Dissecting receptor crosstalk mechanisms that co-ordinate wound healing and drive scar formation

我们将分析整合素和生长因子受体（GFRs）之间促进伤口愈合和驱动疤痕形成的串扰机制。TGF-B和整合素aVB6通过诱导再上皮化、肌成纤维细胞活化、伤口收缩和基质重塑来共同促进伤口愈合。整合素aVB6通过结合并对潜伏的TGF-B施加力来激活TGF-B，诱导结构变化，释放有效的细胞因子。因此，基因缺失或抑制aVB6或TGF-B会导致体内明显的伤口愈合缺陷。然而，尽管aVB6在组织再生中起着重要作用，但aVB6介导的伤口修复的分子基础尚不清楚。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GFR聚集和转运。在体内，MUC1和半乳糖凝集素-3促进伤口修复，我们的iCASE合作伙伴已经开发了靶向半乳糖凝集素-3的药物，可在体内纤维化模型中抑制TGF-B活性。这些数据表明，aVB6、MUC1和半凝集素-3可能协同调节TGF-B激活和aVB6介导的上皮细胞迁移。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：在伤口修复过程中，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、配体接合和迁移的影响：aVB6运输将使用生化内吞噬/回收分析。aVB6聚集和配体结合将通过免疫荧光分析，使用活化报告的aVB6抗体。触致上皮细胞迁移将在抓伤试验和纤维细胞衍生基质上进行分析。将使用生物工程机械调谐的模拟糖蛋白和皮肤上皮细胞中的MUC1外结构域/细胞结构域突变体来分析MUC1压缩张力或信号的作用。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。使用抗aVB6阻断抗体确定aVB6结合的作用。2)分析muc1 -半乳糖凝集素-3是否调节avb6介导的力施加和TGF-B激活：使用潜伏期相关肽作为配体的牵引力显微镜（TFM）和TGF-B1活性荧光素报告试验将证明muc1 -半乳糖凝集素-3的生物物理和信号特性是否调节1)avb6介导的机械力对潜在TGF-B的应用，以及2)avb6依赖性TGF-B的激活。3)检测muc1 -半乳糖凝集素-3相互作用是否协同aVB6漏斗驱动TGF-B活化：地形扫描角度干涉显微镜（T-SAIM）；使用MUC1压缩（压缩应变计传感器）的FRET报告和aVB6激活报告抗体分析膜变形。计算模型将绘制单个MUC1分子内的压缩张力，相对于活性和非活性aVB6。

We will analyse crosstalk mechanisms between integrins and growth factor receptors (GFRs) that promote wound healing and drive scar formation. TGF-B and integrin aVB6 cooperate to drive wound healing by inducing re-epithelialisation, myofibroblast activation, wound contraction and matrix remodelling. Integrin aVB6 activates TGF-B by binding and applying forces to latent TGF-B, inducing a structural change that releases the potent cytokine. Consequently, genetic deletion or inhibition of aVB6 or TGF-B leads to pronounced wound healing defects in vivo. However, despite the fundamental role of aVB6 in tissue regeneration, the molecular basis for aVB6-mediated wound repair is unresolved.We found that: 1) aVB6 signalling complexes recruit a mucin-galectin-3 regulatory module; 2) MUC1-galectin-3 interaction regulates GFR clustering and trafficking. In vivo, MUC1 and galectin-3 promote wound repair and our iCASE partner has developed galectin-3-targeting drugs that inhibit TGF-B activity in in vivo models of fibrosis. These data suggest that aVB6, MUC1 and galectin-3 may co-operate to regulate TGF-B activation and aVB6-mediated epithelial cell migration.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 wound repair.Objectives & Experimental Approach1) Determine impact of MUC1-galectin-3 interaction on aVB6 trafficking, ligand engagement and migration:aVB6 trafficking will be analysed using biochemical endocytosis/recycling assays. aVB6 clustering and ligand-binding will be analysed by immunofluorescence using activation-reporting aVB6 antibodies. Haptotactic epithelial cell migration will be analysed in scratch wound assays and on fibrillar cell-derived matrices.Contributions of MUC1 compressive tension or signalling will be analysed using bio-engineered mechanically-tuned glycoprotein-mimetics and MUC1 ectodomain/cytodomain mutants in skin epithelial cells. Involvement of MUC1-galectin-3 interaction, will be assessed in presence/absence of galectin-3 or galectin-3-targeting drugs. Role of aVB6 engagement will be determined using anti-aVB6 blocking antibodies.2) Analyse whether MUC1-galectin-3 regulates aVB6-mediated force application and TGF-B activation:Traction Force Microscopy (TFM) using latency-associated peptide as a ligand and TGF-B1 activity luciferse-reporter assays will demonstrate whether the biophysical and signalling properties of MUC1-galectin-3 regulate 1) application of aVB6-mediated mechanical forces on latent TGF-B, and 2) aVB6-dependent TGF-B activation.3) Test whether MUC1-galectin-3 interaction co-ordinates aVB6 funnelling to drive TGF-B activation:Topographical-Scanning Angle Interference Microscopy (T-SAIM); analysing membrane deformation using FRET reporters of MUC1 compression (compressive-strain gauge sensors) and aVB6 activation-reporting antibodies. Computational modelling will map compressive tension within individual MUC1 molecules, relative to active and inactive aVB6.