Identification of a Vitamin D response element in the human insulin receptor gene promoter

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Abstract

The present study was designed to explore the possible presence and location of Vitamin D response elements (VDREs) in the human insulin receptor (hIR) gene promoter. To this end, the −1819 to −271 bp fragment of the hIR promoter (wild type promoter) and progressive 5′ deletions of this promoter (up to −1473 and −876 bp) were linked to the luciferase pGL2-basic vector to construct the reported plasmids: phIR (−1819)-GL2, phIR(−1473)-GL2 and phIR(−876)-GL2, respectively. U-937 cells were transiently transfected with these plasmids, and then the cells were either untreated or treated for 24 h with 10−8 M 1,25-dihydroxyvitamin D3 (1,25D3). Luciferase determinations revealed that, while the activity of the wild promoter was increased 1.6-fold by the hormone, the activities of progressive 5′ deletions of this promoter were enhanced 1.7-, and 1.6-fold, respectively. Thus, the region extending from −876 to −271 bp of the hIR promoter, appears to contain VDREs, and to be sufficient for induction by 1,25D3. In order to identify these potential VDREs, we performed a computer search of candidate sequences by homology with a consensus VDRE sequence. This search yielded a sequence located between −761 and −732 bp (5′CGTCGGGCCTGTGGGGCGCCTCCGGGGGTC3′), which includes an overlapping AP-2 like sequence, as a good candidate. Electrophoretic mobility shift assays revealed that the Vitamin D receptor (VDR) specifically recognized this sequence, since a VDR–DNA complex was able to compete with the unlabeled probe and was cleared by the specific anti-VDR antibody 9A7. These data represent the first identification of a VDRE in the hIR 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).

References (42)

  • K.E. Cameron et al.

    Transcriptional regulation of the human insulin receptor promoter

    J. Biol. Chem.

    (1992)
  • K. Yoshizato et al.

    Identification of a cis-acting element and a novel transacting factor of the human insulin receptor gene in HepG2 and rat liver cells

    Biochem. Bioph. Res. Co.

    (2001)
  • E. Araki et al.

    A cluster of four Sp1 binding sites required for efficient expression of the human insulin receptor gene

    J. Biol. Chem.

    (1991)
  • J. Campión et al.

    Inhibition by aldosterone of insulin receptor mRNA levels and insulin binding in U-937 human promonocytic cells

    J. Steroid Biochem. Mol. Biol.

    (1999)
  • K. Hilger-Eversheim et al.

    Regulatory roles of AP-2 transcription factors in vertebrate development, apoptosis and cell-cycle control

    Gene

    (2000)
  • M.F. Holick

    Vitamin D: the underappreciated D-lightful hormone that is important for skeletal and cellular health

    Curr. Opin. Endocr. Diabetes

    (2002)
  • A.J. Brown et al.

    Vitamin D

    Am. J. Physiol.

    (1999)
  • M.R. Haussler et al.

    The nuclear vitamin D receptor: biological and molecular regulatory properties revealed

    J. Bone Miner. Res.

    (1998)
  • A. Aranda et al.

    Nuclear hormone receptors and gene expression

    Physiol. Rev.

    (2001)
  • B. Maestro et al.

    Stimulation by 1,25-dihydroxyvitamin D3 of insulin receptor expression and insulin responsiveness for glucose transport in U-937 human promonocytic cells

    Endocr. J.

    (2000)
  • B. Maestro et al.

    Transcriptional activation of the human insulin receptor gene by 1,25-dihydroxyvitamin D3

    Cell Biochem. Funct.

    (2002)
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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.

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