Skip to main content
SpringerLink
Log in

Characterization, promoter analysis and expression of the interleukin-6 gene in blunt snout bream, Megalobrama amblycephala

  • Published:
Fish Physiology and Biochemistry Aims and scope Submit manuscript

Abstract

Interleukin-6 (IL-6) is one of the most important multifunctional cytokines, playing essential roles in mediating the innate and adaptive immune responses. In this study, il-6 gene and its promoter from blunt snout bream, Megalobrama amblycephala, were characterized, and its expression at the transcript level in healthy fish and after bacterial infection was determined by quantitative real-time PCR. The results showed that the M. amblycephala il-6 (Mamil-6) cDNA had an ORF of 699 bp, encoding 232 amino acids, and contained 9 instable motifs in the 3′ UTR. The deduced MamIL-6 possessed a 24-amino acid signal peptide and was located in the cytoplasm. Although sequence alignment and phylogenetic analysis revealed that IL-6 is poorly conserved in vertebrates, the protein and genomic structure of il-6 gene was well conserved. Analysis of the Mamil-6 promoter revealed the presence of a conserved TATA box and six major cis-regulatory elements, including C/EBPβ (NF-IL6), AP-1, CRE, GRE, GATA and NF-κB binding sites. In healthy fish, Mamil-6 was the most abundant in the spleen. After Aeromonas hydrophila infection, Mamil-6 was significantly up-regulated in all 6 immune-related tissues examined, suggesting that Mamil-6 plays an important role in the blunt snout bream immune system.

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

  • Ammit AJ, Lazaar AL, Irani C, O’Neill GM, Gordon ND, Amrani Y, Penn RB, Panettieri RA Jr (2002) Tumor necrosis factor-alpha-induced secretion of RANTES and interleukin-6 from human airway smooth muscle cells: modulation by glucocorticoids and beta-agonists. Am J Respir Cell Mol Biol 26:465–474

    Article  CAS  PubMed  Google Scholar 

  • Armenante F, Merola M, Furia A, Tovey M, Palmieri M (1999) Interleukin-6 repression is associated with a distinctive chromatin structure of the gene. Nucleic Acids Res 27:4483–4490

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ataie-Kachoie P, Pourgholami MH, Richardson DR, Morris DL (2014) Gene of the month: interleukin 6 (IL-6). J Clin Pathol 67:932–937

    Article  CAS  PubMed  Google Scholar 

  • Bazan JF (1990) Haemopoietic receptors and helical cytokines. Immunol Today 11:350–354

    Article  CAS  PubMed  Google Scholar 

  • Beineke A, Siebert U, van Elk N, Baumgartner W (2004) Development of a lymphocyte-transformation-assay for peripheral blood lymphocytes of the harbor porpoise and detection of cytokines using the reverse-transcription polymerase chain reaction. Vet Immunol Immunopathol 98:59–68

    Article  CAS  PubMed  Google Scholar 

  • Bird S, Zou J, Savan R, Kono T, Sakai M, Woo J, Secombes C (2005) Characterisation and expression analysis of an interleukin 6 homologue in the Japanese pufferfish, Fugu rubripes. Dev Comp Immunol 29:775–789

    Article  CAS  PubMed  Google Scholar 

  • Caput D, Beutler B, Hartog K, Thayer R, Brown-Shimer S, Cerami A (1986) Identification of a common nucleotide sequence in the 3′-untranslated region of mRNA molecules specifying inflammatory mediators. Proc Natl Acad Sci USA 83:1670–1674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Castellana B, Iliev DB, Sepulcre MP, MacKenzie S, Goetz FW, Mulero V, Planas JV (2008) Molecular characterization of interleukin-6 in the gilthead seabream (Sparus aurata). Mol Immunol 45:3363–3370

    Article  CAS  PubMed  Google Scholar 

  • Castellana B, Marin-Juez R, Planas JV (2013) Transcriptional regulation of the gilthead seabream (Sparus aurata) interleukin-6 gene promoter. Fish Shellfish Immunol 35:71–78

    Article  CAS  PubMed  Google Scholar 

  • Chomarat P, Banchereau J, Davoust J, Palucka AK (2000) IL-6 switches the differentiation of monocytes from dendritic cells to macrophages. Nat Immunol 1:510–514

    Article  CAS  PubMed  Google Scholar 

  • Clark A (2000) Post-transcriptional regulation of pro-inflammatory gene expression. Arthritis Res 2:172–174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Clogston CL, Boone TC, Crandall BC, Mendiaz EA, Lu HS (1989) Disulfide structures of human interleukin-6 are similar to those of human granulocyte colony stimulating factor. Arch Biochem Biophys 272:144–151

    Article  CAS  PubMed  Google Scholar 

  • Dendorfer U, Oettgen P, Libermann T (1994) Multiple regulatory elements in the interleukin-6 gene mediate induction by prostaglandins, cyclic AMP, and lipopolysaccharide. Mol Cell Biol 14:4443–4454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ding Z, Wu J, Su L, Zhou F, Zhao X, Deng W, Zhang J, Liu S, Wang W, Liu H (2013) Expression of heat shock protein 90 genes during early development and infection in Megalobrama amblycephala and evidence for adaptive evolution in teleost. Dev Comp Immunol 41:683–693

    Article  CAS  PubMed  Google Scholar 

  • Ebrahimi B, Roy DJ, Bird P, Sargan DR (1995) Cloning, sequencing and expression of the ovine interleukin 6 gene. Cytokine 7:232–236

    Article  CAS  PubMed  Google Scholar 

  • Faggioli L, Merola M, Hiscott J, Furia A, Monese R, Tovey M, Palmieri M (1997) Molecular mechanisms regulating induction of interleukin-6 gene transcription by interferon-gamma. Eur J Immunol 27:3022–3030

    Article  CAS  PubMed  Google Scholar 

  • FBAM (2012) Chinese Fisheries Yearbook. China Agriculture Press, Beijing (in Chinese)

    Google Scholar 

  • Iliev DB, Castellana B, Mackenzie S, Planas JV, Goetz FW (2007) Cloning and expression analysis of an IL-6 homolog in rainbow trout (Oncorhynchus mykiss). Mol Immunol 44:1803–1807

    Article  CAS  PubMed  Google Scholar 

  • Kaiser P, Rothwell L, Avery S, Balu S (2004) Evolution of the interleukins. Dev Comp Immunol 28:375–394

    Article  CAS  PubMed  Google Scholar 

  • Kang SH, Brown DA, Kitajima I, Xu X, Heidenreich O, Gryaznov S, Nerenberg M (1996) Binding and functional effects of transcriptional factor Sp1 on the murine interleukin-6 promotor. J Biol Chem 271:7330–7335

    Article  CAS  PubMed  Google Scholar 

  • Kishimoto T (2003) Interleukin-6. In: Thomson A, Lotze M (eds) The cytokine handbook, 4th edn. Academic Press, San Diego, pp 281–304

    Chapter  Google Scholar 

  • Kong HJ, Nam BH, Kim YO, Kim WJ, Cho HK, Lee CH, Lee SJ, Kim KK (2010) Characterization of the flounder IL-6 promoter and its regulation by the p65 NF-kappaB subunit. Fish Shellfish Immunol 28:961–964

    Article  CAS  PubMed  Google Scholar 

  • Li DH, Kumanogoh A, Cao TM, Parnes JR, Cullen JM (2004) Woodchuck interleukin-6 gene: structure, characterization, and biologic activity. Gene 342:157–164

    Article  CAS  PubMed  Google Scholar 

  • Naka T, Nishimoto N, Kishimoto T (2002) The paradigm of IL-6: from basic science to medicine. Arthritis Res 4(Suppl 3):S233–S242

    Article  PubMed  PubMed Central  Google Scholar 

  • Nam BH, Byon JY, Kim YO, Park EM, Cho YC, Cheong J (2007) Molecular cloning and characterisation of the flounder (Paralichthys olivaceus) interleukin-6 gene. Fish Shellfish Immunol 23:231–236

    Article  CAS  PubMed  Google Scholar 

  • Nicola NA (1994) Guidebook to cytokines and their receptors. Oxford University Press, New York

    Google Scholar 

  • Nielsen ME, Hoi L, Schmidt AS, Qian D, Shimada T, Shen JY, Larsen JL (2001) Is Aeromonas hydrophila the dominant motile Aeromonas species that causes disease outbreaks in aquaculture production in the Zhejiang Province of China? Dis Aquat Organ 46:23–29

    Article  CAS  PubMed  Google Scholar 

  • Øvergård AC, Nepstad I, Nerland AH, Patel S (2012) Characterisation and expression analysis of the Atlantic halibut (Hippoglossus hippoglossus L.) cytokines: IL-1beta, IL-6, IL-11, IL-12beta and IFNgamma. Mol Biol Rep 39:2201–2213

    Article  PubMed  Google Scholar 

  • Premraj A, Sreekumar E, Nautiyal B, Rasool TJ (2006) Molecular characterization and prokaryotic expression of buffalo (Bubalus bubalis) Interleukin-6. Mol Immunol 43:202–209

    Article  CAS  PubMed  Google Scholar 

  • Robb BW, Hershko DD, Paxton JH, Luo GJ, Hasselgren PO (2002) Interleukin-10 activates the transcription factor C/EBP and the interleukin-6 gene promoter in human intestinal epithelial cells. Surgery 132:226–231

    Article  PubMed  Google Scholar 

  • Roca FJ, Cayuela ML, Secombes CJ, Meseguer J, Mulero V (2007) Post-transcriptional regulation of cytokine genes in fish: a role for conserved AU-rich elements located in the 3′-untranslated region of their mRNAs. Mol Immunol 44:472–478

    Article  CAS  PubMed  Google Scholar 

  • Ruderman NB et al (2006) Interleukin-6 regulation of AMP-activated protein kinase. Potential role in the systemic response to exercise and prevention of the metabolic syndrome. Diabetes 55(Suppl 2):S48–S54

    Article  CAS  PubMed  Google Scholar 

  • Sachs AB (1993) Messenger RNA degradation in eukaryotes. Cell 74:413–421

    Article  CAS  PubMed  Google Scholar 

  • Samuel JM, Kelberman D, Smith AJ, Humphries SE, Woo P (2008) Identification of a novel regulatory region in the interleukin-6 gene promoter. Cytokine 42:256–264

    Article  CAS  PubMed  Google Scholar 

  • Sawa Y, Ueki T, Hata M, Iwasawa K, Tsuruga E, Kojima H, Ishikawa H, Yoshida S (2008) LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 expression in human lymphatic endothelium. J Histochem Cytochem 56:97–109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schneider K, Klaas R, Kaspers B, Staeheli P (2001) Chicken interleukin-6. cDNA structure and biological properties. Eur J Biochem 268:4200–4206

    Article  CAS  PubMed  Google Scholar 

  • Sehgal PB (1990) Interleukin 6 in infection and cancer. Proc Soc Exp Biol Med 195:183–191

    Article  CAS  PubMed  Google Scholar 

  • Sehgal PB (1992) Regulation of IL6 gene expression. Res Immunol 143:724–734

    Article  CAS  PubMed  Google Scholar 

  • Simpson RJ, Hammacher A, Smith DK, Matthews JM, Ward LD (1997) Interleukin-6: structure–function relationships. Protein Sci 6:929–955

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sprang SR, Bazan JF (1993) Cytokine structural taxonomy and mechanism of receptor engagement. Curr Opin Struct Biol 3:815–827

    Article  CAS  Google Scholar 

  • Taga T, Fukuda S (2005) Role of IL-6 in the neural stem cell differentiation. Clin Rev Allergy Immunol 28:249–256

    Article  CAS  PubMed  Google Scholar 

  • Tanabe O, Akira S, Kamiya T, Wong GG, Hirano T, Kishimoto T (1988) Genomic structure of the murine IL-6 gene. High degree conservation of potential regulatory sequences between mouse and human. J Immunol 141:3875–3881

    CAS  PubMed  Google Scholar 

  • Tran NT, Gao ZX, Zhao HH, Yi SK, Chen BX, Zhao YH, Lin L, Liu XQ, Wang WM (2015) Transcriptome analysis and microsatellite discovery in the blunt snout bream (Megalobrama amblycephala) after challenge with Aeromonas hydrophila. Fish Shellfish Immunol. doi:10.1016/j.fsi.2015.01.034

    PubMed  Google Scholar 

  • Yan P, Jian J, Wu Z (2013) Cloning and expression analysis of Interleukin-6 in grass carp (Ctenopharyngodon idella). J Guangdong Ocean Univ 3:46–51 (in Chinese with English abstract)

    Google Scholar 

  • Zante MD, Borchel A, Brunner RM, Goldammer T, Rebl A (2015) Cloning and characterization of the proximal promoter region of rainbow trout (Oncorhynchus mykiss) interleukin-6 gene. Fish Shellfish Immunol 43:249–256

    Article  CAS  PubMed  Google Scholar 

  • Zare F, Bokarewa M, Nenonen N, Bergstrom T, Alexopoulou L, Flavell RA, Tarkowski A (2004) Arthritogenic properties of double-stranded (viral) RNA. J Immunol 172:5656–5663

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This project was supported by grants from the Key Technologies R&D Program of China (No. 2012BAD26B00), Fundamental Research Funds for the Central Universities (Nos. 2014PY042, 2013PY067) and Modern Agroindustry Technology Research System entitled “Staple Freshwater Fishery Industry Technology System” (No. CARS-46-05). The authors thank Dr. Hongtao Hu for helpful comments on the manuscript.

Author information

Authors and Affiliations

  1. College of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, 430070, China

    Xiaoqin Fu, Zhujin Ding, Jun Fan, Huanling Wang, Fengjuan Zhou, Lei Cui, Weimin Wang & Hong Liu

  2. Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, 430070, China

    Xiaoqin Fu, Zhujin Ding, Jun Fan, Huanling Wang, Fengjuan Zhou, Lei Cui & Hong Liu

  3. Animal Husbandry and Fisheries Research Center of Haid Group Co., Ltd, Guangzhou, 511400, China

    Chen Boxiang

  4. College of Life Sciences, Hunan University of Arts and Science, Changde, 415006, Hunan, China

    Hong Liu

Authors
  1. Xiaoqin Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhujin Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jun Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Huanling Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fengjuan Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lei Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chen Boxiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Weimin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hong Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hong Liu.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 20 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fu, X., Ding, Z., Fan, J. et al. Characterization, promoter analysis and expression of the interleukin-6 gene in blunt snout bream, Megalobrama amblycephala . Fish Physiol Biochem 42, 1527–1540 (2016). https://doi.org/10.1007/s10695-016-0238-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10695-016-0238-y

Keywords

Search

Navigation