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

特发性肺纤维化（IPF）是一种慢性进行性肺疾病，发病率非常高。转化生长因子- b （TGF-B）和aVB6整合素是IPF的关键驱动因子；aVB6对潜伏TGF-B施加机械力，有效释放活性TGF-B。我们发现：1)aVB6信号复合物募集了粘蛋白-半乳糖凝集素-3调节模块；2) MUC1-galectin-3相互作用调控生长因子受体（GFR）聚集和转运。在体内，MUC1和半乳糖凝集素-3促进肺纤维化，靶向半乳糖凝集素-3的药物抑制TGF-B活性，抑制晚期进展。MUC1空间限制整合素激活；庞大的糖蛋白漏斗受体进入粘连，并施加压缩张力，以促进整合素激活。这种整合素激活的新范式导致了我们的假设：在IPF发病过程中，半凝集素-3和MUC1协调aVB6聚集驱动TGF-B激活。目的和实验方法1)确定muc1 -半乳糖凝集素-3相互作用对aVB6运输和配体接合的影响：aVB6运输将使用生化内吞噬/回收分析。aVB6聚类和配体结合将通过激活特异性抗aVB6抗体的免疫荧光分析。在所有目标中，将使用机械调谐的糖蛋白模拟物和肺上皮细胞中的MUC1外结构域/细胞结构域突变体分析MUC1压缩张力或信号传导的贡献。muc1 -半乳糖凝集素-3相互作用的参与，将在存在/不存在半乳糖凝集素-3或半乳糖凝集素-3靶向药物的情况下进行评估。2)分析muc1 -半乳糖凝集素-3相互作用是否调节avb6给药力的施加和TGF-B的激活：定量牵引力显微镜和TGF-B1活性荧光素报告基因检测将证明muc1 -半乳糖凝集素-3的生物物理和信号特性是否调节avb6介导的机械力和激活TGF-B。3)测试MUC1-半凝集素-3相互作用是否协调aVB6漏斗驱动TGF-B激活：学生将访问Paszek实验室，使用FRET报告MUC1压缩和aVB6激活特异性抗体进行地形扫描角度干涉显微镜分析膜变形。4)确定muc1 -半乳糖凝集素-3介导的aVB6/TGF-B调控是否调节对半乳糖凝集素-3和aVB6靶向药物的反应：器官型IPF模型将用于测试muc1 -半乳糖凝集素-3相互作用在aVB6依赖性疾病的发生/进展中的作用。临床前IPF模型将在给药半乳糖凝集素-3和avb6靶向药物后使用。新颖性和时效性essipf发病率正在上升，迫切需要新的治疗管理途径。这个多学科项目将揭示aVB6、MUC1和半凝集素-3如何协同激活TGF-B；阐明了驱动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.