Starvation in the midst of plenty: making sense of ceramide-induced autophagy by analysing nutrient transporter expression

2009 ◽  
Vol 37 (1) ◽  
pp. 253-258 ◽  
Author(s):  
Aimee L. Edinger
Keyword(s):  
Heat Stress ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Lethal Dose ◽  
Culture Conditions ◽  
Making Sense ◽  
Nutrient Transporter ◽  
Increased Sensitivity ◽  
Methyl Pyruvate ◽  
Transporter Expression

Ceramide induces differentiation, proliferative arrest, senescence and death in mammalian cells. The mechanism by which ceramide produces these outcomes has proved difficult to define. Building on observations that ceramide stimulates autophagy, we have identified a novel mechanism of action for this sphingolipid: ceramide starves cells to death subsequent to profound nutrient transporter down-regulation. In yeast, ceramide generated in response to heat stress adaptively slows cell growth by down-regulating nutrient permeases. In mammalian cells, a lethal dose of ceramide triggers a bioenergetic crisis by so severely limiting cellular access to extracellular nutrients that autophagy is insufficient to meet the metabolic demands of the cell. In keeping with this bioenergetic explanation for ceramide toxicity, methyl pyruvate, a membrane-permeable nutrient, protects cells from ceramide-induced starvation. Also consistent with this model, we have found that the metabolic state of the cell determines its sensitivity to ceramide. Thus the increased sensitivity of cancer cells to ceramide may relate to their inflexible biosynthetic metabolic programme. These studies highlight the value of assessing nutrient transporter expression in autophagic cells and the important role that culture conditions play in determining the cellular response to ceramide.

Growth factors regulate cell survival by controlling nutrient transporter expression

2005 ◽  
Vol 33 (1) ◽  
pp. 225-227 ◽  
Author(s):  
A.L. Edinger
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Cell Survival ◽  
Mammalian Cells ◽  
Mammalian Target Of Rapamycin ◽  
Chemotherapeutic Agents ◽  
Tumour Suppressor ◽  
Nutrient Transporters ◽  
Nutrient Transporter ◽  
Transporter Expression

Growth factors provide permission signals that allow mammalian cells to grow, proliferate and survive. One mechanism by which growth factors maintain this control is through the regulation of cell surface nutrient transporter expression. Following growth factor withdrawal, nutrient transporters are endocytosed and degraded in the lysosome, effectively terminating the cell's ability to obtain nutrients. This results in a state of pseudostarvation in which cells atrophy and initiate a catabolic metabolic programme in the midst of abundant extracellular nutrients. Oncogenic forms of Akt can support growth factor-independent nutrient transporter expression through a mechanism that depends upon mTOR (mammalian target of rapamycin). The ability of activated Akt to support nutrient transporter expression is an essential component of its prosurvival function. When the destruction of nutrient transporters is inhibited, cells are capable of long-term growth-factor-independent cell survival in the absence of receptor-dependent signal transduction. These results imply that proteins involved in nutrient transporter turnover in response to growth factor withdrawal are components of a novel tumour suppressor pathway. Preliminary data suggest that Rab7, a GTPase required for transporter degradation, functions as a tumour suppressor protein, as inhibiting Rab7 activity promotes colony formation in soft agar. These studies indicate that drugs affecting this pathway might have utility as anti-cancer chemotherapeutic agents.

Controlling cell growth and survival through regulated nutrient transporter expression

2007 ◽  
Vol 406 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Aimee L. Edinger
Keyword(s):  
Cell Growth ◽  
Mammalian Cells ◽  
Growth Control ◽  
Cellular Growth ◽  
Growth And Survival ◽  
Tumour Suppression ◽  
Nutrient Transporter ◽  
Phosphoinositide 3 Kinase ◽  
Cell Growth And Proliferation ◽  
Transporter Expression

Although all cells depend upon nutrients they acquire from the extracellular space, surprisingly little is known about how nutrient uptake is regulated in mammalian cells. Most nutrients are brought into cells by means of specific transporter proteins. In yeast, the expression and trafficking of a wide variety of nutrient transporters is controlled by the TOR (target of rapamycin) kinase. Consistent with this, recent studies in mammalian cells have shown that mTOR (mammalian TOR) and the related protein, PI3K (phosphoinositide 3-kinase), play central roles in coupling nutrient transporter expression to the availability of extrinsic trophic and survival signals. In the case of lymphocytes, it has been particularly well established that these extrinsic signals stimulate cell growth and proliferation in part by regulating nutrient transporter expression. The ability of growth factors to control nutrient access may also play an important role in tumour suppression: the non-homoeostatic growth of tumour cells requires that nutrient transporter expression is uncoupled from trophic factor availability. Also supporting a link between nutrient transporter expression levels and oncogenesis, several recent studies demonstrate that nutrient transporter expression drives, rather than simply parallels, cellular metabolism. This review summarizes the evidence that regulated nutrient transporter expression plays a central role in cellular growth control and highlights the implications of these findings for human disease.

Use of Flexible Materials with a Novel Pressure Driven Equibiaxial Cell Stretching Device for Mechanical Stimulation of Single Mammalian Cells

2003 ◽  
Vol 773 ◽  
Author(s):  
James D. Kubicek ◽  
Stephanie Brelsford ◽  
Philip R. LeDuc
Keyword(s):  
Cell Fate ◽  
Mechanical Stimulation ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Single Cells ◽  
Complex Nature ◽  
Cellular Behavior ◽  
Cell Stretching ◽  
Stimulation Of ◽  
Pressure Driven

AbstractMechanical stimulation of single cells has been shown to affect cellular behavior from the molecular scale to ultimate cell fate including apoptosis and proliferation. In this, the ability to control the spatiotemporal application of force on cells through their extracellular matrix connections is critical to understand the cellular response of mechanotransduction. Here, we develop and utilize a novel pressure-driven equibiaxial cell stretching device (PECS) combined with an elastomeric material to control specifically the mechanical stimulation on single cells. Cells were cultured on silicone membranes coated with molecular matrices and then a uniform pressure was introduced to the opposite surface of the membrane to stretch single cells equibiaxially. This allowed us to apply mechanical deformation to investigate the complex nature of cell shape and structure. These results will enhance our knowledge of cellular and molecular function as well as provide insights into fields including biomechanics, tissue engineering, and drug discovery.

scholarly journals Decellularized Porcine Cartilage Scaffold; Validation of Decellularization and Evaluation of Biomarkers of Chondrogenesis

2021 ◽  
Vol 22 (12) ◽  
pp. 6241
Author(s):  
Roxanne N. Stone ◽  
Stephanie M. Frahs ◽  
Makenna J. Hardy ◽  
Akina Fujimoto ◽  
Xinzhu Pu ◽  
...  
Keyword(s):  
Cellular Response ◽  
Cell Attachment ◽  
The United States ◽  
Culture Conditions ◽  
Surgical Approaches ◽  
Cell Matrix ◽  
Cartilage Restoration ◽  
Cell Matrix Interactions ◽  
Decellularized Scaffold ◽  
Extracellular Matrix Scaffold

Osteoarthritis is a major concern in the United States and worldwide. Current non-surgical and surgical approaches alleviate pain but show little evidence of cartilage restoration. Cell-based treatments may hold promise for the regeneration of hyaline cartilage-like tissue at the site of injury or wear. Cell–cell and cell–matrix interactions have been shown to drive cell differentiation pathways. Biomaterials for clinically relevant applications can be generated from decellularized porcine auricular cartilage. This material may represent a suitable scaffold on which to seed and grow chondrocytes to create new cartilage. In this study, we used decellularization techniques to create an extracellular matrix scaffold that supports chondrocyte cell attachment and growth in tissue culture conditions. Results presented here evaluate the decellularization process histologically and molecularly. We identified new and novel biomarker profiles that may aid future cartilage decellularization efforts. Additionally, the resulting scaffold was characterized using scanning electron microscopy, fluorescence microscopy, and proteomics. Cellular response to the decellularized scaffold was evaluated by quantitative real-time PCR for gene expression analysis.

scholarly journals xCELLanalyzer: A Framework for the Analysis of Cellular Impedance Measurements for Mode of Action Discovery

2019 ◽  
Vol 24 (3) ◽  
pp. 213-223 ◽  
Author(s):  
Raimo Franke ◽  
Bettina Hinkelmann ◽  
Verena Fetz ◽  
Theresia Stradal ◽  
Florenz Sasse ◽  
...  
Keyword(s):  
Data Analysis ◽  
Bioactive Compounds ◽  
Mode Of Action ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Label Free ◽  
Synthesis Inhibitor ◽  
Analysis Pipeline ◽  
Bioactive Natural Products ◽  
Data Analysis Pipeline

Mode of action (MoA) identification of bioactive compounds is very often a challenging and time-consuming task. We used a label-free kinetic profiling method based on an impedance readout to monitor the time-dependent cellular response profiles for the interaction of bioactive natural products and other small molecules with mammalian cells. Such approaches have been rarely used so far due to the lack of data mining tools to properly capture the characteristics of the impedance curves. We developed a data analysis pipeline for the xCELLigence Real-Time Cell Analysis detection platform to process the data, assess and score their reproducibility, and provide rank-based MoA predictions for a reference set of 60 bioactive compounds. The method can reveal additional, previously unknown targets, as exemplified by the identification of tubulin-destabilizing activities of the RNA synthesis inhibitor actinomycin D and the effects on DNA replication of vioprolide A. The data analysis pipeline is based on the statistical programming language R and is available to the scientific community through a GitHub repository.

scholarly journals Salmonella Exploits Caspase-1 to Colonize Peyer's Patches in a Murine Typhoid Model

2000 ◽  
Vol 192 (2) ◽  
pp. 249-258 ◽  
Author(s):  
Denise M. Monack ◽  
David Hersh ◽  
Nafisa Ghori ◽  
Donna Bouley ◽  
Arturo Zychlinsky ◽  
...  
Keyword(s):  
Proinflammatory Cytokines ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Lethal Dose ◽  
Cysteine Proteases ◽  
Peyer's Patches ◽  
Peyer’S Patches ◽  
Wild Type ◽  
M Cell ◽  
Caspase 1

Salmonella typhimurium invades host macrophages and induces apoptosis and the release of mature proinflammatory cytokines. SipB, a protein translocated by Salmonella into the cytoplasm of macrophages, is required for activation of Caspase-1 (Casp-1, an interleukin [IL]-1β–converting enzyme), which is a member of a family of cysteine proteases that induce apoptosis in mammalian cells. Casp-1 is unique among caspases because it also directly cleaves the proinflammatory cytokines IL-1β and IL-18 to produce bioactive cytokines. We show here that mice lacking Casp-1 (casp-1−/− mice) had an oral S. typhimurium 50% lethal dose (LD50) that was 1,000-fold higher than that of wild-type mice. Salmonella breached the M cell barrier of casp-1−/− mice efficiently; however, there was a decrease in the number of apoptotic cells, intracellular bacteria, and the recruitment of polymorphonuclear lymphocytes in the Peyer's patches (PP) as compared with wild-type mice. Furthermore, Salmonella did not disseminate systemically in the majority of casp-1−/− mice, as demonstrated by significantly less colonization in the PP, mesenteric lymph nodes, and spleens of casp-1−/− mice after an oral dose of S. typhimurium that was 100-fold higher than the LD50. The increased resistance in casp-1−/− animals appears specific for Salmonella infection since these mice were susceptible to colonization by another enteric pathogen, Yersinia pseudotuberculosis, which normally invades the PP. These results show that Casp-1, which is both proapoptotic and proinflammatory, is essential for S. typhimurium to efficiently colonize the cecum and PP and subsequently cause systemic typhoid-like disease in mice.

Enhanced immunogenicity of a synthetic DNA vaccine expressing consensus SARS-CoV-2 Spike protein using needle-free immunization

2021 ◽  
Author(s):  
Sawsan S Alamri ◽  
Khalid A Alluhaybi ◽  
Rowa Y Alhabbab ◽  
Abdullah Algaissi ◽  
Sarah Almahboub ◽  
...  
Keyword(s):  
Immune Response ◽  
Dna Vaccine ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Igg Antibodies ◽  
Free System ◽  
Mammalian Expression ◽  
Specific Igg ◽  
Needle Injection ◽  
Specific Igg Antibodies

AbstractThe ongoing global pandemic of Coronavirus Disease 2019 (COVID-19) calls for an urgent development of effective and safe prophylactic and therapeutic measures. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is a major immunogenic and protective protein, and plays a crucial role in viral pathogenesis. In this study, we successfully constructed a synthetic codon-optimized DNA-based vaccine as a countermeasure against SARS-CoV-2; denoted as VIU-1005. The design was based on the synthesis of codon-optimized coding sequence for optimal mammalian expression of a consensus full-length S glycoprotein. The successful construction of the vaccine was confirmed by restriction digestion and sequencing, and the protein expression of the S protein was confirmed by western blot and immunofluorescence staining in mammalian cells. The immunogenicity of the vaccine was tested in two mouse models (BALB/c and C57BL/6J). Th1-skewed systemic S-specific IgG antibodies and neutralizing antibodies (nAbs) were significantly induced in both models four weeks post three injections with 100 μg of the VIU-1005 vaccine via intramuscular needle injection but not intradermal or subcutaneous routes. Importantly, such immunization induced long-lasting IgG response in mice that lasted for at least 6 months. Interestingly, using a needle-free system, we showed an enhanced immunogenicity of VIU-1005 in which lower doses such as 25-50 μg or less number of doses were able to elicit significantly high levels of Th1-biased systemic S-specific IgG antibodies and nAbs via intramuscular immunization compared to needle immunization. Compared to the intradermal needle injection which failed to induce any significant immune response, intradermal needle-free immunization elicited robust Th1-biased humoral response similar to that observed with intramuscular immunization. Furthermore, immunization with VIU-1005 induced potent S-specific cellular response as demonstrated by the significantly high levels of IFN-γ, TNF and IL-2 cytokines production in memory CD8+ and CD4+ T cells in BALB/c mice. Together, our results demonstrate that the synthetic VIU-1005 candidate DNA vaccine is highly immunogenic and capable of inducing long-lasting and Th1-skewed immune response in mice. Furthermore, we show that the use of needle-free system could enhance the immunogenicity and minimize doses needed to induce protective immunity in mice, supporting further preclinical and clinical testing of this candidate vaccine.

164 Genome-wide DNA Methylation Profiling in in Utero Heat-stressed Pigs as Measured by Whole-genome Bisulfite Sequencing

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 87-88
Author(s):  
Luiz F Brito ◽  
Jacob M Maskal ◽  
Shi-Yi Chen ◽  
Hinayah R Oliveira ◽  
Jason R Graham ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
Cellular Response ◽  
Bisulfite Sequencing ◽  
Cholesterol Biosynthesis ◽  
In Utero ◽  
Whole Genome ◽  
Whole Genome Bisulfite Sequencing ◽  
Differentially Methylated Genes ◽  
Genome Bisulfite Sequencing

Abstract In utero heat stress (IUHS) has several postnatal consequences in pigs that compromise health, increase stress response, and reduce performance. These phenotypes may be caused by epigenetic modifications such as DNA methylation, which are heritable molecular modifications that impact gene expression and phenotypic outcomes without changing the DNA sequence. Therefore, we aimed to compare the DNA methylation profiles between in-utero thermoneutral (IUTN) and IUHS pigs to identify differentially methylated regions. Twenty-four pregnant gilts were evenly assigned to either a thermoneutral (17.5 ± 2.1°C) or heat stress (cycling 26 to 36°C) chamber from d 0 to 59 of gestation, followed by thermoneutral conditions (20.9 ± 2.3°C) for the rest of gestation and until the piglets were weaned. At 105 d of age, 10 IUTN and 10 IUHS piglets were euthanized and Longissimus dorsi muscle samples were collected and used to perform whole-genome bisulfite sequencing (WGBS). Purified genomic DNA was fragmented and bisulfite conversion was performed. Illumina platforms were used to sequence WGBS libraries. All pigs had similar proportions of methylation at CpG sites. Two-hundred-sixty-eight genomic regions were differentially methylated between IUTN and IUHS pigs. These identified regions are located across all pig chromosomes and ranged from 2 (SSC18) to 40 (SSC10). Eighty-five unique differentially-methylated genes were identified. These genes have been reported to be involved in key biological processes such as transcriptional repressor activity and tRNA processing (e.g., SKOR2,TRMT6, TSEN2), cellular response to heat stress (e.g.,CCAR2), placental vascularization (e.g.,FZD5), central nervous system (e.g.,VEPH1), cholesterol biosynthesis (e.g., CYB5R1), insulin receptor substrate (e.g.,IRS2), synaptic transmission (e.g.,RIMBP2), neurotrophic factor receptor activity (e.g.,LIFR), immune response (e.g., CD84), DNA repair (e.g., CHD1L), and cell proliferation and endocrine signaling (e.g., SSTR1, CYB5R1). These findings contribute to a better understanding of the epigenomic mechanisms underlying postnatal consequences of IUHS in pigs.

scholarly journals MORC2 regulates DNA damage response through a PARP1-dependent pathway

2019 ◽  
Vol 47 (16) ◽  
pp. 8502-8520 ◽  
Author(s):  
Lin Zhang ◽  
Da-Qiang Li
Keyword(s):  
Dna Damage ◽  
Zinc Finger ◽  
Chromatin Remodeling ◽  
Dna Damage Response ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Genotoxic Stress ◽  
Underlying Mechanism ◽  
Dna Lesions ◽  
Damage Response

Abstract Microrchidia family CW-type zinc finger 2 (MORC2) is a newly identified chromatin remodeling enzyme with an emerging role in DNA damage response (DDR), but the underlying mechanism remains largely unknown. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1), a key chromatin-associated enzyme responsible for the synthesis of poly(ADP-ribose) (PAR) polymers in mammalian cells, interacts with and PARylates MORC2 at two residues within its conserved CW-type zinc finger domain. Following DNA damage, PARP1 recruits MORC2 to DNA damage sites and catalyzes MORC2 PARylation, which stimulates its ATPase and chromatin remodeling activities. Mutation of PARylation residues in MORC2 results in reduced cell survival after DNA damage. MORC2, in turn, stabilizes PARP1 through enhancing acetyltransferase NAT10-mediated acetylation of PARP1 at lysine 949, which blocks its ubiquitination at the same residue and subsequent degradation by E3 ubiquitin ligase CHFR. Consequently, depletion of MORC2 or expression of an acetylation-defective PARP1 mutant impairs DNA damage-induced PAR production and PAR-dependent recruitment of DNA repair proteins to DNA lesions, leading to enhanced sensitivity to genotoxic stress. Collectively, these findings uncover a previously unrecognized mechanistic link between MORC2 and PARP1 in the regulation of cellular response to DNA damage.

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