Tumor necrosis factor (TNF) activates pro-inflammatory functions of vascular endothelial cells (EC) through

Tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2 is an intracellular adapter protein, which, upon TNF stimulation, is directly recruited to the intracellular region of TNF receptor 2 (TNFR2) or indirectly, via TRADD, to the intracellular region of TNF receptor 1 (TNFR1). In cultured human umbilical vein endothelial cells, endogenous TRAF2 colocalizes with the membrane-organizing protein caveolin-1 at regions of enrichment subjacent to the plasma membrane as detected by confocal fluorescence microscopy. Both endogenous and transfected TRAF2 protein coimmunoprecipitate with caveolin-1 in the absence of ligand. Upon TNF treatment, the TRAF2-caveolin-1 complex transiently associates with TRADD, and upon overexpression of TNFR2, the TRAF2-caveolin-1 complex stably associates with and causes redistribution of this receptor as detected by confocal fluorescence microscopy. In human embryonic kidney 293 cells, which have minimal endogenous expression of caveolin-1, cotransfection of TRAF2 and caveolin-1 results in spontaneous association of these proteins which can further associate with and redistribute transfected TNFR2 molecules. The association of caveolin-1 with TNFR2 depends upon TRAF2. Cotransfection of caveolin-1 protein increases TRAF2 protein expression levels in HEK 293 cells, which correlates with enhancement of TNF and TRAF2 signaling, measured as transcription of a NF-B promoter-reporter gene, although the caveolin-enhanced response to TNF is attenuated at higher caveolin levels. These findings suggest that intracellular distribution of activated TNF receptors may be regulated by caveolin-1 via its interaction with TRAF2.

SUMMARY:TNF signals are mediated through two different receptors, TNFR1 and TNFR2. In endothelial cells, TNFR1 is predominantly localized in the Golgi apparatus and TNFR2 on the plasma membrane. To investigate structural features responsible for the disparate localization, endothelial cells were transfected with epitope-tagged or green fluorescent protein-fused wild type and mutant receptor molecules. Wild type receptors recapitulated the distribution of endogenous receptors. Deletions of the entire TNFR1 intracellular domain or of the C-terminal death domain (TNFR1 ϪDD ) allowed expression of the receptor on the plasma membrane. However, addition of the death domain to the C-terminus of TNFR2 (TNFR2 ϩDD ) did not lead to Golgi-retention of this chimeric receptor. Overexpressed TNFR1, TNFR2, and TNFR2 ϩDD increased basal expression of a cotransfected NF-B-dependent promotor-reporter gene. Overexpressed TNFR1 ϪDD did not activate NF-B but acted as a ligand-specific dominant negative inhibitor of TNF actions. Unexpectedly, TNF responses were also inhibited by overexpressed TNFR1 and TNFR2 ϩDD , but not TNFR2. We conclude that the death domain of TNFR1 is required for retention of TNFR1 in the Golgi apparatus but is not sufficient to direct Golgi retention of a TNFR2 ϩDD chimera, and that overexpressed receptors that contain the death domain (TNFR1 and TNFR2 ϩDD ) spontaneously activate NF-B while inhibiting TNF responses. (Lab Invest 2000, 80:1185-1194.

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