Abstract
Summary Statement
We have shown that integrin mRNA expression is regulated by the application of mechanical load. This indicates that mechanical loading may modify cell sensitivity to perceive further load through increased interaction with the ECM.
Introduction
Tendinopathies are a range of diseases characterised by pain and insidious degeneration. Although poorly understood, onset is often associated with physical activity. We have previously investigated the regulation by mechanical strain of metalloproteinase gene expression in human tenocyte in a 3D collagen matrix. Integrins are important in cellular interaction with the ECM and are reported to mediate mechanotransduction in various non-tendon tissues. We have reported that TGFbeta activation is a key player in the regulation of metalloproteinases in response to mechanical load, which may be mediated by integrins. This project aims to investigate the effect of cyclic loading and TGFbeta stimulation on integrin expression by human tenocytes, in collagen and fibrin matrices.
Methods
Human tenocytes were seeded at 1.5×106 cells/ml into collagen (rat tail type I, 1mg/ml) or fibrin (fibrinogen 6mg/ml, Thrombin 0.2u/ml) gels and stretched using a sinusoidal waveform of 0–5% at 1Hz using the Flexcell FX4000T(trade mark) system. Cultures were treated with or without 1ng/ml TGFbeta1 and load for 0–48 hours. Taqman Low density Array was used to asses a range of integrin, including ITGA1-6, ITGA10 and ITGA11 as well as ITGB1-5 (n=3).
Results
In collagen cultures all integrins assayed were detectable (Ct < 35). ITGB1 was increased 2 fold with 48 hours of cyclic strain (p=0.006). ITGA6 and ITGA10 were decreased 1.4 and 2 fold with TGFbeta treatment after 24 hours (p=0.019, p=0.006). ITGA3 and ITGB3 were significantly decreased 7.6 and 8.3 fold with TGFbeta treatment after 48 hours (p=0.012, p=0.023). ITGA5 and ITGB1 showed similar responses with strain and TGFbeta, i.e. an increased trend. However, the other integrins showed a dissimilar response to strain and TGFbeta. Here we compare these responses to those in fibrin under the same conditions.
Discussion
We have shown that integrin mRNA expression is regulated by the application of mechanical load. This indicates that mechanical loading may modify cell sensitivity to perceive further load through increased interaction with the ECM. Any differences in the cellular response to load in collagen and fibrin cultures, indicates that cellular interaction with the ECM is an important factor in the detection of load. Due to the differential regulation of some of the integrins with strain and TGFbeta, it appears that TGFbeta may not be responsible for the regulation of all integrins with strain. However this remains unconfirmed and may be explained by a temporal difference. Further analysis of how integrins are regulated in response to mechanical load and how this expression is translated to the protein level is required.
