Institute for Liver and Digestive Health, Division of Medicine, University College London, Rowland Hill Street, London NW3 2PF, UK.
Int J Mol Sci. 2021 Jan 20;22(3):984. doi: 10.3390/ijms22030984.
Excessive cross-linking is a major factor in the resistance to the remodelling of the extracellular matrix (ECM) during fibrotic progression. The role of TGFβ signalling in impairing ECM remodelling has been demonstrated in various fibrotic models. We hypothesised that increased ECM cross-linking by TGFβ contributes to skin fibrosis in Systemic Sclerosis (SSc). Proteomics was used to identify cross-linking enzymes in the ECM of primary human dermal fibroblasts, and to compare their levels following treatment with TGFβ-1. A significant upregulation and enrichment of lysyl-oxidase-like 1, 2 and 4 and transglutaminase 2 were found. Western blotting confirmed the upregulation of lysyl hydroxylase 2 in the ECM. Increased transglutaminase activity in TGFβ-1 treated ECM was revealed from a cell-based assay. We employed a mass spectrometry-based method to identify alterations in the ECM cross-linking pattern caused by TGFβ-1. Cross-linking sites were identified in collagens I and V, fibrinogen and fibronectin. One cross-linking site in fibrinogen alpha was found only in TGFβ-treated samples. In conclusion, we have mapped novel cross-links between ECM proteins and demonstrated that activation of TGFβ signalling in cultured dermal fibroblasts upregulates multiple cross-linking enzymes in the ECM.
过度交联是纤维化进展过程中细胞外基质（ECM）重塑抵抗的一个主要因素。TGFβ 信号在各种纤维化模型中损害 ECM 重塑的作用已得到证实。我们假设 TGFβ 引起的 ECM 交联增加导致系统性硬化症（SSc）中的皮肤纤维化。蛋白质组学用于鉴定原代人真皮成纤维细胞 ECM 中的交联酶，并比较 TGFβ-1 处理后它们的水平。发现赖氨酸氧化酶样 1、2 和 4 和转谷氨酰胺酶 2 的显著上调和富集。Western blot 证实 ECM 中赖氨酸羟化酶 2 的上调。从基于细胞的测定中揭示了 TGFβ-1 处理的 ECM 中转谷氨酰胺酶活性的增加。我们采用基于质谱的方法来确定 TGFβ-1 引起的 ECM 交联模式的改变。在胶原蛋白 I 和 V、纤维蛋白原和纤维连接蛋白中鉴定了交联位点。纤维蛋白原 alpha 中的一个交联位点仅在 TGFβ 处理的样本中发现。总之，我们已经绘制了 ECM 蛋白之间的新交联图谱，并证明了培养的真皮成纤维细胞中 TGFβ 信号的激活上调了 ECM 中的多种交联酶。
