Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454–3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase.

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The effect of in vitro exposure to antisense oligonucleotides on macrophage morphology and function

Journal of Nucleic Acids Investigation ◽

10.4081/jnai.2011.3515 ◽

2011 ◽

Vol 2 (1) ◽

pp. 12 ◽

Cited By ~ 4

Author(s):

Ann Brasey ◽

Raouf Igue ◽

Loubna Djemame ◽

Serge Séguin ◽

Paolo Renzi ◽

...

Keyword(s):

Antisense Oligonucleotides ◽

Morphological Changes ◽

Lower Airway ◽

Functional Responses ◽

Phagocytic Capacity ◽

Proinflammatory Mediators ◽

Functional Consequences ◽

And Function

Antisense oligonucleotides (AON) delivered via inhalation are in drug development for respiratory diseases. In rodents and monkeys, repeated exposure to high doses of inhaled phosphorothioate (PS) AON can lead to microscopic changes in the lungs, including accumulation of alveolar macrophages in the lower airway that have a foamy appearance. The functional consequences that result from this morphological change are unclear as there is controversy whether the vacuoles/inclusion bodies reflect normal clearance of the inhaled AON or are early indicators of lung toxicity. The morphological and functional responses of macrophage to PS AON were characterized in vitro using the comparator drug amiodarone, as a known inducer of foamy macrophages. Morphological changes of increased vacuolization with the presence of lamellated structures were observed in macrophages in response to both amiodarone and AON treatment. Functional responses to the drugs clearly differed with amiodarone treatment leading to apoptosis of cells and cell death, release of proinflammatory mediators IL-1RA, MIP-1α and TNFα, decrease in IP-10, a cytokine shown to be involved in protection against pulmonary fibrosis and altered phagocytosis capacity of the cells. In contrast, AON in concentrations up to 30 μM, had no effect on cell viability or apoptosis, had minimal effects on pro-inflammatory cytokines, increased IP-10 levels and did not alter the phagocytic capacity of the cells. Exposure of macrophages to AON in vitro, led to morphological changes of increased vacuolization, but did not lead to functional consequences which were observed with another vacuolization-inducing drug, suggesting that the in vivo phenotypic changes observed following inhalation of AON may be consistent with a clearance mechanism and not an activation or impairment of macrophages.

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Critical role for Epac1 in inflammatory pain controlled by GRK2-mediated phosphorylation of Epac1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1516036113 ◽

2016 ◽

Vol 113 (11) ◽

pp. 3036-3041 ◽

Cited By ~ 61

Author(s):

Pooja Singhmar ◽

XiaoJiao Huo ◽

Niels Eijkelkamp ◽

Susana Rojo Berciano ◽

Faiza Baameur ◽

...

Keyword(s):

Chronic Pain ◽

Molecular Mechanism ◽

Inflammatory Pain ◽

Critical Role ◽

Specific Inhibitor ◽

Mechanical Hyperalgesia ◽

Dependent Manner ◽

Phosphorylation Event

cAMP signaling plays a key role in regulating pain sensitivity. Here, we uncover a previously unidentified molecular mechanism in which direct phosphorylation of the exchange protein directly activated by cAMP 1 (EPAC1) by G protein kinase 2 (GRK2) suppresses Epac1-to-Rap1 signaling, thereby inhibiting persistent inflammatory pain. Epac1−/− mice are protected against inflammatory hyperalgesia in the complete Freund’s adjuvant (CFA) model. Moreover, the Epac-specific inhibitor ESI-09 inhibits established CFA-induced mechanical hyperalgesia without affecting normal mechanical sensitivity. At the mechanistic level, CFA increased activity of the Epac target Rap1 in dorsal root ganglia of WT, but not of Epac1−/−, mice. Using sensory neuron-specific overexpression of GRK2 or its kinase-dead mutant in vivo, we demonstrate that GRK2 inhibits CFA-induced hyperalgesia in a kinase activity-dependent manner. In vitro, GRK2 inhibits Epac1-to-Rap1 signaling by phosphorylation of Epac1 at Ser-108 in the Disheveled/Egl-10/pleckstrin domain. This phosphorylation event inhibits agonist-induced translocation of Epac1 to the plasma membrane, thereby reducing Rap1 activation. Finally, we show that GRK2 inhibits Epac1-mediated sensitization of the mechanosensor Piezo2 and that Piezo2 contributes to inflammatory mechanical hyperalgesia. Collectively, these findings identify a key role of Epac1 in chronic inflammatory pain and a molecular mechanism for controlling Epac1 activity and chronic pain through phosphorylation of Epac1 at Ser-108. Importantly, using the Epac inhibitor ESI-09, we validate Epac1 as a potential therapeutic target for chronic pain.

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β-Chemokines Enhance Parasite Uptake and Promote Nitric Oxide-Dependent Microbiostatic Activity in Murine Inflammatory Macrophages Infected with Trypanosoma cruzi

Infection and Immunity ◽

10.1128/iai.67.9.4819-4826.1999 ◽

1999 ◽

Vol 67 (9) ◽

pp. 4819-4826 ◽

Cited By ~ 101

Author(s):

Júlio C. S. Aliberti ◽

Fabiana S. Machado ◽

Janeusa T. Souto ◽

Ana P. Campanelli ◽

Mauro M. Teixeira ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Neutralizing Antibodies ◽

Specific Inhibitor ◽

No Production ◽

Dependent Manner ◽

Macrophage Infection ◽

Parasite Replication ◽

Ifn Γ

ABSTRACT In the present study, we describe the ability of Trypanosoma cruzi trypomastigotes to stimulate the synthesis of β-chemokines by macrophages. In vivo infection with T. cruzi led to MIP-1α, RANTES, and JE/MCP1 mRNA expression by cells from peritoneal inflammatory exudate. In addition, in vitro infection with T. cruzi resulted in expression of β-chemokine MIP-1α, MIP-1β, RANTES, and JE mRNA by macrophages. The expression of the β-chemokine MIP-1α, MIP-1β, RANTES, and JE proteins by murine macrophages cultured with trypomastigote forms ofT. cruzi was confirmed by immunocytochemistry. Interestingly, macrophage infection with T. cruzi also resulted in NO production, which we found to be mediated mainly by β-chemokines. Hence, treatment with anti-β-chemokine-specific neutralizing antibodies partially inhibited NO release by macrophages incubated with T. cruzi parasites. Further, the addition of the exogenous β-chemokines MIP-1α, MIP-1β, RANTES, and JE/MCP-1 induced an increased T. cruzi uptake, leading to enhanced NO production and control of parasite replication in a dose-dependent manner. l-NMMA, a specific inhibitor of thel-arginine–NO pathway, caused a decrease in NO production and parasite killing when added to cultures of macrophages stimulated with β-chemokines. Among the β-chemokines tested, JE was more potent in inhibiting parasite growth, although it was much less efficient than gamma interferon (IFN-γ). Nevertheless, JE potentiates parasite killing by macrophages incubated with low doses of IFN-γ. Together, these results suggest that in addition to their chemotactic activity, murine β-chemokines may also contribute to enhancing parasite uptake and promoting control of parasite replication in macrophages and may play a role in resistance to T. cruziinfection.

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Streptococcus gallolyticus Increases Expression and Activity of Aryl Hydrocarbon Receptor-Dependent CYP1 Biotransformation Capacity in Colorectal Epithelial Cells

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.740704 ◽

2021 ◽

Vol 11 ◽

Author(s):

Rahwa Taddese ◽

Rian Roelofs ◽

Derk Draper ◽

Xinqun Wu ◽

Shaoguang Wu ◽

...

Keyword(s):

Epithelial Cells ◽

Aryl Hydrocarbon Receptor ◽

Specific Inhibitor ◽

Intestinal Bacteria ◽

Opportunistic Pathogen ◽

Dependent Manner ◽

Aryl Hydrocarbon ◽

Streptococcus Gallolyticus

ObjectiveThe opportunistic pathogen Streptococcus gallolyticus is one of the few intestinal bacteria that has been consistently linked to colorectal cancer (CRC). This study aimed to identify novel S. gallolyticus-induced pathways in colon epithelial cells that could further explain how S. gallolyticus contributes to CRC development.Design and ResultsTranscription profiling of in vitro cultured CRC cells that were exposed to S. gallolyticus revealed the specific induction of oxidoreductase pathways. Most prominently, CYP1A and ALDH1 genes that encode phase I biotransformation enzymes were responsible for the detoxification or bio-activation of toxic compounds. A common feature is that these enzymes are induced through the Aryl hydrocarbon receptor (AhR). Using the specific inhibitor CH223191, we showed that the induction of CYP1A was dependent on the AhR both in vitro using multiple CRC cell lines as in vivo using wild-type C57bl6 mice colonized with S. gallolyticus. Furthermore, we showed that CYP1 could also be induced by other intestinal bacteria and that a yet unidentified diffusible factor from the S. galloltyicus secretome (SGS) induces CYP1A enzyme activity in an AhR-dependent manner. Importantly, priming CRC cells with SGS increased the DNA damaging effect of the polycyclic aromatic hydrocarbon 3-methylcholanthrene.ConclusionThis study shows that gut bacteria have the potential to modulate the expression of biotransformation pathways in colonic epithelial cells in an AhR-dependent manner. This offers a novel theory on the contribution of intestinal bacteria to the etiology of CRC by modifying the capacity of intestinal epithelial or (pre-)cancerous cells to (de)toxify dietary components, which could alter intestinal susceptibility to DNA damaging events.

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Envelope Gene-Mediated Neurovirulence in Feline Immunodeficiency Virus Infection: Induction of Matrix Metalloproteinases and Neuronal Injury

Journal of Virology ◽

10.1128/jvi.76.6.2622-2633.2002 ◽

2002 ◽

Vol 76 (6) ◽

pp. 2622-2633 ◽

Cited By ~ 24

Author(s):

J. B. Johnston ◽

C. Silva ◽

C. Power

Keyword(s):

Feline Immunodeficiency Virus ◽

Morphological Changes ◽

Neuronal Injury ◽

Envelope Gene ◽

Dependent Manner ◽

Mmp Inhibitor ◽

Feline Immunodeficiency Virus Infection ◽

Immunodeficiency Virus

ABSTRACT The release of neurotoxins by activated brain macrophages or microglia is one mechanism proposed to contribute to the development of neurological disease following infection by lentiviruses, including feline immunodeficiency virus (FIV). Since molecular diversity in the lentiviral envelope gene influences the expression of host molecules implicated in neuronal injury, the role of the envelope sequence in FIV neuropathogenesis was investigated by using the neurovirulent FIV strain V1CSF, the nonneurovirulent strain Petaluma, and a chimera (FIVCh) containing the V1CSF envelope gene in a Petaluma background. All three viruses replicated in primary feline macrophages with equal efficiency, but conditioned medium from V1CSF- or FIVCh-infected cells was significantly more neurotoxic than medium from Petaluma-infected cultures (P < 0.001) and could be attenuated in a dose-dependent manner by treatment with either the matrix metalloproteinase (MMP) inhibitor prinomastat (PMT) or function-blocking antibodies to MMP-2. Although FIV sequences were detectable by PCR in brain tissue from neonatal cats infected with each of the viral strains, immunohistochemistry revealed increased astrogliosis and macrophage activation in the brains of V1CSF- and FIVCh-infected cats relative to the other groups, together with elevated markers of neuronal stress that included morphological changes and increased c-fos immunoreactivity. Similarly, MMP-2, but not MMP-9, mRNA and protein expression was increased in brain tissues of V1CSF- and FIVCh-infected cats relative to Petaluma-infected animals (P < 0.01). Infection with V1CSF or FIVCh was also associated with greater CD4+ cell depletion (P < 0.001) and neurodevelopmental delays (P < 0.005), than in Petaluma-infected animals; these deficits improved following PMT therapy. These findings indicated that diversity in the envelope gene sequence influenced the neurovirulence exhibited by FIV both in vitro and in vivo, possibly through a mechanism involving the differential induction of MMP-2.

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Is Necroptosis a Death Pathway in Aluminum-Induced Neuroblastoma Cell Demise?

International Journal of Immunopathology and Pharmacology ◽

10.1177/039463200802100403 ◽

2008 ◽

Vol 21 (4) ◽

pp. 787-796 ◽

Cited By ~ 14

Author(s):

Q.L. Zhang ◽

Q. Niu ◽

X.L. Ji ◽

P. Conti ◽

P. Boscolo

Keyword(s):

Cell Death ◽

Morphological Changes ◽

Neuroblastoma Cell ◽

Specific Inhibitor ◽

Neural Cell ◽

Mitochondria Membrane Potential ◽

Cell Death Pathway ◽

Apoptosis And Necrosis

Besides being an aggravating factor secondary to major physiological alterations in degenerative diseases, aluminum has also been considered as a risk factor in the etiology. Although many in vivo and in vitro data are in favor of apoptosis and necrosis being involved in Al induced neurodegenerative processes, there is considerable evidence that very complex events may contribute to neural cell death. Necroptosis, a novel cell death pathway, was recently reported to contribute to ischemia brain injury. It is different from, but associated with, apoptosis and necrosis, the two common major pathways of cell demise. In the present study, SH-SY5Y cells were put under stress by Al, a potential degenerative cell death inducer. Nec-1, a specific inhibitor, was used to identify necroptosis. The characteristics observed in Nec-1 and Al treated SH-SY5Y cells showed that necrotic morphological changes were reduced, and a sharp decrease of necrotic rate was detected. Besides, there were Al-induced mitochondria membrane potential decreasing, reactive oxygen species remaining, and autophagosomes declining. The mechanism of Nec-1's effect on cell death may be related to caspases pathways. To our best knowledge, this is the pioneer report on necroptosis in mixed human neural cell death pathways, which might offer a novel therapeutic target for neurodegenerative diseases, and an extended window for neuroprotection.

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gp140v-fms molecules expressed at the surface of cells transformed by the McDonough strain of feline sarcoma virus are phosphorylated in tyrosine and serine.

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4745 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4745-4748 ◽

Cited By ~ 18

Author(s):

T Tamura ◽

E Simon ◽

H Niemann ◽

G T Snoek ◽

H Bauer

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Specific Inhibitor ◽

Dependent Manner ◽

Sodium Orthovanadate ◽

Transmembrane Glycoprotein ◽

Kinase C ◽

Sarcoma Virus

Cells transformed by the McDonough strain of feline sarcoma virus express at their surface a v-fms-specific transmembrane glycoprotein designated gp140v-fms. By labeling with 32Pi, gp140v-fms was shown to be phosphorylated 30-fold more in serine residues than were the cytosolic v-fms polypeptides gp180gag-fms and gp120v-fms. By using the phosphotyrosine phosphatase-specific inhibitor sodium orthovanadate, an additional tyrosine phosphorylation was observed in vivo, again involving predominantly gp140v-fms. In vitro studies showed that the v-fms proteins were phosphorylated by protein kinase C in a calcium- and phosphatidylserine-dependent manner.

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Repositioning Azelnidipine as a Dual Inhibitor Targeting CD47/SIRPα and TIGIT/PVR Pathways for Cancer Immuno-Therapy

Biomolecules ◽

10.3390/biom11050706 ◽

2021 ◽

Vol 11 (5) ◽

pp. 706

Author(s):

Xiuman Zhou ◽

Ling Jiao ◽

Yuzhen Qian ◽

Qingyu Dong ◽

Yixuan Sun ◽

...

Keyword(s):

T Cell ◽

Cancer Immunotherapy ◽

Cd8 T Cell ◽

Dependent Manner ◽

Dual Inhibitor ◽

Macrophage Phagocytosis ◽

Hypertensive Drug ◽

And Function

Strategies boosting both innate and adaptive immunity have great application prospects in cancer immunotherapy. Antibodies dual blocking the innate checkpoint CD47 and adaptive checkpoint PD-L1 or TIGIT could achieve durable anti-tumor effects. However, a small molecule dual blockade of CD47/SIRPα and TIGIT/PVR pathways has not been investigated. Here, an elevated expression of CD47 and PVR was observed in tumor tissues and cell lines analyzed with the GEO datasets and by flow cytometry, respectively. Compounds approved by the FDA were screened with the software MOE by docking to the potential binding pockets of SIRPα and PVR identified with the corresponding structural analysis. The candidate compounds were screened by blocking and MST binding assays. Azelnidipine was found to dual block CD47/SIRPα and TIGIT/PVR pathways by co-targeting SIRPα and PVR. In vitro, azelnidipine could enhance the macrophage phagocytosis when co-cultured with tumor cells. In vivo, azelnidipine alone or combined with irradiation could significantly inhibit the growth of MC38 tumors. Azelnidipine also significantly inhibits the growth of CT26 tumors, by enhancing the infiltration and function of CD8+ T cell in tumor and systematic immune response in the tumor-draining lymph node and spleen in a CD8+ T cell dependent manner. Our research suggests that the anti-hypertensive drug azelnidipine could be repositioned for cancer immunotherapy.

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Prognostic Value and Function of KLF5 in Papillary Thyroid Cancer

Cancers ◽

10.3390/cancers13020185 ◽

2021 ◽

Vol 13 (2) ◽

pp. 185

Author(s):

Poyil Pratheeshkumar ◽

Abdul K. Siraj ◽

Sasidharan Padmaja Divya ◽

Sandeep Kumar Parvathareddy ◽

Sarah Siraj ◽

...

Keyword(s):

Binding Sites ◽

Specific Inhibitor ◽

Papillary Thyroid ◽

Self Renewal ◽

Functional Correlation ◽

Xenograft Growth ◽

Molecular Therapeutic ◽

And Function

The Krüppel-like factor 5 (KLF5), a zinc-finger transcriptional factor, is highly expressed in several solid tumors, but its role in PTC remains unclear. We investigated the expression of KLF5 protein in a large cohort of PTC patient samples and explored its functional role and mechanism in PTC cell lines in vitro and in vivo. KLF5 overexpression was observed in 65.1% of all PTC cases and it was significantly associated with aggressive clinico-pathological parameters and poor outcome. Given the significant association between KLF5 and HIF-1α overexpression in PTC patients, we investigated the functional correlation between KLF5 and HIF-1α in PTC cells. Indeed, the analysis revealed the co-immunoprecipitation of KLF5 with HIF-1α in PTC cells. We also identified KLF5-binding sites in the HIF-1α promoter that specifically bound to KLF5 protein. Mechanistically, KLF5 promoted PTC cell growth, invasion, migration, and angiogenesis, while KLF5 downregulation via specific inhibitor or siRNA reverses its action in vitro. Importantly, the silencing of KLF5 decreases the self-renewal ability of spheroids generated from PTC cells. In addition, the depletion of KLF5 reduces PTC xenograft growth in vivo. These findings suggest KLF5 can be a possible new molecular therapeutic target for a subset of PTC.

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Farnesoid X Receptor Protects Liver Cells from Apoptosis Induced by Serum Deprivation in Vitro and Fasting in Vivo

Molecular Endocrinology ◽

10.1210/me.2007-0527 ◽

2008 ◽

Vol 22 (7) ◽

pp. 1622-1632 ◽

Cited By ~ 43

Author(s):

Yan-Dong Wang ◽

Fan Yang ◽

Wei-Dong Chen ◽

Xiongfei Huang ◽

Lily Lai ◽

...

Keyword(s):

Liver Cell ◽

Mapk Pathway ◽

Specific Inhibitor ◽

Serum Deprivation ◽

Farnesoid X Receptor ◽

Dependent Manner ◽

Induced Apoptosis

Abstract The farnesoid X receptor (FXR) is a key metabolic regulator in the liver by maintaining the homeostasis of liver metabolites. Recent findings suggest that FXR may have a much broader function in liver physiology and pathology. In the present work, we identify a novel role of FXR in protecting liver cell from apoptosis induced by nutritional withdrawal including serum deprivation in vitro or starvation in vivo. Two FXR ligands, chenodeoxycholic acid (CDCA) and GW4064, rescued HepG2 cells from serum deprivation-induced apoptosis in a dose-dependent manner. This effect of FXR on apoptotic suppression was compromised when FXR was knocked down by short interfering RNA. Similarly, the effects of both CDCA and GW4064 were abolished after inhibition of the MAPK pathway by a specific inhibitor of MAPK kinase 1/2. Immunoblotting results indicated that FXR activation by CDCA and GW4064 induced ERK1/2 phosphorylation, which was attenuated by serum deprivation. In vivo, FXR−/− mice exhibited an exacerbated liver apoptosis and lower levels of phosphorylated-ERK1/2 compared to wild-type mice after starvation. In conclusion, our results suggest a novel role of FXR in modulating liver cell apoptosis.

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