Targeting lung fibrosis: Dissecting molecular crosstalk mechanisms that drive disease pathogenesis and therapeutic response

特发性肺纤维化(IPF)是一种慢性、进行性、发病率很高的肺部疾病。转化生长因子-B和aVB6整合素是IPF的关键驱动因子；aVB6对潜伏的转化生长因子-β施加机械力，有效释放活性的转化生长因子-B。我们发现：1)aVB6信号复合体招募粘蛋白-Galectin-3调节模块；2)MUC1-Galectin-3相互作用调节生长因子受体(GFR)的聚集和运输。在体内，MUC1和Galectin-3促进肺纤维化，而Galectin-3靶向药物抑制转化生长因子-B的活性以抑制晚期进展。MUC1在空间上抑制整合素的激活；庞大的糖蛋白将受体输送到粘连中，并应用压缩张力促进整合素的激活。这一新的整合素激活范式导致了我们的假设：在IPF发病过程中，Galectin-3和MUC1协调aVB6聚集来驱动转化生长因子-B的激活。目的与实验方法1)确定MUC1-Galectin-3相互作用对aVB6转运和配体参与的影响：aVB6转运将采用内吞/循环生化分析方法。AVB6聚集和配体结合将使用激活特异性的抗aVB6抗体进行免疫荧光分析。在所有目标中，将使用机械调节的糖蛋白模拟物和肺上皮细胞中的MUC1胞外域/胞外域突变来分析MUC1压缩张力或信号的贡献。参与MUC1-Galectin-3的相互作用，2)分析MUC1-Galectin-3相互作用是否调节aVB6介导力的施加和转化生长因子-β的激活：定量牵引力显微镜和转化生长因子-β1活性荧光素报告分析将证明MUC1-Galectin-3的生物物理和信号特性是否调节aVB6介导的机械力并激活转化生长因子-B。3)测试MUC1-Galectin-3相互作用是否协调aVB6漏斗以驱动转化生长因子-β的激活：学生将访问Paszek实验室进行地形扫描角度干涉显微镜分析膜变形4)确定MUC1-Galectin-3介导的对aVB6/转化生长因子-B的调节是否调制了对Galectin-3和aVB6靶向药物的反应：器官型IPF模型将用于测试MUC1-Galectin-3相互作用在aVB6依赖的疾病发生/发展中的作用。临床前IPF模型将在给予Galectin-3和aVB6靶向药物后使用。新的和及时的IPF发病率正在上升，迫切需要新的治疗管理途径。这个多学科的项目将揭示aVB6、MUC1和Galectin-3如何协同激活转化生长因子-β；阐明推动IPF发病机制和药物反应的机制。

Idiopathic pulmonary fibrosis (IPF) is chronic, progressive, lung disease with very high morbidity. Transforming growth factor-B (TGF-B) and aVB6 integrin are key drivers of IPF; aVB6 applies mechanical force on latent TGF-B to potently release active TGF-B.We have found: 1) aVB6 signalling complexes recruit a mucin-galectin-3 regulatory module; 2) MUC1-galectin-3 interaction regulates growth factor receptor (GFR) clustering and trafficking. In vivo, MUC1 and galectin-3 promote lung fibrosis and galectin-3-targeting drugs inhibit TGF-B activity to suppress late-stage progression.MUC1 spatially-constrains integrin activation; the bulky glycoprotein funnels receptors into adhesions and applies compressive tension to promote integrin activation.This new paradigm of integrin activation leads to our hypothesis: Galectin-3 and MUC1 co-ordinate aVB6 clustering to drive TGF-B activation during IPF pathogenesis.Objectives & Experimental Approach1) Determine impact of MUC1-galectin-3 interaction on aVB6 trafficking and ligand engagement:aVB6 trafficking will be analysed using biochemical endocytosis/recycling assays. aVB6 clustering and ligand-binding will be analysed by immunofluorescence using activation-specific anti-aVB6 antibodies.In all objectives, contributions of MUC1 compressive tension or signalling will be analysed using mechanically-tuned glycoprotein-mimetics and MUC1 ectodomain/cytodomain mutants in lung epithelial cells. Involvement of MUC1-galectin-3 interaction, will be assessed in presence/absence of galectin-3 or galectin-3-targeting drugs.2) Analyse whether MUC1-galectin-3 interaction regulates aVB6-medicated force application and TGF-B activation:Quantitative traction force microscopy and TGF-B1 activity luciferse reporter assays will demonstrate whether biophysical and signalling properties of MUC1-galectin-3 regulate aVB6-mediated mechanical forces and activate TGF-B.3) Test whether MUC1-galectin-3 interaction co-ordinates aVB6 funnelling to drive TGF-B activation:Student will visit Paszek Lab to perform Topographical-Scanning Angle Interference Microscopy to analyse membrane deformation using FRET reporters of MUC1 compression and aVB6 activation-specific antibodies.4) Determine whether MUC1-galectin-3-mediated regulation of aVB6/TGF-B modulates response to galectin-3- and aVB6-targeting drugs:Organotypic IPF models will be used to test involvement of MUC1-galectin-3 interaction on aVB6-dependent disease initiation/progression. Preclinical IPF models will be utilised following administration of galectin-3- and aVB6-targeting drugs.Novelty & TimelinessIPF incidence is rising and in critical need of novel avenues for therapeutic management. This multidisciplinary project will reveal how aVB6, MUC1 and galectin-3 co-operate to activate TGF-B; illuminating the mechanisms driving IPF pathogenesis and drug responses.