J Appl Physiol 91: 1487C1500, 2001 [PubMed] [Google Scholar] 13
J Appl Physiol 91: 1487C1500, 2001 [PubMed] [Google Scholar] 13. ERM activation. In the present study we test the hypothesis that ERM proteins are phosphorylated on this critical threonine residue by thrombin-induced signaling events and explore the role of the ERM family in modulating thrombin-induced cytoskeletal rearrangement and EC barrier function. Thrombin promotes ERM phosphorylation at this threonine residue (ezrin Thr567, radixin Thr564, moesin Thr558) in a PKC-dependent fashion and induces translocation of phosphorylated ERM to the EC periphery. Thrombin-induced ERM threonine phosphorylation Rabbit polyclonal to PACT is likely synergistically mediated by protease-activated receptors PAR1 and PAR2. Using the siRNA approach, depletion of either moesin alone or of all three ERM proteins significantly attenuates thrombin-induced increase in EC barrier permeability (transendothelial electrical resistance), cytoskeletal rearrangements, paracellular gap formation, and accumulation of phospho-myosin light chain. In contrast, radixin depletion exerts opposing effects on these indexes. These data suggest that ERM proteins play important differential roles in the thrombin-induced modulation of EC permeability, with moesin promoting barrier dysfunction and radixin opposing it. ezrin: 5-AACACCGTGGGATGCTCAAAG-3, duplex of sense 5-GAAAUAACCCAGAGACUCUdTdT-3 and antisense 5-AGAGUCUCUGGGUUAUUUCdTdT-3 was used for Desoxyrhaponticin targeting sequences that are part of the coding region for radixin: 5-AAGAAATAACCCAGAGACTCT-3, and duplex of sense 5-GGGAUGUCAACUGACCUAAdTdT-3 and antisense 5-UUAGGUCAGUUGACAUCCCdTdG-3 was used for targeting sequences that are part of the coding region for moesin: 5-CAGGGATGTCAACTGACCTAA-3. Duplex of sense 5-AGAGCUAAG-UAGAUGUGUAdTdT-3 and antisense 5-UACACAUCUACUUAGCUCUdTdG-3 siRNA was used Desoxyrhaponticin for targeting sequences that are part of the coding region for PKCI: 5-CAAGAGCTAAGTAGATGTGTA-3, duplex of sense 5-GAAGCAUGACAGCAUUAAA dTdT-3 and antisense 5-UUUAAUGCUGUCAUGCUUCdCdG-3 was used for targeting sequences that are part of the coding region for PKC: 5-CGGAAGCATGACAGCATTAAA-3, duplex of sense 5-CUCUACCGUGCCACGUUUUdTdT-3 and antisense 5-AAAACGUGGCACGGUAGAGdTdT-3 was used for Desoxyrhaponticin targeting sequences that are part of the coding region for PKC: 5-AACTCTACCGTGCCACGTTTT-3, duplex of sense 5-CAAGAAGUGUAUUGAUAAAdTdT-3 and antisense 5-UUUAUCAAUACACUUCUUGdTdG-3 was used for targeting sequences that are part of the coding region for PKC: 5-CACAAGAAGTGTATTGATAAA-3, duplex of sense 5-CGGAAACACCCGUACCUUAdTdT-3 and antisense 5-UAAGGUACGGGUGUUUCCGdTdG-3 was used for targeting sequences that are part of the coding region for PKC: 5-CACGGAAACACCCGTACCTTA-3. Silencer select predesigned siRNA duplex (Life Technologies, Grand Island, NY) of sense 5-CCCGUAACCUAAUUCCUAUdTdT-3 and antisense 5-AUAGGAAUUAGGUUACGGGdCdC-3 was used for targeting sequences that are part of the coding region for PKC: 5-GGCCCGTAACCTAATTCCTAT-3. Nonspecific, nontargeting AllStars siRNA duplex (Qiagen) was used as negative control treatment. HPAEC were grown to 70% confluence, and the transfection of siRNA (final concentration 50 nM) was performed by using DharmaFECT1 transfection reagent (Dharmacon Research, Lafayette, CO) according to manufacturer’s protocol. Forty-eight hours posttransfection cells were harvested and used for experiments. Additional control experiments using EC transfections with fluorescently labeled nonspecific RNA showed that this protocol Desoxyrhaponticin allowed us to achieve 90C100% transfection efficiency. Plasmid constructs. Moesin constructs (wild-type and phosphorylation-deficient mutant) were prepared as we Desoxyrhaponticin have previously described (9). Immunofluorescent staining. EC were plated on glass coverslips, grown to 70% confluence, and transfected with siRNA followed by stimulation with thrombin. Then cells were fixed in 3.7% formaldehyde solution in PBS for 10 min at 4C, washed three times with PBS, permeabilized with 0.2% Triton X-100 in PBS-Tween (PBST) for 30 min at room temperature and blocked with 2% BSA in PBST for 30 min. Incubation with antibody of interest was performed in blocking solution for 1 h at room temperature followed by staining with either Alexa 488- or Alexa 594-conjugated secondary Ab (Molecular Probes). Actin filaments were stained with Texas red-conjugated phalloidin (Molecular Probes) for 1 h at room temperature. After immunostaining, the glass slides were analyzed by using a Nikon video-imaging system (Nikon Instech, Japan) consisting of a phase-contrast inverted microscope Nikon Eclipse TE2000 connected to Hamamatsu (Hamamatsu Photonics, Japan) digital camera and image processor. The images were recorded and processed with Adobe Photoshop 6.0. Immunoblotting. Protein extracts were separated by.