TNF TRIGGERS TNFR1 REDISTRIBUTION FROM CAVEOLAE IN AN ENDOTHELIAL CELL LINE

Caveolae, which are invaginations of plasma membrane enriched in cholesterol and glycosphingolipids and coated by the scaffolding proteins caveolin-1 and 2, function to juxtapose signaling receptors of various types so as to permit receptor cross talk. We are interested in whether caveolae influence TNF signaling in human endothelial cells (ECs). Human umbilical vein ECs display abundant caveolae in situ, although these organelles are rapidly lost in culture. We have found that the endothelial cell line EAhy.926, produced by fusing human umbilical vein ECs with the permanent human cell line A549 (human lung carcinoma), retains an extensive caveolar network in vitro. By confocal and by immunoelectron microscopy, TNF receptor I (TNFR1/CD120a), the principal signaling receptor for pro-inflammatory responses, colocalizes with caveolin-1 in the region of the plasmalemma. TNFR1 also co-fractionates with caveolin-1 in a sucrose density gradient isolation of caveolae and caveolin-1 co-immunoprecipitates with TNFR1 in detergent lysates of EAhy.926 cell line. Addition of TNF to EAhy.926 cells induces translocation of TNFR1 from the caveolar fraction and dissociation from caveolin-1 within five minutes. TNF treatment also causes TNFR1 to colocalize with endosomal and lysosomal markers by 30–60 minutes. These studies suggest that TNF initiates the rapid translocation of TNFR1 from caveolae followed by a slower process of internalization.