STAT6 Mediates Eotaxin-1 Expression in IL-4 or TNF-α-Induced Fibroblasts

Abstract

Eosinophils are attracted to sites of allergic inflammation by a number of chemoattractants including eotaxin-1. This chemokine can be secreted from epithelial cells and fibroblasts after IL-4 and TNF-α stimulation in a synergistic fashion. TNF-α activated gene expression at the transcriptional level in a STAT6-dependent manner, because: 1) eotaxin-1 promoter luciferase constructs were TNF-α inducible in STAT6-defective HEK293 cells only on cotransfection of STAT6 expression vector, an effect that was partially mediated by activation-induced binding of NF-κB proteins to a composite STAT6/NF-κB element; 2) reporter constructs defective in STAT6 DNA binding did not respond to TNF-α stimulation; 3) eotaxin-1 protein secretion was detected only in STAT6-transfected HEK293 cell supernatants on TNF-α treatment; and 4) a trans-dominant negative STAT6 protein inhibited TNF-α-induced eotaxin-1 secretion in primary fibroblasts. TNF-α inducibility of the IL-8 and monocyte chemoattractant protein-1 genes was not dependent on STAT6 expression in the same experimental systems. The inducing effect of IL-4 and IL-13 was also mediated by STAT6. The synergistic effect of IL-4 and TNF-α observed at the RNA and the protein level was not seen at the promoter level. The data demonstrate that both IL-4 and TNF-α induce eotaxin-1 expression at the level of transcription via a STAT6-mediated pathway.

CD45 isoform expression is associated with different susceptibilities of human naive and effector CD4+ T cells to respond to IL‐4

“…4B demonstrates that IL-4 induced STAT6-mediated gene expression in CD45RA + T cells but not in CD45R0 + T cells. When freshly purified CD45RA + and CD45R0 + T cells were transfected with either a mutant STAT6 lacking the C-terminal transactivation domain (STAT6-DTD), which has been shown to act in a trans-dominant negative fashion [26,27], or full-length STAT6, IL-4/ STAT6-dependent proliferation was observed mainly in naive T cells (Fig. 4C).…”

“…NM_003153) was amplified from activated human T cells and cloned into pcDNA3.1 TOPO (Invitrogen). The C-terminally truncated STAT6 lacking 186 amino acids (STAT6-DTD, cloned in pcDNA3.1), which has been shown to act in a transdominant negative fashion [26], was used as described [27]. Full-length human CD25 (Acc.…”

IL‐4 induces expression of TARC/CCL17 via two STAT6 binding sites

“…The preference of STAT6 for sites in which the half-palindromes (TTC) are separated by four nucleotides (N 4 motif) distinguishes it from other STAT molecules, which preferentially bind to N 3 motifs [24]. STAT6 is critical for the activation of several IL-4-responsive genes, including those for MHC class II molecules, CD23, Eotaxin-1 and -3, germ-line IgE transcripts, and suppressor of cytokine signaling (SOCS)-1 [25][26][27][28][29].…”

“…The preference of STAT6 for sites in which the half-palindromes (TTC) are separated by four nucleotides (N 4 motif) distinguishes it from other STAT molecules, which preferentially bind to N 3 motifs [24]. STAT6 is critical for the activation of several IL-4-responsive genes, including those for MHC class II molecules, CD23, Eotaxin-1 and -3, germ-line IgE transcripts, and suppressor of cytokine signaling (SOCS)-1 [25][26][27][28][29].TARC/CCL17 expression is induced by a variety of stimuli including [30][31][32]. IL-4-dependent regulation of TARC/CCL17 expression has been shown for airway smooth muscle cells [33], the keratinocyte cell line HaCaT [34] and fibroblasts; the mechanism of this regulation, however, remained uncharacterized.…”

“…5). As substitution of two nucleotides in the inverted repeat region of the STAT6 binding site has been shown to abrogate STAT6 binding [28], mutated oligonucleotides (probes MA and MB) featuring these substitutions were tested in the EMSA. These mutated oligonucleotides were unable to interact with the IL-4-induced factor.…”