Article: article from journal or magazin.
CD40-induced signaling in human endothelial cells results in mTORC2- and Akt-dependent expression of vascular endothelial growth factor in vitro and in vivo.
Journal of Immunolog
Publication types: Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov'tPublication Status: ppublish
We have examined CD40-dependent signals in endothelial cells (EC) mediating the expression of vascular endothelial growth factor (VEGF) and VEGF-induced angiogenesis. We treated confluent cultures of EC with soluble CD40L (sCD40L), and by Western blot found a marked increase in the phosphorylation of Akt, 4EBP-1, and S6K1, compared with untreated cells. EC were transfected with a full-length VEGF promoter-luciferase construct and cultured in the absence or presence of rapamycin and sCD40L. We found that rapamycin, which blocks mTORC1 and mTORC2 signaling, inhibited sCD40L-mediated transactivation of VEGF. In addition, by Western blot, we found that the transfection of EC with small interfering RNA (siRNA) to rictor (to inhibit mTORC2), and not raptor (to inhibit mTORC1), inhibited sCD40L-dependent protein expression of VEGF. In additions, we found that basal levels of phosphorylated Akt as well as VEGF were increased in EC transfected with the raptor siRNA. Also, rapamycin failed to inhibit VEGF promoter activation, as well as VEGF protein expression in EC transfected with a constitutively active construct of Akt, further demonstrating that mTORC1 is not necessary for CD40- and Akt-induced expression of VEGF. Finally, we injected human CD40L-transfected fibroblasts or mock transfectants into human skin on SCID mice. We found that the injection of CD40L transfectants, but not mock cells, resulted in VEGF expression and mediated a marked angiogenesis reaction, and this response was reduced in mice treated with rapamycin. Together, these observations indicate that mTORC2 and Akt facilitate CD40-inducible expression of VEGF in EC, which is of clinical importance in tumor growth and the progression of chronic inflammatory diseases.
Adaptor Proteins, Signal Transducing/genetics, Adaptor Proteins, Signal Transducing/immunology, Animals, Anti-Bacterial Agents/pharmacology, Antigens, CD40/genetics, Antigens, CD40/immunology, CD40 Ligand/genetics, CD40 Ligand/immunology, Carrier Proteins/antagonists & inhibitors, Carrier Proteins/genetics, Endothelial Cells/immunology, Endothelial Cells/metabolism, Fibroblasts/immunology, Fibroblasts/metabolism, Humans, Inflammation/genetics, Inflammation/immunology, Mice, Mice, SCID, Neoplasms/genetics, Neoplasms/immunology, Phosphoproteins/genetics, Phosphoproteins/immunology, Phosphorylation/drug effects, Phosphorylation/genetics, Promoter Regions, Genetic/drug effects, Promoter Regions, Genetic/genetics, Protein Biosynthesis/drug effects, Protein Biosynthesis/genetics, Proteins/antagonists & inhibitors, Proteins/genetics, Proto-Oncogene Proteins c-akt/genetics, Proto-Oncogene Proteins c-akt/immunology, RNA, Small Interfering/genetics, RNA, Small Interfering/immunology, Ribosomal Protein S6 Kinases/genetics, Ribosomal Protein S6 Kinases/immunology, Signal Transduction/drug effects, Signal Transduction/genetics, Sirolimus/pharmacology, Transcription Factors/genetics, Transcription Factors/immunology, Transcriptional Activation/drug effects, Transcriptional Activation/genetics, Vascular Endothelial Growth Factor A/biosynthesis, Vascular Endothelial Growth Factor A/genetics
Web of science
Last modification date