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  • Original Paper
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A 5′-distal enhanceosome in the PDGF-A gene is activated in choriocarcinoma cells via ligand-independent binding of vitamin D receptor and constitutive jun kinase signaling

Abstract

Overexpression of platelet-derived growth factor A-chain (PDGF-A) is clearly linked to autocrine and paracrine stimulation of malignant growth in many human cancers. We have shown previously that PDGF-A overexpression in choriocarcinoma, hepatoma and lung carcinoma cell lines is driven by the activity of a 66 bp enhancer element (ACE66) located approximately 7 kb upstream of the PDGF-A transcription start site. In this study, the ACE66 element is shown to be activated in JEG-3 choriocarcinoma cells through synergistic interactions between consensus DNA motifs for binding of vitamin D receptor, AP1 and ELK1. Binding of the vitamin D/retinoid-X receptor (VDR/RXRα) heterodimer to the ACE66 element was reconstituted in vitro with recombinant VDR/RXRα and with JEG-3 nuclear extract, and was verified in living JEG-3 cells by chromatin immunoprecipitation analysis. Transcriptional activity of the ACE66 element, as well as occupancy of the element by VDR/RXRα, was shown to be independent of stimulation with the hormonal VDR ligand, 1,25-dihydroxyvitamin D3. The jun kinase pathway of mitogen-activated protein kinase (MAPK) signaling was shown to activate the ACE66 enhancer, most likely through activation of factors binding to the AP1 element. These results identify a novel mechanism of transcriptional enhancement involving ligand-independent activity of the VDR/RXR heterodimer and MAPK signaling pathways that appears to play an important role in the overexpression of PDGF in many different settings of human malignancy.

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Abbreviations

ACE:

A-chain cell-specific enhancer

ChIP:

chromatin immunoprecipitation

DMEM:

Dulbecco's modified Eagle's medium

DR3:

direct repeat-3

EIF:

ethylation interference footprinting

EMSA:

electrophoretic mobility shift assay

FBS:

fetal bovine serum

MAPK:

mitogen-activated protein kinase

MR:

mobility region

PDGF:

platelet-derived growth factor

RXR:

retinoid-X receptor

VDR:

vitamin D receptor

VDRE:

vitamin D response element

1,25-(OH)2D3, 1:

25-dihydroxyvitamin D3

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Acknowledgements

This work was supported by grants from the National Institutes of Health (HL62877, DK45518 and CA83237) and the Kentucky Lung Cancer Research Board. We thank David Bonthron and Tucker Collins for their kind support in the early phases of this project, particularly for providing many of the PDGF-A genomic clones and promoter vectors employed in this paper. We also thank Lise Binderup of Leo Pharmaceuticals for the gift of 1,25-(OH)2D3 used in these studies, Dennis Templeton for MEKK1 and c-JUN expression plasmids, Hollie Gravatte for technical assistance and Olivier Thibault for assistance with computer graphics.

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Correspondence to David M Kaetzel.

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Pedigo, N., Zhang, H., Bruno, M. et al. A 5′-distal enhanceosome in the PDGF-A gene is activated in choriocarcinoma cells via ligand-independent binding of vitamin D receptor and constitutive jun kinase signaling. Oncogene 24, 2654–2666 (2005). https://doi.org/10.1038/sj.onc.1208336

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