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The Runx1 transcription factor inhibits the differentiation of naive CD4+ T cells into the Th2 lineage by repressing GATA3 expression.

Komine O, Hayashi K, Natsume W, Watanabe T, Seki Y, Seki N, Yagi R, Sukzuki W, Tamauchi H, Hozumi K, Habu S, Kubo M, Satake M - J. Exp. Med. (2003)

Bottom Line: In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression.Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased.We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Biological Sciences, Tokyo University of Science, Noda, Japan.

ABSTRACT
Differentiation of naive CD4+ T cells into helper T (Th) cells is controlled by a combination of several transcriptional factors. In this study, we examined the functional role of the Runx1 transcription factor in Th cell differentiation. Naive T cells from transgenic mice expressing a dominant interfering form of Runx1 exhibited enhanced interleukin 4 production and efficient Th2 differentiation. In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression. Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased. We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation.

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Related in: MedlinePlus

Effect of Runx1 overexpression on TCR- and IL-4R- signaling. (A) Naive CD4+ T cells were isolated from the spleens of BALB/c mice, stimulated with anti-CD3/anti-CD28 antibodies, and infected by retroviruses carrying pMX or pMX-Runx1. After culture in the presence of IL-2, the cells were washed, reactivated via TCR, and processed for flow cytometrical analysis. The red lines represent fluorescence emitted from antibody-stained cells, whereas the gray lines represent fluorescence from cells stained by control IgG. (B) The GFP+ population was sorted from the retrovirus-infected cells and cultured in the absence or presence of IL-4. In the left panel, protein was extracted at 10 min after the addition of IL-4, whereas, in the right panel, protein was extracted at 10, 30, and 60 min after the IL-4 addition. The extract was processed for immunoblot analysis using anti-Jak1, anti-STAT6, anti-phosphotyrosine, and anti-phosphorylated STAT6-specific antibodies. The bands indicated by the bottom arrow probably correspond to phosphorylated Jak1/Jak3, whereas the bands indicated by the top arrow probably represent phosphorylated IRS1/IRS2. (C) Naive CD4+ T cells were TCR-stimulated, infected by retroviruses, and cultured in the presence of IL-2 alone or of both IL-2 and the indicated concentration of IL-4. The GFP+ population was sorted and incubated in the presence of 3H-thymidine. The incorporation of radioactivity into an acid-insoluble fraction of the cells was measured. Data are representative of two independent experiments.
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fig4: Effect of Runx1 overexpression on TCR- and IL-4R- signaling. (A) Naive CD4+ T cells were isolated from the spleens of BALB/c mice, stimulated with anti-CD3/anti-CD28 antibodies, and infected by retroviruses carrying pMX or pMX-Runx1. After culture in the presence of IL-2, the cells were washed, reactivated via TCR, and processed for flow cytometrical analysis. The red lines represent fluorescence emitted from antibody-stained cells, whereas the gray lines represent fluorescence from cells stained by control IgG. (B) The GFP+ population was sorted from the retrovirus-infected cells and cultured in the absence or presence of IL-4. In the left panel, protein was extracted at 10 min after the addition of IL-4, whereas, in the right panel, protein was extracted at 10, 30, and 60 min after the IL-4 addition. The extract was processed for immunoblot analysis using anti-Jak1, anti-STAT6, anti-phosphotyrosine, and anti-phosphorylated STAT6-specific antibodies. The bands indicated by the bottom arrow probably correspond to phosphorylated Jak1/Jak3, whereas the bands indicated by the top arrow probably represent phosphorylated IRS1/IRS2. (C) Naive CD4+ T cells were TCR-stimulated, infected by retroviruses, and cultured in the presence of IL-2 alone or of both IL-2 and the indicated concentration of IL-4. The GFP+ population was sorted and incubated in the presence of 3H-thymidine. The incorporation of radioactivity into an acid-insoluble fraction of the cells was measured. Data are representative of two independent experiments.

Mentions: As overexpression of Runx1 prevented cells from differentiating into Th2 cells, we examined whether Runx1 affects the TCR and IL-4R signaling pathways. We used flow cytometry to analyze cell surface molecules such as the TCRβ chain, CD4, CD25, CD69, and IL-4Rα (Fig. 4 A). The expression profiles of these molecules in the GFP+ fractions were comparable in the pMX- and pMX-Runx1-infected cells. To analyze signaling molecules further, protein was extracted from the GFP+ population and processed for immunoblot analysis (Fig. 4 B). The amounts of Jak1 and STAT6 were not affected by overexpression of Runx1 nor by supplementation with IL-4. Phosphorylation of a 125-kD protein (indicated in Fig. 4 B by the bottom arrow) was induced by the addition of IL-4 and likely represents Jak1 and Jak3. The degree of phosphorylation of this 125-kD protein was comparable in pMX- and pMX-Runx1–infected cells. Phosphorylation of STAT6 was also induced by IL-4 to a similar degree in both the pMX- and pMX-Runx1–infected cells. Thus, overexpression of Runx1 is not likely to affect the IL-4R signaling pathway, at least not through the phosphorylation of STAT6. It must be noted also that overexpression of Runx1 was not inhibitory for IL-4–dependent cell proliferation. Different concentrations of IL-4 were added to the medium and incorporation of 3H-thymidine into an acid-insoluble fraction of the cells was measured (Fig. 4 C). The pMX-Runx1-infected cells proliferated rather better than the pMX-infected cells.


The Runx1 transcription factor inhibits the differentiation of naive CD4+ T cells into the Th2 lineage by repressing GATA3 expression.

Komine O, Hayashi K, Natsume W, Watanabe T, Seki Y, Seki N, Yagi R, Sukzuki W, Tamauchi H, Hozumi K, Habu S, Kubo M, Satake M - J. Exp. Med. (2003)

Effect of Runx1 overexpression on TCR- and IL-4R- signaling. (A) Naive CD4+ T cells were isolated from the spleens of BALB/c mice, stimulated with anti-CD3/anti-CD28 antibodies, and infected by retroviruses carrying pMX or pMX-Runx1. After culture in the presence of IL-2, the cells were washed, reactivated via TCR, and processed for flow cytometrical analysis. The red lines represent fluorescence emitted from antibody-stained cells, whereas the gray lines represent fluorescence from cells stained by control IgG. (B) The GFP+ population was sorted from the retrovirus-infected cells and cultured in the absence or presence of IL-4. In the left panel, protein was extracted at 10 min after the addition of IL-4, whereas, in the right panel, protein was extracted at 10, 30, and 60 min after the IL-4 addition. The extract was processed for immunoblot analysis using anti-Jak1, anti-STAT6, anti-phosphotyrosine, and anti-phosphorylated STAT6-specific antibodies. The bands indicated by the bottom arrow probably correspond to phosphorylated Jak1/Jak3, whereas the bands indicated by the top arrow probably represent phosphorylated IRS1/IRS2. (C) Naive CD4+ T cells were TCR-stimulated, infected by retroviruses, and cultured in the presence of IL-2 alone or of both IL-2 and the indicated concentration of IL-4. The GFP+ population was sorted and incubated in the presence of 3H-thymidine. The incorporation of radioactivity into an acid-insoluble fraction of the cells was measured. Data are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196077&req=5

fig4: Effect of Runx1 overexpression on TCR- and IL-4R- signaling. (A) Naive CD4+ T cells were isolated from the spleens of BALB/c mice, stimulated with anti-CD3/anti-CD28 antibodies, and infected by retroviruses carrying pMX or pMX-Runx1. After culture in the presence of IL-2, the cells were washed, reactivated via TCR, and processed for flow cytometrical analysis. The red lines represent fluorescence emitted from antibody-stained cells, whereas the gray lines represent fluorescence from cells stained by control IgG. (B) The GFP+ population was sorted from the retrovirus-infected cells and cultured in the absence or presence of IL-4. In the left panel, protein was extracted at 10 min after the addition of IL-4, whereas, in the right panel, protein was extracted at 10, 30, and 60 min after the IL-4 addition. The extract was processed for immunoblot analysis using anti-Jak1, anti-STAT6, anti-phosphotyrosine, and anti-phosphorylated STAT6-specific antibodies. The bands indicated by the bottom arrow probably correspond to phosphorylated Jak1/Jak3, whereas the bands indicated by the top arrow probably represent phosphorylated IRS1/IRS2. (C) Naive CD4+ T cells were TCR-stimulated, infected by retroviruses, and cultured in the presence of IL-2 alone or of both IL-2 and the indicated concentration of IL-4. The GFP+ population was sorted and incubated in the presence of 3H-thymidine. The incorporation of radioactivity into an acid-insoluble fraction of the cells was measured. Data are representative of two independent experiments.
Mentions: As overexpression of Runx1 prevented cells from differentiating into Th2 cells, we examined whether Runx1 affects the TCR and IL-4R signaling pathways. We used flow cytometry to analyze cell surface molecules such as the TCRβ chain, CD4, CD25, CD69, and IL-4Rα (Fig. 4 A). The expression profiles of these molecules in the GFP+ fractions were comparable in the pMX- and pMX-Runx1-infected cells. To analyze signaling molecules further, protein was extracted from the GFP+ population and processed for immunoblot analysis (Fig. 4 B). The amounts of Jak1 and STAT6 were not affected by overexpression of Runx1 nor by supplementation with IL-4. Phosphorylation of a 125-kD protein (indicated in Fig. 4 B by the bottom arrow) was induced by the addition of IL-4 and likely represents Jak1 and Jak3. The degree of phosphorylation of this 125-kD protein was comparable in pMX- and pMX-Runx1–infected cells. Phosphorylation of STAT6 was also induced by IL-4 to a similar degree in both the pMX- and pMX-Runx1–infected cells. Thus, overexpression of Runx1 is not likely to affect the IL-4R signaling pathway, at least not through the phosphorylation of STAT6. It must be noted also that overexpression of Runx1 was not inhibitory for IL-4–dependent cell proliferation. Different concentrations of IL-4 were added to the medium and incorporation of 3H-thymidine into an acid-insoluble fraction of the cells was measured (Fig. 4 C). The pMX-Runx1-infected cells proliferated rather better than the pMX-infected cells.

Bottom Line: In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression.Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased.We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Biological Sciences, Tokyo University of Science, Noda, Japan.

ABSTRACT
Differentiation of naive CD4+ T cells into helper T (Th) cells is controlled by a combination of several transcriptional factors. In this study, we examined the functional role of the Runx1 transcription factor in Th cell differentiation. Naive T cells from transgenic mice expressing a dominant interfering form of Runx1 exhibited enhanced interleukin 4 production and efficient Th2 differentiation. In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression. Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased. We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation.

Show MeSH
Related in: MedlinePlus