Vitamin D receptor mRNA measured in leukocytes with the TaqMan fluorogenic detection system: effect of calcitriol administration.
BACKGROUND
The aim of the present study was to investigate the interactions between the circulating concentrations of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and the mRNA concentration of its specific nuclear receptor in human leukocytes.


METHODS
We measured vitamin D receptor (VDR) mRNA extracted from leukocytes by use of TaqMan fluorescence analysis applied to the reverse transcription-PCR (RT-PCR) technique in 16 volunteers before and after calcitriol administration. VDR mRNA was also measured in leukocytes from calcium-stone-formers (37 hypercalciuric and 34 normocalciuric patients). The relationship between VDR mRNA concentrations and genetic VDR polymorphisms was analyzed in these patients.


RESULTS
Imprecision (CV) of RT-PCR was 1.3% within assay (n = 10) and 1.7% between assays (n = 4). Oral 1,25(OH)2D3 increased mean (SE) serum 1,25(OH)2D3 1.6 (0.3)-fold and VDR mRNA 1.6 (0.1)-fold 8 h after administration. The maximum VDR mRNA was reached 3.6 (1.3) h after 1,25(OH)2D3 ingestion. No differences in leukocyte VDR mRNA concentrations were found between normocalciuric and hypercalciuric stone-formers in the absence of stimulation. Finally, no association was found between VDR mRNA concentrations and genetic VDR polymorphisms in stone-formers.


CONCLUSIONS
The TaqMan RT-PCR assay is a rapid and accurate method to measure VDR mRNA, and leukocytes are a useful model to study VDR and 1,25(OH)2D3 interactions. In humans, VDR mRNA is increased by agonist 1,25(OH)2D3, a finding resembling previously reported results obtained in cellular and animal models.
Soldati
Laura
L
Department of Sciences and Biomedical Technologies, University of Milan, Milan, Italy. laura.soldati@unimi.it
Adamo
Donatella
D
Bianchin
Cristiana
C
Arcidiacono
Teresa
T
Terranegra
Annalisa
A
Bianchi
Maria Luisa
ML
Mora
Stefano
S
Cusi
Daniele
D
Vezzoli
Giuseppe
G
D000328:Adult; D002117:Calcitriol / Q000097:blood / Q000494:pharmacology*; D002118:Calcium / Q000652:urine; D005260:Female; D005838:Genotype; D006801:Humans; D007669:Kidney Calculi / Q000652:urine; D007962:Leukocytes / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000097:blood; D018167:Receptors, Calcitriol / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D019914:Taq Polymerase
D012333:RNA, Messenger; D018167:Receptors, Calcitriol; D019914:Taq Polymerase; D002117:Calcitriol; D002118:Calcium