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Abstract
Here we have investigated how lactosylceramide (LacCer) modulates gene expression of adhesion molecules in TNF-α and IFNγ (CM)-stimulated astrocytes. We have observed that stimulation of astrocytes with CM increased the gene expression of ICAM-1 and VCAM-1. D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and N-butyldeoxynojirimycin (NBDNJ), inhibitors of glucosylceramide synthase (GLS) and LacCer synthase (galactosyltransferase, GalT-2), inhibited the gene expression of ICAM-1 and VCAM-1 and activation of their gene promoter induced by CM, which were reversed by exogenously supplied LacCer. Silencing of GalT-2 gene using its antisense oligonucleotides also attenuated CM-induced ICAM-1 and VCAM-1 expression, which were reversed by LacCer. PDMP treatment and silencing of GalT-2 gene significantly reduced CM-induced luciferase activities in NF-κB, AP-1, GAS, and STAT-3 luciferase vectors-transfected cells. In addition, LacCer reversed the inhibition of NF-κB and STAT-1 luciferase activities by PDMP. Taken together, our results suggest that LacCer may play a crucial role in the expression of ICAM-1 and VCAM-1 via modulating transcription factors, such as NF-κB, AP-1, STAT-1, and STAT-3 in CM-stimulated astrocytes.
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Fig. 1.
The expression of ICAM-1 and VCAM-1 in CM-stimulated primary cultured astrocytes. The cells were treated cytokine mixture (CM), TNF-α (10 ng/ml) and IFNγ (10 ng/ml). Total RNA and protein were isolated from 1, 3, 6, 9, 12, 24 and 48 hr in CM-treated astrocytes. Gene expression of ICAM-1 (A) and VCAM-1 (B) were measured by quantitative real-time PCR using Rotor-Gene Q. The expression of each gene was normalized with GAPDH gene expression. Protein levels of ICAM-1 (C) and VCAM-1 (D) were examined by Western blot analysis. Data were obtained from triplicated PCR reactions of three different cultures, and values are mean±SD (∗∗p<0.01, ∗∗∗p<0.001, vehicle vs. CM).
Fig. 2.
Lactosylceramide mediates the expression of ICAM-1 in CM-stimulated astrocytes. The cells were pretreated with PDMP (A) and NBDNJ (B) and LacCer for 0.5 hr before CM. Gene expression of ICAM-1 (A∼C; top) was measured by quantitative real-time PCR at 6 hr after CM stimulation. The expression of each gene was normalized with GAPDH gene expression. For the silencing of GalT-2 gene (C), the cells were transfected with AS-GalT-2 and scrambled DNA oligomer (scramble). At 48 hr after transfection with AS and scrambled oligonucleotides, the cells were stimulated with CM and LacCer. Protein levels of ICAM-1 (A∼C; bottom) was examined by Western blot analysis at 24 hr after CM stimulation. (D) At 24 hr after transient transfection of cells with ICAM-1 promoter-luciferase gene construct, the cells were pretreated with PDMP for 0.5 hr before stimulation with CM. The cellular luciferase activity was measured. All graph indicate mean±SD (A: ∗∗p<0.01, CM vs. CM+PDMP, ++p<0.01, CM+PDMP vs. CM+PDMP+LacCer; B: ∗p<0.05, CM vs. CM+NBDNJ, ++p<0.01, CM+NBDNJ vs. CM+NBDNJ+LacCer; C: ∗p<0.05, scramble-CM vs. AS-GalT-2-CM, +p<0.05, AS-GalT-2-CM vs. AS-GalT-2-CM+LacCer; D: ∗∗∗p<0.001, CM vs. CM+PDMP, +++p<0.001, CM+PDMP vs. CM+PDMP+LacCer, n=3 independent experiments).
Fig. 3.
Lactosylceramide mediates the expression of VCAM-1 in CM-stimulated astrocytes. The cells were pretreated with PDMP (A) and NBDNJ (B) and LacCer for 0.5 hr before CM. Gene expression of VCAM-1 (A∼C; top) was measured by quantitative real-time PCR at 6 hr after CM stimulation. The expression of each gene was normalized with GAPDH gene expression. For the silencing of GalT-2 gene (C), the cells were transfected with AS-GalT-2 and scrambled DNA oligomer (scramble). At 48 hr after transfection with AS and scrambled oligonucleotides, the cells were stimulated with CM and LacCer. Protein levels of VCAM-1 (A∼C; bottom) was examined by Western blot analysis at 24 hr after CM stimulation. (D) At 24 hr after transient transfection of cells with VCAM-1 promoter-luciferase gene construct, the cells were pretreated with PDMP for 0.5 hr before stimulation with CM. The cellular luciferase activity was measured. All graph indicate mean±SD (A: ∗∗∗p<0.001, CM vs. CM+PDMP, +++p<0.001, CM+PDMP vs. CM+PDMP+LacCer; B: ∗∗p<0.01, CM vs. CM+NBDNJ, +p<0.05, CM+NBDNJ vs. CM+NBDNJ+LacCer; C: ∗∗∗p<0.001, scramble-CM vs. AS-GalT-2-CM, +++p<0.001, AS-GalT-2-CM vs. AS-GalT-2-CM+LacCer; D: ∗∗∗p<0.001, CM vs. CM+PDMP, +++p<0.001, CM+PDMP vs. CM+PDMP+LacCer, n=3 independent experiments).
Fig. 4.
Inhibition of GalT-2 attenuates the activation of transcription factors in CM-stimulated astrocytes. At 24 hr after transient transfection of cells with NF-κB (A), AP-1 (C), GAS (E), and STAT-3 (G) luciferase reporter vectors, the cells were pretreated with PDMP (10 and 25 μM) for 0.5 hr before stimulation with CM. For the silencing of GalT-2 gene, the cells were co-transfected with AS-GalT-2 (or scramble) and NF-κB (B), AP-1 (D), GAS (F), and STAT-3 (H) luciferase reporter vectors. At 48 hr after transfection with AS-GalT-2 (or scramble) and luciferase reporter vectors the cells were stimulated with CM. The cellular luciferase activity was measured. All graph indicate mean±S.D (A, C, E, G: ∗∗∗p<0.001, vehicle vs. CM, ++p<0.01, +++p <0.001, CM vs. CM+ PDMP, B, D, F, H: ∗∗∗p<0.001, vehicle vs. CM in scramble-transfected group, +++p<0.001, scamble+CM vs. AS- GalT-2+CM, n=3 independent experiments).
Fig. 5.
Lactosylceramide mediates the activation of NF-kB and STAT-1 in CM-stimulated astrocytes. At 24 hr after transient transfection of cells with NF-kB (A) and GAS (B) luciferase reporter vectors, cells were pretreated with PDMP (20μM) and LacCer (5μM) for 0.5 hr before CM. The cellular luciferase activity was measured as described in Materials and Methods. All graph indicate mean±SD (∗∗∗p<0.001, CM vs. CM+PDMP, +p<0.05, CM+PDMP vs. CM+PDMP+LacCer, n=3 independent experiments).
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