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.

Cloning and sequencing of a new 15-hydroxyprostaglandin dehydrogenase related mRNA.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH) inactivates prostaglandins. We recently reported an mRNA sequence coding for a predicted isomer (PGDH(rI)) of this enzyme. The TT cell line, derived from medullary thyroid carcinoma (MTC), expresses mRNAs for both isomers. We report here the expression by TT cells and MTC of a third 15-PGDH related mRNA (PGDH(rII)), 241 nt shorter than type-I 15-PGDH. RNase protection assays confirmed that TT cells expressed this mRNA (PGDH(rII)). Thus different splicing patterns could be involved in the post-transcriptional regulation of type-I 15-PGDH gene in MTC.

1,25-dihydroxyvitamin D3 induces NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase in human neonatal monocytes.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces the differentiation of monocytes into macrophage-like cells in vitro. To identify the genes expressed during this process, we performed differential display polymerase chain reaction on RNA extracted from cord blood monocytes (CBMs) treated with 1,25-(OH)2D3. Treated CBMs expressed type-I 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH), the key enzyme of prostaglandin E2 (PGE2) catabolism and a 15-PGDH-related mRNA (15-PGDHr). This newly described 15-PGDH-related mRNA was constitutively expressed in adult monocytes. 15-PGDH gene(s) transcription was accompanied by the appearance of the 15-PGDH activity in treated CBMs. In addition, the cyclooxygenase 2 mRNA level was decreased and PGE2 levels in the culture mediums were lowered (50%). Our results stress that 1,25-(OH)2D3, at least in neonatal monocytes, can exert, directly or indirectly, a dual control on key enzymes of PGE2 metabolism. In conclusion, we suggest that modifications in prostaglandin metabolism, induced by the expression of type-I 15-PGDH and the downregulation of cyclooxygenase 2, could be involved in monocytic differentiation.

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.

Identification of a new calcitonin gene in the salmon Oncorhynchus gorbuscha.

Three isoforms of calcitonin (CT) exist in salmonids. Isohormones I and II are expressed in the pink salmon Oncorhynchus gorbuscha. We report here the existence in this species of a CT gene and of its transcripts, which encode for a fourth isohormone, the salmon CT (sCT) IV. This new CT gene was identified by PCR from genomic DNA and by sequencing the amplified DNA. The expression of this CT gene was established in ultimobranchial body and brain, by reverse transcription-PCR, hybridization and sequencing. The sCT IV gene, like the sCT I gene, is a complex transcription unit, containing exons encoding for a CT as a calcitonin gene-related peptide (CGRP) molecule. The predicted peptide, sCT IV, has a greater homology with the eel CT and the sCT II than with the sCT I. Alignment of the sCT IV with other fish and chicken CT showed amino acid modifications in similar positions as those found during evolution. The predicted salmon CGRP IV peptide is highly homologous to the known CGRP molecules in other species, confirming the high conservation of the molecule during evolution. This identification of a new salmon CT gene is interesting both for the therapeutic potential represented by the new molecules encoded by this gene and for phylogenetic studies.

Expression of glucagon-like peptide 1 receptor in a murine C cell line: regulation of calcitonin gene by glucagon-like peptide 1.

We have characterized, by RT-PCR amplification using specific primers, the presence of glucagon-like peptide-1 (GLP-I) receptor mRNA in CA-77 cells, a C cell line derived from a rat medullary thyroid carcinoma. Down-regulation of the GLP-1 receptor mRNA was observed after exposure of CA-77 C cells with GLP-1 (7-37). Increased secretion of both calcitonin gene-related peptide (CGRP) and calcitonin (CT) occurred after treatment with GLP-1 (7-37) associated with elevated steady-state levels of CGRP and CT mRNA. GLP-1 (7-37) increased cAMP formation in CA-77 cells in a dose-dependent manner; exendin (9-39), a GLP-1 receptor antagonist, inhibited cAMP production. The GLP-1 peptide which is produced by intestinal cells could be involved in the control of CT secretion through an entero-thyroidal axis implying GLP-1 receptor and increased CT gene expression.

Modulation of interleukin-1 receptor expression by transforming growth factor-beta in cultured rabbit articular chondrocytes: analysis by reverse transcription-polymerase chain reaction.

Interleukin-1 receptor type I (IL-1RI) expression in cultured rabbit articular chondrocytes (RAC) was studied by reverse transcription-polymerase chain reaction (RT-PCR). A cDNA probe specific for the rabbit IL-1RI gene was constructed using primers derived from the sequence data of the human, murine and chick receptors. Transforming growth factor-beta 1 (TGF beta-1) was shown to transiently increase the level of expected 900-bp PCR product at 1 h of incubation and decrease the expression at 48 and 72 h with no effect at 24 h. In receptor binding assays using [125I]-IL-1 alpha, TGF beta decreased IL-1R bioactivity at all time points. These results suggest that TGF beta-induced down-regulation of IL-1 RI could be responsible for its ability to antagonize the effect of IL-1 and that TGF beta may have a role in the repair of articular cartilage.

Sequence of a novel mRNA coding for a C-terminal-truncated form of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

We amplified, using the polymerase chain reaction (PCR) and NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH)-specific primers, RNA extracted from the HL-60 cell line. Two bands, differing in size by approx. 160 bp, were detected with ethidium bromide staining after electrophoresis of amplification products and hybridization with a 15-PGDH-specific probe. Sequencing these DNA bands revealed that the largest corresponded to the 15-PGDH cloned from human placenta [Ensor et al., J. Biol. Chem. 265 (1990) 14888-14891]. The smaller sequence coded for a predicted C-terminal-truncated form of 15-PGDH. This subtype of the type-I 15-PGDH mRNA was also found using RT-PCR in human liver, placenta and a cell line derived from a human medullary thyroid carcinoma (TT cells). Hybridization studies using specific probes indicated that this new mRNA form probably corresponded to the 3.4-kb mRNA, one of the two 15-PGDH mRNAs previously detected in Northern blot analysis.

Calcitonin secretion, C cell differentiation and proliferation during the spontaneous development of murine medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC), a C cell neoplasm, synthesizes large amounts of calcitonin (CT), its biological marker. However, in some cases with a poor prognosis, MTC is associated with low basal CT levels owing to a decrease in the thyroid CT content. Using a murine model of human MTC, we studied the relationships between CT biosynthesis, C cell proliferation, and the circulating CT level during MTC progression. Cell proliferation was revealed by autoradiography of radioactive thymidine incorporation in dividing nuclei, after CT or CT mRNA detection by immunocytochemistry (ICC) or in situ hybridization (ISH). All rat thyroids showed a severe hyperplasia of C cells containing significant amounts of CT and CT mRNA, and a very low mitotic index. Tumours were found in 68% of the thyroids. In the strongly immunoreactive small nodules (ICC+), many labelled nuclei were observed. Subsequently some nodular cells, still containing detectable CT mRNA (ISH+), were not detected by immunocytochemistry (ICC-) owing to a dramatic decrease in secretory granules. Their mitotic index increased, and a rise of the basal CT plasma level was noted. These ISH+, ICC- tumour MTC cells represent a modified aggressive tumour C cell population exhibiting an increased ability to proliferate and were detected by the rise in the basal circulating CT level.

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.

Evaluation of somatostatin biosynthesis, somatostatin receptors and tumor growth in murine medullary thyroid carcinoma.

Spontaneous medullary thyroid carcinomas (MTCs) of old rat thyroids were analyzed for the expression of somatostatin and somatostatin binding sites in tumoral C cells in relation to the stage of tumor development, the mitotic activity of tumoral tissue and calcitonin biosynthesis as a marker of C cell differentiation. High levels of both immunoreactive somatostatin and its mRNA were detected in a subpopulation of tumoral C cells, gathered in areas suggesting a clonal proliferation and located preferentially at the periphery of the tumor. These cells also displayed high levels of calcitonin and its mRNA. However, many calcitonin immunoreactive cells showed no sign of somatostatin synthesis. The proliferative activity of the somatostatin-containing areas was low and slow compared to the areas lacking somatostatin production. However, it increased during the course of tumor growth. Somatostatin binding sites, measured with in vitro receptor autoradiography using 125I-[Tyr3]-octreotide or 125I-[Leu8, dTrp22, Tyr25]SS-28, were not detected in any of the MTCs tested. In rat MTC cells, somatostatin was associated with differentiation and slow proliferation, two parameters inversely correlated with the progression of malignancy. As expected, owing to the highly regulated secretion of the differentiated endocrine cell type, its presence was correlated with low basal calcitonin levels. However, the absence of somatostatin binding sites on any type of MTC cells does not favor a direct autocrine regulation of this peptide in this murine model of human MTC.

CGRP is expressed in primary cultures of human hepatocytes and in normal liver.

We recently reported that human liver and primary cultures of hepatocytes express calcitonin. We therefore studied the expression of calcitonin gene related peptide (CGRP), the alternative splicing product of the calcitonin gene, in hepatocytes and liver. We used polymerase chain reaction amplification with specific primers to detect the presence of CGRP I and II messengers and a specific radioimmunoassay to measure the peptide. We report here that CGRP is synthesized by primary cultures of hepatocytes and in liver. As liver also possesses specific receptors for CGRP in non-parenchymal cells, a paracrine system could be involved in liver metabolism.

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.

An isoform of the human calcitonin receptor is expressed in TT cells and in medullary carcinoma of the thyroid.

We amplified, using the polymerase chain reaction and calcitonin receptor (CTR) specific primers, RNA extracted from medullary thyroid carcinoma (MTC) and the derived TT cell line. Both secrete large amounts of calcitonin. Electrophoresis of amplification products revealed, in both cases, an ethidium bromide-stained band that hybridized to a CTR probe. Sequencing the band amplified from TT cells revealed an open reading frame identical to the sequence of H-CTR but lacking 16 amino acids in the first intracellular loop. This demonstrates the existence of an mRNA coding for a subtype of H-CTR which is expressed in TT cells and MTC.

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.

Calcitonin gene expression in normal human liver.

Immunoreactive calcitonin (CT) is present in liver. This could represent hormone synthesized by liver cells, degraded or bound to specific receptors reported in this organ. We report here that the calcitonin gene is expressed in liver. We proved this by demonstrating, by PCR amplification using specific primers, the presence of calcitonin messenger in human liver and in primary cultures of human hepatocytes and detected by radioimmunoassay CT in hepatic tissues and cells. The synthesis of hormone by liver that also possesses specific receptors for CT favors the presence of an autocrine or paracrine system involving calcitonin in this organ.

Hormonal study of a human mixed follicular and medullary thyroid carcinoma.

We studied the hormonal secretion of a human mixed follicular and medullary carcinoma. Thyroglobulin (Tg) secretion, especially by large cells and sometimes by small ones, was visualized with immunoenzymatic staining. Calcitonin (CT) was produced by small spindle-shaped cells. Moreover, immunofluorescence double staining performed on the resected thyroid tissue showed the secretion of both Tg and CT in a small number of cells. The cells lost their hormonal secretion after 2 months of culture. Hormonal secretion was modulated by different additives in the medium. Tg secretion was induced when TSH was added to the culture medium; the maximal effect was produced with the addition of 1 mU TSH/ml and 1 microM cortisol, which potentiated the effect of TSH on Tg production. A durable Tg secretion was obtained by embedding the cells in Engelbretch-Hohn-Swarn (EHS) tumour matrix. The CT production was reinduced by the addition of 4 mM Ca2+, 1 microM glucagon and 1 microM cortisol to the culture medium. These findings show that different cells are found in a mixed follicular and medullary carcinoma, some of which can secrete both CT and Tg. They can remain differentiated for a long period after being embedded in EHS tumour matrix with Ca2+ and hormonal components.

An efficient method to detect calcitonin mRNA in normal and neoplastic rat C-cells (medullary thyroid carcinoma) by in situ hybridization using a digoxigenin-labeled synthetic oligodeoxyribonucleotide probe.

We report here an efficient and rapid method for the specific detection of calcitonin in tumor C-cells of medullary thyroid carcinoma (MTC). This occasionally aggressive tumor arises from the endocrine thyroid C-cells. Its principal marker is calcitonin, the predominant C-cell secretion, which is detected in patients and in our animal model by radioimmunoassay of the plasma, as well as by immunohistochemistry of thyroid tissues. Although calcitonin is easily detectable in normal C-cells, its content is greatly reduced in tumor cells owing to the disappearance of the secretory granules that store the mature peptide. This finding suggests cell dedifferentiation correlated with an increasing aggressivity of the tumor. We therefore developed a rapid detection of calcitonin mRNA by in situ hybridization on routine paraffin sections, using a synthetic oligodeoxyribonucleotide probe labeled with digoxigenin-dUTP. The reaction was detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase, and the enzyme catalyzed the appearance of a dark blue color. The signal was exclusively restricted to the normal, hyperplastic, and tumor C-cells. It was specific, as increasing concentrations of the unlabeled oligonucleotide led to progressive disappearance of the reaction. Its sensitivity was slightly diminished as compared with corresponding frozen sections, but the intensity of the signal was quite acceptable. High levels of calcitonin mRNA were found in all normal and hyperplastic C-cells. They were increased in most of the tumor MTC cells, which did not correlate with the amount of intracellular peptide stores but explained the abnormally high basal levels of circulating calcitonin of the tumor-bearing rats. ISH is therefore of greater value than ICC for an early anatomopathological detection of this tumor. Our data show that the tumor cells are not "dedifferentiated." They only lack the granular compartment storing the mature peptide before exocytosis, but CT biosynthesis and the rest of the secretory process seem to be complete. Our results suggest that factors expressed in malignant C-cells affect basic cell mechanisms involved in the storage of the mature calcitonin, rather than the expression of the CALC gene.