Investigation of salmon calcitonin in regulating fibrosis-related molecule production and cell-substrate adhesion in frozen shoulder synovial/capsular fibroblasts.

The purpose of this study was to evaluate the effect of salmon calcitonin (sCT) on improving fibrosis-related indicators in frozen shoulder synovial/capsular fibroblasts (SCFs) and detect the potential downstream pathway. Quantitative real-time polymerase chain reaction and cell-substrate adhesion assays were used to measure alterations in fibrosis-related molecule expression and the cell adhesion ability of frozen shoulder SCFs after treatment with range concentrations of sCT. The presence of calcitonin receptors (CTRs) in shoulder joint synovial/capsular tissue samples was detected by immunohistochemistry (IHC). The downstream pathways of sCT in SCFs were further explored by utilizing three classical pathway inhibitors. With the addition of sCT to the culture medium of frozen shoulder SCFs, the messenger RNA (mRNA) expression of collagen type I (COL1A1), COL3A1, fibronectin 1, laminin 1, transforming growth factor-ß1 (TGF-ß1), and interleukin-1α (IL-1α) showed a descending trend as the sCT concentration increased. Treatment with sCT increased the expression of vascular endothelial growth factor and IL-6 in a dose-dependent manner. The enhanced adhesion ability of frozen shoulder SCFs gradually diminished with increasing concentrations of sCT. By using IHC, the CTR was detected extensively in the frozen shoulder joint synovium and capsule. Blocking the protein kinase C (PKC) pathway reversed the sCT-mediated suppression of COL1A1 production. Blocking the PKC or protein kinase A (PKA) pathway eliminated the sCT-induced inhibition of TGF-ß1 production. This study demonstrated that sCT effectively improved the mRNA expression of fibrosis-related molecules and decreased the enhanced cell-substrate adhesion ability of frozen shoulder SCFs. sCT might achieve these effects by interacting with the CTR that is expressed on the SCF surface and by activating the downstream PKC or PKA pathway.

Estudio de un método inmunoturbidimétrico para la cuantificación de procalcitonina y su capacidad para el diagnóstico de sepsis / Study of an immunoturbidimetric method for quantifying procalcitonin and its ability to diagnose sepsis

Introducción. La procalcitonina (PCT) es un marcador bioquímico para el diagnóstico de sepsis. La electroquimioluminiscencia se considera actualmente el método de referencia para cuantificar PCT. El objetivo de este trabajo es estudiar un método inmunoturbidimétrico para cuantificar la PCT, comprobar su correlación analítica con el método electroquimioluminiscente y establecer su capacidad para el diagnóstico de sepsis bacteriana. Material y métodos. El método inmunoturbidimétrico fue el Diazyme® Procalcitonin Assay (Diazyme Laboratories, Poway, CA, EE. UU.) y el método electroquimioluminiscente fue el Elecsys Brahms PCT (Roche Diagnostics®). El estudio comparativo se realizó con muestras de plasma de pacientes provenientes de diferentes servicios del Hospital Francesc de Borja. Las muestras se analizaron en paralelo en un modular Cobas 6000 (Roche Diagnostics®). Resultados. Se analizaron 97 muestras. Se obtuvo un coeficiente de correlación intraclase de 0,86 (IC 95%: 0,80-0,91). Comparando los dos métodos según los rangos aceptados en la literatura, se observó un acuerdo total del 79,4%, con un coeficiente k no ponderado de 0,72 (IC 95%: 0,61-0,83) y de 0,82 (IC 95%: 0,74-0,90) tras ponderación con pesos lineales. Se obtuvo un área bajo la curva de 0,88 para el método electroquimioluminiscente y de 0,86 para el método inmunoturbidimétrico. Las odds ratio para la PCT fueron 1,19 (IC 95%: 1,06-1,33) y 1,06 (IC 95%: 1,00-1,11) medida por electroquimioluminiscencia y por inmunoturbidimetría, respectivamente. Conclusiones. El método inmunoturbidimétrico de Diazyme® es un método fiable para el diagnóstico y manejo de los pacientes con sospecha de sepsis tal y como lo es en la actualidad el método electroquimioluminiscente de Brahms® (AU)

Introduction. Procalcitonin (PCT) is a biochemical marker for the diagnosis of sepsis. Electrochemiluminescence is currently considered the reference method for quantifying PCT. The aim of this work is to study an immunoturbidimetric method to measure PCT, compare it with electrochemiluminescence method, and establish its capacity for diagnosis of bacterial sepsis. Material and methods. The immunoturbidimetric method was the Diazyme® Procalcitonin Assay (Diazyme Laboratories, Poway, CA, USA), and the electrochemiluminescence method was the Elecsys Brahms PCT. The comparative study was performed with patient plasma samples from different services of the Hospital Francesc de Borja. Samples were analysed in parallel on a Cobas 6000 modular. Results. A total of 97 samples were analysed. Intraclass correlation coefficient of 0.86 (95% CI: 0.80-0.91) was obtained. Comparing the two methods according to the ranges accepted in the literature, total agreement of 79.4% was observed, with an unweighted k coefficient of 0.72 (95% CI: 0.61 - 0.83) and 0.82 (95% CI: 0.74 - 0.90) after weighting with linear weights. An Area under the curve of 0.88 was obtained for the electrochemiluminescence method and 0.86 for the immunoturbidimetric method. The odds ratio for the PCT were 1.19 (95% CI: 1.06 - 1.33) and 1.06 (95% CI: 1.00 - 1.11) measured by electrochemiluminescence and immunoturbidimetry, respectively. Conclusions. Diazyme® immunoturbidimetric method seems to be as reliable a method for the diagnosis and management of patients with suspected sepsis as the current Brahms® electrochemiluminescence method (AU)

Saliva suppresses osteoclastogenesis in murine bone marrow cultures.

Saliva can reach mineralized surfaces in the oral cavity; however, the relationship between saliva and bone resorption is unclear. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro. We used murine bone marrow cultures to study osteoclast formation. The addition of fresh sterile saliva eliminated the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). In line with the histochemical staining, saliva substantially reduced gene expression of cathepsin K, calcitonin receptor, and TRAP. Addition of saliva led to considerably decreased gene expression of receptor activator of nuclear factor kappa-B (RANK) and, to a lesser extent, that of c-fms. The respective master regulators of osteoclastogenesis (c-fos and NFATc1) and the downstream cell fusion genes (DC-STAMP and Atp6v0d2) showed decreased expression after the addition of saliva. Among the costimulatory molecules for osteoclastogenesis, only OSCAR showed decreased expression. In contrast, CD40, CD80, and CD86-all costimulatory molecules of phagocytic cells-were increasingly expressed with saliva. The phagocytic capacity of the cells was confirmed by latex bead ingestion. Based on these in vitro results, it can be concluded that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype.

Glucocorticoid and calcitonin receptor expression in central giant cell lesions: implications for therapy.

Central giant cell lesion is an uncommon benign jaw lesion, with uncertain aetiology, and variable clinical behaviour. Studies of molecular markers may help to understand the nature and behaviour of this lesion, and eventually may represent a target for pharmacological approaches to treatment. The aim of this study was to analyse the expression of glucocorticoid and calcitonin receptors in central giant cell lesions before and after treatment with intralesional steroid. Paraffin-embedded blocks from patients who underwent treatment with intralesional triamcinolone hexacetonide injections were stained immunohistochemically. Biological material from patients who underwent a surgical procedure after treatment were tested immunohistochemically. 18 cases (9 aggressive and 9 non-aggressive) were included. The difference in calcitonin receptor expression was not statistically significant between the aggressive and non-aggressive lesions and between the patients with a good response and those with a moderate/negative response to treatment. Glucocorticoid receptor expression in the multinucleated giant cells was higher in patients with a good response. It can be postulated that immunohistochemical staining for glucocorticoid receptors may provide a tool for selecting the therapeutic strategy. An H-score greater than 48 for glucocorticoid receptors in multinucleated giant cells predicted a good response in this study.

Variability of the paracrine-induced osteoclastogenesis by human breast cancer cell lines.

Breast cancer frequently metastasizes to the bone, often leading to the formation of osteolytic lesions. This work compares the paracrine-induced osteoclastogenesis mediated by four human breast cancer cell lines, the estrogen-receptor positive T47D and MCF-7 and the estrogen-negative SK-BR-3 and Hs-578T cell lines. Human osteoclast precursor cells were cultured in the presence of conditioned media from the breast cancer cell lines (10% and 20%), collected at different culture periods (48 h, 7 days, and 14 days). Cultures performed in the absence or the presence of M-CSF and RANKL served as negative and positive control, respectively. Results showed that the cell lines differentially expressed several osteoclastogenic genes. All cell lines exhibited a significant osteoclastogenic potential, evidenced by a high TRAP activity and number of osteoclastic cells, expression of several osteoclast-related genes, and, particularly, a high calcium phosphate resorption activity. Differences among the osteoclastogenic potential of the cell lines were noted. T47D and MCF-7 cell lines displayed the highest and the lowest osteoclastogenic response, respectively. Despite the variability observed, MEK and NF-κB signaling pathways, and, at a lesser extent, PGE2 production, seemed to have a central role on the observed osteoclastogenic response. In conclusion, the tested breast cancer cell lines exhibited a high osteoclastogenic potential, although with some variability on the cell response profile, a factor to be considered in the development of new therapeutic approaches for breast cancer-induced bone metastasis.

Development of a cyclic adenosine monophosphate assay for Gi-coupled G protein-coupled receptors by utilizing the endogenous calcitonin activity in Chinese hamster ovary cells.

Activation of G(i)-coupled G protein-coupled receptor (GPCRs) by their ligands leads to inhibition of adenylyl cyclase (AC) and reduction of cyclic adenosine monophosphate (cAMP) levels in cells. The traditional cAMP assay for G(i)-coupled GPCRs commonly uses forskolin, a nonspecific AC activator, to increase the basal cAMP level in cells to create an assay window for ligand detection. However, there is still a need to develop a nonforskolin-based cAMP assay because of the challenges inherent in titrating the concentration of forskolin to achieve a reliable assay window, along with issues related to the cAMP-independent effects of forskolin. Herein, we describe such an assay by utilizing the endogenous activity of the calcitonin receptor in Chinese hamster ovary (CHO) cells. The calcitonin receptor is a G(s)-coupled GPCR that, when activated by calcitonin, leads to the stimulation of AC and increases cAMP in cells. Thus, we use calcitonin, instead of forskolin, to increase the basal cAMP level in CHO cells to achieve an assay window. We demonstrated that calcitonin peptides robustly increased cAMP accumulation in several CHO cell lines stably expressing well-known G(i)-coupled GPCRs, such as the Dopamine D2 receptor, the Opioid µ receptor, or the Cannabinoid receptor-1. Agonists of these G(i)-coupled GPCRs attenuated calcitonin-induced cAMP production in their receptor stable cell lines. On the other hand, antagonists and/or inverse agonists blocked the effects of their agonists on calcitonin-induced cAMP production. This calcitonin-based cAMP assay has been demonstrated to be sensitive and robust and exhibited acceptable assay windows (signal/noise ratio) and, thus, can be applied to screen for agonists and antagonists/inverse agonists of G(i)-coupled GPCRs in high-throughput screening formats.

Erosive arthritis in a patient with pycnodysostosis: an experiment of nature.

OBJECTIVE: The excellent poster painter Henri de Toulouse-Lautrec is the most famous patient with cathepsin K-deficient pycnodysostosis. Cathepsin K is believed to play a major role in osteoclast-driven bone resorption. In this study we explored the role of cathepsin K in bone resorption in a patient with a cathepsin K mutation causing pycnodysostosis in whom psoriatic arthritis also developed. We hypothesized that the patient would develop only inflammatory synovitis but would not develop bone erosions or other osteolytic changes. METHODS: Monocytes from the patient with pycnodysostosis and normal control monocytes were isolated and stimulated to fuse and form multinuclear osteoclast-like cells, which were identified by evaluating messenger RNA expression of osteoclast markers. The ability to resorb bone was assessed by determining the extent of pit formation and levels of collagen degradation products generated by cathepsin K (C-terminal crosslinking telopeptide of type I collagen [CTX]) and matrix metalloproteinases (pyridinoline crosslinked C-terminal telopeptide of type I collagen). These experiments were also done in normal control cells after incubation with the cathepsin K inhibitor E64 during bone resorption. RESULTS: In contrast to our a priori hypothesis, the patient developed a mutilating disease with extensive bony erosions associated with lysis of some of the distal phalanges of her hands and feet. After stimulation of monocytes from this patient, the cells formed multinuclear tartrate-resistant acid phosphatase-positive and calcitonin receptor-positive multikaryons, which, however, totally lacked cathepsin K. These multinuclear cells were able to resorb bone but, in contrast to normal control osteoclasts, did not produce CTX. The resorption pattern was abnormal in that, unlike normal control osteoclasts, both osteoclasts from the patient and E64-inhibited osteoclasts did not leave extensive osteoclast trails, but were relatively sessile. CONCLUSION: In this "experiment of nature" we observed that cathepsin K is not necessary for bone degradation. These findings may be pertinent to our understanding of the functions of cathepsin K inhibitors, which are currently being developed as drugs to treat metabolic bone diseases.

Osteoclast differentiation during experimental tooth movement by a short-term force application: an immunohistochemical study in rats.

OBJECTIVE: The origin of osteoclasts responsible for bone resorption during orthodontic tooth movement is not yet clear. Their precursors may reside within the periodontal ligament (PDL) or could be recruited from the circulation or the bone marrow. The aim of this study was to investigate the spatial and sequential distribution of osteoclast precursors during experimental tooth movement by using three differentiation markers: receptor for macrophage colony stimulating factor (c-Fms), receptor activator of nuclear factor-kappaB (RANK), and calcitonin receptor (CTR). MATERIAL AND METHODS: Six-week-old Wistar rats were used. Elastic bands were inserted between the upper 1st and 2nd molars for 1, 2, 3, and 6 days. Immunohistochemical staining for c-Fms, RANK, or CTR was performed on parasagittal sections and positive cells were counted. RESULTS: Before force application, many c-Fms+ and a few RANK+ precursors were present in the bone marrow. No c-Fms+ osteoclast precursors were observed in the PDL. After force application, the number of RANK+ but not c-Fms+ precursors increased rapidly in the PDL. In bone marrow, the number of c-Fms+ and RANK+ precursors also increased rapidly, as did multinuclear c-Fms+, RANK+, and CTR+ cells. Subsequently, the number of c-Fms+, RANK+, and CTR+ multinuclear cells in the PDL increased. After 6 days, the expression profiles tended to return to baseline levels. CONCLUSION: Osteoclast precursors differentiate within the bone marrow and then migrate into the PDL during early tooth movement.

Evidence that human cartilage and chondrocytes do not express calcitonin receptor.

OBJECTIVE: Calcitonin (CT) has been recently shown to exhibit direct protective effects on articular cartilage against joint degenerative disease. It has been proposed that CT might act via the CT receptor (CTR) to activate the cyclic AMP (cAMP) pathway and protect type II collagen degradation. In this study, we investigated the existence of CTR in human articular cartilage and chondrocytes, and examined the potential pharmacological effects and transduction pathway of salmon CT (sCT) in human chondrocytes. METHODS: Five human articular cartilage samples were examined for the expression of the CTR by polymerase chain reaction (PCR), immunostaining and Western blot analysis. cAMP levels in human chondrocyte stimulated with sCT were assessed by ELISA. The effect of sCT on the gene expression profiles, including aggrecan, type II collagen, MMP-1, MMP-3 and MMP-13, of human chondrocytes was also examined by relative quantitative Real-time PCR. RESULTS: We failed to detect the CTR at both the transcriptional and protein levels in human chondrocytes and cartilage tissue by PCR, immunostaining and Western blotting. cAMP levels were significantly elevated in human chondrocytes by forskolin (100muM) to more than 10-fold (P<0.001), however, were not induced by sCT (10(-7)M, 10(-8)M, 10(-9)M). Real-time PCR analysis demonstrated that sCT slightly reduced the gene expression of MMPs, although this effect was not statistically significant. CONCLUSION: In contrary to previous reports, our data indicate that human cartilage and chondrocytes do not express CTR. Furthermore, sCT does not appear to have direct effects on human chondrocytes. We propose that the chondroprotective effect of CT observed in vivo may be indirect via its impact on subchondral bone resorptive activity of osteoclasts.

Knock-down of calcitonin receptor expression induces apoptosis and growth arrest of prostate cancer cells.

Calcitonin (CT) and its receptor (CTR) are expressed only in basal epithelium of benign prostate and in whole epithelium of malignant prostates. Also, CT and CTR mRNA levels in prostate cancers increase with an increase in tumor grade. We tested the role of the CT/CTR autocrine axis on the tumorigenicity of prostate cancer cells. We enforced the expression of CTR in CT-positive/CTR-deficient PC-3 cells. In contrast, we knocked down CTR expression in CT/CTR-positive PC-3M cells. The effect of CTR modulation on the oncogenicity was evaluated by the rate of cell proliferation, invasion, colony formation and in vivo growth in nude mice. Up-regulation of CTR in PC-3 cells and its down-regulation in PC-3M cells significantly altered their tumorigenicity. Intratumorally administered CTR RNAi in preexisting PC-3M xenografts markedly attenuated their further growth. This treatment also led to a remarkable decrease in endothelial cell populations in the tumors and increase in apoptotic, PCNA-negative cell populations. Tumors receiving CTR RNAi treatment displayed markedly lower levels of urokinase-type plasminogen activator, phospho-Akt and survivin, suggesting CTR activates uPA-uPAR axis and PI-3-kinase-Akt-survivin pathway. These results suggest an important role for CT-CTR autocrine axis in the progression of localized prostate tumor to a metastatic phenotype, and offer a potential therapeutic option for invasive cancers.

Adrenomedullin stimulates nitric oxide production from primary rat hypothalamic neurons: roles of calcium and phosphatases.

Adrenomedullin (ADM) in the brain plays important roles in the maintenance of homeostasis. Although in vivo evidence has suggested that nitric oxide (NO) mediates ADM's effects in the brain, mechanisms for ADM stimulation of NO production in neurons have not been identified. In the present study, primary hypothalamic neurons were used to characterize ADM-induced NO production and to study the underlying mechanisms. Using Calcium Orange/4-amino-5-methylamino-2',7'-difluorofluorescein fluorescence live cell imaging, we found that ADM (1 or 10 nM, 5 min) significantly elevated [Ca(2+)](i) and NO production in a concentration-dependent manner. Ca(2+) and NO responses induced by 10 nM ADM were abolished by pretreatment with 50 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), an intracellular Ca(2+) chelator, or protein kinase A (PKA) inhibitors 5 microM N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and 50 microM Rp-cAMP. Furthermore, the ADM-induced NO production was significantly attenuated by a protein phosphatase 1/2A inhibitor, okadaic acid (OA; 0.1 microM), or calcineurin inhibitors, tacrolimus (FK506) (1 microM) and cyclosporin A (CsA; 0.1 microM). Using Western blotting, we found that ADM significantly decreased phosphorylation of neuronal nitric-oxide synthase (nNOS) at serine 847. This dephosphorylation was inhibited by 0.1 microM OA, 1 microM FK506, 0.1 microM CsA, or 5 microM H-89, and attenuated by 50 microM BAPTA-AM. These results suggest that, in hypothalamic neurons, ADM elevates [Ca(2+)](i) via PKA-associated mechanisms. The PKA/Ca(2+) cascade leads to protein phosphatase (PP) 1/PP2A- and calcineurin-mediated dephosphorylation of nNOS. We hypothesize that the Ca(2+) increase and nNOS dephosphorylation contribute to activation of nNOS and production of NO in hypothalamic neurons.

Unaltered mRNA expression of calcitonin-like receptor and receptor activity modifying proteins in human arteries in stroke and myocardial infarction.

Calcitonin-like receptor (CL-R) is a functional CGRP1-receptor when complexed with RAMP1 or an adrenomedullin-receptor or when complexed with RAMP2 or RAMP3. This study was carried out 1. to set up a method to examine the relative quantity of mRNA of CL-R, RAMP1, RAMP2 and RAMP3 in human coronary (CA), pulmonary (PA) and middle cerebral arteries (MCA), and 2. to examine the level of mRNA expression in cerebra- and cardiovascular diseases. The method was validated with respect to the use of postmortem tissue and we compared beta-actin and GAPDH as housekeeping genes. There was no time-dependent change in total RNA and level of mRNA for p-actin or GAPDH could be detected in vessels removed from 1 and 5 days post mortem. The expression of beta-actin appears lower in coronary artery than in pulmonary artery and middle cerebral artery with no significant difference for GAPDH; both worked well. There were some differences in mRNA expression for CL-R (higher) and RAMP3 (lower) in middle cerebral artery compared to coronary artery and pulmonary artery. There was no significant difference in mRNA for RAMP1 and RAMP2 in the three types of arteries. We did not observe any difference in mRNA for CL-R and RAMPs in arteries from patients with hemorrhagic stroke, arteriosclerosis and acute myocardial infarction when compared to patients without these diagnoses. Thus the mRNA expression seems to be unaltered in these disorders.

Quantitative analysis of osteoclast-specific gene markers stimulated by lipopolysaccharide.

Lipopolysaccharide (LPS) in the outer layers of Gram-negative bacteria plays an important role in initiating and sustaining periapical lesions. To understand the mechanisms of osteoclastic bone resorption in periapical lesions induced by LPS, we stimulated osteoclast precursors, RAW 264.7 cells with LPS. LPS stimulated osteoclastogenesis when osteoclast precursors were primed with activator for NF-kappaB ligand (RANKL) as little as 24 h. By employing real-time PCR analysis, we have confirmed that osteoclast-like cells stimulated by LPS express high level of osteoclast-specific gene markers such as TRAP, cathepsin K, and calcitonin receptor. These results suggest that bone-resportive action by LPS is partially independent of RANKL.

Immunohistochemical expression of glucocorticoid and calcitonin receptors as a tool for selecting therapeutic approach in central giant cell granuloma of the jawbones.

Aggressive cases of central giant cell granuloma (CGCG) have been arbitrarily treated with steroids and calcitonin. The aim of this study was to develop a practical tool, based on the relative percentage of positively stained cells for glucocorticoid and/or calcitonin receptors, for selecting the appropriate therapeutic agents to treat CGCG. Forty-one formalin-fixed, paraffin-embedded blocks of CGCG were immunohistochemically stained for glucocorticoid and calcitonin receptors. Percentage of positive lesional mononuclear and giant cells was estimated for each case. Intense staining was considered as staining 50% or more of the cells. Correlations among staining scores were analysed by Spearman's test. All cases stained for glucocorticoid receptor. Heterogeneity among cases showed as intense staining in both cell types (21 lesions), in only one cell type (13 lesions) and weakly in both cell types (7 lesions). Only 23 cases demonstrated staining for calcitonin receptor, of which 15 stained intensely in both cell types and 7 in only one cell type. Among staining scores of both receptors, no significant statistical correlation was found (P>0.05). It can therefore be suggested that the relative percentage of immunohistochemically stained mononuclear and giant cells for glucocorticoid and/or calcitonin receptors can serve as a reliable and practical tool for selecting the appropriate therapeutic agent to treat CGCG. The clinical application of this method should be assessed in well controlled clinical studies, especially in cases of aggressive lesions, before initiating and during therapeutic treatment.

Calcitonin directly attenuates collagen type II degradation by inhibition of matrix metalloproteinase expression and activity in articular chondrocytes.

OBJECTIVE: Calcitonin was recently reported to counter progression of cartilage degradation in an experimental model of osteoarthritis, and the effects were primarily suggested to be mediated by inhibition of subchondral bone resorption. We investigated direct effects of calcitonin on chondrocytes by assessing expression of the receptor and pharmacological effects on collagen type II degradation under ex vivo and in vivo conditions. METHODS: Localization of the calcitonin receptor on articular chondrocytes was investigated by immunohistochemistry, and the expression by reverse transcriptase polymerase chain reaction (RT-PCR). In bovine articular cartilage explants, cartilage degradation was investigated by release of C-terminal telopeptides of collagen type II (CTX-II), induced by tumor necrosis factor-alpha (TNF-alpha) [20 ng/ml] and oncostatin M (OSM) [10 ng/ml], with salmon calcitonin [0.0001-1 microM]. In vivo, cartilage degradation was investigated in ovariectomized (OVX) rats administered with oral calcitonin [2 mg/kg calcitonin] for 9 weeks. RESULTS: The calcitonin receptor was identified in articular chondrocytes by immunohistochemistry and RT-PCR. Calcitonin concentration-dependently increased cAMP levels in isolated chondrocytes. Explants cultured with TNF-alpha and OSM showed a 100-fold increase in CTX-II release compared to vehicle-treated controls (P<0.001). The degradation of type II collagen in these explants was concentration-dependently inhibited by calcitonin, 65% protection at 10 nM calcitonin (P<0.01). TNF-alpha and OSM induced a pronounced increase in matrix metalloproteinase (MMP) activity, which was strongly inhibited by calcitonin. In vivo, administration of salmon calcitonin to OVX rats resulted in significant (P<0.001) decrease in CTX-II levels. CONCLUSION: These results are the first evidence of calcitonin receptor expression on articular chondrocytes and that the chondroprotective effects of calcitonin might involve the inhibition of MMP expression.

Local application of prostaglandin E2 reduces trap, calcitonin receptor and metalloproteinase-2 immunoreactivity in the rat periodontium.

It has been shown that prostaglandin E2 (PGE2) locally released adjacent to the mandible over a 20-day period increases alveolar bone area, in part, due to a reduction in the percentage of eroded surface. To determine the effect of PGE2 on alveolar bone resorption, left mandibles from 24 Lewis rats were treated over a 20-day period with a local application of PGE2 (0.1, 0.05 or 0.025 mg/day) or placebo. The right side served as the non-treated matched control. Tissue sections were stained for tartrate resistant acid phosphatase (TRAP) calcitonin receptor (CTR) and metalloproteinase-2 (MMP-2). Matched samples were analysed by Wilcoxon matched pairs test and, a non-parametric one-way analysis of variance compared groups of treatment. Those tissues treated with PGE2 at doses of 0.1 and 0.05 mg/day showed significantly reduced numbers of TRAP and CTR-positive multinucleated cells compared with matched controls (p<0.005), as well as significantly reduced numbers of TRAP- and CTR-positive multinucleated cells when compared with the placebo-treated group (p<0.001). The number of periodontal ligament cells expressing MMP-2 was also significantly reduced in tissues treated with the two higher doses of PGE2 (p<0.001) comparing with both matched controls and the placebo-treated group. Following a 20-day period, locally released PGE2 at doses of 0.1 and 0.05 mg/day appears to affect alveolar bone resorption in the periodontium of rats, as the number of multinucleated cells expressing TRAP and CTR are significantly reduced. Furthermore, the same doses of PGE2 also significantly reduced the expression of MMP-2 by the periodontal cells.

Evidence for decreased calcitonin gene-related peptide (CGRP) receptors and compromised responsiveness to CGRP of fetoplacental vessels in preeclamptic pregnancies.

Calcitonin gene-related peptide (CGRP) is a potent vasodilatory peptide, and its concentration is increased in both maternal and fetal circulation during late pregnancy. The present study was designed to investigate the expression of CGRP receptor components, calcitonin receptor-like receptor (CRLR), and receptor activity modifying protein 1 (RAMP1), and the relaxation response to CGRP in fetoplacental vessels from normotensive pregnant women and women with preeclampsia. Results showed that: 1) mRNA for both CRLR and RAMP1 was expressed in fetoplacental vessels from normal pregnancies; however, these mRNA expressions were substantially reduced in the vessels from preeclamptic women; 2) CRLR and RAMP1 proteins were abundantly expressed in the endothelium and smooth muscle layer of the fetoplacental vessels, as well as the trophoblast cells in normal placentas. In contrast, both vascular tissues and trophoblasts showed decreased expressions for CRLR and RAMP1 proteins and declined CGRP binding sites in preeclamptic placentas; and 3) CGRP produced a dose-dependent relaxation of serotonin-induced contraction of umbilical and chorionic arteries from normal pregnancies, but the response to CGRP was significantly attenuated in the vessels from preeclampsia. We concluded that CGRP may contribute to the low fetoplacental vascular resistance in normal pregnancies; however, CGRP-dependent vascular relaxation appears to be compromised in preeclamptic pregnancies.

Progesterone induction of calcitonin expression in the murine mammary gland.

Progesterone, via its nuclear receptor, is mandatory not only for the induction and specification of mammary gland ductal side-branching and lobuloalveologenesis but also for carcinogen-induced mammary tumorigenesis. Notwithstanding these recent advances, a more comprehensive molecular explanation of progesterone-induced mammary morphogenesis is contingent upon the identification and characterization of mammary molecular targets that are responsive to the progesterone signal. Toward this goal, we report that calcitonin, a 32 amino acid peptide hormone involved in calcium homeostasis, is exclusively expressed in, and secreted from, luminal epithelial cells within the mammary gland of the pregnant mouse, and, importantly, its expression is progesterone-dependent. Conversely, the calcitonin receptor is present during all stages of post-natal mammary development examined, is localized to the myoepithelial cell lineage, and is not regulated by progesterone. Because calcitonin induction spatiotemporally correlates with increases in progesterone-induced mammary gland proliferation and structural remodeling, we posit that calcitonin - through its receptor - may be involved in one or both of these progesterone-dependent processes.

Calcitonin in human odontoclasts regulates root resorption activity via protein kinase A.

Calcitonin is a known inhibitor of osteoclastic bone resorption, but it remains uncertain whether calcitonin also regulates human odontoclastic activity, particularly during the physiological process of root resorption. In this study, we examined the expression of calcitonin receptors in human odontoclasts and the effect of calcitonin on root resorption, using immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Actin-ring formation was used to assess cytostructural changes during resorption activity. Our results show that calcitonin receptors are expressed in human odontoclasts freshly isolated from deciduous teeth of the periodontal region. Calcitonin inhibited actin-ring formation and resorption activity. This calcitonin-induced inhibition was mimicked by forskolin and dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP), which are protein kinase A (PKA) activators, but not by phorbol 12-myristate 13-acetate, a protein kinase C activator. Pretreatment with adenosine 3',5'-cyclic monophosphothioate Rp diastereomer (Rp-cAMPS), a PKA inhibitor, suppressed the calcitonin-induced inhibition of actin-ring formation. These results indicate that calcitonin receptor activation suppresses odontoclastic root resorption via PKA, a signaling pathway different from that in human osteoclasts.

Immunohistochemical detection of the calcitonin receptor-like receptor protein in the microvasculature of rat endothelium.

Calcitonin-gene-related peptide and adrenomedullin have similar and potent vascular effects, which appear to be mediated by the G protein-coupled calcitonin receptor-like (CRL) receptor. Using immunohistochemical and Western blot analyses, we have obtained novel evidence that CRL receptor is expressed in the rat vascular endothelium using an antibody to rat CRL receptor that we have raised and fully characterised. These results are an important basis for further studies aimed at determining the so far ill-defined functional significance of the extensive distribution of CRL receptor in the vascular endothelium.