The rapid effects of 1,25-dihydroxyvitamin D3 require the vitamin D receptor and influence 24-hydroxylase activity: studies in human skin fibroblasts bearing vitamin D receptor mutations.
If both rapid and genomic pathways may co-exist in the same cell, the involvement of the nuclear vitamin D receptor (VDR) in the rapid effects of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) remains unclear. We therefore studied rapid and long term effects of 1,25-(OH)(2)D(3) in cultured skin fibroblasts from three patients with severe vitamin D-resistant rickets and one age-matched control. Patients bear homozygous missense VDR mutations that abolished either VDR binding to DNA (patient 1, mutation K45E) or its stable ligand binding (patients 2 and 3, mutation W286R). In patient 1 cells, 1,25-(OH)(2)D(3) (1 pm-10 nm) had no effect on either intracellular calcium or 24-hydroxylase (enzyme activity and mRNA expression). In contrast, cells bearing the W286R mutation had calcium responses to 1,25-(OH)(2)D(3) (profile and magnitude) and 24-hydroxylase responses to low (1 pm-100 pm) 1,25-(OH)(2)D(3) concentrations (activity, CYP24, and ferredoxin mRNAs) similar to those of controls. The blocker of Ca(2+) channels, verapamil, impeded both rapid (calcium) and long term (24-hydroxylase activity, CYP24, and ferredoxin mRNAs) responses in patient and control fibroblasts. The MEK 1/2 kinase inhibitor PD98059 also blocked the CYP24 mRNA response. Taken together, these results suggest that 1,25-(OH)(2)D(3) rapid effects require the presence of VDR and control, in part, the first step of 1,25-(OH)(2)D(3) catabolism via increased mRNA expression of the CYP24 and ferredoxin genes in the 24-hydroxylase complex.
Nguyen
Thi-Minh
TM
CNRS UPR 1524/UMR 8104, Hôpital St Vincent de Paul, 75014 Paris, France. cnrs.svp@wanadoo.fr
Lieberherr
Michèle
M
Fritsch
Janine
J
Guillozo
Huguette
H
Alvarez
Maria Laura
ML
Fitouri
Zohra
Z
Jehan
Frederic
F
Garabédian
Michèle
M
D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D002675:Child, Preschool; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D005260:Female; D005288:Ferredoxins / Q000235:genetics; D005347:Fibroblasts; D005419:Flavonoids / Q000494:pharmacology; D006720:Homozygote; D006801:Humans; D007015:Hypophosphatemia, Familial / Q000235:genetics; D007223:Infant; D008297:Male; D009154:Mutation; D020125:Mutation, Missense; D012333:RNA, Messenger / Q000032:analysis; D018167:Receptors, Calcitriol / Q000235:genetics / Q000502:physiology*; D012867:Skin; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D014700:Verapamil / Q000494:pharmacology; D065668:Vitamin D3 24-Hydroxylase
D002121:Calcium Channel Blockers; D005288:Ferredoxins; D005419:Flavonoids; D012333:RNA, Messenger; D018167:Receptors, Calcitriol; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D014700:Verapamil; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D010738:Type C Phospholipases; D002117:Calcitriol; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium