Mechanotransductory regulation of transcription factor activity in fibrosis

纤维化中转录因子活性的机械转导调节

• 批准号: 8784285
• 负责人: Dwight M Chambers
• 金额: $ 4.57万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-02 至 2018-05-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8784285
• 关键词: Acrylamides; Actins; Address; Adhesions; Affect; Area; Atomic Force Microscopy; Attention; Automobile Driving; Binding; Biochemical; Biological Assay; Biological Models; Biology; Biomedical Research; Cell Nucleus; Cells; Cessation of life; Cicatrix; Clinical; Complex; Cytoskeleton; Development; Diagnosis; Disease; Environment; Fibroblasts; Fibrosis; Genetic; Goals; Hamman-Rich syndrome; Human; In Vitro; Incidence; Lamins; Ligation; Link; Lung; Malignant Neoplasms; Measurement; Measures; Mechanical Stress; Mechanics; Mediating; Medical; Membrane Proteins; Mentorship; Metric; Modeling; Molecular; Molecular Target; Morphology; Myofibroblast; Myosin ATPase; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Lamin; Oranges; Pathologic; Pathology; Patients; Phenotype; Physicians; Population; Property; Publishing; Pulmonary Fibrosis; Regenerative Medicine; Regulation; Research; Role; Scientist; Signal Transduction; Stream; Stress; System; Techniques; Technology; Testing; The Sun; Tissue Model; Tissues; Training; Tumor Biology; Work; base; biomaterial development; functional outcomes; in vivo; innovation; insight; interest; mutant; novel; novel therapeutics; optical traps; protein complex; public health relevance; response; skills; tool; transcription factor

DESCRIPTION (provided by applicant): Idiopathic pulmonary fibrosis (IPF) is a fatal scarring of the lungs and has no available medical therapies. Recently, attention has focused on the role of pulmonary fibroblasts, differentiated into contractile, highly synthetic myo-fibroblasts, in the pathological progression of IPF. In vivo, myo-fibroblasts have been linked to active areas of disease in IPF ("fibroblastic foci") and TGF¿ signaling has been shown to be critical in myofibroblastic differentiation. It has been previously demonstrated that the extra-cellular matrix (ECM) regulates the cell's cytoskeleton through its underlying mechanical properties and that this tension can be transmitted to the cell's nucleus. This proposal focuses on a novel hypothesis: cytoskeletal tension transmits fibrotic substrate stiffness to the nuclear membrane and potentiates transcription factor activity by disrupting its interactions with components of the nuclear membrane. Previously, the down-stream effectors of TGF¿ signaling, r-Smads, have been shown to physically associate with MAN1, an inner nuclear membrane integral protein and this complex antagonizes TGF¿ signaling. TGF¿ has been shown in vitro to be a central signaling axis in IPF and has pleiotropic disease-state consequences, including myo-fibroblastic differentiation. The effects of mechanical forces on this r-Smad/MAN1 complex have not been characterized. This proposal will develop a model system to investigate the nuclear membrane deformation associated with the underlying substrate stiffness using innovating genetic tools to disrupt the physical connection between the nuclear membrane and the cytoskeleton. State-of-the-art physical measurements of this system will be conducted using atomic force microscopy and an active micro-rheological technique based on optical trapping technology. Additionally, it will investigate the effects of this deformation on TGF¿/rSmad signaling by using proximity ligation assays to look at the stability and spatial localization on the nuclear membrane of the rSmad/MAN1 complex under stress, and genetic and functional assays to measure TGF¿ signaling. Finally, it will validate the role of substrate stiffness in regulating the r-Smad/MAN1 complex at the nuclear membrane in an advanced, ex vivo model of IPF. This work helps address a possible novel mechanism of the pathobiology of fibrotic disease in the lung. The results of this study should have translational significance by focusing clinical attention on nove pro-fibrotic mechanisms that could form the basis of new therapies for IPF. Additionally, this model of nuclear stiffness as a regulator of transcription factor activity may help inform biomedical research into other disease states, such as cancer/tumor biology and other fibro-proliferative disorders, and development of biomaterial technologies for regenerative medicine approaches. This proposal serves as the lynchpin of a training plan for the applicant and will be the vehicle for imparting technical, scientific and professional skills necessary for his development as a physician-scientist investigating fibrotic diseases.

描述（由申请人提供）：特发性肺纤维化（IPF）是一种致命的肺部疤痕，并且没有可用的医学疗法。最近，人们的注意力集中在肺成纤维细胞（分化为收缩性、高度合成的肌成纤维细胞）在疾病中的作用。这 IPF 的病理进展在体内，肌成纤维细胞与 IPF 疾病的活跃区域（“成纤维细胞病灶”）和 TGF 相关。信号传导已被证明在肌成纤维细胞分化中至关重要，之前已证明细胞外基质。 （ECM）通过其潜在的机械特性调节细胞的细胞骨架，并且这种张力可以传递到细胞核。该提案重点关注一个新的假设：细胞骨架张力将纤维化基质硬度传递到核膜，并通过破坏其转录因子活性来增强。与组件的相互作用 以前，TGF¿的下游效应子。 r-Smads 信号传导已被证明与 MAN1（一种内核膜整合蛋白）有物理关联，并且该复合物可以拮抗 TGF¿ TGF¿已在体外显示为 IPF 的中心信号轴，并具有多效性疾病状态后果，包括肌成纤维细胞分化。该 r-Smad/MAN1 复合物的机械力影响尚未得到表征。模型系统使用创新的遗传工具来研究与底层基底刚度相关的核膜变形，以破坏核膜和细胞骨架之间的物理连接。该系统的最先进的物理测量将使用原子力进行。此外，还将研究这种变形对 TGF 的影响。 /rSmad 信号传导，通过使用邻近连接测定来观察应激下 rSmad/MAN1 复合物核膜上的稳定性和空间定位，并使用遗传和功能测定来测量 TGF¿最后，它将在先进的 IPF 离体模型中验证基质硬度在调节核膜 r-Smad/MAN1 复合物中的作用，这项工作有助于解决肺部纤维化疾病病理学的可能新机制。这项研究的结果应该具有转化意义，将临床注意力集中在新的促纤维化机制上，这些机制可以构成 IPF 新疗法的基础。此外，这种核硬度作为转录因子活性调节剂的模型可能有助于为生物医学研究提供信息。进入其他疾病状态，例如癌症/肿瘤生物学和其他纤维增殖性疾病，以及再生医学方法的生物材料技术的开发该提案是申请人培训计划的关键，并将成为传授技术、作为一名研究纤维化疾病的医师科学家，他需要具备科学和专业技能。