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（由适用提供）：特发性肺纤维化（IPF）是肺的致命疤痕，没有可用的医疗疗法。最近，注意力集中在肺成纤维细胞的作用上，在收缩的，高度合成的肌成纤维细胞中，在 在体内，肌成纤维细胞已与IPF中的疾病活性区域（“成纤维细胞灶”）相关，并且TGF¿信号已证明在肌纤维细胞分化中至关重要。以前已经证明了细胞外基质 （ECM）通过其潜在的机械性能调节细胞的细胞骨架，并可以将这种张力传递到细胞的核。该提案的重点是一个新的假设：细胞骨架张力通过破坏其与其与其与成分的相互作用的相互作用，将纤维化底物刚度传播到核膜和潜在的转录因子活性。 核膜。以前，TGF信号传导（R-SMADS）的下游效应已被证明与MAN1（一种内部核膜积分蛋白）物理缔合，并且该复合物使TGF信号传导拮抗。在体外已显示TGFS是IPF中的一个中心信号轴，并且具有多效性疾病状态后果，包括肌性肌细胞分化。机械力对此R-SMAD/MAN1复合物的影响尚未表征。该建议将开发一个模型系统，以研究与基础底物刚度相关的核膜变形，使用创新的遗传工具破坏核膜与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显微镜和基于光学诱捕技术的主动微生物学技术进行。此外，它将通过使用接近连接测定法研究这种变形对TGF� /RSMAD信号传导的影响，以查看压力下RSMAD /MAN1复合物的核膜的稳定性和空间定位，以及遗传和功能测定法，以测量TGF信号。最后，它将验证底物刚度在核膜上确定R-SMAD/MAN1复合物在IPF的高级外体模型中的作用。这项工作有助于解决肺纤维化疾病病理生物学的新型机制。这项研究的结果应通过将临床注意力集中在新的促纤维化机制上，这可能构成IPF新疗法的基础，具有转化的意义。此外，这种作为转录因子活性调节剂的核僵硬模型可能有助于为其他疾病状态的生物医学研究提供信息，例如癌症/肿瘤生物学和其他纤维增殖疾病，以及开发用于再生医学方法的生物材料技术。该建议是申请人培训计划的林奇平，将成为授予他作为身体科学家调查纤维化疾病所必需的技术，科学和专业技能的工具。