Skip to main content

Advertisement

SpringerLink
Log in

Identification and characterization of functional Smad8 and Smad4 homologues from Echinococcus granulosus

  • Original Paper
  • Published:
Parasitology Research Aims and scope Submit manuscript

Abstract

Smad family proteins are essential cellular mediators of the transforming growth factor-β superfamily. In the present study, we identified two members of the Smad proteins, Smad8 and Smad4 homologues (termed as EgSmadE and EgSmadD, respectively), from Echinococcus granulosus, the causative agent of cystic echinococcosis (CE). Phylogenetic analysis placed EgSmadE in the Smad1, 5, and 8 subgroup of the R-Smad sub-family and EgSmadD in the Co-Smad family. Furthermore, EgSmadE and EgSmadD attained a high homology to EmSmadE and EmSmadD of E. multilocularis, respectively. Both EgSmadE and EgSmadD were co-expressed in the larval stages and exhibited the highest transcript levels in activated protoscoleces, and their encoded proteins were co-localized in the sub-tegumental and tegumental layer of the parasite. As shown by yeast two-hybrid and pull-down analysis, EgSmadE displayed a positive binding interaction with EgSmadD. In addition, EgSmadE localized in the nuclei of Mv1Lu cells (mink lung epithelial cells) upon treatment with human TGF-β1 or human BMP2, indicating that EgSmadE is capable of being translocated into nucleus, in vitro. Our study suggests that EgSmadE and EgSmadD may take part in critical biological processes, including echinococcal growth, development, and parasite-host interaction.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Beall MJ, Pearce EJ (2002) Transforming growth factor-beta and insulin-like signalling pathways in parasitic helminths. Int J Parasitol 32(4):399–404

    Article  PubMed  CAS  Google Scholar 

  • Beall M, McGonigle S, Pearce E (2000) Functional conservation of Schistosoma mansoni Smads in TGF-beta signaling. Mol Biochem Parasitol 111(1):131–142

    Article  PubMed  CAS  Google Scholar 

  • Brehm K (2010a) Echinococcus multilocularis as an experimental model in stem cell research and molecular host-parasite interaction. Parasitology 137(3):537–555

    Article  PubMed  CAS  Google Scholar 

  • Brehm K (2010b) The role of evolutionarily conserved signalling systems in Echinococcus multilocularis development and host-parasite interaction. Med Microbiol Immunol 199(3):247–259

    Article  PubMed  CAS  Google Scholar 

  • Brehm K, Spiliotis M (2008) The influence of host hormones and cytokines on Echinococcus multilocularis signalling and development. Parasite 15(3):286–290

    Article  PubMed  CAS  Google Scholar 

  • Brehm K, Wolf M, Beland H, Kroner A, Frosch M (2003) Analysis of differential gene expression in Echinococcus multilocularis larval stages by means of spliced leader differential display. Int J Parasitol 33(11):1145–1159

    Article  PubMed  CAS  Google Scholar 

  • Calonge MJ, Massague J (1999) Smad4/DPC4 silencing and hyperactive ras jointly disrupt transforming growth factor-beta antiproliferative responses in colon cancer cells. J Biol Chem 274(47):33637–33643

    Article  PubMed  CAS  Google Scholar 

  • Camicia F, Paredes R, Chalar C, Galanti N, Kamenetzky L, Gutierrez A, Rosenzvit MC (2008) Sequencing, bioinformatic characterization and expression pattern of a putative amino acid transporter from the parasitic cestode Echinococcus granulosus. Gene 411:1–9

    Article  PubMed  CAS  Google Scholar 

  • Carlo J, Osman A, Niles EG, Wu W, Fantappie MR, Oliveira FM, LoVerde PT (2007) Identification and characterization of an R-Smad ortholog (SmSmad1B) from Schistosoma mansoni. FEBS J 274(16):4075–4093

    Article  PubMed  CAS  Google Scholar 

  • de Caestecker MP, Yahata T, Wang D, Parks WT, Huang S, Hill CS, Shioda T, Roberts AB, Lechleider RJ (2000) The Smad4 activation domain (SAD) is a proline-rich, p300-dependent transcriptional activation domain. J Biol Chem 275(3):2115–2122

    Article  PubMed  Google Scholar 

  • Ebisawa T, Fukuchi M, Murakami G, Chiba T, Tanaka K, Imamura T, Miyazono K (2001) Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation. J Biol Chem 276(16):12477–12480

    Article  PubMed  CAS  Google Scholar 

  • Epping K, Brehm K (2011) Echinococcus multilocularis: molecular characterization of EmSmadE, a novel BR-Smad involved in TGF-β and BMP signaling. Exp Parasitol 129(2):85–94

    Article  PubMed  CAS  Google Scholar 

  • Freitas TC, Jung E, Pearce EJ (2007) TGF-beta signaling controls embryo development in the parasitic flatworm Schistosoma mansoni. PLoS Pathog 3(4):e52

    Article  PubMed  PubMed Central  Google Scholar 

  • Funaba M, Mathews LS (2000) Identification and characterization of constitutively active Smad2 mutants: evaluation of formation of Smad complex and subcellular distribution. Mol Endocrinol 14(10):1583–1591

    Article  PubMed  CAS  Google Scholar 

  • Gelmedin V, Caballero-Gamiz R, Brehm K (2008) Characterization and inhibition of a p38-like mitogen-activated protein kinase (MAPK) from Echinococcus multilocularis: antiparasitic activities of p38 MAPK inhibitors. Biochem Pharmacol 76(9):1068–1081

    Article  PubMed  CAS  Google Scholar 

  • Gemma A, Hagiwara K, Vincent F, Ke Y, Hancock AR, Nagashima M, Bennett WP, Harris CC (1998) hSmad5 gene, a human hSmad family member: its full length cDNA, genomic structure, promoter region and mutation analysis in human tumors. Oncogene 16(7):951–956

    Article  PubMed  CAS  Google Scholar 

  • Hemer S, Konrad C, Spiliotis M, Koziol U, Schaack D, Förster S, Gelmedin V, Stadelmann B, Dandekar T, Hemphill A, Brehm K (2014) Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development. BMC Biol 12:5. doi:10.1186/1741-7007-12-5

    Article  PubMed  PubMed Central  Google Scholar 

  • Huang S, Flanders KC, Roberts AB (2000) Characterization of the mouse Smad1 gene and its expression pattern in adult mouse tissues. Gene 258(1–2):43–53

    Article  PubMed  CAS  Google Scholar 

  • Huse M, Muir TW, Xu L, Chen YG, Kuriyan J, Massague J (2001) The TGF beta receptor activation process: an inhibitor- to substrate-binding switch. Mol Cell 8(3):671–682

    Article  PubMed  CAS  Google Scholar 

  • Jeong W, Park O, Suh YG, Byun JS, Park SY, Choi E, Kim JK, Ko H, Wang H, Miller AM, Gao B (2011) Suppression of innate immunity (natural killer cell/interferon-γ) in the advanced stages of liver fibrosis in mice. Hepatology 53(4):1342–1351

    Article  PubMed  CAS  Google Scholar 

  • Kawai S, Faucheu C, Gallea S, Spinella-Jaegle S, Atfi A, Baron R, Roman SR (2000) Mouse smad8 phosphorylation downstream of BMP receptors ALK-2, ALK-3, and ALK-6 induces its association with Smad4 and transcriptional activity. Biochem Biophys Res Commun 271(3):682–687

    Article  PubMed  CAS  Google Scholar 

  • Kern P (2003) Echinococcus granulosus infection: clinical presentation, medical treatment and outcome. Langenbecks Arch Surg 388(6):413–420

    Article  PubMed  Google Scholar 

  • Konrad C, Kroner A, Spiliotis M, Zavala-Gongora R, Brehm K (2003) Identification and molecular characterisation of a gene encoding a member of the insulin receptor family in Echinococcus multilocularis. Int J Parasitol 33(3):301–312

    Article  PubMed  CAS  Google Scholar 

  • Kretzschmar M, Liu F, Hata A, Doody J, Massague J (1997) The TGF-beta family mediator Smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase. Genes Dev 11(8):984–995

    Article  PubMed  CAS  Google Scholar 

  • Kretzschmar M, Doody J, Timokhina I, Massague J (1999) A mechanism of repression of TGFbeta/Smad signaling by oncogenic Ras. Genes Dev 13(7):804–816

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Li J, Zhang CS, Lü GD, Wang JH, Wen H, Yan GQ, Wei XF, Lin RY (2011) Molecular characterization of a signal-regulated kinase homolog from Echinococcus granulosus. Chin Med J (Engl) 124(18):2838–2844

    CAS  Google Scholar 

  • Macias-Silva M, Abdollah S, Hoodless P, Pirone R, Attisano L, Wrana J (1996) MADR2 is a substrate of the TGFbeta receptor and its phosphorylation is required for nuclear accumulation and signaling. Cell 87(7):1215–1224

    Article  PubMed  CAS  Google Scholar 

  • Massague J, Wotton D (2000) Transcriptional control by the TGF-beta/Smad signaling system. EMBO J 19(8):1745–1754

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Moustakas A, Souchelnytskyi S, Heldin C-H (2001) Smad regulation in TGF-beta signal transduction. J Cell Sci 114(24):4359–4369

    PubMed  CAS  Google Scholar 

  • Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin CH, Miyazono K, ten Dijke P (1997) TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J 16(17):5353–5362

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Osman A, Niles EG, LoVerde PT (2001) Identification and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor-beta signal transducer. J Biol Chem 276(13):10072–10082

    Article  PubMed  CAS  Google Scholar 

  • Osman A, Niles EG, LoVerde PT (2004) Expression of functional Schistosoma mansoni Smad4: role in Erk-mediated transforming growth factor beta (TGF-beta) down-regulation. J Biol Chem 279(8):6474–6486

    Article  PubMed  CAS  Google Scholar 

  • Osman A, Niles EG, Verjovski-Almeida S, LoVerde PT (2006) Schistosoma mansoni TGF-beta receptor II: role in host ligand-induced regulation of a schistosome target gene. PLoS Pathog 2(6):e54

    Article  PubMed  PubMed Central  Google Scholar 

  • Qin B, Lam SS, Lin K (1999) Crystal structure of a transcriptionally active Smad4 fragment. Structure 7(12):1493–1503

    Article  PubMed  CAS  Google Scholar 

  • Raftery LA, Sutherland DJ (1999) TGF-beta family signal transduction in Drosophila development: from Mad to Smads. Dev Biol 210(2):251–268

    Article  PubMed  CAS  Google Scholar 

  • Rezaei H, Kamato D, Ansari G, Osman N, Little P (2012) Cell biology of Smad2/3 linker region phosphorylation in vascular smooth muscle. Clin Exp Pharmacol Physiol 39(8):661–667

    Article  PubMed  CAS  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425

    PubMed  CAS  Google Scholar 

  • Savage-Dunn C (2001) Targets of TGF beta-related signaling in Caenorhabditis elegans. Cytokine Growth Factor Rev 12(4):305–312

    Article  PubMed  CAS  Google Scholar 

  • Shachar I, Karin N (2013) The dual roles of inflammatory cytokines and chemokines in the regulation of autoimmune diseases and their clinical implications. J Leukoc Biol 93(1):51–61

    Article  PubMed  CAS  Google Scholar 

  • Sharma V, Antonacopoulou AG, Tanaka S, Panoutsopoulos AA, Bravou V, Kalofonos HP, Episkopou V (2011) Enhancement of TGF-β signaling responses by the E3 ubiquitin ligase arkadia provides tumor suppression in colorectal cancer. Cancer Res 71(20):6438–6449

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Shi Y, Wang YF, Jayaraman L, Yang H, Massague J, Pavletich NP (1998) Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling. Cell 94(5):585–594

    Article  PubMed  CAS  Google Scholar 

  • Spiliotis M, Kroner A, Brehm K (2003) Identification, molecular characterization and expression of the gene encoding the epidermal growth factor receptor orthologue from the fox-tapeworm Echinococcus multilocularis. Gene 323:57–65

    Article  PubMed  CAS  Google Scholar 

  • Spiliotis M, Konrad C, Gelmedin V, Tappe D, Brückner S, Mösch HU, Brehm K (2006) Characterisation of EmMPK1, an ERK-like MAP kinase from Echinococcus multilocularis which is activated in response to human epidermal growth factor. Int J Parasitol 36(10–11):1097–1112

    Article  PubMed  CAS  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Tsai IJ, Zarowiecki M, Holroyd N, Garciarrubio A, Sanchez-Flores A, Brooks KL, Tracey A, Bobes RJ, Fragoso G, Sciutto E, Aslett M, Beasley H, Bennett HM, Cai J, Camicia F, Clark R, Cucher M, De Silva N, Day TA, Deplazes P, Estrada K, Fernández C, Holland PW, Hou J, Hu S, Huckvale T, Hung SS, Kamenetzky L, Keane JA, Kiss F, Koziol U, Lambert O, Liu K, Luo X, Luo Y, Macchiaroli N, Nichol S, Paps J, Parkinson J, Pouchkina-Stantcheva N, Riddiford N, Rosenzvit M, Salinas G, Wasmuth JD, Zamanian M, Zheng Y, Taenia solium Genome Consortium, Cai X, Soberón X, Olson PD, Laclette JP, Brehm K, Berriman M (2013) The genomes of four tapeworm species reveal adaptations to parasitism. Nature 496(7443):57–63

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Vuitton DA, Gottstein B (2010) Echinococcus multilocularis and its intermediate host: a model of parasite-host interplay. J Biomed Biotechnol 2010:923–193

    Article  Google Scholar 

  • Watanabe T, Suzuki M, Omori Y, Hishigaki H, Horie M, Kanemoto N, Fujiwara T, Nakamura Y, Takahashi E (1997) Cloning and characterization of a novel member of the human Mad gene family (MADH6). Genomics 42(3):446–451

    Article  PubMed  CAS  Google Scholar 

  • Zavala-Gongora R, Kroner A, Wittek B, Knaus P, Brehm K (2003) Identification and characterisation of two distinct Smad proteins from the fox-tapeworm Echinococcus multilocularis. Int J Parasitol 33(14):1665–1677

    Article  PubMed  CAS  Google Scholar 

  • Zavala-Gongora R, Kroner A, Bernthaler P, Knaus P, Brehm K (2006) A member of the transforming growth factor-beta receptor family from Echinococcus multilocularis is activated by human bone morphogenetic protein 2. Mol Biochem Parasitol 146(2):265–271

    Article  PubMed  CAS  Google Scholar 

  • Zavala-Gongora R, Derrer B, Gelmedin V, Knaus P, Brehm K (2008) Molecular characterisation of a second structurally unusual AR-Smad without an MH1 domain and a Smad4 orthologue from Echinococcus multilocularis. Int J Parasitol 38(2):161–176

    Article  PubMed  CAS  Google Scholar 

  • Zhang Y, Feng X, We R, Derynck R (1996) Receptor-associated Mad homologues synergize as effectors of the TGF-beta response. Nature 383(6596):168–172

    Article  PubMed  CAS  Google Scholar 

  • Zhang WB, Jones MK, Li J, McManus DP (2005) Echinococcus granulosus: pre-culture of protoscoleces in vitro significantly increases development and viability of secondary hydatid cysts in mice. Exp Parasitol 110(1):88–90

    Article  PubMed  Google Scholar 

  • Zheng H, Zhang W, Zhang L, Zhang Z, Li J, Lu G, Zhu Y, Wang Y, Huang Y, Liu J, Kang H, Chen J, Wang L, Chen A, Yu S, Gao Z, Jin L, Gu W, Wang Z, Zhao L, Shi B, Wen H, Lin R, Jones MK, Brejova B, Vinar T, Zhao G, McManus DP, Chen Z, Zhou Y, Wang S (2013) The genome of the hydatid tapeworm Echinococcus granulosus. Nat Genet 45(10):1168–1175

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by NSFC Grant Projects (81260252, 81101271, 81260452, 30960342), the Program for Changjiang Scholars and Innovative Research Team in Universities (IRT1181). We would like to thank Liang Li, Hui Liu, Hanhua Hu, Fenglian Xie, and Jianan Tang for their excellent technical assistance.

Ethic and statement

All animal procedures were approved by the Animal Care and Use Committee and the Ethical Committee of The First Affiliated Hospital of Xinjiang Medical University (IACUC-20130514008).

Conflict of interest

The authors declare that they all read and approved the final version of the manuscript sent for publication to Parasitology Research and that they have no conflict of interest that could influence the conclusions of this paper.

Author information

Authors and Affiliations

  1. Xinjiang Key Laboratory of Echinococcosis, The First Affiliated Hospital of Xinjiang Medical University, No.137 Liyushan South Road, Urumqi, 830054, Xinjiang, China

    Chuanshan Zhang, Limin Wang, Hui Wang, Hongwei Pu, Le Yang, Jing Li, Junhua Wang, Guodong Lü, Xiaomei Lu, Wenbao Zhang & Renyong Lin

  2. State Key Laboratory Incubation Base of Xinjiang Major Diseases Research (2010DS890298), The First Affiliated Hospital of Xinjiang Medical University, No.137 Liyushan South Road, Urumqi, 830054, Xinjiang, China

    Hao Wen

  3. WHO-Collaborating Centre for the Prevention and Treatment of Human Echinococcosis, Department of Digestive Surgery; Jean Minjoz Hospital, University of Franche-Comté and University Hospital, Besançon, France

    Dominique A. Vuitton

  4. Research Unit EA 3181 “Epithelial carcinogenesis: predictive and prognostic factors”, University of Franche-Comté, Besançon, France

    Dominique A. Vuitton

Authors
  1. Chuanshan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Limin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hui Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hongwei Pu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Le Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jing Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Junhua Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Guodong Lü
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Xiaomei Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Wenbao Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Dominique A. Vuitton
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Hao Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Renyong Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Hao Wen or Renyong Lin.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 101 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, C., Wang, L., Wang, H. et al. Identification and characterization of functional Smad8 and Smad4 homologues from Echinococcus granulosus . Parasitol Res 113, 3745–3757 (2014). https://doi.org/10.1007/s00436-014-4040-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-014-4040-4

Keywords

Search

Navigation