PMC:2674207 / 28251-34270 JSON TXT

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2_test

{"project":"2_test","denotations":[{"id":"19436703-15496417-86274497","span":{"begin":213,"end":215},"obj":"15496417"},{"id":"T59134","span":{"begin":213,"end":215},"obj":"15496417"}],"text":"Effect on MKP-1\nDexamethasone inhibits p38 MAPK function in a cell type–specific manner through the rapid induction of the dual kinase phosphatase MKP-1 (MAPK phosphatase-1), and this effect lasts for up to 24 h [28]. FP (10−8 M) treatment of HuT-78 cells activated by anti-CD3/CD28 in vitro significantly decreased p38 MAPK phosphorylation (Figure 3A) and activity measured by phosphorylation of the downstream target ATF-2 (Figure 3B). This effect was detected at 30 min and lasted for at least 14 h (Figure 3B). FP (10−8 M) also significantly reduced GATA-3 serine phosphorylation induced by anti-CD3/CD28 stimulation in both a time- and concentration-dependent manner (Figure 3C). This reduction in GATA-3 phosphorylation was also seen with lower concentrations of FP. We found that FP significantly induced MKP-1 mRNA in both a time- and concentration-dependent manner, reaching a plateau at 10−8 M after 10 min (Figure 3D and 3E). However, the effects of FP on GATA-3 nuclear import, importin-α association and IL-4 mRNA expression are seen at 10,000-fold lower concentrations (10−12 M, see Figure 2).\n10.1371/journal.pmed.1000076.g003 Figure 3 Fluticasone propionate–mediated inhibition of p38 MAP kinase phosphorylation and activation is associated with a marked down-regulation of GATA-3 serine phosphorylation.\n(A) Western blot analysis shows that FP (10−8 M, 30 min) treatment reduced dual phosphorylation (threonine-180 and tyrosine-182) of p38 MAPK in anti-CD3/CD28–co-stimulated HuT-78 cells. (B) Time course of the effect of FP (10−8 M) on phosphorylation of activated transcription factor 2 (ATF-2), a measure of p38 MAPK activity. (C) FP-induced inhibition of p38 MAPK activity is associated with the decrease of anti-CD3/CD28 co-stimulation–induced serine phosphorylation (P-Ser) of GATA-3. For (A–C), quantification of the densitometry data is also shown. Each bar represents mean±SEM of at least three independent experiments. ### p\u003c0.001 compared to control, ***p\u003c0.001 compared to αCD3/CD28-stimulated cells. (D) FP induced MKP-1 mRNA in a concentration-dependent manner. All results are representative of at least three independent experiments and where appropriate expressed as means±SEM, *p\u003c0.05. (E) FP induces MKP-1 mRNA in a time-dependent manner. Results are representative of two independent experiments. All data except (E) were analysed by ANOVA followed by Newman-Keuls post-test. Using an in vitro competition assay (Figure 4A) utilizing purified activated GATA-3, importin-α, and activated GR, we demonstrated that activated GR significantly increased GR-importin-α association in the presence and absence of activated GATA-3 (Figure 4B). This effect is not mutual, since activated GATA-3 did not block GR–importin-α association (Figure 4C). These data also suggest that both activated GR and phospho-GATA-3 can directly associate with importin-α (Figure 4D) and that activated GR attenuates the phospho-GATA-3/importin-α interaction in a concentration-dependent manner (Figure 4E). Together, this suggests that ligand-activated GR may compete with phospho-GATA-3 for importin-α and thereby limit GATA-3 nuclear import.\n10.1371/journal.pmed.1000076.g004 Figure 4 Fluticasone propionate competes with phospho-GATA-3 for importin-α.\n(A) schematic representation of the in vitro binding competition assay. (B) GR isolated from FP (10−8 M) stimulated cells enhances GR–importin-α binding in the presence (•) and absence (▪) of activated GATA-3. * p\u003c0.05 compared to no activated GR. (C) GATA-3 isolated from anti-CD3/CD28–stimulated cells does not attenuate GR–importin-α association. *p\u003c0.05 compared to control. (D) Activated GR blocks the ability of purified phospho-GATA-3 isolated from anti-CD3/CD28–stimulated cells interacting with immobilised importin-α in an in vitro binding assay. *p\u003c0.05 compared to GATA-3 isolated from unstimulated cells. # p\u003c0.05 compared to stimulated GATA-3-importin binding. (E) The effect of activated (•) versus unstimulated (○) GR on attenuation of GATA-3–importin-α association was concentration-dependent. *p\u003c0.05, **p\u003c0.01 between groups. All results are expressed as mean±SEM of three independent experiments and analysed by ANOVA followed by Newman-Keuls post-test. Other possible interpretations of our results could include an effect of FP on GATA-3 nuclear export and/or degradation. Leptomycin B, which inhibits nuclear export, did not affect the ability of FP to block GATA-3 nuclear localization (Figure 5A). Additionally, FP had no effect on whole cell GATA-3 expression during the time course of these experiments (Figure 5B). Nor did addition of FP subsequent to anti-CD3/CD28 nuclear translocation affect GATA-3 nuclear residency (Figure 5C), suggesting that activated GR does not enhance GATA-3 nuclear export. Finally, the effect of FP on GATA-3 nuclear import was not nonspecific, since FP (10−8 M) had no effect on p65 nuclear translocation measured at 60 min (Figure 5D).\n10.1371/journal.pmed.1000076.g005 Figure 5 Fluticasone propionate does not affect GATA-3 nuclear export.\n(A) Western blot analysis showing that the nuclear export inhibitor leptomycin B (2 nM) does not affect the ability of FP (10−8 M) to prevent anti-CD3/CD28–stimulated GATA-3 nuclear localization measured at 60 min. ***p\u003c0.001 compared to unstimulated cells, ### p\u003c0.001 compared to anti-CD3/CD28–stimulated cells. (B) Western blot analysis showing that FP (10−8 M) does not affect whole-cell GATA-3 degradation over 17 h. (C) GFP-tagged GATA-3 is overexpressed and cells stimulated (b–j) or not (a) with anti-CD3/CD28. The effect of treating cells with FP (10−8 M, f–j) after 30 min stimulation with anti-CD3/CD28 is also shown. (D) FP (10−8 M) does not prevent anti-CD3/CD28–stimulated p65 nuclear translocation at 60 min after stimulation. **p\u003c0.01 compared to unstimulated cells. All results are representative of at least four independent experiments and are shown as mean±SEM. Results were analysed by ANOVA followed by Newman-Keuls test.\n\nT"}

pmc-enju-pas

bionlp-st-ge-2016-spacy-parsed

GO-BP

GO-MF

GO-CC

{"project":"GO-CC","denotations":[{"id":"T18542","span":{"begin":3386,"end":3391},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T18543","span":{"begin":3568,"end":3573},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T18544","span":{"begin":3751,"end":3756},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T18545","span":{"begin":3881,"end":3886},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T10715","span":{"begin":62,"end":66},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T10716","span":{"begin":4535,"end":4539},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T18126","span":{"begin":1501,"end":1506},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19161","span":{"begin":5324,"end":5329},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19162","span":{"begin":5380,"end":5385},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19163","span":{"begin":5538,"end":5543},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19164","span":{"begin":5615,"end":5620},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19165","span":{"begin":5849,"end":5854},"obj":"http://purl.obolibrary.org/obo/GO_0005623"}],"text":"Effect on MKP-1\nDexamethasone inhibits p38 MAPK function in a cell type–specific manner through the rapid induction of the dual kinase phosphatase MKP-1 (MAPK phosphatase-1), and this effect lasts for up to 24 h [28]. FP (10−8 M) treatment of HuT-78 cells activated by anti-CD3/CD28 in vitro significantly decreased p38 MAPK phosphorylation (Figure 3A) and activity measured by phosphorylation of the downstream target ATF-2 (Figure 3B). This effect was detected at 30 min and lasted for at least 14 h (Figure 3B). FP (10−8 M) also significantly reduced GATA-3 serine phosphorylation induced by anti-CD3/CD28 stimulation in both a time- and concentration-dependent manner (Figure 3C). This reduction in GATA-3 phosphorylation was also seen with lower concentrations of FP. We found that FP significantly induced MKP-1 mRNA in both a time- and concentration-dependent manner, reaching a plateau at 10−8 M after 10 min (Figure 3D and 3E). However, the effects of FP on GATA-3 nuclear import, importin-α association and IL-4 mRNA expression are seen at 10,000-fold lower concentrations (10−12 M, see Figure 2).\n10.1371/journal.pmed.1000076.g003 Figure 3 Fluticasone propionate–mediated inhibition of p38 MAP kinase phosphorylation and activation is associated with a marked down-regulation of GATA-3 serine phosphorylation.\n(A) Western blot analysis shows that FP (10−8 M, 30 min) treatment reduced dual phosphorylation (threonine-180 and tyrosine-182) of p38 MAPK in anti-CD3/CD28–co-stimulated HuT-78 cells. (B) Time course of the effect of FP (10−8 M) on phosphorylation of activated transcription factor 2 (ATF-2), a measure of p38 MAPK activity. (C) FP-induced inhibition of p38 MAPK activity is associated with the decrease of anti-CD3/CD28 co-stimulation–induced serine phosphorylation (P-Ser) of GATA-3. For (A–C), quantification of the densitometry data is also shown. Each bar represents mean±SEM of at least three independent experiments. ### p\u003c0.001 compared to control, ***p\u003c0.001 compared to αCD3/CD28-stimulated cells. (D) FP induced MKP-1 mRNA in a concentration-dependent manner. All results are representative of at least three independent experiments and where appropriate expressed as means±SEM, *p\u003c0.05. (E) FP induces MKP-1 mRNA in a time-dependent manner. Results are representative of two independent experiments. All data except (E) were analysed by ANOVA followed by Newman-Keuls post-test. Using an in vitro competition assay (Figure 4A) utilizing purified activated GATA-3, importin-α, and activated GR, we demonstrated that activated GR significantly increased GR-importin-α association in the presence and absence of activated GATA-3 (Figure 4B). This effect is not mutual, since activated GATA-3 did not block GR–importin-α association (Figure 4C). These data also suggest that both activated GR and phospho-GATA-3 can directly associate with importin-α (Figure 4D) and that activated GR attenuates the phospho-GATA-3/importin-α interaction in a concentration-dependent manner (Figure 4E). Together, this suggests that ligand-activated GR may compete with phospho-GATA-3 for importin-α and thereby limit GATA-3 nuclear import.\n10.1371/journal.pmed.1000076.g004 Figure 4 Fluticasone propionate competes with phospho-GATA-3 for importin-α.\n(A) schematic representation of the in vitro binding competition assay. (B) GR isolated from FP (10−8 M) stimulated cells enhances GR–importin-α binding in the presence (•) and absence (▪) of activated GATA-3. * p\u003c0.05 compared to no activated GR. (C) GATA-3 isolated from anti-CD3/CD28–stimulated cells does not attenuate GR–importin-α association. *p\u003c0.05 compared to control. (D) Activated GR blocks the ability of purified phospho-GATA-3 isolated from anti-CD3/CD28–stimulated cells interacting with immobilised importin-α in an in vitro binding assay. *p\u003c0.05 compared to GATA-3 isolated from unstimulated cells. # p\u003c0.05 compared to stimulated GATA-3-importin binding. (E) The effect of activated (•) versus unstimulated (○) GR on attenuation of GATA-3–importin-α association was concentration-dependent. *p\u003c0.05, **p\u003c0.01 between groups. All results are expressed as mean±SEM of three independent experiments and analysed by ANOVA followed by Newman-Keuls post-test. Other possible interpretations of our results could include an effect of FP on GATA-3 nuclear export and/or degradation. Leptomycin B, which inhibits nuclear export, did not affect the ability of FP to block GATA-3 nuclear localization (Figure 5A). Additionally, FP had no effect on whole cell GATA-3 expression during the time course of these experiments (Figure 5B). Nor did addition of FP subsequent to anti-CD3/CD28 nuclear translocation affect GATA-3 nuclear residency (Figure 5C), suggesting that activated GR does not enhance GATA-3 nuclear export. Finally, the effect of FP on GATA-3 nuclear import was not nonspecific, since FP (10−8 M) had no effect on p65 nuclear translocation measured at 60 min (Figure 5D).\n10.1371/journal.pmed.1000076.g005 Figure 5 Fluticasone propionate does not affect GATA-3 nuclear export.\n(A) Western blot analysis showing that the nuclear export inhibitor leptomycin B (2 nM) does not affect the ability of FP (10−8 M) to prevent anti-CD3/CD28–stimulated GATA-3 nuclear localization measured at 60 min. ***p\u003c0.001 compared to unstimulated cells, ### p\u003c0.001 compared to anti-CD3/CD28–stimulated cells. (B) Western blot analysis showing that FP (10−8 M) does not affect whole-cell GATA-3 degradation over 17 h. (C) GFP-tagged GATA-3 is overexpressed and cells stimulated (b–j) or not (a) with anti-CD3/CD28. The effect of treating cells with FP (10−8 M, f–j) after 30 min stimulation with anti-CD3/CD28 is also shown. (D) FP (10−8 M) does not prevent anti-CD3/CD28–stimulated p65 nuclear translocation at 60 min after stimulation. **p\u003c0.01 compared to unstimulated cells. All results are representative of at least four independent experiments and are shown as mean±SEM. Results were analysed by ANOVA followed by Newman-Keuls test.\n\nT"}

sentences

events-check-again

bionlp-st-ge-2016-reference-tees

bionlp-st-ge-2016-reference

bionlp-st-ge-2016-uniprot

test2

PMC:2674207 / 28251-34270 JSONTXT

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PMC:2674207 / 28251-34270 JSON TXT