Role for Fox-1/Fox-2 in mediating the neuronal pathway of calcitonin/calcitonin gene-related peptide alternative RNA processing

Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3' splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF(65) binding to the 3' splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.     

Diagnostic relevance of the amino-terminal cleavage peptide of procalcitonin (PAS-57), calcitonin and calcitonin gene-related peptide in medullary thyroid carcinoma patients

We have identified the amino-terminal cleavage peptide of procalcitonin (PAS-57) in the plasma of normal human subjects and of medullary thyroid carcinoma (MTC) patients together with calcitonin (CT) and CT gene-related peptide (CGRP). Major components on reversed-phase high-pressure liquid chromatography had the retention times of synthetic PAS-57, CT and CGRP as well as of precursor proteins. Plasma levels of PAS-57 (290 +/- 50 pgeq/ml; mean +/- S.E.M.), CT (27 +/- 8 pgeq/ml) and CGRP (8.4 +/- 0.8 pgeq/ml) were respectively 2.3-, 1.6- and 1.5-fold higher in normal men (n = 10) than in women (n = 8). In response to 1 min intravenous calcium infusions (2 mg per kilogram body weight) PAS-57 and CT were increased 3.5- and 2.7-fold (P less than 0.001), respectively, but CGRP remained unchanged. In MTC patients (n = 57) with raised levels of PAS-57 and CT, the molar ratio between PAS-57 and CT was 1.7-times higher than in normal subjects (P less than 0.01). We have found that PAS-57 is a predominant CT/CGRP gene derived product in the circulation of normal subjects and of MTC patients and a potential new MTC tumor marker.     

Molecular characterization of calcitonin gene-related peptide (CGRP) in a rat medullary thyroid carcinoma cell line

The rat medullary thyroid carcinoma cell line, CA-77, is known to express the calcitonin gene and the cell line has been used for characterization of procalcitonin. The present investigations concentrate on a molecular characterization of the calcitonin gene-related peptide (CGRP) expressed by a subclone of this cell line. The investigations demonstrate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogeneity for both CGRP and calcitonin, but a significant amount of immunoreactivity elutes corresponding to the elution position for synthetic CGRP and calcitonin, respectively. The gel chromatogram for CGRP demonstrates four immunoreactive peaks with K_d of 0.42, 0.53, 0.68, and 0.85. The immunoreactive peak with K_d 0.42 elutes corresponding to synthetic rat CGRP. The four immunoreactive peaks were characterized by high pressure liquid chromatography followed by sequence analysis and mass spectrometry. The immunoreactive peak with K_d 0.42 was identified as rat -CGRP as was the peak with K_d 0.53. The peak with K_d 0.68 was identified as 19–37 rat -CGRP and the peak with K_d 0.85 as 28–37 rat -CGRP. In summary, we find that the CA-77 cell line expresses large quantities of normally processed amidated -CGRP and specific fragments thereof. However, the cell line does not express detectable levels of rat β-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expression.     

Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid

In this immunohistochemical study, the ontogenic development of calcitonin-gene-related peptide (CGRP) in the rat thyroid was investigated and compared with that of calcitonin using the indirect-immunofluorescence method. Parafollicular cells with immunoreactivity to both CGRP and calcitonin first appeared at an early stage of gestation (days 17 and 18) in the central portion of the thyroid. Cells immunoreactive to CGRP and calcitonin had became numerous by gestational day 22. After postnatal day 7, CGRP- and calcitonin-immunoreactive (C-IR) cells increased rapidly both in number and in the intensity of their fluorescence. In 14- to 90-day old rats, many intensely immunoreactive cells were distributed in the central portion of the thyroid. The cells immunoreactive to CGRP and to calcitonin had an almost identical ontogenic appearance. In 14-day-old and adult rats, C-IR cells also exhibited CGRP immunostaining, suggesting that these cells simultaneously produce and store CGRP during ontogeny.     

Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid

Summary In this immunohistochemical study, the ontogenic development of calcitonin-gene-related peptide (CGRP) in the rat thyroid was investigated and compared with that of calcitonin using the indirect-immunofluorescence method. Parafollicular cells with immunoreactivity to both CGRP and calcitonin first appeared at an early stage of gestation (days 17 and 18) in the central portion of the thyroid. Cells immunoreactive to CGRP and calcitonin had became numerous by gestational day 22. After postnatal day 7, CGRP- and calcitonin-immunoreactive (CIR) cells increased rapidly both in number and in the intensity of their fluorescence. In 14- to 90-day old rats, many intensely immunoreactive cells were distributed in the central portion of the thyroid. The cells immunoreactive to CGRP and to calcitonin had an almost identical ontogenic appearance. In 14-day-old and adult rats, C-IR cells also exhibited CGRP immunostaining, suggesting that these cells simultaneously produce and store CGRP during ontogeny.     

Simultaneous localization of calcitonin mRNA and peptide in a medullary thyroid carcinoma

We report the visualization of calcitonin gene expression products at the mRNA and peptide levels on the same section of a medullary thyroid carcinoma by combined in situ hybridization and immunohistochemistry. mRNA detection was accomplished by hybridization with radioactively labeled antisense RNA probes followed by autoradiography and immunohistochemically using the avidin-biotin complex method. Best results were obtained when in situ hybridization preceded immunohistochemistry, as determined by quantitative analysis of the autoradiographs. When immunohistochemistry was performed prior to in situ hybridization, the RNase inhibitor heparin had to be added to the antibodies to retain hybridizable mRNA. The intensity of the two reactions varied in individual cells, indicating a functional heterogeneity of tumor cells with regard to calcitonin mRNA content and storage of the related immunoreactive peptide. These results, in combination with elevated serum calcitonin levels, suggest significant differences in the rate of secretion of individual tumor cells. Simultaneous localization of mRNA and its peptide within the same cell may, therefore, provide further insight into gene expression and secretory activity at the single cell level.     

Release of calcitonin by cultures of medullary carcinoma of the thyroid.

Three biopsies of medullary carcinoma of the thyroid were grown in monolayer culture. All three cultures initially released high levels of calcitonin into the medium, but the conretion from the culture cells was not stimulated when the medium calcium concentration was increased from 1.8 to 3.6 mEq/L. Four peaks of calcitonin immunoreactivity were found when the culture medium of one cell line was fractionated by gel filtration on Bio-Gel P-10. This closely corresponded to the heterogeneous molecular profile of calcitonin in the serum of this patient and other patients with medullary carcinoma of the thyroid.     

Age-related increase of calcitonin gene-related peptide in rat thyroid and circulation.

Elevated calcitonin levels in thyroid gland extracts and in plasma accompanied by C-cell hyperplasia are frequently found in old rats, in particular those raised in laboratory conditions. In parallel with calcitonin, we demonstrate here that the thyroidal content and plasma levels of immunoreactive calcitonin gene-related peptide (i-CGRP) significantly increase with age in rats (p less than 0.0001). C18 Sep-Pak-extractable i-CGRP level in plasma was 35% of the total i-CGRP. Gel permeation chromatography and rp-HPLC studies revealed a number of immunoreactive molecular forms of CGRP and only 40-50% of the acid-extracted immunoreactivity was coeluted with the synthetic CGRP(1-37). The i-CGRP level measured in plasma was highly correlated with the thyroidal content of CGRP (p less than 0.001) and also with the age of the rat (p less than 0.001), suggesting an age-related increase of contribution of CGRP from thyroid gland into the circulation.     

Stimulation of calcitonin secretion in the pig by calcitonin gene-related peptide

Pig thyroid glands were surgically isolated in situ and perfused with autologous blood to which was added known concentrations of calcitonin gene-related peptide (αCGRP). When thyroids were perfused with measured concentrations of CGRP within the range of 0.6–600 nM, the secretion rate of calcitonin (CT) was stimulated while the release of T₃, T₄, and somatostatin remained unchanged. Specific binding of ¹²⁵I-CGRP to pig thyroid plasma membranes was demonstrated, and binding was inhibited by unlabelled CGRP but not by CT or by other peptides unrelated structurally to CGRP. The findings indicate that the pig thyroid gland contains plasma membrane binding sites for CGRP and that CGRP is capable of stimulating the secretion of CT.