Involvement of Smad proteins in TGF-β 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)
- et al.
A single intraportal administration of follistatin accelerates liver regeneration in partially hepatectomized rats
Gastroenterology
(1995) - et al.
Norepinephrine reverses the effects of activin A on DNA synthesis and apoptosis in cultured rat hepatocytes
Hepatology
(1996) - et al.
Transforming growth factor β1 iuduces nuclear export of inhibitory Smad7
J. Biol. Chem.
(1998) - et al.
Smad7 is an activin-inducible inhibitor of activin-induced growth arrest and apoptosis in mouse B cells
J. Biol. Chem.
(1998) - et al.
Role of growth factors and cytokines in hepatic regeneration
FASEB J.
(1995) - et al.
Liver regeneration
Science
(1997) - et al.
Role of heparin-binding epidermal growth factor-like growth factor as a hepatotrophic factor in rat liver regeneration after partial hepatectomy
Hepatology
(1996) - et al.
Type β transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat
Proc. Natl. Acad. Sci. USA
(1988) - et al.
Transforming growth factor β mRNA increases during liver regeneration: a possible paracrine mechanism of growth regulation
Proc. Natl. Acad. Sci. USA
(1988) - et al.
Cell-specific expression of transforming growth factor-beta in rat liver. Evidence for autocrine regulation of hepatocyte proliferation
J. Clin. Invest.
(1995)
Induction of apoptosis in cultured hepatocytes and in regenerating liver by transforming growth factor β1
Proc. Natl. Acad. Sci. USA
Activin A: an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes
J. Clin. Invest.
Activin induces cell death in hepatocytes in vivo and in vitro
Hepatology
TGF-β signalling from cell membrane to nucleus through SMAD proteins
Nature
TGF-β signal transduction
Annu. Rev. Biochem.
Cited by (38)
Transcription Factor Forkhead Box O1 Mediates Transforming Growth Factor-β1–Induced Apoptosis in Hepatocytes
2023, American Journal of PathologyDown-regulation of miR-23b may contribute to activation of the TGF-β1/Smad3 signalling pathway during the termination stage of liver regeneration
2011, FEBS LettersCitation 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).
Transcriptional effects of progesterone receptor antagonist in rat granulosa cells
2010, Molecular and Cellular EndocrinologyActivin is a potent growth suppressor of epithelial ovarian cancer cells
2009, Cancer LettersCitation 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 EndocrinologyCitation 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