Caveolae participate in tumor necrosis factor receptor 1 signaling and internalization in a human endothelial cell line.
Caveolae are abundant in endothelial cells (ECs) in situ but markedly diminished in cultured cells, making it difficult to assess their role in cytokine signaling. We report here that the human EC line EA.hy926 retains an abundant caveolar system in culture. Tumor necrosis factor (TNF) receptor 1 (TNFR1/CD120a) was enriched in caveolae and co-immunoprecipitated with caveolin-1 from caveolae isolated from these cells. To further investigate the role(s) of caveolae in TNF signaling in ECs, cells were treated with methyl-beta-cyclodextrin to disrupt caveolae. Methyl-beta-cyclodextrin did not alter total cell surface expression of TNFR1 or TNF-induced degradation of IkappaBalpha, a measure of nuclear factor-kappaB activation, but it did inhibit TNF-induced phosphorylation of Akt, a measure of phosphatidylinositol-3 kinase activation. Serum-induced phosphorylation of AKT was unaffected. Treatment with TNF induced disappearance of TNFR1 from caveolae and dissociation from caveolin-1 within 5 minutes. In contrast to transferrin receptor, internalized TNFR1 did not co-localize with clathrin, except possibly in the Golgi, at any time point examined. By 60 minutes of treatment with TNF, TNFR1 appeared in endosomes. We conclude that caveolae function in ECs to allow TNFR1 to activate phosphatidylinositol-3 kinase and Akt, perhaps through receptor cross talk, and that ligand-induced internalization and trafficking of TNFR1 to endosomes may originate directly from this compartment.
D'Alessio
Alessio
A
Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Ave., New Haven, CT 06536-0812, USA.
Al-Lamki
Rafia S
RS
Bradley
John R
JR
Pober
Jordan S
JS
D021941:Caveolae / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D020239:Receptor Cross-Talk / Q000502:physiology; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology
D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C494918:AKT1 protein, human