Expression Of Calcitonin Receptor Research Articles

MiR-148b-5p regulates hypercalciuria and calcium-containing nephrolithiasis

Calcium-containing stones represent the most common form of kidney calculi, frequently linked to idiopathic hypercalciuria, though their precise pathogenesis remains elusive. This research aimed to elucidate the molecular mechanisms involved by employing urinary exosomal microRNAs as proxies for renal tissue analysis. Elevated miR-148b-5p levels were observed in exosomes derived from patients with kidney stones. Systemic administration of miR-148b-5p in rat models resulted in heightened urinary calcium excretion, whereas its inhibition reduced stone formation. RNA immunoprecipitation combined with deep sequencing identified miR-148b-5p as a suppressor of calcitonin receptor (Calcr) expression, thereby promoting urinary calcium excretion and stone formation. Mice deficient in Calcr in distal epithelial cells demonstrated elevated urinary calcium excretion and renal calcification. Mechanistically, miR-148b-5p regulated Calcr through the circRNA-83536/miR-24-3p signaling pathway. Human kidney tissue samples corroborated these results. In summary, miR-148b-5p regulates the formation of calcium-containing kidney stones via the circRNA-83536/miR-24-3p/Calcr axis, presenting a potential target for novel therapeutic interventions to prevent calcium nephrolithiasis.Graphical abstract

Hindbrain REV-ERB nuclear receptors regulate sensitivity to diet-induced obesity and brown adipose tissue pathophysiology

ObjectiveThe dorsal vagal complex (DVC) of the hindbrain is a major point of integration for central and peripheral signals that regulate a wide variety of metabolic functions to maintain energy balance. The REV-ERB nuclear receptors are important modulators of molecular metabolism, but their role in the DVC has yet to be established. MethodsMale REV-ERBα/β floxed mice received stereotaxic injections of a Cre expressing virus to the DVC to create the DVC REV-ERBα/β double knockout (DVC RDKO). Control littermates received stereotaxic injections to the DVC of a green fluorescent protein expressing virus. Animals were maintained on a normal chow diet or a 60% high-fat diet to observe the metabolic phenotype arising from DVC RDKO under healthy and metabolically stressed conditions. ResultsDVC RDKO animals on high-fat diet exhibited increased weight gain compared to control animals maintained on the same diet. Increased weight gain in DVC RDKO animals was associated with decreased basal metabolic rate and dampened signature of brown adipose tissue activity. RDKO decreased gene expression of calcitonin receptor in the DVC and tyrosine hydroxylase in the brown adipose tissue. ConclusionsThese results suggest a previously unappreciated role of REV-ERB nuclear receptors in the DVC for maintaining energy balance and metabolic rate potentially through indirect sympathetic outflow to the brown adipose tissue.

Amylin Modulates a Ventral Tegmental Area–to–Medial Prefrontal Cortex Circuit to Suppress Food Intake and Impulsive Food-Directed Behavior

BackgroundA better understanding of the neural mechanisms regulating impaired satiety to palatable foods is essential to treat hyperphagia linked with obesity. The satiation hormone amylin signals centrally at multiple nuclei including the ventral tegmental area (VTA). VTA–to–medial prefrontal cortex (mPFC) projections encode food reward information to influence behaviors including impulsivity. We hypothesized that modulation of VTA-to-mPFC neurons underlies amylin-mediated decreases in palatable food-motivated behaviors. MethodsWe used a variety of pharmacological, behavioral, genetic, and viral approaches (n = 4–16/experiment) to investigate the anatomical and functional circuitry of amylin-controlled VTA-to-mPFC signaling in rats. ResultsTo first establish that VTA amylin receptor (calcitonin receptor) activation can modulate mPFC activity, we showed that intra-VTA amylin decreased food-evoked mPFC cFos. VTA amylin delivery also attenuated food-directed impulsive behavior, implicating VTA amylin signaling as a regulator of mPFC functions. Palatable food activates VTA dopamine and mPFC neurons. Accordingly, dopamine receptor agonism in the mPFC blocked the hypophagic effect of intra-VTA amylin, and VTA amylin injection reduced food-evoked phasic dopamine levels in the mPFC, supporting the idea that VTA calcitonin receptor activation decreases dopamine release in the mPFC. Surprisingly, calcitonin receptor expression was not found on VTA-to-mPFC projecting neurons but was instead found on GABAergic (gamma-aminobutyric acidergic) interneurons in the VTA that provide monosynaptic inputs to this pathway. Blocking intra-VTA GABA signaling, through GABA receptor antagonists and DREADD (designer receptor exclusively activated by designer drugs)–mediated GABAergic neuronal silencing, attenuated intra-VTA amylin-induced hypophagia. ConclusionsThese results indicate that VTA amylin signaling stimulates GABA-mediated inhibition of dopaminergic projections to the mPFC to mitigate impulsive consumption of palatable foods.

IL-37 alleviates alveolar bone resorption and inflammatory response through the NF-κB/NLRP3 signaling pathway in male mice with periodontitis

ObjectivePeriodontitis is an inflammatory disease, characterized by periodontal pocket formation and alveolar bone resorption, is one of the most common oral diseases. Interleukin-37 (IL-37) is a novel inflammatory suppressor that plays an important role in many inflammatory diseases. This study aimed to investigate the role of IL-37 in periodontitis DesignA mouse model of periodontitis was established by Porphyromonas gingivalis. After four weeks treatment of recombinant human IL-37 (rhIL-37), the effects of IL-37 on the gingival index and tooth loosening degree of periodontitis mice were observed. H&E staining and micro-CT scanning were used to analyze the bone resorption of the maxillary. The number of osteoclasts was counted by TRAP staining and the differentiation of osteoclasts was evaluated by immunohistochemistry. The expression of inflammatory cytokines was detected by ELISA, and the protein expressions of the NF-κB/NLRP3 pathway were analyzed by WB. ResultsRhIL-37 significantly decreased the gingival index and tooth mobility degree, inhibited maxillary bone resorption, decreased the number of osteoclasts and the expression of calcitonin receptor (CTR), periodontal cathepsin K (CTSK) and receptor activator of NF-κB ligand (RANKL), and increased the expression of osteoprotegerin (OPG) in periodontitis mice. At the same time, rhIL-37 significantly decreased the expression of IL-1β, IL-6 and TNF-α, and increased the expression of IL-10 in the gingival tissue of periodontitis mice. In addition, rhIL-37 significantly inhibited the protein expressions of p-p65, NLRP3, ASC, caspase-1 and IL-1β in periodontitis mice. ConclusionIL-37 may alleviate alveolar bone resorption and inflammation response in periodontitis through the NF-κB/NLRP3 pathway.

Unravelling the role of immunohistochemistry in giant cell lesions of jaws: A systematic review.

Controversies exist in literature regarding nature, pathogenesis, and behaviour of giant cell lesions (GCLs) of jaws. Studies were attempted to solve these mysteries with immunohistochemical analysis, using various biological markers. Thus, the aim of this review is to appraise the role of immunohistochemistry (IHC) in evaluating the pathogenesis, cellular phenotype, nature, and behaviour of GCLs of jaws. PubMed, PubMed Central, and Clinical Key (Medline) databases were searched electronically irrespective of date of publication with assortment of several independent terms. Fifty-five articles that fulfilled the eligibility criteria were included in the review. Out of 55 included articles, 49 were associated with nature, pathogenesis, and behaviour and six articles were associated with treatment and outcome prediction. Although IHC solved some of the controversies associated with GCLs of jaws such as the osteoclastic phenotype of multinucleated giant cells, immunoexpression of proliferative markers does not distinct non-aggressive from aggressive central GCL but the nature, histogenesis, pathogenesis, and exact behaviour still remain debatable. With regard to formulation of treatment plan, immunohistochemical analysis revealed that expression of glucocorticoid and calcitonin receptors could act as a tool to decide the therapeutic strategy and aid in therapeutic adjustments according to evolution of the lesion.

Dieckol isolated from Eisenia bicyclis extract suppresses RANKL-induced osteoclastogenesis in murine RAW 264.7 cells

Objective: To demonstrate the effect of dieckol from Eisenia bicyclis on osteoclastogenesis using RAW 264.7 cells. Methods: Murine macrophage RAW 264.7 cells were subjected to dieckol treatment, followed by treatment with receptor activator of nuclear factor kappa-B ligand (RANKL) to induce osteoclastogenesis. Tartrate-resistant acid phosphatase (TRAP) activity was examined using a TRAP activity kit. Western blotting analysis was conducted to examine the level of osteoclast- related factors, including TRAP and calcitonin receptor (CTR), transcriptional factors, including c-Fos, c-Jun, and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), nuclear factor kappa-B (NF-κB), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Immunofluorescence staining was conducted to examine the expression of c-Fos, c-Jun, and NFATc1. Results: Among the four phlorotannin compounds present in Eisenia bicyclis, dieckol significantly hindered osteoclast differentiation and expression of RANKL-induced TRAP and CTR. In addition, dieckol downregulated the expression levels of c-Fos, c-Jun, NFATc1, ERK, and JNK, and suppressed NF-κB signaling. Conclusions: Dieckol can suppress RANKL-induced osteoclastogenesis. Therefore, it has therapeutic potential in treating osteoclastogenesis- associated diseases.

Calcitonin receptor antibody validation and expression in the rodent brain.

Therapeutics that reduce calcitonin gene-related peptide activity are effective migraine treatments. However, gaps remain in our understanding of the molecular mechanisms that link calcitonin gene-related peptide to migraine. The amylin 1 receptor responds potently to calcitonin gene-related peptide, and to the related peptide amylin, but its role in relation to either peptide or to migraine is unclear. We sought to better understand the expression of the amylin 1 receptor protein subunit, the calcitonin receptor, in the rodent brain. We profiled three antibodies for immunodetection of calcitonin receptor, using immunocytochemistry, western blotting, and calcitonin receptor conditional knockout mouse tissue. Selected migraine-relevant rat brain regions were then examined for calcitonin receptor-like immunoreactivity. All three antibodies detected calcitonin receptor protein but only one (188/10) produced robust immunostaining in rodent brain, under the conditions used. Calcitonin receptor-like immunoreactivity was apparent in the rat brainstem and midbrain including the locus coeruleus, periaqueductal grey and spinal trigeminal nucleus. Anti-calcitonin receptor antibodies require comprehensive profiling to ensure confidence in the detection of calcitonin receptor. Using a validated antibody, calcitonin receptor-like immunoreactivity was detected in several brain regions relevant to migraine. Further research is needed to understand the functional consequences of calcitonin receptor expression for calcitonin gene-related peptide or amylin physiology and pathophysiology.

The calcitonin receptor protects against bone loss and excessive inflammation in collagen antibody-induced arthritis

SummaryPharmacological application of teleost calcitonin (CT) has been shown to exert chondroprotective and anti-resorptive effects in patients with rheumatoid arthritis (RA). However, the role of endogenous CT that signals through the calcitonin receptor (CTR) remains elusive. Collagen II antibody-induced arthritis (CAIA) was stimulated in wild type (WT) and CTR-deficient (Calcr−/−) mice. Animals were monitored over 10 or 48 days. Joint inflammation, cartilage degradation, and bone erosions were assessed by clinical arthritis score, histology, histomorphometry, gene expression analysis, and μ-computed tomography. CAIA was accompanied by elevated systemic CT levels and CTR expression in the articular cartilage. Inflammation, cartilage degradation, and systemic bone loss were more pronounced in Calcr−/− CAIA mice. Expression of various pro-inflammatory, bone resorption, and catabolic cartilage markers were exclusively increased in Calcr−/− CAIA mice. Endogenous CT signaling through the mammalian CTR has the potential to protect against joint inflammation, cartilage degradation, and excessive bone remodeling in experimental RA.

Attenuation of CALCR protein expression in stone affected region of kidney biopsy tissue: Case presentation

Calcitonin is one of the most important hormones in human body which regulates calcium homeostasis. It binds with Calcitonin Receptor (CALCR) and reduces serum calcium level by inhibiting bone resorption and proximal renal reabsorption. Alteration in calcium homeostasis promotes diseases like osteoporosis, urolithiasis etc. An interesting finding of reduced CALCR protein expression in a urolithiasis patient motivated us to investigate its level in stone affected region of kidney biopsy tissue and hence in the present study, CALCR expression was checked in urolithiasis patients who had gone through open nephrolithotomy for stone removal. Basic and clinical parameters of the patients were recorded followed by analysis of stone composition, genotypic analysis and studying CALCR protein expression level in the kidney biopsy tissue samples (in the location of stone and an adjacent control area). CALCR protein expression was found to be attenuated in the stone affected region of kidney biopsy tissue as compared to adjacent control tissues (4-6 cm apart from the stone affected region) in all our studied samples (n=5). This case series represents an interesting finding as it is the first time report in the world, regarding expression of CALCR protein in kidney tissues of urolithiasis patients. Keywords: Urolithiasis; CALCR; Protein expression; SNP.

Procalcitonin Exerts a Mediator Role in Septic Shock Through the Calcitonin Gene-Related Peptide Receptor.

Clinically, procalcitonin represents the most widely used biomarker of sepsis worldwide with unclear pathophysiologic significance to date. Pharmacologically, procalcitonin was shown to signal through both calcitonin receptor and calcitonin gene-related peptide receptor in vitro, yet the identity of its biologically relevant receptor remains unknown. Prospective randomized animal investigations and in vitro human blood studies. Research laboratory of a university hospital. C57BL/6J mice and patients with post-traumatic sepsis. Procalcitonin-deficient mice were used to decipher a potential mediator role in experimental septic shock and identify the relevant receptor for procalcitonin. Cecal ligation and puncture and endotoxemia models were employed to investigate septic shock. Disease progression was evaluated through survival analysis, histology, proteome profiling, gene expression, and flow cytometry. Mechanistic studies were performed with cultured macrophages, dendritic cells, and gamma delta T cells. Main findings were confirmed in serum samples of patients with post-traumatic sepsis. Procalcitonin-deficient mice are protected from septic shock and show decreased pulmonary inflammation. Mechanistically, procalcitonin potentiates proinflammatory cytokine expression in innate immune cells, required for interleukin-17A expression in gamma delta T cells. In patients with post-traumatic sepsis, procalcitonin positively correlates with systemic interleukin-17A levels. In mice with endotoxemia, immunoneutralization of interleukin-17A inhibits the deleterious effect of procalcitonin on disease outcome. Although calcitonin receptor expression is irrelevant for disease progression, the nonpeptide calcitonin gene-related peptide receptor antagonist olcegepant, a prototype of currently introduced antimigraine drugs, inhibits procalcitonin signaling and increases survival time in septic shock. Our experimental data suggest that procalcitonin exerts a moderate but harmful effect on disease progression in experimental septic shock. In addition, the study points towards the calcitonin gene-related peptide receptor as relevant for procalcitonin signaling and suggests a potential therapeutic application for calcitonin gene-related peptide receptor inhibitors in sepsis, which warrants further clinical investigation.

Vav1 inhibits RANKL-induced osteoclast differentiation and bone resorption

Vav1 is a Rho/Rac guanine nucleotide exchange factor primarily expressed in hematopoietic cells. In this study, we investigated the potential role of Vav1 in osteoclast (OC) differentiation by comparing the ability of bone marrow mononuclear cells (BMMCs) obtained from Vav1-deficient (Vav1−/−) and wild-type (WT) mice to differentiate into mature OCs upon stimulation with macrophage colony stimulating factor and receptor activator of nuclear kappa B ligand in vitro. Our results suggested that Vav1 deficiency promoted the differentiation of BMMCs into OCs, as indicated by the increased expression of tartrate-resistant acid phosphatase, cathepsin K, and calcitonin receptor. Therefore, Vav1 may play a negative role in OC differentiation. This hypothesis was supported by the observation of more OCs in the femurs of Vav1−/− mice than in WT mice. Furthermore, the bone status of Vav1−/− mice was analyzed in situ and the femurs of Vav1−/− mice appeared abnormal, with poor bone density and fewer number of trabeculae. In addition, Vav1-deficient OCs showed stronger adhesion to vitronectin, an αvβ3 integrin ligand important in bone resorption. Thus, Vav1 may inhibit OC differentiation and protect against bone resorption.

Expression and activity of the calcitonin receptor family in a sample of primary human high-grade gliomas

BackgroundGlioblastoma (GBM) is the most common and aggressive type of primary brain cancer. With median survival of less than 15 months, identification and validation of new GBM therapeutic targets is of critical importance.ResultsIn this study we tested expression and performed pharmacological characterization of the calcitonin receptor (CTR) as well as other members of the calcitonin family of receptors in high-grade glioma (HGG) cell lines derived from individual patient tumours, cultured in defined conditions.Previous immunohistochemical data demonstrated CTR expression in GBM biopsies and we were able to confirm CALCR (gene encoding CTR) expression. However, as assessed by cAMP accumulation assay, only one of the studied cell lines expressed functional CTR, while the other cell lines have functional CGRP (CLR/RAMP1) receptors. The only CTR-expressing cell line (SB2b) showed modest coupling to the cAMP pathway and no activation of other known CTR signaling pathways, including ERK1/2 and p38 MAP kinases, and Ca2+ mobilization, supportive of low cell surface receptor expression.Exome sequencing data failed to account for the discrepancy between functional data and expression on the cell lines that do not respond to calcitonin(s) with no deleterious non-synonymous polymorphisms detected, suggesting that other factors may be at play, such as alternative splicing or rapid constitutive receptor internalisation.ConclusionsThis study shows that GPCR signaling can display significant variation depending on cellular system used, and effects seen in model recombinant cell lines or tumour cell lines are not always reproduced in a more physiologically relevant system and vice versa.

Reduced expression of calcitonin receptor is closely associated with age‐related loss of the muscle stem cell pool

AbstractBackgroundMuscle regenerative capacity reduces during aging. Decline in the muscle stem cell (MuSC) pool is thought to be a cause of age‐related muscle regeneration insufficiency. However, why the number of MuSCs decreases with age remains unclear. We previously found that the calcitonin receptor (Calcr) is expressed by MuSCs and plays a pivotal role in maintaining the MuSC pool. In this study, we examined whether Calcr is involved in the age‐related decrease in MuSC number.Methods and resultsCalcr expression in MuSCs was assessed by immunofluorescence staining using muscle sections during postnatal growth and during aging. The role of Calcr during development, postnatal growth and adult stage was examined using mice conditionally deleted Calcr in myogenic lineage cells. To explore the significance of Calcr signaling on age‐related changes in MuSC number, expression levels of endogenous Calcr ligands were examined. Immunohistological examination revealed that Calcr expression grew stronger as MuSCs became quiescent. Conditional Calcr deletion in early myogenic lineage cells did not affect the initial capacity of the MuSC pool, but promoted age‐dependent MuSC loss. Expression of endogenous Calcr ligands remained unchanged with aging. Calcr expression in MuSCs, however, decreased significantly in aged muscle compared with young muscle in both mice and humans, and this reduction was accompanied by a loss of MuSCs.ConclusionsOur study revealed significant reduction of Calcr in MuSCs with age, possibly leading to age‐related loss of the MuSC pool. Thus, Calcr signaling could represent a promising target for preventing aging‐associated muscle regeneration insufficiency.

Peripheral blood monocytes show increased osteoclast differentiation potential compared to bone marrow monocytes

Bone marrow (BM) and peripheral blood (PB) derived mononuclear cells are precursors of in vitro osteoclast differentiation. However, few studies have compared the phenotypic and functional properties of osteoclasts generated from these sources and the effects of different growth factors on osteoclastogenesis. Both cell types differentiated into functional osteoclasts, but culturing the cells with or without transforming growth factor beta (TGF-β) and dexamethasone revealed differences in their osteoclastogenic capacity. When receptor activator for nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were used for differentiation, we did not observe differences in bone resorption activity or expression of osteoclastogenic genes calcitonin receptor (CR) and nuclear factor of activated T-cells (NFATc1) between the osteoclasts formed from the two sources. Addition of TGF-β and dexamethasone led to higher number of nuclei in multinuclear cells and increased expression of tartrate resistant acid phosphatase (TRACP) 5a and 5b, CR and NFATc1 in PB- derived osteoclasts depicting the higher osteoclastogenic potential and responsiveness to TGF-β and dexamethasone in PB monocytes. These results conclude that the choice of the osteoclast precursor source as well as the choice of osteoclastogenic growth factors are essential matters in determining the phenotypic characteristics of heterogeneous osteoclast populations.

\u03b1-Melanocyte-stimulating hormone directly increases the plasma calcitonin level and involves calcium metabolism in goldfish

The effects of α-melanocyte-stimulating hormone (α-MSH) on calcium metabolism were examined with goldfish. The scales on the left side of goldfish bodies were removed to allow the regeneration of scales under anesthesia. Thereafter, the influences of α-MSH injection (low dose: 0.1 μg/g body weight; high dose: 1 μg/g body weight) on plasma calcitonin (calcium-regulating hormone) and the calcium content of the scales were investigated. Ten days after removing the scales, we measured the plasma calcitonin and calcium content of both regenerating scales on the left side and ontogenic scales on the right side. At both doses of α-MSH injection, plasma calcitonin concentrations in the α-MSH-treated group were significantly higher than those in the control group. The mRNA expressions of α-MSH-receptors were detected in the ultimobranchial glands (secretory organ of calcitonin), indicating that α-MSH directly functions in ultimobranchial glands and promotes calcitonin secretion. Furthermore, we found that the calcium content of regenerating scales in α-MSH-treated goldfish was higher than that in control goldfish, while the calcium content of ontogenic scales on the right side was significantly decreased by α-MSH injection. There was a significant co-relationship between plasma calcitonin and the calcium content of regenerating scales. The mRNA expression of calcitonin receptors in regenerating scales was remarkably higher than that in ontogenic scales. These results imply that calcitonin functions to promote scale regeneration resulting from the inhibition of bone resorption because calcitonin suppresses osteoclastic activity. Thus, we are the first to demonstrate the interaction between α-MSH and calcitonin in teleosts.

Implant‐associated gene expression in the jaw bone of smokers and nonsmokers: A human study using quantitativeqPCR

This study aimed to compare the molecular events in implant-adherent cells and in peri-implant bone during the osseointegration of machined and oxidized titanium implants in smokers and nonsmokers. Twenty-four smokers and 24 nonsmokers each received machined and anodically oxidized mini-implants. The mini-implants and the surrounding bone were retrieved after 1, 7, and 28days, for gene expression analysis of selected factors using quantitative polymerase chain reaction (qPCR). Differences between machined and oxidized implants were more evident in the implant-adherent cells than the peri-implant bone. The machined implants revealed higher expression of proinflammatory cytokines, interleukin-8 (IL-8) (in nonsmokers), and tumor necrosis factor-alpha (in nonsmokers and smokers), compared with the oxidized implants. Conversely, the expression of bone formation genes, alkaline phosphatase and osteocalcin, was generally higher at the oxidized implants. In smokers, the temporal pattern revealed the delayed and initial inhibition of osteoblastic and osteoclastic gene expression, respectively, mainly at the machined implants. In contrast, oxidized implants revealed higher expression of bone remodeling, cathepsin K (CatK) and calcitonin receptor, and coupling, receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin, genes after 7day in smokers. The implant-adherent cells are more sensitive to surface properties and smoking conditions than the cells in the peri-implant bone. Smoking imposes inhibitory effects on the initial molecular events of osseointegration in the human bone-implant interface. The surface properties of oxidized implants appear to have a beneficial effect on osseointegration by mitigating the smoking-induced negative effects.

Immunoexpression of calcitonin and glucocorticoid receptors in central giant cell lesions of the jaws.

This study analyzed the immunoexpression of calcitonin receptor (CTR) and glucocorticoid receptor (GR) in central giant cell lesions (CGCLs) and verified potential associations with patient's response to clinical treatment with intralesional injection of triamcinolone. Fifty-four cases of CGCLs, including 22 non-aggressive, and 32 aggressive, were investigated by immunohistochemistry. Surgery was the therapeutic choice for 53.1% of the aggressive CGCLs, and 46.9% were submitted to the conservative treatment with intralesional triamcinolone injections. Among patients submitted to conservative treatment, 60% (n=9) showed favorable response. CTR expression was observed in 68.51%, and GR in 94.44% of the total sample. There were no differences in the expression of CTR, neither GR in mononucleated stromal cells (MSCs) or multinucleated giant cells (MGCs), in relation to aggressiveness, treatment performed for and the response to conservative treatment. Both markers showed a positive correlation between their expression in MSCs and MGCs in the total sample (P<0.0001). CTR expression on MSCs showed a positive correlation with MGCs in the aggressive and non-aggressive groups (P<0.0001). Calcitonin receptor and GR expression were diffuse and similar in non-aggressive and aggressive cases, and it did not influence the response to clinical treatment with triamcinolone in the sample studied.

Effect of remifentanil on pre-osteoclast cell differentiation in vitro.

BackgroundThe structure and function of bone tissue is maintained through a constant remodeling process, which is maintained by the balance between osteoblasts and osteoclasts. The failure of bone remodeling can lead to pathological conditions of bone structure and function. Remifentanil is currently used as a narcotic analgesic agent in general anesthesia and sedation. However, the effect of remifentanil on osteoclasts has not been studied. Therefore, we investigated the effect of remifentanil on pre-osteoclast (pre-OCs) differentiation and the mechanism of osteoclast differentiation in the absence of specific stimulus.MethodsPre-OCs were obtained by culturing bone marrow-derived macrophages (BMMs) in osteoclastogenic medium for 2 days and then treated with various concentration of remifentanil. The mRNA expression of NFATc1 and c-fos was examined by using real-time PCR. We also examined the effect of remifentanil on the osteoclast-specific genes TRAP, cathepsin K, calcitonin receptor, and DC-STAMP. Finally, we examined the influence of remifentanil on the migration of pre-OCs by using the Boyden chamber assay.ResultsRemifentanil increased pre-OC differentiation and osteoclast size, but did not affect the mRNA expression of NFATc1 and c-fos or significantly affect the expression of TRAP, cathepsin K, calcitonin receptor, and DC-STAMP. However, remifentanil increased the migration of pre-OCs.ConclusionsThis study suggested that remifentanil promotes the differentiation of pre-OCs and induces maturation, such as increasing osteoclast size. In addition, the increase in osteoclast size was mediated by the enhancement of pre-OC migration and cell fusion.

Shikimic Acid Inhibits Osteoclastogenesis in Vivo and in Vitro by Blocking RANK/TRAF6 Association and Suppressing NF-κB and MAPK Signaling Pathways

Background/Aims: Bone homeostasis is associated with the balance between bone-resorbing osteoclasts and bone-forming osteoblasts. Unbalanced bone homeostasis as a result of reduced osteogenesis or excessive osteoclastogenesis can lead to disorders such as osteoporosis, Paget’s disease, and rheumatoid arthritis. Shikimic acid is a cyclohexanecarboxylic acid, reported to exhibit pharmacological properties including anti-inflammatory and antioxidant activities. However, its effects on bone homeostasis remain unknown. Methods: First, the in vitro MTT cell viability assay was performed. Tartrate-resistant acid phosphatase (TRAP) and actin ring formation assays, as well as immunofluorescence staining were then performed to evaluate osteoclastogenesis. Potential signaling pathways were characterized by western blotting and verified in overexpression experiments. Related factors were examined by western blotting, reverse transcription polymerase chain reaction, electrophoretic mobility shift assay, and co-immunoprecipitation. Ovariectomized mice were used for the in vivo study. Results: TRAP staining showed that shikimic acid significantly inhibited osteoclastogenesis and pit resorption in bone marrow monocytes and RAW264.7 cells, and actin ring formation assays showed that shikimic acid suppressed the bone resorption function of osteoclasts. Furthermore, shikimic acid inhibited the receptor activator of nuclear factor-κB RANK/tumor necrosis factor receptor-associated factor 6 (TRAF6) association, suppressed nuclear factor-κB and mitogen-activated protein kinase signaling pathways, and downregulated nuclear factor of activated T-cell cytoplasmic 1. The expression of osteoclastogenesis biomarkers, including TRAF6, calcitonin receptor, TRAP, cathepsin K, and matrix metalloproteinase-9, was inhibited. In vivo, shikimic acid also significantly ameliorated bone loss and prevented osteoclastogenesis in ovariectomized mice. Conclusion: Shikimic acid inhibited osteoclastogenesis and osteoclast function by blocking RANK ligand-induced recruitment of TRAF6, as well as downstream signaling pathways in vitro. Shikimic acid also reduced ovariectomy-induced osteoclastogenesis and bone loss in vivo.

Characterization of prostate cancer cell progression in zebrafish xenograft model

Early diagnosis of prostate cancer (PCa) is critical for the application of efficient treatment to PCa patients. However, the majority of PCas remains indolent from several months to several years before malignancy. Current diagnosis methods have limitations in their reliability and are inefficient in time cost. Thus, an efficient in vivo PCa cell xenograft model is highly desired for diagnostic studies in PCas. In the present study we present a standardized procedure to create a PCa cell xenograft model using zebrafish (Danio rerio) as the host. PC3-CTR cells, a cell line from adenocarcinoma with stable expression of calcitonin receptor (CRT), were subcutaneously injected into zebrafish larvae at 48 h post fertilization. The nursing conditions for the larvae were optimized with stable survival rates of post hatch and post PC3-CTR cell injection. In this system, the progression of PC3-CTR cells in vivo was evaluated by migration and proliferation of the cells. Massive migrations of PC3 cells in vivo were observed at post injection day (PID)3. The injected PC3-CTR cells eventually invaded the whole larval zebrafish at PID5. Quantification of PC3-CTR cell proliferation was done using quantitative PCR (qPCR) analysis targeting the expression profiles of two PCa housekeeping genes, TATA-binding protein (TBP) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) encoding genes. The excessive proliferation of PC3 cells in vivo was detected with both qPCR assays. Expression levels of one non-coding gene, prostate cancer associated 3 gene (pca3), and two other genes encoding transient receptor potential ion channel Melastatin 8 (trpm8) and prostate-specific membrane antigen (psma), showed a significantly enhanced aggressiveness of PC3-CTR cells in vivo. The model established in the present study provides an improved in vivo model for the diagnosis of PCas efficiently. This PCa cell xenograft model can also serve as a tool for high throughput anti-PCa drug screening in therapeutic treatments.