Responses to hypoxia via mTOR : role in endothelial cell proliferation and HIF-1[alpha] stabilization

Abstract

Ischemic cardiovascular disease and cancer are life-threatening disorders in human mortality. Intensive studies on the etiology of and therapeutics for the diseases are ongoing. One of the most intriguing and promising fields for studies in both disorders is angiogenesis. Ischemic myocardium and cancer are closely associated with hypoxia and require de novo blood vessels for tissue survival. However, therapeutically, angiogenesis needs to be induced in the ischemic myocardium whereas it ought to be suppressed in cancer. For both purposes, a thorough understanding of the mechanisms of hypoxia-induced angiogenesis is indispensable. Angiogenesis, in response to ischemia, requires factors that sense hypoxia and that relay the signals to effectors. Mammalian target of rapamycin (mTOR), a key energysensor for cell survival, has recently been shown to be involved in hypoxic signaling. It remains unclear whether mTOR acts as part of the oxygen-sensing machinery and how mTOR regulates the hypoxia-induced signaling transducers. On the other hand, transcription factor hypoxia-inducible factor-1α (HIF-1α) is critical for hypoxic-driven induction of angiogenic molecules. Again, it is unclear how mTOR affects HIF-1α function. To unravel these questions, we have assessed mTOR activity as well as its relationship to HIF-1α in rat aortic endothelial cells (RAECs) in response to hypoxia. Previous studies in the lab had found that hypoxia potentiates angiogenesis of explants from rat aorta in an mTOR dependent way. In this study, we have extended this observation to proliferation of RAEC in vitro and a RAEC-spheroid sprouting assay (angiogenesis in vitro). Rapamycin, an inhibitor of mTOR, inhibited proliferation of RAEC and sprouting of endothelial cells in vitro under hypoxia. Interestingly, upon hypoxic stimulation, mTOR is highly phosphorylated; both mTOR and phospho-mTOR accumulate in cell nucleus, as does HIF-1α. However, S6k and 4E-BP1, two downstream targets of mTOR that are involved in translational control are hypophosphorylated at the same time. In low O2 tension (1% O2), increased nuclear HIF-1α levels are observed over time as well as with decreased O2 saturation. Similarly, the growth factor PDGF-BB induces HIF-1α nuclear accumulation under normoxic conditions. Hypoxia and PDGF-BB synergistically enhance HIF-1α nuclear levels. mTOR inhibition strongly reduces nuclear HIF-1α levels under hypoxia or/and PDGF-BB stimulation while MEK1/2 blockage only reduces PDGF-BB-induced nuclear HIF-1α accumulation in normoxia. Neither JNK nor p38 inhibition alters nuclear HIF-1α protein levels. HIF-1α mRNA levels remain stable under different oxygen saturations and upon mTOR or MEK1/2 inhibition. Notably, rapamycin-decreased HIF-1α nuclear accumulation can be rescued by proteasomal inhibition under hypoxia. Finally, mouse embryonic fibroblasts lacking HIF-1α significantly decreased proliferation rates under hypoxia when compared to wild type cells. However, mTOR over expression restores and further augments hypoxia-triggered proliferation both in HIF-1α wild type and in HIF-1α deficient cells. Taken together, hypoxia activates both HIF-1α-dependent and HIF-1α-independent regulation of cell proliferation and angiogenesis. Hypoxia-induced mTOR activation reduces S6K1 and 4E-BP1 phosphorylation. mTOR activity is required for protecting HIF-1α from proteasomal degradation. Further investigations on mTOR and HIF-1α during hypoxia in RAEC proliferation, spheroid sprouting assays, and angiogenesis in vivo are required. Thus, targeting mTOR to enhance or reduce angiogenesis in response to hypoxia may be clinically relevant.