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J Clin Invest. Nov 1995; 96(5): 2461–2468.
PMCID: PMC185899

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

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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  • 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]
  • Clement B, Grimaud JA, Campion JP, 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. [PubMed]
  • Panduro A, Shalaby F, Weiner FR, Biempica L, Zern MA, Shafritz DA. 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. [PubMed]
  • Pierce RA, Glaug MR, Greco RS, Mackenzie JW, Boyd CD, Deak SB. Increased procollagen mRNA levels in carbon tetrachloride-induced liver fibrosis in rats. J Biol Chem. 1987 Feb 5;262(4):1652–1658. [PubMed]
  • Brenner DA, Chojkier M. Acetaldehyde increases collagen gene transcription in cultured human fibroblasts. J Biol Chem. 1987 Dec 25;262(36):17690–17695. [PubMed]
  • Chojkier M, Houglum K, Solis-Herruzo J, Brenner DA. 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]
  • Houglum K, Brenner DA, 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. [PMC free article] [PubMed]
  • Houglum K, Filip M, Witztum JL, 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. [PMC free article] [PubMed]
  • 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]
  • 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. [PubMed]
  • Bacon BR, Tavill AS, Brittenham GM, Park CH, Recknagel RO. Hepatic lipid peroxidation in vivo in rats with chronic iron overload. J Clin Invest. 1983 Mar;71(3):429–439. [PMC free article] [PubMed]
  • Poh-Fitzpatrick MB. Pathogenesis and treatment of photocutaneous manifestations of the porphyrias. Semin Liver Dis. 1982 May;2(2):164–176. [PubMed]
  • Burk RF, Lane JM, 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. [PMC free article] [PubMed]
  • Friedman SL, Roll FJ, Boyles J, Bissell DM. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8681–8685. [PMC free article] [PubMed]
  • Maher JJ, McGuire RF. 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. [PMC free article] [PubMed]
  • Brenner DA, Alcorn JM, Feitelberg SP, Leffert HL, Chojkier M. Expression of collagen genes in the liver. Mol Biol Med. 1990 Apr;7(2):105–115. [PubMed]
  • Davis BH, Pratt BM, Madri JA. 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]
  • Friedman SL, Roll FJ, Boyles J, Arenson DM, Bissell DM. Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix. J Biol Chem. 1989 Jun 25;264(18):10756–10762. [PubMed]
  • Pinzani M, Gesualdo L, Sabbah GM, Abboud HE. 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. [PMC free article] [PubMed]
  • 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. [PubMed]
  • 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Rockey DC, Boyles JK, Gabbiani G, Friedman SL. 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]
  • Bravo R, Frank R, Blundell PA, Macdonald-Bravo H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature. 1987 Apr 2;326(6112):515–517. [PubMed]
  • Xanthoudakis S, Miao G, Wang F, Pan YC, 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. [PMC free article] [PubMed]
  • Henkel T, Machleidt T, Alkalay I, Krönke M, Ben-Neriah Y, Baeuerle PA. 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. [PubMed]
  • Thompson CB, Challoner PB, Neiman PE, Groudine M. Expression of the c-myb proto-oncogene during cellular proliferation. Nature. 319(6052):374–380. [PubMed]
  • Simons M, Rosenberg RD. Antisense nonmuscle myosin heavy chain and c-myb oligonucleotides suppress smooth muscle cell proliferation in vitro. Circ Res. 1992 Apr;70(4):835–843. [PubMed]
  • 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. [PubMed]
  • Blank RS, McQuinn TC, Yin KC, Thompson MM, Takeyasu K, Schwartz RJ, Owens GK. 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]
  • Chojkier M, Groszmann RJ. Measurement of portal-systemic shunting in the rat by using gamma-labeled microspheres. Am J Physiol. 1981 May;240(5):G371–G375. [PubMed]
  • Parola M, Pinzani M, Casini A, Albano E, Poli G, Gentilini A, Gentilini P, Dianzani MU. 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. [PubMed]
  • 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]
  • Das SK, Fanburg BL. 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. [PubMed]
  • Rossi P, Karsenty G, Roberts AB, Roche NS, Sporn MB, 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. [PubMed]
  • Holt K, Bennett M, Chojkier M. Acetaldehyde stimulates collagen and noncollagen protein production by human fibroblasts. Hepatology. 1984 Sep-Oct;4(5):843–848. [PubMed]
  • Savolainen ER, Leo MA, Timpl R, Lieber CS. Acetaldehyde and lactate stimulate collagen synthesis of cultured baboon liver myofibroblasts. Gastroenterology. 1984 Oct;87(4):777–787. [PubMed]
  • Moshage H, Casini A, Lieber CS. 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. [PubMed]
  • Casini A, Cunningham M, Rojkind M, Lieber CS. 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]
  • Lüscher B, Eisenman RN. New light on Myc and Myb. Part II. Myb. Genes Dev. 1990 Dec;4(12B):2235–2241. [PubMed]
  • Gewirtz AM, Calabretta B. A c-myb antisense oligodeoxynucleotide inhibits normal human hematopoiesis in vitro. Science. 1988 Dec 2;242(4883):1303–1306. [PubMed]
  • Gabrielsen OS, 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. [PubMed]
  • Saikumar P, Murali R, Reddy EP. 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. [PMC free article] [PubMed]
  • Klempnauer KH, Symonds G, Evan GI, Bishop JM. 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. [PubMed]
  • Weston K, Bishop JM. Transcriptional activation by the v-myb oncogene and its cellular progenitor, c-myb. Cell. 1989 Jul 14;58(1):85–93. [PubMed]
  • Biedenkapp H, Borgmeyer U, Sippel AE, Klempnauer KH. Viral myb oncogene encodes a sequence-specific DNA-binding activity. Nature. 1988 Oct 27;335(6193):835–837. [PubMed]
  • Ness SA, 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. [PubMed]
  • Ku DH, Wen SC, Engelhard A, Nicolaides NC, Lipson KE, Marino TA, 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]
  • Evans JL, Moore TL, Kuehl WM, Bender T, Ting JP. 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. [PMC free article] [PubMed]
  • Nicolaides NC, 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. [PMC free article] [PubMed]
  • Furukawa Y, Piwnica-Worms H, Ernst TJ, Kanakura Y, Griffin JD. cdc2 gene expression at the G1 to S transition in human T lymphocytes. Science. 1990 Nov 9;250(4982):805–808. [PubMed]
  • Travali S, Reiss K, Ferber A, Petralia S, Mercer WE, 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. [PMC free article] [PubMed]
  • Simons M, Edelman ER, DeKeyser JL, Langer R, Rosenberg RD. Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature. 1992 Sep 3;359(6390):67–70. [PubMed]
  • Myrset AH, Bostad A, Jamin N, Lirsac PN, Toma F, Gabrielsen OS. DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein. EMBO J. 1993 Dec;12(12):4625–4633. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Ness SA, 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. [PubMed]
  • Katz S, Kowenz-Leutz E, Müller C, Meese K, Ness SA, 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Poli V, Mancini FP, 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. [PubMed]
  • 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. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Cao Z, Umek RM, McKnight SL. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991 Sep;5(9):1538–1552. [PubMed]
  • Stein B, Cogswell PC, Baldwin AS., 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. [PMC free article] [PubMed]
  • Stein B, Baldwin AS., 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. [PMC free article] [PubMed]
  • Burgess TL, Fisher EF, Ross SL, Bready JV, Qian YX, Bayewitch LA, Cohen AM, Herrera CJ, Hu SS, Kramer TB, et al. 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. [PMC free article] [PubMed]

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