title: Flow-dependent Smad2 phosphorylation and TGIF nuclear localization in human aortic endothelial cells.
abstract: 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. 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-β 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.
authors.affiliation: Univ. of Calgary, 2500 Univ. Dr. N.W., Centre for Bioengineering Research and Education, CCIT 208, Calgary AB, Canada T2N 1N4.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Shepherd
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Kos
authors.forename: Kristina D
authors.initials: KD
authors.lastname: Rinker
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005663:Furans / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D051058:Oncogene Protein v-akt / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D012097:Repressor Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051899:Smad2 Protein / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D005663:Furans; D018398:Homeodomain Proteins; D008565:Membrane Proteins; C522973:PI103; D011725:Pyridines; D011743:Pyrimidines; D012097:Repressor Proteins; C497782:SMAD2 protein, human; D051899:Smad2 Protein; C097504:TGIF1 protein, human; D016212:Transforming Growth Factor beta; D012313:RNA; D051058:Oncogene Protein v-akt; D020928:Mitogen-Activated Protein Kinases