Project description:Sepsis, severe sepsis and septic shock are the main cause of mortality in non-cardiac intensive care units. Immunometabolism has been linked to sepsis; however, the precise mechanism by which metabolic reprogramming regulates the inflammatory response is unclear. Here we show that aerobic glycolysis contributes to sepsis by modulating inflammasome activation in macrophages. PKM2-mediated glycolysis promotes inflammasome activation by modulating EIF2AK2 phosphorylation in macrophages. Pharmacological and genetic inhibition of PKM2 or EIF2AK2 attenuates NLRP3 and AIM2 inflammasomes activation, and consequently suppresses the release of IL-1β, IL-18 and HMGB1 by macrophages. Pharmacological inhibition of the PKM2-EIF2AK2 pathway protects mice from lethal endotoxemia and polymicrobial sepsis. Moreover, conditional knockout of PKM2 in myeloid cells protects mice from septic death induced by NLRP3 and AIM2 inflammasome activation. These findings define an important role of PKM2 in immunometabolism and guide future development of therapeutic strategies to treat sepsis.
Project description:The microtubule (MT) cytoskeleton is essential for the formation of morphologically appropriate neurons. The existence of the acentrosomal MT organizing center in neurons has been proposed but its identity remained elusive. Here we provide evidence showing that TPX2 is an important component of this acentrosomal MT organizing center. First, neurite elongation is compromised in TPX2-depleted neurons. In addition, TPX2 localizes to the centrosome and along the neurite shaft bound to MTs. Depleting TPX2 decreases MT formation frequency specifically at the tip and the base of the neurite, and these correlate precisely with the regions where active GTP-bound Ran proteins are enriched. Furthermore, overexpressing the downstream effector of Ran, importin, compromises MT formation and neuronal morphogenesis. Finally, applying a Ran-importin signaling interfering compound phenocopies the effect of TPX2 depletion on MT dynamics. Together, these data suggest a model in which Ran-dependent TPX2 activation promotes acentrosomal MT nucleation in neurons.
Project description:During muscle regeneration, the transcription factor Pax7 stimulates the differentiation of satellite cells (SCs) toward the muscle lineage but restricts adipogenesis. Here, we identify HDAC4 as a regulator of Pax7-dependent muscle regeneration. In HDAC4-deficient SCs, the expression of Pax7 and its target genes is reduced. We identify HDAC4-regulated Lix1 as a Pax7 target gene required for SC proliferation. HDAC4 inactivation leads to defective SC proliferation, muscle regeneration, and aberrant lipid accumulation. Further, expression of the brown adipose master regulator Prdm16 and its inhibitory microRNA-133 are also deregulated. Thus, HDAC4 is a novel regulator of Pax7-dependent SC proliferation and potentially fate determination in regenerating muscle.
Project description:The CCCTC-binding factor (CTCF) works together with the cohesin complex to drive the formation of chromatin loops and topologically associating domains, but its role in gene regulation has not been fully defined. Here, we investigated the effects of acute CTCF loss on chromatin architecture and transcriptional programs in mouse embryonic stem cells undergoing differentiation to neural precursor cells. We identified CTCF-dependent enhancer-promoter contacts genome-wide and found that they disproportionately affect genes that are bound by CTCF at the promoter and are dependent on long-distance enhancers. Disruption of promoter-proximal CTCF binding reduced both long-range enhancer-promoter contacts and transcription, which were restored by artificial tethering of CTCF to the promoter. Promoter-proximal CTCF binding is correlated with the transcription of over 2,000 genes across a diverse set of adult tissues. Taken together, the results of our study show that CTCF binding to promoters may promote long-distance enhancer-dependent transcription at specific genes in diverse cell types.
Project description:Macroautophagy/autophagy is a tightly regulated catabolic process, which contributes at baseline level to cellular homeostasis, and upon its stimulation to the adaptive cellular response to intra- and extracellular stress stimuli. Decrease of autophagy activity is occurring upon aging and thought to contribute to age-related-diseases. Recently, we uncovered, upon autophagy induction, the role of de novo DNMT3A (DNA methyltransferase 3 alpha)-mediated DNA methylation on expression of the MAP1LC3 (microtubule associated protein 1 light chain 3) proteins, core components of the autophagy pathway, which resulted in reduced baseline autophagy activity. Here, we report that serine/threonine kinase ULK3 (unc-51 like kinase 3)-dependent activation of GLI1 (GLI family zinc finger 1) contributes to the transcriptional upregulation of DNMT3A gene expression upon autophagy induction, thereby bringing additional understanding of the long-term effect of autophagy induction and a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions.Abbreviations: CBZ: carbamazepine; ChIP: chromatin immunoprecipitation; Clon: clonidine; DNMT3A: DNA methyltransferase 3 alpha; GLI1: GLI family zinc finger 1; GLI2: GLI family zinc finger 2; MAP1LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PLA: proximity ligation assay; RT-qPCR: quantitative reverse transcription PCR; shRNA: small hairpin RNA; siRNA: small interfering RNA; Treh: trehalose; ULK3: unc-51 like kinase 3.
Project description:Asprosin, a recently identified adipokine, activates agouti-related peptide (AgRP) neurons in the arcuate nucleus of the hypothalamus (ARH) via binding to protein tyrosine phosphatase receptor δ (Ptprd) to increase food intake. However, the intracellular mechanisms responsible for asprosin/Ptprd-mediated activation of AgRPARH neurons remain unknown. Here, we demonstrate that the small-conductance calcium-activated potassium (SK) channel is required for the stimulatory effects of asprosin/Ptprd on AgRPARH neurons. Specifically, we found that deficiency or elevation of circulating asprosin increased or decreased the SK current in AgRPARH neurons, respectively. AgRPARH-specific deletion of SK3 (an SK channel subtype highly expressed in AgRPARH neurons) blocked asprosin-induced AgRPARH activation and overeating. Furthermore, pharmacological blockade, genetic knockdown, or knockout of Ptprd abolished asprosin's effects on the SK current and AgRPARH neuronal activity. Therefore, our results demonstrated an essential asprosin-Ptprd-SK3 mechanism in asprosin-induced AgRPARH activation and hyperphagia, which is a potential therapeutic target for the treatment of obesity.
Project description:The development of antitumor chemotherapy drugs remains a key goal for oncologists, and natural products provide a vast resource for anti-cancer drug discovery. In the current study, we found that the flavonoid dihydromyricetin (DHM) exhibited antitumor activity against liver cancer cells, including primary cells obtained from hepatocellular carcinoma (HCC) patients. In contrast, DHM was not cytotoxic to immortalized normal liver cells. Furthermore, DHM treatment resulted in the growth inhibition and remission of xenotransplanted tumors in nude mice. Our results further demonstrated that this antitumor activity was caused by the activation of the p53-dependent apoptosis pathway via p53 phosphorylation at serine (15Ser). Moreover, our results showed that DHM plays a dual role in the induction of cell death when administered in combination with cisplatin, a common clinical drug that kills primary hepatoma cells but not normal liver cells.
Project description:Mitochondria, a highly metabolically active organelle, have been shown to play an essential role in regulating innate immune function. Mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) is an essential process regulating mitochondrial metabolism by targeting key enzymes involved in the tricarboxylic acid cycle (TCA). Accumulative evidence suggests MCU-dependent mitochondrial Ca2+ signaling may bridge the metabolic reprogramming and regulation of immune cell function. However, the mechanism by which MCU regulates inflammation and its related disease remains elusive. Here we report a critical role of MCU in promoting phagocytosis-dependent activation of NLRP3 (nucleotide-binding domain, leucine-rich repeat containing family, pyrin domain-containing 3) inflammasome by inhibiting phagolysosomal membrane repair. Myeloid deletion of MCU (McuΔmye) resulted in an attenuated phagolysosomal rupture, leading to decreased caspase-1 cleavage and interleukin (IL)-1β release, in response to silica or alum challenge. In contrast, other inflammasome agonists such as adenosine triphosphate (ATP), nigericin, poly(dA:dT), and flagellin induced normal IL-1β release in McuΔmye macrophages. Mechanistically, we demonstrated that decreased NLRP3 inflammasome activation in McuΔmye macrophages was caused by improved phagolysosomal membrane repair mediated by ESCRT (endosomal sorting complex required for transport)-III complex. Furthermore, McuΔmye mice showed a pronounced decrease in immune cell recruitment and IL-1β production in alum-induced peritonitis, a typical IL-1-dependent inflammation model. In sum, our results identify a function of MCU in promoting phagocytosis-dependent NLRP3 inflammatory response via an ESCRT-mediated phagolysosomal membrane repair mechanism.
Project description:The immunological synapse formed between a T-cell and an antigen-presenting cell is important for cell-cell communication during T-cell-mediated immune responses. Immunological synapse formation begins with stimulation of the T-cell receptor (TCR). TCR microclusters are assembled and transported to the center of the immunological synapse in an actin polymerization-dependent process. However, the physical link between TCR and actin remains elusive. Here we show that lymphocyte-specific Crk-associated substrate (Cas-L), a member of a force sensing protein family, is required for transport of TCR microclusters and for establishing synapse stability. We found that Cas-L is phosphorylated at TCR microclusters in an actin polymerization-dependent fashion. Furthermore, Cas-L participates in a positive feedback loop leading to amplification of Ca2+ signaling, inside-out integrin activation, and actomyosin contraction. We propose a new role for Cas-L in T-cell activation as a mechanical transducer linking TCR microclusters to the underlying actin network and coordinating multiple actin-dependent structures in the immunological synapse. Our studies highlight the importance of mechanotransduction processes in T-cell-mediated immune responses.
Project description:Altered metabolism has been implicated in the pathogenesis of inflammatory diseases. NADPH oxidase 4 (NOX4), a source of cellular superoxide anions, has multiple biological functions that may be of importance in inflammation and in the pathogenesis of human metabolic diseases, including diabetes. However, the mechanisms by which NOX4-dependent metabolic regulation affect the innate immune response remain unclear. Here we show that deficiency of NOX4 resulted in reduced expression of carnitine palmitoyltransferase 1A (CPT1A), which is a key mitochondrial enzyme in the fatty acid oxidation (FAO) pathway. The reduced FAO resulted in less activation of the nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome in human and mouse macrophages. In contrast, NOX4 deficiency did not inhibit the activation of the NLR family, CARD-domain-containing 4 (NLRC4), the NLRP1 or the absent in melanoma 2 (AIM2) inflammasomes. We also found that inhibition of FAO by etomoxir treatment suppressed NLRP3 inflammasome activation. Furthermore, Nox4-deficient mice showed substantial reduction in caspase-1 activation and in interleukin (IL)-1β and IL-18 production, and there was improved survival in a mouse model of NLRP3-mediated Streptococcus pneumoniae infection. The pharmacologic inhibition of NOX4 by either GKT137831, which is currently in phase 2 clinical trials, or VAS-2870 attenuated NLRP3 inflammasome activation. Our results suggest that NOX4-mediated FAO promotes NLRP3 inflammasome activation.