TGF-β1的分泌失调和细胞外激活刺激肌成纤维细胞积累无序和僵硬的细胞外基质(ECM),导致纤维化。纤连蛋白固定潜在的TGF-β结合蛋白-1(LTBP-1),从而将TGF-β1存储在ECM中。因为ED-A纤连蛋白剪接变体在纤维化期间显著表达并支持肌成纤维细胞活化,我们调查了ED-A是否促进LTBP-1-纤连蛋白相互作用。使用刚度可调的基质进行人真皮成纤维细胞培养,我们表明,高ECM刚度促进LTBP-1和含ED-A的纤连蛋白的表达和共定位。当用特定的纤连蛋白剪接变体拯救纤连蛋白耗尽的成纤维细胞时,LTBP-1与含ED-A的纤连蛋白的结合比与含ED-B的纤连蛋白和缺乏剪接结构域的纤连蛋白的结合更有效。使用抗体和竞争性肽对ED-A结构域的功能阻断导致LTBP-1与含ED-A的纤连蛋白的结合减少,减少LTBP-1掺入成纤维细胞ECM和减少TGF-β1激活。通过阻断肝素结合片段FNIII12-13-14(HepII)获得了类似的结果,与纤连蛋白中的ED-A结构域相邻。总的来说,我们的结果表明,ED-A结构域通过促进与LTBP-1的弱直接结合以及通过纤连蛋白中的HepII增强肝素介导的蛋白质相互作用,从而增强了潜伏TGF-β1的缔合作用.
Dysregulated secretion and extracellular activation of TGF-β1 stimulates myofibroblasts to accumulate disordered and stiff extracellular matrix (ECM) leading to fibrosis. Fibronectin immobilizes latent TGF-β-binding protein-1 (LTBP-1) and thus stores TGF-β1 in the ECM. Because the ED-A fibronectin splice variant is prominently expressed during fibrosis and supports myofibroblast activation, we investigated whether ED-A promotes LTBP-1-fibronectin interactions. Using stiffness-tuneable substrates for human dermal fibroblast cultures, we showed that high ECM stiffness promotes expression and colocalization of LTBP-1 and ED-A-containing fibronectin. When rescuing fibronectin-depleted fibroblasts with specific fibronectin splice variants, LTBP-1 bound more efficiently to ED-A-containing fibronectin than to ED-B-containing fibronectin and fibronectin lacking splice domains. Function blocking of the ED-A domain using antibodies and competitive peptides resulted in reduced LTBP-1 binding to ED-A-containing fibronectin, reduced LTBP-1 incorporation into the fibroblast ECM and reduced TGF-β1 activation. Similar results were obtained by blocking the heparin-binding stretch FNIII12-13-14 (HepII), adjacent to the ED-A domain in fibronectin. Collectively, our results suggest that the ED-A domain enhances association of the latent TGF-β1 by promoting weak direct binding to LTBP-1 and by enhancing heparin-mediated protein interactions through HepII in fibronectin.