J Korean Med Sci.  2005 Aug;20(4):548-554. 10.3346/jkms.2005.20.4.548.

Epigallocatechin-3-gallate Suppresses Galactose-alpha1,4-galactose-beta1,4-glucose Ceramide Expression in TNF-alpha Stimulated Human Intestinal Epithelial Cells Through Inhibition of MAPKs and NF-kappa B

Affiliations
  • 1Department of Microbiology and Immunology, and Medical Research Institute, and Laboratory of Dendritic Differentiation and Regulation, Pusan National University College of Medicine, Busan, Korea. immunpym@pusan.ac.kr
  • 2School of Applied Marine Science, College of Ocean Science, Cheju National University, Jeju, Korea.
  • 3Department of Microbiology, College of Natural Science, Pusan National University, Busan, Korea.

Abstract

Intestinal epithelial cells (IECs) have been known to produce galactose-alpha1,4-galactose-beta1,4-glucose ceramide (Gb3) that play an important role in the mucosal immune response. The regulation of Gb3 is important to prevent tissue damage causing shiga like toxin. Epigallocatechin-3-gallate (EGCG) has been studied as anti-carcinogenic, anti-oxidant, anti-angiogenic, and anti-viral activities, and anti-diabetic. However, little is known between the expressions of Gb3 on IECs. The aim of this study was to examine the inhibitory effect of EGCG, a major ingredient of green tea, on Gb3 production via mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappa B) in the TNF-alpha stimulated human colon epithelial cells, HT29. To investigate how Gb3 is regulated, ceramide glucosyltransferase (CGT), lactosylceramide synthase (GalT2), and Gb3 synthase (GalT6) were analyzed by RT-PCR in HT 29 cells exposed to TNF-alpha in the presence or absence of EGCG. EGCG dose-dependently manner, inhibits TNF-alpha induced Gb3 expression by blocking in both the MAPKs and NF-kappaB pathways in HT29 cells. TNF-alpha enhanced CGT, GalT2 and GalT6 mRNA levels and EGCG suppressed the level of these enzymes enhanced by TNF-alpha treatment.

Keyword

epigallocatechin gallate; Mitogen-Activated Protein Kinases; NF-kappa B; globotriaosylceramide; Enterocytes; Intestinal Mucosa

MeSH Terms

Apoptosis/drug effects
Blotting, Western
Catechin/*analogs & derivatives/pharmacology
Cell Nucleus/drug effects/metabolism
Cell Proliferation/drug effects
Dose-Response Relationship, Drug
Epithelial Cells/drug effects/metabolism/pathology
Flow Cytometry
Galactosyltransferases/genetics
Gene Expression Regulation, Enzymologic/drug effects
Glucosyltransferases/genetics
HT29 Cells
Humans
Intestinal Mucosa/drug effects/metabolism/pathology
Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism
NF-kappa B/antagonists & inhibitors/*metabolism
Phosphorylation/drug effects
Protein Transport/drug effects
RNA, Messenger/genetics/metabolism
Research Support, Non-U.S. Gov't
Reverse Transcriptase Polymerase Chain Reaction
Trihexosylceramides/*biosynthesis
Tumor Necrosis Factor-alpha/*pharmacology

Figure

  • Fig. 1 Flow cytometric analysis of HT29 cell death. The HT29 cells pretreated with EGCG (25 µM) were cultured for 48 hr with TNF-α (50 ng/mL) and were analyzed by flow cytometric analysis using annexin V-FITC and propidium iodide. LL quadrant: viable cells (annexin V and propidium iodide negative cells); LR quadrant: apoptotic cells (annexin V positive and propidium iodide negative cells); UR quadrant: dead cells (annexin V and propidium iodide positive cells). Numbering refers to the cell percentage of each population. Number of counted cells: 10,000.

  • Fig. 2 Effect of TNF-α on Gb3 induction in HT29 cells. (A) Dose-dependent effect of TNF-α on Gb3 production. TNF-α treated dose-dependently (10, 25, 50, and 100 ng/mL) for 2 days in HT29 cells. And time-dependent effect of TNF-α on Gb3 production. The cells were cultured with TNF-α (50 ng/mL) for 0, 12, 24, 48, and 72 hr. (B) The intensity was measured by densitometry. Isotype control represents the thick lines. The values are the mean±S.E. of duplicate determinations from three separate experiments. The sigmificance was determined by Student's t-test (*, p<0.05). MFI, mean fluorescence intensity.

  • Fig. 3 Effect of EGCG on Gb3 induction in TNF-α stimulated HT29 cells. (A) The HT29 cells pretreated with EGCG (10, 25, and 50 µM) were cultured for 2 days with TNF-α (50 ng/mL), and were analyzed by FACS. (B) The inhibitory effect of EGCG on Gb3 induction by TNF-α was shown in a dose-dependent manner. Isotype control represents the thin lines. Analysis of Gb3 content was carried out using FACS. MFI, mean fluorescence intensity.

  • Fig. 4 Effect of EGCG on ERK1/2, JNK1/2, and p38 phosphorylation in TNF-α-stimulated HT29 cells. (A) The HT29 cells pretreated with EGCG (25 µM) were cultured for 15 min with TNF-α (50 ng/mL), and were analyzed by Western blot using phospho-p38, phospho-JNK1/2 and phospho-ERK1/2 antibodies. (B) The intensity was measured by densitometry. The values are the mean±S.E. from three separate experiments (*p<0.05).

  • Fig. 5 Effect of EGCG on NF-κB p65 nuclear translocation in TNF-α-stimulated HT29 cells. (A) The EGCG (25 µM)-pretreated cells were stimulated for 2 hr with TNF-α (50 ng/mL), and then were analyzed by Western blot using the anti-NF-κB p65 antibody. (B) The optical density unit was measured by densitometry. The values are the mean±S.E. from three separate experiments (*p<0.05).

  • Fig. 6 Scheme of the Gb3 biosynthetic pathway. Depicted are the three enzymes and their respective products of the Gb3 pathway of eukaryotic cells (26).

  • Fig. 7 Effect of EGCG on CGT, GalT2, and GalT6 mRNA expression in TNF-α-stimulated HT29 cells. (A) The HT29 cells pretreated with EGCG (25 µM) were cultured for 24 hr with TNF-α (50 ng/mL), and were analyzed by RT-PCR for CGT, GalT2, GalT6, and α-galactosidase mRNA. (B) A PCR using housekeeping gene β-actin mRNA was carried out in parallel to confirm the equivalency of cDNA preparation. Relative intensity represents mRNA levels of CGT, GalT2, GalT6, and α-galactosidase/β-actin. The values are the mean±S.E. from three separate experiments (*p<0.01).

  • Fig. 8 TNF-α enhanced Gb3 content through MAPK phosphorylation and NF-κB activation, and that then the transcription factor of NF-κB induced the activity of all of the three enzymes involved in Gb3 glycosphingolipid precursor synthesis. EGCG suppresses Gb3 expression in a TNF-α stimulated HT-29 cells via inhibition of NF-κB. TRADD, TNF receptor-associated death domain; RIP, Receptor-interacting protein


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