Partial ATP depletion induces Fas- and caspase-mediated apoptosis in MDCK cells

1999 ◽  
Vol 276 (6) ◽  
pp. F837-F846 ◽  
Author(s):  
L. Richard Feldenberg ◽  
Sundararajah Thevananther ◽  
Marcela del Rio ◽  
Maryely de Leon ◽  
Prasad Devarajan
Keyword(s):  
Dna Cleavage ◽  
Serine Proteases ◽  
Fas Ligand ◽  
Mdck Cells ◽  
Morphological Changes ◽  
Critical Role ◽  
Atp Depletion ◽  
Caspase 8 ◽  
Death Domain

Brief periods of in vitro hypoxia/ischemia induce apoptosis of cultured renal epithelial cells, but the underlying mechanisms remain unknown. We show that partial ATP depletion (≈10–65% of control) results in a duration-dependent induction of apoptosis in Madin-Darby canine kidney (MDCK) cells, as evidenced by internucleosomal DNA cleavage (DNA laddering and in situ nick end labeling), morphological changes (cell shrinkage), and plasma membrane alterations (externalization of phosphatidylserine). The ATP-depleted cells display a significant upregulation of Fas, Fas ligand, and the Fas-associating protein with death domain (FADD). Exogenous application of stimulatory Fas monoclonal antibodies also induces apoptosis in nonischemic MDCK cells, indicating that they retain Fas-dependent pathways of programmed cell death. Furthermore, cleavage of poly(ADP)ribose polymerase (PARP) is evident after ATP depletion, indicating activation of caspases. Indeed, the apoptotic cells display a significant increase in caspase-8 (FLICE) activity. Finally, apoptosis induced by ATP depletion is ameliorated by pretreatment with inhibitors of caspase-8 (IETD), caspase-1 (YVAD), or caspase-3 (DEVD) but is not affected by inhibitors of serine proteases (TPCK). Our results indicate that partial ATP depletion of MDCK cells results in apoptosis and that Fas- and caspase-mediated pathways may play a critical role.

Albumin overload induces apoptosis in LLC-PK1cells

2001 ◽  
Vol 280 (6) ◽  
pp. F1107-F1114 ◽  
Author(s):  
Elif Erkan ◽  
Maryely De Leon ◽  
Prasad Devarajan
Keyword(s):  
Dna Cleavage ◽  
Morphological Changes ◽  
Prognostic Indicator ◽  
Caspase 8 ◽  
Death Domain ◽  
Tubulointerstitial Injury ◽  
Nuclear Condensation ◽  
Glomerular Diseases ◽  
Induced Apoptosis

The degree of albuminuria is a well-known adverse prognostic indicator in human glomerular diseases. However, the mechanisms by which albuminuria by itself contributes to tubulointerstitial injury and progression of renal disease remain unclear. We tested the hypothesis that apoptosis may represent one of the mechanisms by which tubule epithelial cells are damaged after albumin overload in vitro. Cultured LLC-PK1 proximal tubule cells were incubated with varying concentrations of BSA. This resulted in a dose- and duration-dependent induction of apoptosis, as evidenced by internucleosomal DNA cleavage (DNA laddering and nick-end labeling), externalization of plasma membrane phosphatidylserine (annexin labeling), and characteristic morphological changes (cell shrinkage and nuclear condensation). Albumin overload also resulted in a dose-dependent upregulation of Fas and Fas-associated protein with death domain (FADD), and activation of caspase 8. Incubation with the caspase 8 inhibitor IETD ameliorated the albumin-induced apoptosis. Collectively, our results indicate that albumin overload induces apoptosis of cultured LLC-PK1 cells, mediated at least in part by the Fas-FADD-caspase 8 pathway.

scholarly journals Distinct Roles of the Antiapoptotic Effectors NleB and NleF from Enteropathogenic Escherichia coli

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Georgina L. Pollock ◽  
Clare V. L. Oates ◽  
Cristina Giogha ◽  
Tania Wong Fok Lung ◽  
Sze Ying Ong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fas Ligand ◽  
Death Receptor ◽  
Effector Proteins ◽  
Host Cells ◽  
Caspase 8 ◽  
Death Domain ◽  
Content Type ◽  
Fas Signaling

ABSTRACT During infection, enteropathogenic Escherichia coli (EPEC) translocates effector proteins directly into the cytosol of infected enterocytes using a type III secretion system (T3SS). Once inside the host cell, these effector proteins subvert various immune signaling pathways, including death receptor-induced apoptosis. One such effector protein is the non-locus of enterocyte effacement (LEE)-encoded effector NleB1, which inhibits extrinsic apoptotic signaling via the FAS death receptor. NleB1 transfers a single N-acetylglucosamine (GlcNAc) residue to Arg117 in the death domain of Fas-associated protein with death domain (FADD) and inhibits FAS ligand (FasL)-stimulated caspase-8 cleavage. Another effector secreted by the T3SS is NleF. Previous studies have shown that NleF binds to and inhibits the activity of caspase-4, -8, and -9 in vitro. Here, we investigated a role for NleF in the inhibition of FAS signaling and apoptosis during EPEC infection. We show that NleF prevents the cleavage of caspase-8, caspase-3, and receptor-interacting serine/threonine protein kinase 1 (RIPK1) in response to FasL stimulation. When translocated into host cells by the T3SS or expressed ectopically, NleF also blocked FasL-induced cell death. Using the EPEC-like mouse pathogen Citrobacter rodentium, we found that NleB but not NleF contributed to colonization of mice in the intestine. Hence, despite their shared ability to block FasL/FAS signaling, NleB and NleF have distinct roles during infection.

Cadmium is more toxic to LLC-PK1cells than to MDCK cells acting on the cadherin-catenin complex

1998 ◽  
Vol 275 (1) ◽  
pp. F143-F153 ◽  
Author(s):  
L. B. Zimmerhackl ◽  
F. Momm ◽  
G. Wiegele ◽  
M. Brandis
Keyword(s):  
Mdck Cells ◽  
Morphological Changes ◽  
Collecting Duct ◽  
Cadmium Toxicity ◽  
Distal Tubule ◽  
Insoluble Fraction ◽  
Atp Depletion ◽  
Rhodamine Phalloidin ◽  
Apical Side ◽  
E Cadherin

Cadmium toxicity to renal cells was investigated in Madin-Darby canine kidney (MDCK) and LLC-PK1cells as models of the distal tubule/collecting duct and proximal tubule, respectively. Cells were grown on two-compartment filters and exposed to 0.1–50 μM Cd2+. In MDCK cells, Cd2+was more toxic from the basolateral than from the apical side and dependent on the extracellular Ca2+concentration. Toxicity was evident within 24 h, as shown by a decrease in transepithelial resistance (TER), reduced proliferation (bromodeoxyuridine incorporation), reduction in ATP concentration, and morphological changes. On confocal microscopy, E-cadherin and α-catenin staining patterns indicated interference with the cadherin-catenin complex. LLC-PK1cells showed a similar toxicity pattern, which was evident at lower Cd2+concentrations. An increase of E-cadherin and α-catenin molecules in the Triton X-100-insoluble fraction was detectable at high Cd2+concentrations in LLC-PK1cells but not in MDCK cells. Lactate dehydrogenase release indicated membrane leakage in LLC-PK1cells. Rhodamine-phalloidin staining, a probe for F-actin filaments, demonstrated alterations of the actin cytoskeleton in both cell lines. In conclusion, cadmium caused ATP depletion and interfered with the cadherin-catenin complex and probably the tight junctions changing renal cell morphology and function.

scholarly journals P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Feng Jing ◽  
Yixin Zhang ◽  
Ting Long ◽  
Wei He ◽  
Guangcheng Qin ◽  
...  
Keyword(s):  
Chronic Migraine ◽  
Microglial Activation ◽  
Morphological Changes ◽  
Critical Role ◽  
Radiant Heat ◽  
P2y12 Receptor ◽  
Trigeminal Nucleus ◽  
Double Labeling ◽  
Trigeminal Nucleus Caudalis

Abstract Background Microglial activation contributes to the development of chronic migraine (CM). The P2Y12 receptor (P2Y12R), a metabolic purinoceptor that is expressed on microglia in the central nervous system (CNS), has been indicated to play a critical role in the pathogenesis of chronic pain. However, whether it contributes to the mechanism of CM remains unknown. Thus, the present study investigated the precise details of microglial P2Y12R involvement in CM. Methods Mice subjected to recurrent nitroglycerin (NTG) treatment were used as the CM model. Hyperalgesia were assessed by mechanical withdrawal threshold to electronic von Frey and thermal withdrawal latency to radiant heat. Western blot and immunohistochemical analyses were employed to detect the expression of P2Y12R, Iba-1, RhoA, and ROCK2 in the trigeminal nucleus caudalis (TNC). To confirm the role of P2Y12R and RhoA/ROCK in CM, we systemically administered P2Y12R antagonists (MRS2395 and clopidogrel) and a ROCK2 inhibitor (fasudil) and investigated their effects on microglial activation, c-fos, and calcitonin gene-related peptide (CGRP) expression in the TNC. To further confirm the effect of P2Y12R on microglial activation, we preincubated lipopolysaccharide (LPS)-treated BV-2 microglia with MRS2395 and clopidogrel. ELISA was used to evaluate the levels of inflammatory cytokines. Results The protein levels of P2Y12R, GTP-RhoA, ROCK2, CGRP, c-fos, and inducible nitric oxide synthase (iNOS) in the TNC were increased after recurrent NTG injection. A double labeling study showed that P2Y12R was restricted to microglia in the TNC. MRS2395 and clopidogrel attenuated the development of tactile allodynia and suppressed the expression of CGRP, c-fos, and GTP-RhoA/ROCK2 in the TNC. Furthermore, fasudil also prevented hyperalgesia and suppressed the expression of CGRP in the TNC. In addition, inhibiting P2Y12R and ROCK2 activities suppressed NTG-induced microglial morphological changes (process retraction) and iNOS production in the TNC. In vitro, a double labeling study showed that P2Y12R was colocalized with BV-2 cells, and the levels of iNOS, IL-1β, and TNF-α in LPS-stimulated BV-2 microglia were reduced by P2Y12R inhibitors. Conclusions These data demonstrate that microglial P2Y12R in the TNC plays a critical role in the pathogenesis of CM by regulating microglial activation in the TNC via RhoA/ROCK pathway.

scholarly journals Requirements for proteolysis during apoptosis.

1997 ◽  
Vol 17 (11) ◽  
pp. 6502-6507 ◽  
Author(s):  
D L Vaux ◽  
S Wilhelm ◽  
G Häcker
Keyword(s):  
Dna Cleavage ◽  
Morphological Changes ◽  
Cysteine Proteases ◽  
Dna Degradation ◽  
Effector Proteins ◽  
Proteinase K ◽  
Caspase Activation ◽  
Caspase Substrate ◽  
Do So

The key effector proteins of apoptosis are a family of cysteine proteases termed caspases. Following activation of caspases, biochemical events occur that lead to DNA degradation and the characteristic morphological changes associated with apoptosis. Here we show that cytoplasmic extracts activated in vitro by proteinase K were able to cleave the caspase substrate DEVD-7-amino-4-methylcoumarin, while neither proteinase K nor nonactivated extracts were able to do so alone. Caspase-like activity was inhibited by the specific caspase inhibitor DEVD-aldehyde and by the protease inhibitor iodoacetamide, but not by N-ethylmaleimide. When added to isolated nuclei, the activated extracts caused internucleosomal DNA degradation and morphological changes typical of apoptosis. As DNA cleavage and morphological changes could be inhibited by N-ethylmaleimide but not by iodoacetamide, we conclude that during apoptosis, caspase activation causes activation of another cytoplasmic enzyme that can be inhibited by N-ethylmaleimide. Activity of this enzyme is necessary for activation of endonucleases, DNA cleavage, and changes in nuclear morphology.

scholarly journals Normal Thymocyte Negative Selection in TRAIL-deficient Mice

2003 ◽  
Vol 198 (3) ◽  
pp. 491-496 ◽  
Author(s):  
Erika Cretney ◽  
Adam P. Uldrich ◽  
Stuart P. Berzins ◽  
Andreas Strasser ◽  
Dale I. Godfrey ◽  
...  
Keyword(s):  
Negative Selection ◽  
Fas Ligand ◽  
Critical Role ◽  
Death Receptor ◽  
Critical Factor ◽  
Immunological Tolerance ◽  
Trail Signaling ◽  
Selection Of

The molecular basis of thymocyte negative selection, which plays a critical role in establishing and maintaining immunological tolerance, is not yet resolved. In particular, the importance of the death receptor subgroup of the tumor necrosis factor (TNF)-family has been the subject of many investigations, with equivocal results. A recent report suggested that TRAIL was a critical factor in this process, a result that does not fit well with previous studies that excluded a role for the FADD-caspase 8 pathway, which is essential for TRAIL and Fas ligand (FasL) signaling, in negative selection. We have investigated intrathymic negative selection of TRAIL-deficient thymocytes, using four well-established models, including antibody-mediated TCR/CD3 ligation in vitro, stimulation with endogenous superantigen in vitro and in vivo, and treatment with exogenous superantigen in vitro. We were unable to demonstrate a role for TRAIL signaling in any of these models, suggesting that this pathway is not a critical factor for thymocyte negative selection.

scholarly journals The Antioxidant Resveratrol Protects against Chondrocyte Apoptosis by Regulating the COX-2/NF-κB Pathway in Created Temporomandibular Osteoarthritis

2021 ◽  
Vol 2021 ◽  
pp. 1-7
Author(s):  
Wen Li ◽  
Shiyu Hu ◽  
Xuepeng Chen ◽  
Jiejun Shi
Keyword(s):  
Morphological Changes ◽  
Critical Role ◽  
Chondrocyte Apoptosis ◽  
Optimum Dose ◽  
Protective Effects ◽  
Condylar Cartilage ◽  
Temporomandibular Joint Osteoarthritis ◽  
Cox 2 ◽  
Antioxidant Effects

Temporomandibular joint osteoarthritis (TMJOA) is characterized by chronic inflammatory degradation of mandibular condylar cartilage (MCC). Studies have found a positive correlation between inflammation and cyclooxygenase- (COX-) 2 in OA pathology. NF-κB is a crucial transcription factor of inflammatory and immune responses in the cause of TMJOA pathology. Resveratrol (RES) plays a critical role in antioxidation and anti-inflammation. But, studies on the effects of RES on TMJOA are very limited. So, the purpose of this study is to investigate the antioxidant and protective effects of RES against MCC degradation through downregulating COX-2/NF-κB expression. In vitro studies, the MCC cells were divided into three groups: the NC group, OA group, and RES group. The optimum dose of RES (10 μM) was determined. The TMJOA model of mice was created by injection of collagenase. And mice were injected with RES (100 μg/10 μl) 3 times one week for 4 weeks in the RES group. The expressions of COX-2, P65, MMP1, MMP13, COL2, and ACAN were measured by RT-PCR. Morphological changes of MCC were studied with HE staining. The results showed that inflammation could induce MCC degradation in vitro and vivo, while RES could reverse the degradation. Meanwhile, RES could downregulate COX-2/NF-κB/MMP expression and increase cartilage markers in vitro and vivo studies. The results indicated that RES treatment had antioxidant effects against chondrocyte apoptosis by downregulating the COX-2/NF-κB pathway in created TMJOA.

Inhibition of Fas-associated apoptosis in granulation tissue cells accompanies attenuation of postinfarction left ventricular remodeling by olmesartan

2007 ◽  
Vol 292 (5) ◽  
pp. H2184-H2194 ◽  
Author(s):  
Hiromitsu Kanamori ◽  
Genzou Takemura ◽  
Yiwen Li ◽  
Hideshi Okada ◽  
Rumi Maruyama ◽  
...  
Keyword(s):  
Granulation Tissue ◽  
Fas Ligand ◽  
Ventricular Remodeling ◽  
Left Ventricular Remodeling ◽  
Specific Effect ◽  
Left Ventricular ◽  
Death Domain ◽  
Absolute Size ◽  
Tissue Cells

Blockade of angiotensin II type 1 receptor (AT1) signaling attenuates heart failure following myocardial infarction (MI), perhaps through reduction of fibrosis in the noninfarcted myocardium. However, its specific effect on the infarct tissue itself has not been fully clarified, which we examined in the present study. After MI induction in mice, treatment with the AT1 blocker olmesartan, beginning on the 3rd day post-MI, significantly improved survival (94%) 4 wk post-MI, compared with saline (53%) and hydralazine (73%). Olmesartan-treated mice also showed significant attenuation of left ventricular dilatation and dysfunction, as well as significantly greater infarct wall thickness, although the absolute size of the infarct scar was unchanged. In addition, significantly greater numbers of nonmyocytes (mainly vascular cells and myofibroblasts) were present within the infarct scar in olmesartan-treated hearts. Ten days post-MI, apoptosis among granulation tissue cells was significantly suppressed in the olmesartan-treated hearts, where expression of Fas, Bax, procaspase-3, and Daxx and activation of caspase-3, c-Jun NH2-terminal kinase, and c-Jun were all significantly attenuated. By contrast, expression of Fas ligand, Bcl-2, and Fas-associated death domain and activation of caspase-8 were unaffected, suggesting olmesartan exerts a negative regulatory effect on the alternate pathway downstream of Fas receptor. In vitro, olmesartan dose-dependently inhibited Fas-mediated apoptosis in granulation tissue-derived myofibroblasts. The present study proposes this antiapoptotic effect as another important mechanism for an AT1 blocker in improving post-MI ventricular remodeling, as well as its antifibrotic effect, and also suggests a significant link between renin-angiotensin and Fas/Fas ligand systems in postinfarction hearts.

A Critical Role for Thiol, But Not ATP, Depletion in 95% O2-Mediated Injury of Preterm Pneumocytes in Vitro

1994 ◽  
Vol 313 (1) ◽  
pp. 131-138 ◽  
Author(s):  
N.A. Christie ◽  
A.S. Slutsky ◽  
B.A. Freeman ◽  
A.K. Tanswell
Keyword(s):  
Critical Role ◽  
Atp Depletion

scholarly journals Fra-1 Induces Morphological Transformation and Increases In Vitro Invasiveness and Motility of Epithelioid Adenocarcinoma Cells

1998 ◽  
Vol 18 (12) ◽  
pp. 7095-7105 ◽  
Author(s):  
Olga Kustikova ◽  
Dmitrii Kramerov ◽  
Mariam Grigorian ◽  
Vladimir Berezin ◽  
Elisabeth Bock ◽  
...  
Keyword(s):  
Tumor Progression ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Morphological Changes ◽  
Critical Role ◽  
Morphological Transformation ◽  
Morphological Alterations ◽  
Nuclear Extracts

ABSTRACT Two cell lines originating from a common ancestral tumor, CSML0 and CSML100, were used as a model to study AP-1 transcription factors at different steps of tumor progression. CSML0 cells have an epithelial morphology; they express epithelial but not mesenchymal markers and are invasive neither in vitro nor in vivo. CSML100 possesses all characteristics of a highly progressive carcinoma. These cells do not form tight contacts, are highly invasive in vitro, and are metastatic in vivo. AP-1 activity was considerably higher in CSML100 cells than in CSML0 cells. There was a common predominant Jun component, namely, JunD, detected in both cell lines. We found that the enhanced level of AP-1 in CSML100 cells was due to high expression of Fra-1 and Fra-2 proteins, which were undetectable in CSML0 nuclear extracts. Analysis of the transcription of different AP-1 members in various cell lines derived from tumors of epithelial origin revealed a correlation of fra-1 expression with mesenchymal characteristics of carcinoma cells. Moreover, we show here for the first time that the expression of exogenous Fra-1 in epithelioid cells results in morphological changes that resemble fibroblastoid conversion. Cells acquire an elongated shape and become more motile and invasive in vitro. Morphological alterations were accompanied by transcriptional activation of certain genes whose expression is often induced at late stages of tumor progression. These data suggest a critical role of the Fra-1 protein in the development of epithelial tumors.

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