Eglitis, Viesturs; (2020) Mechanoregulation of Adipogenesis and Replicative Senescence in Orbital Fibroblasts through SRC Family Kinases. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Graves orbitopathy is an autoimmune disorder; a complication encountered 40-50% of Graves disease patients. The initiation of the disease is caused by circulating antibodies recognising the thyroid-stimulating hormone receptor, expressed on the surface of orbital fibroblasts. Antibody attachment recruits immune cells initiating a localised immune response which is amplified during the disease. The chronic inflammation causes a phenotypical change in orbital fibroblasts, leading to increased hyaluronan synthesis, adipogenesis and subsequent orbital tissue scarring. Currently, therapeutic options to treat Graves orbitopathy are limited. Using a 3D fibroblast culture model, we previously identified that orbital fibroblasts isolated from patients with Graves orbitopathy spontaneously generate lipid droplets and are more contractile than their healthy counterparts. Both of these phenomenons are reduced by PP2, an Src family kinase (SFK) inhibitor. When downregulated, two SFK members Fyn and Src reduced orbital fibroblast contractility. Src knockdown dramatically increased lipid droplet production in 2D and 3D orbital fibroblast cultures while Fyn abolished it. Furthermore, reduction in Src expression decreased mTORc1 protein expression while reduction of Fyn increased AMPK protein expression and activity. We also discovered that spontaneous lipid droplet generation is specific to orbital fibroblasts cultured on collagen gels and substrates with an elastic modulus close to orbital soft tissue. Soft-gel cultures exhibited reduced Fyn, Src and mTORc1 protein expression and increased AMPK protein expression, and activity. We found that orbital fibroblasts when cultured on collagen gels express lysosomal and adipogenic markers which co-localised with lipid droplets. Our findings suggest that spontaneous lipid droplet production in orbital fibroblasts may occur due to impaired mechanosensing and at least partially are of autophagic origin. Orbital fibroblasts display increased proliferation when cultured on soft substrates and enter a senescence-like state when cultured on stiff substrates, characterised by β-galactosidase activity. Downregulation of Src and Fyn and activation of AMPK reduced this effect, demonstrating a potential functional overlap between signalling components of spontaneous lipid droplet production and replicative senescence. Overall our findings suggest that SFK activity is subject to substrate stiffness and contributes to lipid droplet production via mTORc1 and AMPK pathways. Moreover, the generation of lipid droplets is associated with autophagy.

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