scholarly journals Dimethyl sulfoxide stimulates the AhR-Jdp2 axis to control ROS accumulation in mouse embryonic fibroblasts

2021 ◽  
Author(s):  
Kenly Wuputra ◽  
Ming-Ho Tsai ◽  
Kohsuke Kato ◽  
Ya-han Yang ◽  
Jia-Bin Pan ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Phase I ◽  
Dimethyl Sulfoxide ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Aryl Hydrocarbon ◽  
Ros Accumulation ◽  
Induced Apoptosis ◽  
Ahr Gene

AbstractThe aryl hydrocarbon receptor (AhR) is a ligand-binding protein that responds to environmental aromatic hydrocarbons and stimulates the transcription of downstream phase I enzyme–related genes by binding the cis element of dioxin-responsive elements (DREs)/xenobiotic-responsive elements. Dimethyl sulfoxide (DMSO) is a well-known organic solvent that is often used to dissolve phase I reagents in toxicology and oxidative stress research experiments. In the current study, we discovered that 0.1% DMSO significantly induced the activation of the AhR promoter via DREs and produced reactive oxygen species, which induced apoptosis in mouse embryonic fibroblasts (MEFs). Moreover, Jun dimerization protein 2 (Jdp2) was found to be required for activation of the AhR promoter in response to DMSO. Coimmunoprecipitation and chromatin immunoprecipitation studies demonstrated that the phase I–dependent transcription factors, AhR and the AhR nuclear translocator, and phase II–dependent transcription factors such as nuclear factor (erythroid-derived 2)–like 2 (Nrf2) integrated into DRE sites together with Jdp2 to form an activation complex to increase AhR promoter activity in response to DMSO in MEFs. Our findings provide evidence for the functional role of Jdp2 in controlling the AhR gene via Nrf2 and provide insights into how Jdp2 contributes to the regulation of ROS production and the cell spreading and apoptosis produced by the ligand DMSO in MEFs. Graphical abstract

scholarly journals Critical Role of PDE4D in β2-Adrenoceptor-dependent cAMP Signaling in Mouse Embryonic Fibroblasts

2008 ◽  
Vol 283 (33) ◽  
pp. 22430-22442 ◽  
Author(s):  
Matthew D. Bruss ◽  
Wito Richter ◽  
Kathleen Horner ◽  
S.-L. Catherine Jin ◽  
Marco Conti
Keyword(s):  
Critical Role ◽  
Camp Signaling ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts

scholarly journals Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

10.3791/54372 ◽  
2016 ◽  
Author(s):  
Michael G. Daniel ◽  
Carlos-Filipe Pereira ◽  
Jeffrey M. Bernitz ◽  
Ihor R. Lemischka ◽  
Kateri Moore
Keyword(s):  
Transcription Factors ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts

scholarly journals Quantitative Proteome Analysis of Atg5-Deficient Mouse Embryonic Fibroblasts Reveals the Range of the Autophagy-Modulated Basal Cellular Proteome

mSystems ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Kiran Bala Sharma ◽  
Manish Sharma ◽  
Suruchi Aggarwal ◽  
Amit Kumar Yadav ◽  
Shinjini Bhatnagar ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Inflammatory Response ◽  
Transforming Growth Factor ◽  
Proteome Analysis ◽  
Interferon Response ◽  
Type I ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
The Impact

ABSTRACT Basal autophagy is crucial for maintenance of cellular homeostasis. ATG5 is an essential protein for autophagosome formation, and its depletion has been extensively used as a tool to disrupt autophagy. Here, we characterize the impact of Atg5 deficiency on the cellular proteome of mouse embryonic fibroblasts (MEFs). Using a tandem mass tagging (TMT)-based quantitative proteomics analysis, we observe that 14% of identified proteins show dysregulated levels in atg5−/− MEFs. These proteins were distributed across diverse biological processes, such as cell adhesion, development, differentiation, transport, metabolism, and immune responses. Several of the upregulated proteins were receptors involved in transforming growth factor β (TGF-β) signaling, JAK-STAT signaling, junction adhesion, and interferon/cytokine-receptor interactions and were validated as autophagy substrates. Nearly equal numbers of proteins, including several lysosomal proteins and enzymes, were downregulated, suggesting a complex role of autophagy/ATG5 in regulating their levels. The atg5−/− MEFs had lower levels of key immune sensors and effectors, including Toll-like receptor 2 (TLR2), interferon regulatory factor 3 (IRF3), IRF7, MLKL, and STAT1/3/5/6, which were restored by reexpression of ATG5. While these cells could efficiently mount a type I interferon response to the double-stranded RNA (dsRNA) mimic poly(I·C), they were compromised in their inflammatory response to the bacterial pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and Pam3CSK4. Transcriptional activation and secretion of interleukin-6 (IL-6) in these cells could be recovered by ATG5 expression, supporting the role of autophagy in the TLR2-induced inflammatory response. This study provides a key resource for understanding the effect of autophagy/ATG5 deficiency on the fibroblast proteome. IMPORTANCE Autophagy performs housekeeping functions for cells and maintains a functional mode by degrading damaged proteins and organelles and providing energy under starvation conditions. The process is tightly regulated by the evolutionarily conserved Atg genes, of which Atg5 is one such crucial mediator. Here, we have done a comprehensive quantitative proteome analysis of mouse embryonic fibroblasts that lack a functional autophagy pathway (Atg5 knockout). We observe that 14% of the identified cellular proteome is remodeled, and several proteins distributed across diverse cellular processes with functions in signaling, cell adhesion, development, and immunity show either higher or lower levels under autophagy-deficient conditions. These cells have lower levels of crucial immune proteins that are required to mount a protective inflammatory response. This study will serve as a valuable resource to determine the role of autophagy in modulating specific protein levels in cells.

scholarly journals A Protective Role of Aryl Hydrocarbon Receptor Repressor in Inflammation and Tumor Growth

Cancers ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 589 ◽  
Author(s):  
Christoph F. A. Vogel ◽  
Yasuhiro Ishihara ◽  
Claire E. Campbell ◽  
Sarah Y. Kado ◽  
Aimy Nguyen-Chi ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Aryl Hydrocarbon Receptor ◽  
Inflammatory Responses ◽  
Environmental Pollutants ◽  
Protective Role ◽  
Potential Therapeutic Target ◽  
Embryonic Fibroblasts ◽  
Aryl Hydrocarbon ◽  
Enhancer Binding Protein

The aryl hydrocarbon receptor (AhR) is known for mediating the toxicity of environmental pollutants such as dioxins and numerous dioxin-like compounds, and is associated with the promotion of various malignancies, including lymphoma. The aryl hydrocarbon receptor repressor (AhRR), a ligand-independent, transcriptionally inactive AhR-like protein is known to repress AhR signaling through its ability to compete with the AhR for dimerization with the AhR nuclear translocator (ARNT). While AhRR effectively blocks AhR signaling, several aspects of the mechanism of AhRR’s functions are poorly understood, including suppression of inflammatory responses and its putative role as a tumor suppressor. In a transgenic mouse that overexpresses AhRR (AhRR Tg) we discovered that these mice suppress 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)- and inflammation-induced tumor growth after subcutaneous challenge of EL4 lymphoma cells. Using mouse embryonic fibroblasts (MEF) we found that AhRR overexpression suppresses the AhR-mediated anti-apoptotic response. The AhRR-mediated inhibition of apoptotic resistance was associated with a suppressed expression of interleukin (IL)-1β and cyclooxygenase (COX)-2, which was dependent on activation of protein kinase A (PKA) and the CAAT-enhancer-binding protein beta (C/EBPβ). These results provide mechanistic insights into the role of the AhRR to suppress inflammation and highlight the AhRR as a potential therapeutic target to suppress tumor growth.

scholarly journals TIMELESS mutation alters phase responsiveness and causes advanced sleep phase

2019 ◽  
pp. 201819110 ◽  
Author(s):  
Philip Kurien ◽  
Pei-Ken Hsu ◽  
Jacy Leon ◽  
David Wu ◽  
Thomas McMahon ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Molecular Clock ◽  
Mutant Mice ◽  
Circadian Period ◽  
Negative Regulators ◽  
Mouse Embryonic Fibroblasts ◽  
Sleep Phase ◽  
Embryonic Fibroblasts ◽  
Light Entrainment

Many components of the circadian molecular clock are conserved from flies to mammals; however, the role of mammalian Timeless remains ambiguous. Here, we report a mutation in the human TIMELESS (hTIM) gene that causes familial advanced sleep phase (FASP). Tim CRISPR mutant mice exhibit FASP with altered photic entrainment but normal circadian period. We demonstrate that the mutation prevents TIM accumulation in the nucleus and has altered affinity for CRY2, leading to destabilization of PER/CRY complex and a shortened period in nonmature mouse embryonic fibroblasts (MEFs). We conclude that TIM, when excluded from the nucleus, can destabilize the negative regulators of the circadian clock, alter light entrainment, and cause FASP.

scholarly journals Lack of Bax Prevents Influenza A Virus-Induced Apoptosis and Causes Diminished Viral Replication

2009 ◽  
Vol 83 (16) ◽  
pp. 8233-8246 ◽  
Author(s):  
Jeffrey E. McLean ◽  
Emmanuel Datan ◽  
Demetrius Matassov ◽  
Zahra F. Zakeri
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Mdck Cells ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Induction Of Apoptosis ◽  
Influenza A Virus Infection ◽  
Induced Apoptosis

ABSTRACT The ectopic overexpression of Bcl-2 restricts both influenza A virus-induced apoptosis and influenza A virus replication in MDCK cells, thus suggesting a role for Bcl-2 family members during infection. Here we report that influenza A virus cannot establish an apoptotic response without functional Bax, a downstream target of Bcl-2, and that both Bax and Bak are directly involved in influenza A virus replication and virus-induced cell death. Bak is substantially downregulated during influenza A virus infection in MDCK cells, and the knockout of Bak in mouse embryonic fibroblasts yields a dramatic rise in the rate of apoptotic death and a corresponding increase in levels of virus replication, suggesting that Bak suppresses both apoptosis and the replication of virus and that the virus suppresses Bak. Bax, however, is activated and translocates from the cytosol to the mitochondria; this activation is required for the efficient induction of apoptosis and virus replication. The knockout of Bax in mouse embryonic fibroblasts blocks the induction of apoptosis, restricts the infection-mediated activation of executioner caspases, and inhibits virus propagation. Bax knockout cells still die but by an alternative death pathway displaying characteristics of autophagy, similarly to our previous observation that influenza A virus infection in the presence of a pancaspase inhibitor leads to an increase in levels of autophagy. The knockout of Bax causes a retention of influenza A virus NP within the nucleus. We conclude that the cell and virus struggle to control apoptosis and autophagy, as appropriately timed apoptosis is important for the replication of influenza A virus.

scholarly journals Essential Role of Cyclin-G–associated Kinase (Auxilin-2) in Developing and Mature Mice

2008 ◽  
Vol 19 (7) ◽  
pp. 2766-2776 ◽  
Author(s):  
Dong-won Lee ◽  
Xiaohong Zhao ◽  
Yang-In Yim ◽  
Evan Eisenberg ◽  
Lois E. Greene
Keyword(s):  
Essential Gene ◽  
Inducible Promoter ◽  
Cre Recombinase ◽  
Conditional Knockout ◽  
Mouse Embryonic Fibroblasts ◽  
Coated Pits ◽  
Embryonic Fibroblasts ◽  
Adult Mice ◽  
Cyclin G

Hsc70 with its cochaperone, either auxilin or GAK, not only uncoats clathrin-coated vesicles but also acts as a chaperone during clathrin-mediated endocytosis. However, because synaptojanin is also involved in uncoating, it is not clear whether GAK is an essential gene. To answer this question, GAK conditional knockout mice were generated and then mated to mice expressing Cre recombinase under the control of the nestin, albumin, or keratin-14 promoters, all of which turn on during embryonic development. Deletion of GAK from brain, liver, or skin dramatically altered the histology of these tissues, causing the mice to die shortly after birth. Furthermore, by expressing a tamoxifen-inducible promoter to express Cre recombinase we showed that deletion of GAK caused lethality in adult mice. Mouse embryonic fibroblasts in which the GAK was disrupted showed a lack of clathrin-coated pits and a complete block in clathrin-mediated endocytosis. We conclude that GAK deletion blocks development and causes lethality in adult animals by disrupting clathrin-mediated endocytosis.

scholarly journals Caspase3-deficient cells require fibronectin for protection against autophagy-dependent death

2021 ◽  
Author(s):  
David B. Weir ◽  
Lawrence H. Boise
Keyword(s):  
Cell Death ◽  
Survival Advantage ◽  
Deficient Mouse ◽  
Wild Type ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Serum Withdrawal ◽  
Presence And Absence ◽  
Induced Apoptosis

ABSTRACTCaspases are required for execution of apoptosis. However, in their absence, signals that typically induce apoptosis can still result in cell death. Our laboratory previously demonstrated that Casp3-deficient mouse embryonic fibroblasts (MEFs) have increased fibronectin (FN) secretion, and an adhesion-dependent survival advantage compared to wild type (WT) MEFs. Here, we show that FN is required for survival of Casp3-deficient MEFs following serum withdrawal. Furthermore, when FN is silenced, serum withdrawal-induced death is caspase-independent. However, procaspase-7 is cleaved, suggesting that MOMP is taking place. Indeed, in the absence of FN, cytochrome c release is increased following serum withdrawal in Casp3-deficient MEFs. Yet death does not correspond to cytochrome c release in Casp3-deficient MEFs. This is true both in the presence and absence of FN. Additionally, caspase-independent death is inhibited by Bcl-XL overexpression. These findings suggest that Bcl-XL is not inhibiting death through regulation of Bax/Bak insertion into the mitochondria, but through a different mechanism. One such possibility is autophagy and induction of autophagy is associated with caspase-independent death in Casp3-deficient cells. Importantly, when ATG5 is ablated in Casp3-deficient cells, autophagy is blocked and death is largely inhibited. Taken together, our data indicate that Casp3-deficient cells incapable of undergoing canonical serum withdrawal-induced apoptosis, are protected from autophagy-dependent death by FN-mediated adhesion.

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