Human calcium-sensing receptor gene - Vitamin D response elements in promoters P1 and P2 confer transcriptional responsiveness to 1,25-dihydroxyvitamin D

The calcium-sensing receptor (CASR), expressed in parathyroid chief cells, thyroid C-cells, and cells of the kidney tubule, is essential for maintenance of calcium homeostasis. Here we show parathyroid, thyroid, and kidney CASR mRNA levels increased 2-fold at 15 h after intraperitoneal injection of 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) in rats. Human thyroid C-cell (TT) and kidney proximal tubule cell (HKC) CASR gene transcription increased similar to2-fold at 8 and 12 h after 1,25(OH)(2)D-3 treatment. The human CASR gene has two promoters yielding alternative transcripts containing either exon 1A or exon 1B 5'-untranslated region sequences that splice to exon 2 some 242 by before the ATG translation start site. Transcriptional start sites were identified in parathyroid gland and TT cells; that for promoter P1 lies 27 by downstream of a TATA box, whereas that for promoter P2, which lacks a TATA box, lies in a GC-rich region. In HKC cells, transcriptional activity of a P1 reporter gene construct was 11-fold and of P2 was 33-fold above basal levels. 10(-8) M 1,25(OH)(2)D-3 stimulated P1 activity 2-fold and P2 activity 2.5-fold. Vitamin D response elements (VDREs), in which half-sites (6 lip) are separated by three nucleotides, were identified in both promoters and shown to confer 1,25(OH)(2)D-3 responsiveness to a heterologous promoter. This responsiveness was lost when the VDREs were mutated. In electrophoretic mobility shift assays with either in vitro transcribed/translated vitamin D receptor and retinoid X receptor-a, or HKC nuclear extract, specific protein-DNA complexes were formed in the presence of 1,25(OH)(2)D-3 on oligonucleotides representing the P1 and P2 VDREs. In summary, functional VDREs have been identified in the CASR gene and provide the mechanism whereby 1,25(OH)(2)D up-regulates parathyroid, thyroid C-cell, and kidney CASR expression.