A lipid raft-associated 67 kDa laminin receptor mediates suppressive effect of epigallocatechin-3-O-gallate on FcεRI expression

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Abstract

(−)-Epigallocatechin-3-O-gallate (EGCG), a major green tea polyphenol, has previously exhibited a suppressive effect on the expression of the high-affinity IgE receptor (FcεRI). This effect has been shown to be elicited by interaction with the plasma membrane microdomain lipid rafts. Recently, we have identified the 67 kDa laminin receptor (67LR) as a cell surface EGCG receptor that mediates an anti-cancer action. Here we show that the 67LR is highly associated with lipid rafts on human basophilic KU812 cells. Experiments using 67LR-enhanced and -reduced cells revealed that the EGCG’s ability to downregulate FcεRI expression correlated with the amount of 67LR. Thus, these results suggest that the lipid raft-associated 67LR plays an important role in mediating the FcεRI-suppressive action of EGCG.

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Materials and methods

Reagents. EGCG was purchased from Kurita Water Industries (Tokyo, Japan). Mouse anti-human FcεRI α chain antibody CRA-1 was obtained from Kyokuto seiyaku (Tokyo, Japan). Mouse IgG2b, used as negative control, was bought from Dako Cytomaition (A/S, Denmark). Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody was purchased from Protos Immunoresearch (Burlingame, CA). Mouse anti-phosphorylated ERK1/2 antibody and rabbit anti-ERK1/2 antibody were obtained from Santa Cruz

67LR is extensively associated with the plasma membrane microdomain lipid rafts

Because the 67LR expression in human basophils has not yet been reported, we first examined the expression of 67LR on the cell surface of the human basophilic KU812 cells. Flow cytometric analysis using the 67LR antibody MLuC5 showed that KU812 cells expressed the 67LR on the cell surface (Fig. 1A). To further examine the association of 67LR with lipid rafts, a study of raft integrity was performed using MβCD, a cholesterol-removing agent that disturbs raft structure. After treatment with MβCD,

Discussion

The aim of the present study was to broaden the understanding of EGCG signaling, and a role of the 67LR in the EGCG-induced regulation of FcεRI expression and in the MAPK signaling pathway in human basophilic KU812 cells was examined. Among the many laminin-binding proteins, the 67LR is a high affinity non-integrin laminin receptor [21]. The interaction of cancer cells with laminin has been implicated in tumor metastasis and invasiveness, and the 67LR is thought to be involved in this process

Acknowledgments

This work was supported in part by Grants from Research and Development Program for New Bio-industry Initiatives (to H.T.). We are grateful to Dr. Takeshi Shimomura for helpful discussion. The authors thank Perry Seto for proofreading the manuscript.

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    Abbreviations: EGCG, (−)-epigallocatechin-3-O-gallate; 67LR, 67 kDa laminin receptor; siRNA, small interfering RNA; RNAi, RNA interference; ERK1/2, extracellular signal-regulated kinase 1/2; FcεRI, high-affinity IgE receptor; MAPK, mitogen-activated protein kinase; SPR, surface plasmon resonance; MβCD, methyl-β-cyclodextrin.

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