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Metabolic signals in the hypoxic response

NDRG3 binds to lactate produced in hypoxic cells and stimulates proliferation and angiogenesis. To survive periods of reduced oxygen, cells mount an adaptive response. Stabilization of the transcription factor HIF-1α, which under normal oxygen conditions is degraded by a prolyl hydroxylase (PHD)-E3 ubiquitin ligase (VHL) pathway, initiates a transcriptional response that enables adaptation, partially through altered metabolism, to increase glycolysis instead of oxidative phosphorylation. Lee et al. identified NDRG3 (N-myc downstream-regulated gene 3) as a PHD2-interacting protein that was hydroxylated at Pro294. The abundance of NDRG3 mutants inversely correlated with binding to PHD2 under normoxic conditions, with the N66D mutant having a low affinity and exhibiting high abundance. Coimmunoprecipitation of NDRG3 with VHL also depended on the ability of NDRG3 to interact with PHD2, and hypoxia reduced the ubiquitylation of NDRG3. The hypoxia-induced increase in abundance of NDRG3, which was not associated with changes in transcript abundance, occurred after the hypoxia-induced increase in HIF-1α abundance had begun to decline. Instead, the abundance of NDRG3 correlated with the production of lactate, which resulted from the HIF-1α–transcriptionally induced changes in metabolism. NDRG3 directly bound to radiolabeled lactate. Hypoxia-mediated accumulation of NDRG3 was abolished by knockdown or pharmacological inhibition of lactate dehydrogenase A (LDHA) or by depriving the cells of glucose, a substrate for lactate-producing metabolic pathways. Although lactate binding and the interaction with PHD2 were necessary for hypoxic stabilization and normoxic degradation, respectively, whether lactate binding blocked the prolyl hydroxylation of NDRG3 was not determined. Hypoxia induced the phosphorylation of the kinase Raf and its downstream effectors extracellular signal–regulated kinase 1 and 2 (ERK1/2), which are part of a mitogen-activated protein kinase (MAPK) pathway. Hypoxia-induced phosphorylation of Raf and ERK1/2 depended on lactate production and the presence of NDRG3. Knockdown of NDRG3 in HUVECs (human umbilical vein endothelial cells) reduced hypoxia-induced tube formation. NDRG3 knockdown or inhibition of lactate production in the human liver cancer cell line Huh-7 reduced hypoxia-induced angiogenic responses (marker gene expression and tube-forming assays) in culture. Tumor growth of Huh-7 cells xenografted into mice was reduced by NDRG3 knockdown, and tumors formed from cells with the normoxia-stabilized N66D mutant of NDRG3 exhibited increased Raf and ERK1/2 phosphorylation and transcripts for proteins that promote angiogenesis compared with control Huh-7 tumors. Analysis of human liver cancer samples indicated a correlation in a subset of the samples between NDRG3 abundance and ERK1/2 phosphorylation. This study identifies a mechanism by which hypoxia stimulates the ERK MAPK pathway and also a mechanism by which cells detect hypoxia-induced changes in metabolism to stimulate proliferative and proangiogenic signaling. D. C. Lee, H. A. Sohn, Z.-Y. Park, S. Oh, Y. K. Kang, K.-m. Lee, M. Kang, Y. J. Jang, S.-J. Yang, Y. K. Hong, H. Noh, J.-A. Kim, D. J. Kim, K.-H. Bae, D. M. Kim, S. J. Chung, H. S. Yoo, D.-Y. Yu, K. C. Park, Y. I. Yeom, A lactate-induced response to hypoxia. Cell 161, 595–609 (2015). [PubMed]