Influence of development on the number of calcitonin-containing cells in the mouse thyroid.

Calcitonin-containing cells in serial, 6-micrometer sections of the thyroid glands of Swiss Webster mice, at 1 day, 2 weeks, 4 weeks and 8 weeks of age, were demonstrated by an immunoperoxidase method, using antiserum to human calcitonin. C-cell nuclei were counted in every sixth section of both left and right lobes. The average number of C-cells counted in the thyroid glands of 8-week-old animals was 18-fold, 5.5-fold and 2.5-fold greater than the number observed in 1-day, 2-week and 4-week-old animals, respectively. C-cell concentration was found to be greatest in 4-week-old mice. Mitoses of C-cells were observed in animals which were 1 day, 2 weeks and four weeks old. No mitotic figures were seen in 8-week-old animals. A few C-cells were seen in close association with neurons. The volume of the thyroid glands of 8-week-old animals was about 14-, 4- and 3-fold greater than the volume in the 1-day-old, 2-week-old and 4-week-old mice, respectively. These changes in the C-cell population during development provide a model for the study of C-cell proliferation and storage of calcitonin.     

Immunohistochemical study of the C-cell complex of dog thyroid glands with reference to the reactions of calcitonin,C-thyroglobulin and 19S thyroglobulin

Summary Continued from the previous study in fetal animals (Kameda et al. 1980), the development and maturation of C-cell complexes in postnatal dogs from newborn to adult were investigated by use of an immunoperoxidase method using antisera to calcitonin, C-thyroglobulin (C-Tg) and 19S thyroglobulin, respectively. The younger the animals were, the more numerous were undifferentiated cells and high columnar epithelial cells in the complexes. With increasing age, the constituent elements of the complexes progressively differentiated. In one type of complex there are a large number of C-cells in various developmental stages, as well as undifferentiated cells and cysts. C-cell complexes composed mostly of mature C-cells were regarded as the more highly differentiated structures of this type. A second type contains follicular cells in various stages of differentiation in addition to undifferentiated cells and C-cells, i.e., 19S-positive cell masses not yet organized into follicles, primordial follicles with small lacunae and comparatively larger follicles. The follicular cells in the complexes were similar with respect to immunoreaction and folliculogenesis to the cells of fetal thyroids, but they developed very slowly. In conclusion, the present study indicates that follicular thyroid cells can differentiate within C-cell complexes, i.e., they develop from cells of ultimobranchial body origin.     

Reciprocal epithelial:endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation

The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by >90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation.     

人甲状腺髓样癌TT细胞胃动素基因表达的调控

目的:研究人甲状腺髓样癌TT细胞系中胃动素基因表达调控.方法:通过人甲状腺髓样癌TT细胞体外培养,观察经cAMP,生长激素或甲状腺雌激素诱导后,胃动素表达的改变以及胃动素对TT细胞生长、侵袭和转移的影响.结果:甲状腺髓样癌TT细胞表达胃动素mRNA,胃动素可抑制TT细胞的生长.当胃动素基因沉默后,TT细胞转移和侵袭能力增加.当TT细胞分别经cAMP、胃动素、生长激素或甲状腺刺激素孵育48小时后,胃动素基因转录增加,降钙素基因相关肽与胃动素mRNA比值持续下降.环磷酸腺苷可降低TT细胞增殖和c-myc基因的表达.结论:人甲状腺髓样癌细胞生长活性可能与甲状腺C细胞(低的降钙素基因相关肽与胃动素比率)分化的表型有关.     

Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.     

In vitro splicing analysis of mini-gene constructs of the alternatively processed human calcitonin/CGRP-I pre-mRNA

The human calcitonin/CGRP-I (CALC-I) gene can be alternatively expressed into calcitonin mRNA in thyroid C-cells and into CGRP-I mRNA in particular nerve cells. Formation of calcitonin mRNA requires splicing of exons 1, 2, 3 and 4 and addition of poly(A) at exon 4, whereas splicing of exons 1, 2, 3, 5 and 6 and addition of poly(A) at exon 6 yields CGRP-I mRNA. The calcitonin and CGRP-I mRNA-specific splicing reactions were investigated in vitro, in nuclear extracts of HeLa cells, using model precursor RNAs containing the exon 3 to exon 5 region of the gene. A precursor RNA containing the full-length exon 3 to exon 5 region was only poorly spliced in vitro. Therefore, a systematic analysis was performed of the effect of deletions introduced in the intron 3, exon 4 and intron 4 of this precursor RNA on calcitonin/CGRP mRNA-specific splicing. The deletions increased the efficiency of splicing considerably. In all cases CGRP mRNA-specific splicing is strongly favoured over calcitonin mRNA-specific splicing. In addition, splicing reactions using cryptic 5' splice sites were detected which interfered with the usage of processing signals for calcitonin and CGRP mRNA-specific splicing. The results imply a major regulatory role for the exon 4 poly(A) addition reaction in the generation of calcitonin mRNA.     

Co-occurrence of immunoreactive calcitonin and calcitonin gene-related peptide in neuroendocrine cells of rat lungs

Summary Neuroendocrine cells of the lung, occurring singly or in clusters known as neuroepithelial bodies, contain a variety of biologically active compounds, including several neuropeptides. We have investigated the localization of calcitonin and calcitonin gene-related peptide (CGRP) within single and grouped neuroendocrine cells in the respiratory epithelium of rats by an immunohistochemical double-staining technique which uses specific antisera raised in heterogeneous animal species. Calcitonin- and CGRP-immunoreactivities were nearly totally co-localized in both single neuroendocrine cells and neuroepithelial bodies. CGRP-immunoreactivity was also present in neurons in the jugular, nodose and dorsal root ganglia. The calcitonin-immunoreactivity in neuroendocrine cells, as in thyroid parafollicular (C) cells, was abolished by preincubation of the anticalcitonin serum with synthetic calcitonin. The CGRP-immunoreactivity in neuroendocrine cells and in the neuronal cells was abolished by preincubation of anti-CGRP serum with synthetic CGRP. Thus, while the calcitonin gene is expressed exclusively or predominantly as either calcitonin or CGRP in all other tissues except thyroid C-cells, our results strongly suggest that both peptides are expressed in the rat bronchopulmonary neuroendocrine cells.     

Reactivity of monoclonal islet cell antibodies on normal hyperplastic and neoplastic thyroid C-cells

Summary Thyroid C-cell reactivity to 15 monoclonal antibodies raised against a series of pancreatic islet cells (H[human]ISL, B[bovine]ISL and R[rat]ISL) was evaluated using an indirect immunoperoxidase technique on frozen thyroid sections. Of the monoclonal anti-islet cell antibodies, five reacted specifically with bovine C-cells or human hyperplastic and neoplastic C-cells but not with follicular cells. Two monoclonal antibodies of the bovine series showed strong immunoreactivity with C-cells and only a weakly positive immunostaining of follicular cells. Five monoclonal antibodies reacted with both thyroid C-cells and follicular cells, whereas 3 monoclonal anti-islet cell antibodies did not stain any cell type of the thyroid. In human medullary carcinomas, calcitonin- and somatostatin-producing neoplastic cells were immunoreactive with the same monoclonal antibodies as were normal human C-cells. The protein bands identified by the monoclonal antibodies in human medullary carcinomas had the same molecular weight as those from pancreatic islet extracts. Our study demonstrates the presence of similar differentiation antigens on thyroid C-cells and pancreatic islet cells; this further illustrates common modes of differentiation and specialisation of these embryologically different members of the dispersed neuroendocrine system. The crossreactivity of seven of the monoclonal antibodies investigated with follicular epithelium of the thyroid suggests the existence of common antigenic determinants in different endocrine organs and may partly explain the multiple organ autoimmune response found in patients with polyendocrine diseases.     

Dexamethasone stimulates thyrotropin-releasing hormone production in a C cell line

The hypothalamic peptide hormone TRH is also found in other tissues, including the thyroid. While TRH may be regulated by T3 in the hypothalamus, other regulators of TRH have not been identified and the regulation of TRH in nonhypothalamic tissues is unknown. We recently demonstrated the biosynthesis of TRH in the CA77 neoplastic thyroidal C cell line. We studied the regulation of TRH by dexamethasone in this cell line because glucocorticoids have been postulated to inhibit TSH secretion by decreasing TRH in the hypothalamus. Furthermore, TRH in the thyroid inhibits thyroid hormone release. Thus by regulating thyroidal TRH, glucocorticoids could also directly affect thyroid hormone secretion. Treatment of CA77 cells for 4 days with dexamethasone produced dose-dependent increases in both TRH mRNA and cellular and secreted TRH. Increases in TRH mRNA and peptide levels could be seen with 10(-9) M dexamethasone. A 4.8-fold increase in TRH mRNA and a 4-fold increase in secreted peptide were seen with 10(-7) M dexamethasone. Dexamethasone treatment did not increase beta-actin mRNA levels or cell growth. These results suggest that glucocorticoids may be physiological regulators of TRH in normal C cells. In addition to their inhibitory effects on TSH, glucocorticoids may decrease thyroid hormone levels by increasing thyroidal TRH. Since the glucocorticoid effects on C cell TRH are the converse of what is expected for hypothalamic TRH, glucocorticoid effects in these two tissues may be mediated by different regulators.     

Retinoic acid treatment of human neuroblastoma cells is associated with decreased N-myc expression

Cells from human neuroectodermal tumors (retinoblastoma and neuroblastoma) and from neuroblastoma cell lines express a gene, N-myc, which is frequently amplified in these tumors. We report here that N-myc mRNA content is markedly decreased in cells of a neuroblastoma cell line (LA-N-5) following differentiation induced with retinoic acid. Exposure of the cells to retinoic acid induced morphologic changes consistent with neuronal differentiation, and led to a 75% decrease in expression of N-myc mRNA. These results suggest that N-myc expression is intimately related to an undifferentiated phenotype in neuroblastoma cells, and support other studies which relate N-myc expression to the malignant phenotype in neuroblastoma tumors.     

Thyroid parafollicular cells

Serotonergic neurons play key roles in modulating a wide variety of behavioral and homeostatic processes. However, there is a paucity of good model systems to study these neurons at a molecular level. In this review we will present evidence that cell lines derived from an unexpected source, thyroid parafollicular cells (PF) (also called C cells), fit the criteria for use as models for the study of serotonergic neurons. A strength of PF cell lines over other cell lines is that the parental PF cells have serotonergic properties and a neuronal potential that is consistent with their neural crest origin. Futhermore, PF cells and PF cell lines are capable of expressing the fundamental properties of serotonergic neurons, including: (1) serotonin (5-HT) biosynthesis by tryptophan hydroxylase (TPH), (2) vesicular 5-HT storage and regulated release, (3) expression of a 5-HT autoreceptor, and (4) expression of the 5-HT transporter. In this review, we will focus primarily on the serotonergic and neuronal properties of the rat CA77 PF cell line and the parental rat PF cells. The applicability of CA77 cells for molecular analyses will be described. First, their use for studies on the glucocorticoid regulation of the TPH gene will be discussed. Second, control of the calcitonin/calcitonin gene-related peptide (CT/CGRP) gene will be discussed, with particular emphasis on the application of serotonergic drugs in treating migraine headaches. These examples highlight the versatility of thyroid PF cell lines as a system for studying the control of both serotonin biosynthesis and physiological actions.