RESUMO
Neuromedin B (NB), a bombesin-like peptide, exerts its specific actions by binding to the neuromedin B receptor (NBR), a G protein-coupled receptor. Female NBR-knockout (NBR-KO) mice exhibit resistance to diet-induced obesity, without hyperphagia, suggesting possible increase in energy expenditure. Skeletal muscle (SM) is crucial for whole body energy homeostasis, however, the presence of NB-NBR signaling and its effects in SM are unknown. Here, we show that male and female wild type express Nmbr and Nmb mRNA in SM, with higher levels in females. Female NBR-KO gastrocnemius showed increased Myh7 mRNA level, which characterizes type I fibers (oxidative profile). Their permeabilized gastrocnemius fibers, studied by high-resolution respirometry, exhibited higher consumption of O2 coupled to ATP synthesis and unaltered uncoupled respiration. NBR-KO gastrocnemius had higher protein levels of ATP-synthase and Nduf9 mRNA, corresponding to mitochondrial complex I subunit. NBR-KO gastrocnemius exhibited slight increase in mitochondria number, increased thickness of Z line at electron microscopy, and unaltered mitochondrial dynamics markers. Therefore, in the females' gastrocnemius, a predominantly glycolytic SM, the NBR absence promotes changes that favor mitochondrial oxidative phosphorylation capacity. In addition, in L6 myocytes, NB treatment (5 µg/mL/16 h) promoted lower O2 consumption coupled to ATP synthesis, suggesting direct action at SM cells. Altogether, the study reinforces the hypothesis that inhibition of NB-NBR signaling enhances the capacity for oxidative phosphorylation of white SM, encouraging future studies to elucidate their contribution on other types of SM and whole body energy expenditure, which may lead to a new target to drug development for obesity treatment.NEW & NOTEWORTHY This study describes neuromedin B (NB) and NB receptor as new regulators of skeletal muscle mitochondrial function. The white skeletal muscle mitochondrial oxidative phosphorylation capacity was increased by NB receptor genetic disruption in female mice. These findings may contribute to the resistance to diet-induced obesity, previously found in these mice, which requires future studies. Thus, investigations are necessary to clarify if blockade of NB receptor may be an approach to develop drugs to combat obesity.
Assuntos
Fosforilação Oxidativa , Receptores da Bombesina , Trifosfato de Adenosina/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Obesidade/metabolismo , RNA Mensageiro/metabolismo , Receptores da Bombesina/genética , Receptores da Bombesina/metabolismoRESUMO
PURPOSE: Medulloblastoma (MB) comprises four distinct molecular subgroups, and survival remains particularly poor in patients with Group 3 tumors. Mutations and copy number variations result in altered epigenetic regulation of gene expression in Group 3 MB. Histone deacetylase inhibitors (HDACi) reduce proliferation, promote cell death and neuronal differentiation, and increase sensitivity to radiation and chemotherapy in experimental MB. Bombesin receptor antagonists potentiate the antiproliferative effects of HDACi in lung cancer cells and show promise as experimental therapies for several human cancers. Here, we examined the viability of D283 cells, which belong to Group 3 MB, treated with an HDACi alone or combined with bombesin receptor antagonists. METHODS: D283 MB cells were treated with different doses of the HDACi sodium butyrate (NaB), the neuromedin B receptor (NMBR) antagonist BIM-23127, the gastrin releasing peptide receptor (GRPR) antagonist RC-3095, or combinations of NaB with each receptor antagonist. Cell viability was examined by cell counting. RESULTS: NaB alone or combined with receptor antagonists reduced cell viability at all doses tested. BIM-23127 alone did not affect cell viability, whereas RC-3095 at an intermediate dose significantly increased cell number. CONCLUSION: Although HDACi are promising agents to inhibit MB growth, the present results provide preliminary evidence that combining HDACi with bombesin receptor antagonists is not an effective strategy to improve the effects of HDACi against MB cells.
Assuntos
Apoptose/efeitos dos fármacos , Bombesina/análogos & derivados , Inibidores de Histona Desacetilases/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Receptores da Bombesina/antagonistas & inibidores , Análise de Variância , Antineoplásicos/farmacologia , Bombesina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Meduloblastoma/patologiaRESUMO
Aberrant expression of Neuromedin B (NMB) is associated with the malignant progression of cancer, such as breast cancer, lung cancer and glioma. However, the role of NMB in cervical cancer remains unclear. The present study found that NMB and its receptor NMBR are aberrantly expressed in cervical cancer. NMB activates ERK1/2 and NF-κB signaling pathways, which promote the proliferation of cervical cancer cells and increase the expression of tumor necrosis factor α (TNF-α). The downregulation of NMBR by the specific inhibitor, PD168368, abrogates proliferation and promotes apoptosis of cervical cancer cells. In addition, the NMB/NMBR signaling axis mediates the promoting effect of cancer-associated adipocytes on cervical cancer progression. These findings demonstrate the potential role of NMB/NMBR-regulated ERK1/2 and p65 signaling pathway in cervical cancer progression, which provide new opportunities to diagnose and treat cervical cancer.
RESUMO
Background: Neuromedin B (Nmb) is implicated in the regulation of nociception of sensory neurons. However, the underlying cellular and molecular mechanisms remain unknown. Methods: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of Nmb on the sensory neuronal excitability and peripheral pain sensitivity mediated by Cav3.2 T-type channels. Results: Nmb reversibly and concentration-dependently increased T-type channel currents (IT) in small-sized trigeminal ganglion (TG) neurons through the activation of neuromedin B receptor (NmbR). This NmbR-mediated IT response was Gq protein-coupled, but independent of protein kinase C activity. Either intracellular application of the QEHA peptide or shRNA-mediated knockdown of Gß abolished the NmbR-induced IT response. Inhibition of protein kinase A (PKA) or AMP-activated protein kinase (AMPK) completely abolished the Nmb-induced IT response. Analysis of phospho-AMPK (p-AMPK) revealed that Nmb significantly activated AMPK, while AMPK inhibition prevented the Nmb-induced increase in PKA activity. In a heterologous expression system, activation of NmbR significantly enhanced the Cav3.2 channel currents, while the Cav3.1 and Cav3.3 channel currents remained unaffected. Nmb induced TG neuronal hyperexcitability and concomitantly induced mechanical and thermal hypersensitivity, both of which were attenuated by T-type channel blockade. Moreover, blockade of NmbR signalling prevented mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain, and this effect was attenuated by siRNA knockdown of Cav3.2. Conclusions: Our study reveals a novel mechanism by which NmbR stimulates Cav3.2 channels through a Gßγ-dependent AMPK/PKA pathway. In mouse models, this mechanism appears to drive the hyperexcitability of TG neurons and induce pain hypersensitivity.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Dor/metabolismo , Receptores da Bombesina/metabolismo , Potenciais de Ação , Animais , Canais de Cálcio Tipo T/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Adjuvante de Freund/farmacologia , Gânglios Espinais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neurocinina B/análogos & derivados , Neurocinina B/metabolismo , Dor/fisiopatologia , Receptores da Bombesina/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Transdução de Sinais/efeitos dos fármacos , Gânglio Trigeminal/citologia , Gânglio Trigeminal/metabolismoRESUMO
The precise mechanisms that lead to parturition remain unclear. In our initial complementary DNA (cDNA) microarray experiment, we found that the neuromedin B receptor (NMBR) was differentially expressed in the human myometrium during spontaneous or oxytocin-induced labor. We have previously shown that neuromedin B (NMB) could induce interleukin 6 (IL-6) and type 2 cyclo-oxygenase enzyme (COX-2) expression in the primary human myometrial cells via nuclear factor kappa B (NF-κB) transcription factor p65 (p65) and Jun proto-oncogene, activator protein 1 (AP-1) transcription factor subunit (c-Jun). This study is aimed to investigate whether NMBR is required for NMB-induced effect. Primary myometrial cell culture was established to provide a suitable model to investigate the mechanism of NMB in labor initiation. Immunochemical staining was conducted to validate the NMBR expression in primary myometrial cells. The mRNA and protein expression of NMBR, p65, c-Jun, COX-2 and IL-6 were assessed by Quantitative Real Time PCR (RT-qPCR) and western blotting. Lentiviruses with shRNAs targeting NMBR or containing cDNA sequence of NMBR were transfected to primary myometrial cells to knockdown or overexpress NMBR. Cell death was determined by annexin V and propidium iodide staining and analyzed by flow cytometry. The upregulation of COX-2 and IL-6 and phosphorylation of p65 and c-Jun were significantly attenuated by knockdown of NMBR and enhanced by overexpressed NMBR following NMB treatment, with no significant change in total p65 and c-Jun. In summary, this study showed that NMBR-mediated NMB-induced NF-κB and AP-1 activation, which in turn, induce expression of IL-6 and COX-2 in primary myometrial cells.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Interleucina-6/metabolismo , Miométrio/metabolismo , Neurocinina B/análogos & derivados , Receptores da Bombesina/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2/genética , Feminino , Humanos , Interleucina-6/genética , Miométrio/citologia , Neurocinina B/farmacologia , Trabalho de Parto Prematuro/metabolismo , Trabalho de Parto Prematuro/prevenção & controle , Gravidez , Proto-Oncogene Mas , RNA/análise , RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores da Bombesina/uso terapêutico , Regulação para CimaRESUMO
Neuromedin B, a bombesin-like peptide, and its receptor, are expressed in white adipose tissue with undefined roles. Female mice with disruption of neuromedin B receptor (NB-R) exhibited partial resistance to diet-induced obesity leading to our hypothesis that NB-R is involved in adipogenesis. Here, we showed that adipose stem/stromal cells (ASC) from perigonadal fat of female NB-R-knockout mice, exposed to a differentiation protocol in vitro, accumulated less lipid (45%) than wild type, suggesting reduced capacity to differentiate under adipogenic input. To further explore mechanisms, preadipocytes 3T3-L1 cells were incubated in the presence of NB-R antagonist (PD168368) during the first 3 days in culture. Cells were analyzed in the end of the treatment (Day 3) and later when fully differentiated (Day 21). NB-R antagonist induced lower number of cells at day 3 and 21 (33-39%), reduced cell proliferation at day 3 (-53%) and reduced lipid accumulation at day 21 (-86%). The mRNA expressions of several adipocyte differentiation markers were importantly reduced at both days: Cebpb and Pparg and Fabp4, Plin-1 and Adipoq, and additionally Lep mRNA at day 21. The antagonist had no effect when incubated with mature 3T3-L1 adipocytes. Therefore, genetically disruption of NB-R in mice ASC or pharmacological antagonism of NB-R in 3T3-L1 cells impairs adipogenesis. The mechanisms suggested by results in 3T3-L1 cells involve reduction of cell proliferation and of early gene expressions, leading to decreased number of mature adipocytes. We speculate that NB-R antagonism may be useful to limit the increase in adiposity due to pre-adipocyte differentiation.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia/fisiologia , Receptores da Bombesina/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipogenia/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proliferação de Células/genética , Proliferação de Células/fisiologia , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Indóis/farmacologia , Camundongos , Camundongos Knockout , PPAR gama/genética , PPAR gama/metabolismo , Perilipina-1/genética , Perilipina-1/metabolismo , Piridinas/farmacologia , Receptores da Bombesina/antagonistas & inibidores , Receptores da Bombesina/genéticaRESUMO
The aim of the present study was to investigate the expression and distribution of membrane receptors after bladder outlet obstruction (BOO). Partial bladder outlet obstruction (BOO) was induced in female rats and bladders were harvested after either 10 days or 6 weeks of BOO. The expression of different receptors was surveyed by microarrays and corroborated by immunohistochemistry and western blotting. A microarray experiment identified 10 membrane receptors that were differentially expressed compared to sham-operated rats including both upregulated and downregulated receptors. Four of these were selected for functional experiments on the basis of magnitude of change and relevance to bladder physiology. At 6 weeks of BOO, maximal contraction was reduced for neuromedin B and vasopressin (AVP), consistent with reductions of receptor mRNA levels. Glycine receptor-induced contraction on the other hand was increased and receptor mRNA expression was accordingly upregulated. Maximal relaxation by the ß3-adrenergic receptor agonist CL316243 was reduced as was the receptor mRNA level. Immunohistochemistry supported reduced expression of neuromedin B receptors, V1a receptors and ß3-adrenergic receptors, but glycine receptor expression appeared unchanged. Western blotting confirmed repression of V1a receptors and induction of glycine receptors in BOO. mRNA for vasopressin was detectable in the bladder, suggesting local AVP production. We conclude that changes in receptor expression following bladder outlet obstruction are non-uniform. Some receptors are upregulated, conferring increased responsiveness to agonist, whereas others are downregulated, leading to decreased agonist-induced responses. This study might help to select pharmacological agents that are effective in modulating lower urinary tract symptoms in BOO.