Activin A induces SLC5A8 expression through the Smad3 signaling pathway in human colon cancer RKO cells

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Abstract

SLC5A8 (Solute carrier family 5, member 8), proposed to be a potential tumor suppressor gene, is down-regulated by epigenetic changes in some colorectal cancer cells, and ectopic expression of SLC5A8 in SLC5A8-deficient colon cancer cell lines leads to suppression of the colony-forming ability of these cells. Activin A, a member of the transforming growth factor-β (TGF-β) superfamily, has been shown to inhibit the proliferation of a variety of tumor (and normal) human cell types. However, the mechanism(s) by which activin A exerts its inhibitory effects are not yet understood. In this study, we showed that activin A up-regulated SLC5A8 expression in colorectal cancer RKO cells and human embryonic kidney (HEK) 293T cells. To elucidate the underlying mechanism involved in this process, we investigated the activation of the Smad signaling pathway, and analyzed the effects of dominant negative Smad3 and Smad2 proteins on activin A-induced SLC5A8 expression. The results indicated that activin A-induced SLC5A8 expression was dependent on activation of Smad3. Further analysis showed that activin A induced SLC5A8 expression via transcriptional activation. Deletion analysis indicated that the CAGA elements located within the -273/-222 region of the human SLC5A8 promoter were responsive to activin A. Taken together, our results strongly suggest that activin A up-regulates SLC5A8 expression through the Smad signaling pathway, which also partially explains the inhibitory effects of activin A in RKO cells.

Introduction

Colorectal cancer is the second leading cancer in the United States (Jemal et al., 2007). In China, the incidence of colorectal cancer has significantly risen in recent years. In addition to environmental and dietary factors, changes in the expression of certain genes have been associated with the occurrence of colorectal cancer. SLC5A8 was first identified as a Na+-coupled transporter gene, which is down-regulated in colorectal cancer cell lines and functions as a tumor suppressor (Li et al., 2003). Recently, the expression of SLC5A8 was shown to be down-regulated in cancers of a variety of non-colonic tissues, including kidney, stomach, thyroid, breast, pancreas, and brain (Ueno et al., 2004, Ganapathy et al., 2005, Hong et al., 2005, Porra et al., 2005, Gupta et al., 2006, Thangaraju et al., 2006). Meanwhile, ectopic expression of SLC5A8 in SLC5A8-deficient colon cancer cell lines leads to suppression of colony-forming ability of these cells (Hong et al., 2005). The tumor-suppressive function is closely related to its ability to mediate uptake of butyrate, pyruvate, and propionate, all of which are histone deacetylase inhibitors and protective against colorectal cancer (Miyauchi et al., 2004). Thus, obtaining a better understanding of the mechanisms that regulate SLC5A8 expression as a colorectal cancer marker is critical in the prevention and treatment of this disease.

Activins, which are members of the transforming growth factor-β (TGF-β) superfamily, regulate a number of different cell functions. In cancer, activin A inhibits the proliferation of a variety of human tumor cell types by blocking cell cycle progression from G1 to S phase (Chen et al., 2002, Chen et al., 2006, Burdette et al., 2005). Indeed, overexpression of activin A in human cancer cells can inhibit proliferation, induce apoptosis, and decrease the tumorigenicity of these cells (Zhang et al., 1997). However, the mechanism(s) by which activin A exerts its inhibitory effects are not yet fully understood.

Activin A transduces its signals via binding to activin type II receptors, ActR-II, and ActR-IIB. The ligand/type II receptor complex then recruits, binds, and trans-phosphorylates the type I receptor, which then phosphorylates the intracellular signaling proteins Smad2 and Smad3 on two serine residues in the conserved carboxy-terminal SSXS motif (Nakao et al., 1997). Phosphorylated Smads rapidly dissociate from the receptor complex and bind a co-factor, Smad4, before trans-locating to the nucleus where they interact with DNA binding proteins, co-activators and co-repressors to regulate target gene transcription (Attisano and Wrana, 2002). Although Smads 2 and 3 share significant sequence and structural homology, they have non-redundant roles in affecting growth inhibition and apoptosis. Smad3 has been shown also to be involved in the induction of growth inhibition in response to TGF-β stimulation (Yanagisawa et al., 1998, Brodin et al., 1999, Pardali et al., 2000, Roberts et al., 2006).

Although the Smad pathway represents the canonical signaling pathway stimulated by activin and TGF-β, other intracellular cascades are known to mediate signaling by these growth factor receptors. In particular, the mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs) (Frey and Mulder, 1997, Hartsough and Mulder, 1997), c-Jun N-terminal kinases (JNKs) (Engel et al., 1999, Hocevar et al., 1999), and p38 kinases (Hanafusa et al., 1999, Bakin et al., 2000, Cocolakis et al., 2001) have been shown to act downstream of the TGF-β receptor complex. Activation of these distinct signaling pathways leads to both Smad-dependent and Smad-independent responses in a cell and in a tissue-specific manner (Massague et al., 2000, Derynck and Zhang, 2003).

This general mechanism underlies a large number of TGF-β gene responses controlling cell proliferation, organization, and fate. Therefore, disregulation in this TGF-β/activin signaling pathway would be likely be associated with carcinogenesis. In the present study, we tested the inhibitory effect of activin A in SLC5A8-deficient colon cancer cell lines. We demonstrated that activin A-induced SLC5A8 expression, and the overexpression of SLC5A8 suppressed cell colony formation, which partly explained the inhibitory effect mediated by activin A. We also explored the involvement of the Smad signaling pathway in activin A-mediated up-regulation of SLC5A8 transcriptional activation and expression. Thus, in defining the mechanisms of SLC5A8 gene regulation by activin A, our study will help to explain the role of tumor suppressor genes in tumorigenesis and further the development of colorectal cancer treatment and diagnosis.

Section snippets

Cell lines

HEK 293T, and human colorectal cancer cells (RKO) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, invitrogen, USA) with 10% fetal bovine serum (TBD Science, Tianjin, China), 100 U/ml penicillin and 100 μg/ml streptomycin (Ameresco, USA), at 37 °C, 5% CO2.

Antibodies and reagents

Polyclonal antibody against SLC5A8 was a gift from Aviva Systems Biology (Beijing, China). The mouse monoclonal antibody against GAPDH was purchased from Kangcheng Bio-tech (Shanghai, China), and the polyclonal antibodies

Activin A inhibits the proliferation of RKO cells

Activin A has been shown to inhibit the proliferation of a variety of human tumor (and normal) cell types (Cocolakis et al., 2001, Burdette et al., 2005, Wang et al., 2009). However, the effect of activin A on colorectal cancer cells is still unclear. Therefore, we first examined the effect of activin A on cell viability by treating the human colon cancer RKO cells with three concentrations of activin A (10, 20, and 40 ng/ml). After 24 h of treatment, the viability of the cells was determined by

Discussion

Colorectal cancer is becoming one of the most prevalent cancers in the world. Recently, SLC5A8 was first identified as a putative tumor suppressor gene in colorectal cancers, and down-regulation of SLC5A8 in colorectal cancer was determined to be partially due to aberrant DNA methylation. In our study we found that that 5-aza-dC exhibited anticancer effects, perhaps through restoration of SLC5A8 expression in the RKO colon cancer cells (Fig. 1). Epigenetic changes are highly relevant to colon

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (No. 30873409 and No. 30670220), the Program for New Century Excellent Talents in University (No. NCET-06-0320), the Jilin Technology R&D Program (NO. 20100911) and the Cultivation Fund of the Scientific and Technical Innovation Project of Northeast Normal University (No. NENU-STB07008).

References (41)

  • K. Pardali et al.

    Role of Smad proteins and transcription factor Sp1 in p21(Waf1/Cip1) regulation by transforming growth factor-beta

    J Biol Chem

    (2000)
  • E. Piek et al.

    Functional characterization of transforming growth factor beta signaling in Smad2- and Smad3-deficient fibroblasts

    J Biol Chem

    (2001)
  • A.B. Roberts et al.

    Smad3 is key to TGF-beta-mediated epithelial-to-mesenchymal transition, fibrosis, tumor suppression and metastasis

    Cytokine Growth Factor Rev

    (2006)
  • L.C. Spender et al.

    TGF-beta induces growth arrest in Burkitt lymphoma cells via transcriptional repression of E2F-1

    J Biol Chem

    (2009)
  • B. Wang et al.

    Involvement of ERK, Bcl-2 family and caspase 3 in recombinant human activin A-induced apoptosis in A549

    Toxicology

    (2009)
  • Y. Zhang et al.

    Butyrate induces cell apoptosis through activation of JNK MAP kinase pathway in human colon cancer RKO cells

    Chem Biol Interact

    (2010)
  • Y. Zhang et al.

    Identification and characterization of the human SLC5A8 gene promoter

    Cancer Genet Cytogenet

    (2010)
  • Z. Zhang et al.

    Regulation of growth and prostatic marker expression by activin A in an androgen-sensitive prostate cancer cell line LNCAP

    Biochem Biophys Res Commun

    (1997)
  • L. Attisano et al.

    Signal transduction by the TGF-beta superfamily

    Science

    (2002)
  • Y.L. Bao et al.

    Synergistic activity of activin A and basic fibroblast growth factor on tyrosine hydroxylase expression through Smad3 and ERK1/ERK2 MAPK signaling pathways

    J Endocrinol

    (2005)
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