Enhancement of VDR-mediated transcription by phosphorylation: correlation with increased interaction between the VDR and DRIP205, a subunit of the VDR-interacting protein coactivator complex.
When UMR-106 osteoblastic cells, LLCPK1 kidney cells, and VDR transfected COS-7 cells were transfected with the rat 24-hydroxylase [24(OH)ase] promoter (-1,367/+74) or the mouse osteopontin (OPN) promoter (-777/+79), we found that the response to 1,25dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] could be significantly enhanced 2- to 5-fold by the protein phosphatase inhibitor, okadaic acid (OA). Enhancement of 1,25-(OH)(2)D(3)-induced transcription by OA was also observed using a synthetic reporter gene containing either the proximal 24(OH)ase vitamin D response element (VDRE) or the OPN VDRE, suggesting that the VDRE is sufficient to mediate this effect. OA also enhanced the 1,25-(OH)(2)D(3)-induced levels of 24(OH)ase and OPN mRNA in UMR osteoblastic cells. The effect of OA was not due to an up-regulation of VDR or to an increase in VDR-RXR interaction with the VDRE. To determine whether phosphorylation regulates VDR-mediated transcription by modulating interactions with protein partners, we examined the effect of phosphorylation on the protein-protein interaction between VDR and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex, using glutathione-S-transferase pull-down assays. Similar to the functional studies, OA treatment was consistently found to enhance the interaction of VDR with DRIP205 3- to 4-fold above the interaction observed in the presence of 1,25-(OH)(2)D(3) alone. In addition, studies were done with the activation function-2 defective VDR mutant, L417S, which is unable to stimulate transcription in response to 1,25-(OH)(2)D(3) or to interact with DRIP205. However, in the presence of OA, the mutant VDR was able to activate 24(OH)ase and OPN transcription and to recruit DRIP205, suggesting that OA treatment may result in a conformational change in the activation function-2 defective mutant that creates an active interaction surface with DRIP205. Taken together, these findings suggest that increased interaction between VDR and coactivators such as DRIP205 may be a major mechanism that couples extracellular signals to vitamin D action.
Barletta
Frank
F
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School and Graduate School of Biomedical Sciences, Newark, New Jersey 07103, USA.
Freedman
Leonard P
LP
Christakos
Sylvia
S
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002762:Cholecalciferol / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D046911:Macromolecular Substances; D056913:Mediator Complex Subunit 1; D051379:Mice; D009154:Mutation / Q000235:genetics; D019319:Okadaic Acid / Q000494:pharmacology; D053495:Osteopontin; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D021122:Protein Subunits; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D047488:Retinoid X Receptors; D012795:Sialoglycoproteins / Q000235:genetics; D013250:Steroid Hydroxylases / Q000235:genetics; D013552:Swine; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D030181:Vitamin D Response Element / Q000235:genetics; D065668:Vitamin D3 24-Hydroxylase
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; C538856:Med1 protein, mouse; C538857:Med1 protein, rat; D056913:Mediator Complex Subunit 1; D021122:Protein Subunits; D012333:RNA, Messenger; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; C506897:Spp1 protein, rat; D014157:Transcription Factors; D053495:Osteopontin; D002762:Cholecalciferol; D019319:Okadaic Acid; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D010749:Phosphoprotein Phosphatases