Abstract
On the basis of transepithelial electrical resistance (TER) measurements, we found that capsaicin (100 μM)-treated human intestinal Caco-2 cells show a momentary increase in tight-junction (TJ) permeability (decrease in TER) followed by a complete recovery. We used proteome analysis to search for proteins that are associated with the recovery of TJ permeability in capsaicin-treated Caco-2 cells. A protein with a relative molecular mass of 14 kDa was found to be expressed more highly in capsaicin-treated cells than in nontreated cells. Mass spectrometry and sequence analyses revealed that the protein that is expressed significantly upon capsaicin treatment is the ribosomal protein P2; its cDNA sequence was identical to that found in the human genome database. An increase in the amount of cellular filamentous actin (F-actin) was shown after 8 h of incubation with capsaicin. It has been reported that P2 activates elongation factor 2, which stabilizes F-actin filaments, and that the depolymerization of F-actin is associated with the increase in TJ permeability (decrease in TER). Consequently, these results suggest that P2 plays an important role in the recovery of the TJ permeability in capsaicin-treated human intestinal cells.
Keywords: Caco-2 Cells, Capsaicin, Elongation factor 2, F-actin, Proteome analysis, Ribosomal protein P2, Tight-junction permeability
Full Text
The Full Text of this article is available as a PDF (279.0 KB).
Footnotes
An erratum to this article is available at http://dx.doi.org/10.1007/s10616-005-6709-0.
References
- Alberts B., Johnson A., Lewis J., Raff M., Roberts K., Walter P. Molecular Biology of the Cell. 4. New York: Garland Science (Taylor and Francis Group); 2002. [Google Scholar]
- Bektas M., Nurten R., Gurel Z., Sayers Z., Bermek E. Interaction of eukaryotic elongation factor 2 with actin: a possible link between protein synthetic machinery and cytoskeleton. FEBS Lett. 1994;356:89–93. doi: 10.1016/0014-5793(94)01239-3. [DOI] [PubMed] [Google Scholar]
- Bektas M., Nurten R., Gurel Z., Sayers Z., Bermek E. Interaction of elongation factor 2 with the cytoskeleton and interference with DNase I binding to actin. Eur. J. Biochem. 1998;256:142–147. doi: 10.1046/j.1432-1327.1998.2560142.x. [DOI] [PubMed] [Google Scholar]
- Bruewer M., Hopkins A.M., Hobert M.E., Nusrat A., Madara J.L. RhoARacl, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin. Am. J. Physiol. Cell Physiol. 2004;287:C327–C335. doi: 10.1152/ajpcell.00087.2004. [DOI] [PubMed] [Google Scholar]
- Dadabay C.J., Patton E., Cooper J.A., Pike L.J. Lack of correlation between changes in polyphosphoinositide levels and actin/gelsolin complexes in A431 cells treated with epidermal growth factor. J. Cell Biol. 1991;112:1151–1156. doi: 10.1083/jcb.112.6.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fogh J., Wright W.C., Loveless J.D. Absence of HeLa contamination in 169 cell lines derived from human tumors. J. Natl. Cancer Inst. 1977;58:209–214. doi: 10.1093/jnci/58.2.209. [DOI] [PubMed] [Google Scholar]
- Fuchigami T., Misumi S., Takamune N., Takahashi I., Takama M., Shoji S. Acid-labile formylation of amino terminal proaline of human immunodeficiency virus type 1 p24gag was found by proteomics using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Biochem. Biophys. Res. Commun. 2002;293:1107–1113. doi: 10.1016/S0006-291X(02)00329-7. [DOI] [PubMed] [Google Scholar]
- Furukawa T., Uchiumi T., Tokunaga R., Taketani S. Ribosomal protein P2, a novel iron-binding protein. Arch. Biochem. Biophys. 1992;298:182–186. doi: 10.1016/0003-9861(92)90110-I. [DOI] [PubMed] [Google Scholar]
- Gardner-Thorpe J., Ito H., Ashley S., Whang E.E. Ribosomal protein P2: a potential molecular target for antisense therapy of human malignancies. Anticancer Res. 2003;23:4549–4560. [PubMed] [Google Scholar]
- Gonzalo P., Lavergne J.P., Reboud J.P. Pivotal role of Pl N-terminal domain in the assembly of the mammalian ribosomal stalk and in the proteosynthetic activity. J. Biol. Chem. 2002;276:19762–19769. doi: 10.1074/jbc.M101398200. [DOI] [PubMed] [Google Scholar]
- Han J.K., Isoda H., Maekawa T. Analysis of the mechanism of the tight junctional permeability increase by capsaicin treatment on the Caco-2 cells. Cytotechnology. 2002;40:93–98. doi: 10.1023/A:1023922306968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hashimoto K., Kawagishi H., Nakayama T., Shimizu M. Effect of capsianosidea diterpene glycosideon tight-junctional permeability. Biochim. Biophys. Acta. 1997;1323:281–290. doi: 10.1016/s0005-2736(96)00196-4. [DOI] [PubMed] [Google Scholar]
- Holmes K.C., Popp D., Gebhard W., Kabsch W. Atomic model of the actin filament. Nature. 1990;347:44–49. doi: 10.1038/347044a0. [DOI] [PubMed] [Google Scholar]
- Isoda H., Han J.K., Tominaga M., Maekawa T. Effect of capsaicin on human intestinal cell line Caco-2. Cytotechnology. 2001;36:155–161. doi: 10.1023/A:1014053306343. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lavergne J.P., Conquet F., Reboud J.P., Reboud A.M. Role of acidic phosphoprotein in the parartical reconstitution of the active 60S ribosomal subunit. FEBS Lett. 1987;216:83–88. doi: 10.1016/0014-5793(87)80761-5. [DOI] [PubMed] [Google Scholar]
- Lim S.O., Park S.J., Kim W., Park S.G., Kim H.J., Kim Y.I., Sohn T.S., Noh J.H., Jung G. Proteome analysis of hepatocellular carcinoma. Biochem. Biophys. Res. Commun. 2002;291:1031–1037. doi: 10.1006/bbrc.2002.6547. [DOI] [PubMed] [Google Scholar]
- Nusrat A., Giry M., Turner J.R., Colgan S.P., Parkos C.A., Carnes D., Lemichez E., Boquet P., Madara J.L. Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia. Proc. Natl. Acad. Sci. U.S.A. 1995;92:10629–10633. doi: 10.1073/pnas.92.23.10629. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Poon T.C., Johnson P.J. Poroteome analysis and its impact on the discovery of serological tumor markers. Clinica Chimica Acta. 2001;313:231–239. doi: 10.1016/s0009-8981(01)00677-5. [DOI] [PubMed] [Google Scholar]
- Rayment I., Holden H.M., Whittaker M., Yohn C.B., Lorenz M., Holmes K.C., Milligan R.A. Structure of the actin-myosin complex and its implications for muscle contraction. Science. 1993;261:58–65. doi: 10.1126/science.8316858. [DOI] [PubMed] [Google Scholar]
- Satsu H., Yokoyama T., Ogawa N., Fujiwara-Hatano Y., Shimizu M. Effect of neuronal PC12 cells on the functional properties of intestinal epithelial Caco-2 cells. Biosci. Biotechnol. Biochem. 2003;67:1312–1318. doi: 10.1271/bbb.67.1312. [DOI] [PubMed] [Google Scholar]
- Savala U., Waters C.M. Barrier function of airway epithelium: effects of radiation and protection by keratinocyte growth factor. Radiat. Res. 1998;150:195–203. [PubMed] [Google Scholar]
- Vard C., Guillot D., Bargis P., Lavergne J.P., Reboud J.P. Specific role for the phosphorylation of mammalian acidic ribosomal protein P2. J. Biol. Chem. 1997;272:20259–20262. doi: 10.1074/jbc.272.32.20259. [DOI] [PubMed] [Google Scholar]
- Yap A.S., Mullin J.M., Stevenson B.R. Molecular analyses of tight junction physiology: insights and paradoxes. J. Membr. Biol. 1998;163:159–167. doi: 10.1007/s002329900380. [DOI] [PubMed] [Google Scholar]