Pharmaceutical Research

, Volume 28, Issue 12, pp 3058–3068 | Cite as

PCBP2 siRNA Reverses the Alcohol-induced Pro-fibrogenic Effects in Hepatic Stellate Cells

  • Ravi S. Shukla
  • Bin Qin
  • Yu-Jui Yvonne Wan
  • Kun Cheng
Research Paper



Type I collagen accumulates during liver fibrosis primarily because α-complex protein-2 (αCP2), encoded by the poly(rC) binding protein 2 (PCBP2) gene, binds to the 3′ end of the collagen mRNA and increases its half-life. This study aimed to reverse the pro-fibrogenic effect of alcohol on hepatic stellate cells (HSCs) by silencing the PCBP2 gene with siRNA.


The silencing effects of a series of predesigned PCBP2 siRNAs were evaluated in the rat hepatic stellate cell line, HSC-T6. The pro-fibrogenic effects of alcohol on the expression levels of PCBP2 and type-I collagen were examined by several methods. The effect of PCBP2 siRNA on the stability of type I collagen α1(I) mRNA was investigated by an in vitro mRNA decay assay.


We identified one potent PCBP2 siRNA that reversed the alcohol-induced expression of PCBP2 in HSCs. The decay rate of the collagen α1(I) mRNA increased significantly in HSCs treated with the PCBP2 siRNA.


This study provides the first evidence that alcohol up-regulates the expression of PCBP2, which subsequently increases the half-life of collagen α1(I) mRNA. Silencing of PCBP2 using siRNA may provide a promising strategy to reverse the alcohol-induced pro-fibrogenic effects in HSCs.


alcoholic liver fibrosis mRNA stability PCBP2 siRNA type I collagen 



This project has been supported by a grant (1R21AA017960-01A1) from the National Institute of Alcohol Abuse and Alcoholism (NIAAA) at NIH and CA53596 to Wan. We also would like to express thanks for the financial support from a start-up package at the University of Missouri—Kansas City.


  1. 1.
    Cheng K, Mahato RI. Gene modulation for treating liver fibrosis. Crit Rev Ther Drug Carrier Syst. 2007;24:93–146.PubMedGoogle Scholar
  2. 2.
    Siegmund SV, Dooley S, Brenner DA. Molecular mechanisms of alcohol-induced hepatic fibrosis. Dig Dis. 2005;23:264–74.PubMedCrossRefGoogle Scholar
  3. 3.
    Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005;115:209–18.PubMedGoogle Scholar
  4. 4.
    George J, Chandrakasan G. Molecular characteristics of dimethylnitrosamine induced fibrotic liver collagen. Biochim Biophys Acta. 1996;1292:215–22.PubMedCrossRefGoogle Scholar
  5. 5.
    LaBrecque DR, Gitnick GL, Moody FG. Diseases of the liver and biliary tract. St. Louis: Mosby-Year Book; 1992.Google Scholar
  6. 6.
    Lindquist JN, Marzluff WF, Stefanovic B. Fibrogenesis. III. Posttranscriptional regulation of type I collagen. Am J Physiol Gastrointest Liver Physiol. 2000;279:G471–6.PubMedGoogle Scholar
  7. 7.
    Vuorio E, de Crombrugghe B. The family of collagen genes. Annu Rev Biochem. 1990;59:837–72.PubMedCrossRefGoogle Scholar
  8. 8.
    Joseph J, Kandala JC, Veerapanane D, Weber KT, Guntaka RV. Antiparallel polypurine phosphorothioate oligonucleotides form stable triplexes with the rat alpha1(I) collagen gene promoter and inhibit transcription in cultured rat fibroblasts. Nucleic Acids Res. 1997;25:2182–8.PubMedCrossRefGoogle Scholar
  9. 9.
    Sato M, Suzuki S, Senoo H. Hepatic stellate cells: unique characteristics in cell biology and phenotype. Cell Struct Funct. 2003;28:105–12.PubMedCrossRefGoogle Scholar
  10. 10.
    Lindquist JN, Stefanovic B, Brenner DA. Regulation of collagen alpha1(I) expression in hepatic stellate cells. J Gastroenterol. 2000;35 Suppl 12:80–3.PubMedGoogle Scholar
  11. 11.
    Siegmundand SV, Brenner DA. Molecular pathogenesis of alcohol-induced hepatic fibrosis. Alcohol Clin Exp Res. 2005;29:102S–9S.CrossRefGoogle Scholar
  12. 12.
    Lindquist JN, Parsons CJ, Stefanovic B, Brenner DA. Regulation of alpha1(I) collagen messenger RNA decay by interactions with alphaCP at the 3′-untranslated region. J Biol Chem. 2004;279:23822–9.PubMedCrossRefGoogle Scholar
  13. 13.
    Stefanovic B, Hellerbrand C, Holcik M, Briendl M, Aliebhaber S, Brenner DA. Posttranscriptional regulation of collagen alpha1(I) mRNA in hepatic stellate cells. Mol Cell Biol. 1997;17:5201–9.PubMedGoogle Scholar
  14. 14.
    Makeyev AV, Liebhaber SA. The poly(C)-binding proteins: a multiplicity of functions and a search for mechanisms. RNA. 2002;8:265–78.PubMedCrossRefGoogle Scholar
  15. 15.
    Makeyev AV, Eastmond DL, Liebhaber SA. Targeting a KH-domain protein with RNA decoys. RNA. 2002;8:1160–73.PubMedCrossRefGoogle Scholar
  16. 16.
    Paulding WR, Czyzyk-Krzeska MF. Regulation of tyrosine hydroxylase mRNA stability by protein-binding, pyrimidine-rich sequence in the 3′-untranslated region. J Biol Chem. 1999;274:2532–8.PubMedCrossRefGoogle Scholar
  17. 17.
    Czyzyk-Krzeska MF, Bendixen AC. Identification of the poly(C) binding protein in the complex associated with the 3′ untranslated region of erythropoietin messenger RNA. Blood. 1999;93:2111–20.PubMedGoogle Scholar
  18. 18.
    Ji X, Kong J, Liebhaber SA. In vivo association of the stability control protein alphaCP with actively translating mRNAs. Mol Cell Biol. 2003;23:899–907.PubMedCrossRefGoogle Scholar
  19. 19.
    Yu J, Russell JE. Structural and functional analysis of an mRNP complex that mediates the high stability of human beta-globin mRNA. Mol Cell Biol. 2001;21:5879–88.PubMedCrossRefGoogle Scholar
  20. 20.
    Lindquist JN, Kauschke SG, Stefanovic B, Burchardt ER, Brenner DA. Characterization of the interaction between alphaCP(2) and the 3′-untranslated region of collagen alpha1(I) mRNA. Nucleic Acids Res. 2000;28:4306–16.PubMedCrossRefGoogle Scholar
  21. 21.
    Kato J, Sato Y, Inui N, Nakano Y, Takimoto R, Takada K, et al. Ethanol induces transforming growth factor-alpha expression in hepatocytes, leading to stimulation of collagen synthesis by hepatic stellate cells. Alcohol Clin Exp Res. 2003;27:58S–63S.PubMedCrossRefGoogle Scholar
  22. 22.
    Nieto N, Greenwel P, Friedman SL, Zhang F, Dannenberg AJ, Cederbaum AI. Ethanol and arachidonic acid increase alpha 2(I) collagen expression in rat hepatic stellate cells overexpressing cytochrome P450 2E1. Role of H2O2 and cyclooxygenase-2. J Biol Chem. 2000;275:20136–45.PubMedCrossRefGoogle Scholar
  23. 23.
    Anania FA, Womack L, Jiang M, Saxena NK. Aldehydes potentiate alpha(2)(I) collagen gene activity by JNK in hepatic stellate cells. Free Radic Biol Med. 2001;30:846–57.PubMedCrossRefGoogle Scholar
  24. 24.
    Qin B, Cheng K. Silencing of the IKKepsilon gene by siRNA inhibits invasiveness and growth of breast cancer cells. Breast Cancer Res. 12:R74.Google Scholar
  25. 25.
    Tai W, Qin B, Cheng K. Inhibition of breast cancer cell growth and invasiveness by dual silencing of HER-2 and VEGF. Mol Pharm. 7:543–556.Google Scholar
  26. 26.
    Ahmed N, Dreier R, Gopferich A, Grifka J, Grassel S. Soluble signalling factors derived from differentiated cartilage tissue affect chondrogenic differentiation of rat adult marrow stromal cells. Cell Physiol Biochem. 2007;20:665–78.PubMedGoogle Scholar
  27. 27.
    Grassel S, Rickert M, Opolka A, Bosserhoff A, Angele P, Grifka J, et al. Coculture between periosteal explants and articular chondrocytes induces expression of TGF-beta1 and collagen I. Rheumatology (Oxford). 2010;49:218–30.CrossRefGoogle Scholar
  28. 28.
    Mahato R, Qin B, Cheng K. Blocking IKKalpha expression inhibits prostate cancer invasiveness. Pharm Res. 2010.Google Scholar
  29. 29.
    Brewer G. Regulation of c-myc mRNA decay in vitro by a phorbol ester-inducible, ribosome-associated component in differentiating megakaryoblasts. J Biol Chem. 2000;275:33336–45.PubMedCrossRefGoogle Scholar
  30. 30.
    Opyrchal M, Anderson JR, Sokoloski KJ, Wilusz CJ, Wilusz J. A cell-free mRNA stability assay reveals conservation of the enzymes and mechanisms of mRNA decay between mosquito and mammalian cell lines. Insect Biochem Mol Biol. 2005;35:1321–34.PubMedCrossRefGoogle Scholar
  31. 31.
    Fontana L, Jerez D, Rojas-Valencia L, Solis-Herruzo JA, Greenwel P, Rojkind M. Ethanol induces the expression of alpha 1(I) procollagen mRNA in a co-culture system containing a liver stellate cell-line and freshly isolated hepatocytes. Biochim Biophys Acta. 1997;1362:135–44.PubMedGoogle Scholar
  32. 32.
    Tsutsumi M, Takada A, Takase S, Shimanaka K, Enyama K. Effects of ethanol and its metabolites on collagen synthesis by cultured rat liver cells. J Gastroenterol Hepatol. 1989;4:229–40.PubMedCrossRefGoogle Scholar
  33. 33.
    Maher JJ, Zia S, Tzagarakis C. Acetaldehyde-induced stimulation of collagen synthesis and gene expression is dependent on conditions of cell culture: studies with rat lipocytes and fibroblasts. Alcohol Clin Exp Res. 1994;18:403–9.PubMedCrossRefGoogle Scholar
  34. 34.
    Vogel S, Piantedosi R, Frank J, Lalazar A, Rockey DC, Friedman SL, et al. An immortalized rat liver stellate cell line (HSC-T6): a new cell model for the study of retinoid metabolism in vitro. J Lipid Res. 2000;41:882–93.PubMedGoogle Scholar
  35. 35.
    Cheng K, Yang N, Mahato RI. TGF-beta1 gene silencing for treating liver fibrosis. Mol Pharm. 2009;6:772–9.PubMedCrossRefGoogle Scholar
  36. 36.
    Cheng K, Ye Z, Guntaka RV, Mahato RI. Biodistribution and hepatic uptake of triplex-forming oligonucleotides against type alpha1(I) collagen gene promoter in normal and fibrotic rats. Mol Pharm. 2005;2:206–17.PubMedCrossRefGoogle Scholar
  37. 37.
    Cheng K, Ye Z, Guntaka RV, Mahato RI. Enhanced hepatic uptake and bioactivity of type alpha1(I) collagen gene promoter-specific triplex-forming oligonucleotides after conjugation with cholesterol. J Pharmacol Exp Ther. 2006;317:797–805.PubMedCrossRefGoogle Scholar
  38. 38.
    Ye Z, Cheng K, Guntaka RV, Mahato RI. Targeted delivery of a triplex-forming oligonucleotide to hepatic stellate cells. Biochemistry. 2005;44:4466–76.PubMedCrossRefGoogle Scholar
  39. 39.
    Hamlyn AN, Brown AJ, Sherlock S, Baron DN. Casual blood-ethanol estimations in patients with chronic liver disease. Lancet. 1975;2:345–7.PubMedCrossRefGoogle Scholar
  40. 40.
    Israel Y, Britton RS, Orrego H. Liver cell enlargement induced by chronic alcohol consumption: studies on its causes and consequences. Clin Biochem. 1982;15:189–92.PubMedCrossRefGoogle Scholar
  41. 41.
    Hijioka T, Sato N, Matsumura T, Yoshihara H, Takei Y, Fukui H, et al. Ethanol-induced disturbance of hepatic microcirculation and hepatic hypoxia. Biochem Pharmacol. 1991;41:1551–7.PubMedCrossRefGoogle Scholar
  42. 42.
    Oshita M, Sato N, Yoshihara H, Takei Y, Hijioka T, Fukui H, et al. Ethanol-induced vasoconstriction causes focal hepatocellular injury in the isolated perfused rat liver. Hepatology. 1992;16:1007–13.PubMedCrossRefGoogle Scholar
  43. 43.
    Maatta A, Penttinen RP. A fibroblast protein binds the 3′-untranslated region of pro-alpha 1(I) collagen mRNA. Biochem J. 1993;295(Pt 3):691–8.PubMedGoogle Scholar
  44. 44.
    Maatta A, Penttinen RP. Nuclear and cytoplasmic alpha 1 (I) collagen mRNA-binding proteins. FEBS Lett. 1994;340:71–7.PubMedCrossRefGoogle Scholar
  45. 45.
    Wang X, Kiledjian M, Weiss IM, Liebhaber SA. Detection and characterization of a 3′ untranslated region ribonucleoprotein complex associated with human alpha-globin mRNA stability. Mol Cell Biol. 1995;15:1769–77.PubMedGoogle Scholar
  46. 46.
    Weiss IM, Liebhaber SA. Erythroid cell-specific determinants of alpha-globin mRNA stability. Mol Cell Biol. 1994;14:8123–32.PubMedGoogle Scholar
  47. 47.
    Chkheidze AN, Lyakhov DL, Makeyev AV, Morales J, Kong J, Liebhaber SA. Assembly of the alpha-globin mRNA stability complex reflects binary interaction between the pyrimidine-rich 3′ untranslated region determinant and poly(C) binding protein alphaCP. Mol Cell Biol. 1999;19:4572–81.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Ravi S. Shukla
    • 1
  • Bin Qin
    • 1
  • Yu-Jui Yvonne Wan
    • 2
  • Kun Cheng
    • 1
  1. 1.Division of Pharmaceutical Sciences, School of PharmacyUniversity of Missouri-Kansas CityKansas CityUSA
  2. 2.Department of Pharmacology, Toxicology, and TherapeuticsUniversity of Kansas Medical CenterKansas CityUSA

Personalised recommendations