Alternative Gene Product—CGRP

The CT gene transcript actually encodes a second distinct peptide known as CT gene-related peptide (CGRP), which is produced by tissue-specific alternative splicing of the gene (Fig. 2-3). The mature CGRP and CT mRNAs predict proteins that share sequence identity in the aminoterminal regions, but in the carboxyterminal regions the nucleotide sequences are almost entirely different. The mature, secreted 32 and 37 amino acid CT and CGRP peptides, respectively, result from cleavage of both aminoterminal and carboxyterminal flanking sequences at specific cleavage sites, as depicted in Fig. 2-3.²⁴ CT mRNA is found largely in the thyroid, and CGRP mRNA is found primarily in the nervous system.²⁵ However, aberrant expression of CGRP may be seen in medullary thyroid carcinoma.²⁶ Two different CT/CGRP genes, α and β, have been identified in man and rat.

Processing of the pre-mRNA to the CT mRNA transcript involves usage of exon 4 as a 3′-terminal exon with concomitant polyadenylation at the end of exon 4. Processing to produce the CGRP mRNA involves the exclusion of exon 4 and direct ligation of exon 3 to exon 5, with polyadenylation at the end of exon 6. The hCT/CGRP exon 4, like many differentially incorporated exons, has been characterized as having weak processing signals. Weak differential exons are frequently associated with special enhancer sequences that facilitate exon recognition in the presence of accessory factors that bind to the enhancer. Indeed, such an enhancer, located in the intron downstream of exon 4, has been described for the CT/CGRP gene. In addition, sequences within exon 4 are necessary for the inclusion of exon 4.²⁷

Calcitonin Actions In Bone

New Understanding of The Role of Calcitonin In Bone: Studies In Genetically Manipulated Mice

The preceding discussion of the physiology of CT and its action on bone reflects views that have remained largely unaltered over many years, with few new data to change them. The data on which they are based, particularly since normal and low circulating CT levels cannot be measured with confidence (vide supra), do not provide a convincing argument for a specific physiologic role for calcitonin. Indeed, in a recent review, one of the co-discoverers of calcitonin⁵⁹ argued the case that calcitonin is not involved in calcium homeostasis or in any other important physiologic function, except possibly in protection of the skeleton under conditions of calcium stress. Some recent work has changed this situation. Ablation of the CT/CGRP gene in mice resulted in viable and fertile mice with no production of CT or CGRPα.³² As expected, these mice were much less able than wild-type mice to overcome the hypercalcemia induced by a calcium load,³² and they lost excessive bone during lactation.³³ The great surprise with these mice, however, was that they had increased bone mass, with histomorphometric parameters showing increased bone formation.³² This suggested that a normal role for calcitonin might be that of an inhibitor of bone formation. The finding was unexpected and counterintuitive, and it was possible that the dual ablation of CT and CGRPα might explain it, although there was no obvious mechanism for this. Indeed, the increased bone formation phenotype was not found when CGRPα-deficient mice were examined,⁶⁰ thereby making calcitonin deficiency likely responsible for the increased bone formation. In reviewing the role of calcitonin, Hirsch⁵⁹ had considered the results obtained with the CT/CGRP−/− mice but regarded them as inconclusive; as information accumulates, the new physiology of calcitonin is becoming more apparent. The CT/CGRP-deficient mice were protected against ovariectomy-induced bone loss,^32,60 and, it was striking that after the age of 6 months these mice showed severe cortical porosity, even though indices of increased bone formation were maintained.^61,62 Taken together, the observations are indicative of an inhibitory effect of calcitonin on bone formation, most likely an indirect one, and a direct effect on bone resorption through action on the osteoclast.

The significance and importance of these findings were enhanced however with the outcome of the studies of Dacquin et al⁶³ in mice in which the CT receptor (CTR) was genetically manipulated. CTR−/− mice were embryologically lethal, and this was thought to be due to a placental effect. The CTR+/− mice, however, exhibited a bone phenotype virtually indistinguishable from the CT/CGRP−/− mice, with increased bone mass and increased bone formation on histomorphometry. Thus, the conclusion is that in mice the removal of calcitonin production or action results in an increased amount of bone, implying a physiologic role for calcitonin as a tonic inhibitor of bone formation. The same group prepared mice at least 94% deficient in the CTR and again found evidence of increased indices of bone formation.⁶⁴ How the effect on bone formation comes about remains to be determined. The lack of evidence for specific calcitonin receptors and responses in osteoblasts is compelling, so it is very likely that the physiologic roles of calcitonin in bone are brought about through two pathways: a direct effect on osteoclasts to inhibit resorption and an indirect one resulting in the elaboration of a critical, locally active factor that is necessary for bone formation. This could result from signals through receptors in the osteoclast or in the hypothalamus.^62,65 Resolution of this question will be a matter of very great interest, especially in light of ample recent evidence for central regulation of bone metabolism.⁶⁶

Renal Actions of Calcitonin

When infused into thyroparathyroidectomized rats, CT caused a dose-dependent phosphaturia, but the effect on phosphate excretion was only a minor one in comparison with the phosphaturic effect of PTH.⁶⁷ Although this was demonstrated in human subjects also, in several species CT failed to have any effect on phosphate excretion. Thus, it seems unlikely that the phosphaturic effect is of any major physiologic significance.

A number of other renal effects of CT, including a transient increase in calcium excretion due probably to inhibition of renal tubular calcium reabsorption, have been noted.⁶⁸ Although this has not usually been regarded as an important effect of CT, it has been linked to the calcium-lowering effect of CT in hypercalcemic patients with metastatic bone disease. The use of CT in the treatment of hypercalcemia due to cancer has been based exclusively on the inhibition of osteolysis by CT. Some evidence has been produced that failure of the kidneys to excrete the calcium load derived from bone breakdown is a major contributor to the hypercalcemia. This has prompted careful study of the relative contributions to the hypocalcemic effect of CT of its renal and skeletal components. It was concluded that inhibition of renal tubular reabsorption by CT can induce a rapid fall in serum calcium, and that the magnitude of this effect depends upon the correction of volume depletion, which inevitably accompanies hypercalcemia.⁶⁹ Thus, the calciuretic action of CT may assume greater importance than was hitherto suspected.

CT receptors are present in rat kidney,⁷⁰ and the action of CT upon adenylate cyclase activity has been localized in the human nephron, predominantly to the medullary and cortical portions of the thick ascending limb and to the early portion of the distal convoluted tubule. The co-localization of the CT receptor mRNA expression and cell surface receptors with G protein–sensitive adenylate cyclase is consistent with cAMP being an important mediator of CT action in this organ. A possible role for CT in the kidney is to regulate 1,25(OH)₂D₃ levels, with an original observation of enhanced 1-hydroxylation of 25(OH)D in the proximal straight tubule of the kidney by CT stimulation of the expression of 25(OH)D 1α-hydroxylase.⁷¹ Subsequently, Shinki et al⁷² showed that CT administration to rats induced renal CYP27B1 when serum calcium levels were normal or high, and this was supported by a report that CT treatment in rats increased renal production of CYP27B1 mRNA.⁷³ CT stimulation of the expression of CYP24 in CTR-transfected HEK-293 cells has also been reported.⁷⁴ The authors speculated that, since 1,25(OH)₂D₃ and CT synergistically stimulate CYP24 mRNA production in kidney cells, this latter action of CT could be part of the process by which it regulates serum calcium by controlling renal production of 1,25(OH)₂D₃.

Receptor Distribution

Direct evidence for binding of CT to osteoclasts was obtained from the work of Nicholson et al,³⁸ who used in vitro autoradiography to demonstrate ¹²⁵I-sCT binding specifically to osteoclasts and their precursor cells. The CT receptor became a required phenotypic feature to establish cells as osteoclasts.¹⁰⁰ Radioligand binding studies in tissue sections, membranes, and cultured cells have revealed an extensive extraskeletal distribution of CTRs. These sites include kidney, brain, pituitary, placenta, testis and spermatozoa, lung, and lymphocytes, as well as cancer-derived cells from lung, breast, pituitary, and embryonal carcinoma.⁷⁷

The centrally mediated actions of CT correlate well with the locations of CT binding sites. Autoradiographic mapping revealed high binding densities associated with parts of the ventral striatum and amygdala, the hypothalamic and preoptic areas, as well as most of the circumventricular organs. High-density binding also occurs in parts of the periaqueductal gray, the reticular formation, most of the midline raphe nuclei, parabrachial nuclei, locus coeruleus, and solitary tract nucleus.92,101,102 CTR mRNA has been confirmed in many of these sites, including kidney, brain, lung, placenta, and osteoclasts, along with many cancer cell lines. However, CTR mRNA studies have also provided evidence for previously uncharacterized sites of CTR expression, including prostate, normal and neoplastic breast, and thyroid.⁷⁷ As was discussed earlier, there is no evidence for CTR expression in osteoblasts.

As is discussed later, CT and amylin receptors derive from the same gene product, and consequently care needs to be taken in extrapolating the potential significance of CTR mRNA expression for CT biology. The same is also true for receptor localization studies where ¹²⁵I-sCT is used as the radioligand.

Receptor Cloning

Our knowledge of the molecular basis of CT action, in terms of both ligand binding and postbinding events, was greatly augmented by the molecular cloning of CT receptors. The first cloned receptor was the porcine CTR, which was isolated by expression cloning from a cDNA library derived from the renal epithelial cell line, LLC-PK1.¹⁰³ Subsequently, the sequences of CTR genes from man, rat, mouse, rabbit, guinea pig, and several species of fish have been reported, and the phylogenetic relationships between CTRs of mammalian and nonmammalian species have been well summarized.¹⁰⁴ Analysis of the predicted protein translation product(s) revealed that these receptors comprise approximately 500 amino acids and belong to the class II (family B) subclass of G protein–coupled receptors, which include the receptors for other peptide hormones such as secretin, PTH, glucagon, glucagon-like peptide 1, vasoactive intestinal polypeptide, pituitary adenylate cyclase–activating peptide, and gastric inhibitory peptide.

Receptor Isoforms

Receptor cloning also provided direct evidence for receptor heterogeneity and the existence of multiple receptor isoforms that arise from alternative splicing of the CTR mRNA primary transcript. The human receptor, of which there are at least 5 splice variants, is the most extensively studied. The most common hCTR splice variant occurs in intracellular domain 1, generating a 16 amino acid insert in this domain (I₁₊).105–107 These are now termed CTa (insert negative) and CTb (insert positive).¹⁰⁸ Additional splice variants have been identified in other species. In rodents, alternate splicing leads to two receptor isoforms (these have previously been termed C1a and C1b), which differ by the presence (C1b; E₂₊) or absence (C1a) of an additional 37 amino acids in the second extracellular domain. However, it is unlikely that expression of the rodent C1b isoform occurs in humans. In rabbits, an additional splice variant, in which the exon encoding transmembrane domain 7 is spliced out (ΔTM7; delta e13), has been isolated.

Investigation of the significance of the splice variants revealed that inserts or deletions in the CTR structure lead to alterations in ligand binding (E₂₊, Δamino47), signal transduction (I₁₊), or both (ΔTM7). For the E₂₊ variants, there is a loss of affinity for peptides exhibiting weak α-helical secondary structure, such as human CT, while peptides with strong helical secondary structure, such as salmon CT, maintain high affinity at this receptor.¹⁰⁹ In contrast, the CTb (I₁₊) receptor isoform displays similar affinity to the CTa (I₁₋) variant for CT-like peptides but has markedly altered G protein–coupling efficiency. The presence of the 16 amino acid insert leads to complete loss of intracellular calcium mobilization. Activation by calcitonin of its receptor can result in coupling to multiple different G proteins (see Fig. 2-6). The effect on signaling via Gs is cell type dependent but overall is maintained with decreased efficiency, leading to a 10- to 100-fold reduction in the potency of peptides for stimulation of cAMP production.¹⁰⁶ It is interesting to speculate about the functional significance of the different receptor isoforms, particularly those with apparently impaired signaling capacity. Accumulating evidence suggests that G protein–coupled receptors can form homodimers and heterodimers, and that these interactions can cross-modulate the activity of the partners. Indeed, in the case of the rabbit CTR (delta e13), Seck et al^110,111 reported that this isoform can complex with the CTa isoform and inhibit its cell surface expression and activity, thus exerting an endogenous dominant negative effect on CTR signaling.

Receptor Gene

The CTR gene, like the genes for other class II G protein–coupled receptors, is complex, comprising at least 14 exons with introns ranging in size from 78 nucleotides to >20,000 nucleotides. The total receptor gene is estimated to exceed 70 kb in length.¹¹² The organization and size of the human gene are similar to the pig CTR gene, although with some interspecies differences in organization. For instance, in the pig the I1 insert is generated by selective use of alternate splice sites located in exon 8. However, in humans, the I1 insert occurs on a separate exon, with at least one additional exon proximal to exon 7 in the pig.¹⁰⁶ Isolation of the mouse CTR gene¹¹³ revealed that the locations of introns within the coding region of the mCTR gene (exons E3-E14) are identical to those of the porcine and human CTR genes. The predicted structure of the mefugu fish gene is more complex than the human gene, with an additional nine exons.¹⁰⁴ It is interesting to note that both fish and mammalian genes code for micro-RNA (miR)-489 in intron 3, the function of which remains to be determined.

The regulatory portion of the gene contains three putative promoters (P1, P2, and P3), giving rise to multiple CTR isoforms, which differ in the 5′ region of the gene and generate 5′-untranslated regions of very different lengths, in a tissue specific manner.¹¹³ Promoters P1 and P2 are utilized in osteoclasts, brain, and kidney, and the proximal promoter of the human CTR (hCTRP1) was transcriptionally active in all cell lines tested, with high-level activity dependent on an 11 bp Sp1/Sp3 binding site.¹¹⁴ In contrast, promoter P3 appears to be osteoclast specific and is sensitive to the osteoclast-inducing cytokine, RANKL, as well as the RANKL-induced transcription factor, NFATc1, consistent with a role for the latter in regulating the CTR gene in osteoclasts.¹¹⁵ The human CTR gene is located on chromosome 7 at 7q21.3. In the mouse it is in the proximal region of chromosome 6, while the pig gene is located in chromosomal band 9q11-12. These pig and mouse chromosomal regions are homologous to 7q in humans. It is interesting to note that the mouse CTR gene is expressed preferentially from the maternal allele in brain, with no allelic bias detected in other tissues, indicating that the mouse CTR gene is imprinted in a tissue-specific manner.¹¹⁶

Receptor Polymorphisms and Osteoporosis

An interesting, but as yet unconfirmed, report describes an association between the expression of the CTa receptor mRNA in circulating monocytes of postmenopausal, but not premenopausal, women and their serum levels of bone alkaline phosphatase and urinary deoxypyridinolone.¹¹⁷ The authors therefore suggested a link between CTR expression and increased bone resorption postmenopausally.

Restriction fragment length polymorphism (RFLP) studies have identified a polymorphism within the CTR gene coding sequence; Nakamura et al,¹¹⁸ using the Alu I restriction enzyme, identified a polymorphism arising from a single nucleotide substitution, which leads to either a proline (CC genotype) or a leucine (TT genotype) at amino acid 447 in the human CTa receptor (amino acid 463 of the hCTb receptor), with heterozygotes designated TC. In this Japanese population, the proline heterozygote was the most prevalent (≈70%), with the leucine homozygote accounting for ≈10% of the population and the heterozygote for ≈20%. Additional analyses across different ethnic groups have revealed that the Leu homozygote is most prevalent in Caucasians, with decreased prevalence in African Americans and Hispanics and very low frequency in Asians (0% to 10%).^119–122

The influence of the polymorphism on bone mineral density (BMD) has been studied by multiple investigators. Most studies have identified significant correlations between CTR genotype and osteoporotic markers (BMD, fracture risk); however there is considerable divergence among studies in regard to which genotype is more favorable. High incidence of the T allele (either Leu homozygotes or heterozygotes) has been correlated with both increased BMD119,121,123 and decreased BMD.^119,124,125 The underlying basis for this divergence is unclear. It may be due to linkage disequilibrium of the CTR polymorphism with other genes involved in bone homeostasis. Alternatively, it may be related to the complex nature of action of CT and amylin (likely utilizing the same core receptor) alluded to above from gene knockout studies.⁶³ This work indicates that CT can affect both bone formation and resorption, while amylin may be important for osteoclastogenesis.

The CTR genotype has also been linked to body weight in premenopausal Japanese women,⁸⁷ and to incidence of kidney stones.¹²⁶ To date, in vitro studies have not identified significant functional differences in ligand binding or cAMP generation between the two polymorphic variants.¹²⁷ A large number of other, probably silent, polymorphisms in the hCTR, whose frequency varies among ethnic groups, have been identified.¹²⁷

Amylin and Calcitonin Gene-Related Peptide Receptors

Recent work revealed that the CT receptor–like receptor (CLR), which has highest homology with the cloned CT receptor, is a CGRP receptor. Functionally, however, this receptor requires the coexpression of a novel protein, termed receptor activity modifying protein 1 (RAMP 1). RAMP 1 is a member of a family of three single transmembrane proteins and acts to modify the glycosylation of CLR, enhance the trafficking of the receptor protein to the cell surface and contributing to the cell surface ligand binding and specificity of the receptor. RAMP 2 and RAMP 3 also enhance the trafficking of CLR to the cell surface; association of CLR with RAMP 2 and RAMP 3 gives rise to adrenomedullin-like receptors.128,129 It has been demonstrated^80,81 that the molecular identity of amylin receptors was also founded on RAMP-based hetero-oligomers. In this case, the RAMPs interacted with the CTR to form distinct amylin receptor phenotypes, depending on which RAMP was complexed with the receptor (see Fig. 2-6). It is intriguing that the CTR/RAMP 1 complex, in addition to being a high-affinity amylin receptor, potently interacts with CGRP.⁸⁰ This behavior may contribute to some of the heterogeneity seen in CGRP receptor analyses. Unlike CLR, CTRs do not require RAMPs to translocate to the cell surface and exhibit classic CTR phenotype under these conditions. Both RAMPs and receptors are subject to dynamic regulation, although in the case of CLR-based receptors, RAMPs often appear to be the major component regulated.^82,130

Signaling

Usage of the cAMP pathway in mediating CT action in osteoclasts was discussed earlier. CT also stimulates adenylate cyclase activity in the kidney, with the pattern of CT responsiveness paralleling the distribution of CTRs in this tissue. CT induction of cAMP has now been documented in a large number of cultured CTR-bearing cells that include LLC-PK1 pig kidney cells, and cancers of lung, breast, and bone. Receptor cloning and expression studies have confirmed that cAMP production is an important component of CTR-mediated signaling.103,105,106

It is now apparent that G protein–coupled receptors can interact with and signal through multiple G proteins (Fig. 2-7). Thus, CT action in osteoclasts is probably regulated by alternate signaling pathways, in apparently species-specific ways. In mice, this response appears to be mediated predominantly by the protein kinase A pathway,^131,132 although inhibition of osteoclast-mediated bone resorption by CT can be mimicked by both dibutyryl cAMP and phorbol esters or blocked by protein kinase inhibitors.¹³³ Coupling of the CTR to G_q can also activate phospholipase C, leading to increased cytosolic calcium and triphosphate levels and activation of protein kinase C.¹³⁴ Thus, in human osteoclasts, activation of protein kinase C pathways appears to be the predominant mechanism of CT-mediated osteoclast inhibition,³⁹ although PKA may be more important in human odontoclasts.¹³⁵

Calcitonin induction of interleukin (IL)-6 production in pituitary folliculo-stellate cells required both PKA and PKC signaling pathways.¹³⁶ In hepatocytes, even at very low concentrations, CT is capable of increasing cytosolic calcium.¹³⁷ CT-induced differentiation of early rat embryos is dependent upon intracellular calcium mobilization.¹³⁸ It is interesting to note that although CT inhibits proton extrusion from osteoclasts,¹³⁹ it can increase H⁺ efflux from nonosteoclastic cells, in a PKC-dependent manner.¹⁴⁰ The significance of this activity is not clear. CT-induced changes mediated by cAMP or intracellular calcium in LLC-PK1 pig kidney cells are cell cycle dependent.¹⁴¹ Expression of cloned receptors in a variety of cell types has now conclusively shown that CT receptors of human, rat, and porcine origin are capable of signaling through both cAMP and calcium-activated second messenger systems. It is important to note that comparison of the calcium response in cell lines expressing different CT receptor levels has suggested that the magnitude of the response is proportional to the receptor density, and it is therefore possible that relative receptor density in target tissues may influence the signaling pathway(s) activated.

CT treatment of CTR-bearing cells, in the presence of extracellular calcium, initiates a sustained rise in intracellular calcium, the extent of which is dependent on the concentration of the extracellular calcium and is proportional to the receptor density.¹⁴² Because osteoclasts, which express high levels of CTR, are reportedly exposed to calcium concentrations as high as 26 mM during bone resorption,¹⁴³ this phenomenon may have particular relevance for this cell type. CT and extracellular calcium both can cause intracellular calcium transients in isolated osteoclasts, and each agent greatly augments the signal produced by either agent alone.¹⁴⁴

CTR-mediated activation of the MAPK pathway has been described,145–147 with a role for both Gq and Gi/o proteins—the latter principally via βγ G protein subunits, as is commonly the case for other G protein–coupled receptors. There is also evidence that CT can influence cell attachment by modulating components of focal adhesions and the cytoskeleton.¹⁴⁶ CT treatment of osteoclasts disrupts the F-actin ring that is thought to correspond to the sealing zone^64,148 and increases the tyrosine phosphorylation of paxillin and FAK. In rabbit osteoclasts, these actions were mediated by PKC.¹⁴⁶ These actions of CT, together with decreased osteoclast motility, cytosol retraction, and disassembly of podosomes, have been linked to modulation of the activity and intracellular localization of Pyk2 and Src in osteoclasts.¹⁴⁹ It has also been shown, in nonosteoclastic cells, that CT can cause cell death by aniokis, as the result of loss of cell attachment.¹⁵⁰

It is interesting to note that recent work suggests that coexpression of RAMPs with the CTR leads to pathway-specific modulation of coupling, with strong augmentation of amylin-mediated Gs-coupling, but little effect on Gq- or Gi- mediated signaling, at least for RAMP 1– or RAMP 3–coupled receptors.¹⁵¹

Receptor Regulation

Regulation of the level and/or affinity of cell surface receptors is a key component in the physiologic and pathophysiologic responses to both endogenous and pharmacologically administered agents. The CTR is subject to both homologous (CT-induced) and heterologous regulation. CT-induced CTR downregulation was initially demonstrated in various transformed cell lines and subsequently in primary kidney cell cultures. The induction by CT of resistance to its own action in osteoclasts is such a specific process and correlates so closely in dose dependence to CT efficacy as an agonist,⁴⁸ that it appears to be an important accompaniment of CT action. Proof of this might require genetic experiments, just as genetic manipulation has finally begun to cast light on the physiologic functions of CT.^32,63

Receptor downregulation is mediated by specific loss of cell surface receptors, which occurs via an energy-dependent internalization of the ligand-receptor complex, in which the principal internalization pathway involves processing of the receptor-ligand complex into lysosomes and subsequent degradation of the receptor.¹⁵² Receptor regulatory responses to CT are likely to be cell or tissue dependent. For example, in mouse or rat osteoclasts, a potent downregulation of CTR mRNA appears to be mediated by a cAMP-dependent mechanism, in addition to downregulation of the receptor by internalization.¹⁵³ Similar CT-mediated CTR regulation is also observed in human osteoclasts, but, as stated above, activation of protein kinase C pathways is suggested to be the predominant mechanism of CT-mediated osteoclast inhibition in human osteoclasts.³⁹ In contrast to these observations in osteoclasts, downregulation of CTRs in UMR106-06 cells and T47D cells is not accompanied by changes in CTR mRNA levels.¹¹³ The CT-induced receptor mRNA loss in osteoclasts appears to be due principally to destabilization of receptor mRNA.⁴⁶ The 3′-untranslated region of the mouse and rat CTR mRNA contains four AUUUA motifs, as well as other A/U-rich domains, which can function as signals for rapid mRNA inactivation. Thus, addition of A/U-rich CTR 3′UTR sequences considerably shortens the mRNA half-life of a reporter gene, and evidence shows the involvement of AUF1 p40, HuR in the regulation of CTR mRNA.¹⁵⁴ It is also worth noting that the degree of internalization of human CTRs appears to be isoform specific, with the I₁₊ variant being resistant to internalization.¹⁰⁶ Thus, the regulation of receptors and consequently peptide responses may vary according to the levels of specific receptor isoforms present in each tissue.

Data on regulation of CTR by other agents are limited. In mouse osteoclast cultures, the glucocorticoid dexamethasone increased the level of cell surface CTR following upregulation of receptor mRNA levels, the latter mediated at the level of transcription.⁴⁶ Moreover, the CT-mediated decrease in cell surface receptor and mRNA was attenuated by dexamethasone. Increased production of CTR in response to glucocorticoid stimulation also occurs in the human T47D cell line, where cortisol is required for the expression of CTRs, suggesting that this may be a common regulatory mechanism for induction of CTR expression. It is worth noting the clinical evidence suggesting that glucocorticoids, given together with CT, might prevent to some extent the CT-induced resistance to its own action.¹⁵⁵

Medullary Carcinoma of The Thyroid

The classic syndrome of CT excess is medullary carcinoma of the thyroid (MCT). This tumor clearly differs in origin from all other thyroid cancers, since it is a tumor of the C cells, derived from the ultimobranchial bodies. MCT may occur as a sporadic or a genetic (familial) tumor.156–160 The only effective treatment for MCT is surgical. The earlier this can be performed in the course of this disease, the better is the likelihood of a cure. Once the tumor becomes palpable, any treatment is unlikely to be curative. Genetic testing for the locus of mutation in the responsible gene (the RET proto-oncogene) often has considerable clinical relevance in management, as early surgery is key to survival, especially in the syndrome known as multiple endocrine neoplasia type 2b (MEN-2b).

Calcitonin Receptors In Other Cancers

Specific high-affinity receptors for CT have been demonstrated in human lymphoid cell lines, in a human lung cancer cell line, in several human breast cancer cell lines, including MCF-7, T47D, and ZR-75, and in prostate cancer cells.152,153,161,162 Indeed, CTR mRNA appears to be a frequent accompaniment of human tumors. The first clinical study of CT receptors in surgically obtained human breast cancers identified receptor mRNA production in all of 18 cancers by reverse transcription/polymerase chain reaction (PCR).¹⁶³

In situ hybridization in the same study showed that CT receptor expression was confined to tumor cells in the sections. In contrast, a subsequent study, using laser capture microdissection and PCR, found that the CTR in breast cancer tissue was actually reduced compared with adjacent normal breast tissue.¹⁶⁴ The significance of the CTR in cancer cells is not known; however forced expression of human CT in LNCaP human prostate cancer cells was reported to dramatically enhance their oncogenic characteristics,¹⁶⁵ and CT is expressed endogenously in the prostate.¹⁶⁶ The same group also showed that CT increased hallmarks of invasiveness of prostate cancer cells, including the concentration and activity of MMP-2 and MMP-9.¹⁶⁷ Similar results have been reported for MDA-MB-231 human breast cancer cells.¹⁶⁸

Hypercalcemia

Calcitonin has been used in the treatment of hypercalcemia in many different conditions, especially malignancy, but its effective use is limited by rapid tachyphylaxis, often within 24 hours of use.¹⁷⁷ The mechanism of resistance seems to involve receptor downregulation, which to some extent is blocked by corticosteroid use (see Receptor Regulation earlier).¹⁵⁵

Osteoporosis and Paget’s Disease of Bone

The discussions in this chapter of the mechanism of action of CT provide some background for the use of CT in therapy. One of the most important unanswered questions concerning CT is whether its role as an inhibitor of bone resorption is such that CT deficiency can contribute to the development of osteoporosis. It is the antiresorptive action of CT that is the basis for its use clinically in osteoporosis, for which it is approved in several countries. Although its use remains somewhat controversial, CT administered by injection or by nasal spray has been found to decrease vertebral fracture risk in postmenopausal osteoporotic women.178,179 A reported advantage of calcitonin is its analgesic effect on bone pain, which probably is mediated centrally¹⁷⁹; CT treatment has modest effects on bone quantity (BMD) or bone turnover markers, but new evidence suggests improvement in bone quality parameters after administration of salmon CT nasal spray over 2 years. The results of the QUEST study, in which effects of CT on bone structure were assessed by high-resolution MRI, suggest therapeutic benefit of CT-NS compared with placebo in maintaining trabecular microarchitecture at multiple skeletal sites.¹⁸⁰ Similar protection by CT of bone structure was obtained in ovariectomized sheep, again with MRI used to assess bone changes.¹⁸¹ A problem with CT use clinically is its bioavailability, and attempts are now being made to produce orally bioavailable CT, with promising results.^182,183

CT given by subcutaneous injection was recognized as effective in the treatment of Paget’s disease, with relief of pain, return of biochemical indices of disease activity toward normal, formation of normal lamellar bone, and filling in of osteolytic lesions. In long-term treatment of Paget’s disease with CT, the number of osteoclasts declines progressively, either because of a direct effect on osteoclast precursors or as an indirect consequence of the acute inhibition of osteoclast function by CT. In Paget’s disease, CT needs to be given by injection, since intranasal delivery is relatively ineffective, and its use generally has been supplanted by bisphosphonates, as is reviewed elsewhere in this book.

Osteoarthritis

A number of recent studies have suggested the potential benefit of CT in osteoarthritis. In both in vitro and in vivo animal models,184,185 CT was found to attenuate the indices of progression of osteoarthritis, including degradation of collagen type II, a hallmark of articular cartilage damage. In humans, CT treatment reduced circulating CTX-II¹⁸⁶ and improved functional disability in patients, albeit in a small test group, selected for active disease.¹⁸⁷ The mechanism of action of CT in this putative chondroprotection is not clear. It has been claimed, on the one hand, that CT has direct effects on bovine articular chondrocytes via the CTR,¹⁸⁸ but a recent study was unable to find CTR expression or responsiveness to CT in human chondrocytes.¹⁸⁹ The findings are nonetheless intriguing and benefits could result from the effects of CT on subchondral bone, as have been described for bisphosphonate treatment of animal models of osteoarthritis.¹⁹⁰ Potential benefits of CT have also been suggested for inflammatory arthritis, since it was found to preserve bone morphology in a rat model of rheumatoid arthritis, particularly when used with prednisolone.¹⁹¹

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