MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia
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eLife assessment
The authors find a significant and unexpected consequence of hypoxia in lung fibroblasts and smooth muscle cells - decreased lactate production - a finding that is important in the field of pulmonary hypertension. Additional orthogonal assessments of lactate production will strengthen the conclusions put forward.
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Abstract
Hypoxia requires metabolic adaptations to sustain energetically demanding cellular activities. While the metabolic consequences of hypoxia have been studied extensively in cancer cell models, comparatively little is known about how primary cell metabolism responds to hypoxia. Thus, we developed metabolic flux models for human lung fibroblast and pulmonary artery smooth muscle cells proliferating in hypoxia. Unexpectedly, we found that hypoxia decreased glycolysis despite activation of hypoxia-inducible factor 1α (HIF-1α) and increased glycolytic enzyme expression. While HIF-1α activation in normoxia by prolyl hydroxylase (PHD) inhibition did increase glycolysis, hypoxia blocked this effect. Multi-omic profiling revealed distinct molecular responses to hypoxia and PHD inhibition, and suggested a critical role for MYC in modulating HIF-1α responses to hypoxia. Consistent with this hypothesis, MYC knockdown in hypoxia increased glycolysis and MYC over-expression in normoxia decreased glycolysis stimulated by PHD inhibition. These data suggest that MYC signaling in hypoxia uncouples an increase in HIF-dependent glycolytic gene transcription from glycolytic flux.
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eLife assessment
The authors find a significant and unexpected consequence of hypoxia in lung fibroblasts and smooth muscle cells - decreased lactate production - a finding that is important in the field of pulmonary hypertension. Additional orthogonal assessments of lactate production will strengthen the conclusions put forward.
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Reviewer #1 (Public Review):
The work is largely based on metabolic flux assays of cultured cells, using a combination of metabolite concentration assessments, stable isotope-labeled substrates coupled with mass spectrometry, mathematical modeling, and cell proliferation analysis. The work finds a significant and unexpected phenotype in lung fibroblasts and smooth muscle cells of decreased lactate production in hypoxia which is important in the field of pulmonary hypertension. The evidence is strong and could be assisted with further orthogonal studies.
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Reviewer #2 (Public Review):
Copeland et al. set out to assess the impact of HIF1 activation - either through glycolysis or pharmacological inhibition of prolyl hydroxylase (PHD) - in primary fibroblasts and smooth muscle cells. The goal was to compare the metabolic responses between these two states, and with the scores of papers studying metabolic responses to hypoxia in cancer cells. Using a combination of metabolomics, metabolic flux analysis, and gene expression studies, they surprisingly find that hypoxia induces the expected activation of glycolytic genes, but fails to induce some of the classical metabolic responses reported in cancer cells, including glucose uptake and lactate secretion. Lactate secretion is enhanced by PHD expression, but this is reversed by hypoxia. The authors further find that hypoxia induces the expression …
Reviewer #2 (Public Review):
Copeland et al. set out to assess the impact of HIF1 activation - either through glycolysis or pharmacological inhibition of prolyl hydroxylase (PHD) - in primary fibroblasts and smooth muscle cells. The goal was to compare the metabolic responses between these two states, and with the scores of papers studying metabolic responses to hypoxia in cancer cells. Using a combination of metabolomics, metabolic flux analysis, and gene expression studies, they surprisingly find that hypoxia induces the expected activation of glycolytic genes, but fails to induce some of the classical metabolic responses reported in cancer cells, including glucose uptake and lactate secretion. Lactate secretion is enhanced by PHD expression, but this is reversed by hypoxia. The authors further find that hypoxia induces the expression of MYC, and they use gain and loss of function experiments to show that MYC is involved in the reduction of lactate secretion by hypoxia. The paper's strengths are the detailed and quantitative analysis of metabolic responses in two different models of non-transformed cell proliferation, as well as the combination of gain- and loss-of-function analyses of MYC. Relative limitations include the use of a single chemical compound to activate HIF-1 in normoxia, and the lack of an explanation for why MYC is induced in hypoxia but not in normoxic HIF activation, or why MYC's effects are so different here than what is usually observed in cancer cells. If validated, the findings of the paper would add complexity to the mechanisms by which cells respond to hypoxia; to date, these responses have mostly been studied in cancer cells, and the new data suggest that non-transformed cells respond quite differently. The findings also suggest that MYC's effects on metabolism are determined in part by the cell state, as a large amount of existing data indicates that MYC drives lactate secretion in cancer cells.
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Reviewer #3 (Public Review):
Via a study of metabolic flux of proliferating human primary cells (lung fibroblasts and PASMCs) in vitro, the authors primarily find that MYC uncouples an increase in HIF-dependent glycolytic gene transcription from the glycolytic flux in hypoxia. This finding is surprising and significant, given that prior work in cancer cell lines has indicated that glycolysis is uniformly increased under hypoxic stress. Strengths of the study include the comprehensive rigor of the approach to reach this conclusion, the accounting of multiple confounding variables, and the well-written presentation of the findings. These findings will be of use to the general scientific community, particularly the atlas of molecular alterations seen with their flux analyses. The surprising findings will set the stage for additional work …
Reviewer #3 (Public Review):
Via a study of metabolic flux of proliferating human primary cells (lung fibroblasts and PASMCs) in vitro, the authors primarily find that MYC uncouples an increase in HIF-dependent glycolytic gene transcription from the glycolytic flux in hypoxia. This finding is surprising and significant, given that prior work in cancer cell lines has indicated that glycolysis is uniformly increased under hypoxic stress. Strengths of the study include the comprehensive rigor of the approach to reach this conclusion, the accounting of multiple confounding variables, and the well-written presentation of the findings. These findings will be of use to the general scientific community, particularly the atlas of molecular alterations seen with their flux analyses. The surprising findings will set the stage for additional work on MYC's role in primary vs. transformed cells, the mode of regulation of MYC in primary cells, and the relevance of this mechanism in in vivo contexts of health and disease. A weakness of the study that can be improved upon in future work includes confirmation of findings in more physiologically relevant contexts of primary tissue in the body.
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