1,25-Dihydroxyvitamin D3 stimulates cyclic vitamin D receptor/retinoid X receptor DNA-binding, co-activator recruitment, and histone acetylation in intact osteoblasts.
1,25(OH)2D3 induces gene expression through the VDR. We used chromatin immunoprecipitation techniques to explore this 1,25(OH)2D3-induced process on the 25-hydroxyvitamin D3-24-hydroxylase (Cyp24) and Opn gene promoters in intact osteoblasts. Our studies show that 1,25(OH)2D3-induced transactivation is a dynamic process that involves promoter-specific localization of VDR and RXR, recruitment of histone acetyltransferase complexes, and in the case of the Cyp24 gene, modification of histone 4.


BACKGROUND
The vitamin D receptor (VDR) binds as a retinoid X receptor (RXR) heterodimer to target DNA sequences and facilitates the recruitment of protein complexes that are essential for transcriptional modulation. These complexes include an acetyltransferase component that contains members of the p160 family and p300/CBP as well as human mediator that contains D receptor interacting protein (DRIP205). The objective of this study was to investigate the kinetics of VDR/RXR binding to 25-hydroxyvitamin D3-24-hydroxylase (Cyp24) and osteopontin (Opn) target gene promoters and to explore the recruitment and subsequent activities of co-activator complexes on these target genes in intact cells.


METHODS
Mouse osteoblastic MC3T3-E1 cells and mouse primary calvarial osteoblasts (MOBs) were cultured in alphaMEM medium supplemented with 10% FBS. Confluent cells were treated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or the vitamin D antagonist ZK159222, and the ability of these compounds to induce localization of VDR and RXR to specific regions of Cyp24 and Opn target genes was examined using chromatin immunoprecipitation techniques. The ability of both compounds to induce the recruitment of co-activator proteins such as p160 family members, CBP and DRIP205, and to increase the level of histone acetylation on the two gene promoters in MC3T3-E1 cells was also examined.


RESULTS
1,25(OH)2D3 induces rapid association of the VDR and RXR with both the Cyp24 and the Opn gene promoters in both MC3T3-E1 osteoblasts and MOBs, interactions that are both rapid and cyclic in nature. 1,25(OH)2D3 treatment also induces rapid recruitment of co-regulators such as SRC-1, -2, and -3, CBP, and p300 to both promoters, recruitment that leads to acetylation of histone 4 on Cyp24 but not the Opn. DRIP205 is also recruited to the two promoters in response to hormonal stimulation, an appearance that correlates directly with entry of RNA pol II. Studies with the vitamin D antagonist ZK159222 suggest a complex mode of action of this compound in blocking 1,25(OH)2D3-induced transcription. Our studies indicate that 1,25(OH)2D3-induced transactivation in intact osteoblasts is a dynamic process that involves promoter-specific localization of VDR and RXR as well as the recruitment of a number of co-regulators essential to 1,25(OH)2D3-induced transcription.


CONCLUSIONS
We conclude that co-regulators essential for the transcriptional activity of the steroid receptor gene family are indeed critical for the actions of 1,25(OH)2D3. Selective use of co-regulators by target genes, however, may provide a mechanism for the unique and perhaps gene-selective responses observed with synthetic analogs such as ZK159222.
Kim
Sungtae
S
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Shevde
Nirupama K
NK
Pike
J Wesley
JW
D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D047369:Chromatin Immunoprecipitation; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D050881:E1A-Associated p300 Protein; D051548:Histone Acetyltransferases; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D009687:Nuclear Proteins / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism*; D053495:Osteopontin; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000378:metabolism*; D047488:Retinoid X Receptors / Q000378:metabolism*; D012795:Sialoglycoproteins / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000378:metabolism; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D020798:Two-Hybrid System Techniques; D065668:Vitamin D3 24-Hydroxylase
D006657:Histones; D009687:Nuclear Proteins; D018167:Receptors, Calcitriol; D047488:Retinoid X Receptors; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D015534:Trans-Activators; C410228:ZK159222; D053495:Osteopontin; D012313:RNA; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D000123:Acetyltransferases; D050881:E1A-Associated p300 Protein; C494361:Ep300 protein, mouse; D051548:Histone Acetyltransferases; D002117:Calcitriol