This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ikeda, T.
Right arrow Articles by Funaba, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ikeda, T.
Right arrow Articles by Funaba, M.

 Previous Article  |  Next Article 

Clinical and Diagnostic Laboratory Immunology, November 2004, p. 1189-1191, Vol. 11, No. 6
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.6.1189-1191.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Expression of Tocopherol-Associated Protein in Mast Cells

Teruo Ikeda,1 Masaru Murakami,2 and Masayuki Funaba3*

Azabu University Research Institute of Biosciences,1 Molecular Biology,2 Nutrition, Azabu University School of Veterinary Medicine, Sagamihara, Japan3

Received 19 May 2004/ Accepted 23 July 2004


arrow
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 {alpha}-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.


arrow
TEXT
 
In general, redox status modulates cellular functions. Oxidation stress activates cellular signaling, including the NF-{kappa}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-{alpha}) 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 {alpha}-tocopherol with high affinity (19). Treatment with {alpha}-tocopherol resulted in nuclear translocation of TAP overexpressed in COS7 cells, and TAP acted as a transcriptional regulator in an {alpha}-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 {alpha}-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 1x 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).



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 1. Expression and subcellular localization of TAP in mast cells. (A) Reverse transcription-PCR analyses were performed using RNA from primary cultures of mast cells (BMCMCs and cultured mast cells from mi/mi mice [mi/mi CMCs]), mast cell lines (MC/9, IC-2, and P-815), and melanocytes (B-16). Expression of TAP and the glyceraldehyde-3-phosphate dehydrogenase gene, a housekeeping gene, was examined. (–), negative control. (B) Antiserum against TAP was characterized. COS7 cells were transiently transfected with empty vector or HA-tagged TAP. The cell lysates were subjected to Western blot analyses by use of anti-HA (12CA5) antibody (left panel) or anti-TAP antibody (right panel). (C) Subcellular localization of TAP in BMCMCs was examined by immunofluorescence analyses. BMCMCs were treated (lower panel) or not treated (upper panel) with 50 µM {alpha}-tocopherol for 24 h. (D) The effects of {alpha}-tocopherol and LPS on TNF-{alpha} concentration in culture supernatant of BMCMCs were examined. BMCMCs were pretreated with {alpha}-tocopherol at the indicated concentration for 24 h, followed by LPS treatment. At 24 h after the start of treatment, TNF-{alpha} concentrations of the culture supernatant were measured. *, P < 0.05; **, P < 0.01.

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 immunosorbent assay. The antiserum was characterized by Western blot analyses as described previously (4, 5). For this purpose, a TAP cDNA fragment cut by BamHI and SalI was subcloned into the BamHI and XhoI sites of hemagglutinin (HA)-pcDNA3 (9) to produce C-terminal HA-tagged protein. The plasmid or empty vector (HA-pcDNA3) was transiently transfected into COS7 cells, and at 48 h of transfection, cell lysates were collected as described previously (6). Western blot analyses using anti-TAP antiserum of cell lysates of COS7 cells overexpressing TAP revealed a significant band of the expected size (Fig. 1B, right panel). This band corresponded to that detected with anti-HA antibody (Fig. 1B, left panel), indicating the applicability of the antiserum to further analyses.

Subcellular localization of TAP was examined in BMCMCs by indirect immunofluorescence analyses. TAP was predominantly localized in the cytoplasm (Fig. 1C, upper panel), and treatment with {alpha}-tocopherol (50 µM) for 24 h hardly affected the subcellular localization (Fig. 1C, lower panel). Preincubation of the antiserum with excess synthetic peptide spanning amino acids 1 to 11 abolished significant staining of TAP (data not shown). We also examined the TAP localization in BMCMCs treated with {alpha}-tocopherol at different doses and for different durations, but the subcellular localization of TAP was not altered (data not shown). Furthermore, nuclear translocation of TAP by {alpha}-tocopherol was not observed in HepG2 cells, a hepatoma cell line (data not shown). Thus, we conclude that the subcellular distribution of endogenous TAP is not regulated by {alpha}-tocopherol. These observations contrast with the results obtained by Yamauchi et al. (18), which showed nuclear translocation of overexpressed TAP in response to {alpha}-tocopherol. A possible explanation is that the large amounts of the protein produced in COS7 cells may cause {alpha}-tocopherol-induced nuclear translocation of TAP.

The unchanged subcellular distribution of TAP in response to {alpha}-tocopherol could result from the insensitivity of BMCMCs to {alpha}-tocopherol. To examine this possibility, the effects of {alpha}-tocopherol on lipopolysaccharide (LPS)-induced TNF-{alpha} release were examined in BMCMCs. After pretreatment with {alpha}-tocopherol for 24 h, BMCMCs were stimulated with various doses of LPS. At 24 h of stimulation, the TNF-{alpha} concentration in culture supernatant was measured with a commercial enzyme-linked immunosorbent assay kit (Biosource International, Camarillo, Calif.). Consistent with the previous study (8), the TNF-{alpha} concentration in the culture supernatant was increased by LPS stimulation in a dose-dependent manner (Fig. 1D). Treatment with 5 µM {alpha}-tocopherol increased the TNF-{alpha} concentration in the culture supernatant of BMCMCs. In addition, pretreatment with {alpha}-tocopherol also enhanced the LPS-induced increase in TNF-{alpha} concentration in the culture supernatant, although the effects of {alpha}-tocopherol on TNF-{alpha} concentration with lower doses of LPS are not evident. Overall analysis of variance, including {alpha}-tocopherol, LPS, and the interaction of {alpha}-tocopherol and LPS as the main effects (13), revealed a significant effect of {alpha}-tocopherol (P < 0.05), and Duncan's multiple-range test revealed significant increases in TNF-{alpha} release by pretreatments with {alpha}-tocopherol (5 and 50 µM).

Gueck et al. (7) reported that {alpha}-tocopherol decreased histamine release in mast cells, which was calculated as the ratio of the percentage of histamine in culture supernatant to the percentage of total histamine present in both supernatant and cell pellets. In their study, however, the histamine content of mast cells was also increased by {alpha}-tocopherol treatment, suggesting that {alpha}-tocopherol stimulates the production of histamine (17). The enhanced TNF-{alpha} release by {alpha}-tocopherol may reflect an increased synthesis of inflammatory mediators in mast cells. Although the precise reason for this remains to be clarified, our results clearly showed that BMCMCs are sensitive to {alpha}-tocopherol. Therefore, the result that TAP is localized in the cytoplasm in BMCMCs irrespective of {alpha}-tocopherol treatment suggests different roles of TAP in intracellular metabolism and in the function of {alpha}-tocopherol in mast cells. This is supported by the following findings. (i) TAP is identical to supernatant protein factor, which stimulates squalene monooxygenase, a downstream enzyme in the cholesterol de novo biosynthetic pathway (14). (ii) Activation of squalene monooxygenase is dependent on the phosphorylation state of TAP (15). (iii) TAP is also homologous to Saccharomyces cerevisiae SEC14p, which acts as a phosphatidylinositol transfer protein (1). Further studies are needed to clarify the physiological role of TAP in mast cells.


arrow
ACKNOWLEDGMENTS
 
We thank Naohiro Inohara and Takeyoshi Koseki for providing plasmids.

This work was supported by a Grant-in-Aid for Scientific Research (15580268) from the Japan Society for the Promotion of Science (to M.F.) and grants for graduate schools from the Foundation for Japanese Private School Promotion (to T.I., M.M., and M.F.).


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Nutrition, Azabu University School of Veterinary Medicine, 1-17-71 Fuchinobe, Sagamihara 229-8501, Japan. Phone: 81-42-754-7111(276). Fax: 81-42-754-9930. E-mail: funaba{at}azabu-u.ac.jp. Back


arrow
REFERENCES
 
    1
  1. Bankaitis, V. A., J. R. Aitken, A. E. Cleves, and W. Dowhan. 1990. An essential role for a phospholipid transfer protein in yeast Golgi function. Nature 347:561-562.[CrossRef][Medline]
    2. 2
  2. Bowie, A., and L. A. O'Neill. 2000. Oxidative stress and nuclear factor-kappaB activation: a reassessment of the evidence in the light of recent discoveries. Biochem. Pharmacol. 59:13-23.[CrossRef][Medline]
    3. 3
  3. Funaba, M., T. Ikeda, K. Ogawa, M. Murakami, and M. Abe. 2003. Role of activin A in murine mast cells: modulation of cell growth, differentiation, and migration. J. Leukoc. Biol. 73:793-801.[Abstract/Free Full Text]
    4. 4
  4. Funaba, M., T. Murata, H. Fujimura, E. Murata, M. Abe, M. Takahashi, and K. Torii. 1996. Unique recognition of activin and inhibin by polyclonal antibodies to inhibin subunits. J. Biochem. (Tokyo) 119:953-960.[Abstract/Free Full Text]
    5. 5
  5. Funaba, M., T. Murata, H. Fujimura, E. Murata, M. Abe, and K. Torii. 1997. Immunolocalization of type I or type II activin receptors in the rat brain. J. Neuroendocrinol. 9:105-111.[CrossRef][Medline]
    6. 6
  6. Funaba, M., T. Ikeda, M. Murakami, K. Ogawa, K. Tsuchida, H. Sugino, and M. Abe. 2003. Transcriptional activation of mouse mast cell protease-7 by activin and transforming growth factor-beta is inhibited by microphthalmia-associated transcription factor. J. Biol. Chem. 278:52032-52041.[Abstract/Free Full Text]
    7. 7
  7. Gueck, T., J. R. Aschenbach, and H. Fuhrmann. 2002. Influence of vitamin E on mast cell mediator release. Vet. Dermatol. 13:301-305.[CrossRef][Medline]
    8. 8
  8. Ikeda, T., and M. Funaba. 2003. Altered function of murine mast cells in response to lipopolysaccharide and peptidoglycan. Immunol. Lett. 88:21-26.[CrossRef][Medline]
    9. 9
  9. Inohara, N., T. Koseki, S. Chen, X. Wu, and G. Nunez. 1998. CIDE, a novel family of cell death activators with homology to the 45 kDa subunit of the DNA fragmentation factor. EMBO J. 17:2526-2533.[CrossRef][Medline]
    10. 10
  10. Murakami, M., T. Ikeda, K. Ogawa, and M. Funaba. 2003. Transcriptional activation of mouse mast cell protease-9 by microphthalmia-associated transcription factor. Biochem. Biophys. Res. Commun. 311:4-10.[CrossRef][Medline]
    11. 11
  11. Ogawa, K., M. Funaba, L. S. Mathews, and T. Mizutani. 2000. Activin A stimulates type IV collagenase (matrix metalloproteinase-2) production in mouse peritoneal macrophages. J. Immunol. 165:2997-3003.[Abstract/Free Full Text]
    12. 12
  12. Rimbach, G., A. M. Minihane, J. Majewicz, A. Fischer, J. Pallauf, F. Virgli, and P. D. Weinberg. 2002. Regulation of cell signalling by vitamin E. Proc. Nutr. Soc. 61:415-425.[CrossRef][Medline]
    13. 13
  13. SAS. 1996. SAS/STAT user's guide, release 6.11. SAS Institute Inc., Cary, N.C.
    14. 14
  14. Shibata, N., M. Arita, Y. Misaki, N. Dohmae, K. Takio, T. Ono, K. Inoue, and H. Arai. 2001. Supernatant protein factor, which stimulates the conversion of squalene to lanosterol, is a cytosolic squalene transfer protein and enhances cholesterol biosynthesis. Proc. Natl. Acad. Sci. USA 98:2244-2249.[Abstract/Free Full Text]
    15. 15
  15. Singh, D. K., V. Mokashi, C. L. Elmore, and T. D. Porter. 2003. Phosphorylation of supernatant protein factor enhances its ability to stimulate microsomal squalene monooxygenase. J. Biol. Chem. 278:5646-5651.[Abstract/Free Full Text]
    16. 16
  16. Williams, C. M., and S. J. Galli. 2000. The diverse potential effector and immunoregulatory roles of mast cells in allergic disease. J. Allergy Clin. Immunol. 105:847-859.[CrossRef][Medline]
    17. 17
  17. Wolfreys, K., and D. B. Oliveira. 1997. Alterations in intracellular reactive oxygen species generation and redox potential modulate mast cell function. Eur. J. Immunol. 27:297-306.[Medline]
    18. 18
  18. Yamauchi, J., T. Iwamoto, S. Kida, S. Masushige, K. Yamada, and T. Esashi. 2001. Tocopherol-associated protein is a ligand-dependent transcriptional activator. Biochem. Biophys. Res. Commun. 285:295-299.[CrossRef][Medline]
    19. 19
  19. Zimmer, S., A. Stocker, M. N. Sarbolouki, S. E. Spycher, J. Sassoon, and A. Azzi. 2000. A novel human tocopherol-associated protein: cloning, in vitro expression, and characterization. J. Biol. Chem. 275:25672-25680.[Abstract/Free Full Text]

Clinical and Diagnostic Laboratory Immunology, November 2004, p. 1189-1191, Vol. 11, No. 6
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.6.1189-1191.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ikeda, T.
Right arrow Articles by Funaba, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ikeda, T.
Right arrow Articles by Funaba, M.

Copyright © 2010 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»