scholarly journals Constitutively Activated Akt-1 Is Vital for the Survival of Human Monocyte-Differentiated Macrophages

2001 ◽  
Vol 194 (2) ◽  
pp. 113-126 ◽  
Author(s):  
Hongtao Liu ◽  
Harris Perlman ◽  
Lisa J. Pagliari ◽  
Richard M. Pope
Keyword(s):  
Cell Death ◽  
Ectopic Expression ◽  
Human Monocyte ◽  
Pi3k Pathway ◽  
Dominant Negative ◽  
Human Macrophages ◽  
Pi3k Inhibitor Ly294002 ◽  
Inositol Phosphatase ◽  
Pi3k Inhibition ◽  
Src Homology 2 Domain

Recent data from mice deficient for phosphatase and tensin homologue deleted from chromosome 10 or src homology 2 domain–containing 5′ inositol phosphatase, phosphatases that negatively regulate the phosphatidylinositol 3-kinase (PI3K) pathway, revealed an increased number of macrophages in these animals, suggesting an essential role for the PI3K pathway for macro-phage survival. Here, we focused on the role of the PI3K-regulated serine/threonine kinase Akt-1 in modulating macrophage survival. Akt-1 was constitutively activated in human macrophages and addition of the PI3K inhibitor, LY294002, suppressed the activation of Akt-1 and induced cell death. Furthermore, suppression of Akt-1 by inhibition of PI3K or a dominant negative (DN) Akt-1 resulted in loss of mitochondrial transmembrane potential, activation of caspases-9 and -3, and DNA fragmentation. The effects of PI3K inhibition were reversed by the ectopic expression of constitutively activated Akt-1 or Bcl-xL. Inhibition of PI3K/Akt-1 pathway either by LY294002 or DN Akt-1 had no effect on the constitutive or inducible activation of nuclear factor (NF)-κB in human macrophages. However, after inhibition of the PI3K/Akt-1 pathway, a marked decrease in the expression of the antiapoptotic molecule Mcl-1, but not other Bcl-2 family members was observed, and Mcl-1 rescued macrophages from LY294002-induced cell death. Further, inhibition of Mcl-1 by antisense oligonucleotides, also resulted in macrophage apoptosis. Thus, our findings demonstrate that the constitutive activation of Akt-1 regulates macrophage survival through Mcl-1, which is independent of caspases, NF-κB, or Bad.

scholarly journals SLC25A23 augments mitochondrial Ca2+ uptake, interacts with MCU, and induces oxidative stress–mediated cell death

2014 ◽  
Vol 25 (6) ◽  
pp. 936-947 ◽  
Author(s):  
Nicholas E. Hoffman ◽  
Harish C. Chandramoorthy ◽  
Santhanam Shanmughapriya ◽  
Xueqian Q. Zhang ◽  
Sandhya Vallem ◽  
...  
Keyword(s):  
Cell Death ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Inner Mitochondrial Membrane ◽  
Solute Carriers ◽  
Metabolic Functions ◽  
Evolutionarily Conserved ◽  
Ef Hand ◽  
Solute Carrier ◽  
Negative Phenotype

Emerging findings suggest that two lineages of mitochondrial Ca2+ uptake participate during active and resting states: 1) the major eukaryotic membrane potential–dependent mitochondrial Ca2+ uniporter and 2) the evolutionarily conserved exchangers and solute carriers, which are also involved in ion transport. Although the influx of Ca2+ across the inner mitochondrial membrane maintains metabolic functions and cell death signal transduction, the mechanisms that regulate mitochondrial Ca2+ accumulation are unclear. Solute carriers—solute carrier 25A23 (SLC25A23), SLC25A24, and SLC25A25—represent a family of EF-hand–containing mitochondrial proteins that transport Mg-ATP/Pi across the inner membrane. RNA interference–mediated knockdown of SLC25A23 but not SLC25A24 and SLC25A25 decreases mitochondrial Ca2+ uptake and reduces cytosolic Ca2+ clearance after histamine stimulation. Ectopic expression of SLC25A23 EF-hand–domain mutants exhibits a dominant-negative phenotype of reduced mitochondrial Ca2+ uptake. In addition, SLC25A23 interacts with mitochondrial Ca2+ uniporter (MCU; CCDC109A) and MICU1 (CBARA1) while also increasing IMCU. In addition, SLC25A23 knockdown lowers basal mROS accumulation, attenuates oxidant-induced ATP decline, and reduces cell death. Further, reconstitution with short hairpin RNA–insensitive SLC25A23 cDNA restores mitochondrial Ca2+ uptake and superoxide production. These findings indicate that SLC25A23 plays an important role in mitochondrial matrix Ca2+ influx.

PI3 Kinase Inhibition Induces Apoptotic Cell Death in Acute Lymphoblastic Leukemia Via the Mitochondrial Pathway.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1911-1911
Author(s):  
Karin von Schwarzenberg ◽  
Marco Henkel ◽  
Dennis Conzelmann ◽  
Björn Stork ◽  
Anita Bringmann ◽  
...  
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Lymphoblastic Leukemia ◽  
Death Receptor ◽  
Pi3k Pathway ◽  
Apoptotic Cell Death ◽  
Jurkat Cells ◽  
Parp Cleavage ◽  
Caspase 9 ◽  
Pi3k Inhibition

Abstract The phosphatidylinositol 3-kinase (PI3K) pathway regulates many cellular processes that are involved in tumor progression. Aberrant activation of the PI3K pathway due to an alteration of its elements like PTEN or Akt occurs quite frequently in malignant cells. Thus, inhibition of this pathway represents a promising option for the treatment of cancer patients. The best characterized PI3K inhibitors are LY294002 and wortmannin that were shown to disrupt downstream signaling and induce apoptotic cell death in tumor cells. Acute lymphoblastic leukemia (ALL) is a malignancy mainly found in young children and elderly with constitutive activation of PI3K pathway. In our study we analyzed the effect of PI3K inhibition in cell lines deduced from ALL (Jurkat, BV173, SD1, T-2) and primary leukemic cells by incubating them with increasing concentrations of inhibitors of the PI3K signaling. We found that treatment of ALL cells with LY294002, the mTOR inhibitor rapamycin or Akt inhibitor SH5 induced apoptotic cell death that was accompanied by caspase-3 activation and PARP-cleavage and interfered with intracellular PI3K/Akt signaling as analyzed by phosphorylation and expression of mTOR or P70S6K. In line with these results apoptotic cell death could be inhibited by the pan-caspase inhibitor zVAD. In order to determine the pathway of apoptosis induction we took advantage of Jurkat cells (T-ALL) overexpressing or lacking molecules involved in apoptotic pathways such as FADD, an adaptor molecule recruited to the death receptor upon ligand binding, Caspase-8, Caspase-9 or Bcl-2, an anti-apoptotic protein that prevents the release of cytochrom c from mitochondria. PI3K inhibition by LY294002 induced apoptotic cell death in cells deficient of FADD or caspase-8 with no difference to wild type cells. In contrast, cells overexpressing Bcl2 or lacking caspase-9 were resistant to apoptotic death indicating that PI3-kinase inhibition is independent of the external death receptor signaling and is mediated via the mitochondrial pathway. These results were confirmed by analyzing PARP cleavage and caspase-3 activation in utilized leukemic cell lines. Furthermore, we found that the PI3K inhibitor LY294002 induced apoptosis in ALL cells that could be increased by the etoposide, a topoisomerase inhibitor, or TRAIL. In addition, in contrast to etoposide, treatment of ALL cells with TRAIL could overcome the resistance of ALL cells to PI3K inhibition even in caspase-9 deficient Jurkat cells. Our results provide an interesting approach in designing novel therapeutic strategies to target the PI3K pathway in ALL to overcome the resistance to cytotoxic agents.

scholarly journals Cardiac glycosides cause selective cytotoxicity in human macrophages and ameliorate white adipose tissue homeostasis

2020 ◽  
Author(s):  
Antoni Olona ◽  
Charlotte Hateley ◽  
Ana Guerrero ◽  
Jeong-Hun Ko ◽  
Michael R Johnson ◽  
...  
Keyword(s):  
Cell Death ◽  
Adipose Tissue ◽  
White Adipose Tissue ◽  
Cardiac Glycosides ◽  
Ex Vivo ◽  
Human Monocyte ◽  
Morbidly Obese ◽  
Inflammasome Activation ◽  
Human Macrophages ◽  
Therapeutic Avenue

AbstractCardiac glycosides (CGs) inhibit the Na+,K+-ATPase and are widely prescribed medicines for chronic heart failure and cardiac arrhythmias. Recently, CGs have been described to induce inflammasome activation in human macrophages, suggesting a cytotoxicity that remains to be elucidated in tissues. Here we show that human monocyte-derived macrophages (hMDMs) undergo cell death following incubation with nanomolar concentrations of CGs, and in particular with ouabain (IC50=50 nM). The ouabain-induced cell death is more efficient in hMDMs compared to non-adherent PBMC populations and is through on-target inhibition of Na,K-ATPAse activity, as it causes an intracellular depletion of K+, while inducing accumulation of Na+ and Ca2+ levels. Consistently, the cell-death caused by these ion imbalances can be rescued by addition of potassium chloride in hMDMs. Strikingly, white adipose tissue (WAT) explants from morbidly obese patients cultured with nanomolar concentrations of ouabain causes depletion of macrophages, decreases type VI collagen levels, and ameliorates insulin-sensitivity ex vivo. These results suggest that the usage of nanomolar concentration of CGs can be an attractive therapeutic avenue in metabolic syndrome characterised by pathogenic infiltration and activation of macrophages.

scholarly journals Subamolide B Isolated from Medicinal PlantCinnamomum subaveniumInduces Cytotoxicity in Human Cutaneous Squamous Cell Carcinoma Cells through Mitochondrial and CHOP-Dependent Cell Death Pathways

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Shu-Yi Yang ◽  
Hui-Min Wang ◽  
Tai-Wen Wu ◽  
Yi-Ju Chen ◽  
Jeng-Jer Shieh ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Cell Death ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Ectopic Expression ◽  
Chemotherapeutic Agents ◽  
Cutaneous Squamous Cell Carcinoma ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cell Death Pathways

Subamolide B is a butanolide isolated fromCinnamomum subavenium, a medicinal plant traditionally used to treat various ailments including carcinomatous swelling. We herein reported for the first time that subamolide B potently induced cytotoxicity against diverse human skin cancer cell lines while sparing nonmalignant cells. Mechanistic studies on human cutaneous squamous cell carcinoma (SCC) cell line SCC12 highlighted the involvement of apoptosis in subamolide B-induced cytotoxicity, as evidenced by the activation of caspases-8, -9, -4, and -3, the increase in annexin V-positive population, and the partial restoration of cell viability by cotreatment with the pan-caspase inhibitor z-VAD-fmk. Additionally, subamolide B evoked cell death pathways mediated by FasL/Fas, mitochondria, and endoplasmic reticulum (ER) stress, as supported by subamolide B-induced FasL upregulation, BCL-2 suppression/cytosolic release of cytochrome c, and UPR activation/CHOP upregulation, respectively. Noteworthy, ectopic expression of c-FLIPLor dominant-negative mutant of FADD failed to impair subamolide B-induced cytotoxicity, whereas BCL-2 overexpression or CHOP depletion greatly rescued subamolide B-stimulated cells. Collectively, these results underscored the central role of mitochondrial and CHOP-mediated cell death pathways in subamolide B-induced cytotoxicity. Our findings further implicate the potential of subamolide B for cutaneous SCC therapy or as a lead compound for developing novel chemotherapeutic agents.

scholarly journals Neisseria gonorrhoeae–Induced Inflammatory Pyroptosis in Human Macrophages is Dependent on Intracellular Gonococci and Lipooligosaccharide

2018 ◽  
Vol 11 ◽  
pp. 117906601775090 ◽  
Author(s):  
Jessica Leigh Ritter ◽  
Caroline Attardo Genco
Keyword(s):  
Cell Death ◽  
Neisseria Gonorrhoeae ◽  
Transmitted Disease ◽  
Human Monocyte ◽  
Dependent Manner ◽  
Human Macrophages ◽  
Sexually Transmitted ◽  
Cell Death Pathways ◽  
Dependent Cell ◽  
Obligate Pathogen

Neisseria gonorrhoeae, the human obligate pathogen responsible for the sexually transmitted disease gonorrhea, has evolved several mechanisms to evade the host immune response. One such mechanism is the modulation of host cell death pathways. In this study, we defined cell death pathways induced by N gonorrhoeae in human monocyte-derived macrophages (MDMs). In a dose-dependent manner, N gonorrhoeae stimulation of MDMs resulted in caspase 1 and 4–dependent cell deaths, indicative of canonical and noncanonical pyroptosis, respectively. Internalization of bacteria or stimulation with lipooligosaccharide (LOS) specifically induced pyroptosis in MDMs and increased secretion of IL-1β. Collectively, our results demonstrate that N gonorrhoeae induces inflammatory pyroptosis in human macrophages due in part to intracellular LOS. We propose that this in turn may exacerbate inflammatory outcomes observed during mucosal infection.

Serine phosphorylation of STAT3 is essential for Mcl-1 expression and macrophage survival

Blood ◽  
2003 ◽  
Vol 102 (1) ◽  
pp. 344-352 ◽  
Author(s):  
Hongtao Liu ◽  
Yingyu Ma ◽  
Shawn M. Cole ◽  
Christopher Zander ◽  
Kun-Hung Chen ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Apoptotic Cell ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Serine Phosphorylation ◽  
Mobility Shift ◽  
Raw264.7 Macrophages

Abstract The Bcl-2 family member Mcl-1 is essential for macrophage survival. However, the mechanisms that contribute to the expression of Mcl-1 in these cells have not been fully characterized. The present study focused on the role of signal transducer and activator of transcription 3 (STAT3) in regulation of Mcl-1 in macrophages. Sodium salicylate (NaSal) treatment induced apoptotic cell death in primary human macrophages in a dose- and time-dependent fashion. Incubation with NaSal resulted in the loss of mitochondrial transmembrane potential, the release of cytochromecand second mitochondria-derived activator of caspase/direct IAP binding protein with low pH of isoelectric point (pI) from the mitochondria, and the activation of caspases 9 and 3. Western blot analysis and reverse transcription—polymerase chain reaction demonstrated that NaSal down-regulated the expression of Mcl-1. Electrophoretic mobility shift assay and Western blot analysis for phosphorylated STAT3 demonstrated that STAT3 was constitutively activated in macrophages and that this STAT3 activation was suppressed by NaSal. The activation of STAT3 in macrophages was dependent on Ser727 phosphorylation, in the absence of detectable Tyr705phosphorylation. Ectopic expression of STAT3 in murine RAW264.7 macrophages rescued the inhibition of Mcl-1 promoter-reporter gene activation and the cell death induced by NaSal treatment, while a dominant-negative STAT3 resulted in cell death. To confirm its role in primary macrophages, STAT3 antisense (AS) oligodeoxynucleotides (ODNs) were employed. STAT3 AS, but not control, ODNs decreased STAT3 and Mcl-1 expression and resulted in macrophage apoptosis. These observations demonstrate that the STAT3-mediated expression of Mcl-1 is essential for the survival of primary human in vitro differentiated macrophages. (Blood. 2003;102:344-352)

scholarly journals Human Herpesvirus 6A Induces Dendritic Cell Death and HMGB1 Release without Virus Replication

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 57
Author(s):  
Rasmus Gustafsson
Keyword(s):  
Cell Death ◽  
Dendritic Cells ◽  
High Mobility ◽  
Human Herpesvirus ◽  
Human Monocyte ◽  
Hmgb1 Protein ◽  
Innate And Adaptive Immunity ◽  
Th2 Polarization ◽  
Dependent Cell ◽  
Ifn Γ

Human herpesvirus 6A (HHV-6A) is a common virus that has important immunomodulatory effects. Dendritic cells (DC) are key players in innate and adaptive immunity and are implicated in the pathogenesis of many human diseases, including infections. (1) Background: Previous studies have demonstrated suppressive effects of HHV-6A on key DC functions. (2) Methods: human monocyte derived dendritic cells were inoculated with HHV-6A and viral replication, cell viability, and release of high mobility group box 1 (HMGB1) protein from DC and of the cytokines IL-2, IL-4, IL-6, IL-10, TNF and IFN-γ after co-culture with allogenic CD4+ T cells were assessed. (3) Results: Nonproductive infection of HHV-6A in DC leads to titer-dependent cell death and the release of HMGB1 protein, and a Th2 polarization. (4) Conclusion: These immune responses aimed to clear the infection may also imply risks for inflammatory pathologies associated with HHV-6A such as multiple sclerosis.

scholarly journals RhoA enhances store-operated Ca2+ entry and intestinal epithelial restitution by interacting with TRPC1 after wounding

2015 ◽  
Vol 309 (9) ◽  
pp. G759-G767 ◽  
Author(s):  
Hee Kyoung Chung ◽  
Navneeta Rathor ◽  
Shelley R. Wang ◽  
Jian-Ying Wang ◽  
Jaladanki N. Rao
Keyword(s):  
Cell Migration ◽  
Transient Receptor Potential ◽  
Ectopic Expression ◽  
Receptor Potential ◽  
Dominant Negative ◽  
Intestinal Epithelial ◽  
Epithelial Restitution ◽  
Epithelial Cell Migration ◽  
Transient Receptor ◽  
Wounded Area

Early mucosal restitution occurs as a consequence of epithelial cell migration to resealing of superficial wounds after injury. Our previous studies show that canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca2+ channel (SOC) in intestinal epithelial cells (IECs) and plays an important role in early epithelial restitution by increasing Ca2+ influx. Here we further reported that RhoA, a small GTP-binding protein, interacts with and regulates TRPC1, thus enhancing SOC-mediated Ca2+ entry (SOCE) and epithelial restitution after wounding. RhoA physically associated with TRPC1 and formed the RhoA/TRPC1 complexes, and this interaction increased in stable TRPC1-transfected IEC-6 cells (IEC-TRPC1). Inactivation of RhoA by treating IEC-TRPC1 cells with exoenzyme C3 transferase (C3) or ectopic expression of dominant negative RhoA (DNMRhoA) reduced RhoA/TRPC1 complexes and inhibited Ca2+ influx after store depletion, which was paralleled by an inhibition of cell migration over the wounded area. In contrast, ectopic expression of wild-type (WT)-RhoA increased the levels of RhoA/TRPC1 complexes, induced Ca2+ influx through activation of SOCE, and promoted cell migration after wounding. TRPC1 silencing by transfecting stable WT RhoA-transfected cells with siRNA targeting TRPC1 (siTRPC1) reduced SOCE and repressed epithelial restitution. Moreover, ectopic overexpression of WT-RhoA in polyamine-deficient cells rescued the inhibition of Ca2+ influx and cell migration induced by polyamine depletion. These findings indicate that RhoA interacts with and activates TRPC1 and thus stimulates rapid epithelial restitution after injury by inducing Ca2+ signaling.

scholarly journals Cancer cells activate p53 in response to 10-formyltetrahydrofolate dehydrogenase expression

2005 ◽  
Vol 391 (3) ◽  
pp. 503-511 ◽  
Author(s):  
Natalia V. Oleinik ◽  
Natalia I. Krupenko ◽  
David G. Priest ◽  
Sergey A. Krupenko
Keyword(s):  
Cancer Cells ◽  
Ectopic Expression ◽  
A549 Cells ◽  
Dominant Negative ◽  
G1 Arrest ◽  
Transactivation Domain ◽  
Downstream Target ◽  
Formyltetrahydrofolate Dehydrogenase ◽  
Induced Apoptosis ◽  
Hct116 Cells

A folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase; EC 1.5.1.6), is not a typical tumour suppressor, but it has two basic characteristics of one, i.e. it is down-regulated in tumours and its expression is selectively cytotoxic to cancer cells. We have recently shown that ectopic expression of FDH in A549 lung cancer cells induces G1 arrest and apoptosis that was accompanied by elevation of p53 and its downstream target, p21. It was not known, however, whether FDH-induced apoptosis is p53-dependent or not. In the present study, we report that FDH-induced suppressor effects are strictly p53-dependent in A549 cells. Both knockdown of p53 using an RNAi (RNA interference) approach and disabling of p53 function by dominant-negative inhibition with R175H mutant p53 prevented FDH-induced cytotoxicity in these cells. Ablation of the FDH-suppressor effect is associated with an inability to activate apoptosis in the absence of functional p53. We have also shown that FDH elevation results in p53 phosphorylation at Ser-6 and Ser-20 in the p53 transactivation domain, and Ser-392 in the C-terminal domain, but only Ser-6 is strictly required to mediate FDH effects. Also, translocation of p53 to the nuclei and expression of the pro-apoptotic protein PUMA (Bcl2 binding component 3) was observed after induction of FDH expression. Elevation of FDH in p53 functional HCT116 cells induced strong growth inhibition, while growth of p53-deficient HCT116 cells was unaffected. This implies that activation of p53-dependent pathways is a general downstream mechanism in response to induction of FDH expression in p53 functional cancer cells.

scholarly journals Short-term effects of triiodothyronine on thyroid hormone receptor alpha by PI3K pathway in adipocytes, 3T3-L1

2014 ◽  
Vol 58 (8) ◽  
pp. 833-837 ◽  
Author(s):  
Miriane de Oliveira ◽  
Regiane Marques Castro Olimpio ◽  
Maria Teresa De Sibio ◽  
Fernanda Cristina Fontes Moretto ◽  
Renata de Azevedo Mello Luvizotto ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Mrna Expression ◽  
Signaling Pathway ◽  
Thyroid Hormone Receptor ◽  
Pi3k Pathway ◽  
Control Group ◽  
Pi3k Signaling ◽  
Pi3k Inhibitor Ly294002 ◽  
Receptor Alpha ◽  
Pi3k Signaling Pathway

Objective The present study aimed to examine the effects of thyroid hormone (TH), more precisely triiodothyronine (T3), on the modulation of TH receptor alpha (TRα) mRNA expression and the involvement of the phosphatidyl inositol 3 kinase (PI3K) signaling pathway in adipocytes, 3T3-L1, cell culture. Materials and methods: It was examined the involvement of PI3K pathway in mediating T3 effects by treating 3T3-L1 adipocytes with physiological (P=10nM) or supraphysiological (SI =100 nM) T3 doses during one hour (short time), in the absence or the presence of PI3K inhibitor (LY294002). The absence of any treatment was considered the control group (C). RT-qPCR was used for mRNA expression analyzes. For data analyzes ANOVA complemented with Tukey’s test was used at 5% significance level. Results T3 increased TRα mRNA expression in P (1.91±0.13, p<0.001), SI (2.14±0.44, p<0.001) compared to C group (1±0.08). This increase was completely abrogated by LY294002 in P (0.53±0.03, p<0.001) and SI (0.31±0.03, p<0.001). To examine whether TRα is directly induced by T3, we used the translation inhibitor cycloheximide (CHX). The presence of CHX completely abrogated levels TRα mRNA in P (1.15±0.05, p>0.001) and SI (0.99±0.15, p>0.001), induced by T3. Conclusion These results demonstrate that the activation of the PI3K signaling pathway has a role in T3-mediated indirect TRα gene expression in 3T3-L1 adipocytes.

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