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Title: TGF-β mediated synthesis of degradation-resistant collagen (I) homotrimer in musculoskeletal cells
Author: Gumbs, J. A.
ISNI:       0000 0004 7970 4819
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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Type I collagen is the most abundant structural protein in vertebrates. It is the main component of the extracellular matrix in the majority of musculoskeletal tissues, including bone, muscle, tendon and ligament. Type I collagen usually occurs as a heterotrimer, of two alpha-1 chains and one alpha-2 chain (a1(I))2(a2(I)) encoded by the COL1A1 and COL1A2 genes. However, type I collagen can form homotrimers (a1(I))3 which are resistant to metalloproteinase (MMP) proteolytic cleavage and have been identified in age-related musculoskeletal diseases and fibrosis. Transforming growth factor beta (TGF-ß) is a key fibrogenic cytokine which enhances collagen gene expression, while its effect on homotrimer formation is unknown. The overall aims of the research presented in this thesis, were to analyse the production of type I collagen heterotrimer and homotrimer in response to TGF-b in musculoskeletal cell types, to develop an MMP-1 cleavage protocol for newly synthesised collagenous proteins and to compare and contrast the transcription of regulatory regions of COL1A1 and COL1A2. The methylation status of the proximal promoter of fibrotic and normal cells that had been treated with TGF-b was also examined. TGF-b stimulation was found to not only increase type I collagen production, but also lead to alterations in collagen isoform biosynthesis. Primary cells were isolated and assessed from human, equine and mouse musculoskeletal tissues. In equine energy storing superficial digital flexor tendons(SDFT), there was an age-related increase in the a1(I) chain relative to a2(I) chain at a gene and protein level in response to TGF-ß. The ratio of COL1A1 to COL1A2 mRNA also increased with TGF-b stimulation in mouse tail tenocytes stimulated in vitro. TGF-b treated human Dupuytren disease (DD) fibroblasts show a significant increase in COL1A1 to COL1A2 relative to carpal tunnel cells (as a non-fibrotic control) alongside alterations in the ability to produce fully processed type I collagen. A MMP-1 proteolytic cleavage protocol was developed in order to identify newly synthesised metabolically labelled collagen homotrimer from cells stimulated with TGF-b in vitro. DNA methylation analysis of the proximal promoters near the transcription start site revealed that both COL1A1 and COL1A2 were unmethylated in DD and carpal tunnel cells. The work presented provides greater insight into TGF-b mediated collagen synthesis and the relative transcriptional regulation of type I collagen genes. These results indicate that TGF-b is able to stimulate the production of type I collagen homotrimer in both fibrotic cells and in older cells in vitro.
Supervisor: Laird, Elizabeth ; Clegg, Peter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral