Abstract 14925: Mechanical Tension in Syndecan-1 is Regulated by Extracellular Mechanical Cues and Fluidic Shear Stress

Abstract

Syndecan-1 (SDC1) is a transmembrane proteoglycan that mediates the shear stress-induced signaling and inflammatory phenotypes of endothelial cells. While SDC1 is known to be involved mechanically in regulating the behavior of cells, it remains unknown how SDC1 responds to extracellular mechanical cues on the molecular level. We designed a set of FRET-based SDC1 tension sensors including full-length SDC1 (SDC1TS), SDC1 with deleted ectodomain (ΔEcto), SDC1 with deleted glycosylation sites (ΔGAG) and SDC1 with a deleted cytoplasmic tail (ΔCyto). We transduced these constructs into ECs and validated the constructs by WB and confocal imaging. When cultured on glass-bottom coverslips, we found that the baseline tension in SDC1TS was significantly higher than that in Ecto and GAG mutants. When cultured on engineered substrates, we demonstrated that tension in SDC1 is modulated by micropatterned surfaces and nanotopographical cues. To study if SDC1 directly responds to substrate stiffness, transduced ECs were cultured on 0.2 kPa and 25 kPa substrates. We found that there was significantly decreased tension in the Ecto construct compared to the full-length SDC1TS construct on 0.2 kPa substrates but no significant changes were observed on the 25 kPa substrates. To examine the effect of substrate stiffness on the association of SDC1 with adhesion-related proteins, we performed IP and WB on ECs expressing HA-tagged SDC1 cultured on 0.2 kPa or 25 kPa substrates. We found there were increases in SDC1 binding to action and myosin IIb, while there were decreased in SDC1 binding to Src, PKA, and FAK in cells grown on stiff substrates versus on soft substrate. To study the effect of shear stress on the tension of SDC1, ECs expressing the constructs were cultured on static or under 12dyn/cm 2 fluidic shear stress. We found that a gradient developed in the tension of SDC1 in all four constructs. There was higher tension of SDC1 in the upstream region in comparison to the downstream region. IP and WB results showed that shear stress-induced SDC1 association with actin, Src, myosin IIb, cortactin, calmodulin, and integrin beta3. In summary, our results demonstrate that SDC1 is mechanically responsive to substrate mediated biophysical cues and shear stress.