Molecular characterization of two novel isoforms of the human calcitonin receptor.

Calcitonin inhibits bone resorption by acting on osteoclasts via a specific receptor. The calcitonin receptor (CTR) is also found in many other normal and malignant tissues and cell lines. It has been cloned and sequenced in several species including humans. It belongs to a subclass of seven-transmembrane G protein-coupled receptors. Four human CTR (H-CTR) isoforms generated by alternatively spliced mRNA have previously been described. Two H-CTR encoding DNAs containing an unidentified 50-bp insert are now reported from T47D cells. The 50-bp insert corresponds to a DNA region located between exon 9 and exon 10, and appears to originate from an alternative splicing process. The two H-CTR cDNAs encode 274 and 290 aa long isoforms. Both are deleted from the putative fourth transmembrane domain to C-tail. They differ by the presence (H-CTR5) or absence (H-CTR6) of a previously known 16-aa insert in the putative first intracellular loop. Cell- and tissue-distribution analysis using RT-PCR demonstrates that the shorter one, HCTR6, is more prevalent. The mRNA of both isoforms was detected in giant cell tumor, whereas only H-CTR6 mRNA was detected in TT cells and kidney tissue. Neither H-CTR5 nor H-CTR6 could be detected in peripheral blood mononuclear cells cultured in the presence of RANKL, in MCF7 cells, and in cortical brain and ovarian tissues. When H-CTR6 was transiently expressed in HEK293 cells, CT failed to induce production of cAMP or to bind to the receptor. These suggest either an intrinsic loss of ligand binding function, or an altered intracellular trafficking. Our findings therefore indicate the existence of two novel splice variants of the H-CTR and confirm that multiple splicing patterns could be involved in the post-transcriptional regulation of the gene.

Calcitonin receptor mRNA in mononuclear leucocytes from postmenopausal women: decrease during osteoporosis and link to bone markers with specific isoform involvement.

Calcitonin inhibits bone resorption via its receptor (CTR) on osteoclasts. Two hCTR isoforms, hCTR1 and hCTR2, give proteins that differ in their structure and signaling pathways. We investigated whether specific isoforms or quantitative changes in total hCTR mRNA were associated with high bone resorption and turnover in menopause or osteoporosis. The hCTR mRNA in mononuclear blood cells of premenopausal (PreM), healthy (PostM), and osteoporotic (OsteoP) postmenopausal women was assessed using reverse-transcriptase polymerase chain reaction. hCTR1 and hCTR2 were investigated for 59 total RNA samples, and semiquantitative analysis of total hCTR mRNA was performed for 71. Serum calcitonin, free urinary deoxypyridinoline (D-Pyr), serum bone alkaline phosphatase (SBAP), and osteocalcin (SOC) were also evaluated. Serum calcitonin levels did not differ in PostM and OsteoP. The prevalence of each isoform was similar in the three groups. Healthy postmenopausal women and OsteoP with hCTR2 had lower bone turnover (D-Pyr: 6.79 +/- 0.54, n = 25; SBAP: 11.63 +/- 1.47, n = 26; SOC: 8.31 +/- 0.58, n = 26) than those without hCTR2 (D-Pyr: 9.90 +/- 1.95, n = 5; SBAP: 21 +/- 5.19, n = 5; SOC: 11.9 +/- 2.10, n = 5; p < 0.05). Total hCTR mRNA levels were not different in PreM and PostM. By contrast, values were strikingly lower in OsteoP (0.57 +/- 0.17, n = 28) than in PostM (2. 25 +/- 0.61, n = 19, p < 0.05) and negatively correlated with bone markers values in both. We suggest that a specific isoform and amounts of total hCTR mRNA are linked to increased bone resorption in postmenopausal osteoporosis.

Calcitonin receptor polymorphism is associated with a decreased fracture risk in post-menopausal women.

High bone resorption by the osteoclast results in osteoporosis, a disease affecting 40% of women after the menopause. Calcitonin, used to treat osteoporosis, inhibits bone resorption via receptors located on the osteoclasts. Two alleles of the calcitonin receptor gene ( CTR ) exist: a base mutation T-->C in the third intracellular C-terminal domain changes a proline (CCG) at position 447 to a leucine (CTG). We therefore studied the distribution of these alleles in a cohort of 215 post-menopausal Caucasian women suffering or not from osteoporotic fractures. The region of interest within the point mutation was amplified by PCR and screened for single strand conformation polymorphism. This work was followed by DNA sequencing of the fragments amplified. We found that bone mineral density (BMD) at the femoral neck was significantly higher in heterozygous subjects with the Rr genotype compared with the homozygous leucine (RR) and homozygous proline (rr) genotypes. Also, a decreased fracture risk was observed in heterozygote subjects. In conclusion, our results suggest that polymorphism of CTR could be associated with osteoporotic fractures and BMD in a population of post-menopausal women. CTR heterozygotes could produce both alleles of the receptor. The heterozygous advantage effect of Rr subjects could explain their protection against osteoporosis: higher bone density and decreased fracture risk. Establishing the genotype of the CTR gene in post-menopausal women could be of value in evaluating their risk of developing fractures.

Calcitonin mRNA is produced in liver by two different splicing pathways.

Calcitonin, the hypocalcemic hypophosphatemic hormone is produced in the thyroid by the C-cells. We recently detected the presence of calcitonin and its messenger in hepatic tissue in vivo and in vitro. The calcitonin precursor is composed of the N-terminal peptide, calcitonin and a carboxyterminal peptide. We previously reported that in normal human thyroid and in medullary thyroid carcinoma a second calcitonin messenger is expressed in low quantities. This mRNA differs in its 3' region from the first one. It also codes for the N-terminal peptide, calcitonin and a carboxyterminal peptide which differs by the last eight amino acids from the first. We report here that both calcitonin mRNAs are expressed in normal or tumoral liver. Direct estimation, by a specific immunoassay, of the levels of carboxyterminal peptide II, the specific peptide coded for by calcitonin mRNA II, confirmed that this peptide is synthesized in human liver.

Retinoic acid abolishes the calcitonin gene-related peptide autocrine system in F9 teratocarcinoma cells.

Calcitonin gene-related peptide (CGRP), expressed predominantly in F9 embryonal carcinoma cells, is both a potent chemotactic agent and an autocrine growth factor for these cells. We analyzed the effect of retinoic acid (RA)-induced differentiation of F9 cells into primitive parietal endoderm-like cells, on CGRP production and the CGRP responsiveness of these cells. Poly(A) RNA extracted from F9 cells and analysed by Northern blotting and hybridization with a CGRP probe showed a specific band of about 1200 bases corresponding to mature CGRP mRNA. This band was not detected in F9 cells treated for 6 days with RA (differentiated primitive parietal endoderm-like cells) or in PYS cells (established parietal endoderm-like cell line). During RA-induced differentiation of F9 cells, CGRP mRNA levels fell within 24 h after treatment and were almost undetectable after 2 days. RA treatment also reduced CGRP secretion by F9 cells; the effect was maximal at 3 days and remained stable thereafter. Similarly, RA rapidly reduced adenylate cyclase responsiveness to chicken CGRP (cCGRP) and human CGRP (hCGRP). An 80% fall in cAMP release into the culture medium in the presence of CGRP was observed after 24 h of RA treatment. These results demonstrate that RA rapidly abolishes the CGRP autocrine system involved in the proliferation of F9 cells, at the same time inducing their differentiation into primitive parietal endoderm. They point to the interaction between retinoic acid and growth factors in the regulation of cell proliferation and differentiation.

Production of salmon calcitonin I in Oncorhynchus gorbuscha by alternative polyadenylation of two RNA species.

RNAs of ultimobranchial bodies (U.B.) from the pink salmon, Oncorhynchus gorbuscha, were studied using the polymerase chain reaction (PCR) with specific oligodeoxyribonucleotides (oligos) of the salmon calcitonin (sCT) mRNA selected in exon 2 or 3 and a poly(T) oligo. We observed two amplified DNA fragments, differing by 200 bp which hybridized with a specific exon 4 probe. Sequence analysis indicated that they both encoded exon 4, but differed in the length of their 3' non-coding regions by use of a putative polyadenylation signal situated 200 bp upstream from the established polyadenylation site. These two polyadenylation signals very likely were regulated differently, as the larger expressed transcript was predominant. To date, such use of an alternative polyadenylation signal in a CT mRNA has not been described in other vertebrates, and only the chicken CT mRNA possesses a second classical polyadenylation signal which is not known to be used. This characteristic of sCT biosynthesis appears to be typical in lower vertebrates and is of phylogenic interest. Moreover, it engenders a hypothesis of a relationship between the high concentration of the peptide observed in females of this species and their capacity to produce sCT by different biosynthetic pathways.

The calcitonin gene is expressed in salmon gills.

Calcitonin is an important physiological regulator of salmon gills. Although the calcitonin receptor was found in salmon gills, the critical question concerning the source of the hormone remained unanswered. In this communication, evidence is presented for expression of calcitonin mRNA and its encoded peptide in gills of the pink salmon, Oncorhynchus gorbuscha. The expression of calcitonin gene transcripts was demonstrated by reverse transcription-polymerase chain reaction, Southern hybridization, and sequencing. The sequencing identified a sequence corresponding to that of exon 4 of the salmon calcitonin gene. Expression of the encoded calcitonin gene in gills was detected by radioimmunoassay in gill extracts. This synthesis of calcitonin in gills, which also possess specific receptors to the peptide, suggests function of an autocrine or paracrine process producing calcitonin in this tissue. These observations confirm and extend previous reports on the physiological role of calcitonin in fish gills.

Heterogeneity of circulating calcitonin levels: relations with calcitonin biosynthesis in medullary thyroid carcinomas.

Calcitonin (CT), a hypocalcemic and hypophosphatemic hormone, is produced by the C-cells of the thyroid gland. It is the main tumoral marker of medullary thyroid carcinoma (MTC). Hypersecretion of CT is also associated with other types of tumors. Thus, heterogeneity of circulating CT can play an important role in the accurate determination of hormone levels in blood samples obtained from MTC patients. Further studies will be necessary to establish the predictive value of the several peptides coded by the calcitonin gene family. All of them specifically reflect the ways and the pattern of alternative splicing of the primary transcript of the Calc I gene. Such relations implicate further investigations concerning the relationship between calcitonin circulating levels, biosynthetic activity of C-cells and the expression of gene encoding for this hormone, in normal and neoplastic conditions.

Specific immunostaining for CCP II, a novel calcitonin carboxyl terminal peptide encoded by the calcitonin/CGRP gene.

Alternative splicing of the primary transcript of the CALC I gene in thyroid C-cells results predominantly in calcitonin (CT) mRNA (exons 1-4), whereas CGRP mRNA (exons 1, 2, 3, 5, and 6) is mainly produced in neuronal cells. The CT mRNA encodes for a protein precursor containing an amino terminal peptide, CT, and a carboxyl terminal peptide (CCP I). CGRP precursor is composed of the same amino terminal peptide and CGRP. Recently we reported the presence of a third mature transcript of the CALC I gene in human medullary thyroid carcinoma (MTC) tissues. This transcript encodes for a precursor containing the amino terminal peptide CT and a novel carboxyl terminal peptide, CCP II. This finding was further confirmed in the TT-cell line derived from a human MTC. We produced monoclonal antibodies against CCP II and developed a rapid and specific immunofluorescence method for this peptide. We demonstrated CCP II-specific immunoreactivity in TT-cells and in MTC tissues. CCP II labeling was relatively homogeneous in contrast to CT and CGRP, which presented striking heterogeneity for intensity of labeling. Therefore, CCP II mRNA is translated in tumor cells in an apparently constitutive way.

Expression of CGRP mRNAs in the pink salmon, Oncorhynchus gorbuscha.

We report the isolation of calcitonin gene-related peptide (CGRP) mRNAs and expression of these RNAs in different tissues in the pink salmon, Oncorhynchus gorbuscha. Hybridization of poly(A+) RNAs indicated a mature CGRP RNA of 1.1 kb. The CGRP-like immunoreactivity occurring in tissues and plasma had the same relative molecular weight as the synthetic molecule. Variations in CGRP plasma levels were observed during migration, spawning, and postspawning states. These data suggest that CGRP may play an important role during the reproductive cycle of salmon.

Influence of calcium and vitamin D deficient diet on calcitonin gene expression in the ultimobranchial cells of the developing chicken.

Recently hatched chickens were fed a vitamin D and calcium deficient diet for 4 weeks. Calcitonin (CT) biosynthesis in the ultimobranchial glands (UBG) was studied during the treatment by means of in situ hybridization of specific CT mRNAs and immunocytochemical detection of the CT intracellular stores. Circulating CT levels were measured by radioimmunoassay. Within 1 week of the start of treatment, the deficient animals had significantly lowered plasma calcium concentrations and a dramatic fall of plasma CT levels, but the UBGs were not much affected. From week 2 to week 4, the UBG underwent a gradual atrophy. The tissue became lacunar due to the presence of an abnormally developed cystic component. Although calcemia returned to normal at week 4, the cellular endocrine cords were dramatically reduced, corresponding to the undetectable circulating CT levels. However, the UB glands always contained persistent CT-secreting cells, mainly at the periphery of the tissue or in contact with enlarged parathyroid tissue inclusions. These endocrine UB cells contained large amounts of hybridizable CT mRNA and immunodetectable stores of the mature hormone, and their ultrastructural features were quite unaffected compared to normal ones. Thus, we conclude that, in the chicken, severe calcium malnutrition led to a striking reduction of CT biosynthesis in the UB glands by decreasing the number of secretory cells and not by triggering modifications of the biosynthetic activity of the UB endocrine cells.

Calcitonin gene-related peptide: an autocrine growth factor with regulatory activity in vitro.

We show that an autocrine system for calcitonin gene-related peptide (CGRP) exists in F9 teratocarcinoma cells. Synthesis of CGRP by F9 cells was demonstrated by measuring the peptide concentration in cells and medium and by determining specific mRNA in cells. During six days of culture, CGRP secretion did not vary significantly in the medium, while intracellular CGRP and CGRP mRNA levels increased. F9 cells contained a CGRP-sensitive adenylate cyclase system and CGRP increases the accumulation of cAMP in the culture medium. Interestingly affinity purified antibodies against CGRP specifically inhibited growth of F9 cells by 50%. CGRP therefore stimulates F9 cell growth by an autocrine process, suggesting that CGRP may be a growth factor during early embryogenesis.

CCP II: a novel calcitonin carboxy terminal peptide is expressed in normal thyroid tissue.

We have recently identified in medullary thyroid carcinoma the existence of a second calcitonin messenger, generated by a splicing between the 3' coding region of exon 4 and exon 5 of Calc I gene. It differs from the first one in its 3' coding sequence and codes for a calcitonin precursor which generates the same N terminal peptide, calcitonin and a specific 21 amino acid carboxy terminal peptide differing from Katacalcin by its 8 last amino acids. We searched for the expression of this new messenger in normal human thyroid tissue by Northern and by polymerase chain reaction techniques. This second calcitonin messenger was expressed in 4/4 normal thyroids and 4/5 medullary thyroid carcinoma tissue samples. The expression of this second messenger is apparently a common occurrence in C cells whether normal or tumoral.

A novel calcitonin carboxyl-terminal peptide produced in medullary thyroid carcinoma by alternative RNA processing of the calcitonin/calcitonin gene-related peptide gene.

The calcitonin/calcitonin gene-related peptide gene expresses two different mRNAs by tissue-specific alternative processing. The calcitonin mRNA is produced in thyroid C cells by splicing of the first three exons to the fourth polyadenylated exon. It encodes a protein precursor containing an amino-terminal peptide, calcitonin, and a carboxyl-terminal peptide (CCP I). Calcitonin gene-related peptide (CGRP) mRNA is produced in neuronal cells by splicing of the three common exons to the fifth exon and the polyadenylated sixth exon, leading to the production of a CGRP precursor. Our studies concerning the expression of the calcitonin/CGRP gene in human medullary thyroid carcinoma revealed the presence of a new RNA transcript. Amplification by polymerase chain reaction and direct sequencing showed that the novel transcript is composed of exons 1, 2, and 3, part of exon 4, exon 5, and a polyadenylated exon 6. This transcript contains an open reading frame coding for the known amino-terminal and calcitonin peptides, as well as for a novel calcitonin carboxyl-terminal peptide, CCP II. This third alternative pathway utilizes an internal donor site within the exon coding for calcitonin. The presence of CCP II was demonstrated in plasma and thyroidal tissues of medullary thyroid carcinoma patients, implying that this novel mRNA is actively translated in medullary thyroid carcinoma.

Calcitonin gene-related peptide immunoreactivity in a crustacean Nephrops norvegicus and correlation with calcitonin.

Immunoreactive calcitonin-gene-related peptide (ir-CGRP) was detected in the crustacean Nephrops norvegicus. High levels of ir-CGRP were present in the foregut and hepatopancreas (3 +/- 0.7 and 4.6 +/- 1.0 micrograms eq per 100 mg of fresh organ, respectively). Molecular sieving of acidic extracts of anterior gut of Nephrops norvegicus showed a high molecular weight immunoreactive peptide in the range 15,000 to 25,000 Da. Immunoreactivity related to salmon calcitonin was present in the high molecular weight fraction.

Expression of the calcitonin gene during migration of the Pacific salmon, Oncorhynchus gorbuscha.

Plasma calcitonin levels increase during reproduction in female salmon. Whether these changes in circulating levels of the hormone are due to increased secretion and or increased biosynthesis is not established. We measured total and poly A+ RNAs and calcitonin content of ultimobranchial bodies of male and female pink salmon during the different stages of the reproductive cycle. Calcitonin mRNAs were analysed by hybridization of northern and dot blots with a specific probe for salmon calcitonin. Dot blot autoradiography indicated that female animals had higher levels of CT mRNA than males and these values were correlated with plasma calcitonin levels. In conclusion, sexual hormones still to be identified, are probably implicated in the expression of the calcitonin gene in females during the reproductive cycle.

Distribution of calcitonin gene-related peptide and calcitonin-like immunoreactivity in trout.

Radioimmunoassay and chromatography were used to study the occurrence of calcitonin gene-related peptide in various tissues of the rainbow trout, Salmo gairdnerii. The highest concentrations of the peptide were found in gill (1.68 +/- 0.09 ng/mg protein) and in intestine (1.06 +/- 0.4 ng/mg protein). Significant concentrations were also found in heart and stomach. The level in brain was very low. In trout, the plasma concentration accounted for 283 +/- 82 pg/ml. Chromatographic analysis of the calcitonin gene-related peptide (CGRP)-like immunoreactivity occurring in gills showed that two molecular forms cross-reacted with the anti-human CGRP antibody, one co-eluting with the synthetic human CGRP. In addition, calcitonin in fish is not confined to the ultimobranchial organ but is also present in organs as heart, intestine, kidney, spleen and stomach. The evidence of CGRP in fish emphasizes the role of this hormone in evolution and leads us to investigate its physiological role in this species.

Distribution of renal calcitonin binding sites in mammalian and nonmammalian vertebrates.

Calcitonin (CT), the hypocalcemic hypophosphatemic hormone, is present in many vertebrate species. The principal target organs for CT are, in mammals, kidney and bone, and in fish, bone and gill. We have investigated the presence of renal calcitonin binding sites in a fish (Salmo gairdneri), two amphibians (Bufo bufo and Rana esculenta), two reptiles (Pseudemys scripta and Gekko gecko), and two birds (Gallus domesticus and Cothurnix japonica). We compared their distribution to a mammal, the rat (Wistar strain), using quantitative autoradiography methods. Moreover, CT binding sites were also characterized in the chicken kidney by a membrane assay. No renal 125I-sCT binding sites were detected in the fish, the amphibians, and the reptiles studied. In the rat, binding sites were present in the outer medulla and in the cortex. In the chicken and in the quail, scattered binding sites were also observed in the medulla and in the cortex, whose pattern seemed to follow the distribution of the glomeruli and/or the collecting tubules. Chicken kidney membranes were also purified by sucrose gradient centrifugation. Scatchard analysis of the binding data revealed the presence of one type of 125I-sCT binding site with an apparent dissociation constant of 4.3 nM and a number of sites of 73 fmol/mg/protein. The present results suggest that calcitonin renal receptors appeared late in evolution and thus that a regulation of renal function by calcitonin was only effective in birds.

Actinomycin D inhibits the rapid increase in translatable calcitonin mRNA provoked by acute calcium stimulation.

Calcium, injected to rats, elicits a rapid increase in translatable calcitonin mRNA, by acting probably at the post-transcriptional level, as no change in calcitonin mRNA could be detected by hybridization assay. In this study we have measured calcitonin mRNA extracted from rats subjected or not to acute hypercalcemia and pretreated or not with actinomycin D. Calcitonin mRNA was quantified by its ability to direct the synthesis of calcitonin (CT) precursors in a cell free system and by hybridization to a 32P cDNA probe specific for CT mRNA. Actinomycin D, injected 5 hours before calcium administration, decreased the incorporation of 3H adenine in liver and thyroid, but did not inhibit the rise in plasma levels of calcium and CT (measured by radioimmunoassay). The antibiotic was able to inhibit the eightfold increase in translatable mRNA elicited by calcium administration in the control animals. Hybridizable CT mRNA levels were not modified by the treatments. Thus the increase in translatable CT mRNA after calcium stimulation is independent of CT secretion and is probably due to post-transcriptional modifications involving the expression of other gene(s).

Localization of a salmon calcitonin-like molecule in one type of ultimobranchial cells in the freshwater turtle Pseudemys scripta.

Immunohistochemical methods using affinity adsorbed antibodies raised against the three families of calcitonins (CT) were applied to ultimobranchial (UB) cells in situ to investigate the nature of the Chelonian calcitonin molecule and its distribution in the ultimobranchial bodies of the freshwater turtle, Pseudemys scripta. In this species, the UB glands were present on both sides and consisted of scattered cell clumps between epithelial vesicular structures. The neighboring parathyroid tissue also contained two components, the majority being composed of similar vesicles, with occasional solid cell cords evenly distributed. Calcitonin immunoreactivity was found in the cell clumps of the UB gland and in the cell cords of the associated parathyroid, but not in the epithelial component lining the vesicles or in the amorphous material which sometimes filled the lumen. Turtle calcitonin was exclusively of the salmon type, as determined by the negative results obtained in situ after the use of antibodies raised against human and porcine molecules. The salmon-like calcitonin content of the ultimobranchial area was estimated as 15.2 ng; however, the molecule was undetectable in the circulation. In this work we localize the quantitate a salmon-like CT molecule in one type of ultimobranchial and parathyroid cell of a reptile for the first time.