Enhanced coactivator binding and transcriptional activation of mutant vitamin D receptors from patients with hereditary 1,25-dihydroxyvitamin D-resistant rickets by phosphorylation and vitamin D analogs.
In this study, we report that the function of certain mutant VDRs from patients with hereditary HVDRR can at least be partially restored by phosphorylation and hexafluoro 1,25(OH)2D3 analogs. Our study provides new insights into mechanisms involved in enhancement of mutant VDR function.


BACKGROUND
1,25-Dihydroxyvitamin D-resistant rickets (HVDRR) is a rare genetic disorder caused by inactivating mutations in the vitamin D receptor (VDR). In this study, we examined VDR from patients with HVDRR having mutations in the ligand-binding domain (F251C, I268T, H305Q, E420K). We examined methods of restoring transcriptional activity of these mutants and the mechanisms involved.


METHODS
Reporter gene transcriptional assays were used to examine the activation of mutant VDRs. Western-blot analysis, glutathione S-transferase (GST) pull-down assays, and chromatin immunoprecipitation (ChIP) assays were also used in this study.


RESULTS
Using mutant VDRs, H305Q, F251C, I268T, and 10(-8) M 1,25(OH)2D3, only 10-30% of the activity of wildtype (WT) VDR in activating 24(OH)ase transcription was observed. The transcriptional response of mutant VDR mutants was significantly enhanced 2- to 3-fold by co-treatment of VDR mutant transfected COS-7 cells with 1,25(OH)2D3 and okadaic acid (OA; inhibitor of phosphatase; 50 nM). The H305Q mutant was the most responsive (90% of the response exhibited by WT VDR was restored). The E420K mutant was unresponsive to 1,25(OH)2D3 in the presence or absence of OA. The increased transcriptional response correlated with an increase in the interaction between DRIP205 and the mutant VDR. We further provide evidence that OA induces the phosphorylation of CREB-binding protein (CBP), indicating for the first time a correlation between phosphorylation of CBP and enhanced VDR function. Hexafluoro 1,25(OH)2D3 analogs (RO-26-2198 and RO-4383561) also resulted in at least a partial restoration of the transcriptional responsiveness of mutant VDRs I268T, F251C, and H305Q. Our data indicate that the enhanced potency of the hexafluoro analogs may be caused by increased DRIP205 and glucocorticoid receptor interacting protein 1 (GRIP-1) binding to VDRs and enhanced association of VDRs with DNA, as suggested by results of ChIP assays.


CONCLUSIONS
Our study provides new insights into the mechanisms involved in the enhancement of VDR function by both phosphorylation and hexafluoro analogs and forms a basis for future study of vitamin D analogs or specifically designed kinase activity mediators as potential therapy for the treatment of selected patients with HVDRR.
Liu
Yan
Y
Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School and the Graduate School for Biomedical Sciences, Newark, New Jersey 07103, USA.
Shen
Qi
Q
Malloy
Peter J
PJ
Soliman
Emad
E
Peng
Xiaorong
X
Kim
Sungtae
S
Pike
J Wesley
JW
Feldman
David
D
Christakos
Sylvia
S
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D047369:Chromatin Immunoprecipitation; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D008024:Ligands; D056913:Mediator Complex Subunit 1; D051379:Mice; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010006:Osteoblasts / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018167:Receptors, Calcitriol / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012279:Rickets / Q000188:drug therapy* / Q000235:genetics*; D014157:Transcription Factors / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology
D002352:Carrier Proteins; C491310:GRIP1 protein, human; D008024:Ligands; C538855:MED1 protein, human; C538856:Med1 protein, mouse; C538857:Med1 protein, rat; D056913:Mediator Complex Subunit 1; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D018167:Receptors, Calcitriol; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014807:Vitamin D; D019319:Okadaic Acid; D012313:RNA; C097949:1,25-dihydroxyvitamin D; D005982:Glutathione Transferase