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Proceedings of the National Academy of... Jul 2019Increased glycolysis in the lung vasculature has been connected to the development of pulmonary hypertension (PH). We therefore investigated whether glycolytic regulator...
Increased glycolysis in the lung vasculature has been connected to the development of pulmonary hypertension (PH). We therefore investigated whether glycolytic regulator 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFKFB3)-mediated endothelial glycolysis plays a critical role in the development of PH. Heterozygous global deficiency of protected mice from developing hypoxia-induced PH, and administration of the PFKFB3 inhibitor 3PO almost completely prevented PH in rats treated with Sugen 5416/hypoxia, indicating a causative role of PFKFB3 in the development of PH. Immunostaining of lung sections and Western blot with isolated lung endothelial cells showed a dramatic increase in PFKFB3 expression and activity in pulmonary endothelial cells of rodents and humans with PH. We generated mice that were constitutively or inducibly deficient in endothelial and found that these mice were incapable of developing PH or showed slowed PH progression. Compared with control mice, endothelial -knockout mice exhibited less severity of vascular smooth muscle cell proliferation, endothelial inflammation, and leukocyte recruitment in the lungs. In the absence of , lung endothelial cells from rodents and humans with PH produced lower levels of growth factors (such as PDGFB and FGF2) and proinflammatory factors (such as CXCL12 and IL1β). This is mechanistically linked to decreased levels of HIF2A in lung ECs following knockdown. Taken together, these results suggest that targeting PFKFB3 is a promising strategy for the treatment of PH.
Topics: Animals; Disease Models, Animal; Endothelium; Gene Knockdown Techniques; Glycolysis; Humans; Hypertension, Pulmonary; Hypoxia; Lung; Male; Mice; Mice, Inbred C57BL; Phosphofructokinase-2; Rats; Rats, Sprague-Dawley
PubMed: 31213542
DOI: 10.1073/pnas.1821401116 -
Advances in Cancer Research 2023Phosphofructokinase (PFK) is one of the key enzymes that functions in glycolysis. Studies show that PFKP regulates cell proliferation, apoptosis, autophagy, cell... (Review)
Review
Phosphofructokinase (PFK) is one of the key enzymes that functions in glycolysis. Studies show that PFKP regulates cell proliferation, apoptosis, autophagy, cell migration/metastasis, and stemness through glycolysis and glycolysis-independent functions. PFKP performs its function not only in the cytoplasm, but also at the cell membrane, on the mitochondria, at the lysosomal membrane, and in the nucleus. The functions of PFKP are extensively studied in cancer cells. PFKP is also highly expressed in certain immune cells; nevertheless, the study of the PFKP's role in immune cells is limited. In this review, we summarize how the expression and activity of PFKP are regulated in cancer cells. PFKP may be applied as a prognostic marker due to its overexpression and significant functions in cancer cells. As such, specifically targeting/inhibiting PFKP may be a critical and promising strategy for cancer therapy.
Topics: Humans; Phosphofructokinases; Cell Membrane; Apoptosis; Autophagy; Cell Proliferation
PubMed: 37704285
DOI: 10.1016/bs.acr.2023.03.001 -
Molecular Cell May 2021Oxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for ATP production. The reliance on each varies across tissues and cell states, and can...
Oxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for ATP production. The reliance on each varies across tissues and cell states, and can influence susceptibility to disease. At present, the full set of molecular mechanisms governing the relative expression and balance of these two pathways is unknown. Here, we focus on genes whose loss leads to an increase in OXPHOS activity. Unexpectedly, this class of genes is enriched for components of the pre-mRNA splicing machinery, in particular for subunits of the U1 snRNP. Among them, we show that LUC7L2 represses OXPHOS and promotes glycolysis by multiple mechanisms, including (1) splicing of the glycolytic enzyme PFKM to suppress glycogen synthesis, (2) splicing of the cystine/glutamate antiporter SLC7A11 (xCT) to suppress glutamate oxidation, and (3) secondary repression of mitochondrial respiratory supercomplex formation. Our results connect LUC7L2 expression and, more generally, the U1 snRNP to cellular energy metabolism.
Topics: Amino Acid Transport System y+; Electron Transport Chain Complex Proteins; Gene Expression Regulation; Genome-Wide Association Study; Glutamic Acid; Glycogen; Glycolysis; HEK293 Cells; HeLa Cells; Humans; K562 Cells; Mitochondria; Oxidation-Reduction; Oxidative Phosphorylation; Phosphofructokinase-1, Muscle Type; RNA Precursors; RNA Splicing; RNA, Messenger; RNA-Binding Proteins; Ribonucleoprotein, U1 Small Nuclear
PubMed: 33852893
DOI: 10.1016/j.molcel.2021.02.033 -
Cell Aug 2021In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen...
In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells.
Topics: Adenosine Diphosphate; Adenosine Monophosphate; Allosteric Regulation; Enzyme Activation; Epithelial Cells; Glycolysis; Humans; Intracellular Signaling Peptides and Proteins; Kinetics; Microbial Viability; Models, Molecular; NADPH Oxidases; Neutrophils; Phagocytosis; Phosphate-Binding Proteins; Phosphofructokinase-1, Liver Type; Protein Kinase Inhibitors; Recombinant Proteins; Respiratory Burst; Tetradecanoylphorbol Acetate
PubMed: 34320407
DOI: 10.1016/j.cell.2021.07.004 -
American Journal of Respiratory and... Sep 2019Glycolytic shift is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). It remains unknown how glycolysis is increased and how increased glycolysis...
Glycolytic shift is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). It remains unknown how glycolysis is increased and how increased glycolysis contributes to pulmonary vascular remodeling in PAH. To determine whether increased glycolysis is caused by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and how PFKFB3-driven glycolysis induces vascular remodeling in PAH. PFKFB3 levels were measured in pulmonary arteries of patients and animals with PAH. Lactate levels were assessed in lungs of animals with PAH and in pulmonary artery smooth muscle cells (PASMCs). Genetic and pharmacologic approaches were used to investigate the role of PFKFB3 in PAH. Lactate production was elevated in lungs of PAH rodents and in platelet-derived growth factor-treated PASMCs. PFKFB3 protein was higher in pulmonary arteries of patients and rodents with PAH, in PASMCs of patients with PAH, and in platelet-derived growth factor-treated PASMCs. PFKFB3 inhibition by genetic disruption and chemical inhibitor attenuated phosphorylation/activation of extracellular signal-regulated kinase (ERK1/2) and calpain-2, and vascular remodeling in PAH rodent models, and reduced platelet-derived growth factor-induced phosphorylation/activation of ERK1/2 and calpain-2, collagen synthesis and proliferation of PASMCs. ERK1/2 inhibition attenuated phosphorylation/activation of calpain-2, and vascular remodeling in Sugen/hypoxia PAH rats, and reduced lactate-induced phosphorylation/activation of calpain-2, collagen synthesis, and proliferation of PASMCs. Calpain-2 inhibition reduced lactate-induced collagen synthesis and proliferation of PASMCs. Upregulated PFKFB3 mediates collagen synthesis and proliferation of PASMCs, contributing to vascular remodeling in PAH. The mechanism is through the elevation of glycolysis and lactate that results in the activation of calpain by ERK1/2-dependent phosphorylation of calpain-2.
Topics: Animals; Cell Proliferation; Disease Models, Animal; Humans; Male; Mice; Muscle, Smooth, Vascular; Phosphofructokinase-2; Pulmonary Arterial Hypertension; Rats; Vascular Remodeling
PubMed: 30817168
DOI: 10.1164/rccm.201812-2290OC -
Cell Death & Disease Apr 2022Renal inflammation is a critical pathophysiological characteristic of diabetic kidney disease (DKD). The mechanism of the inflammatory response is complicated, and there...
Renal inflammation is a critical pathophysiological characteristic of diabetic kidney disease (DKD). The mechanism of the inflammatory response is complicated, and there are few effective treatments for renal inflammation that can be used clinically. Insulin-like growth factor-binding protein 5 (IGFBP5) is an important secretory protein that is related to inflammation and fibrosis in several tissues. Studies have shown that the IGFBP5 level is significantly upregulated in DKD. However, the function of IGFBP5 and its mechanism in DKD remain unclear. Here, we showed that IGFBP5 levels were significantly increased in the kidneys of diabetic mice. Ablation of IGFBP5 alleviated kidney inflammation in DKD mice. Mechanistically, IGFBP5 increased glycolysis, which was characterized by increases in lactic acid and the extracellular acidification rate, by activating the transcription factor early growth response 1 (EGR1) and enhancing the expression of PFKFB3 in endothelial cells. Furthermore, a mutation in PFKFB3 attenuated renal inflammation in DKD mice. Taken together, we provided evidence that IGFBP5 enhanced kidney inflammation through metabolic reprogramming of glomerular endothelial cells. Our results provide new mechanistic insights into the effect of IGFBP5 on kidney and highlight potential therapeutic opportunities for IGFBP5 and the metabolic regulators involved in DKD.
Topics: Animals; Carrier Proteins; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Endothelial Cells; Glycolysis; Humans; Inflammation; Mice; Phosphofructokinase-2
PubMed: 35418167
DOI: 10.1038/s41419-022-04803-y -
Revisited Metabolic Control and Reprogramming Cancers by Means of the Warburg Effect in Tumor Cells.International Journal of Molecular... Sep 2022Aerobic glycolysis is an emerging hallmark of many human cancers, as cancer cells are defined as a "metabolically abnormal system". Carbohydrates are metabolically... (Review)
Review
Aerobic glycolysis is an emerging hallmark of many human cancers, as cancer cells are defined as a "metabolically abnormal system". Carbohydrates are metabolically reprogrammed by its metabolizing and catabolizing enzymes in such abnormal cancer cells. Normal cells acquire their energy from oxidative phosphorylation, while cancer cells acquire their energy from oxidative glycolysis, known as the "Warburg effect". Energy-metabolic differences are easily found in the growth, invasion, immune escape and anti-tumor drug resistance of cancer cells. The glycolysis pathway is carried out in multiple enzymatic steps and yields two pyruvate molecules from one glucose (Glc) molecule by orchestral reaction of enzymes. Uncontrolled glycolysis or abnormally activated glycolysis is easily observed in the metabolism of cancer cells with enhanced levels of glycolytic proteins and enzymatic activities. In the "Warburg effect", tumor cells utilize energy supplied from lactic acid-based fermentative glycolysis operated by glycolysis-specific enzymes of hexokinase (HK), keto-HK-A, Glc-6-phosphate isomerase, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase, phosphofructokinase (PFK), phosphor-Glc isomerase (PGI), fructose-bisphosphate aldolase, phosphoglycerate (PG) kinase (PGK)1, triose phosphate isomerase, PG mutase (PGAM), glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase isozyme type M2 (PKM2), pyruvate dehydrogenase (PDH), PDH kinase and lactate dehydrogenase. They are related to glycolytic flux. The key enzymes involved in glycolysis are directly linked to oncogenesis and drug resistance. Among the metabolic enzymes, PKM2, PGK1, HK, keto-HK-A and nucleoside diphosphate kinase also have protein kinase activities. Because glycolysis-generated energy is not enough, the cancer cell-favored glycolysis to produce low ATP level seems to be non-efficient for cancer growth and self-protection. Thus, the Warburg effect is still an attractive phenomenon to understand the metabolic glycolysis favored in cancer. If the basic properties of the Warburg effect, including genetic mutations and signaling shifts are considered, anti-cancer therapeutic targets can be raised. Specific therapeutics targeting metabolic enzymes in aerobic glycolysis and hypoxic microenvironments have been developed to kill tumor cells. The present review deals with the tumor-specific Warburg effect with the revisited viewpoint of recent progress.
Topics: Glycolysis; Hexokinase; Humans; Neoplasms; Phosphofructokinase-1; Phosphoglycerate Kinase; Phosphoglycerate Mutase; Pyruvates; Tumor Microenvironment
PubMed: 36077431
DOI: 10.3390/ijms231710037 -
Circulation Research May 2022Mechanical forces play crucial roles in neointimal hyperplasia after vein grafting; yet, our understanding of their influences on vascular smooth muscle cell (VSMC)...
BACKGROUND
Mechanical forces play crucial roles in neointimal hyperplasia after vein grafting; yet, our understanding of their influences on vascular smooth muscle cell (VSMC) activation remains rudimentary.
METHODS
A cuff mouse model was used to study vein graft hyperplasia. Fifteen percent to 1 Hz uniaxial cyclic stretch (arterial strain), 5% to 1 Hz uniaxial cyclic stretch or a static condition (venous strain) were applied to the cultured VSMCs. Metabolomics analysis, cell proliferation and migration assays, immunoblotting, co-immunoprecipitation, mutagenesis, pull-down and surface plasmon resonance assays were employed to elucidate the potential molecular mechanisms.
RESULTS
RNA-sequencing in vein grafts and the controls identified changes in metabolic pathways and downregulation of mitochondrial protein MFN2 (mitofusin 2) in the vein grafts. Exposure of VSMCs to 15% stretch resulted in MFN2 downregulation, mitochondrial fragmentation, metabolic shift from mitochondrial oxidative phosphorylation to glycolysis, and cell proliferation and migration, as compared with that to a static condition or 5% stretch. Metabolomics analysis indicated an increased generation of fructose 1,6-bisphosphate, an intermediate in the glycolytic pathway converted by PFK1 (phosphofructokinase 1) from fructose-6-phosphate, in cells exposed to 15% stretch. Mechanistic study revealed that MFN2 physically interacts through its C-terminus with PFK1. MFN2 knockdown or exposure of cells to 15% stretch promoted stabilization of PFK1, likely through interfering the association between PFK1 and the E3 ubiquitin ligase TRIM21 (E3 ubiquitin ligase tripartite motif [TRIM]-containing protein 21), thus, decreasing the ubiquitin-protease-dependent PFK1 degradation. In addition, study of mechanotransduction utilizing pharmaceutical inhibition indicated that the MFN2 downregulation by 15% stretch was dependent on inactivation of the SP1 (specificity protein 1) and activation of the JNK (c-Jun N-terminal kinase) and ROCK (Rho-associated protein kinase). Adenovirus-mediated MFN2 overexpression or pharmaceutical inhibition of PFK1 suppressed the 15% stretch-induced VSMC proliferation and migration and alleviated neointimal hyperplasia in vein grafts.
CONCLUSIONS
MFN2 is a mechanoresponsive protein that interacts with PFK1 to mediate PFK1 degradation and therefore suppresses glycolysis in VSMCs.
Topics: Animals; Cell Proliferation; Cells, Cultured; GTP Phosphohydrolases; Hyperplasia; Mechanotransduction, Cellular; Mice; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Phosphofructokinase-1; Ubiquitin-Protein Ligases
PubMed: 35450439
DOI: 10.1161/CIRCRESAHA.122.320846 -
British Journal of Pharmacology Nov 2022Pathological angiogenesis is a major cause of irreversible blindness in individuals with neovascular age-related macular degeneration (nAMD). Macrophages and microglia...
BACKGROUND AND PURPOSE
Pathological angiogenesis is a major cause of irreversible blindness in individuals with neovascular age-related macular degeneration (nAMD). Macrophages and microglia (MΦ) contribute to aberrant ocular angiogenesis. However, the role of glucose metabolism of MΦ in nAMD is still undefined. Here, we have investigated the involvement of glycolysis, driven by the kinase/phosphatase PFKFB3, in the development of choroidal neovascularization (CNV).
EXPERIMENTAL APPROACH
CNV was induced in mice with laser photocoagulation. Choroid/retinal pigment epithelium (RPE) complexes and MΦ were isolated for analysis by qRT-PCR, western blot, flow cytometry, immunostaining, metabolic measurements and angiogenesis assays.
KEY RESULTS
MΦ accumulated within the CNV of murine nAMD models and expressed high levels of glycolysis-related enzymes and M1/M2 polarization markers. This phenotype of hyper-glycolytic and activated MΦ was replicated in bone marrow-derived macrophages stimulated by necrotic RPE in vitro. Myeloid cell-specific knockout of PFKFB3, a key glycolytic activator, attenuated pathological neovascularization in laser-induced CNV, which was associated with decreased expression of MΦ polarization markers and pro-angiogenic factors, along with decreased sprouting of vessels in choroid/RPE complexes. Mechanistically, necrotic RPE increased PFKFB3-driven glycolysis in macrophages, leading to activation of HIF-1α/HIF-2α and NF-κB, and subsequent induction of M1/M2 markers and pro-angiogenic cytokines, finally promoting macrophage reprogramming towards an angiogenic phenotype to facilitate development of CNV. The PFKFB3 inhibitor AZ67 also inhibited activation of HIF-1α/HIF-2α and NF-κB signalling and almost completely prevented laser-induced CNV in mice.
CONCLUSIONS AND IMPLICATIONS
Modulation of PFKFB3-mediated macrophage glycolysis and activation is a promising strategy for the treatment of nAMD.
Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Choroidal Neovascularization; Cytokines; Disease Models, Animal; Glucose; Glycolysis; Mice; Mice, Inbred C57BL; NF-kappa B; Phosphofructokinase-2; Phosphoric Monoester Hydrolases
PubMed: 35830274
DOI: 10.1111/bph.15925 -
Oncogene Aug 2022PFKFB3 (6-phosphofructo-2-kinase) is the rate-limiting enzyme of glycolysis and is overexpressed in several human cancers that are associated with poor prognosis. High...
PFKFB3 (6-phosphofructo-2-kinase) is the rate-limiting enzyme of glycolysis and is overexpressed in several human cancers that are associated with poor prognosis. High PFKFB3 expression in cancer stem cells promotes glycolysis and survival in the tumor microenvironment. Inhibition of PFKFB3 by the glycolytic inhibitor PFK158 and by shRNA stable knockdown in small cell lung carcinoma (SCLC) cell lines inhibited glycolysis, proliferation, spheroid formation, and the expression of cancer stem cell markers CD133, Aldh1, CD44, Sox2, and ABCG2. These factors are also associated with chemotherapy resistance. We found that PFK158 treatment and PFKFB3 knockdown enhanced the ABCG2-interacting drugs doxorubicin, etoposide, and 5-fluorouracil in reducing cell viability under conditions of enriched cancer stem cells (CSC). Additionally, PFKFB3 inhibition attenuated the invasion/migration of SCLC cells by downregulating YAP/TAZ signaling while increasing pLATS1 via activation of pMST1 and NF2 and by reducing the mesenchymal protein expression. PFKFB3 knockdown and PFK158 treatment in a H1048 SCLC cancer stem cell-enriched mouse xenograft model showed significant reduction in tumor growth and weight with reduced expression of cancer stem cell markers, ABCG2, and YAP/TAZ. Our findings identify that PFKFB3 is a novel target to regulate cancer stem cells and its associated therapeutic resistance markers YAP/TAZ and ABCG2 in SCLC models.
Topics: Animals; Cell Line, Tumor; Cell Proliferation; Glycolysis; Hippo Signaling Pathway; Humans; Lung Neoplasms; Mice; Phosphofructokinase-2; Pyridines; Quinolines; Small Cell Lung Carcinoma; Tumor Microenvironment
PubMed: 35804016
DOI: 10.1038/s41388-022-02391-x