Glycolytic enzyme PKM2 regulates cell senescence but not inflammation in the process of osteoarthritis.
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ABSTRACT:
PROVIDER: PRJNA1183791 | ENA |
REPOSITORIES: ENA
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Project description:Chondrocyte senescence is an important mechanism underlying osteoarthritis in the senile population and is characterized by reduced expressions of the extracellular matrix proteins. The involvement of glycolysis and the tricarboxylic acid cycle in the development of osteoarthritis is inclusive. The present study aims to investigate the role of the glycolytic enzyme M2 isoform of pyruvate kinase (PKM2) in chondrocytes in senescence and inflammation. Primary chondrocytes are isolated from the knee joints of neonatal mice. Small interfering RNAs (siRNAs) against PKM2 are transfected using lipofectamine. RNA sequencing is conducted in primary chondrocytes with the PKM2 gene deleted. Cell apoptosis, autophagy, reactive oxygen species measurement, and senescent conditions are examined. The glycolytic rate in cells is measured by Seahorse examination. Interleukin 1-β (IL-1β) increases the protein expressions of matrix metallopeptidases (MMP)13 and PKM2 and reduces the protein expression of collagen type II (COL2A1) in primary chondrocytes. Silencing of PKM2 alters the protein expressions of MMP13, PKM2, and COL2A1 in the same pattern in quiescent and stimulated chondrocytes. RNA sequencing analysis reveals that PKM2 silencing reduces senescent biomarker p16INK4a expression. Compared with low-passage chondrocytes, high-passage chondrocytes exhibit increased expression of p16INK4a and reduced expression of COL2A1. Silencing of PKM2 reduces SA-β-Gal signals and increases COL2A1 expression in high-passage chondrocytes. Seahorse assay reveals that PKM2 deletion favors the tricarboxylic acid cycle in mitochondria in low- but not in high-passage chondrocytes. In summary, the glycolytic enzyme PMK2 modulates chondrocyte senescence but does not participate in the regulation of inflammation.
2024-11-13 | GSE281389 | GEO
Project description:During myocardial infarction (MI), energy production decreases as hypoxia inhibits oxidative metabolism. Our lab has identified an ischemia-regulated alternative splicing event of the glycolytic enzyme pyruvate kinase (muscle, PKM), reducing PKM1 expression and increasing PKM2 expression. We hypothesized the upregulation of PKM2 aids in energy production to maintain heart function after MI. We utilized high-throughput sequencing to evaluate altered gene expression and identify pathways that may be regulated by PKM2.
2024-09-18 | GSE243668 | GEO
Project description:In this study we investigated how manipulation of the glycolytic enzyme pyruvate kinase muscle 2 (PKM2) alters CD8+ T cell gene expression and responsiveness to PD-1 checkpoint blockade in orthotopic mouse lung adenocarcinoma. In this dataset, we performed adoptive co-transfers of antigen-specific OT-I+ Thy1.1+ CD8+ T cells genetically modified by CRISPR/Cas9 ribonucleoproteins to become PKM2 knockout (in Thy1.1+/- cells) or PKM2 wildtype (in Thy1.1+/+ cells). T cells were transferred into orthotopic mouse lung adenocarcinoma HKP1-ova-GFP tumor-bearing syngeneic C57BL/6 mice, and subsequently mice were treated with either IgG or anti-PD-1. Adoptively-transferred T cells were sorted based on Thy1.1 zygosity from tumors at two different time points and bulk RNA sequenced.
2023-10-19 | GSE216675 | GEO
Project description:Metabolic fluxes may be regulated "hierarchically," e.g., by changes of gene expression that adjust enzyme capacities (V(max)) and/or "metabolically" by interactions of enzymes with substrates, products, or allosteric effectors. In the present study, a method is developed to dissect the hierarchical regulation into contributions by transcription, translation, protein degradation, and posttranslational modification. The method was applied to the regulation of fluxes through individual glycolytic enzymes when the yeast Saccharomyces cerevisiae was confronted with the absence of oxygen and the presence of benzoic acid depleting its ATP. Metabolic regulation largely contributed to the approximately 10-fold change in flux through the glycolytic enzymes. This contribution varied from 50 to 80%, depending on the glycolytic step and the cultivation condition tested. Within the 50-20% hierarchical regulation of fluxes, transcription played a minor role, whereas regulation of protein synthesis or degradation was the most important. These also contributed to 75-100% of the regulation of protein levels. Keywords: Condition comparison
2007-10-17 | GSE9232 | GEO
Project description:Alternative splicing of the Pkm gene product generates the PKM1 and PKM2 isoforms of pyruvate kinase, and PKM2 expression is closely linked to embryogenesis, tissue regeneration, and cancer. To interrogate the functional requirement for PKM2 during development and tissue homeostasis, we generated germline PKM2 null mice (Pkm2-/-). Unexpectedly, despite being the primary isoform expressed in most wild-type adult tissues, we found that Pkm2-/- mice are viable and fertile. Thus, PKM2 is not required for embryonic or postnatal development. Loss of PKM2 leads to compensatory expression of PKM1 in the tissues that normally express PKM2. Strikingly, PKM2 loss leads to spontaneous development of hepatocellular carcinoma (HCC) with high penetrance that is accompanied by progressive changes in systemic metabolism characterized by altered systemic glucose homeostasis, inflammation, and hepatic steatosis. Therefore, in addition to its role in cancer metabolism, PKM2 plays a role in controlling systemic metabolic homeostasis and inflammation, thereby preventing HCC by a non-cell-autonomous mechanism. RNA was isolated from flash frozen ground whole liver tissue of 35 week old PKM2 KO and WT mice. Three independent mice from each condition were used as biological replicates.
2016-03-25 | E-GEOD-79535 | biostudies-arrayexpress
Project description:Nuclear translocation of the key metabolic enzyme Pyruvate Kinase Isoform 2 (PKM2) is widely observed in cancer, but its contribution to gene regulation and pathogenesis remains unclear. Here we find that PKM2 specifically recognized folded RNA G-quadruplex (rG4) structures in vitro with high affinity. In human cells, nuclear PKM2 bound thousands of precursor mRNAs at potential rG4 sites and translocation of PKM2 to the nucleus resulted in increased expression of rG4-containing mRNAs.
2024-07-31 | GSE179817 | GEO
Project description:Allosteric regulation is central to the role of the glycolytic enzyme pyruvate kinase M2 (PKM2) in cancer metabolism. Multiple activating and inhibitory allosteric ligands regulate PKM2 activity by controlling the equilibrium between high activity tetramers and low activity dimers and monomers. However, how allosteric inputs from simultaneous binding of different ligands are integrated to regulate PKM2 activity remains elusive. Here, we show that, [PKM2 activation and tetramerisation can be uncoupled as] in the presence of the allosteric inhibitor phenylalanine (Phe), saturating amounts of the activator fructose 1,6-bisphosphate (F-1,6-bP) can induce PKM2 tetramerisation, but fail to maximally increase enzymatic activity. We use a new computational framework to identify residues that mediate FBP-induced allostery and show that, while mutation of A327 and C358 do not abrogate the ability of F-1,6-BP to increase PKM2 activity, it prevents Phe from interfering with it. Our findings demonstrate a role for residues involved in FBP allostery in enabling the integration of allosteric input from Phe and reveal an allosteric cross-talk that underlies the co-ordinate regulation of PKM2 activity by distinct allosteric ligands. The absolute amount of the isoforms of PKM (PKM1/2) were quantified in the cell lines of interested to inform the described model.
2019-11-12 | PXD010334 | Pride
Project description:Altered glycolysis is the most fundamental metabolic change associated with the Warburg effect. However, the precise mechanisms are not completely understood. Here we dissect how MNX1-AS1 reinforces the Warburg effect through facilitating the non-glycolytic actions of PKM2 in the nucleus. We found that MNX1-AS1 expression was frequently overexpressed in hepatocellular carcinoma (HCC). In the context of HCC, we show MNX1-AS1 acts as a scaffold to promote interactions between PKM2 and importin α5. Upon EGFR activation, the resulting ternary complex drives the translocation of PKM2 into the nucleus. In consequence, glycolytic pathway components including key mediators of the Warburg effect (LDHA, Glut1 and PDK1) are upregulated. Manipulating MNX1-AS1 elicited robust effects on glycolysis associated with marked changes in HCC growth in vitro and in vivo, indicative of the significant contribution of MNX1-AS1 to tumorigenesis. Moreover, while MNX1-AS1 expression is driven by c-Myc, its actions associated with PKM2 were shown to be downstream and independent of c-Myc. Given the status of MNX1-AS1 as a pan-cancer upregulated lncRNA, this implicitly highlights the potential of targeting MNX1-AS1 to selectively counter the Warburg effect in a range of tumor types.
2022-11-23 | GSE213064 | GEO
Project description:Purpose: In this study, we compared transcriptome profiling (RNA-seq) between normal mouse embryonic stem cell (E14) and Hexokinase2 (Hk2)/ Pyruvate Kinase M2 (Pkm2) overexpressed E14 cell. Result: Using an optimized data analysis workflow, we mapped over 4 billion sequence reads per sample to the mouse genome (build mm9) and identified 28698 transcripts in 5 samples. Conclusion: Our study represents the first detailed analysis of Hk2/ Pkm2 overexpressed E14 cell transcriptomes, generated by RNA-seq technology We compared transcriptome profiling (RNA-Seq) between normal mouse embryonic stem cell (E14) and E14 cells over-expressing Hexokinase2 (Hk2)/Pyruvate Kinase M2 (Pkm2)
2016-07-03 | E-GEOD-65246 | biostudies-arrayexpress
Project description:Pyruvate kinase M2 (PKM2), as the terminal and last rate-limiting enzyme of the glycolytic pathway, is an ideal enzyme for regulating metabolic phenotype. PKM2 tetramer activation has shown a protective role against diabetic kidney disease (DKD). However, the molecular mechanisms involved in diabetic tubular has not been investigated so far. In this study, we performed transcriptome gene expression profiling in human renal proximal tubular epithelial cell line (HK-2 cells) treated with high D-glucose (HG) for 7 days before the addition of 10-μM TEPP-46, an activator of PKM2 tetramerization, for a further 1 day in the presence of HG. Afterwards, we analyzed the differentially expressed (DE) genes and investigated gene relationships based on weighted gene co-expression network analysis (WGCNA). The results showed that 2,902 DE genes were identified (adjusted P-value ≤ 0.05), where 2,509 DE genes (86.46%) were co-expressed in the key module. Four extremely down-regulated DE genes (HSPA8, HSPA2, HSPA1B and ARRB1) and three extremely up-regulated DE genes (GADD45A, IGFBP3 and SIAH1) enriched in the down-regulated endocytosis (hsa04144) and up-regulated p53 signaling pathway (hsa04115), respectively, were validated by the qRT-PCR experiments. The qRT-PCR results showed that the relative expression levels of HSPA8 (adjusted P-value = 4.45×10-34 & log2(FC) = -1.12), HSPA2 (adjusted P-value = 6.09×10-14 & log2(FC) = -1.27), HSPA1B (adjusted P-value = 1.14×10-11 & log2(FC) = -1.02) and ARRB1 (adjusted P-value = 2.60×10-5 & log2(FC) = -1.13) were significantly different (P-value < 0.05) from case group to control group. Furthermore, the interactions and predicted microRNAs of the key genes (HSPA8, HSPA2, HSPA1B and ARRB1) were visualized in networks. This study identified the key candidate transcriptomic biomarkers and biological pathways in the hyperglycemic HK-2 cells responding to the PKM2 activator TEPP-46. These results will be valuable for further research on PKM2 in DKD.
2022-08-29 | GSE205674 | GEO