Mechanical Control of Mesenchymal Stem Cell Lineage Allocation

描述（由申请人提供）：骨髓中的间充质干细胞（MSCs）为脂肪细胞和成骨细胞提供祖细胞，MSC池的输出反映了这两种谱系之间的相互关系。机械信号促进成骨谱系的能力提出了令人兴奋的可能性，即运动可能能够调节MSC谱系。我们的工作表明，机械输入可以抑制脂肪形成，通过控制¿-catenin信号传导对MSC互操作性施加重要控制。促进MSC脂肪形成的信号包括-catenin信号的减少，随后是PPAR？脂联素和脂质含量。我们有令人信服的数据表明，机械应变通过AKT改变MSC中GSK3的磷酸化，诱导MSC中持续的catenin激活。重复加载增加了-catenin信号持续时间，从而抑制了脂联素和脂滴的上升等下游事件。我们的研究结果表明，即使是强烈的脂肪生成微环境也可以通过重复的机械输入来抵消这种方式。这允许我们假设“机械刺激通过-catenin抑制PPAR抑制脂肪生成转化”。行动”。通过这项拨款提案，我们提议测试这一假设，充分表征机械输入阻止脂肪形成和控制MSC谱系选择的机制。我们将研究信号的时间性质：需要多少次和多少次重复来调节脂肪生成，我们将询问机械输入是否诱导了替代谱系选择，例如，在我们制造MSC克隆用于研究的独特报告小鼠的帮助下，骨祖细胞或肌细胞。将探讨局部细胞之间的相互作用，询问从紧张细胞分泌的可溶性因子是否可以作用于非紧张细胞（SA1）。我们将确定机械应变激活¿-catenin的机制（通过AKT和GSK32），并考虑其他可能施加局部控制的机械靶点（wnt和bmp）。我们将考虑GSK3¿的替代靶点，如NFATc1和mTOR （SA2）。我们将定义机械激活如何干扰PPAR？直接或间接促进脂肪形成。我们的建议对于了解久坐和老龄化人群中MSC的命运具有重要意义。表征与MSC谱系选择的机械调节有关的信号级联将是至关重要的，因为这应该确定抑制脂肪形成和刺激成骨的途径中的可修改步骤。

DESCRIPTION (provided by applicant): Mesenchymal stem cells (MSCs) in bone marrow provide progenitors for both adipocyte and osteoblast cells and the output of the MSC pool reflects a reciprocal relationship between these two lineages. The ability of mechanical signals to promote osteogenic lineage has raised the exciting possibility that exercise might be able to regulate MSC lineage. Our work indicates that mechanical input can inhibit adipogenesis, exerting a significant control over MSC reciprocity through control of ¿-catenin signaling. Signals which promote MSC adipogenesis involve diminution in ¿-catenin signaling, followed by a rise in PPAR?, adiponectin and lipid content. We have compelling data showing that mechanical strain induces persistent ¿-catenin activation in MSC through alteration of GSK3¿ phosphorylation via AKT in MSCs. Repetitive loading bouts increase the ¿-catenin signal duration such that downstream events such as the rise in adiponectin and lipid droplets are inhibited. Our results suggest that even a strongly adipogenic microenvironment can be counteracted in this way by repetitive bouts of mechanical input. This allows us to hypothesize that "mechanical stimulation represses adipogenic conversion through ¿-catenin inhibition of PPAR? action". With this grant proposal we propose to test this hypothesis, fully characterizing the mechanisms by which mechanical input prevents adipogenesis and controls MSC lineage selection. We will investigate the temporal nature of the signal: how much and how many repetitions are required to regulate adipogenesis, and we will ask if mechanical input induces an alternate lineage selection, e.g., osteoprogenitor or myocyte with the help of unique reporter mice from which we make MSC clones for study. Interactions between local cells will be probed asking whether soluble factors secreted from strained cells can act on unstrained cells (SA1). We will ascertain the mechanisms by which mechanical strain activates ¿-catenin (via AKT and GSK32), as well consider other mechanical targets (Wnts and BMPs) that could exert local control. We will consider alternative targets of GSK3¿ such as NFATc1 and mTOR (SA2). We will define how mechanical activation perturbs PPAR? promotion of adipogenesis directly and indirectly in SA3. Our proposal has significance for understanding the fate of MSC in a sedentary and aging population. It will be critically important to characterize the cascade of signals involved in mechanical regulation of MSC lineage selection, as this should identify modifiable steps in pathways that suppress adipogenesis and stimulate osteogenesis. PUBLIC HEALTH RELEVANCE: Evolution has led to interrelationships between bone and fat together to allow individuals to move to food sources (skeleton necessary for locomotion), and store energy (as fat). Clearly our nation's health is impacted by counterproductive "activities" -lack of exercise and caloric excess. Indeed, as the presence of mechanical information prevents emergence of adipocytes from the marrow, exercise may be considered a way to prevent bone marrow senescence as "old" bone marrow mimics unloading with increased fat. As such, understanding the mechanisms by which mechanical input controls lineage selection is highly relevant to an aging population, and should have high importance for understanding the pathophysiology behind the decreased osteoprogenitor pool and how to reverse it. Consensus building now needs to be a targeted goal, examining the basics of how exercise impacts these relationships by defining the fate of mesenchymal stem cells. We propose here a focused investigation of how loading cells can affect lineage selection when that lineage is already directed toward fat (as would be seen in non-exercising individuals, or aged individuals). We seek insights into the type of mechanical input (how much and for how long), and the targets, beginning with ¿-catenin, but considering other factors which determine lineage selection, and what strained cells become. We will examine proximal nodes in strain activation of ¿-catenin and investigate alternative effectors of mechanical strain. We will look at direct effects to prevent adipogenesis (inhibition of PPAR? expression) and indirect ¿- catenin inhibition of PPAR? responses.