The central hypothesis presented here holds that steroid hormone transit generally, and vitamin D metabolite transit specifically, into and within its metabolizing and/or target cell does not depend on the random movement of small, lipid-soluble, easily diffusible molecules. Rather, there exists a family of molecular chaperones that orchestrate the directional movement of vitamin D molecules to specific cellular destinations. It is proposed that this family of molecular shuttle proteins to be the hsp-70-related intracellular vitamin D binding proteins (IDBPs) discovered in this laboratory. Confirmation of this central hypothesis requires that four corollary hypotheses be proved true: 1] IDBPs bind 25-hydroxylased vitamin D metabolites with sufficient capacity; 2] a favorable affinity gradient exists between and among intracellular binding proteins (i.e. megalin- bound serum vitamin D binding protein, other IDBPs, the vitamin- D-hydroxylases, vitamin D receptor) to facilitate the movement of ligand from one protein to the next; 3] a specific intermolecular interaction exists between the binding proteins that would narrow the diffusion distance for ligand exchange; and 4] altered expression of IDBPs must result in a definable effect on vitamin D metabolism or action.
Specific Aim 1 will seek to prove that IDBPs are acceptor and delivery proteins for internalized vitamin D molecules by: 1] investigating the correlative traffic of fluorescently-labeled vitamin D metabolites and IDBPs within the cell; and 2] expanding our preliminary understanding of the specific intermolecular interaction of IDBPs with megalin and the vitamin-D-hydroxylases.
Specific Aim 2 is designed to link the physical interactions described in Specific Aim 1 with a functional consequence by: 1] confirming that 1,25(OH)2D and 24,25(OH)2D production are dependent in part upon IDBP-directed delivery of substrate to the vitamin-D-hydroxylases; and 2] creating in vivo a transgenic mouse model of targeted overexpression of IDBP which recapitulates the functional observations made in vitro. It is anticipated that thus work will lay the foundation for an understanding of a previously unrecognized mode of control over the intracellular trafficking, metabolism and action of sterol/steroid/retinoid/prostanoid hormones that is relevant to both human physiology and disease. Specifically, the current work will help to develop therapeutic strategies to counter common disease states that are worsened by vitamin D deficiency or resistance (i.e., osteoporosis) or that are improved or stabilized by enhancement of local vitamin D metabolite synthesis and action (i.e., leukemia, breast cancer, prostate cancer, psoriasis, etc.).
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