Molecular and Cellular Biochemistry

, Volume 311, Issue 1–2, pp 167–177 | Cite as

The antioxidant effect exerted by TGF-1β-stimulated hyaluronan production reduced NF-kB activation and apoptosis in human fibroblasts exposed to FeSo4 plus ascorbate

  • Giuseppe M. Campo
  • Angela Avenoso
  • Salvatore Campo
  • Angela D’Ascola
  • Paola Traina
  • Dario Samà
  • Alberto Calatroni


Previous studies suggest that Transforming growth factor-1beta (TGF-1β) administration in human fibroblasts exposed to oxidative stress is able to modulate hyaluronan synthases (HASs). HAS modulation in turn increases high molecular weight (Hyaluronan) HA concentration. Nuclear factor kB (NF-kB) is a response transcription factor involved in inflammation and acts by enabling the expression of certain detrimental molecules. Caspases are specific proteases responsible for regulating and programming cell death. HA at medium molecular weight together with chondroitin-4-sulphate proved to be effective on NF-kB and caspases. We investigated whether the protective effect afforded by the high molecular weight HA produced by TGF-1β treatment has any effect on NF-kB and apoptosis activation in fibroblast cultures exposed to oxidative stress. Generation of free radicals gives rise to cell death, increases lipid peroxidation, activates NF-kB, reduces its cytoplasmic inhibitor IkBα, augments caspase-3 and caspase-7 gene expression and their relative protein activity, and depletes catalase (CAT) and glutathione peroxidase (GPx). Treatment of fibroblasts with TGF-1β 12 h before inducing oxidative stress greatly increased HA levels, ameliorated cell survival, inhibited lipid peroxidation, blunted NF-kB translocation, normalized IkBα protein, reduced caspase gene expression and protein levels, and restored the endogenous antioxidants CAT and GPx. Since it was previously reported that antioxidants can work as inhibitors of NF-kB and apoptosis induction we can hypothesize that endogenous HA, by inhibiting lipid peroxidation, may block a step whereby free radical activity converges in the signal transduction pathway leading to NF-kB and caspase activation.


TGF-1β Hyaluronan Oxidative stress Antioxidants NF-kB Caspases Apoptosis 



Butylated hydroxytoluene




Dulbecco’s modified Eagle’s medium


Extracellular matrix


Ethylenediaminetetraacetic acid




Foetal bovine serum


Hyaluronic acid




Hyaluronan synthases


Interferon gamma


Interferon beta


Reduced nicotinamide adenine dinucleotide


Reduced nicotinamide adenine dinucleotide phosphate


Phosphate buffered saline


Polymorphonuclear leukocytes


Sodium dodecylsulphate


Reactive oxygen species


Standard deviation


Tumour necrosis factor alpha



This study was supported in part by a grant ex 40% (COFIN 2004) from the MIUR, Italy and in part by a grant PRA (Research Athenaeum Project 2003) from the University of Messina, Italy.


  1. 1.
    Droge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82:47–95PubMedGoogle Scholar
  2. 2.
    Stern R, Asari AA, Sugahara KN (2006) Hyaluronan fragments: an information-rich system. Eur J Cell Biol 85:699–715PubMedCrossRefGoogle Scholar
  3. 3.
    Campo GM, Avenoso A, Campo S, D’Ascola A, Ferlazzo AM, Calatroni A (2004) The antioxidant and antifibrogenic effects of the glycosaminoglycans hyaluronic acid and chondroitin-4-sulphate in a subchronic rat model of carbon tetrachloride-induced liver fibrogenesis. Chem Biol Interact 148:125–138PubMedCrossRefGoogle Scholar
  4. 4.
    Campo GM, Avenoso A, Campo S, D’Ascola A, Ferlazzo AM, Calatroni A (2004) Reduction of DNA fragmentation and hydroxyl radical production by hyaluronic acid and chondroitin-4-sulphate in iron plus ascorbate-induced oxidative stress in fibroblast cultures. Free Radic Res 38:601–611PubMedCrossRefGoogle Scholar
  5. 5.
    Campo GM, Avenoso A, Campo S, Ferlazzo AM, Altavilla D, Calatroni A (2003) Efficacy of treatment with glycosaminoglycans on experimental collagen-induced arthritis in rats. Arthritis Res Ther 5:R122–R131PubMedCrossRefGoogle Scholar
  6. 6.
    Campo GM, Avenoso A, D’Ascola A, Campo S, Ferlazzo AM, Samà D, Calatroni A (2005) Purified human plasma glycosaminoglycans limit oxidative injury induced by iron plus ascorbate in skin fibroblast cultures. Toxicol In Vitro 19:561–572PubMedCrossRefGoogle Scholar
  7. 7.
    Weigel PH, Hascall VC, Tammi M (1997) Hyaluronan Synthase. J Biol Chem 272:13997–14000PubMedCrossRefGoogle Scholar
  8. 8.
    Itano N, Sawait T, Yoshida M, Lenas P, Yamada Y, Imagawa M, Shinomura T, Hamaguchi M, Yoshida Y, Onhuki Y, Miyauchi S, Spider PA, McDonald AJ, Kimata K (1999) Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties. J Biol Chem 274:25085–25092PubMedCrossRefGoogle Scholar
  9. 9.
    Heldin P, Laurent CT, Heldin CH (1989) Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts. Biochem J 258:919–922PubMedGoogle Scholar
  10. 10.
    Jacobson A, Brinck J, Briskin MJ, Spicer AP, Heldin P (2000) Expression of human hyaluronan synthases in response to external stimuli. Biochem J 348:29–35PubMedCrossRefGoogle Scholar
  11. 11.
    Stuhlmeier KM, Pollaschek C (2004) Differential effect of transforming growth factor beta (TGF-β) on the genes encoding hyaluronan synthases and utilization of the p38 MAPK pathway in TGF-β-induced hyaluronan synthase 1 activation. J Biol Chem 279:8753–8760PubMedCrossRefGoogle Scholar
  12. 12.
    Yamada Y, Itano N, Hata K, Ueda M, Kimata K (2004) Differential regulation by IL-1β and EGF of expression of three different hyaluronan synthases in oral mucosal epithelial cells and fibroblasts and dermal fibroblasts: quantitative analysis using real-time RT-PCR. J Invest Dermatol 122:631–639PubMedCrossRefGoogle Scholar
  13. 13.
    Mantena SK, Katiyar SK (2006) Grape seed proantocyanidis inhibit UV-radiation-induced oxidative stress and activation of MAPK and NF-kappaB signalling in human epidermal keratinocytes. Free Radic Biol Med 40:1603–1614PubMedCrossRefGoogle Scholar
  14. 14.
    Aggarwal BB (2004) Nuclear factor-kB: the enemy within. Cancer Cell 6:203–208PubMedCrossRefGoogle Scholar
  15. 15.
    Nagata S (2001) Apoptotic DNA fragmentation. Exp Cell Res 256:12–18CrossRefGoogle Scholar
  16. 16.
    Ho P, Hawkins J (2005) Mammalian initiator apoptotic caspases. FEBS J 272:5436–5453PubMedCrossRefGoogle Scholar
  17. 17.
    Pryor WA, Houk KN, Foote CS, Fukuto JM, Ignarro LJ, Squadrito GL, Davies KJ (2006) Free radical biology and medicine: it’s a gas, man. Am J Physiol Regul Integr Comp Physiol 291:R491–R511PubMedGoogle Scholar
  18. 18.
    Kajstura J, Bolli R, Sonnenblick EH, Anversa P, Leri A (2006) Cause of death: suicide. J Mol Cell Cardiol 40:425–437PubMedCrossRefGoogle Scholar
  19. 19.
    Hanukoglu I (2006) Antioxidant protective mechanisms against reactive oxygen species (ROS) generated by mitochondrial P450 systems in steroidogenic cells. Drug Metab Rev 38:171–196PubMedCrossRefGoogle Scholar
  20. 20.
    Dimmeler S, Zeiher AM (2000) Reactive oxygen species and vascular cell apoptosis in response to angiotensin II and pro-atherosclerotic factors. Regul Pept 90:19–25PubMedCrossRefGoogle Scholar
  21. 21.
    Campo GM, D’Ascola A, Avenoso A, Campo S, Ferlazzo AM, Micali C, Calatroni A (2004) Glycosaminoglycans reduce oxidative damage induced by copper (Cu+2), iron (Fe+2) and hydrogen peroxide (H2O2) in human fibroblast cultures. Glycoconj J 20:133–141PubMedCrossRefGoogle Scholar
  22. 22.
    Krischel V, Bruch-Gerharz D, Suschek C, Kroncke KD, Ruzicka T, Kolb-Bachofen V (1998) Biphasic effect of exogenous nitric oxide on proliferation and differentiation in skin derived keratinocytes but not fibroblasts. J Invest Dermatol 111:286–291PubMedCrossRefGoogle Scholar
  23. 23.
    Lee HG, Cowman MK (1994) An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution. Anal Biochem 219:278–287PubMedCrossRefGoogle Scholar
  24. 24.
    Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193PubMedCrossRefGoogle Scholar
  25. 25.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  26. 26.
    Iozzo RV (1998) Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem 67:609–652PubMedCrossRefGoogle Scholar
  27. 27.
    Hascall VC, Majors AK, De La Motte CA, Evanko SP, Wang A, Drazba JA, Strong SA, Wight TN (2004) Intracellular hyaluronan: a new frontier for inflammation? Biochim Biophys Acta 1763:3–12Google Scholar
  28. 28.
    Laurent TC, Fraser JRE (1991) Catabolism of hyaluronan. In: Henriksen JH (ed) Degradation of bioactive substances: physiology and pathology. CRC Press, Boca Raton, pp 249–265Google Scholar
  29. 29.
    Laurent TC, Fraser JRE (1992) Hyaluronan. FASEB J 6:2397–2404PubMedGoogle Scholar
  30. 30.
    Iozzo RV, Murdoch AD (1996) Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function. FASEB J 10:598–614PubMedGoogle Scholar
  31. 31.
    Suzuki Y, Yamaguchi T (1993) Effects of hyaluronic acid on macrophage phagocytosis and active oxygen release. Agents Actions 38:32–37PubMedCrossRefGoogle Scholar
  32. 32.
    Kvam BJ, Fragonas E, Degrassi A, Kvam C, Matulova M, Pollesello P, Zanetti F, Vittur F (1995) Oxygen-derived free radical (ODFR) action on hyaluronan (HA), on two HA ester derivatives, and on the metabolism of articular chondrocytes. Exp Cell Res 218:79–86PubMedCrossRefGoogle Scholar
  33. 33.
    Fukuda K, Takayama M, Ueno M, Oh M, Asada S, Kumano F, Tanaka S (1997) Hyaluronic acid inhibits interleukin-1-induced superoxide anion in bovine chondrocytes. Inflamm Res 46:114–117PubMedCrossRefGoogle Scholar
  34. 34.
    Calatroni A (2002) Extraction and purification of glycosaminoglycans (GAGs) from biological fluids. In: Volpi N (eds) Analytical techniques to evaluate the structure and function of natural polysaccharides, glycosaminoglycans. Research Signpost Press, Trivandrum, pp 15–22Google Scholar
  35. 35.
    Laurent TC, Laurent UBG, Fraser JRE (1996) Serum hyaluronan as a disease marker. Ann Med 28:241–253PubMedCrossRefGoogle Scholar
  36. 36.
    Roughley PJ (2001) Articular cartilage and changes in arthritis: noncollagenous proteins and proteoglycans in the extracellular matrix of cartilage. Arthritis Res 3:342–347PubMedCrossRefGoogle Scholar
  37. 37.
    Plevris JN, Haydon GH, Simpson KJ, Dawkes R, Ludlum CA, Harrison DJ, Hayes PC (2000) Serum hyaluronan: a non invasive test for diagnosing liver cirrhosis. Eur J Gastroenterol Hepatol 12:1121–1127PubMedCrossRefGoogle Scholar
  38. 38.
    Gutteridge JMC (1998) Iron in free radical reactions and antioxidant protection. In: T Ozben (eds) Free radicals, oxidative stress, and antioxidants. pathological and physiological significance. Plenum Press, New York and London, pp 1–14Google Scholar
  39. 39.
    Gutteridge JM, Halliwell B (1989) Iron toxicity and oxygen radicals. Baillieres Clin Haematol 2:195–256PubMedCrossRefGoogle Scholar
  40. 40.
    Balogh GT, Illes J, Szekely Z, Forrai E, Gere A (2003) Effect of different metal ions on the oxidative damage and antioxidant capacity of hyaluronic acid. Arch Biochem Biophys 410:76–82PubMedCrossRefGoogle Scholar
  41. 41.
    Merce ALR, Carrera LCM, Romanholi LKS, Recio MAL (2002) Aqueous and solid complexes of iron(III) with hyaluronic acid potentiometric titrations and infrared spectroscopy studies. J Inorg Biochem 89:212–218PubMedCrossRefGoogle Scholar
  42. 42.
    Nagy L, Yamashita S, Yamaguchi T, Sipos P, Wakita H, Nomura M (1998) The local structures of Cu(II) and Zn(II) complexes of hyaluronate. J Inorg Biochem 72:49–55CrossRefGoogle Scholar
  43. 43.
    Elias JA, Jimenez SA, Freundlich B (1987) Recombinant gamma, alpha and beta interferon regulation of human lung fibroblast growth. Am Rev Respir Dis 135:62–65PubMedGoogle Scholar
  44. 44.
    Sugarman BJ, Aggarwal BB, Hass PE, Figari IS, Palladino MA Jr, Shepard HM (1985) Recombinant human tumor necrosis factor-alpha. Effects on proliferation of normal and transformed cells in vitro. Science 230:943–945PubMedCrossRefGoogle Scholar
  45. 45.
    Oguchi T, Ishiguro N (2004) Differential stimulation of three forms of hyaluronan synthase by TGF-β, IL-1β, and TNF. Connect Tissue Res 45:197–205PubMedCrossRefGoogle Scholar
  46. 46.
    Campo GM, Avenoso A, Campo S, D’Ascola A, Ferlazzo AM, Calatroni A (2006) TNF-alpha, IFN-gamma and IL-1beta modulate hyaluronan synthase expression in human skin fibroblasts: synergistic effect by concomital treatment with FeSO(4) plus ascorbate. Mol Cell Biochem . 292:169–178PubMedCrossRefGoogle Scholar
  47. 47.
    Tanimoto K, Ohno S, Fujimoto K, Honda K, Ijuin C, Tanaka N, Doi T, Nakahara M, Tanne K (2001) Proinflammatory cytokines regulate the gene expression of hyaluronic acid synthases in cultured rabbit synovial membrane cells. Connect Tissue Res 42:187–195PubMedCrossRefGoogle Scholar
  48. 48.
    Wilkinson TS, Potter-Perigo S, Tsoi C, Altman CL, Wight TN (2004) Pro- and anti-inflammatory factors cooperate to control hyaluronan synthesis in lung fibroblasts. Am J Respir Cell Mol Biol 31:92–99PubMedCrossRefGoogle Scholar
  49. 49.
    Ducale AE, Ward SI, Dechert T, Yager DR (2005) Regulation of hyaluronan synthase-2 expression in human intestinal mesenchymal cells: mechanisms of interleukin-1β-mediated induction. Am J Physiol Gastrointest Liver Physiol 289:G462–G470PubMedCrossRefGoogle Scholar
  50. 50.
    Liu SF, Malik AB (2006) NF-kappa B activation as a pathological mechanism of septic shock and inflammation. Am J Physiol Lung Cell Mol Physiol 290:L622–L645PubMedCrossRefGoogle Scholar
  51. 51.
    Li HL, Huang Y, Zhang CN, Liu G, Wei YS, Wang AB, Liu YO, Hui RT, Wei C, Williams GM, Liu DP, Liang CC (2006) Epigallocathechin-3 gallate inhibits cardiac hypertrophy through blocking reactive oxidative species-dependent and –independent signal pathways. Free Radic Biol Med 40:1756–1775 PubMedCrossRefGoogle Scholar
  52. 52.
    Alonso M, Collado PS, Gonzalez-Gallego J (2006) Melatonin inhibits the expression of the inducible isoform of nitric oxide and nuclear factor kappa B activation in rat skeletal muscle. J Pineal Res 41:8–14PubMedCrossRefGoogle Scholar
  53. 53.
    Ueda S, Masutani H, Nakamura H, Tanaka T, Ueno M, Yodoi J (2002) Redox control of cell death. Antioxid Redox Signal 4:405–414PubMedCrossRefGoogle Scholar
  54. 54.
    Oral B, Guney M, Demirin H, Ozguner M, Giray SG, Take G, Mungan T, Altuntas I (2006) Endometrial damage and apoptosis in rats induced by dichlorvos and ameliorating effect of antioxidant Vitamins E and C. Reprod Toxicol 22:783–90PubMedCrossRefGoogle Scholar
  55. 55.
    Ham YM, Lim JH, Na HK, Choi JS, Park BD, Yim H, Lee SK (2006) Ginsenoside-Rh2-induced mitochondrial depolarization and apoptosis are associated with ROS-and Ca2+-mediated JNK1 activation in HeLa cells. J Pharmacol Exp Ther 319:1276–1285PubMedCrossRefGoogle Scholar
  56. 56.
    Noble PW, McKee CM, Cowman M, Shin HS (1996) Hyaluronan fragments activate an NF-kappaB/I-kappaB alpha autoregulatory loop in murine macrophages. J Exp Med 183:2373–2378PubMedCrossRefGoogle Scholar
  57. 57.
    Presti D, Scott JE (1994) Hyaluronan-mediated protective effect against cell damage caused by enzymatically produced hydroxyl (OH.) radicals is dependent on hyaluronan molecular mass. Cell Biochem Funct 12:281–288PubMedCrossRefGoogle Scholar
  58. 58.
    Li L, Frei B (2006) Iron chelation inhibits NF-kappaB-mediated adhesion molecole expression by inhibiting p22phox protein expression and NADPH oxidase activity. Arterioscler Thromb Vasc Biol 26:2638–26343PubMedCrossRefGoogle Scholar
  59. 59.
    Cartel NJ, Post M (2005) Abrogation of apoptosis through PDGF-BB-induced sulphated glycosaminoglycans synthesis and secretion. Am J Physiol Lung Cell Mol Physiol 288:L285–L293PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2008

Authors and Affiliations

  • Giuseppe M. Campo
    • 1
  • Angela Avenoso
    • 1
  • Salvatore Campo
    • 1
  • Angela D’Ascola
    • 1
  • Paola Traina
    • 1
  • Dario Samà
    • 1
  • Alberto Calatroni
    • 1
  1. 1.Department of Biochemical, Physiological and Nutritional Sciences, School of MedicineUniversity of Messina, Policlinico UniversitarioMessinaItaly

Personalised recommendations