Elsevier

Hepatology Research

Volume 23, Issue 3, July 2002, Pages 211-219
Hepatology Research

Involvement of Smad proteins in TGF-β and activin A-induced apoptosis and growth inhibition of liver cells

https://doi.org/10.1016/S1386-6346(01)00175-9Get rights and content

Abstract

Transforming growth factor (TGF)-β and activin A inhibit the growth and induce cell death of parenchymal liver cells. Smad proteins have recently been identified as intracellular signaling mediators and modulators of TGF-β family members. This study assessed the role of Smad proteins during the action of TGF-β and activin A on liver cells using a well-differentiated human hepatoma cell line, Hep3B cells. To study the role of Smad proteins in the anti-proliferative, apoptosis-inducing, activities of TGF-β and activin A in liver cells, we stably transfected dominant negative Smad2-3SA or Smad3-3SA mutants in Hep3B cells. Transfection of Smad2-3SA or Smad3-3SA abrogated both TGF-β-induced and activin A-induced growth inhibition and apoptosis of Hep3B cells. Down regulation of Bcl-xL expression by TGF-β was both Smad2 and Smad3 dependent. We also demonstrate that transfection of Smad7, an intracellular antagonist of Smad signaling, inhibited both TGF-β- and activin A-induced apoptosis and growth inhibition of these cells. These results suggest that Smad proteins positively and negatively mediate TGF-β-induced and activin A-induced apoptosis and growth inhibition of liver cells.

Introduction

The transforming growth factor (TGF)-β superfamily elicits diverse effects on cellular growth and differentiation, and modulates various cellular functions in many types of cells including liver cells. Various growth factors are synthesized during liver regeneration [1], [2]. While heparin-binding epidermal growth factor-like growth factor (HB-EGF) [3], hepatocyte growth factor (HGF) and TGF-α stimulate the growth of hepatocytes, a member of the TGF-β superfamily, TGF-β, and activin A inhibit their growth. TGF-β induces apoptosis of hepatocytes and inhibits their proliferation in paracrine and autocrine manners [4], [5], [6], [7]. Activin A is an autocrine negative regulator of DNA synthesis in hepatocytes [8], [9]. It also induces cell death of parenchymal liver cells in vitro and in vivo [10], [11].

Substantial progress has been made in the characterization of the membrane receptors and signal transduction system of ligands belonging to this family. Smad proteins are a group of recently identified molecules that function as intracellular signaling mediators and modulators of TGF-β family members [12], [13]. Smads can be classified into three groups: receptor-regulated Smads (R-Smads), common mediator Smads (Co-Smads), and inhibitory Smads. Upon the binding of a ligand to a type II receptor, type II receptor kinases phosphorylate the GS domain of type I receptors, leading to activation of the type I receptor. The activated type I receptor kinases phosphorylate R-Smads differentially at two serine residues in the SSXS motif at their extreme C termini [14], [15]. R-Smads include Smad1, -2, -3, -5 and -8. Smad1 and -5 mediate the signaling of bone morphogenetic protein (BMP) 2 and -4; Smad2 and -3 mediate the signaling of TGF-β and activins; and Smad8 mediates the signaling of ALK-2 receptor kinases. The phosphorylated R-Smads form oligomeric complexes with a Co-Smad, Smad4; the complexes then translocate into the nucleus. These complexes then activate the transcription of target genes. Inhibitory Smads (I-Smads; Smad6 and Smad7) act in opposition to the signal-transducing R- and Co-Smads, by forming stable associations with activated type I receptors and, thus, preventing the phosphorylation of R-Smads [16], [17], [18].

In this study, we studied the roles of Smad proteins in the apoptosis-inducing and anti-proliferative activities of TGF-β and activin A towards liver cells. We altered the C-terminus motif SSXS of Smad2 and Smad3 to AAXA to generate dominant negative mutants [14], [15]. Then, we transfected these mutants in a well-differentiated hepatoma cell line, Hep3B cells. The results indicated that Smad2 and Smad3 are involved in TGF-β and activin A-induced growth inhibition and apoptosis of these cells. Transfection of Smad7 inhibited TGF-β and activin A-induced apoptosis and growth inhibition of these cells.

Section snippets

Materials

Recombinant human TGF-β1 was purchased from R&D System (Minneapolis, MN). Recombinant human activin A was kindly provided by Dr Eto, Ajinomoto, Inc. (Kawasaki, Japan). Mouse EGF was purchased from Sigma (St. Louis, MO). [3H]-thymidine (25 Ci/mmol) was purchased from Amersham International (Arlington Height, IL). Anti-Flag M2 antibody was purchased from Upstate Lab (Lake Placid, NY), and anti-myc antibody and anti-HA antibody were purchased from Roche Molecular Biochemicals (Mannheim, Germany).

Effect of transfection of Smad2-3SA or Smad3-3SA on TGF-β1 and activin A-induced growth inhibition of Hep3B cells

To characterize the role of R-Smads in liver cells, we stably transfected Smad2-3SA or Smad3-3SA into Hep3B cells. Hep3B cells are highly sensitive to TGF-β apoptotic activity under serum-starved conditions [22], [23]. As shown in Fig. 1B and C, TGF-β and activin A each potently inhibited DNA synthesis in Hep3B cells. These cells thus serve as a suitable system to study the growth and apoptosis of hepatocytes. TGF-β1 and activin A potently inhibit DNA synthesis and induce cell death of

Discussion

We evaluated the roles of Smad proteins in the anti-proliferative and apoptosis-inducing activities of TGF-β and activin A towards liver cells. R-Smads are activated by type I receptor kinases and form oligomeric complexes with a Co-Smad, Smad4. The R-and-Co-Smad complexes then translocate into the nucleus, and function as transcriptional regulators that control the expression of target genes [12], [13]. An R-Smad has two unique phosphorylation domains: a linker region and a C-terminal SSXS

Acknowledgements

We would like to thank Dr Y. Eto for recombinant human activin A, Dr Miyazono for Flag-Smad2, -3, -4 and -6 cDNAs, and Dr P. ten Dijke for Flag-Smad7 cDNA. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan to HY.

References (31)

  • F.A. Oberhammer et al.

    Induction of apoptosis in cultured hepatocytes and in regenerating liver by transforming growth factor β1

    Proc. Natl. Acad. Sci. USA

    (1992)
  • H. Yasuda et al.

    Activin A: an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes

    J. Clin. Invest.

    (1993)
  • R.H. Schwall et al.

    Activin induces cell death in hepatocytes in vivo and in vitro

    Hepatology

    (1993)
  • C.H. Heldin et al.

    TGF-β signalling from cell membrane to nucleus through SMAD proteins

    Nature

    (1997)
  • J. Massagué

    TGF-β signal transduction

    Annu. Rev. Biochem.

    (1998)
  • Cited by (38)

    • Down-regulation of miR-23b may contribute to activation of the TGF-β1/Smad3 signalling pathway during the termination stage of liver regeneration

      2011, FEBS Letters
      Citation Excerpt :

      Therefore, we hypothesise that miR-23b may be linked to LR termination through these pathways by the target genes associated with them. Smads (Smad3, Smad4 and Smad5), which are downstream proteins of TGF-β1 signalling, have been reported to be the target genes of miR-23b in murine fetal liver stem cells (HBC-3 cells) [15,19]. However, results from our real-time PCR and western blot analysis revealed that with treatment of miR-23b mimics (150 pmol in 24-well plate) in BRL-3A cells, Smad3 expression notably decreased in both mRNA and protein level; Smad4 showed no apparent change; Smad5 mRNA expression showed a significant increase, while Smad5 protein expression had no alterations (Fig. 3A and B).

    • Activin is a potent growth suppressor of epithelial ovarian cancer cells

      2009, Cancer Letters
      Citation Excerpt :

      Activin shares its intracellular signalling pathway with TGFβ, a well-documented growth suppressor of many epithelial cancers, including epithelial ovarian cancer (EOC) [3,4]. In agreement with this, activin is a growth inhibitor of cancer cell lines derived from organs such as the prostate [5], liver [6,7] and breast [8]. In addition, inactivating receptor mutations have been reported for ACTRIIA and ACTRIB in gastrointestinal [9] and pancreatic carcinoma [10], respectively.

    • Expression and localization of activin receptor-interacting protein 2 in mouse tissues

      2009, General and Comparative Endocrinology
      Citation Excerpt :

      Activins are widely distributed in embryonic and adult tissues (Kingsley, 1994; Roberts et al., 1996; Liu et al., 1996b; Wu et al., 1999; Mousa and Mousa, 2003; Phillips et al., 2006). They play important roles in the nervous system (Krieglstein et al., 1995; Iwahori et al., 1997; Hughes et al., 1999; Shoji-Kasai et al., 2007), early development of embryos, hematopoiesis, apoptosis and hormone releases as a multifunctional growth and differentiation factor (Thomsen et al., 1990; Oda et al., 1995; Kamamaru et al., 2002; Sugama et al., 2007; Wang et al., 2008). Activin receptors are transmembrane proteins and have the serine/threonine kinase domain in their cytoplasmic regions.

    • The activin axis in liver biology and disease

      2006, Mutation Research - Reviews in Mutation Research
    View all citing articles on Scopus
    View full text