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刚度等多种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特性通过耦合但不清楚的机制被破坏。尽管细胞外基质硬度的变化是骨关节炎最早可检测到的迹象之一，但软骨细胞微环境的这些物理变化在多大程度上导致了软骨细胞稳态的丧失尚不完全清楚。细胞通过富含整合素的局灶黏附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，进而调节细胞增殖和分化等基本细胞过程。例如，细胞骨架张力的变化驱动TGF？诱导Smad3磷酸化和易位来控制软骨基因的表达。尽管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尚不清楚。因此，本项目的目标是确定细胞骨架张力调节TGF？信号和这些机制在软骨对多尺度物理线索的充分记录的反应中的作用。为了实现这一目标，本研究将验证物理线索通过诱导细胞骨架张力的变化来调节软骨细胞行为的假设，而细胞骨架张力的变化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，细胞骨架张力调节TGF？在细胞膜水平信号传导，调节TGF？受体、整合素及其效应器。新的分子工具，超分辨率定量成像和生化方法将被用来追求这种可能性。目的2将确定在多大程度上，细胞骨架张力是软骨细胞对各种物理线索作出反应的共同机制。使用细胞种子3D构建，Aim 2扩展了Aim 1中确定的机制，以了解它们在TGF？信号。这项研究的完成将产生细胞整合物理和生化信号的新分子机制。这一贡献是重要的，因为它将促进鉴定分子靶点，使由于损伤或疾病导致的软骨的物理变性与软骨细胞稳态的丧失脱钩，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 TGF?-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 TGF? 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 TGF? signaling at the level of the cell membrane, regulating physical and functional interactions among TGF? 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 TGF? 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.