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

我们将分析促进伤口愈合和驱动瘢痕形成的整合素和生长因子受体（GFR）之间的串扰机制。TGF-β和整联蛋白aVB 6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协同驱动伤口愈合。整联蛋白aVB 6通过结合并向潜伏的TGF-β施加力来激活TGF-β，诱导释放有效细胞因子的结构变化。因此，aVB 6或TGF-β的遗传缺失或抑制导致体内明显的伤口愈合缺陷。然而，尽管aVB 6在组织再生中的基本作用，但aVB 6介导的伤口修复的分子基础尚未解决。我们发现：1）aVB 6信号传导复合物募集粘蛋白-半乳糖凝集素-3调节模块; 2）MUC 1-半乳糖凝集素-3相互作用调节GFR聚集和运输。在体内，MUC 1和半乳糖凝集素-3促进伤口修复，我们的iCASE合作伙伴已经开发出半乳糖凝集素-3靶向药物，在体内纤维化模型中抑制TGF-B活性。这些数据表明，aVB 6，MUC 1和半乳糖凝集素-3可能合作，以调节TGF-β的激活和aVB 6介导的上皮细胞迁移。MUC 1的空间限制整合素的激活;庞大的糖蛋白漏斗受体进入粘附和施加压缩张力，以促进整合素的激活。这种整合素激活的新范例导致我们的假设：半乳糖凝集素-3和MUC 1协调aVB 6聚类，以驱动创伤修复过程中的TGF-β activation.Objectives & Experimental Approach 1）确定MUC 1-半乳糖凝集素-3相互作用对aVB 6运输，配体接合和迁移的影响：aVB 6运输将使用生化内吞/再循环测定进行分析。aVB 6聚类和配体结合将通过免疫荧光使用激活报告aVB 6抗体进行分析。触觉上皮细胞迁移将在划痕试验和纤维细胞衍生的matrix.Contributions的MUC 1压缩张力或信号将使用生物工程机械调谐糖蛋白模拟物和MUC 1胞外域/胞质结构域突变体在皮肤上皮细胞分析。将在存在/不存在半乳糖凝集素-3或半乳糖凝集素-3靶向药物的情况下评估MUC 1-半乳糖凝集素-3相互作用的参与。aVB 6接合的作用将使用抗aVB 6阻断抗体来确定。2）分析MUC 1-半乳糖凝集素-3是否调节aVB 6介导的力施加和TGF-β激活：使用潜伏相关肽作为配体和TGF-β 1活性探针报告基因测定的牵引力显微镜（TFM）将证明MUC 1-半乳糖凝集素-3的生物物理和信号传导性质是否调节1）aVB 6介导的机械力对潜伏TGF-β的施加，和2）aVB 6依赖性TGF-β激活。3）测试MUC 1-半乳糖凝集素-3相互作用是否协调aVB 6漏斗化以驱动TGF-β激活：拓扑扫描角干涉显微镜（T-SAIM）;使用MUC 1压缩的FRET报告物（压缩应变计传感器）和aVB 6激活报告抗体分析膜变形。计算建模将绘制相对于活性和非活性aVB 6的单个MUC 1分子内的压缩张力。

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.