Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Smad-dependent Expression of Gadd45b Gene during TGF-β-induced Apoptosis in EpH4 Cells.

EpH4 세포에서 TGF-β에 의한 세포사멸시 Smad 단백질에 의존한 Gadd45b 유전자의 발현 변화

  • Cho, Hee-Jun (Department of Microbiology/Research Institute of Life Science, Gyeongsang National University) ;
  • Yoo, Ji-Yun (Department of Microbiology/Research Institute of Life Science, Gyeongsang National University)
  • 조희준 (경상대학교 자연과학대학/생명과학연구원) ;
  • 유지윤 (경상대학교 자연과학대학/생명과학연구원)
  • Published : 2008.04.30
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Abstract

Transforming growth $factor-{\beta}$ ($TGF-{\beta}$)-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. Gadd45b has been known to participate in $TGF-{\beta}-induced$ apoptosis by the activation of p38 kinase. In this report, we show that Gadd45b is an immediate-early response gene for $TGF-{\beta}$ during apoptosis in EpH4 cells. To elucidate the molecular mechanism of $TGF-{\beta}-induced$ Gadd45b gene expression, we cloned the 5'-flanking region of the mouse Gadd45b gene. When transfected into EpH4 cells, this 5'-flanking region conferred promoter activity and inducibility by $TGF-{\beta}$. Deletion analyses demonstrated that the minimal promoter activity was detected in the proximal region 220 bp upstream of the transcription initiation site. We also found that the proximal Gadd45b promoter is activated by $TGF-{\beta}$ through the action of Smad2, Smad3, and Smad4. Finally, we show that the expression of Gadd45b gene by $TGF-{\beta}$ is suppressed in EpRas cells in which $TGF-{\beta}$ could not induce apoptosis, suggesting that Gadd45b may be a crucial target for $TGF-{\beta}-induced$ apoptosis in EpH4 cells.

Transforming growth $factor-{\beta}$ ($TGF-{\beta}$)에 의해 유도되는 세포사멸 과정은 정상 조직에서 손상 받은 조직이나 비정상 적인 조직을 제거하는데 중요한 역할을 담당한다. Gadd45b는 p38 kinase를 활성화시킴으로 $TGF-{\beta}$에 의해 유도되는 세포사멸 과정을 매개한다고 알려져 있다. 본 연구에서는 $TGF-{\beta}$에 의해 세포사멸이 일어나는 EpH4 세포에서 Gadd45b 유전자의 발현이 $TGF-{\beta}$에 의해 촉진됨을 보여주었다. 어떠한 기작으로 $TGF-{\beta}$에 의해 Gadd45b 유전자의 발현이 촉진되는지 알아보기 위해 Gadd45g 유전자의 5'-flanking region을 cloning하였으며, EpH4 세포에서 $TGF-{\beta}$에 의해 그 promoter activity가 증가함을 확인하였다. 여러 가지 deletion mutants를 제조하여 promoter activity를 조사한 결과 전사 개시점으로부터 220 bp upstream 부위 에 promoter activity에 필수적인 sequence가 존재함을 확인하였다. 또한 $TGF-{\beta}$에 의한 Gadd45b 유전자의 promoter activity에 Smad2, Smad3, 그리고 Smad4가 중요한 기능을 담당함도 확인하였다. 마지막으로 ras 유전자가 도입되어 $TGF-{\beta}$에 의한 세포사멸이 억제되어있는 EpRas 세포에서 $TGF-{\beta}$에 의한 Gadd45b 유전자의 발현을 확인한 결과 EpRas 세포에서 $TGF-{\beta}$에 의한 Gadd45b 유전자의 발현이 억제됨을 확인하였다. 이러한 결과는 Gadd45b 유전자가 EpH4 세포에서 $TGF-{\beta}$에 의한 세포사멸을 유도하는데 중요한 기능을 담당할 가능성이 높음을 의미하는 것이다.

Keywords

References

  1. Haufel, T., S. Dormann, J. Hanusch, A. Schwieger and G. Bauer. 1999. Three distinct roles for TGF-beta during intercellular induction of apoptosis: a review. Anticancer Res. 19, 105-111
  2. Hsing, A. Y., K. Kadomatsu, M. J. Bonham and D. Danielpour. 1996. Regulation of apoptosis induced by transforming growth factor-beta1 in nontumorigenic rat prostatic epithelial cell lines. Cancer Res. 56, 5146-5149
  3. Lin, J. K. and C. K. Chou. 1992. In vivo apoptosis in the human hepatoma-cell line induced by transforming growth factor-beta1. Cancer Res. 52, 385-388
  4. Fukuda, K., M. Kojiro and J. F. Chiu. 1993. Induction of apoptosis by transforming growth factor-beta 1 in the rat hepatoma cell line McA-RH7777: a possible association with tissue transglutaminase expression. Hepatology 18, 945-953 https://doi.org/10.1002/hep.1840180428
  5. Oberhammer, F., W. Bursch, R. Tiefenbacher, G. Froschl, M. Pavelka, T. Purchio and R. Schulte-Hermann. 1993. Apoptosis is induced by transforming growth factor-beta1 within 5 hours in regressing liver without significant fragmentation of the DNA. Hepatology 18, 1238-1246 https://doi.org/10.1002/hep.1840180533
  6. Attisano, L. and J. L. Wrana. 2000. Smads as transcriptional co-modulators. Curr. Opin. Cell Biol. 12, 235-243 https://doi.org/10.1016/S0955-0674(99)00081-2
  7. Massague, J. and D. Wotton. 2000. Transcriptional control by the TGF-beta/Smad signaling system. EMBO J. 19, 1745-1754 https://doi.org/10.1093/emboj/19.8.1745
  8. ten Dijke, P., K. Miyazono and C. H. Heldin. 2000. Signaling inputs converge on nuclear effectors in TGF-b signaling. Trends Biochem. Sci. 25, 64-70 https://doi.org/10.1016/S0968-0004(99)01519-4
  9. Zhang, Y. and R. Derynck. 1999. Regulation of Smad signalling by protein associations and signalling crosstalk. Trends Cell Biol. 9, 274-279 https://doi.org/10.1016/S0962-8924(99)01579-2
  10. Sanchez, A., A. M. Alvarez, M. Benito and I. Fabregat. 1996. Apoptosis induced by transforming growth factor- beta in fetal hepatocytes primary cultures: involvement of reactive oxygen intermediates. J. Biol. Chem. 271, 7416-7422 https://doi.org/10.1074/jbc.271.13.7416
  11. Koseki, T., K. Yamato, S. Krajewski, J. C. Reed, Y. Tsujimoto and T. Nishihara. 1995. Activin A-induced apoptosis is suppressed by BCL-2. FEBS Lett. 376, 247-250 https://doi.org/10.1016/0014-5793(95)01290-7
  12. Saltzman, A., R. Munro, G. Searfoss, C. Franks, M. Jaye and Y. Ivashchenko. 1998. Transforming growth factor-beta- mediated apoptosis in the Ramos B-lymphoma cell line is accompanied by caspase activation and Bcl-XL downregulation. Exp. Cell Res. 242, 244-254 https://doi.org/10.1006/excr.1998.4096
  13. Selvakumaran, M., H. K. Lin, R. T. Sjin, J. C. Reed, D. A. Liebermann and B. Hoffman. 1994. The novel primary response gene MyD118 and the proto-oncogenes myb, myc, and bcl-2 modulate transforming growth factor beta 1-induced apoptosis of myeloid leukemia cells. Mol. Cell Biol. 14, 2352-2360 https://doi.org/10.1128/MCB.14.4.2352
  14. Yamamoto, M., K. Fukuda, N. Miura, R. Suzuki, T. Kido and Y. Komatsu. 1998. Inhibition by dexamethasone of transforming growth factor beta1-induced apoptosis in rat hepatoma cells: a possible association with Bcl-xL induction. Hepatology 27, 959-966 https://doi.org/10.1002/hep.510270410
  15. Fukuchi, Y., M. Kizaki, K. Yamato, C. Kawamura, A. Umezawa, Ji. Hata, T. Nishihara and Y. Ikeda. 2001. Mcl-1, an early-induction molecule, modulates activin A-induced apoptosis and differentiation of CML cells. Oncogene 20, 704-713 https://doi.org/10.1038/sj.onc.1204142
  16. Chen, R. H. and T. Y. Chang. 1997. Involvement of caspase family proteases in transforming growth factor-beta- induced apoptosis. Cell Growth Differ. 8, 821-827
  17. Jang, C. W., C. H. Chen, C. C. Chen, J. Y. Chen, Y. H. Su and R. H. Chen. 2002. TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase. Nat. Cell Biol. 4, 51-58 https://doi.org/10.1038/ncb731
  18. Larisch, S., Y. Yi, R. Lotan, H. Kerner, S. Eimeri, W. Tony Parks, Y. Gottfried, S. Birkey Reffey, M. P. de Caestecker, D. Danielpour, N. Book-Melamed, R. Timberg, C, S. Duckett, R, J. Lechleider, H. Steller, J. Orly, S. J. Kim and A. B. Roberts. 2000. A novel mitochondrial septin-like protein, ARTS, mediates apoptosis dependent on its P-loop motif. Nat. Cell Biol. 2, 915-921 https://doi.org/10.1038/35046566
  19. Perlman, R., W. P. Schiemann, M. W. Brooks, H. F. Lodish and R. A. Weinberg. 2001. TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation. Nat. Cell Biol. 3, 708-714 https://doi.org/10.1038/35087019
  20. Valderrama-Carvajal, H., E. Cocolakis, A. Lacerte, E. H. Lee, G. Krystal, S. Ali and J. J. Lebrun. 2002. Activin/TGF-b induce apoptosis through Smad-dependent expression of the lipid phosphatase SHIP. Nat. Cell Biol. 4, 963-969 https://doi.org/10.1038/ncb885
  21. Yoo, J., M. Ghiassi, L. Jirmanova, A. G. Balliet, B. Hoffman, A. J. Fornace Jr., D. A. Liebermann, E. P. Bottinger and A. B. Roberts. 2003. Transforming growth factor-beta-induced apoptosis is mediated by Smad-dependent expression of GADD45b through p38 activation. J. Biol. Chem. 278, 43001-43007 https://doi.org/10.1074/jbc.M307869200
  22. Takekawa, M. and H. A. Saito. 1998. A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell 95, 521-530 https://doi.org/10.1016/S0092-8674(00)81619-0
  23. Janda, E., K. Lehmann, I. Killisch, M. Jechlinger, M. Herzig, J. Downward, H. Beug and S. Grünert. 2002. Ras and TGF-beta cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways. J. Cell Biol. 156, 299-313 https://doi.org/10.1083/jcb.200109037
  24. Oft, M., J. Peli, C. Rudaz, H. Schwarz, H. Beug and E. Reichmann. 1996. TGF-beta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells. Genes Dev. 10, 2462-2477 https://doi.org/10.1101/gad.10.19.2462
  25. Yoo, J., M. J. Jeong, B. M. Kwon, M. W. Hur, Y. M. Park and M. Y. Han. 2002. Activation of dynamin I gene expression by Sp1 and Sp3 is required for neuronal differentiation of N1E-115 cells. J. Biol. Chem. 277, 11904-11909 https://doi.org/10.1074/jbc.M111788200
  26. Balliet, A. G., K. S. Hatton, B. Hoffman and D. A. Liebermann. 2001. Comparative analysis of the genetic structure and chromosomal location of the murine MyD118 (Gadd45beta) gene. DNA Cell Biol. 20, 239-247 https://doi.org/10.1089/104454901750219125
  27. Liberati, N. T., M. B. Datto, J. P. Frederick, X. Shen, C. Wong, E. M. Rougier-Chapman and X. F. Wang. 1999. Smads bind directly to the Jun family of AP-1 transcription factors. Proc. Natl. Acad. Sci. USA 96, 4844-4849 https://doi.org/10.1073/pnas.96.9.4844
  28. Moustakas, A. and D. Kardassis. 1998. Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members. Proc. Natl. Acad. Sci. USA 95, 6733-6738
  29. Zhang, Y. and R. Derynck. 1999. Regulation of Smad signalling by protein associations and signalling crosstalk. Trends Cell Biol. 9, 274-279 https://doi.org/10.1016/S0962-8924(99)01579-2
  30. Wong, C., E. M. Rougier-Chapman, J. P. Frederick, M. B. Datto, N. T. Liberati, J. M. Li and X. F. Wang. 1999. Smad3-Smad4 and AP-1 complexes synergize in transcriptional activation of the c-Jun promoter by transforming growth factor beta. Mol. Cell Biol. 19, 1821-1830 https://doi.org/10.1128/MCB.19.3.1821