Role of force regulated nuclear structure in expression of osteogenesis

摘要：力调节核结构在成骨表达中的作用 骨髓间充质干细胞(MSC)存在于多潜能状态，其中成骨 而成脂基因组在内核小叶的异染色质中沉默。正在激活 成骨分化计划涉及多个调节因素，包括物理因素 力量，这是在动态运动过程中在骨髓空间产生的。MSC体验 通过细胞骨架附着在底物上的机械力，诱导信号 改变基因表达。我们发现，核内肌动蛋白的结构受 胞质细胞骨架和核内肌动蛋白的直接变化 核限制性mDia2(防止核内肌动蛋白聚合)具有深刻的调控作用 对基因表达的控制。此外，尽管动态和静态机械力都被激活 RhoA控制肌动蛋白细胞骨架的形成，这些力量的性质似乎有 基因表达的广泛变异效应-动力抑制脂肪生成并促进脂肪生成 多势性，而静力与成骨有关。动态与静态 应用可能通过在细胞核上产生不同的力来影响基因的表达， 影响机械反应者的核结构和核通路，YAP和β-连环蛋白。 我们在这里假设，通过强制激活的肌动蛋白聚合修饰的核结构， 有助于选择性的MSC分化和命运。为了解决这一假设，我们建议 定义由动态或静态机械力引起的细胞肌动蛋白结构的不同 调控核结构和基因表达。在SA1中，我们将发现动态应变和静态应变 差异修饰核结构(F-肌动蛋白和层蛋白结构，核仁大小和间距， Runx2的细胞和核硬度及FISH定位。我们的数据显示，核内的损失 肌动蛋白聚合减少了核内小叶的层蛋白B1，因此我们将发现力是否发生变化 成骨基因通过层蛋白沉默，以及如果形成蛋白mDia2或laminB1的改变调节 成骨基因组的异染色化。在SA2中，我们将询问动静态力 差异调节YAP或β-连环蛋白的核进入，并与细胞骨架应力传递相关 核膜对这些分子核内通路的变化。公正的RNAseq将 允许我们比较动态和静态作用力后的基因表达，并询问核YAP和β- 连环蛋白是至关重要的。最后，在SA3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mDia2被击倒后定义核结构 (减少核内F-肌动蛋白，诱导成骨)，mDia1基因敲除(减少 细胞质F-肌动蛋白)和改变次级肌动蛋白分支(诱导脂肪生成)。在这些 我们将核结构与Runx2和PPARγ信号通路的激活联系起来。 在我们的目标完成后，我们将能够确定机械力和 核肌动蛋白聚合控制表观遗传诱导成骨和调控核 YAP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.