Flow-dependent Smad2 phosphorylation and TGIF nuclear localization in human aortic endothelial cells.

Endothelial cells respond to fluid flow stimulation through transient and sustained signal pathway activation. Smad2 is a signaling molecule and transcription factor in the Smad signaling pathway, traditionally associated with TGF-ß. Although phosphorylation of Smad2 in the receptor-dependent COOH-terminal region is the most appreciated way Smad2 is activated to affect gene expression, phosphorylation may also occur in the MH1-MH2 linker region (L-psmad2). Here, we show that in human aortic endothelial cells (HAEC), Smad2 was both preferentially phosphorylated in the linker region and localized to the nucleus in a flow-dependent manner. The Smad corepressor transforming growth interacting factor (TGIF) was also found to have flow-dependent nuclear localization. Tissue studies confirmed this L-psmad2 generation trend in rat aorta, indicating likely importance in arterial tissue. HAEC-based inhibitor studies demonstrated that L-psmad2 levels were not related to MAPK phosphorylation, but instead followed the pattern of pAkt(473), both with and without the phosphatidylinositol 3-kinase inhibitor PI-103. Akt and Smad species were also shown to directly interact under flow relative to static controls. To further evaluate impacts of PI-103 treatment, expression profiles for two TGF-ß and shear stress-dependent genes were determined and showed that mRNAs were lower from untreated 10 dyn/cm(2) than 2 dyn/cm(2) average shear stress cultures. However, upon exposure to PI-103, this trend was reversed, with a stronger response observed at 10 dyn/cm(2). Taken together, the results of this work suggest that fluid flow exposure may influence endothelial gene expression by a novel mechanism involving Akt, L-psmad2, and TGIF.

Long term shear stress leads to increased phosphorylation of multiple MAPK species in cultured human aortic endothelial cells.

Fluid dynamics strongly influences endothelial cell function, and participates in the localization of atherosclerotic plaques at blood vessel branches. We investigated the hypothesis that wild-type human aortic endothelial cells (HAEC) exposed to prolonged pulsatile flow stimulation have levels of phosphorylated mitogen-activated protein kinases (MAPK) that are significantly greater than those observed in statically grown cultures. HAEC were exposed to pulsatile laminar shear stress in a parallel-plate flow chamber and analyzed for levels of phosphorylated ERK, JNK and p38 at 1, 10 and 20 h. While some MAPK exhibited alternating patterns of phosphorylation, others were characterized by steady increases or unchanged profiles until the terminal (20 h) time point. However, at 20 h, each MAPK demonstrated an increase in phosphorylation versus statically cultivated cells. Further, 20 h cultures from 10 dyn/cm(2) pulsatile shear stress had higher levels of phosphorylation for each MAPK than those from 2 dyn/cm(2). The finding that MAPK species can be phosphorylated in response to a prolonged pulsatile shear stress in both a time and magnitude dependent manner is an interesting result that may help to explain how the differential behaviors observed between cells from different flow environments can be generated and maintained.