The principal mode of action of 1α,25(OH)2D3 occurs by a steroid hormone receptor mediated mechanism. Definitive bio chemical evidence supports the existence of a receptor for 1α,25(OH)2D3 (VDR) in at least 30 different tis sues. While it is not surprising to find 1α,25(OH)2D3 VDR in the classic target organs of intestine, kidney, and bone linked to calcium homeostasis (Figure 1), the presence of the VDR in such diverse tissues as the parathyroid gland, pancreas, heart and cardiovascu lar system (arteries+veins), cells of the immune system (B and T lymphocytes), muscle, spermatozoa, and brain (Figure 2) emphasizes the wide physiological responsibility of the vitamin D endocrine system and the pleiotropic actions of 1α,25(OH)2D3. While the kidney functions as the major source of plasma 1α,25(OH)2D3, approximately 12 cell types (e.g., macrophages, pancreas, colon, keratinocytes, prostate, spermatozoa, placenta, and brain) have low levels of the 25(OH)D3-1α-hydroxylase (CYP2B1) and thus function as a paracrine source of 1α,25(OH)2D3.
Fig1. A schematic of the vitamin D endocrine system governing calcium homeostasis. The process of calcium homeostasis describes the mechanisms by which the serum Ca2+ concentration is maintained ~constant at 9.5–10.5 mg/100 mL of serum via the integrated actions of the intestine, bone, and kidney; all three of these organs can contribute Ca2+ to the serum compartment based on the actions of three hormones, namely parathyroid hormone (PTH), 1α,25(OH)2D3 and fibroblast growth factor 23 (FGF23). 1α,25(OH)2D3 is responsible for stimulating intestinal Ca2+ absorption in accordance with the circulating level of 1α,25(OH)2D3. If the dietary intake of Ca2+ is inappropriately low, then Ca2+ contribution from the intestine is not adequate and PTH will therefore stimulate the bone to solubilize Ca2+ and HPO4−. At the same time PTH will interact with its kidney receptor and increase the renal tubular reabsorption of Ca2+ and at the same time diminish the renal tubular reabsorption of phosphate (i.e., increase the urinary concentration of phosphate). Also the kidney functions as an endocrine gland in that it enzymatically produces the steroid hormone 1α,25(OH)2D3 according to the stimulatory actions of PTH on the kidney proximal tubule’s 25(OH)2D3-1α-hydroxylase in accordance with the magnitude of the need to elevate the prevailing level of serum Ca2+. The secretion of PTH is governed by the Ca2+receptor of the parathyroid gland which monitors the serum Ca2+ concentration. Finally bone osteocytes secrete the hormone FGF23 when serum Ca2+ is inappropriately low. The FGF23 binds to its receptor in the kidney and inhibits the renal tubular reabsorption of phosphate (i.e., increases the urinary concentration of phosphate).
Fig2. A summary of the contributions of the vitamin D endocrine system to good health. 1α,25(OH)2D3, enzymatically produced by either the kidney or paracrine 25(OH)D3-1α-hydroxylase, is carried throughout the blood compartment by the vitamin D binding protein (DBP). The 1α,25(OH)2D3 enters any of the some 30 cell types that are known to contain the vitamin D receptor (VDR) and stimulates a variety of genomic and rapid responses that can contribute to good health. These responder cell types are organized, in the left-hand column (light green color), into five primary vitamin D-dependent physiological systems. The 1α,25(OH)2D3 brain system is at the present time only studied in rodent systems. The center column tabulates for each of the five major vitamin physiological systems the nature of their biological responses. The right hand column lists known examples of vitamin D deficiency related diseases that are “associated” with each physiological system. In general, only for the calcium homeostasis physiological system have randomized clinical trials (with humans) been carried out showing that correction of the vitamin D deficiency by increased vitamin D3 intake can minimize the disease studied. The blood levels of 25(OH)D have been shown to be correlated with varying extents of vitamin D deficiency. The inset table (bottom left) summarizes, for humans, the changes in the circulating levels of 25(OH)D that are linked to varying degrees of vitamin D deficiency (from severe to insufficient to sufficient, etc.). From a clinical perspective this information can be utilized to determine the recommended daily intake of vitamin D3 to maintain good vitamin D health.
The steroid hormone 1α,25(OH)2D3 generates biological responses via binding to either the VDR located in the cell nucleus to regulate gene transcription, or to the VDR present in the plasma membrane caveolae (VDR), which is coupled to the rapid opening of voltage-gated channels. These topics will be discussed separately.
The 1α,25(OH)2D3 receptor is a protein with a molecular weight of 50,000 daltons. The cellular distribution of the VDR is 75% in the nucleus, 20% in the cell cytosol and 3–5% in the plasma mem brane. (Figure 3 shows that VDR can initiate both genomic effects when in the nucleus of the cell and rapid responses with VDR localized in caveolae at the plasma membrane. Figure 4 illustrates the shapes that 1α,25(OH)2D3 can assume when binding to the VDR with a bowl shape (genomic response) or a planar shape (rapid responses). Figure 5 illustrates how the VDR can “capture” the three hydroxyls of 1α,25(OH)2D3 and form a stable ligand/receptor complex.) The VDR binds 1α,25(OH)2D3 tightly (Kd=1–5×10−10 M) and with great ligand specificity. Absence of the 1α- or 3β-hydroxyl group dramatically reduces interaction with the VDR by > 99.9%. Also, when the eight-carbon side chain is lengthened by two carbons or shortened by one carbon, the interaction with the VDR is reduced by 75%. The parent vitamin D3 blood concentration (~100–500 nM in vitamin D replete individuals) is present at vastly higher concentrations (~1,000×) than the plasma concentration of 1α,25(OH)2D3, (120 pM). However, both vitamin D3 and vitamin D2 are not able to bind to the VDR.
Fig3. Distribution of VDR in a target cell. Target cells for the steroid hormone 1α,25(OH)2D3 have VDR distributed between the nucleus (~80%), the cytosolic compartment (~15%), and the interior surface of the plasma membrane caveolae (~5%). The VDR can move by diffusion to these three compartments. The VDR monomer has hydrophobic bonds with the intracellular surface of the caveolae membrane. The X, Y, and Z red letters are outside the cell. The VDR, arriving from the nucleus and cytosol, docks with the surface of the caveolae facing the cytosol of the cell. When the caveolae becomes associated with a monomeric VDR with a bound 1α,25(OH)2D3 (red triangle), it moves (in this example) to the nearest chloride channel to activate rapid responses. However, the caveolae-associated VDR must have the 1α,25(OH)2D3 bound to the VDR-AP (alternate pocket), and not the VDR-GP (genomic pocket) for this to happen. This VDR/planar ligand is competent to initiate a variety of rapid responses. Illustrated in this figure is the VDR/1α,25(OH)2D3 initiated opening of chloride channels in the plasma membrane. In the nucleus of the cell, VDR with a bound 1α,25(OH)2D3 forms a heterodimer with the retinoid X receptor to activate gene transcription. Gene knock-out (KO) of the VDR in mice results in the loss of both 1α,25(OH)2D3 stimulated gene activation as well as the activation of rapid responses, including the opening of chloride channels, and even exocytosis from the cell.
Fig4. The steroid hormone 1α,25(OH)2D3 is conformationally flexible and presents different 3-D shapes for binding to its receptor, the vitamin D receptor (VDR). Panel A presents the structure of two shapes of 1α,25(OH)2D3. The 6-s-cis presentation shape on the left side is in rapid exchange with the 6-s-trans or extended shape. The change from cis to trans occurs by a 360-degree rotation around the 6-7 single bond (see the two arrows). This interchange process occurs at the rate of several million times per second. This extreme rotation allows the molecule to assume a very large family of three-dimensional shapes. Panel B has two extreme presentations of 1α,25(OH)2D3. The top row is a space-filling three-dimensional representation of 6-s-cis shaped 1α,25(OH)2D3. The two dark images on the left end of the bowl shape each represent the space-filling of the 1α -OH group (bottom surface) and the 3β-OH group (upper surface). At the right-hand end of the bowl shape is the space filling view of carbon-25 OH group. On the right side is the three-dimensional planar shape of 1α,25(OH)2D3. The right-hand three-dimensional ligand shows a 6-s-trans presentation; overall it has a planar shape. The left dark image is the 1α-OH group, while the 3β-OH group is hidden on the “back” surface. At the right end of the planar shape is the dark image of the 25-OH group. The bottom labels for panel B are the VDR-GP (for the VDR genomic pocket engaged in gene transcription; left side) and the VDR-AP (for the VDR alternative pocket engaged in nongenomic responses; right side). In order for the VDR to generate genomic responses, it has been experimentally established that the VDR must have a bound 1α,25(OH)2D3 ligand that is bowl shaped. This can function to turn selected genes either on or off. For the VDR that is localized to the membrane caveolae where it produces rapid responses, it must have a bound 1α,25(OH)2D3 that is in the planar conformational shape. The different shapes of the VDR-GP and the VDR-AP ligands were evaluated by separate determination of the X-ray structure of the VDR-GP with a bound bowl-shape ligand and computer modeling of the VDR-AP with an AP ligand with a planar shape. Panel B has two extreme presentations of 1α,25(OH)2D3. The top row is a space-filling three-dimensional representation of 6-s-cis shaped 1α,25(OH)2D3; it has a bowl-like shape. The right-hand three-dimensional ligand shows a 6-s-trans presentation; it has a planar shape. The second row is a ball-and stick presentation of 1α,25(OH)2D3 in the bowl (left) or planar (right) shape. In order for the VDR to generate genomic responses, i.e., affect the rate of gene transcription, it has been experimentally established that the VDR must have a bound 1α,25(OH)2D3 ligand in the bowl shape. This is referred to as the VDR-GP (VDR genomic pocket). The VDR that is localized to the membrane caveolae and produces “rapid” responses must have a bound 1α,25(OH)2D3 that is in the planar conformational shape. This binding site is referred to as the VDR-AP (VDR alternative pocket).The different shapes of the VDR-GP and VDR-AP ligand were evaluated by separate determination of the X-ray structure of each and computer modeling of the AP ligand.
Fig5. Absence of the 25-hydroxyl of the steroid hormone 1α,25(OH)2D3 reduces the affinity of 1α (OH)D3 to bind to the vitamin D receptor (VDR) by ~660 fold. The schematic model of the VDR illustrates how the receptor for 1α,25(OH)2D3 first “captures” and then forms a stable hydrogen bond (with X) with the 25-hydroxyl group on the end of the conformationally mobile 8-carbon side chain. This is followed by the conformationally mobile A ring’s two hydroxyls of 1α,25(OH)2D3 (the 1β and the 3β) to form a stable (using Y and Z) receptor-ligand complex. The X, Y, and Z labels indicate known binding domains on the receptor’s interior which each form a stabilizing hydrogen bond with the three hydroxyls of 1α,25(OH)2D3 when the ligand is docked inside the VDR. Panel A illustrates the proposed steering effects of the 25-hydroxyl group docking with X which then permits capture of the conformationally active A ring’s two hydroxyls by Y and Z. Panel B illustrates the consequences of the absence of the side chain 25-hydroxyl group on the poor ability of 1α (OH)D3’s 1α- and 3β-hydoxyls to be captured by the VDR. This is reflected by the fact that 1α (OH)D3 binds to the VDR only 0.15% as well as 1α,25(OH)2D3 (100%). Thus the following calculation can be made: [RCI for 1α,25(OH)2D3] / [RCI for 1α (OH)D3] = [100] / [0.15] = 666-fold difference in relative binding in favor of 1α,25(OH)2D3.
