STAT6 transcription factor binding sites with mismatches within the canonical 5′‐TTC…GAA‐3′ motif involved in regulation of δ‐ and µ‐opioid receptors

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Abstract
Opioid receptors are expressed in neuronal and immune cells and regulated in response to immunological processes. Herein, we demonstrate up-regulation of the delta-opioid receptor gene by interleukin-4 in immune cells (primary T and polymorphonuclear leukocytes, Jurkat E6 T cells), and in NG 108-15 neuronal cells. We identified an interleukin-4-responsive element at nt -671 on the murine gene promoter, to which the transcription factor STAT6 binds, as shown by reporter gene analysis and STAT6/DNA interaction studies in living cells with transcription factor decoy oligonucleotides. STAT6 normally binds to palindromic DNA motifs with a 5'-TTC...GAA-3' core. Notably, the delta-opioid receptor STAT6 site (5'-TTC...GGA-3') is an imperfect palindrome with a mismatch within this core sequence. A systematic analysis of possible mismatch 5'-TTC...GAA-3' motifs revealed that STAT6 also binds to the sequence 5'-TTA...GAA-3'. This motif occurs as a polymorphism in the human mu-opioid receptor gene (Kraus et al. 2001 J. Biol. Chem 276, 43901-43908). We show that this mutated element has a significantly reduced STAT6 binding activity which correlates to its reduced interleukin (IL)-4 inducibility. In contrast, the non-canonical STAT6 site of the delta-opioid receptor binds STAT6 with similar high activity as a perfectly palindromic STAT6 site and is strongly inducible by IL-4.