M2 Isoform Research Articles

Disruption of canonical AHR-mediated induction of hepatocyte PKM2 expression compromises antioxidant defenses and increases TCDD-induced hepatotoxicity

Metabolic reprogramming by the pyruvate kinase M2 isoform is associated with cell proliferation and reactive oxygen species (ROS) defenses. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant that induces ROS and hepatotoxicity, dose-dependently induces pyruvate kinase muscle isoform M2 (PKM2) in the liver. To further investigate its role in combating TCDD hepatotoxicity, a PkmΔDRE mouse was constructed lacking the dioxin response element mediating aryl hydrocarbon receptor (AHR) induction. TCDD failed to induce hepatic PKM2 in PkmΔDRE mice and in primary hepatocytes isolated from an AHR knockout model (AHRV375Afl/flAlb-CreERT2), demonstrating induction is AHR dependent. Both wild-type (WT) and PkmΔDRE mice exhibited dose-dependent increases in liver weight after treatment with TCDD every 4 days for 28 days. Glutathione (GSH) levels increased in WT mice while oxidized glutathione (GSSG) levels increased in both models with a 24-fold decrease in the GSH/GSSG ratio in PkmΔDRE mice suggesting lower antioxidant and recycling capacity. Moreover, TCDD-induced fibrosis was more severe in PkmΔDRE mice while PkmΔDRE hepatocytes exhibited greater cytotoxicity following co-treatment with TCDD and hydrogen peroxide. TCDD also induced PKM2 in human HepaRG™ cells with AHR enrichment at a conserved DRE core within the locus. These results suggest AHR-mediated PKM2 induction is a novel antioxidant response to TCDD.

Computational Design, Chemical Synthesis, and Bioevaluation of Pyrimidinyl‐Indoles for Targeting Breast Cancer Cells

AbstractCancer is a global health challenge, despite significant advancements in research. Addressing this issue, we report the design, synthesis and biological evaluation of pyrimidinyl indoles as potential antineoplastic agents. Pyrimidinyl indoles interact with pyruvate kinase isoform M2 (PKM2), which is commonly overexpressed in oral, lung, breast and colorectal cancers. In silico studies indicated that these molecules are having affinity towards PKM2 and targeting key residues such as Phe26, Leu27, Tyr390, Gln393, Leu394 and Glu397 at the interfacial active site region between the two chains of PKM2. Thereafter compounds were designed, synthesized and characterized using nuclear magnetic resonance (NMR), infrared spectroscopy (IR) and mass spectrometry (MS). This was followed by in vitro screening using cell lines and enzyme‐coupled assays. One of the designed PKM2 inhibitor demonstrated potent anticancer activity against MCF‐7 (Breast Cancer) cells while sparing normal MCF‐10 A breast cells. Molecular dynamics (MD) simulation studies showed that the developed inhibitor stabilized the PKM2 active site over a 100 ns simulation.

Tetramerization of PKM2 Alleviates Traumatic Brain Injury by Ameliorating Mitochondrial Damage in Microglia.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Microglial activation and neuroinflammation are key cellular events that determine the outcome of TBI, especially neuronal and cognitive function. Studies have suggested that the metabolic characteristics of microglia dictate their inflammatory response. The pyruvate kinase isoform M2 (PKM2), a key glycolytic enzyme, is involved in the regulation of various cellular metabolic processes, including mitochondrial metabolism. This suggests that PKM2 may also participate in the regulation of microglial activation during TBI. Therefore, the present study aimed to evaluate the role of PKM2 in regulating microglial activation and neuroinflammation and its effects on cognitive function following TBI. A controlled cortical impact (CCI) mouse model and inflammation-induced primary mouse microglial cells in vitro were used to investigate the potential effects of PKM2 inhibition and regulation. PKM2 was significantly increased during the acute and subacute phases of TBI and was predominantly detected in microglia rather than in neurons. Our results demonstrate that shikonin and TEPP-46 can inhibit microglial inflammation, improving mitochondria, improving mouse behavior, reducing brain defect volume, and alleviating pathological changes after TBI. There is a difference in the intervention of shikonin and TEPP-46 on PKM2. Shikonin directly inhibits General PKM2; TEPP-46 can promote the expression of PKM2 tetramer. In vitro experiments, TEPP-46 can promote the expression of PKM2 tetramer, enhance the interaction between PKM2 and MFN2, improve mitochondria, alleviate neuroinflammation. General inhibition and tetramerization activation of PKM2 attenuated cognitive function caused by TBI, whereas PKM2 tetramerization exhibited a better treatment effect. Our experiments demonstrated the non-metabolic role of PKM2 in the regulation of microglial activation following TBI. Both shikonin and TEPP-46 can inhibit pro-inflammatory factors, but only TEPP-46 can promote PKM2 tetramerization and upregulate the release of anti-inflammatory factors from microglia.

The role of NAD-dependent deacetylase sirtuin-2 in liver metabolic stress through regulating pyruvate kinase M2 ubiquitination

NAD-dependent deacetylase Sirt2 is involved in mammalian metabolic activities, matching energy demand with energy production and expenditure, and is relevant to a variety of metabolic diseases. Here, we constructed Sirt2 knockout and adeno-associated virus overexpression mice and found that deletion of hepatic Sirt2 accelerated primary obesity and insulin resistance in mice with concomitant hepatic metabolic dysfunction. However, the key targets of Sirt2 are unknown. We identified the M2 isoform of pyruvate kinase (PKM2) as a key Sirt2 target involved in glycolysis in metabolic stress. Through yeast two-hybrid and mass spectrometry combined with multi-omics analysis, we identified candidate acetylation modification targets of Sirt2 on PKM2 lysine 135 (K135). The Sirt2-mediated deacetylation-ubiquitination switch of PKM2 regulated the development of glycolysis. Here, we found that Sirt2 deficiency led to impaired glucose tolerance and insulin resistance and induced primary obesity. Sirt2 severely disrupted liver function in mice under metabolic stress, exacerbated the metabolic burden on the liver, and affected glucose metabolism. Sirt2 underwent acetylation modification of lysine 135 of PKM2 through a histidine 187 enzyme active site-dependent effect and reduced ubiquitination of the K48 ubiquitin chain of PKM2. Our findings reveal that the hepatic glucose metabolism links nutrient state to whole-body energetics through the rhythmic regulation of Sirt2.

Checkpoint Kinase 1 Stimulates Endogenous Cardiomyocyte Renewal and Cardiac Repair by Binding to Pyruvate Kinase Isoform M2 C-Domain and Activating Cardiac Metabolic Reprogramming in a Porcine Model of Myocardial Ischemia/Reperfusion Injury.

The regenerative capacity of the adult mammalian hearts is limited. Numerous studies have explored mechanisms of adult cardiomyocyte cell-cycle withdrawal. This translational study evaluated the effects and underlying mechanism of rhCHK1 (recombinant human checkpoint kinase 1) on the survival and proliferation of cardiomyocyte and myocardial repair after ischemia/reperfusion injury in swine. Intramyocardial injection of rhCHK1 protein (1 mg/kg) encapsulated in hydrogel stimulated cardiomyocyte proliferation and reduced cardiac inflammation response at 3 days after ischemia/reperfusion injury, improved cardiac function and attenuated ventricular remodeling, and reduced the infarct area at 28 days after ischemia/reperfusion injury. Mechanistically, multiomics sequencing analysis demonstrated enrichment of glycolysis and mTOR (mammalian target of rapamycin) pathways after rhCHK1 treatment. Co-Immunoprecipitation (Co-IP) experiments and protein docking prediction showed that CHK1 (checkpoint kinase 1) directly bound to and activated the Serine 37 (S37)and Tyrosine 105 (Y105) sites of PKM2 (pyruvate kinase isoform M2) to promote metabolic reprogramming. We further constructed plasmids that knocked out different CHK1 and PKM2 amino acid domains and transfected them into Human Embryonic Kidney 293T (HEK293T) cells for CO-IP experiments. Results showed that the 1-265 domain of CHK1 directly binds to the 157-400 amino acids of PKM2. Furthermore, hiPSC-CM (human iPS cell-derived cardiomyocyte) invitro and invivo experiments both demonstrated that CHK1 stimulated cardiomyocytes renewal and cardiac repair by activating PKM2 C-domain-mediated cardiac metabolic reprogramming. This study demonstrates that the 1-265 amino acid domain of CHK1 binds to the 157-400 domain of PKM2 and activates PKM2-mediated metabolic reprogramming to promote cardiomyocyte proliferation and myocardial repair after ischemia/reperfusion injury in adult pigs.

Oridonin attenuates liver ischemia–reperfusion injury by suppressing PKM2/NLRP3-mediated macrophage pyroptosis

BackgroundThe NOD-like receptor protein 3 (NLRP3) mediated pyroptosis of macrophages is closely associated with liver ischemia reperfusion injury (IRI). As a covalent inhibitor of NLRP3, Oridonin (Ori), has strong anti-inflammasome effect, but its effect and mechanisms for liver IRI are still unknown. MethodsMice and liver macrophages were treated with Ori, respectively. Co-IP and LC-MS/MS analysis of the interaction between PKM2 and NLRP3 in macrophages. Liver damage was detected using H&E staining. Pyroptosis was detected by WB, TEM, and ELISA. ResultsOri ameliorated liver macrophage pyroptosis and liver IRI. Mechanistically, Ori inhibited the interaction between pyruvate kinase M2 isoform (PKM2) and NLRP3 in hypoxia/reoxygenation（H/R）-induced macrophages, while the inhibition of PKM2/NLRP3 reduced liver macrophage pyroptosis and liver IRI. ConclusionOri exerted protective effects on liver IRI via suppressing PKM2/NLRP3-mediated liver macrophage pyroptosis, which might become a potential therapeutic target in the clinic.

Abstract 6417: Urinary PKM2 in muscle invasive bladder cancer

Abstract Background & Aims: Biomarkers are commonly used for both detection of cancer and monitoring response to treatment. Although several biomarkers have demonstrated promise in patients with bladder cancer (BCa), only urine cytology is widely utilized. Increased availability of effective biomarkers may improve clinical decision making and reduce the need for more invasive imaging. The M2 isoform of pyruvate kinase (PKM2) is upregulated in a number of cancers, including BCa, and promotes metabolic changes, including a shift to aerobic glycolysis. The aim of this study was to determine whether urine PKM2 levels are useful for detection and monitoring response to cisplatin-based neoadjuvant chemotherapy in patients with muscle invasive BCa. Methods: Urine samples from patients enrolled in the University of Kanas Medical Center Bladder Cancer Biorepository were analyzed for levels of PKM2. Patients were included if they had both T2 stage disease and had received cisplatin-based neoadjuvant chemotherapy and were compared to healthy controls. Patients were categorized based on their pathological response to treatment and recurrence of disease within two years of definitive surgery. Urinary PKM2 levels were measured and evaluated for their ability to predict response to treatment and two-year disease-free survival. Results: Immunohistochemistry revealed high levels of PKM2 within muscle invasive tumors compared to adjacent tissue. Patients with BCa had 150-fold increase in levels of urinary PKM2 compared to controls. However, there were no differences in urine PKM2 levels in patients with complete pathological response compared to those with no response. Urine PKM2 showed limited predictive value for disease recurrence (area under receiver operating characteristic curve = 0.65). Conclusion: Urine levels of PKM2 are significantly elevated in patients with muscle invasive BCa and may be useful for tumor detection. Urinary PKM2 is less effective at monitoring response to treatment and may instead represent a marker of urothelial susceptibility to tumor formation. Citation Format: David Matye, Juliann Leak, Erika Abbott, Benjamin Woolbright, John Taylor. Urinary PKM2 in muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6417.

Interleukin-22 inhibits cardiac fibrosis by regulating fibroblast metabolic reprogramming in myocardial infarction

Cardiac fibrosis, a significant characteristic of cardiovascular diseases, leads to ventricular remodeling and impaired cardiac function. In this study, we aimed to investigate the role of Interleukin-22 (IL-22) in myocardial fibrosis following myocardial infarction (MI) and to explore the underlying metabolic mechanisms. Here we analyzed the single-cell sequencing data and found that the level of aerobic glycolysis was significantly higher in cardiac fibrosis in MI patient, which we validated through in vivo experiments. Utilizing MI mouse model, these experiments revealed decreased serum IL-22 levels and increased levels of AngII and TGF-β1. However, treatment with exogenous IL-22 reversed these changes, reduced infarct size, and fibrosis. In vitro experiments demonstrated that IL-22 inhibited AngII-induced fibroblast-to-myofibroblast transition (FMT) by suppressing the expression of α-SMA, Cola1, and Cola3. Metabolic analysis indicated that IL-22 decreased the expression of glycolytic enzymes and reduced lactate production in cardiac fibroblasts. Further in vivo experiments confirmed the inhibitory effect of IL-22 on Pyruvate kinase isoform M2 (PKM2) levels in heart tissue. Additionally, IL-22 activated the c-Jun N-terminal kinase (JNK) pathway, while inhibition of JNK partially reversed IL-22's effect on PKM2 activity. These findings suggest that IL-22 mitigates cardiac fibrosis and FMT by inhibiting aerobic glycolysis by activating the JNK/PKM2 pathway. Our study highlights IL-22 as a potential therapeutic target for myocardial fibrosis and cardiovascular diseases, providing insights into its role in regulating fibrosis and glycolysis. These findings pave the way for developing targeted therapies and investigating additional metabolic pathways for improved treatment outcomes in the field of cardiovascular diseases.

METTL14/miR-29c-3p axis drives aerobic glycolysis to promote triple-negative breast cancer progression though TRIM9-mediated PKM2 ubiquitination.

The energy metabolic rearrangement of triple-negative breast cancer (TNBC) from oxidative phosphorylation to aerobic glycolysis is a significant biological feature and can promote the malignant progression. However, there is little knowledge about the functional mechanisms of methyltransferase-like protein 14 (METTL14) mediated contributes to TNBC malignant progression. Our study found that METTL14 expression was significantly upregulated in TNBC tissues and cell lines. Silencing METTL14 significantly inhibited TNBC cell growth and invasion invitro, as well as suppressed tumour growth. Mechanically, METTL14 was first found to activate miR-29c-3p through m6A and regulate tripartite motif containing 9 (TRIM9) to promote ubiquitination of pyruvate kinase isoform M2 (PKM2) and lead to its transition from tetramer to dimer, resulting in glucose metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis to promote the progress of TNBC. Taken together, these findings reveal important roles of METTL14 in TNBC tumorigenesis and energy metabolism, which might represent a novel potential therapeutic target for TNBC.

CD8+ T cell metabolic flexibility elicited by CD28-ARS2 axis-driven alternative splicing of PKM supports antitumor immunity

Metabolic flexibility has emerged as a critical determinant of CD8+ T-cell antitumor activity, yet the mechanisms driving the metabolic flexibility of T cells have not been determined. In this study, we investigated the influence of the nuclear cap-binding complex (CBC) adaptor protein ARS2 on mature T cells. In doing so, we discovered a novel signaling axis that endows activated CD8+ T cells with flexibility of glucose catabolism. ARS2 upregulation driven by CD28 signaling reinforced splicing factor recruitment to pre-mRNAs and affected approximately one-third of T-cell activation-induced alternative splicing events. Among these effects, the CD28-ARS2 axis suppressed the expression of the M1 isoform of pyruvate kinase in favor of PKM2, a key determinant of CD8+ T-cell glucose utilization, interferon gamma production, and antitumor effector function. Importantly, PKM alternative splicing occurred independently of CD28-driven PI3K pathway activation, revealing a novel means by which costimulation reprograms glucose metabolism in CD8+ T cells.

Thermodynamics and S-Palmitoylation Dependence of Interactions between Human Aquaporin-4 M1 Tetramers in Model Membranes.

Aquaporin-4 (AQP4) is a water channel protein found primarily in the central nervous system (CNS) that helps to regulate water-ion homeostasis. AQP4 exists in two major isoforms: M1 and M23. While both isoforms have a homotetrameric quaternary structure and are functionally identical when transporting water, the M23 isoform forms large protein aggregates known as orthogonal arrays of particles (OAPs). In contrast, the M1 isoform creates a peripheral layer around the outside of these OAPs, suggesting a thermodynamically stable interaction between the two. Structurally, the M1 isoform has an N-terminal tail that is 22 amino acids longer than the M23 isoform and contains two solvent-accessible cysteines available for S-palmitoylation at cysteine-13 (Cys-13) and cysteine-17 (Cys-17) in the amino acid sequence. Earlier work suggests that the palmitoylation of these cysteines might aid in regulating AQP4 assemblies. This work discusses the thermodynamic driving forces for M1 protein-protein interactions and how the palmitoylation state of M1 affects them. Using temperature-dependent single-particle tracking, the standard state free energies, enthalpies, and entropies were measured for these interactions. Furthermore, we present a binding model based on measured thermodynamics and a structural modeling study. The results of this study demonstrate that the M1 isoform will associate with itself according to the following expressions: 2[AQP4-M1]4 ↔ [[AQP4-M1]4]2 when palmitoylated and 3[AQP4-M1]4 ↔ [AQP4-M1]4 + [[AQP4-M1]4]2 ↔ [[AQP4-M1]4]3 when depalmitoylated. This is primarily due to a conformational change induced by adding the palmitic acid groups at Cys-13 and Cys-17 in the N-terminal tails of the homotetramers. In addition, a statistical mechanical model was developed to estimate the Gibbs free energy, enthalpy, and entropy for forming dimers and trimers. These results were in good agreement with experimental values.

MiR-933 accelerates the growth of liver cancer cells by enhancing pyruvate kinase isoform M2

miR-933 accelerates the growth of liver cancer cells by enhancing pyruvate kinase isoform M2

Omentin-1 induces mechanically activated fibroblasts lipogenic differentiation through pkm2/yap/pparγ pathway to promote lung fibrosis resolution.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by extensive extracellular matrix (ECM) deposition by activated myofibroblasts, which are specialized hyper-contractile cells that promote ECM remodeling and matrix stiffening. New insights on therapeutic strategies aimed at reversing fibrosis by targeting myofibroblast fate are showing promise in promoting fibrosis resolution. Previously, we showed that a novel adipocytokine, omentin-1, attenuated bleomycin (BLM)-induced lung fibrosis by reducing the number of myofibroblasts. Apoptosis, deactivation, and reprogramming of myofibroblasts are important processes in the resolution of fibrosis. Here we report that omentin-1 reverses established lung fibrosis by promoting mechanically activated myofibroblasts dedifferentiation into lipofibroblasts. Omentin-1 promotes myofibroblasts lipogenic differentiation by inhibiting dimerization and nuclear translocation of glycolytic enzymes pyruvate kinase isoform M2 (PKM2) and activation of the downstream Yes-associated protein (YAP) by increasing the cofactor fructose-1,6-bisphosphate (F1, 6BP, FBP). Moreover, omentin-1 activates proliferator-activated receptor gamma (PPARγ) signaling, the master regulator of lipogenesis, and promotes the upregulation of the lipogenic differentiation-related protein perilipin 2 (PLIN2) by suppressing the PKM2-YAP pathway. Ultimately, omentin-1 facilitates myofibroblasts transformation into the lipofibroblast phenotype, with reduced collagen synthesis and enhanced degradation properties, which are crucial mechanisms to clear the ECM deposition in fibrotic tissue, leading to fibrosis resolution. Our results indicate that omentin-1 targets mechanical signal accelerates fibrosis resolution and reverses established lung fibrosis by promoting myofibroblasts lipogenic differentiation, which is closely associated with ECM clearance in fibrotic tissue. These findings suggest that targeting mechanical force to promote myofibroblast lipogenic differentiation is a promising therapeutic strategy against persistent lung fibrosis.

Diagnostic accuracy of tumor M2-pyruvate kinase (tM2-PK) as a non-invasive biomarker in colorectal cancer: A systematic review and meta-analysis

IntroductionThe tumor pyruvate kinase M2 isoform (tM2-PK) is a glycolytic enzyme isoform that is present on the surface of rapidly proliferating cancer cells. The objective of this investigation was to assess the efficacy of the tM2-PK measurement assay in detecting colorectal cancer (CRC) through the analysis of serum/plasma and stool samples obtained from patients. MethodsThe pooled diagnostic performance measures, including sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), the area under the curve (AUC), Q*index, and summary receiver-operating characteristic curve (SROC), were computed using the Meta-Disc V.1.4 and Comprehensive Meta-Analysis V.3.3 software. The statistical methods of I2 and chi-square were employed to assess the presence of heterogeneity. The estimation of publication bias was conducted through the implementation of Begg's rank correlation and Egger's regression asymmetry tests. ResultsA total of 28 studies were found, involving 2900 participants (1560 cases and 1340 controls). The diagnostic accuracy of tM2-PK was calculated in CRC based on the pooled sensitivity of 83.70% (95% CI: 82.0% – 85.30%), specificity of 74.0% (95% CI: 72.0% – 76.0%), PLR of 4.432 (95% CI: 3.33 – 5.60), NLR of 0.187 (95% CI: 0.144 – 0.243), DOR of 30.182 (95% CI: 19.761 – 46.10) as well as AUC at 91.6%, and Q*-index at 85.0%. Publication bias was seen based on Begg's (p = 0.0006) and Egger's (p = 0.00015) tests. ConclusionThe results demonstrate that tM2-PK exhibits promise as a fair marker for CTRC, with the potential to serve as a non-invasive biomarker.

Fibrinogen-like protein 2 regulates macrophage glycolytic reprogramming by directly targeting PKM2 and exacerbates alcoholic liver injury

Background & AimsSwitching of the macrophage activation phenotype affects the pathogenesis of alcoholic liver diseases, and metabolic reprogramming can provide the energy demand for macrophage phenotypes shift. However, the molecular mechanism by which immune metabolism regulates the activation of proinflammatory macrophages remains unclear. ApproachExpression of Fgl2 was examined in patients with alcoholic hepatitis and healthy controls. Mice were fed with a Lieber-DeCarli diet. Livers from mice were used to observe liver injury and macrophage activation. Fgl2 overexpressing THP-1 cell was used to find interacting partners through immunoprecipitation plus mass spectrometry. Naive bone marrow derived macrophages stimulated with LPS and ethanol were used for cell experiments. ResultsExpression of Fgl2 was elevated in macrophages of livers from mice with chronic-binge ethanol feeding or patients with alcoholic hepatitis. Fgl2 depletion ameliorated ethanol diet-induced hepatic steatosis and oxidative injury as well as the levels of proinflammatory cytokines. Fgl2−/− mice exhibited suppressed M1 polarization and glycolysis pathway activation. Fgl2 interacted with the M2 isoform of pyruvate kinase (PKM2) in macrophages and facilitated PKM2 nuclear translocation, thus promoting glycolysis in M1 macrophages and the secretion of proinflammatory cytokines. Furthermore, Fgl2 overexpression in THP-1 cells enhances PKM2-dependent glycolysis and inflammation, which could be reversed by activation of enzymatic PKM2 using DASA58. ConclusionsTaken together, Fgl2 hastens the development of alcoholic liver injury by mediating PKM2 dependent aerobic glycolysis in proinflammatory macrophages. Strategies that inhibiting proinflammatory macrophage activation by silencing Fgl2 might be a potential therapeutic intervention for alcoholic liver injury.

Heterogeneity and mitochondrial vulnerability configurate the divergent immunoreactivity of human induced microglia-like cells

Microglia play versatile roles in progression of and protection against neuroinflammatory diseases. Little is known, however, about the mechanisms underlying the diverse reactivity of microglia to inflammatory conditions. We investigated how human induced microglia-like (iMG) cells respond to innate immune ligands. Quantitative PCR showed that poly-I:C and lipopolysaccharide (LPS) activated the expression of IL1B and TNF. Immunoreactivity of iMG did not differ between controls (n = 11) and patients with neuroinflammatory diseases (n = 24). Flow cytometry revealed that CD14high cells expressed interleukin (IL) -1β after LPS treatment. Immunoblotting showed that poly-I:C and LPS differentially activated inflammatory pathways but commonly induced mitochondrial instability and the expression of pyruvate kinase isoform M2 (PKM2). Furthermore, a potent stimulator of PKM2 (DASA-58) alleviated IL-1β production after LPS treatment. These data indicate that heterogeneous cell populations and mitochondrial stability underlie the divergent immunoreactivity of human iMG in environments.

KITENIN promotes aerobic glycolysis through PKM2 induction by upregulating the c-Myc/hnRNPs axis in colorectal cancer

PurposeThe oncoprotein KAI1 C-terminal interacting tetraspanin (KITENIN; vang-like 1) promotes cell metastasis, invasion, and angiogenesis, resulting in shorter survival times in cancer patients. Here, we aimed to determine the effects of KITENIN on the energy metabolism of human colorectal cancer cells.Experimental designThe effects of KITENIN on energy metabolism were evaluated using in vitro assays. The GEPIA web tool was used to extrapolate the clinical relevance of KITENIN in cancer cell metabolism. The bioavailability and effect of the disintegrator of KITENIN complex compounds were evaluated by LC–MS, in vivo animal assay.ResultsKITENIN markedly upregulated the glycolytic proton efflux rate and aerobic glycolysis by increasing the expression of GLUT1, HK2, PKM2, and LDHA. β-catenin, CD44, CyclinD1 and HIF-1A, including c-Myc, were upregulated by KITENIN expression. In addition, KITENIN promoted nuclear PKM2 and PKM2-induced transactivation, which in turn, increased the expression of downstream mediators. This was found to be mediated through an effect of c-Myc on the transcription of hnRNP isoforms and a switch to the M2 isoform of pyruvate kinase, which increased aerobic glycolysis. The disintegration of KITENIN complex by silencing the KITENIN or MYO1D downregulated aerobic glycolysis. The disintegrator of KITENIN complex compound DKC1125 and its optimized form, DKC-C14S, exhibited the inhibition activity of KITENIN-mediated aerobic glycolysis in vitro and in vivo.ConclusionsThe oncoprotein KITENIN induces PKM2-mediated aerobic glycolysis by upregulating the c-Myc/hnRNPs axis.

Glycolytic enzyme PKM2 regulates cell senescence but not inflammation in the process of osteoarthritis.

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 p16 INK4a expression. Compared with low-passage chondrocytes, high-passage chondrocytes exhibit increased expression of p16 INK4a 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.

Discovery of anti-neuroinflammatory agents from 1,4,5,6-tetrahydrobenzo[2,3]oxepino[4,5-d]pyrimidin-2-amine derivatives by regulating microglia polarization

Neuroinflammation mediated by microglia activation leads to various neurodegenerative and neurological disorders. In order to develop more and better options for this disorders, a series of 3,4-dihydrobenzo[b]oxepin-5(2H)-one derivatives (BZPs, 6–19) and novel 1,4,5,6-tetrahydrobenzo[2,3]oxepino[4,5-d]pyrimidin-2-amine derivatives (BPMs, 20–33) were synthesized and screened the anti-neuroinflamamtion effects. 3,5-bis-trifluoromethylphenyl-substituted BPM 29 showed more potent anti-neuroinflammatory activity and no toxicity to BV2 microglia cells in vitro. 29 significantly reduced the number of M1 phenotype of microglia cells, but significantly increased the number of M2 phenotype of microglia cells in lipopolysaccharide (LPS)-induced BV2 microglia cells. 29 significantly reduced the secretion of inflammatory cytokines (IL-18, IL-1β, TNF-α), but increased the secretion of anti-inflammatory cytokines (IL-10) from LPS-induced BV2 microglia cells. Also, 29 inhibited the NOD-like receptor NLRP3 inflammasome formation, and down-regulated the expression of M2 isoform of pyruvate kinase in LPS-induced BV2 microglia cells. In vivo, 29 reduced the neuroinflammation in cuprizone-induced inflammatory and demyelinating mice by reducing the expression of inducible nitric-oxide synthase, but increased the expression of CD206. Taken together, 29 might be a prospective anti-neuroinflammatory compound for neuroinflammatory and demyelinating disease by alleviating microglia activation.

LincRNA-EPS Alleviates Inflammation in TMJ Osteoarthritis by Binding to SRSF3

Temporomandibular joint osteoarthritis (TMJOA) is a common inflammatory disease that can cause pain, cartilage degradation, and subchondral bone loss. However, the key regulatory factors and new targets for the treatment of TMJOA have yet to be determined. Long noncoding RNAs (lncRNAs) have shown remarkable potential in regulating tissue homeostasis and disease development. The long intergenic RNA–erythroid prosurvival (lincRNA-EPS) is reported to be an effective inhibitor of inflammation, but its role in TMJOA is unexplored. Here, we found that lincRNA-EPS is downregulated and negatively correlated with inflammatory factors in the condyles of TMJOA mice. LincRNA-EPS knockout aggravated inflammation and tissue destruction after TMJOA modeling. The in vitro studies confirmed that loss of lincRNA-EPS facilitated inflammatory factor expression in condylar chondrocytes, while recovered expression of lincRNA-EPS showed anti-inflammatory effects. Mechanistically, RNA sequencing revealed that the inflammatory response pathway nuclear factor–kappa B (NF-κB) was mostly affected by lincRNA-EPS deficiency. Moreover, lincRNA-EPS was proved to effectively bind to serine/arginine-rich splicing factor 3 (SRSF3) and inhibit its function in pyruvate kinase isoform M2 (PKM2) formation, thus restraining the PKM2/NF-κB pathway and the expression of inflammatory factors. In addition, local injection of the lincRNA-EPS overexpression lentivirus significantly alleviated inflammation, cartilage degradation, and subchondral bone loss in TMJOA mice. Overall, lincRNA-EPS regulated the inflammatory process of condylar chondrocytes by binding to SRSF3 and showed translational application potential in the treatment of TMJOA.