Transcriptional Regulation of Mouse δ-Opioid Receptor Gene: IKAROS-2 AND UPSTREAM STIMULATORY FACTOR SYNERGIZE IN TRANS-ACTIVATING MOUSE δ-OPIOID RECEPTOR GENE IN T CELLS

Ping Sun,Horace H Loh

doi:10.1074/jbc.m208162200

Ping Sun, Horace H Loh

Open Access

https://doi.org/10.1074/jbc.m208162200

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Abstract

Considerable evidence indicates that transcription of the delta-opioid receptor (dor) gene is correlated with both the expression of DOR on T cells and the capacity of DOR agonists to modulate the immunological functions of the T cell. We previously reported that increased Ikaros (Ik) binding activity over an Ik-binding site at -378 to -374 (with the translation start site designated as +1) in the mouse dor promoter was required for the enhanced transcription of dor gene in phytohemagglutinin-activated EL-4 cells, a mouse T cell line that constitutively expresses DOR. In the present study, we have analyzed further the mouse dor promoter in EL-4 cells and have demonstrated that Ik-2 homodimers bind to the -378/-374 Ik-binding site and exerts a position-dependent trans-activation effect on the dor promoter. Moreover, an E box (-185 to -180) that binds upstream stimulatory factor is essential for the dor promoter activity in both resting and phytohemagglutinin-activated T cells. Furthermore, we have demonstrated that Ik-2 and upstream stimulatory factor synergize in trans-activating the dor promoter via the putative Ik-binding site and the E box, respectively.

Highlights

Endogenous and synthetic ␦-opioids have been shown to modulate T-cell proliferation, cytokine production, and calcium mobilization through the ␦-opioid receptor (DOR)1 on T cells [1,2,3,4]
We previously reported that increased Ikaros (Ik) binding activity over an Ik-binding site at ؊378 to ؊374 in the mouse dor promoter was required for the enhanced transcription of dor gene in phytohemagglutinin-activated EL-4 cells, a mouse T cell line that constitutively expresses DOR
Further analyses were carried out in EL-4 cells. Through both in vivo and in vitro experiments, we have demonstrated that Ik-2 homodimers bind to the Ϫ378/Ϫ374 Ik-binding site and exerts a position-dependent trans-activation effect on the dor promoter

Summary

Introduction

Endogenous and synthetic ␦-opioids have been shown to modulate T-cell proliferation, cytokine production, and calcium mobilization through the ␦-opioid receptor (DOR) on T cells [1,2,3,4]. ␤-endorphin was shown to enhance intracellular calcium mobilization in murine splenic T cells, which was inhibited by naltrindole, a selective DOR antagonist, whereas the selective ␮-opioid receptor antagonist was ineffective [2]. Subsequent studies revealed that the minimum dor promoter was sufficient to confer constitutive dor promoter activity in EL-4 cells, a mouse T cell line that constitutively expresses DOR. Increased binding activity of Ikaros (Ik) at an Ik-binding site (Ϫ378 to Ϫ374) was demonstrated to account for the significantly enhanced dor promoter activity in phytohemagglutinin (PHA)-activated EL-4 cells [13]. Further analyses were carried out in EL-4 cells Through both in vivo and in vitro experiments, we have demonstrated that Ik-2 homodimers bind to the Ϫ378/Ϫ374 Ik-binding site and exerts a position-dependent trans-activation effect on the dor promoter. We have demonstrated that Ik-2 and USF synergize in trans-activating the dor promoter via the putative Ikbinding site and the E box, respectively

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