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. 1995 Nov;96(5):2461–2468. doi: 10.1172/JCI118304

Activation of hepatic stellate cells by TGF alpha and collagen type I is mediated by oxidative stress through c-myb expression.

K S Lee 1, M Buck 1, K Houglum 1, M Chojkier 1
PMCID: PMC185899  PMID: 7593635

Abstract

Excessive production of collagen type I is a major contributor to hepatic fibrosis. Activated (myofibroblastic), but not quiescent, hepatic stellate cells (lipocytes) have a high level of collagen type I and alpha-smooth muscle actin expression. Therefore, stellate cell activation is a critical step in hepatic fibrosis. Here we show that quiescent stellate cells were activated by the generation of free radicals with ascorbate/FeSO4 and by malondialdehyde, a product of lipid peroxidation. In addition, stellate cell activation by collagen type I matrix and TGF alpha was blocked by antioxidants, such as d-alpha-tocopherol and butylated hydroxytoluene. Moreover, oxidative stress, TGF alpha and collagen type I markedly stimulated stellate cell entry into S-phase, NFkB activity, and c-myb expression, which were prevented by antioxidants. c-myb antisense oligonucleotide blocked the activation and proliferation of stellate cells induced by TGF alpha. Nuclear extracts from activated, but not from quiescent, stellate cells formed a complex with the critical promoter E box of the alpha-smooth muscle actin gene, which was disrupted by c-myb and NFkB65 antibodies, and competed by c-myb and NFkB cognate DNA. c-Myb expression was also stimulated in activated stellate cells in carbon tetrachloride-induced hepatic injury and fibrogenesis. This study indicates that oxidative stress plays an essential role, through the induction of c-myb and NFkB, on stellate cell activation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akira S., Isshiki H., Sugita T., Tanabe O., Kinoshita S., Nishio Y., Nakajima T., Hirano T., Kishimoto T. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 1990 Jun;9(6):1897–1906. doi: 10.1002/j.1460-2075.1990.tb08316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bacon B. R., Tavill A. S., Brittenham G. M., Park C. H., Recknagel R. O. Hepatic lipid peroxidation in vivo in rats with chronic iron overload. J Clin Invest. 1983 Mar;71(3):429–439. doi: 10.1172/JCI110787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Badiani P., Corbella P., Kioussis D., Marvel J., Weston K. Dominant interfering alleles define a role for c-Myb in T-cell development. Genes Dev. 1994 Apr 1;8(7):770–782. doi: 10.1101/gad.8.7.770. [DOI] [PubMed] [Google Scholar]
  4. Bedossa P., Houglum K., Trautwein C., Holstege A., Chojkier M. Stimulation of collagen alpha 1(I) gene expression is associated with lipid peroxidation in hepatocellular injury: a link to tissue fibrosis? Hepatology. 1994 May;19(5):1262–1271. [PubMed] [Google Scholar]
  5. Biedenkapp H., Borgmeyer U., Sippel A. E., Klempnauer K. H. Viral myb oncogene encodes a sequence-specific DNA-binding activity. Nature. 1988 Oct 27;335(6193):835–837. doi: 10.1038/335835a0. [DOI] [PubMed] [Google Scholar]
  6. Blank R. S., McQuinn T. C., Yin K. C., Thompson M. M., Takeyasu K., Schwartz R. J., Owens G. K. Elements of the smooth muscle alpha-actin promoter required in cis for transcriptional activation in smooth muscle. Evidence for cell type-specific regulation. J Biol Chem. 1992 Jan 15;267(2):984–989. [PubMed] [Google Scholar]
  7. Bravo R., Frank R., Blundell P. A., Macdonald-Bravo H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature. 1987 Apr 2;326(6112):515–517. doi: 10.1038/326515a0. [DOI] [PubMed] [Google Scholar]
  8. Brenner D. A., Alcorn J. M., Feitelberg S. P., Leffert H. L., Chojkier M. Expression of collagen genes in the liver. Mol Biol Med. 1990 Apr;7(2):105–115. [PubMed] [Google Scholar]
  9. Brenner D. A., Chojkier M. Acetaldehyde increases collagen gene transcription in cultured human fibroblasts. J Biol Chem. 1987 Dec 25;262(36):17690–17695. [PubMed] [Google Scholar]
  10. Buck M., Turler H., Chojkier M. LAP (NF-IL-6), a tissue-specific transcriptional activator, is an inhibitor of hepatoma cell proliferation. EMBO J. 1994 Feb 15;13(4):851–860. doi: 10.1002/j.1460-2075.1994.tb06328.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Burgess T. L., Fisher E. F., Ross S. L., Bready J. V., Qian Y. X., Bayewitch L. A., Cohen A. M., Herrera C. J., Hu S. S., Kramer T. B. The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):4051–4055. doi: 10.1073/pnas.92.9.4051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Burk R. F., Lane J. M., Patel K. Relationship of oxygen and glutathione in protection against carbon tetrachloride-induced hepatic microsomal lipid peroxidation and covalent binding in the rat. Rationale for the use of hyperbaric oxygen to treat carbon tetrachloride ingestion. J Clin Invest. 1984 Dec;74(6):1996–2001. doi: 10.1172/JCI111621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cao Z., Umek R. M., McKnight S. L. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991 Sep;5(9):1538–1552. doi: 10.1101/gad.5.9.1538. [DOI] [PubMed] [Google Scholar]
  14. Casini A., Cunningham M., Rojkind M., Lieber C. S. Acetaldehyde increases procollagen type I and fibronectin gene transcription in cultured rat fat-storing cells through a protein synthesis-dependent mechanism. Hepatology. 1991 Apr;13(4):758–765. [PubMed] [Google Scholar]
  15. Chojkier M., Groszmann R. J. Measurement of portal-systemic shunting in the rat by using gamma-labeled microspheres. Am J Physiol. 1981 May;240(5):G371–G375. doi: 10.1152/ajpgi.1981.240.5.G371. [DOI] [PubMed] [Google Scholar]
  16. Chojkier M., Houglum K., Solis-Herruzo J., Brenner D. A. Stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts. A role for lipid peroxidation? J Biol Chem. 1989 Oct 5;264(28):16957–16962. [PubMed] [Google Scholar]
  17. Clement B., Grimaud J. A., Campion J. P., Deugnier Y., Guillouzo A. Cell types involved in collagen and fibronectin production in normal and fibrotic human liver. Hepatology. 1986 Mar-Apr;6(2):225–234. doi: 10.1002/hep.1840060212. [DOI] [PubMed] [Google Scholar]
  18. Das S. K., Fanburg B. L. TGF-beta 1 produces a "prooxidant" effect on bovine pulmonary artery endothelial cells in culture. Am J Physiol. 1991 Oct;261(4 Pt 1):L249–L254. doi: 10.1152/ajplung.1991.261.4.L249. [DOI] [PubMed] [Google Scholar]
  19. Davis B. H., Pratt B. M., Madri J. A. Retinol and extracellular collagen matrices modulate hepatic Ito cell collagen phenotype and cellular retinol binding protein levels. J Biol Chem. 1987 Jul 25;262(21):10280–10286. [PubMed] [Google Scholar]
  20. Descombes P., Chojkier M., Lichtsteiner S., Falvey E., Schibler U. LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein. Genes Dev. 1990 Sep;4(9):1541–1551. doi: 10.1101/gad.4.9.1541. [DOI] [PubMed] [Google Scholar]
  21. Evans J. L., Moore T. L., Kuehl W. M., Bender T., Ting J. P. Functional analysis of c-Myb protein in T-lymphocytic cell lines shows that it trans-activates the c-myc promoter. Mol Cell Biol. 1990 Nov;10(11):5747–5752. doi: 10.1128/mcb.10.11.5747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Friedman S. L., Roll F. J., Boyles J., Arenson D. M., Bissell D. M. Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix. J Biol Chem. 1989 Jun 25;264(18):10756–10762. [PubMed] [Google Scholar]
  23. Friedman S. L., Roll F. J., Boyles J., Bissell D. M. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8681–8685. doi: 10.1073/pnas.82.24.8681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Furukawa Y., Piwnica-Worms H., Ernst T. J., Kanakura Y., Griffin J. D. cdc2 gene expression at the G1 to S transition in human T lymphocytes. Science. 1990 Nov 9;250(4982):805–808. doi: 10.1126/science.2237430. [DOI] [PubMed] [Google Scholar]
  25. Gabrielsen O. S., Sentenac A., Fromageot P. Specific DNA binding by c-Myb: evidence for a double helix-turn-helix-related motif. Science. 1991 Sep 6;253(5024):1140–1143. doi: 10.1126/science.1887237. [DOI] [PubMed] [Google Scholar]
  26. Gewirtz A. M., Calabretta B. A c-myb antisense oligodeoxynucleotide inhibits normal human hematopoiesis in vitro. Science. 1988 Dec 2;242(4883):1303–1306. doi: 10.1126/science.2461588. [DOI] [PubMed] [Google Scholar]
  27. Henkel T., Machleidt T., Alkalay I., Krönke M., Ben-Neriah Y., Baeuerle P. A. Rapid proteolysis of I kappa B-alpha is necessary for activation of transcription factor NF-kappa B. Nature. 1993 Sep 9;365(6442):182–185. doi: 10.1038/365182a0. [DOI] [PubMed] [Google Scholar]
  28. Holt K., Bennett M., Chojkier M. Acetaldehyde stimulates collagen and noncollagen protein production by human fibroblasts. Hepatology. 1984 Sep-Oct;4(5):843–848. doi: 10.1002/hep.1840040508. [DOI] [PubMed] [Google Scholar]
  29. Houglum K., Bedossa P., Chojkier M. TGF-beta and collagen-alpha 1 (I) gene expression are increased in hepatic acinar zone 1 of rats with iron overload. Am J Physiol. 1994 Nov;267(5 Pt 1):G908–G913. doi: 10.1152/ajpgi.1994.267.5.G908. [DOI] [PubMed] [Google Scholar]
  30. Houglum K., Brenner D. A., Chojkier M. d-alpha-tocopherol inhibits collagen alpha 1(I) gene expression in cultured human fibroblasts. Modulation of constitutive collagen gene expression by lipid peroxidation. J Clin Invest. 1991 Jun;87(6):2230–2235. doi: 10.1172/JCI115258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Houglum K., Buck M., Adir V., Chojkier M. LAP (NF-IL6) transactivates the collagen alpha 1(I) gene from a 5' regulatory region. J Clin Invest. 1994 Aug;94(2):808–814. doi: 10.1172/JCI117400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Houglum K., Filip M., Witztum J. L., Chojkier M. Malondialdehyde and 4-hydroxynonenal protein adducts in plasma and liver of rats with iron overload. J Clin Invest. 1990 Dec;86(6):1991–1998. doi: 10.1172/JCI114934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Katz S., Kowenz-Leutz E., Müller C., Meese K., Ness S. A., Leutz A. The NF-M transcription factor is related to C/EBP beta and plays a role in signal transduction, differentiation and leukemogenesis of avian myelomonocytic cells. EMBO J. 1993 Apr;12(4):1321–1332. doi: 10.1002/j.1460-2075.1993.tb05777.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Klempnauer K. H., Symonds G., Evan G. I., Bishop J. M. Subcellular localization of proteins encoded by oncogenes of avian myeloblastosis virus and avian leukemia virus E26 and by chicken c-myb gene. Cell. 1984 Jun;37(2):537–547. doi: 10.1016/0092-8674(84)90384-2. [DOI] [PubMed] [Google Scholar]
  35. Ku D. H., Wen S. C., Engelhard A., Nicolaides N. C., Lipson K. E., Marino T. A., Calabretta B. c-myb transactivates cdc2 expression via Myb binding sites in the 5'-flanking region of the human cdc2 gene. J Biol Chem. 1993 Jan 25;268(3):2255–2259. [PubMed] [Google Scholar]
  36. Lüscher B., Eisenman R. N. New light on Myc and Myb. Part II. Myb. Genes Dev. 1990 Dec;4(12B):2235–2241. doi: 10.1101/gad.4.12b.2235. [DOI] [PubMed] [Google Scholar]
  37. Maher J. J., McGuire R. F. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo. J Clin Invest. 1990 Nov;86(5):1641–1648. doi: 10.1172/JCI114886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Martinez-Hernandez A. The hepatic extracellular matrix. II. Electron immunohistochemical studies in rats with CCl4-induced cirrhosis. Lab Invest. 1985 Aug;53(2):166–186. [PubMed] [Google Scholar]
  39. Moshage H., Casini A., Lieber C. S. Acetaldehyde selectively stimulates collagen production in cultured rat liver fat-storing cells but not in hepatocytes. Hepatology. 1990 Sep;12(3 Pt 1):511–518. doi: 10.1002/hep.1840120311. [DOI] [PubMed] [Google Scholar]
  40. Myrset A. H., Bostad A., Jamin N., Lirsac P. N., Toma F., Gabrielsen O. S. DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein. EMBO J. 1993 Dec;12(12):4625–4633. doi: 10.1002/j.1460-2075.1993.tb06151.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ness S. A., Kowenz-Leutz E., Casini T., Graf T., Leutz A. Myb and NF-M: combinatorial activators of myeloid genes in heterologous cell types. Genes Dev. 1993 May;7(5):749–759. doi: 10.1101/gad.7.5.749. [DOI] [PubMed] [Google Scholar]
  42. Ness S. A., Marknell A., Graf T. The v-myb oncogene product binds to and activates the promyelocyte-specific mim-1 gene. Cell. 1989 Dec 22;59(6):1115–1125. doi: 10.1016/0092-8674(89)90767-8. [DOI] [PubMed] [Google Scholar]
  43. Nicolaides N. C., Gualdi R., Casadevall C., Manzella L., Calabretta B. Positive autoregulation of c-myb expression via Myb binding sites in the 5' flanking region of the human c-myb gene. Mol Cell Biol. 1991 Dec;11(12):6166–6176. doi: 10.1128/mcb.11.12.6166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Panduro A., Shalaby F., Weiner F. R., Biempica L., Zern M. A., Shafritz D. A. Transcriptional switch from albumin to alpha-fetoprotein and changes in transcription of other genes during carbon tetrachloride induced liver regeneration. Biochemistry. 1986 Mar 25;25(6):1414–1420. doi: 10.1021/bi00354a034. [DOI] [PubMed] [Google Scholar]
  45. Parola M., Pinzani M., Casini A., Albano E., Poli G., Gentilini A., Gentilini P., Dianzani M. U. Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen alpha 1 (I) gene expression in human liver fat-storing cells. Biochem Biophys Res Commun. 1993 Aug 16;194(3):1044–1050. doi: 10.1006/bbrc.1993.1927. [DOI] [PubMed] [Google Scholar]
  46. Pierce R. A., Glaug M. R., Greco R. S., Mackenzie J. W., Boyd C. D., Deak S. B. Increased procollagen mRNA levels in carbon tetrachloride-induced liver fibrosis in rats. J Biol Chem. 1987 Feb 5;262(4):1652–1658. [PubMed] [Google Scholar]
  47. Pinzani M., Gesualdo L., Sabbah G. M., Abboud H. E. Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells. J Clin Invest. 1989 Dec;84(6):1786–1793. doi: 10.1172/JCI114363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Poh-Fitzpatrick M. B. Pathogenesis and treatment of photocutaneous manifestations of the porphyrias. Semin Liver Dis. 1982 May;2(2):164–176. doi: 10.1055/s-2008-1040706. [DOI] [PubMed] [Google Scholar]
  49. Poli V., Mancini F. P., Cortese R. IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell. 1990 Nov 2;63(3):643–653. doi: 10.1016/0092-8674(90)90459-r. [DOI] [PubMed] [Google Scholar]
  50. Rockey D. C., Boyles J. K., Gabbiani G., Friedman S. L. Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture. J Submicrosc Cytol Pathol. 1992 Apr;24(2):193–203. [PubMed] [Google Scholar]
  51. Rossi P., Karsenty G., Roberts A. B., Roche N. S., Sporn M. B., de Crombrugghe B. A nuclear factor 1 binding site mediates the transcriptional activation of a type I collagen promoter by transforming growth factor-beta. Cell. 1988 Feb 12;52(3):405–414. doi: 10.1016/s0092-8674(88)80033-3. [DOI] [PubMed] [Google Scholar]
  52. Saikumar P., Murali R., Reddy E. P. Role of tryptophan repeats and flanking amino acids in Myb-DNA interactions. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8452–8456. doi: 10.1073/pnas.87.21.8452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Savolainen E. R., Leo M. A., Timpl R., Lieber C. S. Acetaldehyde and lactate stimulate collagen synthesis of cultured baboon liver myofibroblasts. Gastroenterology. 1984 Oct;87(4):777–787. [PubMed] [Google Scholar]
  54. Shibanuma M., Kuroki T., Nose K. Release of H2O2 and phosphorylation of 30 kilodalton proteins as early responses of cell cycle-dependent inhibition of DNA synthesis by transforming growth factor beta 1. Cell Growth Differ. 1991 Nov;2(11):583–591. [PubMed] [Google Scholar]
  55. Simons M., Edelman E. R., DeKeyser J. L., Langer R., Rosenberg R. D. Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature. 1992 Sep 3;359(6390):67–70. doi: 10.1038/359067a0. [DOI] [PubMed] [Google Scholar]
  56. Simons M., Rosenberg R. D. Antisense nonmuscle myosin heavy chain and c-myb oligonucleotides suppress smooth muscle cell proliferation in vitro. Circ Res. 1992 Apr;70(4):835–843. doi: 10.1161/01.res.70.4.835. [DOI] [PubMed] [Google Scholar]
  57. Stein B., Baldwin A. S., Jr Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-kappa B. Mol Cell Biol. 1993 Nov;13(11):7191–7198. doi: 10.1128/mcb.13.11.7191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Stein B., Cogswell P. C., Baldwin A. S., Jr Functional and physical associations between NF-kappa B and C/EBP family members: a Rel domain-bZIP interaction. Mol Cell Biol. 1993 Jul;13(7):3964–3974. doi: 10.1128/mcb.13.7.3964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Thompson C. B., Challoner P. B., Neiman P. E., Groudine M. Expression of the c-myb proto-oncogene during cellular proliferation. 1986 Jan 30-Feb 5Nature. 319(6052):374–380. doi: 10.1038/319374a0. [DOI] [PubMed] [Google Scholar]
  60. Trautwein C., Caelles C., van der Geer P., Hunter T., Karin M., Chojkier M. Transactivation by NF-IL6/LAP is enhanced by phosphorylation of its activation domain. Nature. 1993 Aug 5;364(6437):544–547. doi: 10.1038/364544a0. [DOI] [PubMed] [Google Scholar]
  61. Trautwein C., van der Geer P., Karin M., Hunter T., Chojkier M. Protein kinase A and C site-specific phosphorylations of LAP (NF-IL6) modulate its binding affinity to DNA recognition elements. J Clin Invest. 1994 Jun;93(6):2554–2561. doi: 10.1172/JCI117266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Travali S., Reiss K., Ferber A., Petralia S., Mercer W. E., Calabretta B., Baserga R. Constitutively expressed c-myb abrogates the requirement for insulinlike growth factor 1 in 3T3 fibroblasts. Mol Cell Biol. 1991 Feb;11(2):731–736. doi: 10.1128/mcb.11.2.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Weston K., Bishop J. M. Transcriptional activation by the v-myb oncogene and its cellular progenitor, c-myb. Cell. 1989 Jul 14;58(1):85–93. doi: 10.1016/0092-8674(89)90405-4. [DOI] [PubMed] [Google Scholar]
  64. Xanthoudakis S., Curran T. Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity. EMBO J. 1992 Feb;11(2):653–665. doi: 10.1002/j.1460-2075.1992.tb05097.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Xanthoudakis S., Miao G., Wang F., Pan Y. C., Curran T. Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme. EMBO J. 1992 Sep;11(9):3323–3335. doi: 10.1002/j.1460-2075.1992.tb05411.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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