Fig. 5

Upregulation of interleukin (IL)-13 gene expression by transforming growth factor (TGF)-β occurs via GATA-3 transcription factor regulation: implication of Smad3 and mitogen-activated protein kinase (MAPK) signaling pathways. a GATA-3 mRNA levels in peripheral blood lymphocytes (PBLs) from patients with systemic sclerosis (SSc) (n = 11) and healthy donors (n = 8) were measured by RT-PCR analysis. b Jurkat T cells were cotransfected with pGL3 2666-bp IL-13 promoter construct/Luciferase (2666 bp-IL13-Lux) promoter and either a small interfering RNA (siRNA) specific for GATA-3 or siRNA control. Jurkat transfected cells were then treated or not with TGF-β for 24 h in 0.5 % fetal calf serum (FCS)-containing medium. The results are presented as mean percentage (±SD) of 2666 bp-IL13-Lux promoter activity in the presence (dark bars) or absence (white bars) of TGF-β relative to control siRNA activity without TGF-β taken as 100 (left panel). Expression of GATA-3 and β-actin in total lysates from control or GATA-3 siRNA was measured by Western blotting (right panel). The results are representative of three independent experiments. **p < 0.01. c Expression of GATA-3 and β-actin was measured by Western blotting in nuclear lysates from patients with SSc (left panel), healthy donors (middle panel) and Jurkat T cells (right panel) cultured in 0.5 % FCS-containing medium with SB431542 (activin receptor-like kinase ALK5 inhibitor), specific Smad3 inhibitor (SIS3) or SB203580 (specific p38 inhibitor) for 1 h before addition of TGF-β for 4 h. The results are representative of four independent experiments. Semiquantitative analysis of the blots was performed by using ImageJ software with β-actin levels for normalization. Normalized data are schematized as bars under the Western blots. d Jurkat T cells were cultured in 0.5 % FCS- containing medium for 16 h before addition of human recombinant TGF-β for 4 h (left panel) or for 30 min or 4 h (right panel). e PBLs from patients with SSc (n = 3) (left panel) and healthy donors (n = 3) (right panel) were stimulated with anti-CD3/anti-CD28 antibodies and IL-2 for 5 days and then treated or not with TGF-β for the last 4 h. d and e After precipitation of the protein–DNA complexes with specific antibody to GATA-3 or control immunoglobulin G (IgG), PCR amplification of the IL-13 fragment was performed using IL-13 promoter primers. GATA-3 chromatin immunoprecipitation (ChIP) enrichment = ChIP/input × 100 was determined by quantitative RT-PCR. The results are presented as mean (±SD) GATA-3 ChIP enrichment (GATA-3/IgG ratio) in the IL-13 gene after 4-h incubation with TGF-β (dark bars) and relative to respective expression in the absence of TGF-β (white bars) and reported to 100 to allow comparison of enrichment. The amplified SERPINF1 and CXCL4 promoter regions were used as positive controls for GATA-3 binding in Jurkat T-cells. *p < 0.05, **p < 0.01