CD40-induced signaling in human endothelial cells results in mTORC2- and Akt-dependent expression of vascular endothelial growth factor in vitro and in vivo.
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.
Dormond
Olivier
O
Department of Medicine, Division of Nephrology, Transplantation Research Center, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA.
Contreras
Alan G
AG
Meijer
Esther
E
Datta
Dipak
D
Flynn
Evelyn
E
Pal
Soumitro
S
Briscoe
David M
DM
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019013:CD40 Antigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D023201:CD40 Ligand / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018797:Cell Cycle Proteins; D042783:Endothelial Cells / Q000276:immunology* / Q000378:metabolism; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D016513:Mice, SCID; D046912:Multiprotein Complexes; D009369:Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D011506:Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000276:immunology; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D000076223:Regulatory-Associated Protein of mTOR; D019893:Ribosomal Protein S6 Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
D048868:Adaptor Proteins, Signal Transducing; D000900:Anti-Bacterial Agents; D019013:CD40 Antigens; C493245:CRTC2 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D011506:Proteins; C492571:RICTOR protein, human; D034741:RNA, Small Interfering; C463659:RPTOR protein, human; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D000076223:Regulatory-Associated Protein of mTOR; D014157:Transcription Factors; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D023201:CD40 Ligand; D000076222:Mechanistic Target of Rapamycin Complex 1; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus