Expression of Tocopherol-Associated Protein in Mast Cells

Abstract

Tocopherol-associated protein (TAP) was expressed in mouse mast cells. TAP was predominantly localized in the cytoplasm, and the subcellular localization was not changed by ␣-tocopherol. The results suggest that the physiological role of TAP in mast cells is not regulation of tocopherol function but an as-yet-unidentified activity.In general, redox status modulates cellular functions. Oxidation stress activates cellular signaling, including the NF-B pathway and the mitogen-associated protein kinase pathway (2, 12). These reactions activate mast cells, leading to the release of inflammatory mediators, including tumor necrosis factor alpha (TNF-␣) and histamine (16,17). Vitamin E, a mixture of tocopherols and tocotrienols, is a lipid-soluble antioxidant that inhibits inflammatory reactions in vitro (12). Tocopherol-associated protein (TAP) is a cytosolic protein that associates with ␣-tocopherol with high affinity (19). Treatment with ␣-tocopherol resulted in nuclear translocation of TAP overexpressed in COS7 cells, and TAP acted as a transcriptional regulator in an ␣-tocopherol-dependent manner (18). These results suggest that TAP plays a crucial role in the intracellular metabolism and function of tocopherol (12). In the present paper, we report on the expression of TAP in mast cells. In contrast to the results obtained with COS7 cells (18), it was found that subcellular localization of endogenous TAP in mast cells is not regulated by ␣-tocopherol. This result suggests that the physiological role of TAP in mast cells is not regulation of tocopherol function but an as-yet-unidentified activity.Gene expression of a human TAP homologue in mouse mast cells was examined first. Total RNA was isolated from primary cultures of mouse mast cells, mouse mast cell lines, and mouse melanoma cells, and reverse transcription-PCR analyses were performed as described previously (10). PCR primers for the glyceraldehyde-3-phosphate dehydrogenase gene, a housekeeping gene, were described previously (3, 11). For the mouse TAP gene transcript (GenBank accession no. BC005759), oligonucleotides coding for nucleotide positions 758 through 778 and 1162 through 1141 were used as PCR primers. The PCR products were separated on 2% agarose gels in 1ϫ Tris-acetate EDTA buffer and visualized with ethidium bromide. Gene expression of TAP was detected in primary cultured cells, including bone marrow-derived cultured mast cell progenitors (BMCMCs) and cultured mast cells from mi/mi mice (Fig. 1A). In addition, TAP expression was detected in mast cell lines (MC/9, IC-2, and P-815) and melanoma cells (B-16), although the expression in MC/9 cells tended to be lower (Fig. 1A).To examine expression of TAP in mast cells at the protein level, antiserum directed against TAP was generated. A synthetic peptide spanning amino acids 1 to 11 of mouse TAP, coupled to keyhole limpet hemocyanin with glutaraldehyde, was administered to New Zealand White rabbits as described previously (4, 5). The reactivity of antiserum was monitored by enzyme-linked immunosorben...

Characterization of three human sec14p-like proteins: α-Tocopherol transport activity and expression pattern in tissues

“…However, two cell lines appeared to express higher levels of hTAP1 mRNA, the HMC-1 mastocytoma and the H295R human adrenocortical carcinoma cells. In line with the results of Ikeda et al [30], HMC-1 expressed mainly hTAP1 (Fig. 1A).…”

The Physiological Roles of Vitamin E and Hypovitaminosis E in the Transition Period of High-Yielding Dairy Cows

“…This report suggested that TAP might be associated with intracellular metabolism, non-antioxidative function, and the regulation of gene expression of α-Toc [ 92 ]. However, TAP, expressed in mouse mast cells, was predominantly localised in the cytoplasm and its subcellular localisation was not changed by α-Toc [ 93 ]. These results suggest that the physiological role of TAP in mast cells is not α-Toc-related, while as an α-Toc binding protein, TAP can promote α-Toc retention and thus increase its concentration in breast cancer cells [ 94 ].…”