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Abstract
This study was designed to examine the cellular and molecular response of tendon fibroblasts to growth/differentiation factor-5 (GDF-5). Rat Achilles tendon fibroblasts (ATFs) were treated in culture with varying concentrations of GDF-5 (0–1000 ng/ml) over varying periods of time (0–12 days). Cell proliferation, evaluated through use of a standard MTT colorimetric assay, confirmed that GDF-5 stimulates ATF proliferation in a concentration- and time-dependent fashion. Temporal and concentration analysis revealed that GDF-5 increases total DNA, glycosaminoglycan (GAG), and hydroxyproline (HYP) content. Ratios of HYP/DNA and GAG/DNA increased with increasing concentrations of GDF-5 (0–1000 ng/ml). Expression of the following 12 extracellular matrix (ECM) and cell-adhesion-related genes was assessed using real-time reverse transcriptase polymerase chain reaction (RT-PCR): collagen I (col I), collagen III (col III), matrix metalloproteinases (MMP)-3 and -13, aggrecan, tissue inhibitor of matrix metalloproteinase (TIMP)-2, syndecan-4, N-cadherin, tenascin-C, biglycan, versican, and decorin. RT-PCR data revealed an increase in the expression of col I, col III, MMP-3, MMP-13, TIMP-2, syndecan-4, N-cadherin, tenascin-C, and aggrecan genes by day 6. A statistically significant decrease in TIMP-2 and MMP-13 was observed on day 12. Decorin expression was depressed at all time points in cells treated with GDF-5. There was no significant change in biglycan expression in ATFs supplemented with GDF-5. These findings suggest that GDF-5 induces cellular proliferation and ECM synthesis as well as expression of ECM and cell-adhesion-related genes in ATFs. This study further defines the influence of GDF-5 on rat ATFs through its action on the expression of genes that are associated with tendon ECM.