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Ets1 and heat shock factor 1 regulate transcription of the Transformer 2β gene in human colon cancer cells

Abstract

Background

Transformer (Tra) 2β is a member of the serine/arginine-rich (SR)-like protein family that regulates alternative splicing of numerous genes in a concentration-dependent manner. Several types of cancer cells up-regulate Tra2β expression, while the regulatory mechanism of Tra2β expression remains to be elucidated. In this study, we examined the transcriptional regulation and possible functions of Tra2β in human colon cancer cells.

Methods

We cloned 959 bp-upstream of the human TRA2β 5′-flank into luciferase constructs. Chromatin immunoprecipitation (ChIP) was employed to identify crucial cis element(s) and trans activator(s) of the TRA2β promoter. Tra2β expression in the human colon and colon cancer tissues was examined by immunohistochemistry.

Results

In response to sodium arsenite, colon cancer cells (HCT116) increased levels of TRA2β1 mRNA encoding a functional, full-length Tra2β with a peak around 6 h without changing its mRNA stability. Transient expression assays using a reporter gene driven by serially truncated TRA2β promoters and Chip assay demonstrated that an Ets1-binding site present at −64 to −55 bp was crucial for basal transcription, while three heat shock elements (HSEs) located at −145 to −99 bp mediated the oxidant-induced transactivation of TRA2β. Tra2β knockdown caused apoptosis of HCT116 cells. Tra2β were preferentially expressed in proliferative compartment of normal human colonic glands and adenocarcinomas, where Ets1 and heat shock factor 1 were also highly expressed.

Conclusions

Our results suggest that oxidative stress-responsive Tra2β may play an important role in colon cancer growth.

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References

  1. Matsuo N, Ogawa S, Imai Y, et al. Cloning of a novel RNA binding polypeptide (RA301) induced by hypoxia/reoxygenation. J Biol Chem. 1995;270:28216–22.

    Article  CAS  PubMed  Google Scholar 

  2. Beil B, Screaton G, Stamm S. Molecular cloning of htra2-beta-1 and htra2-beta-2, two human homologs of tra-2 generated by alternative splicing. DNA Cell Biol. 1997;16:679–90.

    CAS  PubMed  Google Scholar 

  3. Tacke R, Tohyama M, Ogawa S, et al. Human Tra2 proteins are sequence-specific activators of pre-mRNA splicing. Cell. 1998;93:139–48.

    Article  CAS  PubMed  Google Scholar 

  4. Hertel KJ, Maniatis T. The function of multisite splicing enhancers. Mol Cell. 1998;1:449–55.

    Article  CAS  PubMed  Google Scholar 

  5. Qi J, Su S, McGuffin ME, et al. Concentration dependent selection of targets by an SR splicing regulator results in tissue-specific RNA processing. Nucleic Acids Res. 2006;34:6256–63.

    Article  CAS  PubMed  Google Scholar 

  6. Nayler O, Cap C, Stamm S. Human transformer-2-beta gene (SFRS10): complete nucleotide sequence, chromosomal localization, and generation of a tissue-specific isoform. Genomics. 1998;53:191–202.

    Article  CAS  PubMed  Google Scholar 

  7. Hofmann Y, Lorson CL, Stamm S, et al. Htra2-beta 1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 (SMN2). Proc Natl Acad Sci USA. 2000;97:9618–23.

    Article  CAS  PubMed  Google Scholar 

  8. Singh NN, Androphy EJ, Singh RN. In vivo selection reveals combinatorial controls that define a critical exon in the spinal muscular atrophy genes. RNA. 2004;10:1291–305.

    Article  CAS  PubMed  Google Scholar 

  9. Martins de Araujo M, Bonnal S, Hastings ML, et al. Differential 3′ splice site recognition of SMN1 and SMN2 transcripts by U2AF and U2 snRNP. RNA 2009;15:515–23.

    Google Scholar 

  10. Jiang Z, Tang H, Havlioglu N, et al. Mutations in tau gene exon 10 associated with FTDP-17 alter the activity of an exonic splicing enhancer to interact with Tra2 beta. J Biol Chem. 2003;278:18997–9007.

    Article  CAS  PubMed  Google Scholar 

  11. Kondo S, Yamamoto N, Murakami T, et al. Tra2 beta, SF2/ASF and SRp30c modulate the function of an exonic splicing enhancer in exon 10 of tau pre-mRNA. Genes Cells. 2004;9:121–30.

    Article  CAS  PubMed  Google Scholar 

  12. Watermann DO, Tang Y, Zur Hausen A, et al. Splicing factor Tra2-beta1 is specifically induced in breast cancer and regulates alternative splicing of the CD44 gene. Cancer Res. 2006;66:4774–80.

    Article  CAS  PubMed  Google Scholar 

  13. Tsukamoto Y, Matsuo N, Ozawa K, et al. Expression of a novel RNA-splicing factor, RA301/Tra2beta, in vascular lesions and its role in smooth muscle cell proliferation. Am J Pathol. 2001;158:1685–94.

    Article  CAS  PubMed  Google Scholar 

  14. Kiryu-Seo S, Matsuo N, Wanaka A, et al. A sequence-specific splicing activator, tra2beta, is up-regulated in response to nerve injury. Brain Res Mol Brain Res. 1998;62:220–3.

    Article  CAS  PubMed  Google Scholar 

  15. Gabriel B, Zur Hausen A, Bouda J, et al. Significance of nuclear hTra2-beta1 expression in cervical cancer. Acta Obstet Gynecol Scand. 2009;88:216–21.

    Article  CAS  PubMed  Google Scholar 

  16. Fischer DC, Noack K, Runnebaum IB, et al. Expression of splicing factors in human ovarian cancer. Oncol Rep. 2004;11:1085–90.

    CAS  PubMed  Google Scholar 

  17. Takeo K, Kawai T, Nishida K, et al. Oxidative stress-induced alternative splicing of transformer 2beta (SFRS10) and CD44 pre-mRNAs in gastric epithelial cells. Am J Physiol Cell Physiol. 2009;297:C330–8.

    Article  CAS  PubMed  Google Scholar 

  18. Daoud R, Da Penha Berzaghi M, Siedler F, et al. Activity-dependent regulation of alternative splicing patterns in the rat brain. Eur J Neurosci. 1999;11:788–802.

    Article  CAS  PubMed  Google Scholar 

  19. Nakayama T, Ito M, Ohtsuru A, et al. Expression of the ets-1 proto-oncogene in human colorectal carcinoma. Mod Pathol. 2001;14:415–22.

    Article  CAS  PubMed  Google Scholar 

  20. Stoilov P, Daoud R, Nayler O, et al. Human tra2-beta1 autoregulates its protein concentration by influencing alternative splicing of its pre-mRNA. Hum Mol Genet. 2004;13:509–24.

    Article  CAS  PubMed  Google Scholar 

  21. Wang ET, Sandberg R, Luo S, et al. Alternative isoform regulation in human tissue transcriptomes. Nature. 2008;456:470–6.

    Article  CAS  PubMed  Google Scholar 

  22. Yoshida K, Sanada M, Shiraishi Y, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. 2011;478:64–9.

    Article  CAS  PubMed  Google Scholar 

  23. Rockenstein EM, McConlogue L, Tan H, et al. Levels and alternative splicing of amyloid beta protein precursor (APP) transcripts in brains of APP transgenic mice and humans with Alzheimer’s disease. J Biol Chem. 1995;270:28257–67.

    Article  CAS  PubMed  Google Scholar 

  24. Fugier C, Klein AF, Hammer C, et al. Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy. Nat Med. 2011;17:720–5.

    Article  CAS  PubMed  Google Scholar 

  25. Venables JP, Klinck R, Koh C, et al. Cancer-associated regulation of alternative splicing. Nat Struct Mol Biol. 2009;16:670–6.

    Article  CAS  PubMed  Google Scholar 

  26. Meira LB, Bugni JM, Green SL, et al. DNA damage induced by chronic inflammation contributes to colon carcinogenesis in mice. J Clin Invest. 2008;118:2516–25.

    CAS  PubMed  Google Scholar 

  27. Ozaki M, Deshpande SS, Angkeow P, et al. Rac1 regulates stress-induced, redox-dependent heat shock factor activation. J Biol Chem. 2000;275:35377–83.

    Article  CAS  PubMed  Google Scholar 

  28. Jacobs AT, Marnett LJ. Heat shock factor 1 attenuates 4-Hydroxynonenal-mediated apoptosis: critical role for heat shock protein 70 induction and stabilization of Bcl-XL. J Biol Chem. 2007;282:33412–20.

    Article  CAS  PubMed  Google Scholar 

  29. Vilaboa NE, Galan A, Troyano A, et al. Regulation of multidrug resistance 1 (MDR1)/P-glycoprotein gene expression and activity by heat-shock transcription factor 1 (HSF1). J Biol Chem. 2000;275:24970–6.

    Article  CAS  PubMed  Google Scholar 

  30. Sharrocks AD. The ETS-domain transcription factor family. Nat Rev Mol Cell Biol. 2001;2:827–37.

    Article  CAS  PubMed  Google Scholar 

  31. Oikawa T, Yamada T. Molecular biology of the Ets family of transcription factors. Gene. 2003;303:11–34.

    Article  CAS  PubMed  Google Scholar 

  32. Hsu T, Trojanowska M, Watson DK. Ets proteins in biological control and cancer. J Cell Biochem. 2004;91:896–903.

    Article  CAS  PubMed  Google Scholar 

  33. Singh AK, Swarnalatha M, Kumar V. c-ETS1 facilitates G1/S-phase transition by up-regulating cyclin E and CDK2 genes and cooperates with hepatitis B virus X protein for their deregulation. J Biol Chem. 2011;286:21961–70.

    Article  CAS  PubMed  Google Scholar 

  34. Seth A, Watson DK. ETS transcription factors and their emerging roles in human cancer. Eur J Cancer. 2005;41:2462–78.

    Article  CAS  PubMed  Google Scholar 

  35. Ito Y, Takeda T, Okada M, et al. Expression of ets-1 and ets-2 in colonic neoplasms. Anticancer Res. 2002;22:1581–4.

    CAS  PubMed  Google Scholar 

  36. Foulds CE, Nelson ML, Blaszczak AG, et al. Ras/mitogen-activated protein kinase signaling activates Ets-1 and Ets-2 by CBP/p300 recruitment. Mol Cell Biol. 2004;24:10954–64.

    Article  CAS  PubMed  Google Scholar 

  37. Wilson LA, Gemin A, Espiritu R, et al. ets-1 is transcriptionally up-regulated by H2O2 via an antioxidant response element. FASEB J. 2005;19:2085–7.

    CAS  PubMed  Google Scholar 

  38. Venables JP, Bourgeois CF, Dalgliesh C, et al. Up-regulation of the ubiquitous alternative splicing factor Tra2beta causes inclusion of a germ cell-specific exon. Hum Mol Genet. 2005;14:2289–303.

    Article  CAS  PubMed  Google Scholar 

  39. Rochat-Steiner V, Becker K, Micheau O, et al. FIST/HIPK3: a Fas/FADD-interacting serine/threonine kinase that induces FADD phosphorylation and inhibits fas-mediated Jun NH(2)-terminal kinase activation. J Exp Med. 2000;192:1165–74.

    Article  CAS  PubMed  Google Scholar 

  40. Novoyatleva T, Heinrich B, Tang Y, et al. Protein phosphatase 1 binds to the RNA recognition motif of several splicing factors and regulates alternative pre-mRNA processing. Hum Mol Genet. 2008;17:52–70.

    Article  CAS  PubMed  Google Scholar 

  41. Benderska N, Becker K, Girault JA, et al. DARPP-32 binds to tra2-beta1 and influences alternative splicing. Biochim Biophys Acta. 2010;1799:448–53.

    Article  CAS  PubMed  Google Scholar 

  42. Shi Y, Manley JL. A complex signaling pathway regulates SRp38 phosphorylation and pre-mRNA splicing in response to heat shock. Mol Cell. 2007;28:79–90.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This study was supported by Grants-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (#22790649, 24659370 to Y.K., #22659142 to K.R.).

Conflict of interest

The authors declare that they have no conflict of interest.

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Correspondence to Yuki Kuwano.

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Kajita, K., Kuwano, Y., Kitamura, N. et al. Ets1 and heat shock factor 1 regulate transcription of the Transformer 2β gene in human colon cancer cells. J Gastroenterol 48, 1222–1233 (2013). https://doi.org/10.1007/s00535-012-0745-2

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  • DOI: https://doi.org/10.1007/s00535-012-0745-2

Keywords

  • Transformer 2β
  • Colon cancer
  • Oxidative stress
  • Ets1
  • Heat shock factor 1