Co-chaperone potentiation of vitamin D receptor-mediated transactivation: a role for Bcl2-associated athanogene-1 as an intracellular-binding protein for 1,25-dihydroxyvitamin D3.
The constitutively expressed member of the heat shock protein-70 family (hsc70) is a chaperone with multiple functions in cellular homeostasis. Previously, we demonstrated the ability of hsc70 to bind 25-hydroxyvitamin D3 (25-OHD3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Hsc70 also recruits and interacts with the co-chaperone Bcl2-associated athanogene (BAG)-1 via the ATP-binding domain that resides on hsc70. Competitive ligand-binding assays showed that, like hsc70, recombinant BAG-1 is able to bind 25-OHD3 (Kd=0.71+/-0.25 nM, Bmax 69.9+/-16.1 fmoles/microg protein) and 1,25(OH)2D3 (Kd=0.16+/-0.07 nM, Bmax = 38.1+/-3.5 fmoles/microg protein; both n=3 separate binding assays, P<0.001 for Kd and Bmax). To investigate the functional significance of this, we transiently overexpressed the S, M, and L variants of BAG-1 into human kidney HKC-8 cells stably transfected with a 1,25(OH)2D3-responsive 24-hydroxylase (CYP24) promoter-reporter construct. As HKC-8 cells also express the enzyme 1alpha-hydroxylase, both 25-OHD3 (200 nM) and 1,25(OH)2D3 (5 nM) were able to induce CYP24 promoter activity. This was further enhanced following overexpression of all the three BAG-1 isoforms. By contrast, BAG-1 isoforms had no effect on metabolism of 25-OHD3 by HKC-8 cells (either via 1alpha- or 24-hydroxylase activities). Further studies showed that a mutant form of BAG-1S exhibited decreased binding of 1,25(OH)2D3 and this resulted in a concomitant loss of potentiation of CYP24 promoter transactivation. Similar effects were not observed for 25-OHD3. These data highlight a novel role for BAG-1 as an intracellular-binding protein for 1,25(OH)2D3 and further suggest that BAG-1 is able to potentiate vitamin D receptor-mediated transactivation by acting as a nuclear chaperone for 1,25(OH)2D3.
Chun
R F
RF
Division of Endocrinology, Diabetes and Metabolism, Burns and Allen Research Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA.
Gacad
M
M
Nguyen
L
L
Hewison
M
M
Adams
J S
JS
D015153:Blotting, Western; D002112:Calcifediol / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D018167:Receptors, Calcitriol / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics*; D014162:Transfection; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D065668:Vitamin D3 24-Hydroxylase
C091732:BCL2-associated athanogene 1 protein; D004268:DNA-Binding Proteins; D018832:Molecular Chaperones; D020033:Protein Isoforms; D018167:Receptors, Calcitriol; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D008156:Luciferases; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002112:Calcifediol