Negative regulation of human parathyroid hormone gene promoter by vitamin D3 through nuclear factor Y

doi:10.1016/j.bbrc.2005.01.033

Biochemical and Biophysical Research Communications

Volume 328, Issue 4, 25 March 2005, Pages 831-837

https://doi.org/10.1016/j.bbrc.2005.01.033 Get rights and content

Abstract

The negative regulation of the human parathyroid hormone (PTH) gene by biologically active vitamin D₃ (1,25-dihydroxyvitamin D₃; 1,25(OH)₂D₃) was studied in rat pituitary GH4C1 cells, which express factors needed for the negative regulation. We report here that NF-Y binds to sequences downstream of the site previously reported to bind the vitamin D receptor (VDR). Additional binding sites for NF-Y reside in the near vicinity and were shown to be important for full activity of the PTH gene promoter. VDR and NF-Y were shown to exhibit mutually exclusive binding to the VDRE region. According to our results, sequestration of binding partners for NF-Y by VDR also affects transcription through a NF-Y consensus binding element in GH4C1 but not in ROS17/2.8 cells. These results indicate that 1,25(OH)₂D₃ may affect transcription of the human PTH gene both by competitive binding of VDR and NF-Y, and by modulating transcriptional activity of NF-Y.

Section snippets

Materials and methods

Constructs. The single-stranded 27-deoxynucleotide-long PTH-VDRE (27-VDRE, Fig. 1) [3] and the NF-Y consensus oligodeoxynucleotide (Ycons, Fig. 1) [5], [6], [7], [8] were synthesized with MluI or HindIII overhangs, annealed, and cloned into the respective site of the luciferase plasmid pGL3 (Promega, Madison, USA). The extended 44-deoxynucleotide-long single-stranded sequences (44-VDRE, Fig. 1) were annealed and blunt-end ligated into the SmaI site of the same plasmid. The pGL3 construct

Characterization of protein–DNA interactions of the PTH-VDRE region

To characterize protein–DNA interactions at the PTH-VDRE sequences, we performed EMSA with the different PTH-VDRE probes. The 27-VDRE previously reported to bind VDR [2], [3] formed several protein–DNA complexes with nuclear proteins from GH4C1 cells (Fig. 2). From these complexes, the most slowly migrating complex (arrowhead) was recognized by the VDR N-terminal antibody, whereas none of the complexes was recognized by the Sp1 (Fig. 2A) or NF-Y (Fig. 2B) antibodies. Binding reactions with the

Discussion

Nuclear factor Y (NF-Y) is an abundant transcription factor, which regulates expression of about 30% of all eukaryotic genes. It has a very high requirement for the sequence of CCAAT and its flanking nucleotides for efficient DNA binding. Only nine sequences out of the 178 sequences studied had one nucleotide difference in the CCAAT core and, for these sequences, the binding affinity of NF-Y was lower than that for the intact sequence [7]. In this study, we found that NF-Y bound to the extended

Acknowledgments

We thank Dr. Roberto Mantovani for the NF-YAmut construct, Dr. Henry Kronenberg for the pc301 plasmid, and Dr. Lise Binderup for 1,25(OH)₂D₃. We also thank Mrs. Maija Hiltunen, Mrs. Hanna Eskelinen, and Miss Liisa Röntynen for skilful technical assistance. This work was supported in part by the grants from the Research and Science Foundation of Farmos Co. (T.J.) and from the Academy of Finland (Grant No. 30566, P.M.).

References (14)

T. Okazaki et al.
5′-Flanking region of the parathyroid hormone gene mediates negative regulation by 1,25-(OH)₂ vitamin D₃
J. Biol. Chem.
(1988)
H. Darwish et al.
Identification of a transcription factor that binds to the promoter region of the human parathyroid hormone gene
Arch. Biochem. Biophys.
(1999)
P. Bucher
Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences
J. Mol. Biol.
(1990)
W. Bi et al.
DNA binding specificity of the CCAAT-binding factor CBF/NF-Y
J. Biol. Chem.
(1997)
R. Mantovani
The molecular biology of the CCAAT-binding factor NF-Y
Gene
(1999)
R. Mantovani et al.
Dominant negative analogs of NF-YA
J. Biol. Chem.
(1994)
Y. Xu et al.
Transcriptional activity of the SHP-1 gene in MCF7 cells is differentially regulated by binding of NF-Y factor to two distinct CCAAT-elements
Gene
(2001)

There are more references available in the full text version of this article.

Cited by (15)

Involvement of GCMB in the transcriptional regulation of the human parathyroid hormone gene in a parathyroid-derived cell line PT-r. Effects of calcium and 1,25(OH)<inf>2</inf>D<inf>3</inf>
2010, Bone
Citation Excerpt :
Within this region we found putative binding sites for Pbx1, GATA3, Sp1, Mafs, and NF-Y (Fig. 3A), and one or some of these transcription factors may be working to maintain the basal transcriptional activity of PTH gene [15–18,33–35]. Besides the tissue-specific regulation, PTH gene also receives short-term and dynamic transcriptional regulation by extracellular calcium and 1,25(OH)2D [5,7–12,14,16,20,21,45,46]. Among the factors, plasma calcium is known to exert a strict negative control on PTH secretion, but the effect on PTH gene expression is much less clear.
Expression of the PTH gene is known to be under strict tissue-specific control and is also regulated by extracellular calcium and 1,25(OH)₂D. However, the precise mode of transcriptional regulation remains to be elucidated, because of the unavailability of appropriate cell lines derived from the parathyroid gland. We tried to identify the transcription factor(s) regulating the human PTH gene transcription using the PT-r cell line. We found that PT-r cells endogenously express PTH and several parathyroid-related genes. Using the cells, we investigated the transcriptional regulation of human PTH gene. We found that GCMB binds to the PTH gene 5′-promoter (−390/−383 bp) and positively regulates its transcription. On the other hand, 1,25(OH)₂D₃, and, in the presence of the calcium sensing receptor, high extracellular calcium, exerted inhibitory effects on PTH gene expression. These results indicate that GCMB and vitamin D receptor are involved in the positive and negative regulation of PTH gene expression, respectively. Our data also suggest that PT-r cells retain some of the characteristics of parathyroid cells.
Predominant role of 25OHD in the negative regulation of PTH expression: Clinical relevance for hypovitaminosis D
2008, Life Sciences
Although severe deficiency of bioactive vitamin D (1,25OH2D) causes rickets, mild insufficiency of the hormone, known as hypovitaminosis D, is responsible for the occurrence of secondary hyperparathyroidism and osteoporosis. To clarify the pathophysiology of the disease, we studied the negative feedback effect of 1,25OH2D and its precursor 25OHD on the transcriptional activity of parathyroid hormone (PTH) gene using the PT-r parathyroid cell line. We found that PT-r cells express endogenous 1α-hydroxylase as well as PTH mRNAs. We also found the potent suppressive effect of physiological concentration of 25OHD on the transcriptional activity of PTH gene. A similar effect was obtained with 1,25OH2D but only with pharmacological concentration. Interestingly, the effect of 25OHD was completely abolished when the cells were treated with 1α-hydroxylase inhibitor ketoconazole. These results suggest that the negative feedback regulation of vitamin D on PTH gene transcription occurs not by the end-product 1,25OH2D but by its prohormone 25OHD via intracellular activation by 1α-hydroxylase within the parathyroid cells.
Parathyroid Hormone: Structure, Function and Dynamic Actions
2006, Dynamics of Bone and Cartilage Metabolism: Principles and Clinical Applications
Parathyroid Hormone: Structure, Function and Dynamic Actions
2006, Dynamics of Bone and Cartilage Metabolism
The Measurement and Interpretation of Fibroblast Growth Factor 23 (FGF23) Concentrations
2023, Calcified Tissue International
Local NF-κB Activation Promotes Parathyroid Hormone Synthesis and Secretion in Uremic Patients
2021, Endocrinology (United States)

View all citing articles on Scopus

^☆: Abbreviations: 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; CRBP-RXRE, retinoic acid responsive element of the cellular retinol-binding protein II gene; EMSA, electrophoretic mobility shift assay; NF-Y, nuclear factor Y; PTH, parathyroid hormone; RXR, retinoid X receptor; VDR, vitamin D receptor; VDRE, vitamin D response element.

¹: These authors should be considered as equal first authors.

²: Present address: Department of Medical Biochemistry, University of Kuopio, FIN-70211 Kuopio, Finland.

³: Present address: University Hospital of Oulu, FIN-90220 Oulu, Finland.

View full text

Negative regulation of human parathyroid hormone gene promoter by vitamin D3 through nuclear factor Y☆

Abstract

Section snippets

Materials and methods

Characterization of protein–DNA interactions of the PTH-VDRE region

Discussion

Acknowledgments

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Mol. Biol.

J. Biol. Chem.

Gene

J. Biol. Chem.

Gene

Negative regulation of human parathyroid hormone gene promoter by vitamin D₃ through nuclear factor Y☆