The mechanobiology of TGF-beta signaling in chondrocytes

描述（由申请人提供）：软骨细胞对从压缩到细胞外基质硬度等多个长度尺度的物理信号非常敏感。然而，在骨关节炎中，关节软骨的物理和生物学特性通过耦合但不清楚的机制被破坏。虽然细胞外基质硬度的变化是骨关节炎最早可检测到的体征之一，但软骨细胞微环境中这些物理变化导致软骨细胞稳态丧失的程度尚未完全了解。细胞通过富含整合素的粘着斑和肌动球蛋白产生的细胞骨架张力来感知和响应微环境中的物理信号。细胞骨架张力的变化影响细胞信号传导和基因表达，这反过来又调节基本的细胞过程，如增殖和分化。例如，细胞骨架张力的变化驱动TGF β诱导的Smad3磷酸化和易位以控制软骨形成基因的表达。虽然细胞骨架张力通过几种生长因子信号通路改变细胞对信号的反应，但这种敏感性的分子机制仍不清楚。因此，本项目的目标是确定新的分子机制，细胞骨架张力调节TGF β信号和这些机制的作用，在软骨的多尺度物理线索的充分记录的反应。为了实现这一目标，拟议的研究将测试的假设，物理线索调节软骨细胞的行为，诱导细胞骨架张力的变化，这反过来又影响生长因子受体的定位和功能。目的1将确定细胞骨架张力改变细胞对生长因子反应的机制。初步数据表明，细胞骨架张力在细胞膜水平调节TGF β信号传导，调节TGF β受体、整合素及其效应物之间的物理和功能相互作用。新的分子工具，超分辨率定量成像和生物化学方法将被用来追求这种可能性。目的2将确定在何种程度上细胞骨架张力是一个共同的机制，软骨细胞响应不同的物理线索。使用细胞接种的3D构建体，Aim 2扩展了Aim 1中确定的机制，以了解它们在通过TGF β信号传导维持或丧失软骨细胞稳态中的作用。这项研究的完成将产生新的分子机制，细胞通过这些机制整合物理和生物化学线索。这一贡献是重要的，因为它将推进分子靶点的鉴定，这些分子靶点将由于损伤或疾病引起的软骨的物理变性与软骨细胞稳态的丧失分开，以预防或阻断骨关节炎。

DESCRIPTION (provided by applicant): Chondrocytes are acutely sensitive to physical cues across multiple length scales ranging from compression to extracellular matrix stiffness. However, in osteoarthritis, the physical and biological properties of articular cartilage are disrupted through mechanisms that are coupled but unclear. Although changes in extracellular matrix stiffness are among the earliest detectable signs of osteoarthritis, the extent to which these physical changes in the chondrocyte microenvironment contribute to the loss of chondrocyte homeostasis is not fully understood. Cells sense and respond to physical cues in their microenvironment through integrin-rich focal adhesions and actomyosin-generated cytoskeletal tension. Changes in cytoskeletal tension affect cell signaling and gene expression, which in turn, regulate basic cellular processes such as proliferation and differentiation. For example, changes in cytoskeletal tension drive TGFß-induced Smad3 phosphorylation and translocation to control chondrogenic gene expression. Although cytoskeletal tension modifies the cellular response to signaling by several growth factor signaling pathways, the molecular mechanisms responsible for this sensitivity remain unclear. Therefore, the goal of this project is to identify novel molecular mechanisms by which cytoskeletal tension regulates TGFß signaling and the role of these mechanisms in the well-documented response of cartilage to multi-scale physical cues. To achieve this goal, the proposed research will test the hypothesis that physical cues regulate chondrocyte behavior by inducing changes in cytoskeletal tension, which, in turn, influences growth factor receptor localization and function. Aim 1 will identify mechanisms by which cytoskeletal tension alters the cellular response to growth factors. Preliminary data suggest that cytoskeletal tension regulates TGFß signaling at the level of the cell membrane, regulating physical and functional interactions among TGFß receptors, integrins, and their effectors. New molecular tools, super-resolution quantitative imaging, and biochemical approaches will be employed to pursue this possibility. Aim 2 will determine the extent to which cytoskeletal tension is a common mechanism by which chondrocytes respond to diverse physical cues. Using cell-seeded 3D constructs, Aim 2 extends mechanisms identified in Aim 1 to understand their role in the maintenance or loss of chondrocyte homeostasis by TGFß signaling. The completion of this research will yield new molecular mechanisms by which cells integrate physical and biochemical cues. This contribution is significant because it will advance the identification of molecular targets that uncouple the physical degeneration of cartilage, due to injury or disease, from the loss of chondrocyte homeostasis, to prevent or block osteoarthritis.