Caveolin-1 Regulates P2Y2 Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma

Caveolae-associated protein caveolin-1 (Cav-1) plays key roles in cellular processes such as mechanosensing, receptor coupling to signaling pathways, cell growth, apoptosis, and cancer. In 1321N1 astrocytoma cells Cav-1 interacts with the P2Y2 receptor (P2Y2R) to modulate its downstream signaling. P2Y2R and its signaling machinery also mediate pro-survival actions after mechanical injury. This study determines if Cav-1 knockdown (KD) affects P2Y2R signaling and its pro-survival actions in the 1321N1 astrocytoma cells mechanical injury model system. KD of Cav-1 decreased its expression in 1321N1 cells devoid of or expressing hHAP2Y2R by ~88% and ~85%, respectively. Cav-1 KD had no significant impact on P2Y2R expression. Post-injury densitometric analysis of pERK1/2 and Akt activities in Cav-1-positive 1321N1 cells (devoid of or expressing a hHAP2Y2R) revealed a P2Y2R-dependent temporal increase in both kinases. These temporal increases in pERK1/2 and pAkt were significantly decreased in Cav-1 KD 1321N1 (devoid of or expressing a hHAP2Y2R). Cav-1 KD led to an ~2.0-fold and ~2.4-fold decrease in the magnitude of the hHAP2Y2R-mediated pERK1/2 and pAkt kinases’ activity, respectively. These early-onset hHAP2Y2R-mediated signaling responses in Cav-1-expressing and Cav-1 KD 1321N1 correlated with changes in cell viability (via a resazurin-based method) and apoptosis (via caspase-9 expression). In Cav-1-positive 1321N1 cells, expression of hHAP2Y2R led to a significant increase in cell viability and decreased apoptotic (caspase-9) activity after mechanical injury. In contrast, hHAP2Y2R-elicited changes in viability and apoptotic (caspase-9) activity were decreased after mechanical injury in Cav-1 KD 1321N1 cells expressing hHAP2Y2R. These findings support the importance of Cav-1 in modulating P2Y2R signaling during mechanical injury and its protective actions in a human astrocytoma cell line, whilst shedding light on potential new venues for brain injury or trauma interventions.

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Caveolin-1 Regulates the P2Y2Receptor Signaling in Human 1321N1 Astrocytoma Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m116.730226 ◽

2016 ◽

Vol 291 (23) ◽

pp. 12208-12222 ◽

Cited By ~ 11

Author(s):

Namyr A. Martinez ◽

Alondra M. Ayala ◽

Magdiel Martinez ◽

Freddyson J. Martinez-Rivera ◽

Jorge D. Miranda ◽

...

Keyword(s):

Caveolin 1 ◽

Astrocytoma Cells ◽

1321N1 Astrocytoma

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Direct Effect of Septic Plasma in Human Cell Lines Viability

Blood Purification ◽

10.1159/000494597 ◽

2018 ◽

Vol 47 (1-3) ◽

pp. 270-276

Author(s):

Grazia Maria Virzì ◽

Chiara Borga ◽

Chiara Pasqualin ◽

Silvia Pastori ◽

Alessandra Brocca ◽

...

Keyword(s):

Cytochrome C ◽

Cell Viability ◽

Cell Lines ◽

Caspase 3 ◽

Test Group ◽

Organ Injury ◽

Enzyme Linked Immunosorbent Assay ◽

Caspase 9 ◽

Renal Tubular Cells

Background: Sepsis is a life-threatening condition often associated with a high incidence of multiple organs injury. Several papers suggested the immune response by itself, with the production of humoral inflammatory mediators, is crucial in determining organ injury. However, little is known of how sepsis directly induces organ injury at the cellular levels. To assess this point, we set up an in vitro study to investigate the response of renal tubular cells (RTCs), monocytes (U937) and hepatocytes (HepG2) after 24 h-incubation with septic patients’ plasma. Methods: We enrolled 26 septic patients (“test” group). We evaluated cell viability, apoptosis and necrosis by flow cytometer. Caspase-3,-8,-9 and cytochrome-c concentrations have been analyzed using the Human enzyme-linked immunosorbent assay kit. Results: We found that a decrease of cell viability in all cell lines tested was associated to the increase of apoptosis in RTCs and U937 (p < 0.0001) and increase of necrosis in HepG2 (p < 0.5). The increase of apoptosis in RTCs and U937 cells was confirmed by higher levels of caspase-3 (p < 0.0001). We showed that apoptosis in both RTCs and U937 was triggered by the activation of the intrinsic pathway, as caspase-9 and cytochrome-c levels significantly increased (p < 0.0001), while caspase-8 did not change. This assumption was strengthened by the significant correlation of caspase-9 with both cytochrome-c (r = 0.73 for RTCs and r = 0.69 for U937) and caspase-3 (r = 0.69 for RTCs and r = 0.63 for U937). Conclusion: Humoral mediators in septic patients’ plasma induce apoptosis. This fact suggests that apoptosis inhibitors should be investigated as future strategy to reduce sepsis-induced organ damages.

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Red nucleus IL-33 facilitates the early development of mononeuropathic pain in male rats by inducing TNF-α through activating ERK, p38 MAPK, and JAK2/STAT3

Journal of Neuroinflammation ◽

10.1186/s12974-021-02198-9 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Hao-Nan Li ◽

Qing-Qing Yang ◽

Wen-Tao Wang ◽

Xue Tian ◽

Fan Feng ◽

...

Keyword(s):

P38 Mapk ◽

Early Development ◽

Red Nucleus ◽

Neutralizing Antibody ◽

Male Rats ◽

Post Injury ◽

Pain Model ◽

Mechanical Hypersensitivity ◽

Downstream Signaling ◽

Tnf Α

Abstract Background Our recent studies have identified that the red nucleus (RN) dual-directionally modulates the development and maintenance of mononeuropathic pain through secreting proinflammatory and anti-inflammatory cytokines. Here, we further explored the action of red nucleus IL-33 in the early development of mononeuropathic pain. Methods In this study, male rats with spared nerve injury (SNI) were used as mononeuropathic pain model. Immunohistochemistry, Western blotting, and behavioral testing were used to assess the expressions, cellular distributions, and actions of red nucleus IL-33 and its related downstream signaling molecules. Results IL-33 and its receptor ST2 were constitutively expressed in the RN in naive rats. After SNI, both IL-33 and ST2 were upregulated significantly at 3 days and peaked at 1 week post-injury, especially in RN neurons, oligodendrocytes, and microglia. Blockade of red nucleus IL-33 with anti-IL-33 neutralizing antibody attenuated SNI-induced mononeuropathic pain, while intrarubral administration of exogenous IL-33 evoked mechanical hypersensitivity in naive rats. Red nucleus IL-33 generated an algesic effect in the early development of SNI-induced mononeuropathic pain through activating NF-κB, ERK, p38 MAPK, and JAK2/STAT3, suppression of NF-κB, ERK, p38 MAPK, and JAK2/STAT3 with corresponding inhibitors markedly attenuated SNI-induced mononeuropathic pain or IL-33-evoked mechanical hypersensitivity in naive rats. Red nucleus IL-33 contributed to SNI-induced mononeuropathic pain by stimulating TNF-α expression, which could be abolished by administration of inhibitors against ERK, p38 MAPK, and JAK2/STAT3, but not NF-κB. Conclusions These results suggest that red nucleus IL-33 facilitates the early development of mononeuropathic pain through activating NF-κB, ERK, p38 MAPK, and JAK2/STAT3. IL-33 mediates algesic effect partly by inducing TNF-α through activating ERK, p38 MAPK and JAK2/STAT3.

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Hyperphosphorylated FAK Delocalizes from Focal Adhesions to Membrane Ruffles

Journal of Oncology ◽

10.1155/2010/932803 ◽

2010 ◽

Vol 2010 ◽

pp. 1-10 ◽

Cited By ~ 11

Author(s):

Abdelkader Hamadi ◽

Therese B. Deramaudt ◽

Kenneth Takeda ◽

Philippe Rondé

Keyword(s):

Tumor Metastasis ◽

Focal Adhesions ◽

Downstream Signaling ◽

Astrocytoma Cells ◽

Src Inhibitor ◽

Human Astrocytoma ◽

Cell Adhesion And Migration ◽

And Migration ◽

Downstream Signaling Pathway ◽

Human Astrocytoma Cells

Cell adhesion and migration are key determinants in tumor metastasis. Adherence of tumor cell to the extracellular matrix is mediated via integrin containing focal adhesions (FAs). Binding of integrins to ECM triggers phosphorylation of two major components of FAs, focal adhesion kinase (FAK) and Src, activating downstream signaling pathway which leads to FA disassembly and cell migration. In this paper, we analyze how phosphorylation of FAK regulates its trafficking at FAs in living human astrocytoma cells. Upon pervanadate-induced FAK phosphorylation, phosphorylated FAK appeared highly expressed at newly formed membrane ruffles. This effect was abolished in presence of the specific Src inhibitor PP2. Our findings demonstrate that upon phosphorylation, FAK delocalizes from FAs to membrane ruffles.

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Exosomes from Adipose-Derived Mesenchymal Stem Cells Overexpressing Stanniocalcin-1 Promote Reendothelialization after Carotid Endarterium Mechanical Injury

10.21203/rs.3.rs-115771/v1 ◽

2020 ◽

Author(s):

Kun Liu ◽

Huihua Shi ◽

Zhiyou Peng ◽

Xiaoyu Wu ◽

Weimin Li ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Mesenchymal Stem Cells ◽

Mechanical Injury ◽

Molecular Probe ◽

Blue Staining ◽

Control Group ◽

Lateral Migration ◽

Post Injury

Abstract Background and objective: Endothelial cell inflammation caused by mechanical injury of endovascular treatment remains the major obstacle to reendothelialization, which leads to arterial restenosis. We investigated the reendothelialization effect of exosomes from adipose-derived mesenchymal stem cells (ADSC) overexpressing Stanniocalcin-1 (STC-1).Methods: Primary ADSCs were extracted from the adipose tissue of the inguinal area of C57/BL mice. ADSCs were transfected with lentivirus vectors containing STC-1. Exosomes were purified from culture medium using the Exo-Quick kit and characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot. PHK-26 as molecular probe was used to track the exosomes engulfed by mice arterial endothelial cells (MAEC). The role of STC-1-ADSC-Exosome (S-ADSC-Exo) in MAECs was verified through scratch test and tube forming experiment. Carotid endarterium mechanical injury was induced by insertion with a guidewire into the common carotid artery lumen. Exosomes were administered by tail vein injection. Content of Reactive oxygen species (ROS) was measured using commercial kits. Carotid arteries were harvested for histological examination, immunofluorescence staining, and Evan’s blue staining.Results: Transfection of STC-1 significantly enhanced STC-1 levels in ADSCs, their exosomes, and MAECs. Compared with the control group and the ADSC-Exo group, STC-1 enriched exosomes markedly enhanced STC-1 level, inhibited the expression of NLRP3, Caspase-1, and IL-1β in MAECs, exhibited good lateral migration capacity, and promoted angiogenesis. Exosome-pretreating groups exhibited lower levels of ROS than those of controls. In vivo administration of S-ADSC-Exo had reendothelialization effect on post-injury carotid endarterium as evidenced by thinner arterial wall, low-expressed NLRP3, and more living endothelial cells.Conclusions: The reendothelialization effect of exosomes from ADSCs on post-injury carotid endarterium can be enhanced by genetic modification to contain elevated STC-1.

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Intramolecular Diels-Alder Cycloaddition: 7-Isocyanoamphilecta- 11(20),15-diene (Miyaoka), (–)-Scabronine G (Kanoh), Basiliolide B (Stoltz), Hirsutellone B (Uchiro), Echinopine A (Chen)

Organic Synthesis ◽

10.1093/oso/9780190200794.003.0078 ◽

2015 ◽

Author(s):

Douglass F. Taber

Keyword(s):

Neurotrophic Factors ◽

Claisen Rearrangement ◽

Diels Alder ◽

Relative Configuration ◽

Intramolecular Cycloaddition ◽

Quaternary Centers ◽

Astrocytoma Cells ◽

National University ◽

Seoul National University ◽

1321N1 Astrocytoma

The amphilectane diterpenes, exemplified by 7-isocyanoamphilecta-11(20),15-diene 3, have been little investigated. In the course of a synthesis of 3, Hiroaki Miyaoka of the Tokyo University of Pharmacy and Life Sciences took advantage (Synlett 2011, 547) of the kinetic enolization and silylation of 1 to convert it into a trienone that spontaneously cyclized to 2. Scabronine G 6, isolated from the mushroom Sarcodon scabrosus, was found to enhance the secretion of neurotrophic factors from 1321N1 astrocytoma cells. To set the absolute configuration of the two quaternary centers that are 1, 4 on the cyclohexane ring of 6, Naoki Kanoh and Yoshiharu Iwabuchi of Tohoku University cyclized (Org. Lett. 2011, 13, 2864) 4 to 5. Although described by the authors as a double Michael addition, this transformation has the same connectivity as an intramolecular Diels-Alder cycloaddition. The diterpenes isolated from the genus Thapsia, represented by basiliolide B 9, induce rapid mobilization of intracellular Ca2+ stores. Brian M. Stoltz of Caltech effected (Angew. Chem. Int. Ed. 2011, 50, 3688) Claisen rearrangement of 7 to give an intermediate that cyclized to 8 as a mixture of diastereomers. A significant challenge in the synthesis was the assembly of the delicate enol ether/lactone of 9. Hirsutellone B 12, isolated from Hirsutella nivea, shows significant antituberculosis activity. Hiromi Uchiro of the Tokyo University of Science found it useful (Org. Lett. 2011, 13, 6268) to protect the intermediate unsaturated keto ester by intermolecular cycloaddition with pentamethylcyclopentadiene before constructing the triene of 10. Simple thermolysis reversed the intermolecular addition, opening the way to intramolecular cycloaddition to give 11. The tetracyclic ring system of the diterpene echinopine A 15 represents a substantial synthetic challenge. David Y.-K. Chen of Seoul National University approached this problem (Org. Lett. 2011, 13, 5724) by Pd-mediated cyclization of 13 to the diene, which then underwent intramolecular Diels-Alder cycloaddition to give 14, with control of the relative configuration of two of the three ternary centers of 15. Double bond migration followed by oxidative cleavage of the resulting cyclohexenone then set the stage for the intramolecular cyclopropanation that completed the synthesis of 15.

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Thrombin receptors modulate insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate accumulation in 1321N1 astrocytoma cells

Biochemical Journal ◽

10.1042/bj3170347 ◽

1996 ◽

Vol 317 (2) ◽

pp. 347-351 ◽

Cited By ~ 25

Author(s):

Ian H. BATTY ◽

C. Peter DOWNES

Keyword(s):

Insulin Receptors ◽

Thrombin Receptor ◽

Astrocytoma Cells ◽

Thrombin Receptors ◽

Transient Activation ◽

Ligand Type ◽

Insulin Receptor Signalling ◽

Human Thrombin ◽

Tethered Ligand ◽

1321N1 Astrocytoma

Thrombin and insulin receptor signalling via phosphoinositide (PI)-specific phospholipase C (PLC) and PI 3-kinase was studied in [3H]inositol-labelled 1321N1 cells. Thrombin stimulated a dramatic, transient activation of PLC which is probably mediated via receptors of the ‘tethered-ligand’ type, since it was both reproduced by, and abolished following, pretreatment of cells with a synthetic peptide (SFLLRN) corresponding to the ligand domain of the human thrombin receptor. However, neither thrombin nor SFLLRN stimulated PI 3-kinase. By contrast, insulin did not influence [3H]InsP3 concentrations but stimulated accumulation of [3H]PtdIns(3,4,5)P3 and [3H]PtdIns(3,4)P2, the relative steady-state concentrations of which may indicate degradation of [3H]PtdIns(3,4,5)P3 by 5- and 3-phosphatases. The independent coupling of thrombin and insulin receptors to PLC and PI 3-kinase respectively in 1321N1 cells allowed interactions between these systems to be examined. Thus insulin-stimulated [3H]PtdIns(3,4,5)P3 accumulation was attenuated on co-stimulation of the thrombin receptor, whereas concentrations of [3H]PtdIns(3,4)P2 were transiently enhanced but then reduced. These results indicate that thrombin receptors in 1321N1 cells do not activate PI 3-kinase, but can modulate signalling by this enzyme.

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Dexmedetomidine attenuates lipopolysaccharide induced acute lung injury in rats by inhibition of caveolin-1 downstream signaling

Biomedicine & Pharmacotherapy ◽

10.1016/j.biopha.2019.109314 ◽

2019 ◽

Vol 118 ◽

pp. 109314 ◽

Cited By ~ 4

Author(s):

Jing Liu ◽

Xinhua Huang ◽

Siping Hu ◽

Huanzhong He ◽

Zhipeng Meng

Keyword(s):

Acute Lung Injury ◽

Lung Injury ◽

Caveolin 1 ◽

Downstream Signaling

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Critical role of caveolin-1 in aflatoxin B1-induced hepatotoxicity via the regulation of oxidation and autophagy

Cell Death and Disease ◽

10.1038/s41419-019-2197-6 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 5

Author(s):

Qingqiang Xu ◽

Wenwen Shi ◽

Pan Lv ◽

Wenqi Meng ◽

Guanchao Mao ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Viability ◽

Aflatoxin B1 ◽

Cell Apoptosis ◽

Critical Role ◽

Hepatic Cell ◽

Hepatocellular Injury ◽

Caveolin 1 ◽

Induced Apoptosis

AbstractAflatoxin B1 (AFB1) is a potent hepatocarcinogen in humans and exposure to AFB1 is known to cause both acute and chronic hepatocellular injury. As the liver is known to be the main target organ of aflatoxin, it is important to identify the key molecules that participate in AFB1-induced hepatotoxicity and to investigate their underlying mechanisms. In this study, the critical role of caveolin-1 in AFB1-induced hepatic cell apoptosis was examined. We found a decrease in cell viability and an increase in oxidation and apoptosis in human hepatocyte L02 cells after AFB1 exposure. In addition, the intracellular expression of caveolin-1 was increased in response to AFB1 treatment. Downregulation of caveolin-1 significantly alleviated AFB1-induced apoptosis and decreased cell viability, whereas overexpression of caveolin-1 reversed these effects. Further functional analysis showed that caveolin-1 participates in AFB1-induced oxidative stress through its interaction with Nrf2, leading to the downregulation of cellular antioxidant enzymes and the promotion of oxidative stress-induced apoptosis. In addition, caveolin-1 was found to regulate AFB1-induced autophagy. This finding was supported by the effect that caveolin-1 deficiency promoted autophagy after AFB1 treatment, leading to the inhibition of apoptosis, whereas overexpression of caveolin-1 inhibited autophagy and accelerated apoptosis. Interestingly, further investigation showed that caveolin-1 participates in AFB1-induced autophagy by regulating the EGFR/PI3K-AKT/mTOR signaling pathway. Taken together, our data reveal that caveolin-1 plays a crucial role in AFB1-induced hepatic cell apoptosis via the regulation of oxidation and autophagy, which provides a potential target for the development of novel treatments to combat AFB1 hepatotoxicity.

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