The Journal of Steroid Biochemistry and Molecular Biology
Identification of a Vitamin D response element in the human insulin receptor gene promoter☆
Introduction
1,25-Dihydroxyvitamin D3 (1,25D3), the most active Vitamin D metabolite, is known to regulate plasma calcium and phosphorus concentrations to levels required for normal skeleton mineralization and neuromuscular function [1], [2]. Further physiological functions of this steroid hormone include effects on cell proliferation, differentiation and immunosupression, secretion of hormones, and regulation of gene expression in different cells [3], [4].
1,25D3 acts as a ligand for the Vitamin D receptor (VDR, NR1I1). This receptor is a member of the superfamily of nuclear receptors, which regulates gene expression as a Vitamin D-dependent transcription factor, and exerts this action by binding, preferentially as a heterodimer with the retinoid X receptor (RXR), to Vitamin D response elements (VDREs) in the promoter regions of target genes [5].
A VDRE generally consists of two direct imperfect repeats of six nucleotides separated by a three nucleotide spacer. The VDR occupies the 3′ half-site, while the RXR binds to the 5′ half-site. Several sequence variations had been detected in the 3′ half-element, the 5′ half-element, the spacer, and in the sequences flanking the VDREs [4], [6]. These differences appear to be important in determining receptor-binding efficiency [7].
Although many genes have been reported to be regulated by 1,25D3, transcriptional regulation by this hormone has only been described in a small proportion of them, and the identification of VDREs has only been possible in a very limited number of these genes [4], [6].
We recently reported the first demonstration that 1,25D3 increased human insulin receptor (hIR) mRNA levels, insulin binding, and insulin responsiveness of U-937 human promonocytic cells via mechanisms involving direct transcriptional activation of the hIR gene [8], [9], [10]. These effects involved no change in IR mRNA stability [8] and were mediated by an increase in VDR expression, both at the RNA and protein levels [9]. These findings suggest that the activated VDR, behaving as a Vitamin D transcription factor, binds to potential VDREs in the hIR gene promoter. However, the existence of VDREs that could account for this transcriptional induction of hIR mRNA levels by 1,25D3 has not yet been demonstrated in this promoter.
The aim of the present study was thus to investigate the possible existence and location of VDREs in the hIR promoter. The results indicate that a sequence, from −761 to −732 bp of this promoter, including an AP-2 like sequence, specifically binds VDR.
Section snippets
Cell culture and treatments
U-937 human promonocytic cells (mycoplasma-free) were grown in RPMI-1640 medium, supplemented with 10% (v/v) heat-inactivated fetal calf serum and antibiotics at 37 °C in a humidified 5% CO2 atmosphere as previously described [11]. 1,25D3 (Roche) was dissolved in absolute ethanol at 10−5 M, and applied to the cells at a final concentration of 10−8 M. Untreated cells received only the vehicle. After 24 h of treatment, the cells were collected by centrifugation, and washed three times in
Results
In the present work, we initially determined the relative activities of the −1819 to −271 bp of the hIR promoter (wild type promoter), and that of a series of constructs of this promoter corresponding to the plasmids: phIR(−1819)-GL2, phIR(−1473)-GL2, phIR(−876)-GL2 and phIR(−577)-GL2. U-937 cells were transiently transfected with these plasmids. Luciferase determinations revealed that the basal promoter activity of the wild type promoter, considered as 100%, gradually decreased in the
Discussion
Our research group is presently engaged in exploring hIR gene expression regulation by different hormones, in particular, steroid hormones such as glucocorticoids [11], [33], mineralocorticoids [34], [35], estrogens [36] and 1,25D3 [8], [9], [10] in U-937 human promonocytic cells.
Concerning 1,25D3, we were previously able to demonstrate that the capacity of this hormone to potenciate hIR expression and insulin action in U-937 cells was transcriptionally regulated [10]. This was tested by
Acknowledgements
The authors are indebted to Drs. S.Y. Tsai and G. Elberg for their generous gift of the hIR promoter. B. Maestro holds a fellowship at the Complutense University of Madrid.
This work was supported by research funds from the DGES, grant (BMC2000/0765); and the CAM, grant (08.6/0010/98).
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Poster paper presented at the 15th International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, “Recent Advances in Steroid Biochemistry and Molecular Biology” Munich, Germany, 17–20 May 2002.