Immune Netw.  2014 Dec;14(6):321-327. 10.4110/in.2014.14.6.321.

SUMO Proteins are not Involved in TGF-beta1-induced, Smad3/4-mediated Germline alpha Transcription, but PIASy Suppresses it in CH12F3-2A B Cells

• Lee SH
• Kim PH
• Oh SM
• Park JH
• Yoo YC
• Lee J
• Park SR

• KMID: 2150822
• DOI: http://doi.org/10.4110/in.2014.14.6.321

Abstract

TGF-beta induces IgA class switching by B cells. We previously reported that Smad3 and Smad4, pivotal TGF-beta signal-transducing transcription factors, mediate germline (GL) alpha transcription induced by TGF-beta1, resulting in IgA switching by mouse B cells. Post-translational sumoylation of Smad3 and Smad4 regulates TGF-beta-induced transcriptional activation in certain cell types. In the present study, we investigated the effect of sumoylation on TGF-beta1-induced, Smad3/4-mediated GLalpha transcription and IgA switching by mouse B cell line, CH12F3-2A. Overexpression of small ubiquitin-like modifier (SUMO)-1, SUMO-2 or SUMO-3 did not affect TGF-beta1-induced, Smad3/4-mediated GLalpha promoter activity, expression of endogenous GLalpha transcripts, surface IgA expression, and IgA production. Next, we tested the effect of the E3 ligase PIASy on TGF-beta1-induced, Smad3/4-mediated GLalpha promoter activity. We found that PIASy overexpression suppresses the GLalpha promoter activity in cooperation with histone deacetylase 1. Taken together, these results suggest that SUMO itself does not affect regulation of GLalpha transcription and IgA switching induced by TGF-beta1/Smad3/4, while PIASy acts as a repressor.

Keyword

IgA; germline alpha transcripts; SUMO; PIASy; HDAC1

MeSH Terms

Animals
B-Lymphocytes*
Cell Line
Histone Deacetylase 1
Immunoglobulin A
Immunoglobulin Class Switching
Mice
Small Ubiquitin-Related Modifier Proteins*
SUMO-1 Protein*
Sumoylation
Transcription Factors
Transcriptional Activation
Transforming Growth Factor beta
Transforming Growth Factor beta1
Ubiquitin-Protein Ligases
Histone Deacetylase 1
Immunoglobulin A
SUMO-1 Protein
Small Ubiquitin-Related Modifier Proteins
Transcription Factors
Transforming Growth Factor beta
Transforming Growth Factor beta1
Ubiquitin-Protein Ligases

Figure

• 

  Figure 1 Effects of SUMO and Ubc9 on TGF-β1-induced, Smad3/4-mediated GLα promoter activity. (A) CH12F3-2A cells were transfected with GLα promoter reporter construct (pGL3-GLα [-448/+72], 10µg) and the indicated expression plasmids (5µg each). TGF-β1 (0.5 ng/ml) was added, and luciferase activity was determined 16 h later. Transfection efficiency was normalized to β-gal activities. Data shown are average luciferase activities of three independent transfections with SEM (bars) (left panel). Sumoylated proteins were detected by immunoblotting analysis for HA from extracts of CH12F3-2A cells transfected with SUMO-1 and SUMO-3 (right panel). (B) CH12F3-2A cells were transfected with pGL3-GLα [-448/+72] (10µg) and the indicated expression plasmids (5µg each) including Ubc9 (5µg). Induction and analysis were performed as shown in (A). *p<0.05, **p<0.01.

• 

  Figure 2 SUMO-1 is not involved in TGF-β/Smad3-induced GLα transcription and IgA isotype switching. CH12F3-2A cells were transfected with Smad3 (5µg) and SUMO-1 (5µg), and the cells were stimulated with TGF-β1 (0.5 ng/ml). (A) After 36 h of culture, RNA was isolated, and levels of GLTα and β-actin mRNA were measured by RT-PCR. (B) Following 3 days incubation, cells were stained with FITC-labeled anti-mouse IgA and PE-labeled anti-mouse IgM and analyzed by flow cytometry. (C) Culture supernatants were harvested after incubation for 4 days, and IgA levels were determined by ELISA. Data are means±SEM of three cultures.

• 

  Figure 3 Effects of PIASy and HDAC1 on TGF-β1-induced, Smad3/4-mediated GLα promoter activity. (A) CH12F3-2A cells were transfected with pGL3-GLα [-448/+72] (10µg) and the indicated doses of PIASy alone (left panel) or PIASy (30µg) and Smad3/4 (5µg each) (right panel). Induction and analysis were performed as shown in Fig. 1A. (B) CH12F3-2A cells were transfected with pGL3-GLα [-448/+72] (10µg) and empty vector (pcDNA3, 30µg) or PIASy (30µg). TGF-β1 (0.5 ng/ml) and the indicated doses of TSA were added (upper panel). PIASy (30µg) and HDAC1 (15µg) were transfected along with the GLα promoter reporter (10µg) into CH12F3-2A cells, and cells were treated with TGF-β1 (lower left panel). The GLα promoter reporter (10µg), Smad3/4 (5µg each), and PIASy (30µg) were transfected into CH12F3-2A cells, and TGF-β1 (0.5 ng/ml) and TSA (5 ng/ml) were added (lower right panel). Luciferase activity was determined 16 h later. *p<0.05, **p<0.01.

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