Calcitonin mRNA Research Articles

Calcitonin Gene-Related Peptide and Calcitonin mRNA Expression in Periaqueductal Gray Area in the Formalin Test in Rats

Background: Calcitonin gene-related peptide (CGRP) and rat calcitonin (rCT) play critical roles in descending pain control systems. Objectives: The present research aimed to evaluate the effect of the intracerebroventricular (ICV) administration of CGRP and rCT on the mRNA expression of CGRP and rCT peptides in the periaqueductal gray (PAG) of healthy rats in the formalin test. Methods: A total of 24 male Sprague Dawley rats were categorized into four groups (n = 6). One week after stereotaxic surgery, 1.5 nmol CGRP or rCT peptides were injected (ICV) once daily for 7 days. After 20 min from the last injection, the right foot of the animal was injected subcutaneously with 2.5% formalin. Pain-related behaviors were recorded immediately for 60 min. The PAG nucleus was then removed to assess the changes made in the mRNA expression of the CGRP and rCT. Results: ICV injection of CGRP or rCT reduced pain in the different phases of the trial. ICV injection of rCT induced the expression of rCT mRNA in the PAG area (P < 0.05). However, ICV injection of rCT had no significant effect on the CGRP mRNA expression in the PAG area. Moreover, following the ICV injection of CGRP, the expression of rCT mRNA increased in the PAG area (P < 0.05). It is noteworthy that the ICV injection of CGRP caused a significant effect on the CGRP mRNA expression in the PAG area (P < 0.05). Conclusions: The ICV injections of CGRP and rCT peptides decreased pain in the formalin test. Higher mRNA expression of these peptides in the PAG might be a possible mechanism for this observation.

Abstract 11079: Calcitonin Signaling Controls Disease-Induced Left Ventricular Remodeling

Introduction: We recently discovered that the well-known thyroid hormone calcitonin (CT) is produced by atrialcardiomyocytes (CMs) and regulates atrial fibrogenesis and arrhythmia susceptibility via CT receptors (CTRs) inatrial cardiofibroblasts (CFs). Whether the CT-CTR system regulates ventricular function and structure isunknown and was the object of this study. Method and Results: Human studies were performed on left ventricular (LV) samples from 27 patients who hadcardiac surgery (Oxford, UK; ethical approval 18/SC/0404). Animal studies used 8-10 week-old male control(CTR flx/flx ; n=150) and CTR -/- (n=131) mice subjected to transverse aortic constriction (TAC) and myocardialinfarction (MI), with selected analyses in wild type mice (n=5). Cardiac function was assessed byechocardiography at baseline, 3 and 6 weeks post-TAC, and 1 and 4 weeks post-MI.Expression profiling detected CTR and CT mRNA (qPCR; Fig. A-B) and protein (immunoblotting) in human LVtissue and mouse LV CMs and CFs, with a higher CT abundance in CMs. CTR protein was decreased in LV tissue from patients with hypertension vs non-hypertensive controls (Fig. C).CTR deletion enhanced atrial and LV fibrosis (Masson’s Trichrome; Fig. D-E), significantly exacerbated post-TAC LV ejection fraction reduction by 15.2% (Fig. F), and atrial (by 23.7%, Fig. G) and LV dilation (by 19.9%),while increasing induced-AF duration by 6-fold (Fig. H) and inducibility by 15% vs post-TAC controls.In CTR -/- mice, acute MI produced greater fibrosis in the LV risk-region (by 18%; Fig. I), ejection fractionreduction (by 14%, Fig. J) vs CTR flx/flx controls. Mortality, atrial dilation and AF-inducibility were similar inCTR -/- and control MI-mice. Conclusion: CTR deletion in mice exacerbates adverse LV remodeling and dysfunction in clinically-relevantdisease models, pointing towards an important protective role of CT-CTR signaling in ventricular myocardium.

Suppressive effect of melatonin on osteoclast function via osteocyte calcitonin.

Many studies have investigated the actions of melatonin on osteoblasts and osteoclasts. However, the underlying mechanisms, especially regarding osteocyte function, remain largely unknown. Therefore, this study aimed to clarify the underlying mechanisms of melatonin action on bone tissue via osteocyte function. Chick calvariae were employed as a model. In ovo injection of melatonin (5, 50 and 500 µg) dose-dependently decreased the mRNA expression levels of cathepsin K and matrix metalloproteinase 9 (MMP9) in chick calvariae without affecting the expression levels of receptor activator of NF-κB ligand or osteoprotegerin. Surprisingly enough, the expression of calcitonin mRNA in chick calvariae was significantly raised. After 3 days of in vitro treatment of melatonin (10-7 and 10-5 M) on newly hatched chick calvariae, both calcitonin mRNA expression in calvariae and the concentration of calcitonin in cultured medium were augmented in a dose-dependent manner, coincident with the decreased mRNA expression levels of cathepsin K and MMP9. Immunohistochemical analyses revealed expression of melatonin receptors and calcitonin by osteocytes buried in bone matrix. Moreover, the mRNA expression levels of melatonin receptors, calcitonin and sclerostin (a marker of osteocyte), were strongly and positively correlated. In conclusion, we demonstrated the expression of melatonin receptors and calcitonin expression in osteocytes for the first time and suggest a new mechanism underlying the suppressive effect of melatonin on osteoclasts via upregulation of calcitonin secretion by osteocytes.

Melatonin is a potential drug for the prevention of bone loss during space flight.

Astronauts experience osteoporosis‐like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O‐methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast‐inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

Fasting and refeeding induce differential changes in hypothalamic mRNA abundance of appetite-associated factors in 7 day-old Japanese quail, Coturnix japonica

There is little information regarding effects of fasting on feeding behavior and hypothalamic physiology in young Japanese quail. The aim was thus to measure food intake and hypothalamic mRNA in response to fasting and refeeding. Five d-old quail ate little during the dark cycle. Food intake was greatest during the first 2 h of the light cycle. Six day-old quail fasted for 6 h ate the most during the first 15 min of refeeding. In 7 d-old quail, 3 h of fasting up-regulated hypothalamic neuropeptide Y (NPY), NPY receptor sub-type 2 (NPYR2), agouti-related peptide (AgRP), orexin receptor 2 (ORXR2), melanocortin receptors 3 and 4 (MC3R and MC4R, respectively), and neuropeptide S (NPS) and decreased corticotropin-releasing factor receptor sub-type 1 (CRFR1) mRNA. Quail fasted for 3 h and refed for 1 h had greater NPY, AgRP, POMC, and MC3R but less CRFR1 mRNA than fed quail. Quail fasted for 6 h expressed more NPY, NPYR1, NPYR2, and MC3R and less ORXR2, prolactin releasing peptide (PrRP), cocaine- and amphetamine-regulated transcript (CART), and calcitonin (CAL) mRNA than fed quail. Quail fasted for 6 h and refed for 1 h expressed more NPY, NPYR1, NPYR2, AgRP, MC3R, MC4R, and NPS and less galanin, ORXR2, PrRP, CART, and CAL mRNA than fed birds. Hence, fasting induced changes in hypothalamic mRNA, with the largest changes occurring in genes associated with NPY and melanocortin signaling. Most genes remained elevated or downregulated after refeeding, suggesting that there was a time lag for transcription to respond to compensatory feeding.

Primary high-grade calcitonin-negative neuroendocrine carcinoma of the thyroid: a very rare cancer.

SummaryMost medullary thyroid carcinomas (MTCs) are low grade and produce calcitonin. There are some calcitonin-negative MTCs that produce only calcitonin gene-related peptide (CGRP). Rarely, MTCs are negative for calcitonin and CGRP peptides, but contain their corresponding mRNAs. Primary thyroid neuroendocrine neoplasms other than MTCs are extremely rare. We describe a primary high-grade neuroendocrine carcinoma that was negative for CGRP and calcitonin at both the protein and mRNA levels. A 42-year-old woman presented with a rapidly enlarging thyroid mass replacing most of the left lobe and isthmus. A computed tomography-guided core-needle biopsy was performed. The tumor was composed of sheets of small-to-medium sized epithelial cells. The cells were immunoreactive for pancytokeratin, synaptophysin, CD56 and thyroid transcription factor-1, but negative for CK7, CK20, CD45, CD99, ERG, chromogranin A, thyroglobulin, calcitonin, CGRP and carcinoembryonic antigen. The Ki-67 proliferation index was ~90%. In situ hybridization was negative for calcitonin mRNA. The patient was initially diagnosed as having a small cell carcinoma. She was treated with cisplatin and etoposide (VP16), followed by radiation therapy. Given the excellent clinical course, the tumor was reviewed and reclassified as a high-grade neuroendocrine carcinoma (non-small-cell type). Heretofore, only a few other similar high-grade neuroendocrine tumors with negative markers of C-cell derivation have been reported. In our case, the patient is cancer free five years after diagnosis, but in the other cases, the outcome was poor.Learning points:There are neuroendocrine carcinomas of the thyroid that do not produce calcitonin or calcitonin gene-related peptide.This category of calcitonin-negative neuroendocrine carcinomas is heterogeneous, consisting of low- and high-grade tumors.The high-grade neuroendocrine carcinomas of the thyroid are rare and generally have a poor prognosis. They are divided into small cell and non-small cell or large cell types.

Calcitonin is expressed in the submaxillary glands of rats

Calcitonin is usually produced by the parafollicular cells of the thyroid. However in an immunohistochemistry experiment we observed that the cells of the serous acini of rat submaxillary gland tissue were stained positive with calcitonin antibodies. We further used immunocytochemistry and nucleic acid hybridization to localize the distribution of calcitonin protein and calcitonin mRNA respectively in cultivated cells of rat submaxillary glands. The results showed that the cytoplasm of the epithelial cells of the submaxillary glands had positive staining in immunocytochemistry using calcitonin monoclonal antibody and positive reaction in nucleic acid hybridization using calcitonin mRNA complementary DNA probe. For the first time we found that the cells of the submaxillary glands of rats can produce the hormone calcitonin.

Reactive Oxygen Species Induce Procalcitonin Expression in Trigeminal Ganglia Glia

To examine calcitonin gene-related peptide (CGRP) gene expression under inflammatory conditions using trigeminal ganglia organ cultures as an experimental system. These cultures have increased proinflammatory signaling that may mimic neurogenic inflammation in the migraine state. The trigeminal nerve sends peripheral pain signals to the central nervous system during migraine. Understanding the dynamic processes that occur within the trigeminal nerve and ganglion may provide insights into events that contribute to migraine pain. A neuropeptide of particular interest is CGRP, which can be elevated and play a causal role in migraine. However, most studies have overlooked a second splice product of the Calca gene that encodes calcitonin (CT), a peptide hormone involved in calcium homeostasis. Importantly, a precursor form of CT called procalcitonin (proCT) can act as a partial agonist at the CGRP receptor and elevated proCT has recently been reported during migraine. We used a trigeminal ganglion whole organ explant model, which has previously been demonstrated to induce pro-inflammatory agents in vitro. Quantitative polymerase chain reaction and immunohistochemistry were used to evaluate changes in messenger ribonucleic acid (mRNA) and protein levels of CGRP and proCT. Whole mouse trigeminal ganglia cultured for 24 hours showed a 10-fold increase in CT mRNA, with no change in CGRP mRNA. A similar effect was observed in ganglia from adult rats. ProCT immunoreactivity was localized in glial cells. Cutting the tissue blunted the increase in CT, suggesting that induction required the close environment of the intact ganglia. Consistent with this prediction, there were increased reactive oxygen species in the ganglia, and the elevated CT mRNA was reduced by antioxidant treatment. Surprisingly, reactive oxygen species were increased in neurons, not glia. These results demonstrate that reactive oxygen species can activate proCT expression from the CGRP gene in trigeminal glia by a paracrine regulatory mechanism. We propose that this glial recruitment pathway may occur following cortical spreading depression and neurogenic inflammation to increase CGRP nociceptive actions in migraine.

Cervical lymph node metastases from thyroid cancer: does thyroglobulin and calcitonin measurement in fine needle aspirates improve the diagnostic value of cytology?

BackgroundMeasurement of thyroglobulin (Tg) protein in the washout of the needle used for fine needle aspiration biopsy cytology (FNAB-C) has been shown to increase the sensitivity of FNAB-C in identifying cervical lymph node (CLN) metastasis from well-differentiated thyroid cancer (TC). In this study, we evaluated whether routine measurement of Tg protein (FNAB-Tgp), Tg mRNA (FNAB-Tgm) and calcitonin (CT) mRNA (FNAB-CTm) in the FNAB washout of CLN increases the accuracy of FNAB-C in the diagnosis of suspicious metastatic CLN.MethodsIn this prospective study 35 CLN from 28 patients were examined. Histology showed metastatic papillary TC (PTC) in 26 CLN, metastatic medullary TC (MTC) in 3 CLN, metastatic anaplastic TC (ATC) in 3 CLN and 3 metastatic CLN from extra-thyroidal cancers.ResultsThe overall accuracy of FNAB-C was 84.4%, reaching 95.7% when the analysis was restricted to PTC. Both FNAB-Tgp and FNAB-Tgm compared favorably with FNAB-C and shown diagnostic performances not statistically different from that of FNAB-C. However, FNAB-Tgp and FNAB-Tgm/FNAB-CTm were found useful in cases in which cytology results were inadequate or provided diagnosis inconsistent with patient's clinical parameters.ConclusionsWe demonstrated that FNAB-C, Tg/CT mRNA and Tg protein determination in the fine-needle washout showed similar accuracy in the diagnosis of metastatic CLN from TC. The results of this study suggest that samples for Tg protein and Tg/CT mRNA measurements from CLN suspicious for metastatic TC should be collected, but their measurements should be restricted to cases in which FNAB-C provides uninformative or inconsistent diagnosis with respect to patient's clinical parameters.

Calcitonin gene-related peptide (CGRP)

Calcitonin gene-related peptide (CGRP) is a 37 amino acid vasoactive neuropeptide that is widely distributed in central and peripheral nervous systems in mammals. CGRP was discovered in 1982 by molecular cloning of calcitonin (CT) gene. CGRP-specific mRNA appears to predominate in the hypothalamus while CT mRNA predominates in the thyroid. CGRP has an amphiphilic α-helical secondary structure in the amino acid sequence between 8-25. Half-life of CGRP in mammalian plasma is about 10 min. CGRP is immunohistochemically co-localized with substance P in cardiovascular system and it has similar role to that of substance P. CGRP is secreted by primary afferents and causes primary hyperalgesia, and its expression increases in dorsal horn under sensitization conditions. CGRP plays important role in blood pressure system. (Nature and Science. 2004;2(3):41-47).

Epigenetic regulation of the calcitonin gene–related peptide gene in trigeminal glia

The neuropeptide calcitonin gene-related peptide (CGRP) plays a key role in migraine. CGRP gene expression involves an enhancer that is active in neurons, yet inactive in glia. In this report, we analyze epigenetic modifications that allow enhancer activation in glia. DNA methylation and histone acetylation states were measured in rat and human- model cell lines and primary cultures of rat trigeminal ganglia glia. The functional consequence of altering the chromatin state was determined by quantitative measurements of both calcitonin (CT) and CGRP mRNAs. A hypermethylated CpG island flanking the enhancer was identified in glia and non-expressing cell lines. In addition, the chromatin was hypoacetylated. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine induced CT mRNA ~30-fold in glial cultures. Treatment with a histone deacetylase inhibitor alone had little effect; however, the combination of inhibitors yielded a synergistic ~80-fold increase in CT and ~threefold increase in CGRP mRNA. Treated glia contained CT precursor (pro-CT) immunoreactivity. Epigenetic modulation is sufficient to induce the CGRP gene in glia. Because the CGRP gene is systemically activated by inflammatory conditions, this suggests that glial pro-CT may be an unexplored biomarker during migraine.

Involvement of the Cyclic-AMP-dependent Protein Kinase A Pathway in Thyroxine Effects on Calcitonin Secretion from TT Cells

Previous studies have demonstrated that plasma calcitonin is lower in hypothyroid patients and that thyroxine stimulates the human thyroid to release calcitonin. Therefore, thyroid hormones may regulate the secretion of calcitonin, but further work is needed to address this possibility in more detail. TT cells, a model of human thyroid C cells, were incubated in a medium containing vehicle, thyroxine, or thyroxine methyl-hemisuccinate-bovine serum albumin (BSA-L-T(4), thyroxine was immobilized and linked to BSA); then, the levels of secreted calcitonin (hCT), calcitonin mRNA, and cAMP were measured. To study links that connect the cAMP-dependent protein kinase A (PKA) pathway to the observed thyroxine effects, cells were treated with either vehicle or thyroxine plus SQ22536 [an adenylyl cyclase (AC) inhibitor], KT5720 (a PKA inhibitor), or 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor). The activity levels of AC and PKA, and secreted calcitonin were then measured. The results indicate that thyroxine increases calcitonin secretion, cellular cAMP accumulation, and the activities of AC and PKA, but does not increase hCT mRNA levels in TT cells. BSA-L-T(4) also increases calcitonin secretion. These effects are inhibited by SQ22536, and KT5720 and suggest that the nongenomic thyroxine effects that stimulate calcitonin secretion from TT cells involve the cAMP-dependent PKA pathway.

Expression Profile and Localization of Mouse Calcitonin (CT) Sense/Antisense Transcripts in Pre- and Postnatal Tissue Development

Calcitonin (CT) has been shown to have various functions including osteoclast activity and calcium and phosphorus metabolism in mammals. In the present study, we measured the amounts of CT mRNA in the mouse brain, liver, kidney, heart and testis at various development stages, 14 days post-coitum (dpc), 17-dpc, newborn, 1 week and 8 weeks (adult), using real-time PCR. In the brain and kidney, the amount of CT mRNA decreased with development. In the testis, elevated amounts were observed at 17-dpc and 8 weeks. In the liver, the amount increased from the 14 dpc embryo to newborn stage and then decreased. In the heart, elevated amounts were observed at 17-dpc. Additionally, the CT antisense transcript was determined using a modified RT-PCR and nucleotide sequencing in the present study. Organs with high mRNA expressions were examined for localization of transcripts using in situ hybridization. The CT sense and antisense transcripts in the 14 dpc brain were mainly localized in the mesencephalon. In the pre- and postnatal stages, sense and antisense transcripts were shown to exist rather uniformly in the kidney, heart, liver and testis. In the 17-dpc rib and thyroid lobe and the adult ovary, the sense and antisense transcripts were found to be densely localized.

The Murine Glucagon-Like Peptide-1 Receptor Is Essential for Control of Bone Resorption

Gastrointestinal hormones including gastric inhibitory polypeptide (GIP), glucagon-like peptide (GLP)-1, and GLP-2 are secreted immediately after meal ingestion, and GIP and GLP-2 have been shown to regulate bone turnover. We hypothesize that endogenous GLP-1 may also be important for control of skeletal homeostasis. We investigated the role of GLP-1 in the regulation of bone metabolism using GLP-1 receptor knockout (Glp-1r(-/-)) mice. A combination of bone density and histomorphometry, osteoclast activation studies, biochemical analysis of calcium and PTH, and RNA analysis was used to characterize bone and mineral homeostasis in Glp-1r(-/-) and Glp-1r(+/+) littermate controls. Glp-1r(-/-) mice have cortical osteopenia and bone fragility by bone densitometry as well as increased osteoclastic numbers and bone resorption activity by bone histomorphometry. Although GLP-1 had no direct effect on osteoclasts and osteoblasts, Glp-1r(-/-) mice exhibited higher levels of urinary deoxypyridinoline, a marker of bone resorption, and reduced levels of calcitonin mRNA transcripts in the thyroid. Moreover, calcitonin treatment effectively suppressed urinary levels of deoxypyridinoline in Glp-1r(-/-), mice and the GLP-1 receptor agonist exendin-4 increased calcitonin gene expression in the thyroid of wild-type mice. These findings establish an essential role for endogenous GLP-1 receptor signaling in the control of bone resorption, likely through a calcitonin-dependent pathway.

The cyclic AMP-dependent protein kinase A pathway is involved in progesterone effects on calcitonin secretion from TT cells

It is well known that gonadal steroid hormones influence the level of plasma calcitonin (CT), but the mechanism by which progesterone affects CT secretion is not clear. Immortalized TT cells are a reliable model system for studying the endocrine function of human parafollicular cells. In the present study, the effects of progesterone on CT secretion were examined in TT cells. TT cells were incubated in medium containing vehicle (DMSO), progesterone or BSA-progesterone for 60 or 150 min, and then the levels of CT in the medium, progesterone receptors, cAMP accumulation and CT mRNA expression were measured. To study the correlation between progesterone effects and the cAMP-dependent protein kinase A (PKA) pathway, cell lysates or cells in 24-well plates were treated with either vehicle or progesterone plus RU486, SQ22536, KT5720, or 3-isobutyl-1-methylxanthine. Then, adenylyl cyclase and protein kinase A (PKA) activities were measured in the cell lysates, and the CT levels were measured in the medium from the 24-well plate. The activated cAMP response element binding protein (P-CREB) was also measured by immunofluorescence. Administration of 1 μM progesterone or 500 nM BSA-progesterone increased the secretion of CT by 381% and 100%, respectively. Progesterone receptors A and B were downregulated by progesterone treatment. The cAMP concentration, adenylyl cyclase and PKA activity, CT mRNA expression, and nuclear P-CREB concentrations all showed an increase after progesterone treatment. RU486, SQ22536 and KT5720 inhibited the progesterone-stimulated effects. These results suggest that a cAMP-dependent PKA pathway is involved in progesterone-stimulated effects on CT secretion from TT cells.

The RET Kinase Inhibitor NVP-AST487 Blocks Growth and Calcitonin Gene Expression through Distinct Mechanisms in Medullary Thyroid Cancer Cells

The RET kinase has emerged as a promising target for the therapy of medullary thyroid cancers (MTC) and of a subset of papillary thyroid cancers. NVP-AST487, a N,N'-diphenyl urea with an IC(50) of 0.88 mumol/L on RET kinase, inhibited RET autophosphorylation and activation of downstream effectors, and potently inhibited the growth of human thyroid cancer cell lines with activating mutations of RET but not of lines without RET mutations. NVP-AST487 induced a dose-dependent growth inhibition of xenografts of NIH3T3 cells expressing oncogenic RET, and of the MTC cell line TT in nude mice. MTCs secrete calcitonin, a useful indicator of tumor burden. Human plasma calcitonin levels derived from the TT cell xenografts were inhibited shortly after treatment, when tumor volume was still unchanged, indicating that the effects of RET kinase inhibition on calcitonin secretion were temporally dissociated from its tumor-inhibitory properties. Accordingly, NVP-AST487 inhibited calcitonin gene expression in vitro in TT cells, in part, through decreased gene transcription. These data point to a previously unknown physiologic role of RET signaling on calcitonin gene expression. Indeed, the RET ligands persephin and GDNF robustly stimulated calcitonin mRNA, which was blocked by pretreatment with NVP-AST487. Antagonists of RET kinase activity in patients with MTC may result in effects on plasma calcitonin that are either disproportionate or dissociated from the effects on tumor burden, because RET kinase mediates a physiologic pathway controlling calcitonin secretion. The role of traditional tumor biomarkers may need to be reassessed as targeted therapies designed against oncoproteins with key roles in pathogenesis are implemented.

Expression of Thyroglobulin and Calcitonin in Spontaneous Thyroid Gland Tumors in the Han Wistar Rat

Spontaneous follicular and C-cell tumors of the thyroid gland in the Han Wistar rat were examined using two morphologic procedures. Firstly, in situ hybridization (ISH) was used to localize thyroglobulin (TG) and calcitonin (CT) mRNAs. Secondly, the proteins for these markers were detected using immunohistochemistry (IHC). The aim was to study the morphology of the tumors and to examine the usefulness of TG and CT markers in the differential diagnosis of these lesions. Follicular tumors with cystic, papillary and follicular patterns showed relatively consistent expression of TG mRNA by ISH, thereby confirming the diagnostic value of this technique. However, no staining for TG markers was observed in solid lesions. In general, C-cell tumors comprised well-differentiated cells that continued to express CT mRNA and peptides even after embolic spread and metastasis. Therefore, the performance of either ISH or IHC for CT markers can be used for diagnostic confirmation. Additional features noted in C-cell tumors included the appearance of tumor emboli or metastases in association with small primary lesions (less than 5 average follicular diameters in size) and the presence of eosinophilic (amyloid-like) material showing immunopositivity for CT peptides. Finally, evidence is provided for the sequestration of TG protein by proliferating C-cells.

Calcitonin is expressed in the chicken pituitary gland: influence of gonadal steroids and sexual maturation

Calcitonin (CT) is primarily produced by the thyroid C cells in mammals or by the ultimobranchial gland in chickens. CT is also expressed by the pituitary gland in rats in which it functions as a paracrine factor causing decreased lactotroph proliferation and prolactin (PRL) secretion. Gonadal steroids influence CT expression in the rat pituitary gland. However, the expression of the CT gene in the pituitary gland of chickens or of any other avian species has not previously been reported. We have tested the hypotheses that CT is expressed in the chicken pituitary gland, and that its expression is influenced by sexual maturation or in response to ovarian steroid administration. We have detected robust expression of CT cDNA in the chicken pituitary gland by reverse transcription/polymerase chain reaction (PCR). The sequence of the pituitary-derived CT cDNA is identical to that of the ultimobranchial gland. CT-immunoreactive (ir) cells have been observed throughout the anterior pituitary gland by confocal microscopy. Many of the PRL-ir cells show co-localization with CT-ir cells. Quantitative real-time PCR analysis has revealed an inverse relationship between the quantities of PRL mRNA and CT mRNA in the pituitary gland: sexually mature hens contain lower amounts of CT mRNA but larger quantities of PRL mRNA compared with sexually immature chickens. Estradiol and/or progesterone treatment of sexually immature chickens leads to a significant decrease in the quantity of pituitary CT mRNA relative to that in the vehicle-treated chickens. We conclude that pituitary CT plays an important paracrine/autocrine role in the control of lactotroph function and PRL secretion in the chicken.

Calcitonin induces apoptosis resistance in prostate cancer cell lines against cytotoxic drugs via the Akt/Survivin pathway

The expression of calcitonin (CT) and CT-receptor (CTR) mRNA in primary prostate tumors increase with tumor progression. Since advanced prostate tumors display chemoresistance, we tested a hypothesis that CT increases apoptosis resistance of prostate cells against cytotoxic drugs.We examined the effect of CT on etoposide-induced apoptosis in PC-3M, LNCaP and NRP-152 cell lines. The cytoprotective actions of CT were then tested on paclitaxel-, dexamethasone- and selenite-induced apoptosis. We also examined cytotoxic actions of these drugs in CTR-silenced PC-3M cells. Since the role of Akt and inhibitors of apoptosis proteins (IAPs) in chemoresistance of advanced prostate cancers has been established, we tested the effect of CT on phospho-Akt and survivin levels in PC-3M cells. Finally, the cytoprotective effect of CT on PC-3M cells was tested in the presence of PI3K inhibitors such as LY 294002 and wortmannin.Acutely added CT significantly attenuated apoptosis of PC cell lines in response to etoposide, dexamethasone and selenite treatment, but could not reduce paclitaxel-induced apoptosis. CT potently stimulated phospho-Akt and survivin synthesis in PC-3M cells in a sustained manner, and LY 294002 attenuated CT-induced survivin synthesis as well as apoptosis resistance.These results suggest that CT induces chemoresistance to etoposide, dexamethasone and selenite but not to paclitaxel in prostate cells. Cytoprotective action of CT is mediated by CTR-induced activation of Akt-survivin pathway. Since CT/CTR expression in prostate cancers increases with tumor progression, the suppression of “CT System” may enhance the effectiveness of chemotherapy.

Calcitonin increases invasiveness of prostate cancer cells: Role for cyclic AMP-dependent protein kinase A in calcitonin action

Calcitonin (CT) is synthesized and secreted in prostate epithelium, and its secretion from malignant prostates is several-fold higher than from benign prostates. CT receptor (CTR) is expressed in malignant prostate epithelium, and its activation stimulates growth of prostate cancer (PC) cells via activation of adenylyl cyclase and calcium/phospholipid pathways. To identify the role of "CT System" in prostate cancer, we tested the expression of CT and CTR mRNAs in invading tumor cells of prostate cancer specimens. The effect of CT on in vitro invasion of PC cell lines and on activation of gelatinases was also examined. The cells of primary tumors and those invading stroma co-expressed CT/CTR mRNAs. Exogenously added CT increased in vitro invasion of PC cell lines and caused a rapid, several-fold but transient increase in protein kinase A activity. In contrast, anti-CT serum caused a dose-dependent inhibition of in vitro invasion of PC-3M cells. CT also increased the concentration and activities of MMP-2 and MMP-9. Rp.cAMP, a competitive inhibitor of cAMP-dependent protein kinase A, myristoylated protein kinase A inhibitory peptide (PKI) as well as the expression of dominant negative form of PKA all attenuated basal in vitro invasion of PC-3M cells, and CT could not increase in vitro invasiveness in their presence. These results suggest that overexpression of "CT System" in invasive PC tumors significantly contributes to increased invasiveness of prostate cancer cells. The action of CT may be mediated by protein kinase A signaling, which subsequently leads to increased cell invasion and secretion of gelatinases.