Abstract
Ionizing radiation is one of the types of oxidative stress that has a number of damaging effects on cutaneous tissues. One of the histological features of radiation-induced cutaneous fibrosis is the accumulation of extracellular matrix (ECM) components, including heparan sulfate proteoglycan (HSPG), which are required for the repair of tissue damage, and operate by interacting with a variety of growth factors. In this study, we established a model of human HaCaT keratinocytes overexpressing anti-oxidative enzyme genes to elucidate the mechanism of oxidative stress leading to the accumulation of HSPG and the role of its accumulation. Catalase overexpression induced an increase in anti-HS antibody (10E4) epitope expression in these cells. Western blotting showed that the smeared bands of HSPG were obviously shifted to a higher molecular weight in the catalase transfectants due to glycosylation. After heparitinase I treatment, the core proteins of HSPG were expressed in the catalase transfectants to almost the same extent as in the control cells. In addition, the transcript levels of all the enzymes required for the synthesis of the heparan sulfate chain were estimated in the catalase transfectant clones. The levels of five enzyme transcripts — xylosyltransferase-II (XT-II), EXTL2, D-glucuronyl C5-epimerase (GLCE), HS2-O-sulfotransferase (HS2ST), and HS6-O-sulfotransferase (HS6ST) — were significantly increased in the transfectants. Moreover, hydrogen peroxide was found to down-regulate the levels of these enzymes. By contrast, siRNA-mediated repression of catalase decreased 10E4 epitope expression, the transcript level of HS2ST1, and the growth rate of HaCaT cells. These findings suggested that peroxide-mediated transcriptional regulation of HS metabolism-related genes modified the HS chains in the HaCaT keratinocytes.
[1] Esko, J.D. and Selleck, S.B. Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu. Rev. Biochem. 71 (2002) 435–471. http://dx.doi.org/10.1146/annurev.biochem.71.110601.13545810.1146/annurev.biochem.71.110601.135458Search in Google Scholar
[2] Flaumenhaft, R., Moscatelli, D. and Rifkin, D.B. Heparin and heparan sulfate increase the radius of diffusion and action of basic fibroblast growth factor. J. Cell Biol. 111 (1990) 1651–1659. http://dx.doi.org/10.1083/jcb.111.4.165110.1083/jcb.111.4.1651Search in Google Scholar
[3] Yayon, A., Klagsbrun, M., Esko, J.D., Leder, P. and Ornitz, D.M. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64 (1991) 841–848. http://dx.doi.org/10.1016/0092-8674(91)90512-W10.1016/0092-8674(91)90512-WSearch in Google Scholar
[4] Aviezer, D., Levy, E., Safran, M., Svahn, C., Buddecke, E., Schmidt, A., David, G., Vlodavsky, I. and Yayon, A. Differential structural requirements of heparin and heparan sulfate proteoglycans that promote binding of basic fibroblast growth factor to its receptor. J. Biol. Chem. 269 (1994) 114–121. Search in Google Scholar
[5] Götting, C., Kuhn, J., Zahn, R., Brinkmann, T. and Kleesiek, K. Molecular cloning and expression of human UDP-D-Xylose: proteoglycan core protein β-d-xylosyltransferase and its first isoform XT-II. J. Mol. Biol. 304 (2000) 517–528. http://dx.doi.org/10.1006/jmbi.2000.426110.1006/jmbi.2000.4261Search in Google Scholar PubMed
[6] Pönighaus, C., Ambrosius, M., Casanova, J.C., Prante, C., Kuhn, J., Esko, J.D., Kleesiek, K. and Götting, C. Human xylosyltransferase II is involved in the biosynthesis of the uniform tetrasaccharide linkage region in chondroitin sulfate and heparan sulfate proteoglycans. J. Biol. Chem. 282 (2007) 5201–5206. http://dx.doi.org/10.1074/jbc.M61166520010.1074/jbc.M611665200Search in Google Scholar PubMed
[7] Almeida, R., Levery, S.B., Mandel, U., Kresse, H., Schwientek, T., Bennett, E. P. and Clausen, H. Cloning and expression of a proteoglycan UDP-galactose: β-xylose β1,4-galactosyltransferase I. A seventh member of the human β4-galactosyltransferase gene family. J. Biol. Chem. 274 (1999) 26165–26171. http://dx.doi.org/10.1074/jbc.274.37.2616510.1074/jbc.274.37.26165Search in Google Scholar PubMed
[8] Bai, X., Zhou, D., Brown, J.R., Crawford, B.E., Hennet, T. and Esko, J.D. Biosynthesis of the linkage region of glycosaminoglycans: cloning and activity of galactosyltransferase II, the sixth member of the β1,3-galactosyltransferase family (β3GalT6). J. Biol. Chem. 276 (2001) 48189–48195. Search in Google Scholar
[9] Kitagawa, H., Tone, Y., Tamura, J., Neumann, K.W., Ogawa, T., Oka, S., Kawasaki, T. and Sugahara, K. Molecular cloning and expression of glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans. J. Biol. Chem. 273 (1998) 6615–6618. http://dx.doi.org/10.1074/jbc.273.12.661510.1074/jbc.273.12.6615Search in Google Scholar PubMed
[10] Zak, B.M., Crawford, B.E. and Esko, J.D. Hereditary multiple exostoses and heparan sulfate polymerization. Biochim. Biophys. Acta 1573 (2002) 346–355. Search in Google Scholar
[11] Habuchi, H., Habuchi, O. and Kimata, K. Sulfation pattern in glycosaminoglycan: does it have a code? Glycoconj J. 21 (2004) 47–52. http://dx.doi.org/10.1023/B:GLYC.0000043747.87325.5e10.1023/B:GLYC.0000043747.87325.5eSearch in Google Scholar
[12] Orellana, A., Hirschberg, C.B., Wei, Z., Swiedler, S.J. and Ishihara, M. Molecular cloning and expression of a glycosaminoglycan N-acetylglucosaminyl N-deacetylase/N-sulfotransferase from a heparinproducing cell line. J. Biol. Chem. 269 (1994) 2270–2276. Search in Google Scholar
[13] Eriksson, I., Sandbäck, D., Ek, B., Lindahl, U. and Kjellén, L. cDNA cloning and sequencing of mouse mastocytoma glucosaminyl N-deacetylase/N-sulfotransferase, an enzyme involved in the biosynthesis of heparin. J. Biol. Chem. 269 (1994) 10438–10443. Search in Google Scholar
[14] Aikawa, J. and Esko, J.D. Molecular cloning and expression of a third member of the heparan sulfate/heparin GlcNAc N-deacetylase/N-sulfotransferase family. J. Biol. Chem. 274 (1999) 2690–2695. http://dx.doi.org/10.1074/jbc.274.5.269010.1074/jbc.274.5.2690Search in Google Scholar PubMed
[15] Aikawa, J., Grobe, K., Tsujimoto, M. and Esko, J.D. Multiple isozymes of heparan sulfate/heparin GlcNAc N-deacetylase/GlcN N-sulfotransferase. Structure and activity of the fourth member, NDST4. J. Biol. Chem. 276 (2001) 5876–5882. http://dx.doi.org/10.1074/jbc.M00960620010.1074/jbc.M009606200Search in Google Scholar PubMed
[16] Habuchi, H., Tanaka, M., Habuchi, O., Yoshida, K., Suzuki, H., Ban, K. and Kimata, K. The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine. J. Biol. Chem. 275 (2000) 2859–2868. http://dx.doi.org/10.1074/jbc.275.4.285910.1074/jbc.275.4.2859Search in Google Scholar
[17] Shworak, N.W., Liu, J., Fritze, L.M., Schwartz, J.J., Zhang, L., Logeart, D. and Rosenberg, R.D. Molecular cloning and expression of mouse and human cDNAs encoding heparan sulfate d-glucosaminyl 3-O-sulfotransferase. J. Biol. Chem. 272 (1997) 28008–28019. http://dx.doi.org/10.1074/jbc.272.44.2800810.1074/jbc.272.44.28008Search in Google Scholar
[18] Shworak, N.W., Liu, J., Petros, L. M., Zhang, L., Kobayashi, M., Copeland, N. G., Jenkins, N.A. and Rosenberg, R.D. Multiple isoforms of heparan sulfate d-glucosaminyl 3-O-sulfotransferase. Isolation, characterization, and expression of human cDNAs and identification of distinct genomic loci. J. Biol. Chem. 274 (1999) 5170–5184. http://dx.doi.org/10.1074/jbc.274.8.517010.1074/jbc.274.8.5170Search in Google Scholar
[19] Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.P., Malmstrom, A., Shukla, D. and Liu, J. Heparan sulfate 3-O-sulfotransferase isoform 5 generates both an antithrombin-binding site and an entry receptor for herpes simplex virus, type 1. J. Biol. Chem. 277 (2002) 37912–37919. http://dx.doi.org/10.1074/jbc.M20420920010.1074/jbc.M204209200Search in Google Scholar
[20] Ward, J.F. DNA damage produced by ionizing radiation in mammalian cells: identities, mechanisms of formation, and reparability. Prog. Nucleic Acid Res. Mol. Biol. 35 (1988) 95–125. http://dx.doi.org/10.1016/S0079-6603(08)60611-X10.1016/S0079-6603(08)60611-XSearch in Google Scholar
[21] Reth, M. Hydrogen peroxide as second messenger in lymphocyte activation. Nat. Immunol. 3 (2002) 1129–1134. http://dx.doi.org/10.1038/ni1202-112910.1038/ni1202-1129Search in Google Scholar PubMed
[22] Preston, T.J., Muller, W.J. and Singh, G. Scavenging of extracellular H2O2 by catalase inhibits the proliferation of HER-2/Neu-transformed rat-1 fibroblasts through the induction of a stress response. J. Biol. Chem. 276 (2001) 9558–9564. http://dx.doi.org/10.1074/jbc.M00461720010.1074/jbc.M004617200Search in Google Scholar PubMed
[23] Nakayama, F., Teraki, Y., Kudo, T., Togayachi, A., Iwasaki, H., Tamatani, T., Nishihara, S., Mizukawa, Y., Shiohara, T. and Narimatsu, H. Expression of cutaneous lymphocyte-associated antigen regulated by a set of glycosyltransferases in human T cells: involvement of α1,3-fucosyltransferase VII and β1,4-galactosyltransferase I. J. Invest. Dermatol. 115 (2000) 299–306. http://dx.doi.org/10.1046/j.1523-1747.2000.00032.x10.1046/j.1523-1747.2000.00032.xSearch in Google Scholar PubMed
[24] Nakayama, F., Nishihara, S., Iwasaki, H., Kudo, T., Okubo, R., Kaneko, M., Nakamura, M., Karube, M., Sasaki, K. and Narimatsu, H. CD15 expression in mature granulocytes is determined by α1,3-fucosyltransferase IX, but in promyelocytes and monocytes by α1,3-fucosyltransferase IV. J. Biol. Chem. 276 (2001) 16100–16106. http://dx.doi.org/10.1074/jbc.M00727220010.1074/jbc.M007272200Search in Google Scholar PubMed
[25] Hachiya, M. and Akashi, M. Catalase regulates cell growth in HL60 human promyelocytic cells: evidence for growth regulation by H2O2. Radiat. Res. 163 (2005) 271–282. http://dx.doi.org/10.1667/RR330610.1667/RR3306Search in Google Scholar PubMed
[26] Clare, D.A., Duong, M.N., Darr, D., Archibald, F. and Fridovich, I. Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase. Anal. Biochem. 140 (1984) 532–537. http://dx.doi.org/10.1016/0003-2697(84)90204-510.1016/0003-2697(84)90204-5Search in Google Scholar
[27] Uyama, T., Kitagawa, H., Tamura, J. and Sugahara, K. Molecular cloning and expression of human chondroitin N-acetylgalactosaminyltransferase: the key enzyme for chain initiation and elongation of chondroitin/dermatan sulfate on the protein linkage region tetrasaccharide shared by heparin/heparan sulfate. J. Biol. Chem. 277 (2002) 8841–8846. http://dx.doi.org/10.1074/jbc.M11143420010.1074/jbc.M111434200Search in Google Scholar PubMed
[28] Gotoh, M., Sato, T., Akashima, T., Iwasaki, H., Kameyama, A., Mochizuki, H., Yada, T., Inaba, N., Zhang, Y., Kikuchi, N., Kwon, Y.D., Togayachi, A., Kudo, T., Nishihara, S., Watanabe, H., Kimata, K. and Narimatsu, H. Enzymatic synthesis of chondroitin with a novel chondroitin sulfate N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine to glucuronic acid in initiation and elongation of chondroitin sulfate synthesis. J. Biol. Chem. 277 (2002) 38189–38196. http://dx.doi.org/10.1074/jbc.M20361920010.1074/jbc.M203619200Search in Google Scholar PubMed
[29] Sato, T., Gotoh, M., Kiyohara, K., Akashima, T., Iwasaki, H., Kameyama, A., Mochizuki, H., Yada, T., Inaba, N., Togayachi, A., Kudo, T., Asada, M., Watanabe, H., Imamura, T., Kimata, K. and Narimatsu, H. Differential roles of two N-acetylgalactosaminyltransferases, CSGalNAcT-1, and a novel enzyme, CSGalNAcT-2. Initiation and elongation in synthesis of chondroitin sulfate. J. Biol. Chem. 278 (2003) 3063–3071. http://dx.doi.org/10.1074/jbc.M20888620010.1074/jbc.M208886200Search in Google Scholar PubMed
[30] Kitagawa, H., Shimakawa, H. and Sugahara, K. The tumor suppressor EXT-like gene EXTL2 encodes an α1,4-N-acetylhexosaminyltransferase that transfers N-acetylgalactosamine and N-acetylglucosamine to the common glycosaminoglycan-protein linkage region. The key enzyme for the chain initiation of heparan sulfate. J. Biol. Chem. 274 (1999) 13933–13937. http://dx.doi.org/10.1074/jbc.274.20.1393310.1074/jbc.274.20.13933Search in Google Scholar PubMed
[31] Kim, B.T., Kitagawa, H., Tamura, J., Saito, T., Kusche-Gullberg, M., Lindahl, U. and Sugahara, K. Human tumor suppressor EXT gene family members EXTL1 and EXTL3 encode α1,4-N-acetylglucosaminyltransferases that likely are involved in heparan sulfate/heparin biosynthesis. Proc. Natl. Acad. Sci. USA 98 (2001) 7176–7181. http://dx.doi.org/10.1073/pnas.13118849810.1073/pnas.131188498Search in Google Scholar PubMed PubMed Central
[32] Turnbull, J.E., Fernig, D.G., Ke, Y., Wilkinson, M.C. and Gallagher, J.T. Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate. J. Biol. Chem. 267 (1992) 10337–10341. Search in Google Scholar
[33] Kreuger, J., Salmivirta, M., Sturiale, L., Gimenez-Gallego, G. and Lindahl, U. Sequence analysis of heparan sulfate epitopes with graded affinities for fibroblast growth factors 1 and 2. J. Biol. Chem. 276 (2001) 30744–30752. http://dx.doi.org/10.1074/jbc.M10262820010.1074/jbc.M102628200Search in Google Scholar PubMed
[34] Liu, J., Shworak, N.W., Sinaÿ, P., Schwartz, J.J., Zhang, L., Fritze, L.M. and Rosenberg, R.D. Expression of heparan sulfate d-glucosaminyl 3-O-sulfotransferase isoforms reveals novel substrate specificities. J. Biol. Chem. 274 (1999) 5185–5192. http://dx.doi.org/10.1074/jbc.274.8.518510.1074/jbc.274.8.5185Search in Google Scholar PubMed
[35] Baker, M.S., Feigan, J. and Lowther, D.A. Chondrocyte antioxidant defences: the roles of catalase and glutathione peroxidase in protection against H2O2 dependent inhibition of proteoglycan biosynthesis. J. Rheumatol. 15 (1988) 670–677. Search in Google Scholar
[36] Bates, E.J., Johnson, C.C. and Lowther, D.A. Inhibition of proteoglycan synthesis by hydrogen peroxide in cultured bovine articular cartilage. Biochim. Biophys. Acta 838 (1985) 221–228. Search in Google Scholar
[37] Schalkwijk, J., van den Berg, W.B., van de Putte, L. and Joosten, L.A. Hydrogen peroxide suppresses the proteoglycan synthesis of intact articular cartilage. J. Rheumatol. 12 (1985) 205–210. Search in Google Scholar
© 2008 University of Wrocław, Poland
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.