Abstract
Purpose: :
A potential aryl hydrocarbon receptor (AhR) binding site [xenobiotic responsive element (XRE)-like; at positions -238/-216] exists in the mouse small heat shock protein/αB-crystallin enhancer. Here we examine whether AhR can regulate αB-crystallin promoter activity.
Methods: :
αB-crystallin promoter activity was tested in a dual-luciferase reporter plasmid driven by the -959/+42bp fragment in transient and stable co-transfection experiments. AhR and ARNT(AhR nuclear translocator) expression plasmids were gifts of Dr. Alvaro Puga (University of Cincinnati Medical Center, Ohio) and Dr. Oliver Hankinson(University of California, CA). Reporter gene tests, electrophoretic mobility gel shift assays (EMSAs) and point mutations were executed by conventional methods.
Results: :
AhR/ARNT caused dose-dependent increases (up to 8.5-fold) in αB-crystallin promoter activity in co-transfected HeLa cells. Added TCDD (10 nM) or 3-MC (1uM) resulted in <2-fold further increase in αB-crystallin promoter activity. AhR/ARNT also stimulated αB-crystallin promoter activity in co-transfected COS7 cells (5.1-fold), NIH3T3 cells (3.4-fold), and transiently and stabley transfected lens αTN4 cells (2-fold and 4.6-fold, respectively). EMSAs using HeLa, αTN4 and Hepa-1 cell nuclear extracts and non-radioactive probe competition and antibody supershift assays revealed specific binding of AhR/ARNT to the XRE-like element (core sequence: CATGCGA) in the absence and presence of added TCDD. Mutations of the core sequence to a consensus XRE I core sequence (CACGCAA) increased AhR/ARNT binding in the gel shift assay but did not increase αB-crystallin promoter activity in the reporter gene assays. Mutation of the core sequence to a non-XRE sequence (CGGACGA) greatly decreased AhR/ARNT binding in the gel shift assay and significantly decreased AhR/ARNT-induced reporter activity. Finally, co-transfected GATA1 or GATA3 repressed αB-crystallin promoter activity in HeLa cells and abolished AhR/ARNT activation of the αB-crystallin promoter. Multiple potential GATA binding sites were found in αB-crystallin promoter/enhancer region.
Conclusions: :
Our data suggest that AhR-ARNT can activate the αB-crystallin promoter in a ligand independent fashion.
Keywords: crystallins • gene/expression • transcription factors