Immune Netw.  2009 Dec;9(6):248-254. 10.4110/in.2009.9.6.248.

Tiul1 and TGIF are Involved in Downregulation of TGFbeta1-induced IgA Isotype Expression

Affiliations
  • 1Department of Molecular Bioscience, School of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701, Korea. phkim@kangwon.ac.kr

Abstract

TGF-beta1 is well known to induce Ig germ-line alpha (GLalpha) transcription and subsequent IgA isotype class switching recombination (CSR). Homeodomain protein TG-interacting factor (TGIF) and E3-ubiquitin ligases TGIF interacting ubiquitin ligase 1 (Tiul1) are implicated in the negative regulation of TGF-beta signaling. In the present study, we investigated the roles of Tiul1 and TGIF in TGFbeta1-induced IgA CSR. We found that over-expression of Tiul1 decreased TGFbeta1-induced GLalpha promoter activity and strengthened the inhibitory effect of Smad7 on the promoter activity. Likewise, overexpression of TGIF also diminished GLalpha promoter activity and further strengthened the inhibitory effect of Tiul1, suggesting that Tiul1 and TGIF can down-regulate TGFbeta1-induced GLalpha expression. In parallel, overexpression of Tiul1 decreased the expression of endogenous IgA CSR-predicitive transcripts (GLT(alpha), PST(alpha), and CT(alpha)) and TGFbeta1-induced IgA secretion, but not GLT(gamma3) and IgG3 secretion. Here, over-expressed TGIF further strengthened the inhibitory effect of Tiul1. These results suggest that Tiul1 and TGIF act as negatively regulators in TGFbeta1-induced IgA isotype expression.

Keyword

TGF-beta1; Tiul1; TGIF; IgA; TbetaRI; Smad

MeSH Terms

Down-Regulation
Immunoglobulin A
Immunoglobulin Class Switching
Immunoglobulin G
Ligases
Recombination, Genetic
Transforming Growth Factor beta
Transforming Growth Factor beta1
Ubiquitin
Immunoglobulin A
Immunoglobulin G
Ligases
Transforming Growth Factor beta
Transforming Growth Factor beta1
Ubiquitin

Figure

  • Figure 1 Effects of Smad7, Tiul1, and TGIF on TGFβ1-induced GLα promoter activity. (A) A20.3 B lymphoma cells were transfected with expression plasmids (10 µg) for Smad7, Tiul1 and GLα-Luc reporter (15 µg). TGF-β1 (1 ng/ml) was added and luciferase activity measured 16 h later. Transfection efficiency was normalized to β-gal activities. (B) A20.3 B lymphoma cells were transfected with expression plasmids (10 µg) for Tiul1, TGIF and GLα-Luc reporter (15 µg). TGF-β1 (1 ng/ml) was added and luciferase activity measured 16 h later. Transfection efficiency was normalized to β-gal activities. Data represent average luciferase activity from three independent transfections with SEMs (bars).

  • Figure 2 Effects of Smad7 and Tiul1 on the levels of Ig GLTs and Ig secretion by mouse B lymphoma cells. (A) Diagram of DNA recombination occurring during switching to IgA. Rectangles and ovals represent exons and S regions, respectively. RNA transcripts are indicated beneath the DNA diagrams. (B) CH12F3-2A B lymphoma cells were transfected with expression plasmid for Smad7, Tiul1 or pcDNA3 (30 µg of each). They were then cultured with LPS (12.5 µg/ml) and TGF-β1 (1 ng/ml), and after 24 h, total RNA isolated and measured levels of endogenous GLTα, GLTγ3, PSTα, and CTα transcripts by RT-PCR. (C) After 3 days of culture, supernatant were collected and secretion of IgA and IgG3 was determined by isotype-specific ELISA. Data are means of triplicate samples±SEM.

  • Figure 3 Effects of Tiul1 and TGIF on TGFβ1-induced GLTα transcription and IgA secretion in mouse B cells. CH12F3-2A B lymphoma cells were transfected with expression plasmid for Tiul1, TGIF or pcDNA3 (30 µg of each). They were then cultured with LPS (12.5 µg/ml) and TGF-β1 (1 ng/ml), and after 24 h, total RNA isolated and measured levels of endogenous GLTα and GLTγ3 transcripts by RT-PCR (Panel A). After 3 days of culture, supernatant were collected and secretion of IgA and IgG3 was determined by ELISA (Panel B). Data are means of triplicate samples±SEM.

  • Figure 4 Proposed mechanisms by which Tiul1 and TGIF downregulate TGFα1-induced IgA isotype expression. (I) Upon stimulation by TGFβ1, Smad3 becomes phosphorylated by the activated TGF-β1 receptors and forms complexes with Smad4. These Smads complexes translocate to the nucleus where they bind SBEs in the GLα promoter, thereby activating transcription. (II) In the cytoplasm, Smad7 can inhibit TGF-β1 signaling by deactivating Smad3 phosphorylation. Tiul1 interacts with Smad7 leading to ultimate degradation of the activated type I receptor. On the other hand, TGIF prevents the ligand-dependent phosphorylation of Smad3. In addition, Tiul1 interacts with TGIF in the nucleus resulting in degradation of R-Smads such as Smad2 and Smad3.


Cited by  1 articles

SUMO Proteins are not Involved in TGF-β1-induced, Smad3/4-mediated Germline α Transcription, but PIASy Suppresses it in CH12F3-2A B Cells
Sang-Hoon Lee, Pyeung-Hyeun Kim, Sang-Muk Oh, Jung-Hwan Park, Yung-Choon Yoo, Junglim Lee, Seok-Rae Park
Immune Netw. 2014;14(6):321-327.    doi: 10.4110/in.2014.14.6.321.


Reference

1. Lagna G, Hata A, Hemmati-Brivanlou A, Massagué J. Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways. Nature. 1996. 383:832–836.
Article
2. Wu RY, Zhang Y, Feng XH, Derynck R. Heteromeric and homomeric interactions correlate with signaling activity and functional cooperativity of Smad3 and Smad4/DPC4. Mol Cell Biol. 1997. 17:2521–2528.
Article
3. Zhang Y, Musci T, Derynck R. The tumor suppressor Smad4/DPC 4 as a central mediator of Smad function. Curr Biol. 1997. 7:270–276.
Article
4. Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin CH, Miyazono K, ten Dijke P. TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J. 1997. 16:5353–5362.
Article
5. Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA Jr, Topper JN, Gimbrone MA Jr, Wrana JL, Falb D. The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling. Cell. 1997. 89:1165–1173.
Article
6. Imamura T, Takase M, Nishihara A, Oeda E, Hanai J, Kawabata M, Miyazono K. Smad6 inhibits signalling by the TGF-beta superfamily. Nature. 1997. 389:622–626.
7. Nakao A, Afrakhte M, Morén A, Nakayama T, Christian JL, Heuchel R, Itoh S, Kawabata M, Heldin NE, Heldin CH, ten Dijke P. Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling. Nature. 1997. 389:631–635.
Article
8. Ebisawa T, Fukuchi M, Murakami G, Chiba T, Tanaka K, Imamura T, Miyazono K. Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation. J Biol Chem. 2001. 276:12477–12480.
Article
9. Kavsak P, Rasmussen RK, Causing CG, Bonni S, Zhu H, Thomsen GH, Wrana JL. Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Mol Cell. 2000. 6:1365–1375.
Article
10. Kim KI, Baek SH. SUMOylation code in cancer development and metastasis. Mol Cells. 2006. 22:247–253.
11. Zhu H, Kavsak P, Abdollah S, Wrana JL, Thomsen GH. A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. Nature. 1999. 400:687–693.
Article
12. Zhao M, Qiao M, Oyajobi BO, Mundy GR, Chen D. E3 ubiquitin ligase Smurf1 mediates core-binding factor alpha1/Runx2 degradation and plays a specific role in osteoblast differentiation. J Biol Chem. 2003. 278:27939–27944.
Article
13. Jin YH, Jeon EJ, Li QL, Lee YH, Choi JK, Kim WJ, Lee KY, Bae SC. Transforming growth factor-beta stimulates p300-dependent RUNX3 acetylation, which inhibits ubiquitination-mediated degradation. J Biol Chem. 2004. 279:29409–29417.
Article
14. Lin X, Liang M, Feng XH. Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-beta signaling. J Biol Chem. 2000. 275:36818–36822.
Article
15. Zhang Y, Chang C, Gehling DJ, Hemmati-Brivanlou A, Derynck R. Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin ligase. Proc Natl Acad Sci U S A. 2001. 98:974–979.
Article
16. Koinuma D, Shinozaki M, Komuro A, Goto K, Saitoh M, Hanyu A, Ebina M, Nukiwa T, Miyazawa K, Imamura T, Miyazono K. Arkadia amplifies TGF-beta superfamily signalling through degradation of Smad7. EMBO J. 2003. 22:6458–6470.
Article
17. Seo SR, Lallemand F, Ferrand N, Pessah M, L'hoste S, Camonis J, Atfi A. The novel E3 ubiquitin ligase Tiul1 associates with TGIF to target Smad2 for degradation. EMBO J. 2004. 23:3780–3792.
Article
18. Wotton D, Lo RS, Lee S, Massague J. A Smad transcriptional corepressor. Cell. 1999. 97:29–39.
Article
19. Wotton D, Lo RS, Swaby LA, Massagué J. Multiple modes of repression by the Smad transcriptional corepressor TGIF. J Biol Chem. 1999. 274:37105–37110.
Article
20. Seo SR, Ferrand N, Faresse N, Prunier C, Abécassis L, Pessah M, Bourgeade MF, Atfi A. Nuclear retention of the tumor suppressor cPML by the homeodomain protein TGIF restricts TGF-beta signaling. Mol Cell. 2006. 23:547–559.
Article
21. Park SR, Lee JH, Kim PH. Smad3 and Smad4 mediate transforming growth factor-beta1-induced IgA expression in murine B lymphocytes. Eur J Immunol. 2001. 31:1706–1715.
Article
22. Park SR, Lee EK, Kim BC, Kim PH. p300 cooperates with Smad3/4 and Runx3 in TGFbeta1-induced IgA isotype expression. Eur J Immunol. 2003. 33:3386–3392.
Article
23. Choi SH, Seo GY, Nam EH, Jeon SH, Kim HA, Park JB, Kim PH. Opposing effects of Arkadia and Smurf on TGFbeta1-induced IgA isotype expression. Mol Cells. 2007. 24:283–287.
24. Li SC, Rothman PB, Zhang J, Chan C, Hirsh D, Alt FW. Expression of I mu-C gamma hybrid germline transcripts subsequent to immunoglobulin heavy chain class switching. Int Immunol. 1994. 6:491–497.
Article
25. Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell. 2000. 102:553–563.
Article
26. Kinoshita K, Harigai M, Fagarasan S, Muramatsu M, Honjo T. A hallmark of active class switch recombination: transcripts directed by I promoters on looped-out circular DNAs. Proc Natl Acad Sci U S A. 2001. 98:12620–12623.
Article
Full Text Links
  • IN
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr