Parathyroid anatomy, hormone synthesis, secretion, action, and receptors
Parathyroid embryology, anatomy, and morphology
Humans have two pairs of parathyroid glands lying in the anterior cervical region. The fetal parathyroid glands begin developing at 5 weeks from the third and fourth pharyngeal pouches. The third pharyngeal pouch, which contains tissue that will become the thymus and parathyroid, migrates downward and gives rise to the two inferior parathyroid glands normally located at the lower poles of the thyroid. The fourth pharyngeal pouch does not migrate and gives rise to the two upper parathyroid glands, which normally are attached to the upper poles of the thyroid (1).
Eighty-five per cent of normal adults have four parathyroid glands, but the number can vary markedly in some individuals. The location of the glands is also variable with the upper glands sometimes located behind the pharynx or the oesophagus. The lower glands may be found close to or within the thymus in the superior mediastinum. Because of the variability in location surgical exploration of the neck can be problematic, especially in hyperparathyroidism of chronic kidney disease (2). Thus referral to an experienced parathyroid surgeon is essential to maximize localization of affected glands and minimize complications. Conversely, hypoparathyroidism most commonly occurs as a result of surgical excision of, or damage to, the parathyroid glands during non-parathyroid surgery, e.g. total thyroidectomy for thyroid cancer and radical neck dissection for laryngeal or oesophageal carcinoma, as well as repeated surgery for hyperparathyroidism.
Most patients with primary hyperparathyroidism, about 80%, have a single benign adenoma (3). Multiple (so-called) adenomas are rarely found and probably represent asynchronous parathyroid hyperplasia. Hyperplasia accounts for 15–20% of cases, and malignant parathyroid carcinoma is extremely rare, less than 1% of cases. In secondary hyperparathyroidism, all four glands are enlarged.
The chief cell is the predominant cell type in humans, with some oxyphil cells, which have an acidophilic cytoplasm and mitochondria are also present. Parathyroid cells have limited numbers of secretory granules containing parathyroid hormone (PTH), indicating that relatively little hormone is stored in the gland. Parathyroid cells normally divide at an extremely slow rate—mitoses are rarely observed.
Knowledge of the embryological formation of the parathyroids has been gained by study of mouse models in which deletion of specific genes has led to lack of parathyroid gland development (4), on the one hand, and of human familial hypoparathyroidism, in which the defective formation of the parathyroid glands is inherited in an autosomal-dominant, autosomal-recessive, or X-linked manner, on the other (1).
The mouse has a single pair of parathyroid glands, and at day e10 both the precursor thymus and parathyroid cells in the third pharyngeal pouch endoderm, express the four transcription factors, Hoxa3, Pax1, Eya1, and Pax9. The conjoined thymus and parathyroid rudiment develops at day e11 and the primordium also expresses transcription factors Six1 and Pbx1. At day e12, separate pathways distinct for the different parts of the rudiment that will develop into the thymus and parathyroid become apparent. Signalling molecules, including sonic hedgehog, bone morphogenetic protein-4, noggin, and fibroblast growth factor-8, act in a complex fashion to affect the outgrowth of the parathyroid precursor. By day e13.5, the parathyroid cell mass and thymus cell mass are separate. The thymic cells express Foxn1 that is not present in the parathyroid cells that in turn specifically express glial cells missing-2 (Gcm2). Gcm2 expression continues into adulthood; it transactivates the calcium-sensing receptor (Casr) gene and thereby influences the expression of the parathyroid calciostat (5).
In humans, hypoparathyroidism is part of the DiGeorge’s syndrome, which occurs because of a 22q11 microdeletion. Congenital defects arise as a result of the failure to develop the derivatives of the third and fourth pharyngeal pouches, leading to agenesis or hypoplasia of the parathyroid glands and thymus (6). Haploinsufficiency of the TBX1 transcription factor gene appears to play an important role although loss of other contiguous genes such as CRKL, encoding a tyrosine kinase signalling adaptor protein, probably contributes to the full expression of the syndrome. Hypoparathyroidism is a part of the Barakat’s or HDR (hypoparathyroidism, nerve deafness, and renal dysplasia) syndrome, which maps to 10p14–10pter. HDR is due to haploinsufficiency and loss-of-function mutations in the GATA3 gene, which encodes a zinc finger transcription factor (7). GATA3 is essential for normal embryonic development of the parathyroids, auditory system, and kidney in humans. Hypoparathyroidism together with growth and mental retardation, and characteristic dysmorphism (HRD) occur in autosomal recessive Kenny–Caffey and Sanjad–Sakati syndromes. The HRD syndrome is due to mutations in the tubulin chaperone E (TBCE) gene, which maps to 1q42–43 (8). In an X-linked recessive form of hypoparathyroidism there is an interstitial deletion–insertion involving chromosomes 2p25.3 and Xq27.1 near the SOX3 gene, which encodes a high mobility group box transcription factor. It is proposed that the hypoparathyroidism is caused by disruption of regulatory elements of the SOX3 gene (1). Rare cases of primary hypoparathyroidism inherited in either an autosomal recessive or dominant manner due to mutations in the GCM2 gene on chromosome 6p24 have been identified. In the latter case the mutated GCM2 acts in a dominant-negative fashion (5).
Parathyroid hormone synthesis
PTH is the product of a single-copy gene and, in mammals, has 84 amino acids (9, 10) (Fig. 4.1.1). The gene, which encodes a larger precursor molecule of 115 amino acids, preproPTH, is organized into three exons. Exon I encodes the 5′ untranslated region of the messenger RNA, exon II encodes the NH2-terminal pre- or signal peptide and a part of the short propeptide, and exon III encodes the Lys−2–Arg−1 of the prohormone cleavage site, the 84 amino acids of the mature hormone, and the 3′ untranslated region of the mRNA (Fig. 4.1.2). The importance of correct splicing of the primary PTH gene transcript, or premessenger RNA, was emphasized by the identification of a donor splice mutation in the PTH gene in affected members of a family with autosomal recessive isolated hypoparathyroidism, resulting in the loss of exon II, which encodes the initiation codon and signal peptide (1).
The second member of the PTH gene family encodes the parathyroid hormone-related peptide (PTHrP), which is the causal factor responsible in the majority of cases of hypercalcaemia associated with malignancies. PTHrP plays a critical role in fetal development, especially skeletogenesis (11, 12), but is not involved in normal calcium homoeostatic control in the adult. In postnatal life, PTHrP regulates the epithelial mesenchymal interactions that are critical for development of the mammary gland, skin, and hair follicle. The PTH and PTHrP genes map to chromosome 11p15 and chromosome 12p12.1–11.2, respectively. These two human chromosomes are thought to have arisen by an ancient duplication of a single chromosome, and their respective gene clusters have been maintained as syntenic groups across the genomes of several species. Because of the similarity in NH2-terminal sequence of their mature peptides, their gene organization, and chromosomal locations, it is likely that the PTH and PTHrP genes evolved from a single ancestral gene, with PTHrP being the more ancient gene.
The gene for tuberoinfundibular peptide of 39 residues (TIP39), a more distantly related member of the gene family, resides on chromosome 19q13.33. TIP39 is a neuropeptide (13). The TIP39 gene shares organizational features with the PTH and PTHrP genes, having one exon encoding the 5′ untranslated region, one encoding the precursor leader sequence, and one encoding the prohormone cleavage site and the mature peptide (Fig. 4.1.2).
Transcription of the PTH gene occurs almost exclusively in the endocrine cells of the parathyroid gland, and is subject to strong repressor activity in all other cells. Ectopic PTH synthesis (i.e. synthesis outside parathyroid tissue) has been documented in only a very few cases of malignancies associated with hypercalcaemia. Activation of genes in a particular tissue is often related to demethylation of cytosine residues, and the PTH gene in parathyroid cells is hypomethylated at CpG residues relative to other tissues. In one of the few cases of true ectopic PTH production, involving a pancreatic tumour, the upstream regions of the PTH gene were abnormally hypomethylated (14). The human PTH gene has two functional TATA box-controlled transcription start sites, a cyclic AMP response element (CRE), and a negative vitamin D response element (VDRE) in its proximal promoter. While PTH gene transcription is negatively regulated by the hormonally active metabolite of vitamin D, 1,25-dihydroxvitamin D (1,25(OH)2D), any regulation by extracellular calcium remains to be established. Also located distally are sequences that function to silence transcription in nonparathyroid cells. In a further case of ectopic PTH production, an ovarian carcinoma, this repressor regulatory region was replaced by a foreign sequence, which allowed inappropriate transcription of the PTH gene to take place (3).
The human PTH produced by patients with hyperparathyroidism is structurally normal (9, 10). In a small number of parathyroid tumours examined, the PTH gene sequence is rearranged, and the 5′ flanking region of the PTH gene is placed upstream of the cyclin D1 (CCND1) gene located on the long arm of chromosome 11. This is thought to lead to deregulated expression of the CCND1 gene, which contributes to tumour development (3). However, this type of gene arrangement occurs very infrequently in parathyroid tumours. A more common event involves the loss or inactivation of the multiple endocrine neoplasia type 1 (MEN1) gene, also on the long arm of chromosome 11. The protein encoded by the MEN1 gene (15, 16) called menin, is a 610-amino acid nuclear protein (17). Germ-line mutations in the MEN1 gene cause familial and sporadic MEN 1 and are found in 20% of non-MEN 1 parathyroid adenomas. Loss of heterozygosity at 11q13 is found in MEN 1 tumours and sporadic parathyroid adenomas, consistent with MEN1 being a tumour suppressor gene.
A target of the Wnt pathway, β-catenin, encoded by the CTNNB1 gene, is a candidate for involvement in parathyroid neoplasia. Very few of the parathyroid adenomas examined so far have stabilizing missense CTNNB1 mutations, suggesting that mutation of the β-catenin gene itself is unlikely to be involved in the initiation or early progression of parathyroid adenomatosis. However, other components of the Wnt signalling pathway, e.g. a constitutively active LRP5 receptor derived from an alternatively spliced mRNA, may be implicated in parathyroid tumorigenesis (18).
Early onset recurrent parathyroid tumours occur as part of the uncommon autosomal dominant hyperparathyroidism and jaw tumour syndrome, in which parathyroid carcinoma is frequent. The responsible gene, HRPT2, at 1q31.2, encodes a novel transcription factor, parafibromin, of 531 amino acids (19). Sporadic parathyroid carcinomas very commonly contain somatic mutations of the HRPT2 gene and some of these patients harbour germline mutations. In these cases, genetic testing in family members provides for early diagnosis (6). Loss of heterozygosity at chromosome 1q occurs in carcinomas of the familial and sporadic disorder, usually by intragenic mutations.
PTH follows a pattern of biosynthesis and of vectorial transport through organelles of the cell similar to that of many other peptide hormones. It is biosynthesized on the polyribosomes of the rough endoplasmic reticulum of the parathyroid endocrine cell. The gene for PTH encodes a precursor, preproPTH, which is extended at the N-terminus of PTH 1–84 by 31 residues. The NH2-terminal 25-residue portion, characterized by its hydrophobicity, is called the signal, leader, or pre sequence, and it facilitates entry of the nascent hormone into the cisternae of the endoplasmic reticulum. One patient with autosomal dominant hypoparathyroidism had a mutation within the protein coding region of the PTH gene in which there was a single base substitution (T→C) in exon II, resulting in the replacement of arginine (CGT) for cysteine (TGT) in the signal peptide. This places a charged amino acid in the hydrophobic core of the signal peptide, leading to inefficient processing of the mutant preproPTH to PTH (6). Further studies have suggested that the mutant polypeptide acts in a dominant-negative fashion by promoting endoplasmic reticulum stress leading to apoptosis (20).
Normally, as the signal sequence of the synthesized hormone emerges from the ribosome, it binds to a signal recognition particle, which stops further synthesis of the nascent protein. The signal recognition particle carrying the ribosome then binds to an integral membrane protein of the endoplasmic reticulum, called the docking protein or signal recognition particle receptor. This protein releases the block in protein synthesis, and the nascent peptide is transported across the membrane into the cisternae of the endoplasmic reticulum. The signal sequence is simultaneously removed at the inner surface of the endoplasmic reticulum, at a glycyl–lysyl bond, by a signalase enzyme. The resultant precursor molecule, proPTH, is extended at the NH2-terminus of PTH 1–84 by only six amino acids. The pro sequence is necessary for efficient translocation and cleavage of the signal peptide. Once formed, proPTH is transported to the Golgi apparatus.
The prohormone hexapeptide has several basic residues, which serve as a recognition sequence to yield the mature hormone. Unlike many other prohormones, proPTH does not contain another sequence at the COOH-terminus and has not been detected within the circulation even in states of parathyroid gland hyperfunction. ProPTH has little biological activity until cleaved to create the hormonal form (21). The conversion of proPTH to PTH takes place within the trans-Golgi network rather than the secretory granules as occurs with other prohormones such as proinsulin. The enzymes involved include furin and PC7, mammalian proprotein convertases, which are related to bacterial subtilisins (22). Little proPTH is stored within the gland.
The resultant mature 84-amino acid form of the hormone is packaged in secretory granules and transported to the region of the plasma membrane. The hormone is released by exocytosis in response to the principal stimulus to secretion hypocalcaemia. The calcium ion does not influence the enzymatic cleavages involved in the processing of preproPTH or proPTH.
Parathyroid hormone secretion
Relatively little PTH is stored in secretory granules within the parathyroid glands. In the absence of a stimulus for release, intraglandular metabolism occurs, causing complete degradation to its constituent amino acids or partial degradation to fragments (Fig. 4.1.3). This has been postulated to occur through a specific calcium-regulated enzymatic mechanism. In the case of hypercalcaemia, the predominant hormonal entities released from the gland are fragments comprising midregion or COOH-terminal sequences. In response to hypocalcaemia, degradation of PTH within the parathyroid cell is minimized, and the major hormonal entity released is the bioactive PTH 1–84 molecule. Thus, in the presence of hypocalcaemia, increased amounts of bioactive PTH are secreted, even in the absence of additional synthesis of hormone. Hormone stores are insufficient, however, to maintain secretion for more than a few hours in the presence of a sustained, severe hypocalcaemic stimulus, and other mechanisms—transcriptional and posttranscriptional—come into play to increase hormone production. For example, hypocalcaemia promotes stabilization of the preproPTH mRNA, leading to increased PTH synthesis. In the presence of a sustained, severe hypocalcaemic stimulus, additional PTH secretion depends on an increase in the number of parathyroid cells. Such an increase may also be stimulated by the reduction in circulating 1,25-dihydroxvitamin D (1,25(OH)2D) that often accompanies hypocalcaemia. Normally, the sterol inhibits parathyroid cell proliferation by inhibiting expression of early immediate response genes, such as the MYC proto-oncogene.
A circadian rhythm has been reported for PTH secretion, with increased blood levels occurring at night and small-amplitude pulses of PTH secretion occurring at much shorter intervals. This suggests neural or central nervous system influences on PTH secretion, or reflects circadian alterations in the levels of extracellular calcium.
Calcium
There is an inverse relationship between ambient calcium levels and PTH release that is curvilinear rather than proportional (23). This relationship between PTH and extracellular calcium contrasts with the influence of the calcium ion as a secretagogue in most other secretory systems in which elevations in this ion enhance release of the secretory product. This distinction between the parathyroid cell and other secretory cells is maintained intracellularly, where elevations rather than decreases in cytosolic calcium correlate with decreased PTH release. Alterations in extracellular fluid calcium levels are transmitted through a parathyroid plasma membrane calcium-sensing receptor (CaSR) that couples through a Gq/11-protein complex to phospholipase C. Increases in extracellular calcium lead to increases in inositol 1,4,5-trisphosphate (IP3) and mobilization of intracellular calcium stores. The CaSR also couples to a Gi-protein complex thereby inhibiting cyclic AMP production. The precise mechanisms whereby activation of the CaSR inhibits PTH secretion and synthesis and parathyroid cell proliferation are not known.
The human CaSR has 1078 amino acids with a large extracellular domain (ECD) (c. 600 amino acids) and a seven transmembrane-spanning domain and cytoplasmic tail (24). The CaSR is a member of group C of the G protein-coupled receptor (GPCR) superfamily that includes the metabotropic glutamate, γ-aminobutyric acid-B, and vomeronasal odorant receptors. These receptors function as dimers with the ECDs of each monomer having a so-called Venus flytrap domain consisting of two lobes, which close upon the ligand leading to conformation changes in the transmembrane domain of the receptor, allowing coupling of G proteins to the intracellular loops and the cytoplasmic tail. The CaSR has a low affinity for Ca2+ appropriate for it monitoring the relatively high levels of the mineral ion in the blood. Besides the parathyroid, the CaSR is also expressed in other cells having Ca2+-sensing functions, such as those of the kidney tubule, the calcitonin-secreting thyroid C-cells, and in diverse other organs and tissues such as brain, bone and cartilage, haematopoietic stem cells, keratinocytes, gastrointestinal tract, mammary gland, placenta, and vascular smooth muscle. Neomycin binds the receptor, which may account for the toxic renal effects of aminoglycoside antibiotics.
Inherited abnormalities of the CASR gene located on chromosome 3q13.3–21 can lead to either hypercalcaemia or hypocalcaemia depending upon whether they are inactivating or activating, respectively (25). Heterozygous loss-of-function mutations give rise to familial (benign) hypocalciuric hypercalcaemia (FHH) in which the lifelong hypercalcaemia is asymptomatic. The homozygous condition manifests itself as neonatal severe hyperparathyroidism (NSHPT), a rare disorder characterized by extreme hypercalcaemia and the bony changes of hyperparathyroidism. Several cases of NSHPT have normocalcaemic parents and seem to be sporadic. The disorder autosomal dominant hypocalcaemia (ADH) is due to gain-of-function mutations in the CASR gene. ADH may be asymptomatic or present with neonatal or childhood seizures. Because of the overactive CaSR in the nephron, these patients are at a greater risk of developing renal complications during vitamin D therapy than patients with idiopathic hypoparathyroidism. A common polymorphism in the intracellular tail of the CaSR, Ala to Ser at position 986, has a modest effect on the serum calcium concentrations in healthy individuals (26). CASR polymorphisms might also affect urinary calcium excretion and therefore CASR is a candidate gene for involvement in disorders such as idiopathic hypercalciuria and primary hyperparathyroidism.
The CaSR is a target for phenylalkylamine compounds—so-called calcimimetics—which are allosteric stimulators of the CaSR’s affinity for cations. These orally active compounds have been approved for use in patients with uraemic secondary hyperparathyroidism and parathyroid cancer and by their direct action on the parathyroid gland CaSR they provide an effective medical means of lowering PTH secretion (27). Cinacalcet HCl is marketed as Sensipar in North America and Australia and Mimpara in the European Union. Ongoing clinical trials in patients with mild primary hyperparathyroidism (PHPT) have shown that calcimimetics reduce serum calcium and PTH levels and increase serum phosphate levels but do not significantly affect bone turnover or bone mineral density (BMD). While calcimimetics provide an important addition to the armamentarium of drugs to treat the secondary hyperparathyroidism of chronic kidney disease, their more widespread use in the medical management of PHPT is uncertain at present.
CaSR allosteric antagonists, calcilytics, are also being evaluated in clinical trials as a treatment of osteoporosis (27). As intermittent administration of exogenous PTH produces increases in BMD, it is proposed that once-daily administration of a short-acting calcilytic could achieve a similar result by producing a pulse of endogenous PTH secretion.
The CaSR expressed in the developing parathyroid glands—and in the placenta—plays an important role in regulating fetal calcium concentrations. Normally, the fetal blood calcium level is elevated above the maternal level. This depends upon the action of PTHrP released from the fetal parathyroids and placenta on placental calcium transport. Disruption of the CaSR, as shown by studies in CaSR-deficient mice, causes fetal hyperparathyroidism and hypercalcaemia due to fetal bone resorption. The transfer of calcium across the placenta is reduced and renal calcium excretion is increased.
Some patients with anti-CaSR autoantibodies (of the inactivating type) associated with autoimmune disorders such as sprue or autoimmune thyroid disease present as an FHH phenocopy, termed acquired hypocalciuric hypercalcaemia (AHH). The anti-CaSR antibodies are directed against the ECD and interfere with elevated extracellular Ca2+-mediated suppression of PTH release and perturb Ca2+ sensing in the kidney, thereby closely mimicking FHH (25). Autoantibodies from a subset of patients with autoimmune hypoparathyroidism that inhibited PTH secretion were identified several years ago. More recently, the CaSR has been identified as a self-antigen in patients with autoimmune polyendocrine syndrome type 1 (APS 1) or acquired hypoparathyroidism associated with autoimmune hypothyroidism or idiopathic hypoparathyroidism. The activating antibodies are directed against epitopes in the ECD of the receptor and inhibit PTH secretion from parathyroid cells.
In vivo, PTH mRNA levels are markedly stimulated by decreased circulating calcium concentrations. This occurs, in part, by a post-transcriptional mechanism whereby hypocalcaemia stabilizes and hypercalcaemia destabilizes the PTH mRNA. Prolonged hypocalcaemia in vivo may stimulate DNA replication, cell division, and the production of increased numbers of parathyroid cells or parathyroid hyperplasia. This would increase the synthesis of proteins, including PTH, within the hypercellular parathyroid gland and ultimately would increase PTH release. In primary parathyroid gland hyperfunction resulting in hyperparathyroidism, alterations in the calcium-sensing mechanism may manifest as a set-point error, producing a shift to the right of the curve relating PTH secretion to extracellular calcium levels. Consequently, elevated concentrations of extracellular fluid calcium may be required to reduce PTH secretion, resulting in an adenomatous or hyperplastic parathyroid gland that is incompletely suppressed by calcium. Such a mechanism may underlie the observation that an increase in the mass of parathyroid tissue like that produced by transplantation, can be associated with hypercalcaemia. The parathyroid glands of patients with primary and severe uraemic secondary hyperparathyroidism have reduced CaSR expression as assessed by immunostaining. Loss of a functional CaSR, as in humans with NSHPT or in mice in which the Casr gene has been ablated, leads to severe parathyroid hyperplasia. If basal secretion per cell produces a significant amount of bioactive PTH, the cumulative increase in this basal or non-calcium-suppressible secretion arising from an increase in parathyroid cells could also be responsible for the hypercalcaemia. The precise mechanistic relationship of extracellular calcium to parathyroid cell growth remains to be determined.
1,25-dihydroxyvitamin D
Vitamin D metabolites modulate PTH release. There is a feedback loop between PTH-induced increase in 1,25(OH)2D and vitamin D metabolite-induced decrease of PTH levels (28). This latter effect is achieved by a direct action on PTH gene transcription, thus altering the quantities of hormone available for immediate release by secretagogues. ‘Low calcaemic analogues’ of vitamin D have been developed that appear to diminish PTH secretion in vitro and in vivo and that serve as therapeutics for hyperparathyroidism in chronic kidney disease.
Other factors
In addition to calcium and vitamin D metabolites, several other factors influence the release of PTH from parathyroid glands. The cation magnesium affects PTH release like calcium, although with reduced efficacy. (The CaSR is also a magnesium sensor.) High concentrations of aluminium also suppress PTH release. Hyperphosphataemia is associated with increased levels of PTH, an effect that is most often indirect and a result of the hypocalcaemia and/or the decreased 1,25(OH)2D production that accompanies the rise in serum phosphate. However, the anion can exert a more direct effect on PTH synthesis with hyperphosphataemia stabilizing and hypophosphataemia destabilizing PTH mRNA levels. Glucocorticoids (in some studies) increase PTH secretion. Agents such as biogenic amines, which increase parathyroid gland cAMP levels, induce PTH secretion, and those that lower cAMP levels within the parathyroid gland decrease PTH secretion.
PTH measurement
Circulating PTH is heterogeneous. The major circulating bioactive moiety is similar or identical to intact PTH(1–84). This is metabolized by the liver, which releases midregion and COOH-terminal fragments into the circulation for subsequent clearance by the kidney. These biologically inert moieties generated by metabolism and secretion from the parathyroid gland are cleared more slowly than intact PTH. Circulating bioactive PTH is best measured by sensitive immunometric assays that simultaneously recognize NH2 and COOH epitopes on the PTH molecule, and detect intact PTH(1–84). This is the method of choice for the accurate diagnosis of patients with hypercalcaemia, especially in distinguishing patients with primary hyperparathyroidism from those with hypercalcaemia of malignancy and in assessing hyperparathyroidism in chronic kidney disease.
Actions of PTH
The major function of PTH is the maintenance of a normal level of extracellular fluid calcium (23, 28) (Fig. 4.1.4). The hormone exerts important effects on bone and kidney and indirectly influences the gastrointestinal tract. In response to a fall in the extracellular fluid ionized calcium concentration, PTH is released from the parathyroid cell and acts directly on the kidney to enhance renal calcium reabsorption and promote the conversion of 25-hydroxyvitamin D to 1,25(OH)2D. The latter metabolite increases gastrointestinal absorption of calcium and, with PTH, induces skeletal resorption, causing the restoration of extracellular fluid calcium and the neutralization of the signal initiating PTH release. The opposite series of homoeostatic events occur in response to a rise in extracellular fluid calcium levels.
Although this scheme outlines the overall events that occur after a fall in calcium, aspects of the response may vary. Certain actions of PTH, such as renal calcium retention, may predominate at relatively low circulating concentrations of PTH. Furthermore, PTH appears to be essential as a bone anabolic factor in the fetus (29) and neonate (30) but may be predominantly resorptive in older animals (31) when the source of external calcium changes. PTH and PTHrP regulate osseous cellular differentiation, proliferation, and development, and are now considered to be anabolic skeletal agents when administered periodically rather than continuously in vivo. Thus, intermittent doses of PTH(1–34)—and PTHrP(1–34) and related analogues—promote bone formation. Daily injections of PTH(1–34) increase hip and spine bone mineral density, and prevent vertebral and non-vertebral fractures in osteoporosis, and human PTH is now used clinically as a bone anabolic agent.
Besides regulating calcium homoeostasis, PTH elicits various other responses. Among these responses are perturbations of other ions, the most marked of which are those involving phosphate. As a consequence of PTH-enhanced 1,25(OH)2D production, the gastrointestinal absorption of phosphate is facilitated to some extent, and with PTH-induced skeletal lysis, phosphate and calcium are released. These effects increase the extracellular fluid phosphate levels, but the predominant effect of PTH on phosphate homeostasis is to inhibit renal phosphate reabsorption and produce phosphaturia. Consequently, a net decrease in extracellular fluid phosphate concentration occurs, which is adjunctive to the role of PTH in raising calcium levels.
PTH receptors
Like other peptide hormones, PTH interacts through a receptor on the plasma membrane of target cells. This same receptor binds PTHrP (32). The PTH/PTHrP receptor (PTHR1) is a seven-transmembrane G-protein linked receptor that has the ‘signature’ GPCR topology, a seven-membrane-spanning, ‘serpentine’ domain, as well as an extracellular ligand-binding domain and an intracellular COOH-terminal domain (33). It is a member of group B of the GPCR superfamily. The receptor can couple to the stimulatory G protein, Gs, leading to increased adenylate cyclase activity, the generation of cAMP, and activation of the protein kinase A (PKA) pathway, and can couple to Gq, leading to an increase in the protein kinase C (PKC) pathway and to an increase in IP3, diacylglycerol, and intracellular Ca2+ (33). As with other GPCRs, PTHR1 undergoes cyclical receptor activation, desensitization, and internalization (34). After ligand binding and endocytosis, the PTHR1 is either recycled to the cell membrane or targeted for degradation. High circulating levels of PTH in hyperparathyroid states have been associated with hormonal desensitization in target tissues. Arrestins contribute to the desensitization of both Gs and Gq mediated PTHR1 signalling. PTHR1 activation and internalization can be selectively dissociated (35). PTHR1 signalling can be modified by scaffolding proteins such as the Na+/H+ exchanger regulatory factor (NHERF) 1 and 2 through PDZ1 and PDZ2 domains (36). PTHR1 signalling via the cAMP pathway, leading to PKA activation, results in phosphorylation of the cyclic AMP response element binding protein (CREB). CREB binds to the cyclic AMP response element (CRE) in the promoter region of many genes and transcriptionally modulates their expression.
The PTHR1 is highly expressed in kidney and bone, the primary target tissues of PTH, but is also expressed in a wide variety of embryonic and adult tissues, including cartilage, liver, brain, smooth muscle, spleen, testis, and skin. In most of these tissues, the receptor appears to mediate the autocrine/paracrine actions of locally produced and secreted PTHrP. Nevertheless, PTHrP may also exert some of its bioactivity through domains of the molecule that do not interact with PTHR1 (37).
The human PTH/PTHrP receptor gene (PTHR1) localizes to chromosome 3p21.1–22. A second related receptor, which is the product of a distinct gene (PTHR2 on chromosome 2q33), and which binds PTH, TIP39, but not PTHrP, has been identified (38). It is expressed in brain, pancreas, testis and placenta and its endogenous ligand is TIP39.
Direct evidence that the PTHR1 mediates the calcium homoeostatic actions of PTH and the skeletal growth plate actions of PTHrP in humans has come from the study of rare genetic disorders. Jansen’s metaphyseal chondrodysplasia (JMC) is inherited in an autosomal dominant fashion although most reported cases are sporadic (6). The disorder comprises short-limbed dwarfism secondary to severe growth plate abnormalities, asymptomatic hypercalcaemia, and hypophosphataemia. There is increased bone resorption similar to that in primary hyperparathyroidism and urinary cAMP levels are elevated, but circulating PTH and PTHrP levels are low or undetectable. Although PTHR1 is found widely in fetal and adult tissues, it is most abundant in three major organs, the kidney, bone, and metaphyseal growth plate. The changes in mineral ion homoeostasis and the growth plate in JMC are caused by heterozygous gain-of-function mutations (Fig. 4.1.5) in the PTHR1 giving rise to constitutively active receptors.
Inactivating or loss-of-function mutations in the PTHR1 have been implicated in the molecular pathogenesis of Blomstrand’s lethal chondrodysplasia (BLC) (6). This rare disease is characterized by advanced endochondral bone maturation, short-limbed dwarfism, abnormal breast and tooth morphogenesis, and fetal death, thus mimicking the phenotype of Pthr1-less mice (39). The majority of BLC cases were born to phenotypically normal, consanguineous parents, suggesting an autosomal recessive mode of inheritance. Mutant PTHR1s (Fig. 4.1.5) identified in BLC fetuses fail to bind ligand or stimulate cAMP or inositol phosphate production. A milder form of recessively inherited skeletal dysplasia, known as Eiken’s syndrome, has been linked to mutations of PTHR1, suggesting a wider range of skeletal phenotypes to this gene. Dominantly acting heterozygous PTHR1 mutations have been identified in familial, nonsyndromic primary failure of tooth eruption (40). Heterozygous PTHR1 mutations have been identified in endochondromas of patients with endochondromatosis (Ollier’s disease), a familial disorder with evidence of autosomal dominance characterized by multiple benign cartilage tumours, and a predisposition to malignant osteocarcinoma (41). As many patients with Ollier’s disease do not apparently have PTHR1 mutations, the condition may be genetically heterogeneous.
Heterozygous inactivating mutations in the GNAS1 gene encoding Gαs cause an approximately 50% reduction in amount/ activity of the protein leading to resistance to PTH and other hormones in the disorder, pseudohypoparathyroidism (PHP) type 1a (42). In contrast, patients with PHP type 1b have end-organ resistance to PTH without the typical physical stigmata—termed Albright’s hereditary osteodystrophy—of PHP type 1a. Linkage to chromosome 20q13.3, which includes the GNAS1 locus, was established in kindreds with PHP type 1b (43). In addition, the genetic defect is imprinted paternally and is inherited in the same fashion as the PTH resistance in kindreds with PHP type 1a, and in a mouse model heterozygous for ablation of the Gnas gene (44). In PHP type 1b patients, mutations some distance upstream of the GNAS1 coding regions affect the normal differential methylation of maternal and paternal alleles leading to silencing of the GNAS gene specifically in the renal proximal tubules (45).
PTH controls renal phosphate reabsorption. Mutations in the genes encoding the two renal sodium phosphate co-transporters, NPT2a and NPT2c, have been identified in a few patients with hyperphosphaturia. The NHERF1 interacts with the PTHR1 and NPT2a. Study of hyperphosphaturic patients referred initially for nephrolithiasis or osteopenia identified a few cases having NHERF1 mutations that could contribute to the renal phosphate loss (46).
Summary
PTH is responsible for the minute-to-minute maintenance of calcium homoeostasis. PTH secretion is controlled via the parathyroid CaSR, and inactivating or activating mutations in this receptor lead to inherited hypercalcaemic and hypocalcaemic disorders, respectively. Both PTH (and the related gene family member, PTHrP) act through the PTHR1 that is widely expressed and signals through multiple second messenger pathways. Inactivating mutations in the PTHR1 cause Blomstrand’s lethal chondrodysplasia, whereas activating mutations are found in Jansen’s metaphyseal chondrodysplasia.
References
1. Thakker RV. Genetic regulation of parathyroid gland development. In: Bilezikian JP, Martin TJ, Raisz LG, eds. Principles of Bone Biology. 3rd edn. San Diego: Academic Press, 2008: 1415–29.
Find This Resource
2. Meakins JL, Milne CA, Hollomby DJ, Goltzman D. Total parathyroidectomy: parathyroid hormone levels and supernumerary glands in hemodialysis patients. Clin Invest Med, 1984; 7: 21–5.
Find This Resource
3. Hendy GN, Arnold A. Molecular basis of PTH overexpression. In: Bilezikian JP, Martin TJ, Raisz LG, eds. Principles of Bone Biology. 3rd edn. San Diego: Academic Press, 2008: 1311–26.
Find This Resource
4. Miao D, He B, Karaplis AC, Goltzman D. Parathyroid hormone is essential for normal fetal bone formation. J Clin Invest, 2002; 109: 1173–82.
Find This Resource
5. Canaff L, Zhou X, Mosesova I, Cole DEC, Hendy GN. Glial cells missing-2 transactivates the calcium-sensing receptor gene: effect of a dominant-negative GCM2 mutant associated with autosomal dominant hypoparathyroidism. Hum Mutat, 2009; 30: 85–92.
Find This Resource
6. Hendy GN, Cole DEC. Parathyroid disorders. In: Rimoin DL, Connor JM, Pyeritz RE, Korf BE, eds. Emery and Rimoin’s Principles and Practice of Medical Genetics. Vol. 2. 5th edn. Edinburgh: Churchill Livingstone, 2007: 1951–79.
Find This Resource
7. Ali A, Christie PT, Grigorieva IV, Harding B, Van Esch H, Ahmed SF, et al. Functional characterization of GATA3 mutations causing the hypoparathyroidism-deafness-renal (HDR) dysplasia syndrome: insight into the mechanisms of DNA binding by the GATA3 transcription factor. Hum Mol Genet, 2007; 16: 265–75.
Find This Resource
8. Parvari R, Diaz GA, Hershkovitz E. Parathyroid development and the role of tubulin chaperone E. Horm Res, 2007; 67: 12–21.
Find This Resource
Keutmann HT, Sauer MM, Hendy GN, O’Riordan JLH, Potts JT Jr. Complete amino acid sequence of human parathyroid hormone. Biochemistry, 1978; 17: 243–4.
Find This Resource
10. Hendy GN, Kronenberg HM, Potts JT Jr, Rich A. Nucleotide sequence of cloned DNAs encoding human preproparathyroid hormone. Proc Natl Acad Sci U S A, 1981; 78: 7365–9.
Find This Resource
11. Karaplis AC, Luz A, Glowacki J, et al. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Gene Develop, 1994; 8: 277–89.
Find This Resource
12. Amizuka N, Warshawsky H, Henderson JE, Goltzman D, Karaplis AC. Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation. J Cell Biol, 1994; 126: 1611–23.
Find This Resource
13. Fegley DB, Holmes A, Riordan T, Faber CA, Weiss JR, Ma S, et al. Increased fear- and stress-related anxiety-like behavior in mice lacking tuberoinfundibular peptide of 39 residues. Genes Brain Behav, 2008; 7: 933–42.
Find This Resource
14. VanHouten JN, Yu N, Rimm D, Dotto J, Arnold A, Wysolmerski JJ, Udelsman R, et al. Hypercalcemia of malignancy due to ectopic transactivation of the parathyroid hormone gene. J Clin Endocrinol Metab, 2006; 91: 580–3.
Find This Resource
15. Chandrasekharappa SC, Guru SC, Manickam P, Olufemi SE, Collins FS, Emmert-Buck MR, et al. Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science, 1997; 276: 404–7.
Find This Resource
16. Lemmens I, Van der Ven WJ, Kas K, Zhang CX, Giraud S, Wautot V, et al. Identification of the multiple endocrine neoplasia type 1 (MEN1) gene. European Consortium on MEN1. Hum Mol Genet, 1997; 6: 1177–83.
Find This Resource
17. Hendy GN, Kaji H, Canaff L. Cellular functions of menin. Adv Exp Med Biol, 2009; 668: 37–50.
Find This Resource
18. Bjorklund P, Akerstrom G, Westin G. An LRP5 receptor with internal deletion in hyperparathyroid tumors with implications for deregulated WNT/β-catenin signalling. PLoS Medicine, 2007; 4: e328.
Find This Resource
19. Carpten JD, Robbins CM, Villablanca A, Forsberg L, Presciuttini S, Bailey-Wilson J, et al. HRPT2, encoding parafibromin, is mutated in hyperparathyroidism-jaw tumor syndrome. Nat Genet, 2002; 32: 676–80.
Find This Resource
20. Datta R, Waheed A, Shah GN, Sly WS. Signal sequence mutation in autosomal dominant form of hypoparathyroidism induces apoptosis that is corrected by a chemical chaperone. Proc Natl Acad Sci USA, 2007; 104: 19989–94.
Find This Resource
21. Rabbani SA, Kaiser SM, Henderson JE, et al. Synthesis and characterization of extended and deleted recombinant analogues of parathyroid hormone-(1–84): correlation of peptide structure with function. Biochemistry, 1990; 29: 10080–9.
Find This Resource
22. Hendy GN, Bennett HPJ, Gibbs BF, Lazure C, Day R, Seidah NG. Proparathyroid hormone (ProPTH) is preferentially cleaved to parathyroid hormone (PTH) by the prohormone convertase furin: a mass spectrometric analysis. J Biol Chem, 1995; 270: 9517–25.
Find This Resource
23. Brown EM. Physiology of calcium metabolism. In: Becker KL, Bilezikian JP, eds. Principles and Practice of Endocrinology and Metabolism. 3rd edn. Philadelphia: JB Lippincott, 2000: 478–89.
Find This Resource
24. Brown EM. Biology of the extracellular Ca2+-sensing receptor. In: Bilezikian JP, Martin TJ, Raisz LG, eds. Principles of Bone Biology. 3rd edn. San Diego: Academic Press, 2008: 533–53.
Find This Resource
25. Hendy GN, Guarnieri V, Canaff L. Calcium-sensing receptor and associated diseases. Prog Mol Biol Transl Sci, 2009; 89: 31–95.
Find This Resource
26. Cole DEC, Peltekova VD, Rubin LA, et al. A986S polymorphism of the calcium-sensing receptor and circulating calcium concentrations. Lancet, 1999; 353: 112–5.
Find This Resource
27. Nemeth EF. Drugs acting on the calcium receptor. Calcimimetics and calcilytics. In: Bilezikian JP, Martin TJ, Raisz LG, eds. Principles of Bone Biology. 3rd edn. San Diego: Academic Press, 2008: 1711–35.
Find This Resource
28. Hendy GN. Calcium regulating hormones. Vitamin D and parathyroid hormone. In: Melmed S, Conn PM, eds. Endocrinology. Basic and Clinical Principles. Totowa, NJ: Humana Press Inc, 2005: 283–99.
Find This Resource
29. Miao D, He B, Karaplis AC, Goltzman D. Parathyroid hormone is essential for normal fetal bone formation. J Clin Invest, 2002; 109: 1173–82.
Find This Resource
30. Miao D, He B, Lanske B, et al. Skeletal abnormalities in Pth-null mice are influenced by dietary calcium. Endocrinology, 2004; 145: 2046–53.
Find This Resource
31. Xue Y, Karaplis AC, Hendy GN, Goltzman D, Miao D. Genetic models show that parathyroid hormone and 1,25-dihydroxyvitamin D3 play distinct and synergistic roles in postnatal mineral ion homeostasis and skeletal development. Hum Mol Genet, 2005; 14: 1515–28.
Find This Resource
32. Goltzman D. Interactions of PTH and PTHrP with the PTH/PTHrP receptor and with downstream signaling pathways: exceptions that provide the rules. J Bone Miner Res, 1999; 14: 173–7.
Find This Resource
33. Mannstadt M, Jüppner H, Gardella TJ. Receptors for PTH and PTHrP: their biological importance and functional properties. Am J Physiol, 1999; 277: F665–75.
Find This Resource
34. Weinman EJ, Hall RA, Friedman PA, Liu-Chen LY, Shenolikar S. The association of NHERF adaptor proteins with G protein-coupled receptors and receptor tyrosine kinases. Ann Rev Physiol, 2006; 68: 491–505.
Find This Resource
35. Sneddon WB, Syme CA, Bisello A, Magyar CE, Rochdi MD, Parent JL, et al. Activation independent parathyroid hormone receptor internalization is regulated by NHERF1 (EBP50). J Biol Chem, 2003; 278: 43787–96.
Find This Resource
36. Mahon MJ, Donowitz M, Yun CC, Segre GV. Na+/H+ exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling. Nature, 2002; 417: 858–61.
Find This Resource
37. Miao D, Su H, He B, Gao J, Xia Q, Zhu M, et al. Severe growth retardation and early lethality in mice lacking the nuclear localization sequence and C-terminus of PTH-related protein. Proc Natl Acad Sci U S A, 2008; 105: 20309–14.
Find This Resource
38. Usdin TB, Gruber C, Bonner TI. Identification and functional expression of a receptor selectively recognizing parathyroid hormone, the PTH2 receptor. J Biol Chem, 1995; 270: 15455–8.
Find This Resource
39. Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, et al. PTH/PTHrP receptor in early development and Indian Hedgehog-regulated bone growth. Science, 1996; 273: 663–6.
Find This Resource
40. Decker E, Stellzig-Eisenhauer A, Fiebig BS, Rau C, Kress W, Saar K, et al. PTHR1 loss-of-function mutations in familial, nonsyndromic primary failure of tooth eruption. Am J Hum Genet, 2008; 83: 781–6.
Find This Resource
41. Couvineau A, Wouters V, Bertrand G, Rouyer C, Gérard B, Boon LM, et al. PTHR1 mutations associated with Ollier diseases result in receptor loss of function. Hum Mol Genet, 2008; 17: 2766–75.
Find This Resource
42. Bastepe M. The GNAS locus and pseudohypoparathyroidism. Adv Exp Med Biol, 2008; 626: 27–40.
Find This Resource
43. Juppner H, Schipani E, Bastepe M, Cole DE, Lawson ML, Mannstadt M, et al. The gene responsible for pseudohypoparathyroidism type 1b is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3. Proc Natl Acad Sci U S A, 1998; 95: 11798–803.
Find This Resource
44. Yu S, Yu D, Lee E, Eckhaus M, Lee R, Corria Z, et al. Variable and tissue-specific hormone resistance in heterotrimeric Gs protein α-subunit (Gsα) knockout mice is due to tissue-specific imprinting of the Gsα gene. Proc Natl Acad Sci U S A, 1998; 95: 8715–20.
Find This Resource
45. Bastepe M, Frolich LF, Hendy GN, Indridason OS, Josse RG, Koshiyama H, et al. Autosomal pseudohypoparathyroidism type 1b is associated with a heterozygous microdeletion that likely disrupts a putative imprinting control element of GNAS. J Clin Invest, 2003; 112: 1255–63.
Find This Resource
46. Karim Z, Gérard B, Bakouh N, Alili R, Leroy C, Beck L, et al. NHERF1 mutations and responsiveness of renal parathyroid hormone. N Engl J Med, 2008; 359: 1128–35.
Find This Resource