scholarly journals Nuclear signalling by Rac GTPase: essential role of phospholipase A2

1997 ◽  
Vol 326 (2) ◽  
pp. 333-337 ◽  
Author(s):  
Byung-Chul KIM ◽  
Jae-Hong KIM
Keyword(s):  
Phospholipase A2 ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Major Product ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Inhibitory Protein ◽  
Rac Gtpase

Rac, one member of Rho family GTPases, stimulates c-fos serum response element (SRE)–luciferase reporter gene in Rat-2 fibroblast cells. By transient transfection analysis, we demonstrated that the activation of phospholipase A2 (PLA2) and the subsequent production of arachidonic acid (AA) are essential for Rac-induced c-fos SRE activation, implying a critical role for PLA2 in the Rac-signalling pathway to the nucleus. Either pretreatment with mepacrine, a specific inhibitor of PLA2, or co-transfection with the expression plasmid of lipocortin-1, a proposed inhibitory protein of PLA2, selectively abolished RacV12-induced SRE activation. Further, we demonstrated that subsequent metabolism of AA, a major product of Rac-activated PLA2, by lipoxygenase (LO) is essential for Rac-induced c-fos SRE activation. In agreement with the role of the PLA2–AA–LO cascade as a potential mediator of Rac signalling to the nucleus, the addition of exogenous AA stimulated c-fos SRE-luciferase activity in an LO-dependent manner. Together, our results demonstrate that ‘Rac-activated PLA2 and subsequent AA metabolism by LO’ constitute a novel and specific pathway in Rac GTPase-induced c-fos SRE activation.

Pleiotropic role of VEGF-A in regulating fetal pulmonary mesenchymal cell turnover

2006 ◽  
Vol 290 (6) ◽  
pp. L1183-L1192 ◽  
Author(s):  
S. Majka ◽  
K. Fox ◽  
B. McGuire ◽  
J. Crossno ◽  
P. McGuire ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Critical Role ◽  
Subsequent Development ◽  
Mesenchymal Cell ◽  
Normal Lung ◽  
Vegf Receptor ◽  
Dependent Manner ◽  
Inhibitory Protein

Tight regulation of VEGF-A production and signaling is important for the maintenance of lung development and homeostasis. VEGF null mice have provided little insight into the role of VEGF during the later stages of lung morphogenesis. Therefore, we examined the in vitro effects of autocrine and paracrine VEGF-A production and the inhibition of VEGF-A signaling on a Flk-1-negative subset of fetal pulmonary mesenchymal cells (pMC). We hypothesized that VEGF-A receptor signaling regulates turnover of fetal lung mesenchyme in a cell cycle-dependent manner. VEGF receptor blockade with SU-5416 caused cell spreading and decreased proliferation and bcl-2 localization. Nuclear expression of the cell cycle inhibitory protein, p21, was increased with SU-5416 treatment, and p27 was absent. Autocrine VEGF production by pMC resulted in proliferation and p21/p27-dependent contact inhibition. In contrast, exogenous VEGF-A increased cell progression through the cell cycle. Selective activation of Flt by placental growth factor demonstrated the importance of this receptor/kinase in the VEGF-A responsiveness of pMC. The expression and localization of the survival factor bcl-2 was dependent on VEGF. These results provide evidence that VEGF-A plays a critical role in the regulation of fetal pulmonary mesenchymal proliferation, survival, and the subsequent development of normal lung architecture through bcl-2 and p21/p27-dependent cell cycle control.

scholarly journals Exogenous C2-ceramide activates c-fos serum response element via Rac-dependent signalling pathway

1998 ◽  
Vol 330 (2) ◽  
pp. 1009-1014 ◽  
Author(s):  
Byung-Chul KIM ◽  
Jae-Hong KIM
Keyword(s):  
Phospholipase A2 ◽  
Reporter Gene ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Response Element ◽  
Serum Response ◽  
Negative Mutant

Ceramide is an important regulatory molecule implicated in a variety of biological processes in response to stress and cytokines. To understand the signal transduction pathway of ceramide to the nucleus, in the present study, we examined whether C2-ceramide, a cell permeable ceramide, activates c-fos serum response element (SRE). Treatment of Rat-2 fibroblast cells with C2-ceramide caused the stimulation of c-fos SRE-dependent reporter gene activity in a dose- and time-dependent manner by transient transfection analysis. Next, we examined the role of Rho family GTPases in the ceramide-induced signalling to SRE activation. By reporter gene analysis following transient transfections with various plasmids expressing a dominant negative mutant form of Cdc42, Rac1 or RhoA, C2-ceramide-induced SRE activation was shown to be selectively repressed by pEXV-RacN17 encoding a dominant negative mutant of Rac1, suggesting that Rac activity is essential for the signalling cascade of ceramide to the nucleus. In a further study to analyse the downstream mediator of Rac in the ceramide-signalling pathway, we observed that either pretreatment with mepacrine, a potent and specific inhibitor of phospholipase A2, or co-transfection with antisense cytosolic phospholipase A2 (cPLA2) oligonucleotide repressed the C2-ceramide-induced SRE activation selectively, implying a critical role of cPLA2 in C2-ceramide-induced signalling to nucleus. Consistent with these results, the translocation of cPLA2 protein as well as the release of arachidonic acid, a principal product of phospholipase A2, was rapidly induced by the addition of C2-ceramide in a Rac-dependent manner. Together, our findings suggest the critical role of ‘Rac and subsequent activation of phospholipase A2’ in ceramide-signalling to nucleus.

scholarly journals The critical role of myeloperoxidase in Streptococcus pneumoniae clearance and tissue damage during mouse acute otitis media

2017 ◽  
Vol 23 (3) ◽  
pp. 296-306 ◽  
Author(s):  
Yun Xiang ◽  
Chunfang Jin ◽  
Wei Wang ◽  
Zimeng Wang ◽  
Yifei Huang ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Otitis Media ◽  
Tissue Damage ◽  
Early Stage ◽  
Critical Role ◽  
Acute Otitis Media ◽  
Specific Inhibitor ◽  
Dependent Manner ◽  
Innate Defense

We have recently reported that neutrophils play a pivotal role in innate defense against Streptococcus pneumoniae ( Spn) during mouse acute otitis media (AOM). However, the underlying mechanism remains unclear. By constructing models of pneumococcal AOM in C57BL/6 mice and using a specific inhibitor in vivo, we investigated the role of myeloperoxidase (MPO), one of the most important protein components of neutrophils. Experiment results showed a significant increase in MPO production of the recruited neutrophils in Spn-infected mice. Neutrophils killed Spn in a MPO-dependent manner. MPO facilitated the generation of reactive oxygen species (ROS), and consequently promoted Spn clearance at an early stage and exacerbated tissue damage. Moreover, MPO induced neutrophil apoptosis and necrosis, which, in turn, worsened tissue damage. In summary, our study demonstrates that neutrophil MPO plays a paradoxical role in bacterial clearance and tissue damage in pneumococcal AOM.

Exosomal miR-214-5p Released from Glioblastoma Cells Modulates Inflammatory Response of Microglia after Lipopolysaccharide Stimulation through Targeting CXCR5

2019 ◽  
Vol 18 (1) ◽  
pp. 78-87 ◽  
Author(s):  
Jian-kai Yang ◽  
Hong-jiang Liu ◽  
Yuanyu Wang ◽  
Chen Li ◽  
Ji-peng Yang ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Critical Role ◽  
Luciferase Reporter ◽  
Clinical Prognosis ◽  
Direct Target ◽  
And Migration ◽  
High Level ◽  
Cxcr5 Expression ◽  
Proliferation And Migration

Background and Objective: Exosomes communicate inter-cellularly and miRNAs play critical roles in this scenario. MiR-214-5p was implicated in multiple tumors with diverse functions uncovered. However, whether miR-214-5p is mechanistically involved in glioblastoma, especially via exosomal pathway, is still elusive. Here we sought to comprehensively address the critical role of exosomal miR-214-5p in glioblastoma (GBM) microenvironment.Methods:The relative expression of miR-214-5p was determined by real-time PCR. Cell viability and migration were measured by MTT and transwell chamber assays, respectively. The secretory cytokines were measured with ELISA kits. The regulatory effect of miR-214-5p on CXCR5 expression was interrogated by luciferase reporter assay. Protein level was analyzed by Western blot.Results:We demonstrated that miR-214-5p was aberrantly overexpressed in GBM and associated with poorer clinical prognosis. High level of miR-214-5p significantly contributed to cell proliferation and migration. GBM-derived exosomal miR-214-5p promoted inflammatory response in primary microglia upon lipopolysaccharide challenge. We further identified CXCR5 as the direct target of miR-214- 5p in this setting.Conclusion:Overexpression of miR-214-5p in GBM modulated the inflammatory response in microglia via exosomal transfer.

scholarly journals Sevoflurane inhibits proliferation, invasion, but enhances apoptosis of lung cancer cells by Wnt/β-catenin signaling via regulating lncRNA PCAT6/ miR-326 axis

2020 ◽  
Vol 15 (1) ◽  
pp. 159-172
Author(s):  
Guoning Su ◽  
Zhibing Yan ◽  
Min Deng
Keyword(s):  
Lung Cancer ◽  
Cancer Cells ◽  
Volatile Anesthetic ◽  
Lung Cancer Cells ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Western Blot Assay ◽  
Gene Assay ◽  
Cancer Tissues

AbstractSevoflurane was frequently used as a volatile anesthetic in cancer surgery. However, the potential mechanism of sevoflurane on lung cancer remains largely unclear. In this study, lung cancer cell lines (H446 and H1975) were treated by various concentrations of sevoflurane. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assessment and colony formation assay were performed to detect the cell viability and proliferation, separately. Also, transwell assay or flow cytometry assay was applied as well to evaluate the invasive ability or apoptosis in lung cancer cells, respectively. Western blot assay was employed to detect the protein levels of β-catenin and Wnt5a. Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) was used to examine the expression level of prostate cancer-associated transcript 6 (PCAT6) and miR-326 in lung cancer tissues and cells. The target interaction between miR-326 and PCAT6 or Wnt5a was predicted by bioinformatics analysis and verified by the dual-luciferase reporter gene assay. Sevoflurane inhibited the abilities on viability, proliferation, invasion, and activation of Wnt/β-catenin signaling, but promoted apoptosis of H446 and H1975 cells in a dose-dependent manner. The expression of PCAT6 was increased in lung cancer tissues and cells, except for that of miR-326. Besides, sevoflurane could lead to expressed limitation of PCAT6 or improvement of miR-326. This process presented a stepwise manner. Up-regulation of PCAT6 restored the suppression of sevoflurane on abilities of proliferation, invasion, rather than apoptosis, and re-activated the Wnt5a/β-catenin signaling in cells. Moreover, the putative binding sites between miR-326 and PCTA6 or Wnt5a were predicted by starBase v2.0 software online. PCAT6 suppressing effects on cells could be reversed by pre-treatment with miR-326 vector. The promotion of Wnt5a inverted effects led from miR-326 or sevoflurane. Our study indicated that sevoflurane inhibited the proliferation, and invasion, but enhanced the apoptosis in lung cancer cells by regulating the lncRNA PCAT6/miR-326/Wnt5a/β-catenin axis.

scholarly journals Knockdown of lncRNA X inactive specific transcript (XIST) radiosensitizes non-small cell lung cancer (NSCLC) cells through regulation of miR-16-5p/WEE1 G2 checkpoint kinase (WEE1) axis

2021 ◽  
Vol 35 ◽  
pp. 205873842096608
Author(s):  
Ran Du ◽  
Feng Jiang ◽  
Yanhua Yin ◽  
Jinfen Xu ◽  
Xia Li ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Western Blot ◽  
Small Cell ◽  
Luciferase Reporter ◽  
Small Cell Lung ◽  
Luciferase Reporter Gene ◽  
G2 Checkpoint ◽  
Qrt Pcr ◽  
Specific Transcript

Long non-coding RNA (lncRNA) X inactive specific transcript (XIST) is reported to play an oncogenic role in non-small cell lung cancer (NSCLC). However, the role of XIST in regulating the radiosensitivity of NSCLC cells remains unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expressions of XIST and miR-16-5p in NSCLC in tissues and cells, and Western blot was used to assess the expression of WEE1 G2 checkpoint kinase (WEE1). Cell counting kit-8 (CCK-8), colony formation and flow cytometry assays were used to determine cell viability and apoptosis after NSCLC cells were exposed to different doses of X-rays. The interaction between XIST and miR-16-5p was confirmed by StarBase database, qRT-PCR and dual-luciferase reporter gene assays. TargetScan database was used to predict WEE1 as a target of miR-16-5p, and their targeting relationship was further validated by Western blot, qRT-PCR and dual-luciferase reporter gene assays. XIST was highly expressed in both NSCLC tissue and cell lines, and knockdown of XIST repressed NSCLC cell viability and cell survival, and facilitated apoptosis under the irradiation. MiR-16-5p was a target of XIST, and rescue experiments demonstrated that miR-16-5p inhibitors could reverse the role of XIST knockdown on radiosensitivity in NSCLC cells. WEE1 was validated as a target gene of miR-16-5p, and WEE1 could be negatively regulated by XIST. XIST promotes the radioresistance of NSCLC cells by regulating the expressions of miR-16-5p and WEE1, which can be a novel target for NSCLC therapy.

scholarly journals The binding of NCAM to FGFR1 induces a specific cellular response mediated by receptor trafficking

2009 ◽  
Vol 187 (7) ◽  
pp. 1101-1116 ◽  
Author(s):  
Chiara Francavilla ◽  
Paola Cattaneo ◽  
Vladimir Berezin ◽  
Elisabeth Bock ◽  
Diletta Ami ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cellular Response ◽  
Critical Role ◽  
Biological Significance ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Fgf Receptor ◽  
Neural Cell Adhesion ◽  
Sustained Activation

Neural cell adhesion molecule (NCAM) associates with fibroblast growth factor (FGF) receptor-1 (FGFR1). However, the biological significance of this interaction remains largely elusive. In this study, we show that NCAM induces a specific, FGFR1-mediated cellular response that is remarkably different from that elicited by FGF-2. In contrast to FGF-induced degradation of endocytic FGFR1, NCAM promotes the stabilization of the receptor, which is recycled to the cell surface in a Rab11- and Src-dependent manner. In turn, FGFR1 recycling is required for NCAM-induced sustained activation of various effectors. Furthermore, NCAM, but not FGF-2, promotes cell migration, and this response depends on FGFR1 recycling and sustained Src activation. Our results implicate NCAM as a nonconventional ligand for FGFR1 that exerts a peculiar control on the intracellular trafficking of the receptor, resulting in a specific cellular response. Besides introducing a further level of complexity in the regulation of FGFR1 function, our findings highlight the link of FGFR recycling with sustained signaling and cell migration and the critical role of these events in dictating the cellular response evoked by receptor activation.

The P-element-induced silencing effect of KP transposons is dose dependent in Drosophila melanogaster

Genome ◽  
2011 ◽  
Vol 54 (9) ◽  
pp. 752-762 ◽  
Author(s):  
Alireza Sameny ◽  
John Locke
Keyword(s):  
Drosophila Melanogaster ◽  
Critical Role ◽  
Mutagenic Effect ◽  
P Element ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
P Elements ◽  
Single Well ◽  
Dose Dependent

Transposable elements are found in the genomes of all eukaryotes and play a critical role in altering gene expression and genome organization. In Drosophila melanogaster, transposable P elements are responsible for the phenomenon of hybrid dysgenesis. KP elements, a deletion-derivative of the complete P element, can suppress this mutagenic effect. KP elements can also silence the expression of certain other P-element-mediated transgenes in a process called P-element-dependent silencing (PDS), which is thought to involve the recruitment of heterochromatin proteins. To explore the mechanism of this silencing, we have mobilized KP elements to create a series of strains that contain single, well-defined KP insertions that show PDS. To understand the quantitative role of KP elements in PDS, these single inserts were combined in a series of crosses to obtain genotypes with zero, one, or two KP elements, from which we could examine the effect of KP gene dose. The extent of PDS in these genotypes was shown to be dose dependent in a logarithmic rather than linear fashion. A logarithmic dose dependency is consistent with the KP products interacting with heterochromatic proteins in a concentration-dependent manner such that two molecules are needed to induce gene silencing.

scholarly journals Critical Role of TLR2 and MyD88 for Functional Response of Macrophages to a Group IIA-Secreted Phospholipase A2 from Snake Venom

PLoS ONE ◽  
2014 ◽  
Vol 9 (4) ◽  
pp. e93741 ◽  
Author(s):  
Elbio Leiguez ◽  
Karina Cristina Giannotti ◽  
Vanessa Moreira ◽  
Márcio Hideki Matsubara ◽  
José María Gutiérrez ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Functional Response ◽  
Snake Venom ◽  
Critical Role ◽  
Secreted Phospholipase A2

scholarly journals Functional modulation of PTH1R activation and signalling by RAMP2

2021 ◽  
Author(s):  
Katarina Nemec ◽  
Hannes Schihada ◽  
Gunnar Kleinau ◽  
Ulrike Zabel ◽  
Eugene O. Grushevskyi ◽  
...  
Keyword(s):  
Homology Modelling ◽  
Critical Role ◽  
Ion Homeostasis ◽  
Optical Biosensors ◽  
Dependent Manner ◽  
Activated State ◽  
Class B ◽  
Receptor Activity Modifying Proteins ◽  
G Protein Coupled

Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein-coupled receptors (GPCRs) including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR, and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function. Using different optical biosensors to measure the activation of PTH1R and its downstream signalling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique pre-activated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signalling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signalling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered β-arrestin2 recruitment to PTH1R. Employing homology modelling we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signalling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.

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