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

• Park KH
• Nam EH
• Seo GY
• Seo SR
• Kim PH

• KMID: 2150653
• DOI: http://doi.org/10.4110/in.2009.9.6.248

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

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  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.

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