Deciphering the Molecular Mechanisms of Stem Cell Mechanotransduction: A New Avenue for the Development of Therapeutics for Osteoporosis

Abstract

Osteoporosis is characterised by reduced bone density and weakened bone architecture leading to high fracture risk. The changes in bone result from an imbalance in the bone remodelling cycle whereby the rate of resorption increases along with a decrease in bone formation by MSC derived osteoblasts. The most common treatments for osteoporosis inhibit resorption and have adverse side effects. Bone tissue adapts to the surrounding mechanical environment, increasing bone formation in response to loading which requires the osteogenic differentiation of MSCs. It is appreciated that MSCs respond to mechanical stimuli but the mechanisms by which loading is transduced into MSC lineage commitment lacks understanding. The delineation of these mechanotransduction mechanisms is sought to identify novel anabolic therapeutic targets to correct net bone loss in osteoporosis. In the first study of this thesis, it was shown that fluid shear induced calcium signalling in MSCs is dependent on the transmembrane channel TRPV4. Furthermore, the fluid shear induced upregulation of the osteogenic gene expression is abrogated by antagonising TRPV4. Treating MSCs with the specific TRPV4 agonist, GSK101, can mimic the fluid shear induced calcium signal and upregulation of osteogenic matrix deposition, demonstrating the importance of TRPV4 in MSC osteogenic lineage commitment. Although TRPV4 is expressed throughout the MSC membrane, it is heavily localised to the primary cilium. MSCs where the cilium is abrogated show an altered response to biochemical TRPV4 activation suggesting an important role for the cilia-localised TRPV4. The second study sought to enhance primary cilium mediated MSC osteogenesis. Three known ciliotherapies were investigated of which both lithium chloride (LiCl) and fenoldopam enhanced MSC cilium incidence and length. LiCl negatively affected osteogenic markers while fenoldopam maintained or increased early expression. Both treatments increased the fluid shear induced Cox2 upregulation demonstrating enhanced mechanosensitivity. Fenoldopam upregulated loading-induced mineralisation and was determined as the optimal ciliotherapy to enhance osteogenic differentiation of MSCs. In the third study MSCs were isolated from the bone marrow aspirates of healthy and osteoporotic human donors and their bone regeneration capacity evaluated. Osteoporotic MSCs display an inhibited migration, an inhibited upregulation of COX2, OPN and RUNX2 in response to fluid shear as well as diminished proliferation. Furthermore, calcium deposition under biochemically induced osteogenic differentiation was decreased and the quality of osteogenic matrix was compromised in osteoporotic MSCs. Outlining deficits in the bone forming capacity of osteoporotic MSCs especially the reduced mechanoresponse in osteoporotic MSCs highlight potential targets for therapeutic design. The final study applied the protocols developed for the biochemical activation of the TRPV4 and primary cilium mechanotransduction to healthy and osteoporotic MSCs. Interestingly there is a loss in fluid shear-induced upregulation of TRPV4 in osteoporotic MSCs and osteoporotic cilium incidence and length are significantly diminished. Applying GSK101 maintained but did not enhance TRPV4 expression. The fenoldopam protocol elongated osteoporotic MSC primary cilia and displayed a similar trend in healthy counterparts. Moreover, GSK101 increased the fluid shear induced collagen formation of osteoporotic MSCs, while fenoldopam increased the fluid shear induced calcium formation in healthy MSCs. Together these results suggest that with further development targeting TRPV4 and the primary cilium could enhance the osteogenic response of MSCs to mechanical stimuli. In conclusion, this thesis has delineated two MSC mechanotransduction mechanisms through TRPV4 and the cilium, that can be targeted therapeutically. It was shown the MSCs have a diminished mechanosensitivity and regenerative potential in the setting of osteoporosis. Finally, it was shown that these new mechanotherapeutics show promise in correcting this defect in MSC behaviour, and with further optimisation, could be used to enhance bone formation, correcting net bone loss in osteoporosis.