The Role of the Discoidin Domain Receptor-1 (DDR1) in Vascular Smooth Muscle Cell (VSMC) Mechanosensing

Mechanotransduction is the process by which extracellular physical cues are converted into intracellular biochemical responses. The stiffness of the matrix influences numerous processes such as cell differentiation, cell migration, fibrosis, and tumorigenesis. Stiffness is sensed by matrix-binding receptors and transmitted through the cytoskeleton by actomyosin contractility. The work presented in this thesis identifies how Discoidin Domain Receptor-1 (DDR1) mediates stiffness sensing and how this influences vascular smooth muscle cell (VSMC) calcification. In the first study, experiments in primary murine Ddr1+/+ or Ddr1-/- VSMCs revealed that DDR1 promotes VSMC calcification in a stiffness-dependent manner by a positive feedback loop between DDR1 and actomyosin contractility. Increased matrix stiffness promoted increased nuclear RUNX2 levels, increased DDR1 activation and expression, increased Vav2-RhoA activity, and increased stress fibre formation in Ddr1+/+ but not Ddr1-/- VSMCs. RhoA activation increased DDR1 expression and activity independent of ligand, whereas inhibition of RhoA and actomyosin contractility were inhibitory. RUNX2 activity and expression, and VSMC calcification were increased by actomyosin contractility. The second study shows that regulation of stiffness-sensitive genes is mediated by a DDR1-YAP/TAZ nuclear complex. Ddr1-/- VSMCs had reduced nuclear YAP/TAZ, and loss of YAP/TAZ resulted in decreased DDR1 expression. DDR1 activation and increased substrate stiffness promoted DDR1 and YAP/TAZ nuclear localization. DDR1 and YAP/TAZ nuclear translocation were dependent on actomyosin contractility and caveolae. Increased stiffness, collagen stimulation, and RhoA activation all promoted DDR1 and YAP/TAZ localization to the chromatin. Collagen stimulation and RhoA activation stimulated the formation of a DDR1-YAP/TAZ-RNA polymerase II nuclear complex. Finally, DDR1 was found to localize to the promoters of Ddr1 and the canonical YAP/TAZ target gene Ctgf. In summary, these studies show a key role for DDR1 as a stiffness sensor in vascular smooth muscle cells to influence vascular pathologies like vascular calcification. DDR1 can mediate mechanosignaling via a positive feedback loop between DDR1 and actomyosin contractility, and through direct regulation of mechanosensitive genes like Ddr1 itself by a nuclear DDR1-YAP/TAZ complex. This research highlights the role of matrix stiffness in promoting vascular calcification, but also uncovers novel downstream pathways by which DDR1 mechanosignaling occurs at the membrane and in the nucleus.