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Carboxypeptidase E (CPE) has recently been described as a multifunctional protein that regulates proliferation, migration and survival in several tumor entities. In glioblastoma (GBM), the most malignant primary brain tumor, secreted CPE (sCPE) was shown to modulate tumor cell migration. In our current study, we aimed at clarifying the underlying molecular mechanisms regulating anti-migratory as well as novel metabolic effects of sCPE in GBM. Here we show that sCPE activates mTORC1 signaling in glioma cells detectable by phosphorylation of its downstream target RPS6. Additionally, sCPE diminishes glioma cell migration associated with a negative regulation of Rac1 signaling via RPS6, since both inhibition of mTOR and stimulation of Rac1 results in a reversed effect of sCPE on migration. Knockdown of CPE leads to a decrease of active RPS6 associated with increased GBM cell motility. Apart from this, we show that sCPE enhances glucose flux into the tricarboxylic acid cycle at the expense of lactate production, thereby decreasing aerobic glycolysis, which might as well contribute to a less invasive behavior of tumor cells. Our data contributes to a better understanding of the complexity of GBM cell migration and sheds new light on how tumor cell invasion and metabolic plasticity are interconnected.
Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair.
Objective: To determine the role of the AMPKα2 subunit in vascular repair.
Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2-/- versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice.
Conclusions: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia.