Immune Netw.  2011 Aug;11(4):196-202. 10.4110/in.2011.11.4.196.

Activin A Stimulates Mouse APCs to Express BAFF via ALK4-Smad3 Pathway

• Kim JH
• Seo GY
• Kim PH

• KMID: 1449810
• DOI: http://doi.org/10.4110/in.2011.11.4.196

Abstract

BACKGROUND
B cell-activating factor belonging to the TNF family (BAFF) is primarily expressed by macrophages and dendritic cells, and stimulates B cell proliferation, differentiation, survival, and Ig production. In the present study, we explored the effect of activin A on BAFF expression by APCs.
METHODS
To investigate the effect of activin A on BAFF expression by mouse APCs, we measured the level of BAFF expression at the transcriptional and protein levels using RT-PCR and ELISA.
RESULTS
Activin A markedly enhanced BAFF expression in mouse macrophages and dendritic cells at both the transcriptional and protein levels. SB431542, an activin receptor-like kinase 4 (ALK4) inhibitor, completely abrogated activin A-induced BAFF transcription. Furthermore, overexpression of DN-Smad3 abolished activin-induced BAFF expression at the transcriptional and protein levels.
CONCLUSION
These results demonstrate that activin A can enhance BAFF expression through ALK4-Smad3 pathway.

Keyword

Activin A; BAFF; APCs; ALK4; Smad3; Ig class switch recombination

MeSH Terms

Activin Receptors
Activins
Animals
Benzamides
Cell Proliferation
Dendritic Cells
Dioxoles
Humans
Macrophages
Mice
Activin Receptors
Activins
Benzamides
Dioxoles

Figure

• 

  Figure 1 Activin A stimulates mouse DCs to express BAFF. (A) Effect of activin A on BAFF transcriptional levels in DC2.4 cells. DC2.4 cells (a mouse DC line) were treated with activin A (5, 10, and 20 ng/ml) for 24 h (left panel). Activin A (10 ng/ml) was added to the cell cultures for the indicated times (right panel). In all experiments, BAFF mRNA levels were determined by RT-PCR. Fold increase (Fold I.) values represent relative amounts of BAFF cDNA normalized to the expression of β-actin cDNA using Kodak Molecular Imaging software. (B) Effect of activin A on BAFF promoter activity. DC2.4 cells (1×106) were transfected with the pBAFF reporter (1µg). Cells were then stimulated with activin A (10 ng/ml) and luciferase activity was determined 24 h later. Transfection efficiency was normalized by β-gal activity. Data are average luciferase activities of two independent transfections with ranges (bars). (C) Effect of activin A on BAFF secretion. DC2.4 cells were treated activin A (10 ng/ml). After 48 h of culture, supernatants were collected and the secreting form of BAFF was measured by ELISA. Data are means of triplicate samples±SEM. *p<0.05; **p<0.01. (D) Effect of activin A on the growth kinetics of DC2.4 cells. DC2.4 cells were cultured with activin A (10 ng/ml). Numbers of viable cells were enumerated by trypan blue exclusion. Data are means of triplicate samples±SEM.

• 

  Figure 2 Effect of activin A on BAFF expression by bone marrow-derived DCs. Freshly isolated bone marrow stem cells were differentiated and incubated with activin A (10 ng/ml). After 24 h of culture, levels of BAFF transcripts were determined by RT-PCR (left panel). After 48 h of culture, BAFF secretion was measured by ELISA (right panel). Data are means of triplicate samples±SEM. *p<0.05.

• 

  Figure 3 Activin A stimulates BAFF expression in mouse macrophage cells. (A) Effect of activin A on BAFF transcription in RAW264.7 cells. RAW264.7 cells (a mouse macrophage cell line) were treated with TGF-β (1 ng/ml) and activin A (10 ng/ml) for 24 h (left panel). Effect of activin A on BAFF transcription as a function of time (right panel). Activin A (10 ng/ml) was added to the cell cultures for the indicated times. Levels of BAFF mRNA were determined by RT-PCR. (B) Effect of activin A on BAFF expression by mouse peritoneal macrophages. Freshly isolated mouse peritoneal macrophages were incubated with activin A (10 ng/ml) for 24 h. Levels of BAFF transcripts were determined by RT-PCR (left panel). After 48 h of culture, BAFF secretion was measured by ELISA (right panel). Data are means of triplicate samples±SEM. **p<0.01.

• 

  Figure 4 Activin A enhances BAFF expression through ALK4 and Smad3. (A) Activin A induces BAFF mRNA expression via ALK4. DC2.4 cells were pre-treated with 10µM SB431542, 10µM SB203580, 10µM SP600125, or 5µM PD98059 for 1 h and then stimulated with activin A (10 ng/ml) for 24 h. Levels of BAFF transcripts were determined by RT-PCR. (B) DC2.4 cells (1×106) were transfected with DN-Smad3 expression vectors (2, 4, 8µg) and stimulated with activin A (10 ng/ml) for 24 h. Levels of BAFF transcripts were determined by RT-PCR. (C) BAFF secretion by DN-Smad3-transfected DC 2.4 was measured by ELISA after 48 h incubation with activin A. Data are means of triplicate samples±SEM. **p<0.01. (D) Proposed mechanisms underlying activin A-induced BAFF expression in mouse APCs. Our results indicate that the ALK4 and Smad3/4 mediate activin A-induced BAFF expression.

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