RESUMO
Cholinergic receptor muscarinic 3 (CHRM3)-mediated focal adhesion kinase/YES-associated protein (YAP) signalling is essential for the growth of castration-resistant prostate cancer (CRPC) cells. Here, we evaluated the molecular mechanisms through which CHRM3 overexpression facilitates castration-resistant growth. Small RNA sequencing combined with in silico analyses revealed that CHRM3 was a putative target of miR-15b-5p. Notably, androgen deprivation suppressed miR-15b-5p expression and increased CHRM3 expression. Moreover, miR-15b-5p bound directly to CHRM3 and inhibited YAP activation induced by CHRM3 stimulation. Furthermore, miR-15b-5p abolished the growth of CRPC cells induced by CHRM3 stimulation. We conclude that the miR-15b-5p/CHRM3/YAP signalling axis promotes the castration-resistant growth of prostate cancer.
Assuntos
MicroRNAs , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , MicroRNAs/genética , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Antagonistas de Androgênios , Proliferação de Células/fisiologia , Castração , Linhagem Celular Tumoral , Receptores Colinérgicos/metabolismo , Colinérgicos , Regulação Neoplásica da Expressão Gênica , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismoRESUMO
Emerging evidence suggested that long non-coding RNAs (lncRNAs) were involved in Parkinson's disease (PD) pathogenesis. Herein, we used gene expression profiles from GEO database to construct a PD-specific ceRNA network. Functional enrichment analysis suggested that ceRNA network might participate in the development of PD. PPI networks were constructed, and the ceRNA subnetwork based on five hub genes was set up. In a cohort of 32 PD patients and 31 healthy controls, the expression of 10 DElncRNAs (TTC3-AS1, LINC01259, ZMYND10-AS1, CHRM3-AS1, MYO16-AS1, AGBL5-IT1, HOTAIRM1, RABGAP1L-IT1, HLCS-IT1, and LINC00393) were further verified. Consistent with the microarray data, LINC01259 expression was significantly lower in PD patients compared with controls (P = 0.008). Intriguingly, such a difference was only observed among male patients and male controls when dividing study participants based on their gender (P = 0.016). However, the expression of other lncRNAs did not differ significantly between the two groups. Receiver operating characteristic (ROC) curve analysis revealed that the diagnostic power of LINC01259 was 0.694 for PD and 0.677 for early-stage PD. GSEA enrichment analysis revealed that LINC01259 was mainly enriched in biological processes associated with immune function and inflammatory response. Moreover, LINC01259 expression was not correlated with age of patients, disease duration, disease stage, MDS-UPDRS score, MDS-UPDRS III score, MMSE score, and MOCA score. The current study provides further evidence for the dysregulation of lncRNAs in circulating leukocytes of PD patients, revealing that LINC01259 has clinical potential as a novel immune and inflammatory biomarker for PD and early-stage PD diagnosis.
Assuntos
Doença de Parkinson , RNA Longo não Codificante , Humanos , Masculino , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Doença de Parkinson/genética , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , FemininoRESUMO
Overexpression of M3 subtype muscarinic receptors (M3R) hastens colon cancer progression. As microRNA (miRNA) expression is commonly dysregulated in cancer, we used microarrays to examine miRNA profiles in muscarinic receptor agonist-treated human colon cancer cells. We used quantitative RT-PCR (qPCR) to validate microarray results and examine miRNA expression in colon cancers and adjacent normal colon. These assays revealed that acetylcholine (ACh) treatment robustly induced miR-222 expression; miR-222 levels were three-fold higher in cancer compared to normal colon. In kinetic studies, ACh induced a 4.6-fold increase in pri-miR-222 levels within 1 h, while mature miR-222 increased gradually to 1.8-fold within 4 h. To identify post-M3R signaling mediating these actions, we used chemical inhibitors and agonists. ACh-induced increases in pri-miR-222 were attenuated by pre-incubating cells with atropine and inhibitors of protein kinase C (PKC) and p38 MAPK. Treatment with a PKC agonist, phorbol 12-myristate 13-acetate, increased pri-miR-222 levels, an effect blocked by PKC and p38 MAPK inhibitors, but not by atropine. Notably, treatment with ACh or transfection with miR-222 mimics increased cell proliferation; atropine blocked the effects of ACh but not miR-222. These findings identify a novel mechanism whereby post-M3R PKC/p38 MAPK signaling stimulates miR-222 expression and colon cancer cell proliferation.
Assuntos
Neoplasias do Colo , MicroRNAs , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Derivados da Atropina , Colinérgicos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Humanos , Cinética , MicroRNAs/genética , Antagonistas Muscarínicos/farmacologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Stroke profoundly disrupts cortical excitability which impedes recovery, but how it affects the function of specific inhibitory interneurons, or subpopulations therein, is poorly understood. Interneurons expressing vasoactive intestinal peptide (VIP) represent an intriguing stroke target because they can regulate cortical excitability through disinhibition. Here we chemogenetically augmented VIP interneuron excitability in a murine model of photothrombotic stroke and show that it enhances somatosensory responses and improves recovery of paw function. Using longitudinal calcium imaging, we discovered that stroke primarily disrupts the fidelity (fraction of responsive trials) and predictability of sensory responses within a subset of highly active VIP neurons. Partial recovery of responses occurred largely within these active neurons and was not accompanied by the recruitment of minimally active neurons. Importantly, chemogenetic stimulation preserved sensory response fidelity and predictability in highly active neurons. These findings provide a new depth of understanding into how stroke and prospective therapies (chemogenetics), can influence subpopulations of inhibitory interneurons.
Assuntos
Interneurônios/fisiologia , Acidente Vascular Cerebral/terapia , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Clozapina/análogos & derivados , Clozapina/uso terapêutico , Humanos , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Camundongos , Inibição Neural/efeitos dos fármacos , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Recuperação de Função Fisiológica , Córtex Somatossensorial/citologia , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/fisiologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Despite great advances in our understanding of the pathobiology of colorectal cancer and the genetic and environmental factors that mitigate its onset and progression, a paucity of effective treatments persists. The five-year survival for advanced, stage IV disease remains substantially less than 20%. This review examines a relatively untapped reservoir of potential therapies to target muscarinic receptor expression, activation, and signaling in colorectal cancer. Most colorectal cancers overexpress M3 muscarinic receptors (M3R), and both in vitro and in vivo studies have shown that activating these receptors stimulates cellular programs that result in colon cancer growth, survival, and spread. In vivo studies using mouse models of intestinal neoplasia have shown that using either genetic or pharmacological approaches to block M3R expression and activation, respectively, attenuates the development and progression of colon cancer. Moreover, both in vitro and in vivo studies have shown that blocking the activity of matrix metalloproteinases (MMPs) that are induced selectively by M3R activation, i.e., MMP1 and MMP7, also impedes colon cancer growth and progression. Nonetheless, the widespread expression of muscarinic receptors and MMPs and their importance for many cellular functions raises important concerns about off-target effects and the safety of employing similar strategies in humans. As we highlight in this review, highly selective approaches can overcome these obstacles and permit clinicians to exploit the reliance of colon cancer cells on muscarinic receptors and their downstream signal transduction pathways for therapeutic purposes.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/metabolismo , Receptores Muscarínicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Biomarcadores , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/patologia , Gerenciamento Clínico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Inibidores de Metaloproteinases de Matriz/farmacologia , Inibidores de Metaloproteinases de Matriz/uso terapêutico , Metaloproteinases da Matriz/metabolismo , Terapia de Alvo Molecular , Agonistas Muscarínicos/farmacologia , Agonistas Muscarínicos/uso terapêutico , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Receptores Muscarínicos/classificação , Receptores Muscarínicos/genéticaRESUMO
OBJECTIVES: Pancreatic acinar necrosis is a typical feature in the early phase of severe acute pancreatitis (SAP). Muscarinic acetylcholine receptor M3 (CHRM3) has been reported to play important roles in promoting insulin secretion and tumor cell proliferation, but its effect on necrosis remains unknown. This study revealed the important role of CHRM3 in regulating L-arginine-induced SAP and the molecular mechanisms. METHODS: To verify the function of CHRM3, pancreatic tissues and primary acinar cells of CRISPR/Cas9-mediated Chrm3 knockout mice were used in CHRM3 knockdown experiments, and to ascertain the CHRM3 overexpression, PLV-EGFP-Chrm3 plasmids were transfected in acinar cells in vitro. RESULTS: In L-arginine-induced SAP, CHRM3 is activated and regulates SAP through the mitogen-activated protein kinase/p38 pathway. Moreover, the expression of miR-31-5p decreased in the SAP model both in vitro and in vivo. Mir-31-5p effects the necrosis of acinar cells in SAP by upregulating the target gene RIP3, and miR-31-5p is a downstream miRNA of CHRM3. CONCLUSIONS: Necrosis in L-arginine-induced SAP is promoted by CHRM3 through the mitogen-activated protein kinase-p38/miR-31-5p/RIP3 axis.
Assuntos
Células Acinares/enzimologia , MicroRNAs/metabolismo , Pâncreas/enzimologia , Pancreatite/prevenção & controle , Receptor Muscarínico M3/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células Acinares/patologia , Animais , Arginina , Células Cultivadas , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Necrose , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/enzimologia , Pancreatite/patologia , Fosforilação , Receptor Muscarínico M3/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de SinaisRESUMO
Microglia, the brain's resident macrophages, help to regulate brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival1. Here we show that microglia are also critical modulators of neuronal activity and associated behavioural responses in mice. Microglia respond to neuronal activation by suppressing neuronal activity, and ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular ATP, which is released upon neuronal activation by neurons and astrocytes. ATP triggers the recruitment of microglial protrusions and is converted by the microglial ATP/ADP hydrolysing ectoenzyme CD39 into AMP; AMP is then converted into adenosine by CD73, which is expressed on microglia as well as other brain cells. Microglial sensing of ATP, the ensuing microglia-dependent production of adenosine, and the adenosine-mediated suppression of neuronal responses via the adenosine receptor A1R are essential for the regulation of neuronal activity and animal behaviour. Our findings suggest that this microglia-driven negative feedback mechanism operates similarly to inhibitory neurons and is essential for protecting the brain from excessive activation in health and disease.
Assuntos
Retroalimentação Fisiológica , Microglia/fisiologia , Inibição Neural , Neurônios/fisiologia , 5'-Nucleotidase/metabolismo , Potenciais de Ação , Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Cálcio/metabolismo , Corpo Estriado/citologia , Corpo Estriado/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Inibição Neural/genética , Receptor A1 de Adenosina/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Fatores de TempoRESUMO
Glial fibrillary acidic protein expressing (GFAP+) glia modulate nociceptive neuronal activity in both the peripheral nervous system (PNS) and the central nervous system (CNS). Resident GFAP+ glia in dorsal root ganglia (DRG) known as satellite glial cells (SGCs) potentiate neuronal activity by releasing pro-inflammatory cytokines and neuroactive compounds. In this study, we tested the hypothesis that SGC Gq-coupled receptor (Gq-GPCR) signaling modulates pain sensitivity in vivo using Gfap-hM3Dq mice. Complete Freund's adjuvant (CFA) was used to induce inflammatory pain, and mechanical sensitivity and thermal sensitivity were used to assess the neuromodulatory effect of glial Gq-GPCR activation in awake mice. Pharmacogenetic activation of Gq-GPCR signaling in sensory SGCs decreased heat-induced nociceptive responses and reversed inflammation-induced mechanical allodynia via peripheral adenosine A1 receptor activation. These data reveal a previously unexplored role of sensory SGCs in decreasing afferent excitability. The identified molecular mechanism underlying the analgesic role of SGCs offers new approaches for reversing peripheral nociceptive sensitization.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Hiperalgesia/prevenção & controle , Inflamação/fisiopatologia , Neuroglia/enzimologia , Nociceptividade/fisiologia , Receptor A1 de Adenosina/fisiologia , Receptor Muscarínico M3/fisiologia , Animais , Benzilatos/farmacologia , Clozapina/análogos & derivados , Clozapina/farmacologia , Adjuvante de Freund/toxicidade , Genes Sintéticos , Temperatura Alta , Hiperalgesia/fisiopatologia , Inflamação/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Agonistas Muscarínicos/farmacologia , Neuroglia/fisiologia , Nortropanos/farmacologia , Regiões Promotoras Genéticas , Agonistas do Receptor Purinérgico P1/farmacologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , Receptor A1 de Adenosina/efeitos dos fármacos , Receptor Muscarínico M3/efeitos dos fármacos , Receptor Muscarínico M3/genética , Receptores Acoplados a Proteínas G , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Teofilina/análogos & derivados , Teofilina/farmacologia , Tato , Xantinas/farmacologiaRESUMO
Animals can remember a situation associated with an aversive event. Contextual fear memory is initially encoded and consolidated in the hippocampus and gradually consolidated in multiple brain regions over time, including the medial prefrontal cortex (PFC). However, it is not fully understood how PFC neurons contribute to contextual fear memory formation during learning. In the present study, neuronal activity was increased in PFC neurons utilizing the pharmacogenetic hM3Dq-system in male mice. We show that fear expression and memory formation are enhanced by increasing neuronal activity in PFC during conditioning phase. Previous studies showed that the activation of hM3Dq receptor in a subset of amygdala neurons enhanced fear memory formation and biased which neurons are allocated to a memory trace, in which immediate early gene c-fos was preferentially expressed following memory retrieval in these pre-activated neurons. In this study, hM3Dq activation in PFC could not change the probability of c-fos expression in pre-activated neurons flowing memory retrieval. Instead, the number c-fos positive neurons following memory retrieval was significantly increased in the basolateral amygdala. Our results suggest that neuronal activity in PFC at the time of learning modulates fear memory formation and downstream cellular activity at an early phase.
Assuntos
Medo , Memória , Neurônios/metabolismo , Córtex Pré-Frontal/metabolismo , Animais , Complexo Nuclear Basolateral da Amígdala/metabolismo , Condicionamento Clássico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismoRESUMO
Prostate cancer (PCa) innervation contributes to the progression of PCa. However, the precise impact of innervation on PCa cells is still poorly understood. By focusing on muscarinic receptors, which are activated by the nerve-derived neurotransmitter acetylcholine, we show that muscarinic receptors 1 and 3 (m1 and m3) are highly expressed in PCa clinical specimens compared with all other cancer types, and that amplification or gain of their corresponding encoding genes (CHRM1 and CHRM3, respectively) represent a worse prognostic factor for PCa progression free survival. Moreover, m1 and m3 gene gain or amplification is frequent in castration-resistant PCa (CRPC) compared with hormone-sensitive PCa (HSPC) specimens. This was reflected in HSPC-derived cells, which show aberrantly high expression of m1 and m3 under androgen deprivation mimicking castration and androgen receptor inhibition. We also show that pharmacological activation of m1 and m3 signaling is sufficient to induce the castration-resistant growth of PCa cells. Mechanistically, we found that m1 and m3 stimulation induces YAP activation through FAK, whose encoding gene, PTK2 is frequently amplified in CRPC cases. Pharmacological inhibition of FAK and knockdown of YAP abolished m1 and m3-induced castration-resistant growth of PCa cells. Our findings provide novel therapeutic opportunities for muscarinic-signal-driven CRPC progression by targeting the FAK-YAP signaling axis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptor Muscarínico M1/biossíntese , Receptor Muscarínico M3/biossíntese , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Quinase 1 de Adesão Focal/genética , Humanos , Masculino , Proteínas de Neoplasias/genética , Células PC-3 , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Receptor Muscarínico M1/genética , Receptor Muscarínico M3/genética , Fatores de Transcrição/genética , Proteínas de Sinalização YAPRESUMO
Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by IgG autoantibodies (AuAbs) binding to epidermal keratinocytes and inducing a devastating blistering disease affecting oral and/or esophageal surfaces and, sometimes, also the skin. Anti-keratinocyte AuAbs developed by the desmoglein (Dsg) 1/3 AuAb-negative acute PV patients are pathogenic, as they induced acantholysis and epidermal split in the experimental models of PV in vitro and in vivo. These PV patients have various combinations of AuAbs to keratinocyte muscarinic acetylcholine receptor subtype M3 (M3AR), the secretory pathway Ca2+/Mn2+-ATPase isoform 1 (SPCA1), and desmocollin 3 whose relative concentrations correlate with the disease activity. In this study, we identified new molecular mechanisms of the synergistic cooperation of AuAbs to M3AR and SPCA1 in inducing acantholysis in the anti-Dsg 1/3 AuAb-negative PV patients. Anti-M3AR AuAb was found to play an important role in determining the level of intraepidermal split just above the basal cells, caspase to mediate early pro-apoptotic events triggered by anti-SPCA1 AuAb, and the neonatal Fc receptor (FcRn) to contribute to the pathobiological actions of both anti-M3AR and anti-SPCA1 AuAbs. Altogether, these novel results support our original hypothesis that pemphigus acantholysis is a complex disease process (also known as apoptolysis) initiated by AuAbs directed against different keratinocyte proteins that play important roles in supporting cell viability and regulating vital cell functions.
Assuntos
Autoanticorpos/imunologia , ATPases Transportadoras de Cálcio/imunologia , Queratinócitos/imunologia , Pênfigo/imunologia , Receptor Muscarínico M3/imunologia , Animais , Linhagem Celular , Desmogleína 1/imunologia , Desmogleína 3/imunologia , Humanos , Camundongos Knockout , Pênfigo/patologia , Receptor Muscarínico M3/genética , Pele/patologiaRESUMO
This study analyzed the effect of inflammation on acetylcholine (ACh)-induced muscarinic receptors (MR)2 and MR3 conducted contractility of the porcine uterus. On Day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) was injected into uterine horns or laparotomy was performed (CON group). Eight days later, infected gilts developed severe acute endometritis. Compared to the period before ACh treatment, ACh (10-5â¯M) increased the tension in myometrium (MYO) and endometrium/myometrium (ENDO/MYO) of the CON group (Pâ¯<â¯0.01) and in ENDO/MYO of the SAL group (Pâ¯<â¯0.01), the amplitude in strips of the CON (Pâ¯<â¯0.05) and SAL (MYO: Pâ¯<â¯0.05, ENDO/MYO: Pâ¯<â¯0.001) groups and the frequency in strips of the CON (MYO: Pâ¯<â¯0.01, ENDO/MYO: Pâ¯<â¯0.001) and SAL (Pâ¯<â¯0.01) groups. In the E.coli group, ACh (10-5â¯M) reduced the amplitude in MYO (Pâ¯<â¯0.05) and ENDO/MYO (Pâ¯<â¯0.001), increased the frequency in MYO (Pâ¯<â¯0.01) and ENDO/MYO (Pâ¯<â¯0.001) and did not change (Pâ¯>â¯0.05) the tension. ACh (10-5â¯M) in ENDO/MYO of the E.coli group, reduced the tension compared to the CON group (Pâ¯<â¯0.05) and the amplitude compared to other groups (Pâ¯<â¯0.001), while increased the frequency in relation to the SAL group (Pâ¯<â¯0.05). MR2 antagonist (AF-DX 44â¯116) and ACh (10-5â¯M) reduced (by 16.92%, Pâ¯<â¯0.01) the tension in MYO of the CON group and increased (Pâ¯<â¯0.01) it in the E.coli group compared to the period before antagonist and ACh addition. In MYO of the SAL group, the tension was increased (Pâ¯<â¯0.01) in response to MR3 antagonist (4-DAMP) and ACh (10-7, 10-6â¯M). In the E.coli group, these substances did not change (Pâ¯>â¯0.05) the tension, but it was lower (Pâ¯<â¯0.001) in MYO (ACh: 10-7â¯M) and ENDO/MYO (ACh: 10-5â¯M) than in the SAL group. MR2 or MR3 antagonists and ACh (10-5â¯M) increased (Pâ¯<â¯0.05-0.001) the amplitude in strips of the CON and SAL groups and reduced it in the E.coli group (Pâ¯<â¯0.001) compared to the period before antagonists and ACh use. This parameter in the E.coli group was lower (Pâ¯<â¯0.001) after using MR2 or MR3 antagonists and ACh (10-6, 10-5â¯M) than in other groups. Both antagonists and ACh (10-5â¯M) reduced the frequency in the CON, SAL (Pâ¯<â¯0.05) and E.coli (MR2 antagonist: Pâ¯<â¯0.01, MR3 antagonist: Pâ¯<â¯0.05) groups compared to period before antagonists and ACh addition. Data show that ACh reduces the contractility of the inflamed porcine uterus by MR2 and MR3, which suggests that pharmacological modulation of these receptors can be used to raise the contractility of an inflamed uterus.
Assuntos
Acetilcolina/farmacologia , Doenças dos Suínos/fisiopatologia , Contração Uterina/efeitos dos fármacos , Doenças Uterinas/veterinária , Animais , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/fisiopatologia , Inflamação/veterinária , Miométrio/efeitos dos fármacos , Miométrio/metabolismo , Pirenzepina/análogos & derivados , Pirenzepina/farmacologia , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Suínos , Doenças Uterinas/patologia , Doenças Uterinas/fisiopatologiaRESUMO
Given the global epidemic in type 2 diabetes, novel antidiabetic drugs with increased efficacy and reduced side effects are urgently needed. Previous work has shown that M3 muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic ß cells play key roles in stimulating insulin secretion and maintaining physiological blood glucose levels. In the present study, we tested the hypothesis that a positive allosteric modulator (PAM) of M3R function can improve glucose homeostasis in mice by promoting insulin release. One major advantage of this approach is that allosteric agents respect the ACh-dependent spatiotemporal control of M3R activity. In this study, we first demonstrated that VU0119498, a drug known to act as a PAM at M3Rs, significantly augmented ACh-induced insulin release from cultured ß cells and mouse and human pancreatic islets. This stimulatory effect was absent in islets prepared from mice lacking M3Rs, indicative of the involvement of M3Rs. VU0119498 treatment of wild-type mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These effects were mediated by ß-cell M3Rs, since they were absent in mutant mice selectively lacking M3Rs in ß cells. Moreover, acute VU0119498 treatment of obese, glucose-intolerant mice triggered enhanced insulin release and restored normal glucose tolerance. Interestingly, doses of VU0119498 that led to pronounced improvements in glucose homeostasis did not cause any significant side effects due to activation of M3Rs expressed by other peripheral cell types. Taken together, the data from this proof-of-concept study strongly suggest that M3R PAMs may become clinically useful as novel antidiabetic agents.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Receptor Muscarínico M3/efeitos dos fármacos , Acetilcolina/metabolismo , Adulto , Regulação Alostérica/efeitos dos fármacos , Animais , Glicemia/análise , Glicemia/metabolismo , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Feminino , Intolerância à Glucose/sangue , Intolerância à Glucose/tratamento farmacológico , Intolerância à Glucose/metabolismo , Humanos , Hipoglicemiantes/uso terapêutico , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Obesos , Camundongos Transgênicos , Pessoa de Meia-Idade , Agonistas Muscarínicos/uso terapêutico , Obesidade/sangue , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Cultura Primária de Células , Estudo de Prova de Conceito , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Adulto JovemRESUMO
AIMS: The prevalence of urinary dysfunction increases with age, yet therapies are often suboptimal. Incomplete understanding of the linkages between system, organ, and tissue domains across lifespan remains a knowledge gap. If tissue-level changes drive the aging bladder phenotype, parallel changes should be observed across these domains. In contrast, a lack of inter-domain correlation across age groups would support the hypothesis that urinary performance is a measure of the physiologic reserve, dependent on centrally-mediated adaptive mechanisms in the aging system. METHODS: Male and female mice across four age groups underwent sequential voiding spot assays, pressure/flow cystometry, bladder strip tension studies, histology, and quantitative PCR analyses. The primary objective of this study was to test the impact of age on the cortical, autonomic, tissue functional and structural, and molecular domains, and identify inter-domain correlations among variables showing significant changes with age within these domains. RESULTS: Behavior revealed diminished peripheral voiding and spot size in aged females. Cystometry demonstrated increased postvoid residual and loss of volume sensitivity, but the preservation of voiding contraction power, with almost half of oldest-old mice failing under cystometric stress. Strip studies revealed no significant differences in adrenergic, cholinergic, or EFS sensitivity. Histology showed increased detrusor and lamina propria thickness, without a change in collagen/muscle ratio. Adrb2 gene expression decreased with age. No consistent inter-domain correlations were found across age groups. CONCLUSIONS: Our findings are consistent with a model in which centrally-mediated adaptive failures to aging stressors are more influential over the aging bladder phenotype than local tissue changes.
Assuntos
Envelhecimento/fisiologia , Contração Muscular/fisiologia , Bexiga Urinária/fisiopatologia , Micção/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Envelhecimento/genética , Envelhecimento/patologia , Animais , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Estimulação Elétrica , Feminino , Isoproterenol/farmacologia , Masculino , Camundongos , Mucosa/patologia , Miografia , Fenótipo , Receptor Muscarínico M3/genética , Receptores Adrenérgicos beta 2/genética , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Bexiga Urinária/patologiaRESUMO
AIM: Muscarinic acetylcholine receptors (AChMR1-5) are fundamental for cellular responses upon release of the neurotransmitter acetylcholine (ACh) from parasympathetic nerve fibers. ACh is the prototypical agonist stimulating endothelium-dependent dilation, but most blood vessels lack parasympathetic innervation, raising the question as to the physiologic function of endothelial AChMR in vivo. Global deletion of AChM3R revealed a role in ACh-induced vasodilation in vitro and food uptake, but overall cardiovascular homeostasis has not been examined thoroughly. METHODS: To characterize the function of endothelial AChM3R in vivo, we deleted AChM3R specifically in endothelial cells with an inducible or a non-inducible Cre-loxP system, driven by the endothelium-specific promoters VE-cadherin (indEC-M3R-/- ) or TIE2 (tek2; EC-M3R-/- ) and examined arteriolar dilation in the cremaster microcirculation, arterial pressure and cardiac function in these mice in vivo. RESULTS: In both EC-M3R-/- , ACh-induced dilation was strongly impaired in arterioles in vivo, while responses to other dilators were mostly preserved. However, arterial pressure (indEC-M3R-/- ) and arteriolar tone as a surrogate for peripheral vascular resistance did not differ between EC-M3R-/- and control mice. Aged EC-M3R-/- mice (74-78 weeks) did not differ in body weight, heart weight, cardiac structure or contractile function from controls. CONCLUSION: We conclude that AChM3R elicits the endothelium-dependent dilation upon ACh also in arterioles in vivo. Despite this prominent role, the endothelial deletion of AChM3R does not affect overall cardiovascular homeostasis. Thus, their physiologic function in endothelial cells remains obscure.
Assuntos
Acetilcolina/toxicidade , Endotélio Vascular/metabolismo , Receptor Muscarínico M3/metabolismo , Receptor TIE-2/metabolismo , Vasodilatação/efeitos dos fármacos , Animais , Endotélio Vascular/efeitos dos fármacos , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase , Masculino , Camundongos , Camundongos Transgênicos , Receptor Muscarínico M3/genética , Receptor TIE-2/genética , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/farmacologiaRESUMO
Cancer cells are considered to have high morphological heterogeneity in human melanoma tissue. Here, we report that epithelial cancer cells are dominant in different development stages of human melanoma tissues. The cellular and molecular mechanisms that maintain melanoma cells in the epithelial state are further investigated in the A2058 cell line. We find that micropore (8 µm) transwell invasion, but not superficial migration in the scratch assay, can induce remarkable morphological changes between epithelial and mesenchymal melanoma cells within 4 days. The morphological switch is associated with dynamic changes of epithelial-mesenchymal transition (EMT) hallmarks E-cadherin and vimentin. Further immunoflurencent staining and co-immunoprecipitation assay showed the uncoupling of the M3 muscarinic acetylcholine receptor (mAChR) and the p75 neurotrophin receptor (p75NTR) in epithelial melanoma cells. Specific knockdown of M3 mAChR by small interfering RNA (siRNA) significantly abrogates the transition of spindle-shaped mesenchymal cells to epithelial cells. Collectively, we report a cellular model of invasiveness-triggered state transition (ITST) in which melanoma cell invasion can induce morphological changes between epithelial and mesenchymal cells. ITST is one of the biological basis for maintaining metastatic melanoma cells in the epithelial state. Furthermore, M3 mAChR receptor-mediated ITST provides a novel therapeutic strategy to inhibit the development of malignant melanoma.
Assuntos
Movimento Celular , Transição Epitelial-Mesenquimal , Melanoma/patologia , Neoplasias Cutâneas/patologia , Antígenos CD/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Forma Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/metabolismo , Invasividade Neoplásica , Proteínas do Tecido Nervoso/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Vimentina/metabolismoRESUMO
Prostate cancer (PCa) testing is currently based on measurement of serum prostatespecific antigen levels and digital rectal examination, which are limited by a low predictive value and the adverse effects associated with overdiagnosis and overtreatment. Recent studies have reported that the abnormal expression of microRNAs (miRNAs) is associated with the mechanism underlying the development of PCa. Thus, the aim of the present study was to investigate the effects of miR30e and its target gene, M3 muscarinic acetylcholine receptor (CHRM3), on the adhesion, migration, invasion and cell cycle distribution of PCa cells via the mitogenactivated protein kinase (MAPK) signaling pathway. The differentially expressed genes were screened in the Gene Expression Omnibus database from a gene expression microarray (GSE55945) of PCa. PCa tissues and adjacent tissues were collected from patients with PCa. The PC3 and DU145 human PCa cell lines were treated with activator, inhibitor and siRNAs. The effects of miR30e on cell adhesion, migration, invasion and cell cycle distribution with the involvement of CHRM3 and the MAPK signaling pathway were investigated. The bioinformatics results demonstrated that the CHRM3 gene and the MAKP signaling pathway were involved in the progression of PCa, and hasmiR30e was selected for further study. The levels of miR30e were significantly downregulated, while the levels of CHRM3 were obviously upregulated in PCa. CHRM3 was verified as a target gene of miR30e. Upregulation of miR30e and downregulation of CHRM3 decreased the levels of pP38, pextracellular signalregulated kinase, pcJun Nterminal kinase, pcfos and pcJUN, cell adhesion, migration and invasion ability, and the number of cells in the S phase, while they increased the number of cells in the G0 and G1 phases. The findings of the present study suggest that miR30e inhibited the adhesion, migration, invasion and cell cycle entry of PCa cells by suppressing the activation of the MAPK signaling pathway and inhibiting CHRM3 expression. Thus, miR30e may serve as a candidate target for the treatment of PCa.
Assuntos
Adesão Celular/genética , MicroRNAs/genética , Neoplasias da Próstata/genética , Receptor Muscarínico M3/genética , Idoso , Ciclo Celular/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Masculino , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias da Próstata/patologia , Transdução de SinaisRESUMO
BACKGROUND: We explored the genetic variants that were associated with polycystic ovary syndrome (PCOS) by genome-wide association study (GWAS) and evaluated the association of genetic risk scores (GRS) of the selected genetic variants with insulin resistance and the interaction of the GRS with nutrient intake to develop insulin resistance. METHODS: The 6 genetic variants involved in brain and nervous system (acid sensing ion channel subunit 2 rs8071961, rs1988598, and rs16589, macro domain containing 2 rs7262810 CHRM3 rs1867265 and chromosome 2 open reading frame 83 rs11889798) were selected from a GWAS of the PCOS study. GRS of the 6 single nucleotide polymorphisms was calculated in 3,723 Korean women in the Ansan/Ansung cohort of KARE study. RESULTS: Fasting serum insulin and C-reactive protein (CRP) levels, homeostasis model assessment of insulin resistance (HOMA-IR), and psychological stress levels were significantly higher in the high-GRS group than the low-GRS group. However, serum-free T4 levels were significantly lower in the high-GRS group. HOMA-IR and CRP were higher OR (1.129 and 1.382) in the high-GRS group than the low-GRS group after adjusted for covariates. There was a significant interaction between GRS and daily energy intake (p = 0.004). The OR (1.233) for HOMA-IR was higher in the high-GRS group than the low-GRS group only in the group with lower energy intakes based on estimated energy requirement. CONCLUSION: Women with high-GRS for PCOS had increased risk of insulin resistance and low energy intake did not protect against the elevation of insulin resistance in women with high GRS. Low energy intake might be protective against PCOS in carriers with low and medium GRS.
Assuntos
Canais Iônicos Sensíveis a Ácido/genética , Enzimas Reparadoras do DNA/genética , Hidrolases/genética , Resistência à Insulina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Síndrome do Ovário Policístico/genética , Receptor Muscarínico M3/genética , Adulto , Glicemia/análise , Índice de Massa Corporal , Proteína C-Reativa/análise , Estudos de Coortes , Ingestão de Energia , Jejum , Feminino , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Humanos , Insulina/sangue , Pessoa de Meia-Idade , Nutrigenômica , Síndrome do Ovário Policístico/sangue , Polimorfismo de Nucleotídeo Único/genética , República da Coreia , Tiroxina/sangueRESUMO
Primary dysmenorrhea affects the quality of life in young women, particularly school and work performance. This study investigated the mechanisms of penehyclidine hydrochloride (PHC) efficacy on a rat model of primary dysmenorrhea. The model was induced by injecting both estradiol benzoate and oxytocin. Different doses of PHC were administrated intraperitoneally following estradiol benzoate administration. Writhing scores were assessed, and pathological changes of the uterus were observed via hematoxylin and eosin staining. Western blot and real-time PCR were used to evaluate the expression level of the M3 receptor, both TLR3 and TLR4 in uterine tissue, and the level of Ca2+ was measured in uterine tissues. Writhing scores significantly decreased in the PHC treatment group compared to model, and PHC alleviated the occurrence of edema or necrosis in the uteri compared to model group. PHC can decrease the M3 receptor, TLR3 , TLR4 expression, and the Ca2+ level compared to the model group. PHC is a potential candidate for the future treatment of primary dysmenorrhea due to its ability to attenuate muscarinic receptors and TLRs. Preclinical Research & Development.
Assuntos
Dismenorreia/tratamento farmacológico , Quinuclidinas/uso terapêutico , Animais , Comportamento Animal/efeitos dos fármacos , Cálcio/metabolismo , Modelos Animais de Doenças , Dismenorreia/induzido quimicamente , Dismenorreia/genética , Dismenorreia/metabolismo , Estradiol/análogos & derivados , Feminino , Ocitocina , Dor/tratamento farmacológico , Quinuclidinas/farmacologia , Ratos Sprague-Dawley , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Útero/efeitos dos fármacos , Útero/metabolismo , Útero/patologiaRESUMO
Purpose: As only some smokers develop COPD with emphysema, we explored the molecular pathogenesis of early-stage COPD with emphysema using gene expression profiling of human lung tissues. Patients and methods: First, 110 subjects who had smoked more than ten pack-years were classified into three groups: COPD with emphysema, COPD without emphysema, and healthy smokers. COPD and emphysema were confirmed by post-bronchodilator forced expiratory volume in 1 second/forced vital capacity <0.7 and by chest computed tomography. Lung tissues obtained surgically from the 110 subjects were processed and used for RNA-Seq analysis. Results: Among the 110 subjects, 29 had COPD with emphysema, 21 had COPD without emphysema, and 60 were healthy smokers; their mean post-bronchodilator forced expiratory volume in 1 second values were 78%, 80%, and 94%, respectively. Using RNA-Seq, we evaluated 16,676 genes expressed in lung tissues. Among them, 1,226 genes in the COPD with emphysema group and 434 genes in the COPD without emphysema group were differentially expressed genes compared to the expression in healthy smokers. In the COPD with emphysema group, ACER2 and LMAN2L were markedly increased and decreased, respectively. In the COPD without emphysema group, the CHRM3 gene, previously reported to be associated with COPD, and HDAC10 were markedly increased and decreased, respectively. Conclusion: Our study identified differences in gene expression in subjects with COPD according to emphysema status using RNA-Seq transcriptome analysis. These findings may have mechanistic implications in COPD.