Development of a synthetic gene network to modulate gene expression by mechanical forces

Mechanotransduction, the ability of cells to sense, process and respond to mechanical forces, is increasingly recognised to play a crucial role in a variety of physiological processes including tissue formation and regeneration, angiogenesis, hearing and sensing1,2,3. In addition to physiological processes, mechanical forces have been shown to play an important role in diseases such as osteoarthritis, atherosclerosis, tumour growth and metastasis4,5,6,7,8,9,10,11,12,13,14,15. Mechanotransduction starts with the sensing of mechanical force by a group of cell membrane molecules, collectively known as mechanosensors7. These mechanosensitive molecules include G-protein coupled receptors (G-PCRs), ion channels and receptor tyrosine kinases16. Following sensing, the mechanical signal is converted into biochemical signals inside the cell, which then activate a cascade of signalling events, ultimately leading to gene expression up- or down-regulation. For example, in the case of endothelial shear stress sensing, it is estimated that 1000–2000 genes are involved in the mechanotransduction process, regulating 8–10 transcription factors15,17,18,19,20,21. Despite the emerging importance of the mechanotransduction field, specific interventions to modulate mechanotransduction are currently not available, substantially hampering progress towards treatment of the above mentioned mechanosensitivity-related diseases. Thus, there is a need to investigate how varying levels of a particular mechanical stimulus affect the degree of activation or deactivation of signaling molecules within mechanotransduction signaling pathways. Additionally, it is of crucial importance to be able to modulate gene expression (e.g., therapeutic genes and transcription factors) by mechanical input signals in cells exposed to particular (pathological) mechanical environments. To address these needs, we developed a novel synthetic gene network to monitor shear stress sensing activity and regulate gene expression in a graded manner in mammalian cells. The network was inserted into cells via a bespoke hybrid transfection procedure that overcame difficulties in inserting large DNA constructs into hard-to-transfect mammalian cells. Once transfected, cells were seeded into a custom flow chamber that imposed a range of linearly increasing shear stress over the length of the chamber, which demonstrated increased activation of the transfected gene network as measured by GFP signal with increased shear stress. Future work will employ this modular network to monitor pharmaceutical targets of specific mechanotransduction molecules to attenuate pathological mechanical signaling.