Role of force regulated nuclear structure in expression of osteogenesis

力调节核结构在成骨表达中的作用

• 批准号: 10632101
• 负责人: Janet E Rubin
• 金额: $ 45.78万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10632101
• 关键词: Actins; Address; Affect; Architecture; Biomechanics; Bone Marrow; Cell Lineage; Cell Nucleus; Cells; Chromatin; Cytoplasm; Cytoskeleton; DNA; DNA Repair; Data; EZH2 gene; Epigenetic Process; Euchromatin; Exercise; F-Actin; Focal Adhesions; Gene Activation; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genome; Heterochromatin; Lamin B1; Lamins; Link; Marrow; Measures; Mechanics; Mediating; Mesenchymal; Mesenchymal Stem Cells; Modeling; Nature; Nuclear; Nuclear Envelope; Nuclear Import; Nuclear Pore; Nuclear Structure; Nuclear Translocation; Osteoblasts; Osteogenesis; PPAR gamma; Physical condensation; Polymers; Positioning Attribute; Proliferating; Publishing; Regulator Genes; Regulatory Pathway; Role; Signal Induction; Signal Pathway; Signal Transduction; Small Interfering RNA; Stimulus; Stress; Stromal Cells; Structure; Testing; Variant; beta catenin; bone; bone marrow mesenchymal stem cell; epigenetic regulation; experience; histone methylation; inhibitor; knock-down; lipid biosynthesis; loss of function; mechanical force; mechanical stimulus; novel; nuclear transfer; osteogenic; polymerization; prevent; programs; promoter; response; stem; stem cell differentiation; stem cell fate; stem cells; stemness; transcription factor; transcriptome; transcriptome sequencing

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.

摘要：力调节核结构在成骨的表达中的作用 骨髓间充质干细胞（MSC）存在于多能状态，其中成骨 在内部核小叶的异染色质中沉默的脂肪基因组被沉默。激活 成骨分化计划涉及多种调节因素，包括物理 力，在动态运动过程中在骨髓空间中产生。 MSC经验 通过其细胞骨架附着在底物上的机械力，引起的信号传导 改变基因表达。我们表明，插图的肌动蛋白结构受到变化的影响 细胞质细胞骨架，并因敲低 核受限的MDIA2（预防静脉肌动蛋白聚合）发挥深刻的调节性 控制基因表达。尽管动态和静态机械力都激活 Rhoa控制肌动蛋白细胞骨架的形成，这些力的性质似乎具有 对基因表达的广泛影响 - 动态力抑制脂肪形成并促进 多能性，而静态力与成骨相关。动态与静态 应用可能会通过在细胞核上产生不同的力来影响基因表达， 影响机制，YAP和β-catenin的核结构和核通道。 我们在这里假设该核结构通过力激活的肌动蛋白聚合而修饰， 有助于选择性MSC分化和命运。为了解决这一假设，我们建议 定义细胞肌动蛋白结构如何由动态或静态机械力差异化产生 调节核结构和基因表达。在SA1中，我们会发现动态和静态应变是否 差异修饰核结构（F-肌动蛋白和层粘连蛋白结构，核仁大小和间距， runx2的细胞和核刚度和鱼的定位）。我们的数据表明，插图的丢失 肌动蛋白聚合在内部核小叶处下降层lamin b1，因此我们将发现力是否改变 成骨基因通过lamins沉默，以及formin mDia2或laminb1调节的改变 成骨基因组的异晶体化。在SA2中，我们将询问动态和静态力量是否 差异调节YAP或β-catenin的核进入，并将细胞骨架应激转移与 这些分子的核通路变化的核膜。无偏的RNASEQ会 允许我们比较动态和静态力后的基因表达，并询问核YAP和β-是否是 Catenin至关重要。最后，在SA3中，我们确定Interansclear肌动蛋白结构是否直接控制 进入成骨基因组。我们将在MDIA2撞倒后定义核结构 （减少插图的F-肌动蛋白，诱导成骨），MDIA1敲低（减少 细胞质F-肌动蛋白）和次级肌动蛋白分支（诱导的脂肪形成）。在这些 我们将将核结构与Runx2和PPARγCISTROMES的激活联系起来。 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成骨的表观遗传诱导并调节核 YAP和β-catenin的转移。