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.

R F Chun, M Gacad, L Nguyen, M Hewison, J S Adams

Division of Endocrinology, Diabetes and Metabolism, Burns and Allen Research Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA.

Journal of molecular endocrinology 2007 Aug

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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.

Citation

R F Chun, M Gacad, L Nguyen, M Hewison, J S Adams. 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. Journal of molecular endocrinology. 2007 Aug;39(2):81-9