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-β. While phosphorylation of Smad2 in the receptor dependent C-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 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 PI3K 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-β and shear stress dependent genes were determined, and showed that mRNAs were lower from untreated 10 dyn/cm2 than 2 dyn/cm2 average shear stress cultures. However, upon exposure to PI-103, this trend was reversed, with a stronger response observed at 10 dyn/cm2. 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.
- mitogen activated protein kinases
- Copyright © 2010, American Journal of Physiology - Heart and Circulatory Physiology