TGFβ-1 Induced Cross-Linking of the Extracellular Matrix of Primary Human Dermal Fibroblasts

• Published in: International journal of molecular sciences 2021-01, Vol.22 (3), p.984
• Main Authors: Semkova, Mariya E, Hsuan, J Justin
• Format: Article
• Language: English
• Subjects: Amino Acid Oxidoreductases - metabolism; Cells, Cultured; Collagen; Collagen - chemistry; Collagen - metabolism; Cross-Linking Reagents - chemistry; Crosslinking; Cytokines; dermal fibroblasts; Dermis - cytology; Disease; Enzymes; Expected values; Experiments; Extracellular matrix; Extracellular Matrix - chemistry; Extracellular Matrix - drug effects; Extracellular Matrix - metabolism; Female; Fibrinogen; Fibrinogen - chemistry; Fibrinogen - metabolism; Fibroblasts; Fibroblasts - metabolism; Fibronectin; Fibronectins - chemistry; Fibronectins - metabolism; Fibrosis; Growth factors; GTP-Binding Proteins - metabolism; Humans; Hydroxylase; Hypotheses; Identification methods; lysyl hydroxylase 2; lysyl-oxidase-like enzymes; Mass spectrometry; Mass spectroscopy; Mortality; Procollagen-lysine 5-dioxygenase; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase - metabolism; Proteins; Proteomics; Resistance factors; Scleroderma; Skin; Systemic sclerosis; Transforming Growth Factor beta - metabolism; Transforming Growth Factor beta - pharmacology; Transglutaminase 2; transglutaminase cross-linking; transglutaminases; Transglutaminases - metabolism; Western blotting
• ISSN: 1422-0067; 1661-6596; 1422-0067
• 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.