PMC:2222968 / 20242-23455 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2222968","sourcedb":"PMC","sourceid":"2222968","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2222968","text":"GATA3 Is a Negative Regulator of FOXP3 Expression\nFOXP3 expression decreased once GATA3 expression is high; therefore, we hypothesized a potential role for GATA3 in repressing FOXP3. Besides GATA3′s well-known positive effect on gene regulation, GATA3′s repressive capabilities were previously shown to restrict Th1 commitment by inhibiting STAT4 expression [2,21], and therefore GATA3 prevents differentiation into Th1 cells. To investigate whether GATA3 can directly inhibit FOXP3 induction, we transduced GATA3 or a truncated GATA3 lacking the DNA-binding domain in human primary CD4+CD45RA+ T cells using a TAT-fused, recombinantly expressed GATA3. After transduction, the cells were activated with soluble anti-CD3/CD28 in the presence or absence of TGF-β. TAT-GATA3 was successfully transduced in a homogeneous and dose-dependent manner into human CD4+ T cells (Figure 7A, upper panel). TAT-GATA3 reduced FOXP3 expression in a dose-dependent manner, whereas a DNA-binding domain truncated version (TAT-ΔDBD-GATA3) did not affect FOXP3 expression as compared with expression in untransduced cells (Figure 7A). In addition, we analyzed the inhibitory effect of GATA3 on FOXP3 in transgenic DO11.10 mice, constitutively overexpressing GATA3 under the control of the CD2 locus control region (DO11.10xCD2-GATA3). The thymic selection into the CD4 lineage is largely intact in DO11.10xCD2-GATA3 (RW Hendriks, unpublished data). These mice develop lymphomas at an older age, but signs of autoimmune disease were not described [22]. To investigate the effect of GATA3 on iTreg, CD4+CD62L+CD25– cells were isolated, activated with OVA in the presence or absence of TGF-β, and Foxp3 expression was analyzed after 4 d. The naive CD4+CD25– cells were Foxp3– (unpublished data). As described for the human cells, TGF-β dramatically up-regulated Foxp3 in the DO11.10 littermate control mice. In contrast, cells from the CD2-GATA3xDO11.10 mice showed dramatically reduced Foxp3 expression when activated with TGF-β and OVA (Figure 7B). All mice produced similar amounts of TGF-β; in addition, Smad7 was equally expressed [23] in T cells of both mice strains (Figure 7C), indicating intact TGF-β signaling.\nFigure 7 GATA3 Acts as a Negative Regulator of FOXP3 Expression\n(A) Human naive CD4+CDRA+ T cells were transduced with 0, 20, 100, and 500 nM of TAT-GATA3 protein, and intracellular presence of GATA3 was analyzed using FACS following anti-CD3/CD28 activation of the cells. GFP-positive cells were gated and analyzed for intracellular FOXP3 expression following a 2-d incubation period (lower panel). Data are representative of four independent experiments.\n(B) CD4+CD25− T cells were isolated from D011.10 and D011.10xCD2-GATA3 mice and treated with OVA and TGF-β for 96 h. Surface CD4 and intracellular FOXP3 were measured by FACS. These data are representative of three independent experiments.\n(C) The cells treated as in (B) were harvested and mRNA was quantified by real-time PCR for SMAD7 and TGF-β expression. Bars show the mean ± SD of three independent experiments. Taken together, these results demonstrated a repressive role of IL-4-induced GATA3 transcription factor in the generation of iTreg cells.","divisions":[{"label":"Title","span":{"begin":0,"end":49}},{"label":"Figure 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