PTH and Vitamin D

Syed Jalal Khundmiri; Rebecca D Murray; Eleanor Lederer

doi:10.1002/cphy.c140071

. Author manuscript; available in PMC: 2024 Jun 10.

Published in final edited form as: Compr Physiol. 2016 Mar 15;6(2):561–601. doi: 10.1002/cphy.c140071

PTH and Vitamin D

Syed Jalal Khundmiri ^1,², Rebecca D Murray ^1,², Eleanor Lederer ^1,^2,^3,^*

PMCID: PMC11163478 NIHMSID: NIHMS1995435 PMID: 27065162

Abstract

PTH and Vitamin D are two major regulators of mineral metabolism. They play critical roles in the maintenance of calcium and phosphate homeostasis as well as the development and maintenance of bone health. PTH and Vitamin D form a tightly controlled feedback cycle, PTH being a major stimulator of vitamin D synthesis in the kidney while vitamin D exerts negative feedback on PTH secretion. The major function of PTH and major physiologic regulator is circulating ionized calcium. The effects of PTH on gut, kidney, and bone serve to maintain serum calcium within a tight range. PTH has a reciprocal effect on phosphate metabolism. In contrast, vitamin D has a stimulatory effect on both calcium and phosphate homeostasis, playing a key role in providing adequate mineral for normal bone formation. Both hormones act in concert with the more recently discovered FGF23 and klotho, hormones involved predominantly in phosphate metabolism, which also participate in this closely knit feedback circuit. Of great interest are recent studies demonstrating effects of both PTH and vitamin D on the cardiovascular system. Hyperparathyroidism and vitamin D deficiency have been implicated in a variety of cardiovascular disorders including hypertension, atherosclerosis, vascular calcification, and kidney failure. Both hormones have direct effects on the endothelium, heart, and other vascular structures. How these effects of PTH and vitamin D interface with the regulation of bone formation are the subject of intense investigation.

Parathyroid Hormone

Introduction

Parathyroid hormone (PTH), a product of the parathyroid glands, embedded in the thyroid (in rodents) or located behind the thyroid (in humans), is a key regulator of calcium and phosphorus homeostasis through its effects on bone, kidney and intestine, and by regulating 1α, 25-dihydroxyvitamin D (372). The serum concentration of PTH is derived both from the release of PTH stored in secretory granules and from de novo synthesis of PTH in response to alterations in the serum levels of calcium, phosphorus, and vitamin D (277). Acute regulation of PTH is accomplished by the release of stored PTH in response to ambient calcium level through the calcium sensing receptor expressed on the chief cells of the parathyroid glands while long-term synthesis and release is dependent upon de novo synthesis through transcription and translation of mRNA encoding pre-pro-PTH (520, 521). PTH restores serum calcium by three different mechanisms: (i) release of calcium and phosphorus from the bones through stimulation of osteoclastic activity; (ii) decrease in calcium excretion and a concomitant decrease in phosphate reabsorption in the kidney; and (iii) increase dietary absorption of calcium and phosphorus in the gut (271) (Fig. 1).

Regulation of serum Ca²⁺ by PTH. Low serum Ca²⁺ stimulates the release of PTH from the parathyroid gland. PTH acts on the bone to increase bone resorption, releasing Ca²⁺ and HPO₄²⁻ into the bloodstream. At the kidney, PTH increases Ca²⁺ reabsorption and decreases HPO₄²⁻ reabsorption, maintaining the elevated serum Ca²⁺ from the resorption of bone. Vitamin D becomes activated in the kidney by 1α-hydroxylase, leading to increased Ca²⁺ absorption from the gut. The restored serum Ca²⁺ provides a negative feedback signal to the parathyroid glands, discontinuing the release of PTH.

Substantial advances made in the late 1970s and early 1980s to understand the biochemical and cellular regulation of PTH metabolism and mechanisms of action (278-282) led to the development of assays for the detection of PTH in the blood of humans and animals. These efforts uncovered the presence of multiple forms of immunoreactive PTH molecules in circulation adding a previously unappreciated complexity to PTH metabolism. Evidence suggests that the hormone is subjected to proteolysis both in the parathyroid gland and in end organs including liver and kidney. This review will focus on the structure, synthesis, secretion, and functions of the hormone and consider the pathophysiological, pharmacological, and treatment of abnormalities of biosynthesis and secretion of PTH.

History

Although evidence of pathologies now associated with the parathyroid glands can be documented as far back as ancient Egypt (164, 332), the existence of the parathyroid glands was not discovered until the second half of the nineteenth century, and their function not definitively explored until well into the twentieth century. The parathyroid glands went through numerous cycles of naming and discovery, and took several decades to gain traction in the scientific field. It was during the necropsy of a Great Indian Rhinoceros in 1852 that Sir Richard Owen of the Royal College of Surgeons first described the parathyroid glands (219, 497). Remak (544) and Wirchow (690) found the parathyroid glands in humans. Although it had been well established by the beginning of the twentieth century that removal of the glands from humans and animals caused death from tetany, intense debate still existed as to the function of the glands. It was even suggested that the parathyroid glands serve a detoxification purpose, much akin to the liver (409-411). At the turn of the twentieth century, the German pathologist Erdheim determined that patients undergoing thyroid surgery who developed tetany had undergone simultaneous accidental removal of the parathyroid glands (201, 655). In 1915, Schlagenhaufer, a Viennese physician, was the first to draw the conclusion that the osteitis fibrosa cystica observed in his patient was in fact due to an enlarged parathyroid, and not the other way around (332). While previous researchers had implicated a role of the parathyroid in calcium homeostasis, injection of parathyroid extract into animals with tetany failed to relieve the symptoms; thus, the precise relationship between parathyroid glands and calcium homeostasis remained unclear. It was not until Collip developed a method for extracting biologically active parathyroid hormone that the role of the parathyroid glands in calcium homeostasis began to emerge. Collip used a hot acid extraction method to purify potent parathyroid extracts that when injected back into parathyroidectomized animals restored muscle excitability to normal levels (160-162, 522, 524). Although effective, the hot acid extraction was also harsh, and yielded fragmented portions of parathyroid hormone, which frustrated efforts to sequence the polypeptide in the 1950s. In 1954, Handler et al. (284) summarized the frustrations of several scientists in a report by stating that “(i) the active material in the gland may be large protein which in the course of isolation is degraded into fractions of varying size, each of which still has activity; (ii) the active molecule may not be a large molecule at all, but instead a small molecule which adheres to each one of these fractions.” In 1959, Aurbach (35) and Rasmussen and Craig (534) independently isolated the polypeptide and individual fragments without degradation using organic solvent extraction methods. Using standard protein sequencing techniques, they were able to determine the structure of bovine and human PTH. In the 1970s with the development of molecular techniques, it became possible to determine the mechanisms for hormone synthesis, processing, and metabolism.

PTH gene regulation

Several proteins play a critical role in parathyroid gland development. These include glial cells missing (GCM), eyes absent (Eya1), and Hoxa3/Pax1 compound genes. However, there is very limited information available identifying activating and repressing factors that control the transcription of the PTH gene. Early studies suggested that extracellular calcium inhibited PTH gene transcription through a conserved negative calcium-response element (371, 487-489). More recent studies have elucidated an additional role for calcium in regulation of PTH through posttranscriptional repression. Rats that were fed low calcium diets were found to have increased levels of PTH mRNA, whereas rats that were fed low phosphate diets had decreased PTH mRNA expression (457). Low serum calcium and high serum phosphorus are signals that both increase PTH secretion by increasing PTH gene expression posttranscriptionally (60, 61). Additionally, 1,25-dihydroxy-vitamin D decreases PTH expression by decreasing PTH mRNA (471, 472). Vitamin D deficiency increases the PTH mRNA expression through two processes, (i) impaired calcium absorption leading to decreased extracellular calcium and (ii) removal of a known repressor of PTH gene transcription (369). Initial experiments determined that specific sequences in the 3′ UTR of PTH mRNA determine its rate of degradation (646). Calcium and phosphate were identified as regulating PTH posttranscriptionally, through alteration in the interaction of RNA binding protein with the 3′-UTR of the PTH transcript. Two of these proteins are AU-rich element binding factor 1 (AUF1) and KSRP (372). AUF1, an RNA-binding protein, enhances the PTH transcript stability by binding to the 3′UTR region in response to phosphate and calcium concentrations in the serum (61). KSRP destabilizes the PTH transcript through KSRP’s interactions with the 3’-UTR of PTH mRNA (239). Other proteins involved in PTH gene expression include hepatocyte nuclear factor 1β (HNF1β), which binds to the PTH promoter and acts as a translational repressor, as patients with a mutated HNF1β display hyperparathyroidism (223).

Several consensus sequences have been identified in PTH promoter region that regulate its gene expression. A cyclic AMP response element was identified at the transcription start site of the human, bovine, and murine PTH genes. A unique DNA repressor element that binds to the vitamin D receptor has also been identified in the human PTH gene promoter. Alimov et al. (19, 20) identified a highly conserved Sp1 element and a Sp3 element in the human and bovine PTH promoter. Sp1 strongly stimulates the transcription of wild-type bovine and human PTH promoters. The role of Sp3 promoter is not known.

Structure and biochemical properties of PTH

The biosynthetic pathways involved in the synthesis, cellular transport, and metabolism of PTH has been extensively studied. These studies demonstrated that the mRNA encoding the PTH is translated as a 115 amino acid pre-pro-PTH on the rough endoplasmic reticulum (277, 343-346) (Fig. 2). The first two methionines are cleaved during translation by a methionyl amino peptidase releasing the signal peptide such that the protein is directed to a membrane vesicle. During the transit to the Golgi, the N-terminal signal sequence of 23 amino acids is cleaved at the glycyl-lysyl bond to form an intermediate protein of 90 amino acids called the pro-PTH, which is an inactive precursor. The N-terminal six amino acids of the pro-PTH are proteolytically cleaved in the Golgi yielding the mature 84 amino acid PTH which is stored in granules to be released into the circulation by exocytosis after appropriate stimulus (282, 371, 598). Analysis of the region-specific radioimmunoassays demonstrated that pro-PTH constitutes only 7% of the total PTH in normal parathyroid glands and the rest is mostly mature PTH (283). The protein is further cleaved into smaller fragments by cathepsin-B in the parathyroid glands (227, 412, 413). The hormone and its fragments are removed from circulation by receptors predominantly in kidney but also in the bone (282).

Primary sequence of human parathyroid hormone (sequence from Ensembl.org). The pre- and prosequences are cleaved prior to secretion into the bloodstream. Residues 1 and 2 (diagonally striped) are required for PTH receptor activation. Residues 28-34 (checkered pattern) interact with the extracellular amino domain of the PTH receptor. The C-fragment may be cleaved either prior to or after release from the parathyroid gland.

The naturally occurring PTH 1-37 and 1-34 fragments of PTH maintain full activity of the intact PTH 1-84. Osteoporosis patients treated with PTH1-34 have increased bone density suggesting that this fragment of PTH has all the anabolic activities of the intact 1-84 PTH. Mutation analyses of the PTH molecule have identified amino acids critical for receptor signaling. Truncation of the first two amino acids (PTH 3-34) results in a partially active PTH while removal of the first six amino acids (PTH 7-34) results in a low-affinity antagonist. Further studies have demonstrated that the 17 to 34 amino acid residues are critical for high affinity receptor binding (468).

The structure of PTH has been partially defined through X-ray crystallography and NMR spectroscopy techniques (323). The secondary structure of PTH 1-34 differs depending upon whether it is in aqueous solutions, lipid solutions, or in the presence of secondary structure-inducing solvents such as trifluorethanol (47, 91, 357, 478, 505, 614). X-ray crystallography studies have identified critical amino acids associated with specific structural qualities of PTH. PTH 1-34 has a multihelical structure with a bend between amino acid residues 12 and 21 (323). Mutation analysis demonstrated that the helical structure around Gly12 is important for biological activity and binding of the peptide to its receptor (149, 150). NMR studies have shown three helices between Ser3-Asn10, Ser17 to Lys27, and Asp30 to Leu37 in the N-terminal. An additional poorly defined helix between Asn57 and Ser62 was observed in the C-terminal with evidence of interaction between helix 1 and helix 2. The NMR studies also demonstrated a “U” or “V” shaped tertiary structure formed by the interaction between the N- and C-terminal helices which form a hydrophobic core (47, 141, 270, 699).

Regulation of PTH synthesis, secretion, and metabolism

Mature PTH is stored in granules close to the plasma membrane. Electron microscopy of the parathyroid gland demonstrated that the granules containing mature PTH are limited while there are abundant immature vesicles present near the Golgi. These vesicles are transported to the cell surface without incorporation into mature granules. Extracellular ionized calcium concentration is the major physiologic regulator of the synthesis and secretion of PTH (Fig. 3A and B). Decreasing ionized calcium concentration by infusion of the calcium chelator, EGTA, in cows increases the synthesis and secretion of PTH within 20 s of infusion. The initial response to a 0.1 mg/dL decrease in calcium concentration is to release the preformed vesicles from the parathyroid gland (100). Chronic decreases in serum calcium will increase the rate of synthesis and release of PTH (101). Apart from serum calcium, epinephrine, calcitonin, vitamin D, magnesium, and phosphate regulate the synthesis and release of PTH in humans (105, 226). (Fig. 3C) An extracellular calcium receptor (CaSR) acting as a sensor for ionized calcium levels provides the critical link between circulating ionized calcium concentration and PTH secretion, maintaining calcium within a narrow range. High extracellular calcium levels sensed by the CaSR results in decreased PTH secretion and increased Ca⁺⁺ excretion by the kidney (148, 474). Conversely, lower levels of plasma calcium stimulate PTH secretion and Ca⁺⁺ reabsorption by the kidney (474-477, 671).

(A) Effect of serum [Ca²⁺] on PTH synthesis and secretion. Ca²⁺ binds to the CaSR and activates G_q and G_i. G_q activation leads to increased [Ca²⁺]_i via PLC signaling and IP₃ formation. Increased [Ca²⁺]_i inhibits exocytosis of PTH secretory vesicles. G_i activation inhibits adenylyl cyclase, leading to less cAMP, and therefore less transcription of the PTH gene as well as decreased stimulus for secretory vesicle exocytosis. (B) Effect of low serum [Ca²⁺] on PTH synthesis and secretion. In the absence of serum Ca²⁺, CaSR signaling terminates, allowing [cAMP]_i to increase, which drives transcription of PTH and secretory vesicle exocytosis, leading to increased serum PTH levels. (C) Effect of Vitamin D on PTH secretion. Vitamin D enters the cell through diffusion across the plasma membrane. Once inside, Vitamin D is bound by the vitamin D receptor (VDR). In the nucleus, the Vit D-VDR complex binds to RXR and the vitamin D response element (VDRE) within the PTH promoter. This promoter complex inhibits transcription of the PTH gene, leading to less preproPTH production. The Vit D-VDR complex also binds to the VDRE within the promoter of p21, activating the transcription of the p21 gene. Increased p21 expression inhibits parathyroid cell proliferation.

Plasma PTH levels exhibit significant fluctuations during the course of the day and about 20% to 30% of its secretion is pulsatile (565). The circadian rhythm of PTH reaches a maximum at late morning, followed by two prominent peaks one in the afternoon and another in early morning (569). However, the maximum PTH secretion occurs at nighttime when the bone resorption activity is highest (202). Both nocturnal increases in PTH secretion and bone resorption can be prevented by administration of calcium in the evening (426). PTH concentration time profiles have revealed a rhythm of secretion consisting of seven secretory pulses per hour, accounting for approximately 30% of spontaneous PTH secretion and regulated by extracellular calcium (572). The mechanisms for the pulsatile secretion of PTH are not very well understood, nor are the functional consequences. It is proposed that the dense autonomic innervation of the parathyroid gland acts as neuronal pacemaker for PTH secretion (123, 208, 613). Acute disruption of sympathetic input by β-adrenergic receptor blockers increases the PTH pulse two fold and increases plasma PTH levels but does not eliminate the pulsatile oscillations (570, 571). It has been suggested that this cyclic release of PTH may be critical for the anabolic effects of bone mass by activating the cyclic AMP pathway while chronically elevated levels of PTH lead to bone destruction through PKC and RGS2-dependent pathways (312, 342). This is based, at least in part, on the observation that daily recombinant PTH injections, which results in exaggerated peaks of serum PTH levels, are an effective treatment for osteoporosis and bone repair, stimulating new bone production in postmenopausal women (75, 209). In contrast, sustained high levels of PTH seen in primary and secondary hyperparathyroidism tend to result in bone destruction. PTH secretion also is dependent upon seasonal fluctuations. It decreases by about 20% in summer and increases by about 20% in winter season (693).

Berson and Yalow demonstrated immunochemical heterogeneity of plasma PTH for the first time and suggested that this may be due to postsecretory modifications of the hormone (70-72). Later studies confirmed the observations of Berson and Yalow and by using RIA demonstrated that the majority of fractions are C-terminal fragments of the hormone (468). Habener et al. demonstrated that direct intravenous injection of intact bovine PTH into calves led to the accumulation of C-terminal fragments generated by peripheral metabolism of the administered hormone (278). In 1973 Canterbury et al. identified N-terminal fragments of PTH that were biologically active (117). This observation was later confirmed by several studies. The N-terminal fragments were found to be more short-lived than the C-terminal fragments. The discovery of biologically active N-terminal fragments led to the hypothesis that peripheral metabolism of secreted PTH is required for biological activity (503). However, this was disproved by the observations of Glotzman et al. who demonstrated that intact PTH could activate adenylate cyclase (260, 507). Fang and Tashjian (215) were the first to demonstrate the contribution of liver in clearance of intact PTH from circulation. This was later confirmed by several studies [reviewed in (468)]. Recent studies demonstrated that the Kupffer cells take up intact PTH, a process that is dependent on amino acids 28-48, and generate C-terminal fragments by proteolysis of the intact hormone (173, 174). Daugaard et al. (183, 184) demonstrated that only C-terminal biologically inactive fragments were generated during liver perfusion. Bringhurst et al. (110-112) demonstrated that the N-terminal fragments are degraded in the Kupffer cells. The demonstration that both intact and fragmented circulating forms of PTH are increased in patients with renal disease (71, 179, 443) and nephrectomized animals (118, 313, 431, 432) suggests that kidneys play a major role in PTH clearance. A portion of PTH is cleared independent of glomerular filtration, through peritubular uptake by binding to PTH1R and involve receptor-mediated endocytosis at the basolateral membrane of the tubule cells (185, 432). Recent studies suggest that megalin/cubulin-dependent endocytosis plays an important role in PTH clearance from urine independent of PTH1R (273, 298).

PTH receptors

Three distinct receptors for PTH viz., PTH1R, PTH2R, and PTH3R have been described in literature. The most common and the classical receptor, the PTH1R, a type II G-protein coupled receptor, is expressed widely, both in the classic PTH target tissues, bone and kidney, as well as many others. PTH1R is activated by both PTH and the PTH-related peptide, a protein that shares the name with PTH but is not derived from the same gene. PTH2R is expressed in very low levels in most tissues, except for the limbic system and the hypothalamus (148). PTH2R is activated by PTH and an unrelated protein, the tuberoinfundibular peptide of 39 amino acids (TIP39). The PTH3R was cloned from zebrafish and is activated 20 times more potently by PTHrP than PTH. The mammalian homologues of PTH3R have not yet been discovered.

The PTH1R is the best studied PTH receptor. Jüppner et al. in 1991 identified a 585 amino acids protein from COS7 and opossum kidney (OK) cells using 125I-[Tyr36]hPTHrP (1-35)NH2 binding (329). This protein was characterized as type II G-protein-coupled receptor, characterized by the presence of an ~ 150 amino acid N-terminal extracellular domain with four N-glycosylation sites, eight conserved extracellular cysteine residues forming four disulfide bridges, seven transmembrane regions, and a large (150-190 amino acid) intracellular C-terminal tail (148, 362, 578) (Fig. 4A). The PTH1R gene resides on the short (p) arm of chromosome 3 between positions 22 and 21.1 (3p22-p21.1). The PTH1R is encoded by a rather large 22 kb gene that contains 15 exons and 14 introns. The mature transcripts exhibit an extensive poly A tail at the 3′ end (366). Examination of the genomic map of the PTH1R gene has led to the identification of potential splice donor and acceptor sequences (148, 327, 366), and multiple promoters in the 5′ regulatory regions of the mouse, rat, human, and porcine PTH1R resulting in the production of multiple transcripts which show regulation at the developmental and tissue level (327, 441, 442, 595). The mouse, rat, and porcine transcripts have two promoter regions (P1 and P2) while the human transcript has three promoter regions (P1, P2, and P3) (75, 148, 327, 442, 604). They all lack the conventional TATA box homology. However, they contain the GC rich Sp1 motifs and are regulated by ubiquitously expressed transcription factors Sp1, myc-associated zinc-finger protein, and embryonic TEA domain-containing factor (34, 442, 450). The P1 and P3 promoter regions are methylated in a tissue specific manner and the differential methylation patterns are important during development (74). Transcription of the PTH1R is regulated by vitamin D, retinoic acid, and glucocorticoids (23, 335, 643, 701). Vitamin D downregulates the transcription of PTH1R in osteoblasts by inhibiting the activity of P2 promoter while retinoic acid and dexamethasone increases the transcription in mouse embryonal carcinoma P19 cells and ROS 17/2.8 cells respectively but not in OK cells. Glycosylation is an important posttranslational modification of GPCR that regulates the intracellular folding, stabilization, intracellular trafficking, and function (23, 335, 643, 644, 701). The four N-glycosylation sites (N-151, 160, 165, and 175) are located in the putative ligand-binding extracellular amino terminal domain of the PTH1R. Initial studies using inhibition of glycosylation by tunicamycin or by treatment with endoglycosidase F in OK and HEK 293 cells suggested that glycosylation is not required for proper folding, expression, and ligand binding (91, 93, 382, 722). However, more recent data using site-directed mutagenesis of the Asn to Gln showed decreased membrane expression and ligand binding in transiently transfected COS-7 cells suggesting that glycosylation plays an important role in intracellular trafficking, membrane expression, and function of PTH1R (721). Mutations in the six extracellular amino terminal cysteine residues also showed decreased membrane expression and ligand binding of the PTH1R. Two amino acid residues, R233 in the second and Q451 in the seventh transmembrane domain, are highly conserved in type II GPCR, and critical for effective PTH interaction and signaling as mutations in these two sites reduce ligand binding and transmembrane signaling by PTH1R (148, 381) (Fig. 4B).

(A) PTH receptor topology and ligand binding. (A)The PTH1R consists of an extracellular amino terminus, a J domain consisting of transmembrane domains as well as intra- and extracellular loops, and an intracellular carboxy terminus. The extracellular amino terminus is 150 residues long, with 4 N-glycosylation sites and 4 disulfide bridges. Less is known regarding PTH2R topology as compared to PTH1R topology. Importantly, residue variations in two of the extracellular loops (highlighted in red) decrease the affinity of the receptor for PTHrP while maintaining specificity for PTH. (B) PTH receptor topology and ligand binding. (B) PTH(1-34) (rainbow structure) interacts with both the extracellular amino terminus of the PTH1R as well as the J domain. Docking of PTH to the PTH1R is thought to occur through initial binding of the C-terminus of PTH (residues 15-34) to the N-terminus (blue regions) of the PTH1R. This interaction is closely followed by the binding of the N-terminus of PTH (residues 1-14) to the J domain (red regions) of the PTH1R, initiating G protein recruitment and intracellular signaling cascade activation. (C) PTH-receptor binding and intracellular signaling. (1) Two-site model of PTH-receptor docking. (2) The N-domain of the PTH receptor (PTHR1) binds the C-domain of PTH. (3) The J-domain binds the amino-terminal region of PTH. (4) Binding of the ligand to the receptor increases the association with the J-domain, while also increasing the affinity of the intracellular beta-gamma binding region of the C-terminal region of the PTH receptor for G proteins, resulting in their subsequent activation and initiation of downstream signaling cascades. G_αs activates adenylate cyclase (AC), which increases intracellular [cAMP], resulting in activation of Epac and PKA. G_q activates phospholipase C (PLC), forming diacylglycerol (DAG) and inositol triphosphate (IP₃). DAG directly activates PKC, whereas IP₃ indirectly activates PKC by releasing Ca²⁺ from the ER.

The PTH2R was identified by homology screening based on conserved sequences from calcitonin, secretin, and PTH1R from a cerebral cortex cDNA library. The gene for PTH2R, an 88kb gene with 13 exons and several large introns, has been located on chromosome 2q33. Like the PTH1R, the PTH2R is a class II GPCR and 51% identical to PTH1R. Similar to PTH1R, PTH2R has a large hydrophilic amino-terminal domain containing 120 amino acid residues containing four glycosylation sites and six extracellular cysteine residues, seven transmembrane domains, and a large intracellular C-terminal domain. The PTH2R is highly expressed in brain particularly in the limbic system and parts of hypothalamus. Peripherally, the PTH2R is also expressed in pancreatic islet D cells, parafollicular C cells of the thyroid, cells that produce gastrointestinal peptide, cartilage, and heart muscle cells (148). In contrast to the PTH1R, PTH2R selectively binds to PTH but not to PTHrP. The natural ligand of PTH2R in the CNS is the 39 amino acid tubero-infundibular peptide (TIP-39) in the bovine hypothalamus, described for human, rat, and zebrafish PTH2R. PTH also activates human PTH2R but not the rat and zebrafish PTH2R. TIP-39 and PTH do not share sequence homology. Only five residues of bovine PTH and TIP-39 are similar and align with each other. However, NMR studies have demonstrated that the two peptides are structurally very similar with regard to the orientation of polar and nonpolar amino acid residues in the amino-terminal region (57, 245, 300, 645).

The third and the most recently identified PTH receptor, the PTH3R was cloned using genomic PCR from zebrafish DNA. PTH3R is 69% similar and 61% identical to the PTH1R and shares only 48% similarity with PTH2R. PTH3R is almost exclusively activated by PTHrP and is the least studied of the three PTH receptors. The mammalian ortholog of PTH3R has not yet been identified. Similar to PTH1R and PTH2R, PTH3R has been classified as a class II GPCR but little else about its structure-function relationships is known (557, 702, 703).

Recent evidence suggests the presence of PTH receptors specific for the C-terminal of PTH which may play a role in gluconeogenesis, leukocyte migration, and pancreatic secretions [reviewed in (468)].

PTH-PTH1R interactions

Photo-affinity cross-linking studies of the interactions between a modified PTH 1-34 and its cognate receptor (PTH1R) suggest that the position 13 of the PTH (1-34) docks at the positions 169-198 in the N-terminal region of the PTH1R (3, 91, 722) and position 27 of PTH1-34 docks at position 241-285 of the receptor (267, 512). The 1, 3, 23, and 27 positions of the PTH interact with the receptor residues M425, R186, T33/Q87, and L261 (91, 148). Molecular modeling demonstrated that the N-terminal region of hPTH1-34 binds to an invagination of the PTH receptor formed between TM3, TM4, and TM6. (Fig. 4B) Mutation analysis revealed that the residues Trp23, Leu24, and Leu28 of PTH1-34 interact with Phe173 and Leu174 of the PTH receptor through hydrophobic interactions. Hydrophilic interactions are formed between Arg20 of PTH1-34 and residues Glu177, Glu180 of the receptor, and between Lys27 of PTH1-34 and Glu169 of the receptor (250). Mutation of Leu24 and Leu28 results in 4000- and 1600-fold decrease in binding affinity, respectively, while mutation of Asp30 to Lys has no effect on receptor binding (246). The hydrophilic interactions are less important for binding as compared to hydrophobic interactions (246). The human PTH1-37 and 1-34 fragments retain full activity of the intact PTH1-84 and activate both adenylyl cyclase and phospholipase C activity. However, PTH1-31 is nearly equipotent but predominantly stimulates adenylyl cyclase activity, while removal of 2 N-terminal amino acids (PTH3-34) results in loss of adenylyl cyclase activity but activation of phospholipase C activity is retained (45, 46, 231, 685).

Based on molecular modeling, Hoare et al. (299) described a “two-site” dynamic model of interaction between PTH and PTH1R. According to their model, the extracellular N-terminal domain of PTH1R is the docking site for the C-terminal portion of PTH 1-34. The N-terminal of PTH 1-34 interacts with the J domain of the PTH1R which includes extra and intracellular loops and the transmembrane domains. The PTH1R N-domain provides the binding interactions between PTH and PTH1R while the J domain provides a stabilization function involved in receptor activation, G-protein coupling, and signal transduction (65, 330). Residues 15-34 of PTH function primarily as the binding domain (131) while residues 1-14 domain are responsible for the initiation of intracellular signaling through adenylate cyclase and PKC (65, 242, 246, 521). The binding of the N- and C-termini of PTH to the N- and J-domains of the receptor leads to increased affinity of the ligand-receptor complex, G protein complex formation, and activation of the downstream signaling pathways (65, 243, 244, 330). The current data suggest that the PTH receptor and PTH complex is present in an intermediate preactive R⁰ confirmation where G proteins are not coupled to the receptor ligand complex. The PTH-PTHR1 interaction stimulates the binding of heteromeric G proteins and the complex switches to an active confirmation (RG). Competition binding assays suggest that PTH1-34 binds with greater affinity to the R⁰ state than the RG state (299).

Signaling mechanisms of PTH1R

When PTH binds to the PTHR, the PTHR undergoes conformational change that promotes binding of G proteins (Gαβγ) to the receptor, followed by exchange of GDP for GTP on the α-subunit, and dissociation of the Gα from Gβγ subunits. (Fig. 4C) Gαs activates adenylyl cyclases to synthesize cyclic-AMP resulting in activation of protein kinase A (PKA). Gαq activates phospholipase C (PLC) which converts PIP2 to diacyl glycerol (DAG) and inositol (1,4,5)-triphosphate (IP3). IP3 stimulates calcium release from the endoplasmic reticulum to the cytosol. Increased Ca⁺⁺ allows translocation of protein kinase C to the plasma membrane where it is activated by DAG (658, 660). Identification of signal selective peptides has led to better understanding of the PTH-PTHR interactions and downstream signaling mechanisms. For example, PTH1-28 is a cAMP-selective agonist that stimulates PKA activation but does not activate PLC-dependent and -independent PKC activation. This agonist also does not cause receptor internalization or recruitment of β-arrestin. Removal of the first two amino acids of PTH results in loss of cAMP signaling (92). Mutation analysis has demonstrated that the conserved valine at position 2 is critical for signaling through both arms (589). PTH 7-34 acts as a weak antagonist of PTHR, does not stimulate either Gs or Gq-mediated signaling pathways but does cause receptor internalization. The N-terminal truncated PTH fragments like PTH7-34 and PTH39-84 may have inhibitory effects (479). The C-terminal fragments of PTH blunt bone resorption and vitamin-D dependent osteoclastogenesis (196). These actions of PTH are thought to be independent of PTHR but are dependent upon yet an elusive carboxy-terminal PTH fragment receptor and stimulate alkaline phosphatase activity and induce expression of mRNA for both alkaline phosphatase and osteocalcin (195, 617). It is important to note that the ability of PTH to activate cAMP and/or PLC/PKC pathways is cell specific. For example, PTH stimulates cAMP-PKA pathway but not the PLC/PKC pathway in vascular smooth muscle cells (415) while in keratinocytes (495, 684), cardiac myocytes (531, 568), and lymphocytes (359), PTH activates PLC/PKC pathway but not the cAMP/PKA pathway. The development of FRET based assays allowed kinetic studies of binding of PTH to its receptor and signaling in live cells. These studies demonstrated that binding of PTH to its receptor involves two steps. The first step, rapid binding of PTH to the N-terminus of the receptor, requires 150 ms at saturating concentrations of PTH. The second step where the C-terminal of PTH binds to the J-domain of the receptor is considerably slower, requiring about 1 s. The subsequent interaction between PTHR and Gs depends upon the expression levels of Gs and can be completed in about 0.96 s. In about 10 s following receptor coupling with Gs, cAMP production is initiated (130, 222, 656).

The actions of PTH on bone are very well documented and have been the subject of recent excellent reviews (62, 135, 165, 209, 459, 528, 543, 591, 691, 719). PTH interacts directly with osteoblasts and osteocytes through PTH1R to stimulate a number of different pathways including cAMP/PKA, PLC/PKC, β arrestin translocation, and ERK1/2. In bone, the downstream signaling from these pathways is heavily regulated by RGS2 (regulator of G protein signaling 2), one of a family of proteins that modulates G protein activity stimulated by G protein-coupled receptors. Depending on whether the hormone presence is continuous or pulsatile, the overall effect of PTH signaling on bone metabolism will be catabolic or anabolic, respectively. Key to determining which effect predominates is the differential control of the osteoprotegerin-receptor activator of NFκB ligand-receptor activator of NFκB (OPG-RANKL-RANK) pathway. OPG, which is a bone-derived cytokine, and RANK, which is a receptor located on the preosteoclast, compete for binding with RANKL, another bone derived cytokine. Interaction between RANK and RANKL stimulates osteoclastogenesis while OPG prevents that interaction by binding itself to RANKL. It is this pathway that controls the PTH-stimulated interaction with the osteoclast precursor cell, which can mature into a functional osteoclast and mediate bone resorption. Continuous presence of PTH leads to an increase in the mRNA for RANKL and a decrease in the mRNA for OPG through PKA dependent pathways, the result of which is enhanced binding of RANKL to RANK and enhanced osteoclast maturation. PTH stimulates osteoblast differentiation, decreases osteoblast apoptosis, and activates lining cells. The effects of PTH to stimulate osteoblastogenesis can be seen in bone progenitor cells, mediated through changes in the transcriptional program that result in the expression of characteristic bone proteins such as alkaline phosphatase, type I collagen, RUNX2, and others. Recently, several downstream mediators of PTH action on bone have been identified including monocyte chemoattractant protein-1, sclerostin, dickkopf1, and EphrinB2/EphB4 (591) Investigation into the mechanisms of PTH regulation of bone is a dynamic area of inquiry as witnessed by the recent explosion of findings highlighting the intricacies of the process.

The other major target for PTH is the kidney. In human kidneys, the PTH receptors are expressed in proximal tubules, cortical ascending limbs of the Loop of Henle, and distal tubules. The receptors are expressed on both the apical and basolateral membranes of proximal tubules (291, 340, 353, 546, 633). Activation of the receptors on the basolateral membranes activates the PLC/PKC pathway while activation of the receptors on the apical membranes activates cAMP-PKA pathway (546, 633). Both pathways contribute to endocytosis of the type IIa sodium-phosphate cotransporter, leading to inhibition of phosphate reabsorption (350, 351, 380, 467). The differences in the activation of PLC/PKC pathway or cAMP-PKA pathway has been attributed to the binding of PTHR to sodium-hydrogen exchanger regulatory factor-1 (NHERF1), a scaffolding protein that exhibits two internal PDZ domains and an ezrin binding domain at the C terminal (677). Mahon and Segre recognized that the intracellular C-terminal domain of PTHR expresses a PDZ recognition motif D/E-S/T-X-ϕ that preferentially binds to PDZ1 domain of NHERF1 and PDZ2 domain of NHERF2 (418), and confirmed that PTHR binds to both proteins, NHERF1 and NHERF2. NHERF1 and NHERF2, through binding with ezrin, link membrane proteins with the actin cytoskeleton and recruit several proteins including receptors, ion transporters, and signaling proteins to the plasma membrane (97, 167, 676, 677). Using heterologous expression of NHERF2 and PTHR in PS120 fibroblast cells, Mahon and Segre identified that PTH activates PLC/PKC pathway. In the absence of NHERF2, PTH activated cAMP-PKA without affecting PLC/PKC pathway (418, 419). These studies suggest that NHERF provides signaling switch that could explain cell specific functions of PTHR independent of PTHR expression levels and expression of splice variants of PTHR (237). The expression patterns of NHERF1 and PTHR have important physiological consequences for renal phosphate and calcium handling. NHERF1 null mice exhibit phosphaturia and hypophosphatemia in part due to decreased apical membrane expression of type IIa sodium phosphate cotransporter without any changes in serum calcium (121, 167-170, 583, 584, 663, 675, 678-681). Phosphate wasting without changes in serum calcium has also been seen in patients who express NHERF1 polymorphisms or mutations (64, 166, 334, 545). The absence of NHERF1 expression in distal nephrons may explain the differences in the renal regulation of phosphate and calcium by PTH (237).

PTH1R trafficking and desensitization

Following binding and activation, the PTHR undergoes internalization and desensitization similar to most GPCRs (251, 252, 386). PTHR is phosphorylated in the C-terminus by G-protein-coupled receptor kinases (GRKs), resulting in increased association with β-arrestin and internalization through clathrin-coated pit dependent pathways (129, 606, 618, 624, 625, 659). Some of the receptors undergo rapid recycling while the rest are degraded in the lysosomes. The PTHR desensitization is more complex due to its associations with cytoplasmic adapter proteins NHERF1, NHERF2, and disheveled (Dvl2) (552, 607). Binding of these adapter proteins to the C-terminal domain of PTHR alters the selectivity and specificity of PTHR signaling through cell and ligand-dependent effects (273, 416, 417, 420). The laboratory of Peter Friedman extensively studied the internalization of PTHR in kidney cells. Using EGFP tagged PTH1R they demonstrated that GRK2-dependent phosphorylation of PTH1R is required for endocytosis in mouse proximal and distal tubule cells (92, 657). Their studies revealed that the PTH fragments that do not activate the receptor like PTH7-34 cause endocytosis in distal convoluted tubules but not in proximal tubules (237, 660). When they stimulated NHERF1- transfected distal tubule cells with PTH 7-34, they demonstrated blunting of internalization of PTH1R. Similarly, proximal tubule cells expressing dominant negative NHERF1 showed increased internalization of the PTHR in response to PTH7-34. These data suggest that interaction of PTHR with NHERF1 plays an important role in receptor internalization (237, 660). Mutations in the PDZ binding motif of PTHR prevented binding of NHERF1 to the PTHR but did not interfere with signaling or internalization of the receptor. These results suggest that the intact PDZ motif is required for association with NHERF1, but additional studies will be needed to define the nature of association and the impact on receptor internalization. Mutations in the NHERF1 ezrin binding domain prevented NHERF1-PTHR-actin interaction but failed to prevent internalization in response to PTH7-34. Similar results were shown in the presence of actin destabilizing agent, cytochalasin-D. Together these studies suggest that degradation of any component of the interaction between NHERF1-PTHR-actin cytoskeleton allows internalization of the PTHR in response to PTH7-34 (237).

Recent studies from Friedman laboratory suggest that PTHR also associates with the PDZ adaptor protein disheveled 2 (Dvl2). Unlike, the interaction with NHERF1, association with Dvl2 occurs between residues 470 and 480 of the PTHR. Using immunoprecipitation, they demonstrated that PTH1-34 increases interaction between PTHR and Dvl2 transiently. Binding of PTHR with Dvl2 increases its association with AP2 and β-arrestins resulting in internalization of the receptor (140, 552, 710).

Recent studies suggest that internalization of the PTHR in response to PTH1-34 does not completely blunt cAMP-PKA pathway. These observations led to the hypothesis that persistent calcemic responses in animals, prolonged increases in serum 1,25-dihydroxyvitamin D, bone resorption and prolonged cAMP-PKA activation results from signaling through intracellular PTH bound to PTHR in the early endocytic compartment. These provocative studies suggest that GPCR stimulated signaling is not confined to the plasma membrane (116, 192, 222, 360). Further studies are required to determine the physiological consequences of intracellular signaling through endocytosed GPCRs.

Functions of PTH

Several very careful cellular, molecular, and in vivo studies helped to understand the physiological roles of PTH. PTH plays diverse roles in the body from maintaining whole body calcium homeostasis to maintaining bone density. In the kidney, PTH regulates phosphate homeostasis by decreasing expression of sodium-phosphate cotransporters NpT2a (38, 39, 96, 121, 188, 203, 294, 348, 350, 351, 467) and NpT2c (109, 274, 377, 379, 454, 455, 576, 577), thus inhibiting reabsorption of filtered phosphate by the proximal tubules. PTH may also decrease phosphate absorption in the intestine by decreasing membrane expression of NpT2b (115, 255, 562). These two actions that tend to decrease total body phosphate content contrast with the pro-absorptive effects of PTH on calcium and ensure that maintenance of adequate calcium stores is not accompanied by accumulation of excessive phosphate. PTH increases ammoniagenesis (146) and gluconeogenesis (347, 573, 664) in the kidney. In the proximal tubules PTH induces inhibition of bicarbonate partly through inhibition of Na⁺/H⁺ exchanger activity (235). The actions of PTH on Na⁺/H⁺ exchanger are complex and different from the actions of Na-Pi cotransporters. PTH inhibits the activity of Na⁺/H⁺ exchanger but does not decrease the number of transporters whereas it causes endocytosis and degradation of Na-Pi cotransporters (390, 707, 720). The activity of Na⁺/H⁺ exchanger returns quickly to normal levels once PTH is removed unlike the activity of Na-Pi cotransporters which requires de novo synthesis (292, 462). PTH activates 25 vitamin D 1α-hydroxylase, thus stimulating conversion of 25-hydroxy vitamin D to its active form, 1, 25-dihydroxy vitamin D (373, 586). The active vitamin D increases calcium reabsorption in the intestine. In the thick ascending limb of the Loop of Henle PTH increases transepithelial transport of sodium, calcium, and magnesium. PTH stimulates distal renal tubular calcium reabsorption by regulating the expression of proteins involved in calcium reabsorption, viz; sodium calcium exchanger, Ca-ATPase, calbindin, TRPV5, and TRPV6 (187, 235, 321, 651). PTH promotes apical calcium entry through dihydropyridine-sensitive calcium channels. Apical calcium entry in this segment of the nephron is favored due to basolateral increase in chloride conductance resulting in increased chloride efflux and fall of intracellular chloride concentrations. Calbindin also plays a crucial role in calcium homeostasis by binding to reabsorbed calcium inside the cells and therefore prevents increases in free intracellular calcium. Expression of calbindin is regulated by PTH-dependent calcitriol synthesis (321). However, the most important function of PTH is to regulate bone mineralization. PTH affects all bone cells; stimulation of osteoblasts enhances bone formation while stimulation of osteoclast maturation increases bone resorption. The stimulatory effects of PTH on bone turnover have been successfully and effectively used pharmacologically to treat osteoporosis (520).

Interaction of PTH with other hormones

The most well studied interaction of PTH with other hormones is the interaction with fibroblast growth factor 23 (FGF23)/klotho complex (328). FGF23 is produced by osteocytes and osteoblasts (68, 429, 525, 527, 588, 597, 704). Both PTH and FGF23 are phosphaturic hormones and regulate phosphate homeostasis by inhibiting phosphate absorption in the intestines and reabsorption in the kidneys through decreasing membrane expression of sodium-phosphate cotransporters NpT2a (26, 247, 248) and NpT2c (66, 247, 248). PTH increases 1α-hydroxylase activity, active vitamin D, and calcium absorption in the intestines and reabsorption in the kidneys whereas FGF23 decreases 1α hydroxylase activity, active vitamin D thereby decreasing the absorption of calcium (289). PTH and FGF23 regulate the synthesis of each other. Higher serum phosphate increases the synthesis of FGF23 and lowers serum calcium, which will trigger synthesis of PTH. While PTH increases calcium reabsorption and indirectly increases FGF23 synthesis. Both of these mechanisms will eventually result in bone disease (328).

In recent years, an association between PTH and aldosterone has been described. New data demonstrate the expression of PTH in the aldosterone secreting zona glomerulosa cells of the adrenal glands and the expression of the mineralocorticoid receptor in parathyroid cells (632). Studies suggest that PTH directly regulate the secretion of aldosterone from the zona glomerulosa cells of adrenal glands and aldosterone in turn regulates PTH secretion from the parathyroid cells (631, 632). Isales et al. (316) and Olgaard et al (490) demonstrated that PTH stimulated aldosterone secretion in a dose dependent manner and potentiated angiotensin 2 stimulated aldosterone secretion. Rosenberg et al confirmed that adrenal glands are a novel target of PTH (554, 555). The effects of PTH on aldosterone secretion were mediated through PTH1R activation of cAMP/PKA and PLC/PKC pathways (213, 214). In patients with primary hyperparathyroidism (pHPT), elevated levels of aldosterone have been demonstrated (249), and parathyroidectomy has resulted in decreased aldosterone levels, decreased blood pressure, decreased risk of metabolic syndrome, and improvement in several parameters of vascular function (21, 216, 217, 407, 496). A prospective study in 226 patients with essential hypertension demonstrated a positive correlation between aldosterone and PTH levels (631, 632). Primary aldosteronism (PA) has been associated with higher PTH and lower calcium. Treatment with spironolactone decreased PTH levels in PA patients (556). Taken together these data suggest aldosterone and PTH cooperatively would cause vascular damage to multiple organs and support the hypothesis that regulation of PTH and aldosterone are associated, though whether these are linked or separate pathways remains unclear. These primarily epidemiologic and associative studies require more investigation to confirm a true cause and effect relationship and to identify the prevalence of these combined disorders.

Pathophysiology

Once it was recognized that parathyroid glands secrete a hormone, PTH, in response to changes in ionized calcium levels, studies were conducted to understand the consequences of hyper- or hyposecretion of PTH. Even in the absence of reliable imaging techniques or assays for the measurement of mineral ions in the early twentieth century, pioneering work of several investigators identified clinical sequelae of excessive (hyperparathyroidism) or insufficient (hypoparathyroidism) PTH.

Hyperparathyroidism

The insights gained from pathological and mechanism-based studies identified and defined the clinical features of hyperparathyroidism (16, 17). Hyperparathyroidism may be primary or secondary (448). pHPT can result from an adenoma, multigland hyperplasia, or carcinoma. In most (about 90%) adults a single adenoma causes pHPT while others (about 5%) may have double or multiple adenomas of the parathyroid glands. About 5% of patients present with glandular hyperplasia and 1% with carcinoma (558). Mechanisms for the development and growth of parathyroid adenomas in the setting of primary and secondary hyperparathyroidism have been investigated extensively. The size of the adenomas is inversely proportional to the nutritional status of vitamin D (532), suggesting a role for vitamin D in regulating the growth of these adenomas, presumably through the VDR. On the other hand, studies of the expression of vitamin D receptor in the adenomas have not demonstrated a consistent pattern (368, 616, 654). Similarly, expression of the CaSR in adenomas has been described as increased, decreased, or unchanged; however, recently Koh and colleagues identified RGS5 as highly expressed in parathyroid adenomas and have suggested that the increased expression of this protein could alter CaSR signaling to decrease its effect on PTH secretion (361). Proteomic analysis comparing normal and adenomatous tissue suggests that proteins involved in apoptosis are decreased in parathyroid adenomas compared to normal parathyroid glands (653). The genetic basis for the development of sporadic hyperparathyroidism has not been fully established. In some circumstances, mutations in the MEN1 gene, the RET gene, or the CaSR gene which are associated with familial forms of hyperparathyroidism have been identified, but not universally (630). Clearly, multiple different mechanisms appear to result in the clinical syndrome of hyperparathyroidism, suggesting that identification of these mechanisms in the individual patient could lead to more directed and effective therapy, perhaps even nonsurgical therapy (585).

Clinically, most patients present with asymptomatic hypercalcemia on routine lab work and a high circulating PTH level. Older literature classically refers to the manifestations of hyperparathyroidism as “stones, bones, abdominal groans, and moans” (58). Stones are due to nephrocalcinosis, hypercalcuria, and renal tubular reabsorption disturbances. Bone pain can be experienced due to fractures resulting from enhanced bone resorption leading to osteoporosis and osteitis fibrosa cystica. Abdominal groans are due to nausea, constipation, anorexia, and pain. Moans refer to both neuromuscular and neuropsychiatric symptoms including depression, anxiety, cognitive dysfunction, and fatigue. Other symptoms include headache, emesis, polydipsia, diarrhea, and joint pains (58). Symptoms are frequently more severe in children than in adults but this may be due in part to delay in diagnosis as serum calcium is not monitored regularly in children (364). Recent data suggest that cyclin D1 mutations also cause pHPT (32, 33, 422, 423).

pHPT can be normocalcemic, asymptomatic, or hereditary (533). Normocalcemic hyperparathyroidism is a fairly newly recognized condition wherein patients present with normal calcium (including ionized calcium) levels and high serum PTH levels with no secondary causes like renal disease, medications, gastrointestinal illness, idiopathic hypercalciuria, or vitamin D deficiency (592). A percentage of these individuals later develop hypercalcemia. It is often diagnosed in patients found to have low bone mass or nephrolithiasis. This two-phase process for the development of full-blown hyperparathyroidism is incompletely understood. One theory holds that initial target organ resistance to the actions of PTH, as would be seen perhaps in premenopausal women with higher circulating estrogen levels, mask the hypercalcemic response to PTH, which then becomes apparent after menopause when estrogen levels decline precipitously. However, most patients with normocalcemic hyperparathyroidism actually are post-menopausal, somewhat negating this theory (18). The epidemiology and natural history of this disorder are not well understood as yet [reviewed in (172)].

Asymptomatic hyperparathyroidism is characterized by mild hypercalcemia, low to deficient vitamin D, and normal serum phosphate levels. It is more prevalent in women than in men and manifests within the first decade after menopause. In asymptomatic patients, densitometric and histomorphometric analyses demonstrate reduced bone mineral density in the distal one-third radius while lumbar region is often preserved and the hip region is intermediate between distal and lumbar regions. PTH levels are high with low 25-hydroxyvitamin D levels [reviewed in (593)].

Familial hyperparathyroidism is a group of inherited autosomal dominant parathyroid disorders (256). These include multiple endocrine neoplasia (MEN) type I MEN1, type 2 MEN2a, type 4 MEN4, familial hypocalciuric hypercalcemia (FHH), neonatal severe hyperparathyroidism (NSHPT), autosomal dominant moderate hyperparathyroidism (ADMH), hyperparathyroidism-jaw tumor syndrome (HPT-JT), and familial isolated hyperparathyroidism (FIHPT). MEN1 is caused by mutations in the MEN1 gene on chromosome 11q13 (388, 389). Patients develop parathyroid adenomas but carcinomas are rare. Patients show multiglandular parathyroid disease with asynchronously and asymmetrically enlarged parathyroid glands (211, 212, 236, 424). MEN2a syndrome is an autosomal dominant condition with high risk of developing medullary thyroid carcinoma. The average onset of PHPT is 38 years of age (256). MEN2 is caused by mutations in RET gene localized to chromosome 10 that encodes a tyrosine kinase (286, 458, 463-465). MEN2 is characterized by parathyroid adenoma or hyperplasia (256). MEN4 is a rare disorder, the result of mutations in CDKN1B gene leading to dysfunctional cell cycle inhibitor p27 (383, 384, 425, 504). Little is known about this illness.

FHH and NSHPT are associated with inactivating mutations in CaSR gene. FHH patients express a mutation in one allele and have hypercalcemia, mild hypermagnesemia, and hypophosphatemia. Generally, these individuals are completely asymptomatic; however, parathyroid glands may be moderately enlarged. This condition is differentiated from the usual sporadic pHPT by very low urine calcium, generally less than 100 mg/day, and requires no treatment. NSHPT is a homozygous form of FHH, resulting in the very rapid development of PHPT at birth or shortly thereafter. Patients have severe hypercalcemia, bone demineralization, and neurodevelopmental disorders. In this disorder, parathyroid glands should be surgically removed within first few days of life to prevent fatal outcome (434-437).

ADMH is caused by mutations in cytoplasmic C-terminal tail of CaSR and is characterized by parathyroid hyperplasia or adenoma. The treatment of choice is surgical removal of the parathyroid gland (126, 127).

HPT-JT is an autosomal dominant disorder caused by mutations in the HRPT2 gene encoding parafibromin, a critical protein for cell growth (128). HPT-JT is associated with a variety of manifestations including fibrous-osseous tumors of the jaw, Wilm’s tumor, papillary renal carcinoma, polycystic kidney disease, renal cysts, and pHPT (132, 133, 136, 582). Sporadic parathyroid carcinomas are very common in the HPT-JT patients (256).

FIHPT is another rare autosomal dominant disorder associated with mutations in CaSR, MEN1, and HRPT2 genes. The disease is characterized by uni- or multiglandular lesions of parathyroid glands, treated by simple surgical removal of adenomas (285, 449, 626, 672).

Secondary hyperparathyroidism is quite common and is caused by decreased levels of vitamin D, hypocalcemia, or in chronic renal disease (233). Patients may present with low bone density, osteoporosis, or fragility fractures. Hypocalcemia of any cause can result in increased PTH secretion. Common clinical situations include intestinal malabsorption or poor diet, which limit calcium intake; pancreatitis or rhabdomyolysis, which sequester calcium; or vitamin D deficiency due to poor diet, lack of sun exposure, nephrotic syndrome, or liver failure. In contrast to pHPT, correction of the underlying disorder will normalize PTH levels. Individuals with secondary hyperparathyroidism more commonly have diffusely hyperplastic glands than those with sporadic pHPT, who more likely will have an adenoma. Secondary hyperparathyroidism complicates the clinical course of nearly all patients with chronic kidney disease, although it is generally not manifest until late in the course. The etiology of secondary hyperparathyroidism associated with chronic kidney disease is complex (210, 275). Early in the development of chronic kidney disease, levels of FGF23 begin to rise, a phenomenon attributed at least in part to the loss of renal expression of klotho and to a diminished ability of the kidney to excrete phosphorus. The rise in FGF23 is mirrored by a decrease in 1,25-dihydroxyvitamin D, resulting in decreased intestinal calcium absorption and hypocalcemia. Clinically, this hypocalcemia may be subtle, asymptomatic, and not recognized. As kidney failure progresses, frank hyperphosphatemia becomes more prominent. The combination of hypocalcemia, decreased active vitamin D, and hyperphosphatemia results in progressive secondary hyperparathyroidism, which is not easily reversible. These individuals may exhibit parathyroid gland hyperplasia or multiple adenomas composed of monoclonal or polyclonal clusters of parathyroid cells. The secondary hyperparathyroidism of chronic kidney disease is implicated in a variety of complications of kidney disease including accelerated vascular disease, vascular calcification, and fractures.

Hypoparathyroidism

Patients with hypoparathyroidism present with severe hypocalcemia, hyperphosphatemia, tetany, hypomagnesemia, and lower levels of vitamin D. Basal ganglia calcifications are another very common feature of this syndrome. The most common cause of hypoparathyroidism is damage or removal of parathyroid glands during neck surgery, especially complicated thyroid surgery. However, hypoparathyroidism may occur as a congenital disorder or as an autoimmune condition, in isolation or in conjunction with other organ failure. The reader is referred to several recent excellent clinical reviews of this rare condition (73, 89, 590). A number of parathyroid-specific autoantibodies have been identified and implicated in the development of hypoparathyroidism in the autoimmune polyendocrinopathy syndrome type I, including antibodies directed against the calcium sensing receptor, tryptophan hydroxylase, interferon omega, and the NACHT leucine rich repeat protein 5 (NALP5) to name a few. However, the autoantibodies responsible for isolated autoimmune hypoparathyroidism and for other forms of autoimmune polyendocrinopathy syndromes are still in question. Standard treatment for symptomatic patients has been high-dose active vitamin D and calcium, but recently, clinical trials of recombinant parathyroid hormone have been initiated (171). Another rare syndrome is pseudohypoparathyroidism where there is resistance to PTH hormone action, either globally or confined to the proximal tubule of the kidney. This condition is caused by mutations in the gene for the Gsα subunit, resulting in abnormal Gsα function or in abnormal Gsα transcription (448, 629).

Human chondrodysplasias

Two devastating forms of chondrodysplasia, Blomstrand’s lethal chondrodysplasia (BLC) and Jansen’s metaphyseal chondrodysplasia (JMC), resulting from mutations in PTH1R gene have been described (567).

BLC was first described by Blomstrand et al in 1985 (99) and is prenatally lethal due to premature bone mineralization, ossification, shortened limbs, and abnormal tooth and mammary gland development (567, 700). Three different inactivating PTH1R mutations have been identified in patients with BLC, which result in failure of PTH to bind to the receptor, diminished PTH1R expression, or impaired signal transduction [reviewed in (567)].

JMC is a rare autosomal dominant disorder associated with severe abnormalities of the growth plate. Clinically, the patients have short stature, disproportionate limbs, and micrognathia. Patients have severe asymptomatic hypercalcemia, hypophosphatemia, increased phosphate and cAMP excretion in urine, and elevated levels of vitamin D with normal or undetectable PTH. JMC is caused by gain of activity single point mutations in PTH1R including H223R, T410P, and I458R that result in PTH-independent activation of cAMP/PKA pathway [reviewed in (567)].

Enchondromatosis is caused due to common solitary or multiple benign tumors of bone. Recently, missense mutation (R150C) in PTH1R has been identified in two patients with enchondromatosis. The mutation results in a constitutively active receptor leading to increased cAMP levels. This mutation is less severe than the mutations observed in JMC [reviewed in (567)].

Conclusions

PTH is a hormone critical for many cell processes, primarily focused on mineral metabolism. This tightly regulated hormone is critical for regulation of calcium and phosphate homeostasis as well as bone metabolism. Dysfunction in the regulation results in dramatic clinical pictures characterized by poor bone mineralization and increased soft tissue mineralization. These abnormalities in turn lead to cardiovascular disease and kidney failure. Key gaps in our understanding of PTH include the role of intracellular signaling, the interaction of PTH with other hormones involved in mineral metabolism, and the mechanisms by which PTH can influence cardiovascular health.