TNF-induced activation of pulmonary microvessel endothelial cells: a role for GSK3beta.
The hypothesis tested was PKCalpha mediates the phosphorylation of glycogen synthetase kinase 3beta (GSK3beta) and that the GSK3beta inhibition modulates the response to tumor necrosis factor-alpha (TNF) in rat pulmonary microvessel endothelial cells (PMEC). PMEC were treated with TNF for 4.0 h (100 ng/ml) or vehicle. First, to assess the role of PKCalpha in the phosphorylation of GSK3beta (i.e., an indicator of GSK3beta inhibition), PMEC were pretreated with 1) nonsense-RNA-PKCalpha, 2) siRNA-PKCalpha, and 3) the PKC inhibitor Gö6983. In the nonsense RNA-PKCalpha+TNF and TNF groups, there was increased phosphorylated GSK3beta-Ser9 that did not occur in the Gö6983+TNF group. In the TNF groups, there was a significant correlation between PKCalpha protein and phosphorylated GSK3beta-Ser9 that did not occur in the groups without TNF. Second, to assess the role of GSK3beta in beta-catenin activity, PMEC were pretreated with 1) wild-type (w) GSK3beta plasmid to enhance GSK3beta activity, 2) kinase dead (kd)-GSK3beta plasmid, and 3) the GSK3beta inhibitor SB-216763. In the TNF group, there was increased unphosphorylated beta-catenin-Ser37/33 compared with the control group. In the GSK3beta-inhibited groups (i.e., SB-216763 and kdGSK3beta) +/- TNF, the unphosphorylated beta-catenin-Ser37/33 was similar to the TNF group. In the GSK3beta-enhanced group +/- TNF, the unphosphorylated beta-catenin-Ser37/33 was similar to the control. Finally, PMEC were also treated with TOPflash, a beta-catenin-dependent promoter luciferase reporter, or the mutant construct FOPflash, 2 days before treatment with TNF. In the TNF group, there was an increased TOPflash/FOPflash activity ratio compared with the control group. In the GSK3beta-inhibited groups (i.e., SB-216763 and kdGSK3beta) +/- TNF, the TOPflash/FOPflash activity ratio was similar to the TNF group. In the GSK3beta-enhanced group +/- TNF, the TOPflash/FOPflash activity ratio was similar to the control. The data indicate that TNF induces endothelial activation that is modulated by a PKCalpha-dependent inhibition of GSK3beta.
Johnson
Arnold
A
Department of Pharmaceutical Science, Albany College of Pharmacy and Health Sciences, Albany, NY 12208, USA. arnie.johnson@acphs.edu
D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008168:Lung / Q000098:blood supply* / Q000166:cytology* / Q000201:enzymology; D008301:Maleimides / Q000494:pharmacology; D055806:Microvessels / Q000166:cytology* / Q000201:enzymology; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D051571:Protein Kinase C-alpha / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D012694:Serine / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D051176:beta Catenin / Q000378:metabolism
D007211:Indoles; D008301:Maleimides; C417521:SB 216763; D014409:Tumor Necrosis Factor-alpha; D051176:beta Catenin; D012694:Serine; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D051571:Protein Kinase C-alpha; D038362:Glycogen Synthase Kinase 3