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.

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.

Influences of fixatives on the immunodetection of calcitonin in mouse "C" cells during pre- and post- natal development.

The presence of calcitonin in fetal and postnatal mouse "C" cell was demonstrated by radioimmunoassays of fetal and adult thyroid extracts and by immunohistochemistry following the use of Bouin's fixative from 18 days postcoitum onwards. However, after fixation with formaldehyde no calcitonin could be detected in fetal or early postnatal samples, although it was still easily detectable in adult tissues treated with this fixative, raising the possibility that there may be age-dependent differences in the chemistry or physiological state of intracellular calcitonin. Experiments conducted with several other fixatives and the results of the radioimmunoassays suggest that the loss of calcitonin reactivity is not due to differences in the anti-genic sites of the molecule at the two stages in development. Neither can the positive detection of fetal material treated with Bouin's fixative be due to any action of this liquid. However, the inhibiting action of the aldehydes could be related to the preservation of the granule membranes in the fetal "C" cells: our ultrastructural studies show that the granules can be morphologically classified in dense-core and homogeneous matrix. Such granule membranes are abundant in the young and adult "C" cell and would be reactive, while the small dense-core granules are prevalent in the fetus and would be unreactive, confirming the hypothesis of a difference of structure of the granule membrane and not of calcitonin between fetal and late postnatal "C" cells.

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.

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.

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.

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.

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.

[Heterogeneity of human calcitonin]. / Hétérogénéité de la calcitonine humaine

Chromatographic analysis of plasma, urine and tumour tissues extracted from medullary cancer patients demonstrates the existence of immunoreactive calcitonins with higher molecular weight than that of the monomer hormone.

Calcitonin secretion during the estrous cycle of the rat.

Plasma levels of calcitonin (CT), parathyroid hormone (PTH), and estradiol (E2) were measured in the rat during the estrous cycle. Changes in the circulating levels of CT were observed, in particular a significant fall during estrus. No significant changes in PTH levels were recorded using a N terminal assay. E2 levels were not correlated with CT or PTH levels.

Purification and characterization of calcitonin receptors in rat kidney membranes by covalent cross-linking techniques.

We have characterized the binding parameters of renal receptors (Scatchard analysis revealed the presence of two binding sites: site I, Ka1 = 1.29 X 10(9) M-1, number of binding sites = 9.9 X 10(6)/micrograms protein; site II, Ka2 = 0.93 X 10(8) M-1, number of binding sites = 4.27 X 10(8)/micrograms protein) and studied the effect of solubilization. The high-affinity sites are preserved during affinity chromatography and the process results in a 6080-fold purification of those sites. The lower-affinity sites are also preserved but the overall purification factor is about 40% lower than that obtained using molecular sieving. The purification of the renal calcitonin receptor by molecular sieving (Sephacryl S-200) is accompanied by total loss of the high-affinity site; however, the low-affinity site is enriched over 1642-fold. Binding parameters were obtained for the purified fractions. Synthetic salmon calcitonin was also bound to renal membranes using the bifunctional reagent disuccinimidyl suberate and photo-affinity cross-linking using hydroxysuccinimidyl azidobenzonate reagent. Cross-linked receptor eluted in the same volume as solubilized membranes specifically binding salmon calcitonin (S-200 chromatography). Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of purified fractions showed several protein bands with apparent molecular masses ranging from 18 000 Da to 100 000 Da in the presence or absence of a reducing agent (2-mercaptoethanol). Autoradiography of polyacrylamide gels of cross-linked calcitonin receptor showed only three protein bands specifically binding salmon calcitonin. Their molecular masses were 70 000 Da, 40 000 Da and 33 000 Da respectively. The 40 000-Da molecule represents a major band (47% total binding species). This suggests that these three proteins are the principal components of the calcitonin receptor and that S-S bonds are not involved in the assembly of the receptor subunits.

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.

[Tests of stimulation of calcitonin secretion. Value in medullary cancer of the thyroid]. / Epreuves de stimulation de la sécrétion de calcitonine. Intérêt dans les cancers médullaires de la thyroïde

The detection of high circulating levels of calcitonin is a most valuable procedure to diagnose advanced cases of medullary carcinoma of the thyroid. However, the diagnosis of a primitive tumor in the early stages of development (as in cases of the familial form of the disease) or of a metastasis following ablation of the tumor is more difficult. In the latter cases, the levels of circulating calcitonin may be within normal limits, and for diagnosis one must then resort to tests to stimulate the secretion of calcitonin. We are reporting from our personal experiences the advantages, disadvantages and inconveniences of the three most well-known tests. All patients responded positively to administration of calcium or pentagastrin. The alcohol test, however, produced inconsistent results.

[Heterogeneity of immunoreactive calcitonin in the plasma of patients with bone marrow cancer]. / Hétérogénéité de la calcitonine immunoréactive dans le plasma de sujets avec cancer médullaire

Plasma from patients with medullary carcinoma containing very high levels of immunoreactive calcitonin were fractionated by filtration on Sephadex gel. In all cases the elution gave four immunoreactive fractions. Two of these fractions correspond to the volume of elution of the monomere and of the dimere of human calcitonin. The two other fractions emerge at an elution volume corresponding to much higher molecular weights. After stimulation of calcitonin secretion in vivo, by dynamic tests, the fractions corresponding to the monomere and dimere increase more strongly than the two other fractions. Preliminary studies of secretion, in vitro, of calcitonin by medullary carcinoma tissue, show the presence in the incubate of four immunoreactive forms having the same elution characteristics as those found in the plasma. The significance of these results is discussed.

Calcium regulating hormones during the estrous cycle of the rat.

Plasma levels of calcium (Ca), phosphate (P), calcitonin (CT), parathyroid hormone (PTH), and prolactin (PRL) were measured at 8, 13, and 17 hr during the 4-day estrous cycle of the rat. Ca levels fell throughout the day during proestrus (PE) and estrus (E). In contrast Ca rose transiently during diestrus (D1, D2). P levels fluctuated inconsistently at all stages of the cycle with the exception of E where P levels were significantly higher at 13 hr. CT levels showed an increase during D1 and D2 and fell to their lowest values during E, at 13 hr. Daily fluctuations in each stage were also recorded. Variations of PTH levels during the estrous cycle were minor. PRL levels increased sharply during PE. No direct relationship between PRL secretion and CT secretion could be established. These results indicate that CT but not PTH varies specifically in relation to the estrous cycle. They suggest that there is a link between sexual hormones and CT, apparently independent of plasma calcium levels.

[Immunohistochemical demonstration of calcitonin cells in the fetal thyroid of mice]. / Démonstration immunohistochimique des cellules à calcitonine de la thyroïde foetale de souris.

Calcitonin was revealed by means of an indirect immunohistochemical method using a serum against synthetic human calcitonin. After the use of the Bouin-Hollande fixative liquid, the hormone was detected in cells located in the central part of the thyroïdian wings. They are few at the 18th day of fetal life, their number increases regularly until reaching ten times the initial number at birth. The immunological reaction is negative if only aldehyde fixatives are used.

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 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.

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.