Abstract 536: Lysosome Insufficiency in Atherosclerosis Prone DBA/2 Mouse Macrophages Associated With Impaired Autolysosome Formation and Lipid Drop Clearance

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Peggy Robinet ◽  
Jonathan D Smith
Keyword(s):  
Cathepsin L ◽  
Foam Cells ◽  
Lysosomal Degradation ◽  
Over Expression ◽  
Protein Levels ◽  
Lysosome Biogenesis ◽  
Mouse Macrophages ◽  
Macrophage Foam Cells ◽  
And Function

In a previous study, we identified autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the atherosclerosis sensitive DBA/2 strain compared to the atherosclerosis resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we wanted to evaluate the role of lysosome biogenesis and function on autolysosome formation in AKR and DBA/2 cells. The transcription factor TFEB is a key regulator for lysosome biogenesis and function that positively regulates the expression of lysosomal enzymes and structural proteins, and controls lysosomes number. For all our studies, we cultured AKR and DBA/2 macrophages with or without acetylated LDL (AcLDL) for 24h. First, we analyzed TFEB protein expression by western blot. Upon loading, TFEB was increased in AKR (48%, p<0.01) but not DBA/2 cells leading to a 45% higher TFEB level in AKR vs. DBA/2 foam cells (p<0.05), suggesting that lysosome number and function may be impaired in DBA/2 foam cells. To assess lysosome function and number, cells were labeled with Lysotracker red DND-99 (LyT) and analyzed by flow cytometry. We found that AcLDL loading did not affect LyT intensity. However, in both unloaded and loaded conditions, DBA/2 cells exhibited a 30 to 50% lower LyT intensity suggesting that they have intrinsically decreased lysosome number/function. Lysosomal degradation capacity was assayed by incubation with DQ-ovalbumin and we observed a 27% decrease in lysosome function in DBA/2 vs. AKR foam cells (p<0.01). In addition, upon loading, the mature form of cathepsin L was increased in AKR (43%, p<0.05) but not DBA/2 cells. Together these data suggest an impairment of lysosomal degradation capacity in DBA/2 foam cells. Finally, we investigated the role of TPC2, a lysosomal membrane protein which over expression has been previously linked to a defect in autolysosome formation. We found that upon AcLDL loading TPC2 protein levels were increased by 35% in DBA/2 cells, which are defective in autolysosome formation, while they were unchanged in AKR cells. In conclusion, we found that DBA/2 vs. AKR foam cells express more TPC2 and have fewer and/or dysfunctional lysosomes that may explain the autolysosome formation defect in these cells.

Abstract 550: TFEB Expression, Turnover, and Nuclear Localization are Altered in DBA/2 Mouse Macrophages Associated With Impaired Autolysosome Formation and Lipid Droplet Clearance

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Peggy Robinet ◽  
Jonathan D Smith
Keyword(s):  
Western Blot ◽  
Nuclear Translocation ◽  
Cathepsin L ◽  
Foam Cells ◽  
Preliminary Evaluation ◽  
Lysosomal Degradation ◽  
Exact Test ◽  
Lysosome Biogenesis ◽  
Mouse Macrophages ◽  
And Function

In a previous study, we identified autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the atherosclerosis sensitive DBA/2 strain compared to the atherosclerosis resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we wanted to evaluate the role of lysosomes in autolysosome formation in AKR and DBA/2 cells. For all our studies, we cultured AKR and DBA/2 bone marrow-derived macrophages with or without acetylated LDL (AcLDL) for 24h. Lysosome function and number, assessed by flow cytometry on Lysotracker red DND-99 (LyT) labeled cells, was not affected by AcLDL loading in AKR and DBA/2 cells. However, in both unloaded and loaded conditions, DBA/2 cells exhibited a 30 to 50% lower LyT intensity suggesting that they have intrinsically decreased lysosome number/function. Lysosomal degradation capacity was assayed by incubation with DQ-ovalbumin and we observed a 27% decrease in lysosome function in DBA/2 vs. AKR foam cells (p<0.01). The impairment of lysosomal degradation was confirmed by 44.8% decreased levels of cathepsin L mature form in DBA/2 vs. AKR foam cells (p<0.05). As the transcription factor TFEB is a key regulator for lysosome biogenesis and function, we studied this factor in our system. First, we analyzed TFEB protein expression by western blot. Upon cholesterol loading, TFEB was induced in AKR (48%, p<0.01) but not DBA/2 cells leading to a 45% higher TFEB levels in AKR vs. DBA/2 foam cells (p<0.05). A preliminary evaluation of TFEB turnover by western blot also revealed that, upon loading, TFEB half-life is twice as long in AKR vs. DBA/2 macrophages (10.1h and 4.8h, respectively). Finally, we investigated TFEB nuclear translocation by immunofluorescence. AKR and DBA/2 macrophages were labeled with an antibody against TFEB and the percentage of TFEB positive nuclei was assessed. After 24h AcLDL loading, only 26% of DBA/2 nuclei contained TFEB vs. 48% for AKR nuclei (p=0.0002 by Fisher’s exact test). In conclusion, we found that DBA/2 vs. AKR foam cells have altered TFEB processing that may explain the altered lysosome number and function. This may result in the autolysosome formation defect in the DBA/2 foam cells.

scholarly journals T-bet represses collagen-induced arthritis by suppressing Th17 lineage commitment through inhibition of RORγt expression and function

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masaru Shimizu ◽  
Yuya Kondo ◽  
Reona Tanimura ◽  
Kotona Furuyama ◽  
Masahiro Yokosawa ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
T Helper 1 ◽  
Collagen Induced Arthritis ◽  
Over Expression ◽  
Th17 Differentiation ◽  
And Function ◽  
Arthritic Joints

AbstractT-bet is a key transcription factor for the T helper 1 lineage and its expression level is negatively correlated to inflammation in patients with rheumatoid arthritis (RA). Our previous study using T-bet transgenic mice revealed over-expression of T-bet completely suppressed collagen-induced arthritis (CIA), a murine model of RA, indicating a potential suppressive role of T-bet in the pathogenesis of autoimmune arthritis. Here, we show T-bet-deficiency exacerbated CIA. T-bet in CD4 + T cells, but not in CD11c + dendritic cells, was critical for regulating the production of IL-17A, IL-17F, IL-22, and TNFα from CD4 + T cells. T-bet-deficient CD4 + T cells showed higher RORγt expression and increased IL-17A production in RORγt-positive cells after CII immunization. In addition, T-bet-deficient naïve CD4 + T cells showed accelerated Th17 differentiation in vitro. CIA induced in CD4-Cre T-betfl/fl (cKO) mice was more severe and T-bet-deficient CD4 + T cells in the arthritic joints of cKO mice showed higher RORγt expression and increased IL-17A production. Transcriptome analysis of T-bet-deficient CD4 + T cells revealed that expression levels of Th17-related genes were selectively increased. Our results indicate that T-bet in CD4 + T cells repressed RORγt expression and function resulting in suppression of arthritogenic Th17 cells and CIA.

microRNA-422a Inhibits DCC Expression in a Manner Dependent on SNP rs12607853

2020 ◽  
Vol 160 (2) ◽  
pp. 63-71
Author(s):  
Yunxiao Li ◽  
Xugang Shi ◽  
Xintong Cai ◽  
Yongsheng Zhu ◽  
Yuanyuan Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Opioid Addiction ◽  
Heroin Addiction ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Protein Levels ◽  
Gene Assay ◽  
New Biomarkers ◽  
And Function

DCC netrin 1 receptor (DCC) affects the structure and function of the dopamine circuitry, which in turn affects the susceptibility to developing addiction. In a previous study, we found that single nucleotide polymorphism (SNP) rs12607853 in the 3′ untranslated region (3′-UTR) of DCC was significantly associated with heroin addiction. In the current study, we first used bioinformatics prediction to identify the DCC rs12607853 C allele as a potential hsa-miR-422a and hsa-miR-378c target site. We then used vector construction and dual-luciferase reporter assays to investigate the targeting relationship of DCC rs12607853 with hsa-miR-422a and hsa-miR-378c. The dual-luciferase reporter gene assay confirmed that the C allele of rs12607853 in combination with hsa-miR-422a led to repressed dual-luciferase gene expression. Moreover, gene expression assays disclosed that hsa-miR-422a inhibited DCC expression at both the mRNA and protein levels. We also found that morphine inhibited the expression of hsa-miR-422a but increased the expression of DCC mRNA, and this change in the expression of hsa-miR-422a could not be reversed by naloxone, which suggested that the role of DCC in opioid addiction might be regulated by hsa-miR-422a. In summary, this study improves our understanding of the role of hsa-miR-422a and identifies the genetic basis of rs12607853, which might contribute to the discovery of new biomarkers or therapeutic targets for opioid addiction.

scholarly journals SMCR8 negatively regulates AKT and MTORC1 signaling to modulate lysosome biogenesis and tissue homeostasis

2018 ◽  
Author(s):  
Yungang Lan ◽  
Peter M. Sullivan ◽  
Fenghua Hu
Keyword(s):  
Frontotemporal Lobar Degeneration ◽  
Physiological Function ◽  
Chromosome Region ◽  
Tissue Homeostasis ◽  
Spine Density ◽  
Protein Levels ◽  
Lysosome Biogenesis ◽  
Mtorc1 Signaling ◽  
Lateral Sclerosis

AbstractThe intronic hexanucleotide expansion in the C9orf72 gene is one of the leading causes of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), 2 devastating neurodegenerative diseases. C9orf72 forms a heterodimer with SMCR8 (Smith-Magenis syndrome chromosome region, candidate 8) protein. However, the physiological function of SMCR8 remains to be characterized. Here we report that ablation of SMCR8 in mice results in splenomegaly with autoimmune phenotypes similar to that of C9orf72 deficiency. Furthermore, SMCR8 loss leads to a drastic decrease of C9orf72 protein levels. Many proteins involved in the macroautophagy-lysosome pathways are downregulated upon SMCR8 loss due to elevated activation of MTORC1 and AKT, which also leads to increased spine density in the Smcr8 knockout neurons. In summary, our studies demonstrate a key role of SMCR8 in regulating MTORC1 and AKT signaling and tissue homeostasis.

scholarly journals Elevated CLOCK and BMAL1 Contribute to the Impairment of Aerobic Glycolysis from Astrocytes in Alzheimer’s Disease

2020 ◽  
Vol 21 (21) ◽  
pp. 7862
Author(s):  
Ik Dong Yoo ◽  
Min Woo Park ◽  
Hyeon Woo Cha ◽  
Sunmi Yoon ◽  
Napissara Boonpraman ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Circadian Rhythm ◽  
Aerobic Glycolysis ◽  
Lactate Production ◽  
Over Expression ◽  
Protein Levels ◽  
Human Astrocytes ◽  
Altered Glucose

Altered glucose metabolism has been implicated in the pathogenesis of Alzheimer’s disease (AD). Aerobic glycolysis from astrocytes is a critical metabolic pathway for brain energy metabolism. Disturbances of circadian rhythm have been associated with AD. While the role of circadian locomotor output cycles kaput (CLOCK) and brain muscle ARNT-like1 (BMAL1), the major components in the regulation of circadian rhythm, has been identified in the brain, the mechanism by which CLOCK and BMAL1 regulates the dysfunction of astrocytes in AD remains unclear. Here, we show that the protein levels of CLOCK and BMAL1 are significantly elevated in impaired astrocytes of cerebral cortex from patients with AD. We demonstrate that the over-expression of CLOCK and BMAL1 significantly suppresses aerobic glycolysis and lactate production by the reduction in hexokinase 1 (HK1) and lactate dehydrogenase A (LDHA) protein levels in human astrocytes. Moreover, the elevation of CLOCK and BMAL1 induces functional impairment by the suppression of glial fibrillary acidic protein (GFAP)-positive filaments in human astrocytes. Furthermore, the elevation of CLOCK and BMAL1 promotes cytotoxicity by the activation of caspase-3-dependent apoptosis in human astrocytes. These results suggest that the elevation of CLOCK and BMAL1 contributes to the impairment of astrocytes by inhibition of aerobic glycolysis in AD.

scholarly journals PHD2 Is a Regulator for Glycolytic Reprogramming in Macrophages

2016 ◽  
Vol 37 (1) ◽  
Author(s):  
Annemarie Guentsch ◽  
Angelika Beneke ◽  
Lija Swain ◽  
Katja Farhat ◽  
Shunmugam Nagarajan ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Critical Role ◽  
Hypoxia Inducible Factor ◽  
Pyruvate Dehydrogenase Kinase ◽  
Metabolic Phenotype ◽  
Protein Levels ◽  
Dehydrogenase Enzyme ◽  
Pyruvate Dehydrogenase Kinase 1 ◽  
And Function

ABSTRACT The prolyl-4-hydroxylase domain (PHD) enzymes are regarded as the molecular oxygen sensors. There is an interplay between oxygen availability and cellular metabolism, which in turn has significant effects on the functionality of innate immune cells, such as macrophages. However, if and how PHD enzymes affect macrophage metabolism are enigmatic. We hypothesized that macrophage metabolism and function can be controlled via manipulation of PHD2. We characterized the metabolic phenotypes of PHD2-deficient RAW cells and primary PHD2 knockout bone marrow-derived macrophages (BMDM). Both showed typical features of anaerobic glycolysis, which were paralleled by increased pyruvate dehydrogenase kinase 1 (PDK1) protein levels and a decreased pyruvate dehydrogenase enzyme activity. Metabolic alterations were associated with an impaired cellular functionality. Inhibition of PDK1 or knockout of hypoxia-inducible factor 1α (HIF-1α) reversed the metabolic phenotype and impaired the functionality of the PHD2-deficient RAW cells and BMDM. Taking these results together, we identified a critical role of PHD2 for a reversible glycolytic reprogramming in macrophages with a direct impact on their function. We suggest that PHD2 serves as an adjustable switch to control macrophage behavior.

scholarly journals BAG2 promotes tumorigenesis through enhancing mutant p53 protein levels and function

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Xuetian Yue ◽  
Yuhan Zhao ◽  
Juan Liu ◽  
Cen Zhang ◽  
Haiyang Yu ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Tumor Growth ◽  
Poor Prognosis ◽  
P53 Protein ◽  
Underlying Mechanism ◽  
Mutant P53 ◽  
Tumor Suppressor P53 ◽  
Protein Levels ◽  
And Function

Tumor suppressor p53 is the most frequently mutated gene in tumors. Many mutant p53 (mutp53) proteins promote tumorigenesis through the gain-of-function (GOF) mechanism. Mutp53 proteins often accumulate to high levels in tumors, which is critical for mutp53 GOF. Its underlying mechanism is poorly understood. Here, we found that BAG2, a protein of Bcl-2 associated athanogene (BAG) family, promotes mutp53 accumulation and GOF in tumors. Mechanistically, BAG2 binds to mutp53 and translocates to the nucleus to inhibit the MDM2-mutp53 interaction, and MDM2-mediated ubiquitination and degradation of mutp53. Thus, BAG2 promotes mutp53 accumulation and GOF in tumor growth, metastasis and chemoresistance. BAG2 is frequently overexpressed in tumors. BAG2 overexpression is associated with poor prognosis in patients and mutp53 accumulation in tumors. These findings revealed a novel and important mechanism for mutp53 accumulation and GOF in tumors, and also uncovered an important role of BAG2 in tumorigenesis through promoting mutp53 accumulation and GOF.

scholarly journals JMJD6 Dysfunction Due to Iron Deficiency in Preeclampsia Disrupts Fibronectin Homeostasis Resulting in Diminished Trophoblast Migration

2021 ◽  
Vol 9 ◽  
Author(s):  
Sruthi Alahari ◽  
Abby Farrell ◽  
Leonardo Ermini ◽  
Chanho Park ◽  
Julien Sallais ◽  
...  
Keyword(s):  
Time Lapse ◽  
Lysosomal Degradation ◽  
Related Disorder ◽  
Novel Target ◽  
And Function ◽  
Time Lapse Imaging ◽  
Fibronectin Assembly ◽  
Degradation Time ◽  
Trophoblast Migration

The mechanisms contributing to excessive fibronectin in preeclampsia, a pregnancy-related disorder, remain unknown. Herein, we investigated the role of JMJD6, an O2- and Fe2+-dependent enzyme, in mediating placental fibronectin processing and function. MALDI-TOF identified fibronectin as a novel target of JMJD6-mediated lysyl hydroxylation, preceding fibronectin glycosylation, deposition, and degradation. In preeclamptic placentae, fibronectin accumulated primarily in lysosomes of the mesenchyme. Using primary placental mesenchymal cells (pMSCs), we found that fibronectin fibril formation and turnover were markedly impeded in preeclamptic pMSCs, partly due to impaired lysosomal degradation. JMJD6 knockdown in control pMSCs recapitulated the preeclamptic FN phenotype. Importantly, preeclamptic pMSCs had less total and labile Fe2+ and Hinokitiol treatment rescued fibronectin assembly and promoted lysosomal degradation. Time-lapse imaging demonstrated that defective ECM deposition by preeclamptic pMSCs impeded HTR-8/SVneo cell migration, which was rescued upon Hinokitiol exposure. Our findings reveal new Fe2+-dependent mechanisms controlling fibronectin homeostasis/function in the placenta that go awry in preeclampsia.

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