Abstract
Introduction
Tendons are critical to mobility, and are susceptible to degeneration through injury and ageing. Type I collagen is the most abundant protein in vertebrates; it is the main structural protein of the extracellular matrix in numerous musculoskeletal tissues, including tendons. Type I collagen predominantly is a heterotrimer, which consists of two alpha-1 chains and one alpha-2 chain (α1)2(α2) encoded by the COL1A1 and COL1A2 genes, respectively. However, type I collagen can form homotrimers (α1)3 which are protease-resistant, and are associated with age-related musculoskeletal diseases, fibrotic and connective tissue pathologies. Transforming growth factor beta (TGFβ) enhances collagen (I) gene expression, is involved in tendon mechanobiology and repair processes, while its effect on homotrimer formation is unknown. Our aim is to investigate the relative expressions of collagen (I) α1 and α2 polypeptide chains in tenocytes (tendon fibroblasts) stimulated with TGFβ.
Materials and Methods
Included RT-qPCR to measure the relative expression of COL1A1 and COL1A2 genes. [14C]-proline metabolic labelling was used to measure the expression of the collagen (I) α1 and α2 polypeptide chains. These techniques were performed in equine superficial digital flexor tendon (SDFT) tenocytes (n=3) and murine tail tendon tenocytes (n=3) with different concentrations of TGFβ (0.01 ng/ml-100 ng/ml).
Results
There was an increase in both COL1A1 and COL1A2 gene expression when stimulated with TGFβ in both cell types. In equine tenocytes the gene expression ratio of COL1A1:COL1A2 increased from 1.73 ± 0.75 to 7.87 ± 2.9 (p=0.003) when stimulated with 100 ng/ml of TGFβ3. TGFβ upregulated collagen (I) protein in both cell types. In equine tenocytes (n=3) when stimulated with 100 ng/ml of TGFβ3, the α1:α2 protein chain ratio increased from 1.93 ± 0.54 to 3.02 ± 0.32 (p=0.059) in comparison with serum-starved cells, which alongside the changes in gene expression, may be indicative of collagen (I) homotrimer production.
Discussion
There were biosynthetic alterations in collagen production, and putative collagen (I) homotrimer when equine tenocytes were stimulated with 100 ng/ml TGFβ3. Future work will focus isolating different collagens by repeated differential salt precipitation. The level of TGFβ receptors and Smad signaling molecules will be also analysed using RT-qPCR and western blotting.
