| Research Abstract |
We investigated signal transduction leading to shear stress responsive element-mediated transcription. Bovine endothelial cells were transfected with luciferase reporter vectors containing tandem repeats of TRE, CRE or NFkB element, and exposed to a shear stress of 15 dynes/cm^2. Transcriptional activity via each element began to increase significantly at 3 h after the onset of shear stress and increased further with time, peaking at 12h. These responses to shear stress were significantly suppressed by the treatment of cells with a tyrosine kinase inhibitor (Herbimycin A, 1 mM). Shear stress induced tyrosine phosphorylation of proteins of around 30, 45-65, and 120 kDa including focal adhesion kinase (FAK) in endothelial cells. Overexpression of FRNK (FAK-related non-kinase), which acts as an inhibitor of FAK, markedly inhibited the shear stress-induced increase in transcription via TRE, CRE or NFkB element. These results suggest that tyrosine kinase and FAK signaling are critical for s
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hear stress induction of TRE-, CRE- and NFkB element-mediated transcription in endothelial cells.
We also studied molecular mechanism of Ca^<2+> signaling, including the ion channels responsible for the shear stress-induced Ca^<2+> responses in endothelial cells. Human umbilical vein ECs (HUVECs) loaded with the Ca^<2+> indicator lndo-1/AM were exposed to laminar flow of Hanks' balanced salt solution (HBSS) at various concentrations of ATP and changes in intraceliular Ca^<2+> concentrations ([Ca^<2+>]i) were monitored by confocal laser scanning microscopy. A stepwise increase in flow rate elicited a corresponding stepwise-increase in [Ca^<2+>]i at 250 nmol/L ATP.The flow rate-dependent increase in [Ca^<2+>]i disappeared after the chelation of extracellular Ca^<2+> with EGTA.Antisense oligonucleotides designed to knockout P2X4 purinoceptor expression abolished the flow-induced Ca^<2+> influx. Human embryonic kidney 293 cells showed no Ca^<2+> response to flow at 2μmol/L ATP, but when transfected with P2X4 cDNA they began to show flow-rate-dependent Ca^<2+> influx. P2X4 purinoceptors may have a "shear-transducer" property by which shear stress is perceived directly or indirectly and transmitted into the cell interior via Ca^<2+> signaling. Less
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