F-肌动蛋白和核纤层蛋白结构，核仁大小和间距， 细胞和核硬度以及Runx 2的FISH定位）。我们的数据显示， 肌动蛋白聚合减少了内核小叶的核纤层蛋白B1，因此我们将发现，如果力改变， 成骨基因沉默通过核纤层蛋白，如果改变在β-dia 2或核纤层蛋白B1调节 成骨基因组的异染色质化。在SA 2中，我们将询问动态和静态力是否 差异调节雅普或β-连环蛋白的核进入，并将细胞骨架应力转移与 核膜对这些分子进入细胞核的变化的反应。无偏RNAseq将 让我们比较动态与静态力后的基因表达，并询问是否核雅普和β- catenin是关键的。最后，在SA 3中，我们确定核内肌动蛋白结构是否直接控制 获取成骨基因组。我们将在mDia 2敲除后定义核结构 （减少核内F-肌动蛋白，诱导骨生成），mDia 1敲低（减少 细胞质F-肌动蛋白）和改变次级肌动蛋白分支（诱导脂肪形成）。在这些 我们将把核结构与Runx 2和PPARγ顺反系统的激活联系起来。 在完成我们的目标后，我们将能够建立机械力和 核肌动蛋白聚合控制表观遗传诱导成骨和调节核 雅普和β-连环蛋白的转移。

Abstract: Role of force regulated nuclear structure in expression of osteogenesis Bone marrow mesenchymal stem cells (MSC) exist in a multipotential state, where osteogenic and adipogenic genomes are silenced in heterochromatin at the inner nuclear leaflet. Activating the osteogenic differentiation program involves multiple regulatory factors, including physical force, which is generated in the marrow space during dynamic exercise. MSC experience mechanical force through their cytoskeletal attachments to substrate, inducing signaling that alters gene expression. We showed that intranuclear actin structures are affected by changes in the cytoplasmic cytoskeleton, and direct changes in intranuclear actin due to knock down of nuclear restricted mDia2 (preventing intranuclear actin polymerization) exert profound regulatory control on gene expression. Further, although both dynamic and static mechanical force activate RhoA to control formation of the actin cytoskeleton, the nature of these forces appear to have widely variant effects on gene expression – dynamic force inhibits adipogenesis and promotes multipotentiality, while static force is associated with osteogenesis. Dynamic versus static applications may affect gene expression through generating different forces on the nucleus, affecting nuclear structure and nuclear access of the mechanoresponders, Yap and β-catenin. We here hypothesize that nuclear structure, modified by force activated actin polymerization, contributes to selective MSC differentiation and fate. To address this hypothesis, we propose to define how cellular actin structure resulting from dynamic or static mechanical force differentially regulate nuclear architecture and gene expression. In SA1 we will find if dynamic and static strain differentially modify nuclear architecture (F-actin and lamin structure, nucleoli size and spacing, cell and nuclear stiffness and FISH localization of Runx2). Our data shows that loss of intranuclear actin polymerization decreases lamin B1 at the inner nuclear leaflet, thus we will find if force alters osteogenic gene silencing through lamins, and if alterations in formin mDia2 or laminB1 modulate heterochromatization of the osteogenic genome. In SA2 we will ask if dynamic and static force differentially regulate nuclear entry of Yap or β-catenin, and relate cytoskeletal stress transfer to the nuclear membrane to changes in nuclear access of these molecules. Unbiased RNAseq will allow us to compare gene expression after dynamic vs static force and ask if nuclear Yap and β- catenin are critical. Lastly in SA3 we determine if intranuclear actin structure directly controls access to the osteogenic genome. We will define nuclear structure after mDia2 knock down (decrease intranuclear F-actin, induces osteogenesis), mDia1 knock down (decreasing cytoplasmic F-actin) and altering secondary actin branching (induces adipogenesis). In these conditions we will relate nuclear structure to activation of Runx2 and PPARγ cistromes. Upon completion of our objectives we will be able to establish how mechanical forces and nuclear actin polymerization control epigenetic induction of osteogenesis and regulate nuclear transfer of YAP and β-catenin.