Hypoxia-induced endothelial proliferation requires both mTORC1 and mTORC2.
A central regulator of cell growth that has been implicated in responses to stress such as hypoxia is mTOR (mammalian Target Of Rapamycin). We have shown previously that mTOR is required for angiogenesis in vitro and endothelial cell proliferation in response to hypoxia. Here we have investigated mTOR-associated signaling components under hypoxia and their effects on cell proliferation in rat aortic endothelial cells (RAECs). Hypoxia (1% O(2)) rapidly (>30 minutes) and in a concentration-dependent manner promoted rapamycin-sensitive and sustained phosphorylation of mTOR-Ser2448 followed by nuclear translocation in RAECs. Similarly, hypoxia induced phosphorylation of the mTORC2 substrate Akt-Ser473 (3 to 6 hours at 1% O(2)) and a brief phosphorylation peak of the mTORC1 substrate S6 kinase-Thr389 (10 to 60 minutes). Phosphorylation of Akt was inhibited by mTOR knockdown and partially with rapamycin. mTOR knockdown, rapamycin, or Akt inhibition specifically and significantly inhibited proliferation of serum-starved RAECs under hypoxia (P<0.05; n> or =4). Similarly, hypoxia induced Akt-dependent and rapamycin-sensitive proliferation in mouse embryonic fibroblasts. This response was partially blunted by hypoxia-inducible factor-1alpha knockdown and not affected by TSC2 knockout. Finally, mTORC2 inhibition by rictor silencing, especially (P<0.001; n=7), and mTORC1 inhibition by raptor silencing, partially (P<0.05; n=7), inhibited hypoxia-induced RAEC proliferation. Thus, mTOR mediates an early response to hypoxia via mTORC1 followed by mTORC2, promoting endothelial proliferation mainly via Akt signaling. mTORC1 and especially mTORC2 might therefore play important roles in diseases associated with hypoxia and altered angiogenesis.
Li
Weimin
W
Department of Research, University Hospital Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland.
Petrimpol
Marco
M
Molle
Klaus D
KD
Hall
Michael N
MN
Battegay
Edouard J
EJ
Humar
Rok
R
D000818:Animals; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology*; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D000860:Hypoxia / Q000378:metabolism* / Q000473:pathology*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D051379:Mice; D009994:Osmolar Concentration; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012694:Serine; D058570:TOR Serine-Threonine Kinases; D013912:Threonine; D014157:Transcription Factors / Q000378:metabolism*; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000172:deficiency
C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020033:Protein Isoforms; D014157:Transcription Factors; C000624654:Tsc2 protein, mouse; C000624655:Tsc2 protein, rat; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D013912:Threonine; D012694:Serine; D011494:Protein Kinases; C546843:mTOR protein, mouse; C546845:mTOR protein, rat; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases