Activation of Eotaxin-3/CCL26 Gene Expression in Human Dermal Fibroblasts Is Mediated by STAT6

Abstract

Allergic inflammatory conditions such as asthma are characterized by an accumulation of eosinophils at sites of inflammation. Eotaxin-3/CCL26 is a member of the family of CC chemokines, which are known to be potent chemoattractants for eosinophils. This chemokine was shown to be up-regulated by IL-4 and IL-13 in endothelial cells. This study demonstrates that eotaxin-3 transcription and eotaxin-3 protein expression are stimulated by IL-4 and IL-13 in a time- and dose-dependent fashion in human dermal fibroblasts. In contrast to eotaxin-1/CCL11, TNF-α could not act as inducer on its own nor did it synergize with IL-4. The activities of eotaxin-3 promoter luciferase constructs were significantly increased by IL-4 and IL-13 in human dermal fibroblasts. This effect was mediated by a binding site for the transcription factor STAT6 in the eotaxin-3 promoter sequence. Mutations in the STAT6 binding site abrogated up-regulation of eotaxin-3 promoter activity. In STAT6-defective human embryonic kidney 293 cells, the wild-type luciferase construct, but not the STAT6 binding mutant, was inducible by IL-4 only upon cotransfection of STAT6 expression vector. In addition, eotaxin-3 protein was detectable in the supernatants of STAT6-transfected human embryonic kidney 293 cells upon IL-4 or IL-13 stimulation. In the same experiments, TNF-α induced activation of the monocyte chemoattractant protein-1/CCL2 gene was independent of STAT6 transfection. These results indicate that IL-4 and IL-13 activate eotaxin-3 gene expression in a STAT6-dependent fashion. Although both eotaxin-1 and -3 are regulated by this transcription factor, the response of the eotaxin-3 gene to TNF-α stimulation appears to be different.

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

“…Increased levels of TARC/CCL17 mRNA became detectable as early as 2 h after IL-4 stimulation. This swift induction appears to be typical, as similar kinetics are seen with many IL-4-induced genes including SOCS-1 [29,38], FIZZ1 [39], Eotaxin/CCL11 [28] and Eotaxin-3/CCL26 [27]. Most other cell types that were examined for IL-4 inducibility of TARC/CCL17 require simultaneous stimulation with TNF-a to up-regulate its expression [8,21,30,33,[40][41][42].…”

“…Albeit they were shown to express TARC/ CCL17 mRNA and to express TARC/CCL17 protein upon allergen challenge [36,37], T cells have thus far not been demonstrated to express TARC/CCL17 after stimulation with IL-4. Hence, the present work was intended to study the IL-4 inducibility of the expression of TARC/ [27]. Most other cell types that were examined for IL-4 inducibility of TARC/CCL17 require simultaneous stimulation with TNF-a to up-regulate its expression [8,21,30,33,[40][41][42].…”

“…These were unable to block binding to the radio-labeled probes. Further, we used oligonucleotides containing a known STAT6 binding site of the Eotaxin-3/CCL26 promoter [27] as a control. These completely blocked binding of the radio-labeled probes when added as cold competitors.…”

“…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.…”