The influence on the transcriptional regulation of chemokine receptor 3 (CCR3)

Abstract

Chemokine receptor 3 (CCR3), the major chemokine receptor expressed on eosinophils, binds promiscuously to several ligands, mainly the eotaxin family of chemokines which are up-regulated in inflammatory response. CCR3 expression in airway epithelial cells has also been proposed to play an important role in airway inflammation by promoting epithelial wound repair and subsequent tissue remodeling. The promoter region of CCR3 gene has recently been characterized in the literature and contains promoter elements which include a TAT A box and motifs for transcription factors such as NF-KB, AP-l and GATA-I. In this study, we investigated the effects of drugs whose action include an anti-inflammatory component, and for which literature evidence for a transcriptional mechanism exists, on the transcriptional regulation of CCR3 expression. pGL3E luciferase-based reporter deletion constructs were generated for the 1.6kb CCR3 promoter region, using standard cloning approaches in DH5α E. Coli cells. Each promoter construct was transfected in a pulmonary epithelial cell line (A549) in microwell plate format and stimulated with drugs (glucocorticoid dexamethasone, endogenous glucocorticoid cortisol, and theophylline) in a dose dependent manner. This study has shown that CCR3 transcription in cytokine unstimulated A549 cells can be regulated by glucocorticoids and theophylline. A tri-phasic response (i.e. activating at low concentration, repressive at medium concentration and activating at high concentration) in CCR3 transcription response to dexamethasone was observed, indicating a complex transcriptional regulatory mechanism. Dexamethasone induced an nF-κB independent transcriptional repression of CCR3 in the unstimulated A549, possibly involving the interaction of the activated glucocorticoid receptor with AP-l in a trans-repressive molecular mechanism. Furthermore, CCR3 transcription response to the endogenous glucocorticoid cortisol was significantly different than that observed for dexamethasone. Our results have also shown that theophylline significantly represses CCR3 transcription in the absence of glucocorticoids.