Skip to main content
  • Research Article
  • Published:

Hydrogen peroxide as a potential mediator of the transcriptional regulation of heparan sulphate biosynthesis in keratinocytes

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.

Abbreviations

CS:

chondroitin sulfate

FGF:

fibroblast growth factor

GAG:

glycosaminoglycan

Gal:

galactose

GalNAc:

N-acetylgalactosamine

GlcA:

glucuronic acid

GLCE:

d-glucuronyl C5-epimerase

GlcNAc:

N-acetylglucosamine

HSPG:

heparan sulfate proteoglycan

HS2ST:

heparan sulfate 2-O-sulfotransferase

HS6ST:

heparan sulfate 6-O-sulfotransferase

PDGF:

platelet-derived growth factor

ROS:

reactive oxygen species

SOD:

superoxide dismutase

XT:

xylosyltransferase

Xyl:

xylose

References

  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.

    Article  PubMed  CAS  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.

    Article  PubMed  CAS  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.

    Article  PubMed  CAS  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.

    PubMed  CAS  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  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.

    Article  PubMed  CAS  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.

    PubMed  CAS  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.

    PubMed  CAS  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  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.

    Article  PubMed  CAS  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.

    Article  PubMed  CAS  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.

    Article  PubMed  CAS  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.

    Article  PubMed  CAS  Google Scholar 

  21. Reth, M. Hydrogen peroxide as second messenger in lymphocyte activation. Nat. Immunol. 3 (2002) 1129–1134.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  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.

    PubMed  CAS  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.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Fumiaki Nakayama.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nakayama, F., Hagiwara, A., Yamamoto, T. et al. Hydrogen peroxide as a potential mediator of the transcriptional regulation of heparan sulphate biosynthesis in keratinocytes. Cell Mol Biol Lett 13, 475–492 (2008). https://doi.org/10.2478/s11658-008-0016-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-008-0016-7

Key words