Chondrocyte

REABSORPTION OF Ca2+ & Pi -

ABSORPTION OF Ca2+ -

"MOBILIZATION / ACCRETION OF Ca2* & Pi

FIGURE 9-8 Summary of the vitamin D endocrine system. In addition to production of lct,25(OH)2D3 and 24R,25(OH)2D3 by the endocrine gland function of the kidney, small amounts of la,25(OH)2D3 are also produced in a paracrine fashion and by the placenta during pregnancy. Target organs and cells for la,25(OH)2D3 by definition contain nuclear receptors for lc*,25(OH)2D3 (nVDR). Also, la,25(OH)2D3 generates biological effects involving rapid signal transduction pathways utilizing a putative membrane receptor. The precise biological roles of 24,25(OH)2D3 are not yet defined although it is believed to function in bone and cartilage.

involved in Ca2+ binding (sensing?) coupled to a seven-membrane-spanning domain like those in the G-protein-coupled receptor superfamily.

The most important biological actions of PTH are the following: (1) to increase the rate of conversion of 25(OH)D3 to la,25(OH)2D3 in kidney tissue and, thus, increase the serum concentration of la,25(OH)2D3; (2) to increase the plasma calcium concentrations; (3) to increase the extent of osteoclastic and osteocytic osteolysis in bone (bone resorption and remodeling); and (4) to increase the urinary excretion of phosphate and hydroxyproline-containing peptides and to decrease the urinary excretion of calcium.

The opossum kidney cell PTH receptor has been cloned, sequenced, and found to be a member of a distinct family of G-protein-coupled receptors with seven-transmembrane-spanning domains (see Figures 1-19C and 1-20). The mature receptor protein contains 585 amino acids. Both PTH and PTHrP bind with equal affinity to the cloned and expressed receptor, and both ligands equivalently stimulate adenyl cyclase.

Intriguingly, there is close structural homology between the PTH receptor, the PTHrP receptor, and the calcitonin receptor, as well as with the secretin, vasoactive intestinal polypeptide (VIP), and glucagon receptors. All of these receptors couple receptor occu-

Serum calcium (mg/dL)

FIGURE 9-9 Changes in plasma levels of immunoreactive parathyroid hormone (iPTH) and calcitonin (iCT) as a function of plasma total calcium. The data were obtained in pigs given EDTA to decrease plasma calcium or calcium infusions to increase plasma calcium. Note that, as serum calcium increases, iPTH falls and serum iCT increases; as serum calcium decreases the reverse occurs. Reproduced with permission of Arnaud, C. D. et al. (1970). In "Calcitonin: Proceedings of the Second International Symposium" (S. Taylor, ed.). Heinemann, London, p. 236.

Serum calcium (mg/dL)

FIGURE 9-9 Changes in plasma levels of immunoreactive parathyroid hormone (iPTH) and calcitonin (iCT) as a function of plasma total calcium. The data were obtained in pigs given EDTA to decrease plasma calcium or calcium infusions to increase plasma calcium. Note that, as serum calcium increases, iPTH falls and serum iCT increases; as serum calcium decreases the reverse occurs. Reproduced with permission of Arnaud, C. D. et al. (1970). In "Calcitonin: Proceedings of the Second International Symposium" (S. Taylor, ed.). Heinemann, London, p. 236.

pancy to activation of adenyl cyclase and, in some instances, increases in the concentration of intracellular Ca2+.

The PTH receptor has been found to be expressed in both kidney and osteoblast cells, which are prime targets for PTH action, and also in aorta, brain, heart, ileum, liver, placenta, skin, uterus, and testes. The functional significance of these apparent new target organs for PTH remains to be established.

The actions of PTH on bone are complex and are an area of intense investigation. The response of bone to PTH is biphasic; the immediate action is largely that of bone mineral mobilization (i.e., an elevation of the blood levels of both calcium and phosphorus). These effects may be seen within minutes following hormone administration and have, in fact, been used in parathy-roidectomized animals as a bioassay for PTH.

A second or slower action of PTH is its effect upon bone cell activity. [PTH is believed to increase the size and number of the bone-resorbing cells, namely, the osteoclasts. This, then, upsets the balance between osteoblasts (bone-forming cells) and osteoclasts and can lead to an increased remodeling rate of bone.] The net effect is normally that resorption is somewhat greater than accretion, so that a net negative skeletal balance of Ca2+ and Pi is observed. Although PTH is a potent bone-resorbing agent, receptors for PTH are not found on osteoclasts and are only found on osteoblasts (see later discussion on bone remodeling). Also associated with prolonged bone resorption is an increased release of lysosomal enzymes, so that there is a mobilization of the bone matrix that results in both increased blood levels and urinary excretion of the amino acid hydroxy-proline.

The principal actions of PTH on the kidney are to stimulate the renal excretion of phosphate and to enhance the renal tubular reabsorption of calcium. The site of action of PTH in the kidney has been localized to be in the proximal tubule (see Figure 15-4). Also, there are well documented physiologically important effects of PTH that lead to increased excretion of potassium, bicarbonate, sodium, and amino acids and decreased excretion of ammonia and calcium.

B. Parathyroid Hormone-Related Protein

The existence of PTHrP was first encountered as a consequence of the disease of humoral hypercalcemia of malignancy (HHM). In HHM, patients have significantly elevated serum Ca2+ levels and a tumor that secretes PTHrP. Although PTH and PTHrP are quite different proteins, because of their high structural homology over the first 13 amino acids (see Figure 9-3) PTHrP is able to interact with the PTH receptor and generate PTH-like biological responses in bone and kidney. As discussed earlier, the N-terminus of PTH is crucial for interaction with its membrane receptor and activation of adenylate cyclase. It has not yet been unequivocally established whether there is a separate and distinct membrane receptor for PTHrP. PTHrP is known to be an effective agonist for the PTH receptor.

The PTHrP is known to be synthesized in several tissues (growth plate of bone, placenta, uterus, skin) with specific temporal and spatial profiles appropriate for its location; it is believed that PTHrP has largely paracrine functions.

Current research into the biological actions of PTHrP suggests that it is involved in regulating the rate of cartilage differentiation. During vertebrate embryogenesis, the first element of the skeleton to appear is a template comprised of cartilage cells (chondrocytes). These chondrocytes are ultimately replaced by mineralized bone in a process termed endochondral ossification. PTHrP has been shown to be a key regulator of the rate of chondrocyte differentiation. It has been postulated that if chondrocyte differentiation proceeds too slowly or too quickly, abnormalities in the length of the long bones will appear, which may explain some forms of dwarfism.

In other target tissues, PTHrP has been shown to increase placental calcium transport and inhibit contraction of uterine muscle and, as well, has been suggested to play a role in the onset of labor.

C. Calcitonin

The dominant biological action of calcitonin is to mediate a lowering of serum calcium levels. Calcitonin is secreted in response to elevated blood levels of ionized calcium; the rate of calcitonin secretion is a direct function of the plasma calcium concentration (Figure 9-9). Extensive studies have demonstrated that one or more hormones of the digestive tract have the ability to elicit increased calcitonin secretion. The most widely tested hormones in this regard have been gastrin and its synthetic analog pentagastrin. These results are consistent with the observation that the secretion of calcitonin is stimulated shortly after the ingestion of high dietary levels of calcium.

In addition to the onset of hypocalcemia, there is also normally an accompanying hypophosphatemia after the administration of calcitonin. Also, in experimental animals, the blood level of calcitonin is elevated in pregnancy and lactation.

The biological effects of calcitonin are mediated as a consequence of the interaction of CT with a receptor present in the outer membrane of target cells of both skeletal and renal tissues. The human calcitonin receptor has been cloned and sequenced. Like the PTH receptor, the calcitonin receptor belongs to a distinct family of G-protein-coupled receptors with seven-transmembrane-spanning domains. The mature receptor comprises 490 amino acids. When the cloned CT receptor was transfected into COS cells, the resulting cells bound both salmon CT and human CT and displayed dose-dependent production of cAMP. Also, calcitonin, but not calcitonin gene-related peptide, can generate a receptor-mediated increase in intracellular Ca2+ and IP3, suggesting activation of the phospholi-pase C signal transduction pathway as well as the adenyl cyclase pathway. As yet no detailed description of the mechanism of action of calcitonin is available.

Evidence exists for multiple isoforms of the CT receptor, and the CT receptor is known to be expressed in porcine kidney cells, human osteoblasts, mouse brain cells, and human ovary cells. The functional significance of these various isoforms of the calcitonin receptor remains to be established.

The hypocalcemic and hypophosphatemic effects of calcitonin are believed to be due to an inhibition of PTH-mediated calcium resorption from bone, mediated by cyclic AMP. In addition, calcitonin has inde pendent actions in the kidney, where it can stimulate calcium and phosphate excretion.

At least three important biological functions for calcitonin have been proposed: (1) protection of the young animal or newborn against postprandial hypercalcemia; (2) blocking of the actions of PTH in mobilizing bone calcium and phosphorus; and (3) stimulation of the urinary excretion of both calcium and phosphate in the kidney. The net effect of these three actions is to mediate a reduction in serum calcium levels.

D. Vitamin D and Its Metabolites

The vitamin D molecule itself has no intrinsic biological activity. All biological responses attributed to vitamin D are now known to arise only as a consequence of the metabolism of this secosteroid into its biologically active daughter metabolites, namely, la,25(OH)2D3 and 24,25(OH)2D3.

Figure 9-8 summarizes the scope of the vitamin D endocrine system. The steroid hormone la,25(OH)2D3 is produced only in accord with strict physiological signals dictated by the calcium "demand" of the organism; a bimodal mode of regulation has been suggested. On a time scale of minutes, changes in the ionic environment of the kidney mitochondria resulting from the accumulation and release of calcium and / or inorganic phosphate may alter the enzymatic activity of the 1-hydroxylase. In addition, parathyroid hormone has been shown, on a time scale of hours, to be capable of stimulating the production of la,25(OH)2D3, possibly by stimulating the biosynthesis of the 1-hydroxylase. It is also relevant that la,25(OH)2D3 is a stimulant for the renal production of 24,25(OH)D3. Thus, under normal physiological circumstances, both renal dihydrox-ylated metabolites are secreted and are circulating in the plasma. There is evidence of a "short feedback loop" for both of these metabolites to modulate and/or reduce the secretion of PTH. There is also some evidence that other endocrine modulators such as estrogens, androgens, growth hormone, prolactin, and insulin may affect the renal production of la,25(OH)2D3 (see Figure 9-8). Thus, the kidney is clearly an endocrine gland, in the classic sense, that is capable of producing, in a physiologically regulated manner, appropriate amounts of la,25(OH)2D3.

The plasma compartment contains a specific protein, termed the vitamin D-binding protein (DBP), that is utilized to transport vitamin D secosterols. DBP is similar in function to the corticosteroid-binding globulin (CBG), which carries glucocorticoids (see Chapter 10), and the steroid hormone-binding globulin (SHBG), which transports estrogens or androgens (see Chapter 12). DBP is a slightly acidic (pi = 5.2) monomeric glyco-

protein of 53,000 Da, which is synthesized and secreted by the liver as a major plasma constituent. From analysis of the cloned cDNA, it has been determined that DBP is structurally homologous to albumin and a-fetoprotein; these three plasma proteins are members of the same multigene family, which likely is derived from the duplication of a common ancestral gene. DBP, originally called group-specific component (Gc), was initially studied electrophoretically as a polymorphic marker in the a-globulin region of human serum.

DBP appears to be a multifunctional protein. It possesses one ligand-binding site for secosterols of the vitamin D family; thus, it may carry the parent, D3, or the daughter metabolites: 25(OH)D3, la,25(OH)2D3, and 24,25(OH)D3. DBP is also able to bind with high affinity to monomers of actin, thereby preventing their polymerization in the blood compartment. The suggestion has been made that DBP may also function as a precursor of a macrophage-activating factor (MAF). Preliminary evidence has been presented that MAF is a cytokine that increases the production of osteoclasts. Since the total plasma concentration of vitamin D sterols is only —0.2 ¡jlM, while DBP circulates at 9-13 /iM, under normal circumstances only 12% of the sterol-binding sites on DBP are occupied.

The principal mode of action of the vitamin D metabolites is believed to occur by a steroid hormone-like mechanism. Definitive biochemical evidence supports

FIGURE 9-10 Schematic model of how the nuclear la,25(OH)2D3 receptor (VDR) "captures" the conforma-tionally mobile A ring to form a stable receptor-ligand complex. Panel A illustrates the proposed steering effects of the 25-hydroxyl group to permit capture of the conformationally active A ring, while panel B illustrates the proposed consequences of the absence of the 25-hydroxyl group on vitamin D ligand capture by the VDR. X, Y, and Z indicate postulated binding domains on the VDR. [Reproduced with permission from A. W. Norman, A. W., and Henry, H. L. (1979). Vitamin D to 1,25-dihydroxycholecaIciferol: Evolution of a steroid hormone. Trends Biochem. Sci. 4, 14-18.]

FIGURE 9-10 Schematic model of how the nuclear la,25(OH)2D3 receptor (VDR) "captures" the conforma-tionally mobile A ring to form a stable receptor-ligand complex. Panel A illustrates the proposed steering effects of the 25-hydroxyl group to permit capture of the conformationally active A ring, while panel B illustrates the proposed consequences of the absence of the 25-hydroxyl group on vitamin D ligand capture by the VDR. X, Y, and Z indicate postulated binding domains on the VDR. [Reproduced with permission from A. W. Norman, A. W., and Henry, H. L. (1979). Vitamin D to 1,25-dihydroxycholecaIciferol: Evolution of a steroid hormone. Trends Biochem. Sci. 4, 14-18.]

the existence of nuclear receptors for la,25(OH)2D3 (nVDR) in at least «30 different tissues (see Figure 98). While it is not surprising to find la,25(OH)2D3 nVDR in the classic target organs of intestine, kidney, and bone, the presence of the nVDR in such diverse tissues as the pancreas, eggshell gland, parathyroid gland, pituitary, reticuloendothelial system cells, and cerebellum emphasizes the diversity of the vitamin D endocrine system and the pleiotropic actions of la,25(OH)2D3.

Evidence has been presented that the steroid hormone is able to generate biological responses via signal transduction pathways, which involve its interaction with a nuclear la,25(OH)2D3 receptor (nVDR) to regulate gene transcription, or via a putative membrane receptor (mVDR), which is believed to be coupled to the opening of voltage-gated Ca2+ channels. These topics will be discussed separately.

The nuclear la,25(OH)2D3 receptor is a protein with a molecular weight of 50,000 (see Table 1-5). Extensive evidence exists that supports the view that the unoccupied VDR is largely associated with the nuclear chromatin fraction. The VDR binds la,25(OH)2D3 tightly (Kd = (1-5) X 10 :i M) and with great ligand specificity. Table 9-4 summarizes the structural features of the ligand that optimize its tight binding to the VDR. Figure 9-10 presents a schematic model that emphasizes the importance of the simultaneous presence of the la-, 3/8-, and 25-hydroxyl groups on the ligand for stabilization of the conformationally mobile la,25(OH)2D3. Absence of the la- or 3/3-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 76%. The data of Table 9-4 also emphasize that neither the parent vitamin D3 nor the intermediate 25(OH)D3, both of which are present in the blood at vastly higher concentrations than la,25(OH)2D3, is able to significantly interact with the nVDR.

Preliminary evidence has suggested the existence of protein receptors for 24,25(OH)D3 in chondrocytes (bone cartilage cells). As yet no specific function for the 24,25(OH)D3 receptor has been identified, although there is a possibility of its involvement in some aspects of mineralization and in bone fracture healing.

As can be appreciated from an examination of the scope of the vitamin D endocrine system (Figure 9-8), the biological actions of la,25(OH)2D3 are known to extend far beyond the classical actions of vitamin D in the intestine, kidney, and bone. The nuclear VDR is present in at least 32 target tissues. In each of these tissues, the la,25(OH)2D3-nuclear receptor complex is involved in selective regulation of gene transcription; this includes genes associated with mineral homeosta-

TABLE 9-4 Ligand Specificity of the Nuclear la,25(OH)2D3 Receptor

Ligand

Structural modification

RCI"

la,25(OH)2D3

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