RESUMO
Receptor activity modifying protein 1 (RAMP1) facilitates the localization of the calcitonin-like receptor (CLR) to the plasma membrane, but its role in osteosarcoma (OS) remains unclear. We evaluated the RAMP1 expression and prognostic value across different cancers, studying tumor immune infiltration. The prognostic value was analyzed using the GSE39058 and TARGET datasets. Differential gene expression was evaluated. a protein-protein interaction network was constructed, and gene set enrichment analysis was performed. The function of RAMP1 in the tumor microenvironment was analyzed, and its expression in OS cell lines was validated using quantitative real-time PCR. High RAMP1 expression correlated with poor prognosis relative to low RAMP1 expression (p < 0.05). Low RAMP1 expression correlated with an abundance of CD4+ memory-activated T cells. whereas a high expression level correlated with a high proportion of gamma-delta T cells (γδ T cells). Differentially expressed genes from TARGET was enriched in olfactory transduction pathways (normalized enrichment scores [NES] = 1.6998, p < 0.0001). RAMP1 expression negatively correlated with CD44 expression but positively correlated with TNFSF9 expression. The RAMP1 gene is substantially expressed in OS cells compared to the normal osteoblast cell line hFOB1.19. Thus, RAMP1 may be a prognostic biomarker and potential therapeutic target in OS.
Assuntos
Osteossarcoma , Receptores da Calcitonina , Humanos , Proteína 1 Modificadora da Atividade de Receptores/genética , Prognóstico , Linhagem Celular , Receptores da Calcitonina/genética , Receptores da Calcitonina/metabolismo , Osteossarcoma/genética , Biomarcadores , Microambiente TumoralRESUMO
The calcitonin (CT)/CT gene-related peptide (CGRP) family is a peptide gene family that is widely found in bilaterians. CT, CGRP, adrenomedullin (AM), amylin (AMY), and CT receptor-stimulating peptide (CRSP) are members of the CT/CGRP family. In mammals, CT is involved in calcium homeostasis, while CGRP and AM primarily function in vasodilation. AMY and CRSP are associated with anorectic effects. Diversification of the molecular features and physiological functions of the CT/CGRP family in vertebrate lineages have been extensively reported. However, the origin and diversification mechanisms of the vertebrate CT/CGRP family of peptides remain unclear. In this review, the molecular characteristics of CT/CGRP family peptides and their receptors, along with their major physiological functions in mammals and teleosts, are introduced. Furthermore, novel candidates of the CT/CGRP family in cartilaginous fish are presented based on genomic information. The CT/CGRP family peptides and receptors in urochordates and cephalochordates, which are closely related to vertebrates, are also described. Finally, a putative evolutionary scenario of the CT/CGRP family peptides and receptors in chordates is discussed.
Assuntos
Depressores do Apetite , Cordados , Neuropeptídeos , Hormônios Peptídicos , Adrenomedulina , Animais , Calcitonina/genética , Peptídeo Relacionado com Gene de Calcitonina/química , Peptídeo Relacionado com Gene de Calcitonina/genética , Cálcio , Peixes/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Mamíferos , Proteínas Modificadoras da Atividade de Receptores , Receptores da Calcitonina/genética , Tomografia Computadorizada por Raios X , VertebradosRESUMO
The peptide hormone amylin reduces food intake and body weight and is an attractive candidate target for novel pharmacotherapies to treat obesity. However, the short half-life of native amylin and amylin analogs like pramlintide limits these compounds' potential utility in promoting sustained negative energy balance. Here, we evaluate the ability of the novel long-acting amylin/calcitonin receptor agonist ZP5461 to reduce feeding and body weight in rats, and also test the role of calcitonin receptors (CTRs) in the dorsal vagal complex (DVC) of the hindbrain in the energy balance effects of chronic ZP5461 administration. Acute dose-response studies indicate that systemic ZP5461 (0.5-3 nmol/kg) robustly suppresses energy intake and body weight gain in chow- and high-fat diet (HFD)-fed rats. When HFD-fed rats received chronic systemic administration of ZP5461 (1-2 nmol/kg), the compound initially produced reductions in energy intake and weight gain but failed to produce sustained suppression of intake and body weight. Using virally mediated knockdown of DVC CTRs, the ability of chronic systemic ZP5461 to promote early reductions in intake and body weight gain was determined to be mediated in part by activation of DVC CTRs, implicating the DVC as a central site of action for ZP5461. Future studies should address other dosing regimens of ZP5461 to determine whether an alternative dose/frequency of administration would produce more sustained body weight suppression.
Assuntos
Agonistas dos Receptores da Amilina/farmacologia , Depressores do Apetite/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Receptores da Calcitonina/agonistas , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/efeitos dos fármacos , Rombencéfalo/efeitos dos fármacos , Nervo Vago/efeitos dos fármacos , Aumento de Peso/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Ingestão de Energia/efeitos dos fármacos , Masculino , Ratos Sprague-Dawley , Receptores da Calcitonina/genética , Receptores da Calcitonina/metabolismo , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genética , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Rombencéfalo/metabolismo , Transdução de Sinais , Fatores de Tempo , Nervo Vago/metabolismoRESUMO
Based upon its interactions with amyloid ß peptide (Aß), the amylin receptor, a class B G protein-coupled receptor (GPCR), is a potential modulator of Alzheimer's disease (AD) pathogenesis. However, past pharmacological approaches have failed to resolve whether activation or blockade of this receptor would have greater therapeutic benefit. To address this issue, we generated compound mice expressing a human amyloid precursor protein gene with familial AD mutations in combination with deficiency of amylin receptors produced by hemizygosity for the critical calcitonin receptor subunit of this heterodimeric GPCR. These compound transgenic AD mice demonstrated attenuated responses to human amylin- and Aß-induced depression of hippocampal long-term potentiation (LTP) in keeping with the genetic depletion of amylin receptors. Both the LTP responses and spatial memory (as measured with Morris water maze) in these mice were improved compared to AD mouse controls and, importantly, a reduction in both the amyloid plaque burden and markers of neuroinflammation was observed. Our data support the notion of further development of antagonists of the amylin receptor as AD-modifying therapies.
Assuntos
Doença de Alzheimer/genética , Aprendizagem em Labirinto/fisiologia , Receptores da Calcitonina/genética , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/genética , Memória Espacial/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/psicologia , Precursor de Proteína beta-Amiloide/genética , Animais , Células Cultivadas , Endotélio Vascular/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Receptores da Calcitonina/deficiência , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/deficiênciaRESUMO
The adrenomedullin (AM) family is involved in diverse biological functions, including cardiovascular regulation and body fluid homeostasis, in multiple vertebrate lineages. The AM family consists of AM1, AM2, and AM5 in tetrapods, and the receptor for mammalian AMs has been identified as the complex of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2) or RAMP3. However, the receptors for AM in amphibians have not been identified. In this study, we identified the cDNAs encoding calcrl (clr), ramp2, and ramp3 receptor components from the western clawed frog (Xenopus tropicalis). Messenger RNAs of amphibian clr and ramp2 were highly expressed in the heart, whereas that of ramp3 was highly expressed in the whole blood. In HEK293T cells expressing clr-ramp2, cAMP response element luciferase (CRE-Luc) reporter activity was activated by am1. In HEK293T cells expressing clr-ramp3, CRE-Luc reporter activity was increased by the treatment with am2 at the lowest dose, but with am5 and am1 at higher dose. Our results provided new insights into the roles of AM family peptides through CLR-RAMP receptor complexes in the tetrapods.
Assuntos
Adrenomedulina , Hormônios Peptídicos , Receptores da Calcitonina , Adrenomedulina/genética , Animais , Proteína Semelhante a Receptor de Calcitonina/genética , Células HEK293 , Humanos , Proteína 2 Modificadora da Atividade de Receptores/genética , Proteína 3 Modificadora da Atividade de Receptores/genética , Receptores de Adrenomedulina/genética , Receptores da Calcitonina/genética , XenopusRESUMO
The area postrema (AP), located in the caudal hindbrain, is one of the primary binding sites for the endocrine satiation hormone amylin. Amylin is co-secreted with insulin from pancreatic ß-cells and binds to heterodimeric receptors that consist of a calcitonin core receptor (CTR) paired with receptor-activity modifying protein (RAMP) 1 or 3. In this study, we aim to validate a CTR-floxed (CTRfl/fl) mouse model for the functional and site-specific depletion of amylin/CTR signaling in the AP and the nucleus tractus solitarius (NTS). CTRfl/fl mice were injected in the NTS with adeno-associated virus (AAV) containing a green fluorescent protein tag (GFP) and Cre recombinase to create a locally restricted knockout of CTR in the caudal hindbrain. KO mice showed a lack of c-Fos expression, a marker for neuronal activation, in the AP, NTS and LPBN after amylin injection. The effect of amylin and salmon calcitonin (sCT), an amylin receptor agonist, on food intake was blunted in KO mice, confirming a functional reduction of amylin signaling in the hindbrain.
Assuntos
Área Postrema , Receptores da Calcitonina , Animais , Área Postrema/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Camundongos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores da Calcitonina/genética , Receptores da Calcitonina/metabolismo , Núcleo Solitário/metabolismoRESUMO
Amylin is a pancreatic peptide, which acts as a key controller of food intake and energy balance and predominately binds to three receptors (AMY 1-3). AMY 1-3 are composed of a calcitonin core receptor (CTR) and associated receptor-activity modifying proteins (RAMPs) 1-3. Using RAMP1, RAMP3 and RAMP1/3 global KO mice, this study aimed to determine whether the absence of one or two RAMP subunits affects food intake, glucose homeostasis and metabolism. Of all the RAMP-deficient mice, only high-fat diet fed RAMP1/3 KO mice had increased body weight. Chow-fed RAMP3 KO and high-fat diet fed 1/3 KO male mice were glucose intolerant. Fat depots were increased in RAMP1 KO male mice. No difference in energy expenditure was observed but the respiratory exchange ratio (RER) was elevated in RAMP1/3 KO. RAMP1 and 1/3 KO male mice displayed an increase in intermeal interval (IMI) and meal duration, whereas IMI was decreased in RAMP3 KO male and female mice. WT and RAMP1, RAMP3, and RAMP1/3 KO male and female littermates were then assessed for their food intake response to an acute intraperitoneal injection of amylin or its receptor agonist, salmon calcitonin (sCT). RAMP1/3 KO were insensitive to both, while RAMP3 KO were responsive to sCT only and RAMP1 KO to amylin only. While female mice generally weighed less than male mice, only RAMP1 KO showed a clear sex difference in meal pattern and food intake tests. Lastly, a decrease in CTR fibers did not consistently correlate with a decrease in amylin- induced c-Fos expression in the area postrema (AP). Ultimately, the results from this study provide evidence for a role of RAMP1 in mediation of fat utilization and a role for RAMP3 in glucose homeostasis and amylin's anorectic effect.
Assuntos
Ingestão de Alimentos , Metabolismo Energético , Glucose , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Proteína 1 Modificadora da Atividade de Receptores , Proteína 3 Modificadora da Atividade de Receptores/genética , Animais , Feminino , Masculino , Camundongos , Proteína 1 Modificadora da Atividade de Receptores/genética , Receptores da Calcitonina/genética , Receptores da Calcitonina/metabolismoRESUMO
ABSTRACT Purpose It has been reported that calcitonin receptor (CALCR) gene polymorphisms might be associated with calcium stone urolithiasis. Owing to mixed and inconclusive results, we conducted a meta-analysis to summarize and clarify this association. Materials and Methods A systematic search of studies on the association between CALCR gene polymorphisms and calcium stone urolithiasis susceptibility was conducted in databases. Results Odds ratios and 95% confidence intervals were used to pool the effect size. Five articles were included in our meta-analysis. Conclusions CALCR rs1801197 might be associated with increased risk of calcium stone urolithiasis. There is insufficient data to fully confirm the association between CALCR rs1042138 and calcium stone urolithiasis susceptibility. Well-designed studies with larger sample size and more subgroups are required to validate the risk identified in the current meta-analysis.
Assuntos
Humanos , Masculino , Feminino , Receptores da Calcitonina/genética , Polimorfismo de Nucleotídeo Único , Urolitíase/genética , Cálcio/metabolismo , Fatores de Risco , Medição de Risco , Estudos de Associação GenéticaRESUMO
Axon guidance molecules, originally found to mediate the positioning of axons during nerve development, have been receiving great attention due to their critical roles in regulating bone metabolism. Recently, SLITs, another group of neuronal guidance proteins, were found to be significantly expressed in bone cells. Furthermore, we had provided experimental evidence that SLIT3 is an osteoclast-secreted coupling factor playing an osteoprotective role. Therefore, we hypothesized that SLIT2, a member of the SLIT family, may also affect bone homeostasis. SLIT2 suppressed osteoclast differentiation in a dose-dependent manner and in vitro bone resorption by more than 80%. Consistently, the expression of osteoclast differentiation markers, such as tartrate-resistant acid phosphatase (Trap) and calcitonin receptor (Ctr), was decreased by SLIT2. The migration and fusion of preosteoclasts were markedly reduced in the presence of SLIT2, suggesting that SLIT2 mainly functions in the early stage of osteoclastogenesis. SLIT2 suppressed Cdc42 activity among small GTPases, whereas Cdc42 overexpression almost completely reversed the SLIT2-mediated suppression of osteoclast differentiation. Among ROBO1-4, the SLIT receptors, ROBO1 and ROBO3 were known to be predominantly expressed in osteoclast lineages. A binding ELISA experiment in mouse bone marrow-derived macrophages showed that ROBO1, rather than ROBO3, was directly associated with SLIT2, and gene silencing with Robo1 siRNA blocked the SLIT2-mediated suppression of osteoclastogenesis. Taken together, our results indicated that SLIT2 inhibits osteoclastogenesis and the resultant bone resorption by decreasing Cdc42 activity, suggesting that this was a potential therapeutic target in metabolic bone diseases related to high bone turnover states.
Assuntos
Reabsorção Óssea/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas do Tecido Nervoso/genética , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Proteína cdc42 de Ligação ao GTP/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Reabsorção Óssea/prevenção & controle , Diferenciação Celular , Proliferação de Células , Fêmur/citologia , Fêmur/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Osteoblastos/citologia , Osteoclastos/patologia , Cultura Primária de Células , Células RAW 264.7 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores da Calcitonina/genética , Receptores da Calcitonina/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais , Fosfatase Ácida Resistente a Tartarato/genética , Fosfatase Ácida Resistente a Tartarato/metabolismo , Tíbia/citologia , Tíbia/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas RoundaboutRESUMO
BACKGROUND: Glioblastoma (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. RESULTS: In 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. CONCLUSIONS: This 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.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Receptores da Calcitonina/genética , Receptores da Calcitonina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/mortalidade , Proteína Semelhante a Receptor de Calcitonina/genética , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Técnicas de Cultura de Células , Proliferação de Células , Glioblastoma/mortalidade , Humanos , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteína 1 Modificadora da Atividade de Receptores/genética , Proteína 2 Modificadora da Atividade de Receptores/genética , Transdução de Sinais , Análise de Sobrevida , Transcriptoma , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: 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. MATERIALS AND METHODS: Fifty-four cases of CGCLs, including 22 non-aggressive, and 32 aggressive, were investigated by immunohistochemistry. RESULTS: 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). CONCLUSIONS: 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.
Assuntos
Células Gigantes/metabolismo , Granuloma de Células Gigantes/tratamento farmacológico , Granuloma de Células Gigantes/metabolismo , Imuno-Histoquímica , Doenças Maxilomandibulares/tratamento farmacológico , Doenças Maxilomandibulares/metabolismo , Receptores da Calcitonina/metabolismo , Receptores de Glucocorticoides/metabolismo , Triancinolona , Adolescente , Adulto , Criança , Feminino , Expressão Gênica , Humanos , Injeções Intralesionais , Masculino , Pessoa de Meia-Idade , Receptores da Calcitonina/genética , Receptores de Glucocorticoides/genética , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
Osteoporosis, a common and multifactorial disease, is influenced by genetic factors and environments. However, the pathogenesis of osteoporosis has not been fully elucidated yet. Recently, emerging evidence suggests that epigenetic modifications may be the underlying mechanisms that link genetic and environmental factors with increased risks of osteoporosis and bone fracture. MicroRNA (miRNA), a major category of small noncoding RNA with 20-22 bases in length, is recognized as one important epigenetic modification. It can mediate post-transcriptional regulation of target genes with cell differentiation and apoptosis. In this review, we aimed to profile the role of miRNA in bone remodeling and its therapeutic implications for osteoporosis. A deeper insight into the role of miRNA in bone remodeling and osteoporosis can provide unique opportunities to develop a novel diagnostic and therapeutic approach of osteoporosis.
Assuntos
Remodelação Óssea/genética , Epigênese Genética , Fraturas Ósseas/genética , MicroRNAs/genética , Osteogênese/genética , Osteoporose/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fraturas Ósseas/epidemiologia , Fraturas Ósseas/metabolismo , Fraturas Ósseas/patologia , Humanos , MicroRNAs/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteoporose/epidemiologia , Osteoporose/metabolismo , Osteoporose/patologia , Receptores da Calcitonina/genética , Receptores da Calcitonina/metabolismo , Transdução de SinaisRESUMO
OBJECTIVES: To investigate the molecular and structural patterns of bone healing during guided bone regeneration (GBR), comparing two resorbable non-cross-linked collagen membranes. MATERIALS AND METHODS: Trabecular bone defects in rat femurs were filled with deproteinized bovine bone (DBB) and covered with either a membrane comprising collagen and elastin (CXP) or collagen (BG). Samples were harvested after 3 and 21 days for histology/histomorphometry and gene expression analysis. Gene expression analysis was performed on the membrane (at 3 days) and the underlying defect compartment (at 3 and 21 days). RESULTS: At the total defect level, no differences in bone area percentage were found between the CXP and BG. When evaluating the central area of the defect, a higher percentage of de novo bone formation was seen for the CXP membrane (34.9%) compared to BG (15.5%) at 21 days (p = .01). Gene expression analysis revealed higher expression of bone morphogenetic protein-2 (Bmp2) in the membrane compartment at 3 days in the BG group. By contrast, higher Bmp2 expression was found in the defect compartment treated with the CXP membrane, both at 3 and 21 days. A significant temporal increase (from 3 to 21 days) in the remodeling activity, cathepsin K (Catk) and calcitonin receptor (Calcr), was found in the CXP group. Molecular analysis demonstrated expression of several growth factors and cytokines in the membrane compartment irrespective of the membrane type. Bmp2 expression in the membrane correlated positively with Bmp2 expression in the defect, whereas fibroblast growth factor-2 (Fgf2) expression in the membrane correlated positively with inflammatory cytokines, tumor necrosis factor-alpha (Tnfa) and interleukin-6 (Il6) in the defect. CONCLUSIONS: The results provide histological and molecular evidence that different resorbable collagen membranes contribute differently to the GBR healing process. In the BG group, bone formation was primarily localized to the peripheral part of the defect. By contrast, the CXP group demonstrated significantly higher de novo bone formation in the central portion of the defect. This increase in bone formation was reflected by triggered expression of potent osteogenic growth factor, Bmp2, in the defect. These findings suggest that the CXP membrane may have a more active role in regulating the bone healing dynamics.
Assuntos
Regeneração Óssea/fisiologia , Colágeno , Fêmur/fisiologia , Regeneração Tecidual Guiada , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Catepsina K/genética , Catepsina K/metabolismo , Citocinas/genética , Citocinas/metabolismo , Elastina , Fêmur/anatomia & histologia , Fêmur/metabolismo , Perfilação da Expressão Gênica , Masculino , Modelos Animais , Osteocalcina/genética , Osteocalcina/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores da Calcitonina/genética , Receptores da Calcitonina/metabolismoRESUMO
Purpose: Despite significant advances in the understanding of the biology, the prognosis of glioblastoma (GBM) remains dismal. The objective was to carry out whole-exome sequencing (WES) of Indian glioma and integrate with that of TCGA to find clinically relevant mutated pathways.Experimental Design: WES of different astrocytoma samples (n = 42; Indian cohort) was carried out and compared with that of TCGA cohort. An integrated analysis of mutated genes from Indian and TCGA cohorts was carried out to identify survival association of pathways with genetic alterations. Patient-derived glioma stem-like cells, glioma cell lines, and mouse xenograft models were used for functional characterization of calcitonin receptor (CALCR) and establish it as a therapeutic target.Results: A similar mutation spectrum between the Indian cohort and TCGA cohort was demonstrated. An integrated analysis identified GBMs with defective "neuroactive ligand-receptor interaction" pathway (n = 23; 9.54%) that have significantly poor prognosis (P < 0.0001). Furthermore, GBMs with mutated calcitonin receptor (CALCR) or reduced transcript levels predicted poor prognosis. Exogenously added calcitonin (CT) inhibited various properties of glioma cells and pro-oncogenic signaling pathways in a CALCR-dependent manner. Patient-derived mutations in CALCR abolished these functions with the degree of loss of function negatively correlating with patient survival. WT CALCR, but not the mutant versions, inhibited Ras-mediated transformation of immortalized astrocytes in vitro Furthermore, calcitonin inhibited patient-derived neurosphere growth and in vivo glioma tumor growth in a mouse model.Conclusions: We demonstrate CT-CALCR signaling axis is an important tumor suppressor pathway in glioma and establish CALCR as a novel therapeutic target for GBM. Clin Cancer Res; 24(6); 1448-58. ©2017 AACR.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Glioblastoma/genética , Glioblastoma/mortalidade , Mutação com Perda de Função , Receptores da Calcitonina/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Exoma , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Humanos , Avaliação de Resultados da Assistência ao Paciente , Prognóstico , Receptores da Calcitonina/metabolismo , Transdução de SinaisRESUMO
The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is a therapeutic target for the treatment of hypercalcaemia of malignancy, Paget's disease and osteoporosis. In primates, the CTR is subject to alternative splicing, with a unique, primate-specific splice variant being preferentially expressed in reproductive organs, lung and kidney. In addition, humans possess a common non-synonymous single-nucleotide polymorphism (SNP) encoding a proline/leucine substitution in the C-terminal tail. In low power studies, the leucine polymorphism has been associated with increased risk of osteoporosis in East Asian populations and, independently, with increased risk of kidney stone disease in a central Asian population. The CTR is pleiotropically coupled, though the relative physiological importance of these pathways is poorly understood. Using both COS-7 and HEK293 cells recombinantly expressing human CTR, we have characterized both splice variant and polymorphism dependent response to CTs from several species in key signalling pathways and competition binding assays. These data indicate that the naturally occurring changes to the intracellular face of CTR alter ligand affinity and signalling, in a pathway and agonist dependent manner. These results further support the potential for these primate-specific CTR variants to engender different physiological responses. In addition, we report that the CTR exhibits constitutive internalization, independent of splice variant and polymorphism and this profile is unaltered by peptide binding.
Assuntos
Receptores da Calcitonina/metabolismo , Transdução de Sinais/fisiologia , Animais , Células COS , Chlorocebus aethiops , Regulação da Expressão Gênica , Células HEK293 , Humanos , Polimorfismo Genético , Isoformas de Proteínas , Receptores da Calcitonina/genéticaRESUMO
Fluorosis, caused by ingestion of excess fluoride, is endemic in at least 25 countries across the globe, China and India being the worst affected among them. Dental, skeletal and non-skeletal are the major types of fluorosis affecting millions of people in these countries. A number of genetic epidemiological studies carried out by investigators have shown the evidence for association between genetic polymorphisms in candidate genes and differences in the susceptibility pattern of different types of fluorosis among individuals living in the same community and having the same environmental exposure. These studies have pointed out that genetic variants in some candidate genes like COL1A2 (Collagen type 1 alpha 2), CTR (Calcitonin receptor gene), ESR (Estrogen receptor), COMT (Catechol-o-methyltransferase), GSTP1 (Glutathione S-transferase pi 1), MMP-2 (Matrix metallopeptidase 2), PRL (Prolactin), VDR (Vitamin D receptor) and MPO (Myeloperoxidase) could increase or decrease the risk of fluorosis among the exposed individuals in endemic areas. So, it is increasingly becoming evident that an individual's genetic background could play a major role in influencing the risk to fluorosis when other factors like specific environmental exposures including dietary patterns of fluoride intake and other nutrients remain the same. The current manuscript presents an up-to-date critical review on fluorosis, focusing mainly on the genetic association studies that have looked at the possible involvement of genetic factors in fluorosis.
Assuntos
Exposição Ambiental/efeitos adversos , Fluorose Dentária/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único , Catecol O-Metiltransferase/genética , China/epidemiologia , Colágeno Tipo I/genética , Feminino , Fluorose Dentária/epidemiologia , Fluorose Dentária/etiologia , Glutationa S-Transferase pi/genética , Humanos , Índia/epidemiologia , Lectinas Tipo C/genética , Metaloproteinase 2 da Matriz/genética , Peroxidase/genética , Receptores da Calcitonina/genética , Receptores de Calcitriol/genética , Receptores de Estrogênio/genéticaRESUMO
Staphylococcus aureus (S. aureus) is the most common organism causing osteomyelitis, and Staphylococcus aureus protein A (SpA) is an important virulence factor anchored in its cell wall. However, the precise mechanisms underlying the bone loss caused by SpA have not been well understood. The present study aimed to investigate the effect of SpA on osteoclast differentiation, and the probable mechanism was investigated. Raw264.7 cells were treated with SpA in the absence or presence of receptoractivated (NF)κB ligand for 5 days, and morphological and biochemical assays were used to assess osteoclastogenesis and explore the underlying mechanisms. Data demonstrated that SpA induced osteoclast differentiation and promoted bone resorption in a dosedependent manner in the absence or presence of RANKL. In addition, the expression of osteoclastspecific genes, such as the tartrate resistant acid phosphatase, matrix metalloproteinase9, cathepsin K, calcitonin receptors and d2 isoform of the vacuolar ATPase Vo domain, were enhanced by SpA. Furthermore, the SpAinduced osteoclast differentiation was associated with the degradation of inhibitor of κBα, phosphorylation of NFκB p65 and increased expression of nuclear factor of activated Tcells. However, by treatment with JSH23, an NFκB inhibitor, the formation of osteoclastlike cells and resorption pits was significantly reduced, and the expression of osteoclastspecific genes was also inhibited. Collectively, in the present study SpA induced osteoclast differentiation, promoted bone resorption, and the NFκB signaling pathway was involved in this process.
Assuntos
NF-kappa B/metabolismo , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína Estafilocócica A/farmacologia , Animais , Catepsina K/genética , Catepsina K/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , NF-kappa B/genética , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/genética , Fenilenodiaminas/farmacologia , Ligante RANK/farmacologia , Células RAW 264.7 , Receptores da Calcitonina/genética , Receptores da Calcitonina/metabolismo , Proteína Estafilocócica A/isolamento & purificação , Staphylococcus aureus/química , Fosfatase Ácida Resistente a Tartarato/genética , Fosfatase Ácida Resistente a Tartarato/metabolismoRESUMO
The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited â¼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI- cells, which yield core N-glycans (Man5GlcNAc2). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity â¼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N â Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.
Assuntos
Asparagina/metabolismo , Calcitonina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Receptores da Calcitonina/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Substituição de Aminoácidos , Asparagina/química , Sítios de Ligação , Calcitonina/química , Glicosilação , Células HEK293 , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Cinética , Ligantes , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Conformação Molecular , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteína 2 Modificadora da Atividade de Receptores/agonistas , Proteína 2 Modificadora da Atividade de Receptores/química , Proteína 2 Modificadora da Atividade de Receptores/genética , Receptores da Calcitonina/agonistas , Receptores da Calcitonina/química , Receptores da Calcitonina/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
There is considerable inter-individual variabil¬ity in chemoradiotherapy responses in nasopharyngeal carcinoma (NPC) patients receiv¬ing the same or similar treatment protocols. In this study we evaluated the association between the gene polymorphisms in endoplasmic reticulum (ER) stress pathway and chemoradiation responses in Chinese NPC patients. A total of 150 patients with histopathologically conformed NPC and treated with concurrent chemoradiotherapy were enrolled. Genotypes in ER stress pathway genes, including VCP (valosin-containing protein) rs2074549, HSP90B1 rs17034943, CANX (calnexin) rs7566, HSPA5 [heat shock protein family A (Hsp70) member 5] rs430397, CALCR (calcitonin receptor) rs2528521, and XBP1 (X-box binding protein 1) rs2269577 were analyzed by Sequenom MassARRAY system. The short-term effects of primary tumor and lymph node after radiotherapy were assessed based on the Response Evaluation Criteria in Solid Tumors (RECIST) of WHO. And acute radiation-induced toxic reactions were evaluated according to the Radiation Therapy Oncology Group or European Organization for Research and Treatment of Cancer (RTOG/EORTC). The effects of gene polymorphisms on clinical outcomes of chemoradiotherapy were assessed by chi-square test, univariate and multivariate logistic regression analyses. We found that CT and CT+CC genotypes of CANX rs7566 was significantly correlated with primary tumor treatment efficacy at 3 months after chemoradiotherapy and with occurrence of radiation-induced myelosuppression in Chinese NPC patients. CT and CT+CC genotypes of CALCR rs2528521 were significantly correlated with cervical lymph node efficacy at 3 months after chemoradiotherapy. And CC and CT+CC genotypes of VCP rs2074549 were significantly associated with occurrence of myelosuppression. In conclusion, SNPs of VCP rs2074549, CANX rs7566 and CALCR rs2528521 in ER stress pathway genes may serve as predictors for clinical outcomes of chemoradiotherapy in Chinese NPC patients.
Assuntos
Carcinoma/terapia , Estresse do Retículo Endoplasmático/genética , Neoplasias Nasofaríngeas/terapia , Adenosina Trifosfatases/genética , Adulto , Povo Asiático , Calnexina/genética , Carcinoma/metabolismo , Proteínas de Ciclo Celular/genética , Quimiorradioterapia , Chaperona BiP do Retículo Endoplasmático , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Polimorfismo Genético , Receptores da Calcitonina/genética , Transdução de Sinais , Proteína com ValosinaRESUMO
Osteoclasts, which are specialized bone multinuclear cells, are responsible for bone lytic diseases such as osteoporosis. 3',4',7,8-tetrahydroxyflavone is a flavonoid from Acacia confusa. In the present study, we found that 3',4',7,8-tetrahydroxyflavone markedly inhibited receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastic differentiation from mouse bone marrow-derived macrophages (BMMs). 3',4',7,8-tetrahydroxyflavone also reduced the mRNA expression levels of osteoclastic marker genes including the calcitonin receptor (CTR) and cathepsin K. In addition, 3',4',7,8-tetrahydroxyflavone decreased the bone resorption activity of osteoclasts on dentin slices. We found that 3',4',7,8-tetrahydroxyflavone inhibited RANKL-induced expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), a key transcription factor of osteoclast differentiation. Furthermore, ectopic overexpression of a constitutively active form of NFATc1 completely rescued the anti-osteoclastogenic effect of 3',4',7,8-tetrahydroxyflavone, suggesting that the anti-osteoclastogenic effect was mainly attributed to the reduction in NFATc1 expression. Taken together, our data suggest that 3',4',7,8-tetrahydroxyflavone inhibits osteoclast differentiation and bone loss and may therefore be considered a promising drug candidate for treating or preventing bone-lytic diseases.