Transcriptional regulation and renal localization of 1,25-dihydroxyvitamin D3-24-hydroxylase gene expression: effects of the Hyp mutation and 1,25-dihydroxyvitamin D3.
X-Linked hypophosphatemic (HYP) mice respond to low phosphate (Pi) intake with a fall in the serum concentration of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and an increase in the renal activity of 1,25-(OH)2D3-24-hydroxylase (24-hydroxylase), the first enzyme in the C-24 oxidation pathway that degrades 1,25-(OH)2D3 to its final inactivation product. In contrast, normal mice respond to a low Pi diet with an adaptive increase in serum 1,25-(OH)2D3 levels and no change in renal 24-hydroxylase. The low Pi response in Hyp mice involves a 3-fold increase in renal 24-hydroxylase maximum velocity and a corresponding increase in 24-hydroxylase immunoreactive protein and messenger RNA (mRNA). To determine the mechanism for the increase in 24-hydroxylase mRNA in the mutant strain, we examined the effect of actinomycin D on renal 24-hydroxylase mRNA abundance and measured renal 24-hydroxylase gene transcription by nuclear run-off assay in Hyp mice fed control and low Pi diets for 4 h. Vehicle and 1,25-(OH)2D3-treated normal mice were also studied 2-4 h post-treatment. Actinomycin D abrogated the increase in renal 24-hydroxylase mRNA elicited by a low Pi diet in Hyp mice and by 1,25-(OH)2D3 in both normal and Hyp mice. 24-Hydroxylase gene transcription, relative to that of glyceraldehyde-3-phosphate dehydrogenase, was increased 2-fold by feeding the low Pi diet to Hyp mice (n = 4; P < 0.05) and 5.4-fold by 1,25-(OH)2D3 administration to normal mice (n = 3; P < 0.01). In situ hybridization localized 24-hydroxylase transcripts to the proximal tubule of normal and mutant mice fed control and low Pi diets and showed that the 1,25(OH)2D3-induced increase in 24-hydroxylase mRNA occurred in the same nephron segment. The present study demonstrates that 1) transcriptional activation can account for the increase in renal 24-hydroxylase mRNA in Pi-deprived Hyp mice and for 24-hydroxylase mRNA induction by 1,25-(OH)2D3; and 2) the renal proximal tubule is the primary site of increased expression of 24-hydroxylase mRNA.
Roy
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Medical Research Council Genetics Group, McGill University-Montreal Children's Hospital Research Institute, Montreal, Quebec, Canada.
Tenenhouse
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D000818:Animals; D016375:Antisense Elements (Genetics); D001483:Base Sequence; D002117:Calcitriol / Q000494:pharmacology*; D002467:Cell Nucleus / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System; D003609:Dactinomycin / Q000494:pharmacology; D004386:Duodenum / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017674:Hypophosphatemia / Q000235:genetics; D007668:Kidney / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D016226:Phosphorus, Dietary / Q000494:pharmacology; D015347:RNA Probes; D012016:Reference Values; D013250:Steroid Hydroxylases / Q000096:biosynthesis*; D014158:Transcription, Genetic / Q000187:drug effects; D065668:Vitamin D3 24-Hydroxylase; D014960:X Chromosome
D016375:Antisense Elements (Genetics); D016226:Phosphorus, Dietary; D015347:RNA Probes; D003609:Dactinomycin; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol