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This Article Full Text Full Text (PDF) All Versions of this Article: 296/4/L700    most recent 90566.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Johnson, A. Search for Related Content PubMed PubMed Citation Articles by Johnson, A.

TNF-induced activation of pulmonary microvessel endothelial cells: a role for GSK3β

Department of Pharmaceutical Science, Albany College of Pharmacy and Health Sciences, and Research Service of the Stratton Veterans Affairs Medical Center, Albany, New York

Submitted 17 November 2008 ; accepted in final form 9 February 2009

The hypothesis tested was PKC mediates the phosphorylation of glycogen synthetase kinase 3β (GSK3β) and that the GSK3β inhibition modulates the response to tumor necrosis factor- (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 PKC in the phosphorylation of GSK3β (i.e., an indicator of GSK3β inhibition), PMEC were pretreated with 1) nonsense-RNA-PKC, 2) siRNA-PKC, and 3) the PKC inhibitor Gö6983. In the nonsense RNA-PKC+TNF and TNF groups, there was increased phosphorylated GSK3β-Ser9 that did not occur in the Gö6983+TNF group. In the TNF groups, there was a significant correlation between PKC protein and phosphorylated GSK3β-Ser9 that did not occur in the groups without TNF. Second, to assess the role of GSK3β in β-catenin activity, PMEC were pretreated with 1) wild-type (w) GSK3β plasmid to enhance GSK3β activity, 2) kinase dead (kd)-GSK3β plasmid, and 3) the GSK3β inhibitor SB-216763. In the TNF group, there was increased unphosphorylated β-catenin-Ser37/33 compared with the control group. In the GSK3β-inhibited groups (i.e., SB-216763 and kdGSK3β) ± TNF, the unphosphorylated β-catenin-Ser37/33 was similar to the TNF group. In the GSK3β-enhanced group ± TNF, the unphosphorylated β-catenin-Ser37/33 was similar to the control. Finally, PMEC were also treated with TOPflash, a β-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 GSK3β-inhibited groups (i.e., SB-216763 and kdGSK3β) ± TNF, the TOPflash/FOPflash activity ratio was similar to the TNF group. In the GSK3β-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 PKC-dependent inhibition of GSK3β.

glycogen synthetase kinase; permeability