RESUMO
The diabetes-cancer association remains underexplained. Here, we describe a glucose-signaling axis that reinforces glucose uptake and glycolysis to consolidate the Warburg effect and overcome tumor suppression. Specifically, glucose-dependent CK2 O-GlcNAcylation impedes its phosphorylation of CSN2, a modification required for the deneddylase CSN to sequester Cullin RING ligase 4 (CRL4). Glucose, therefore, elicits CSN-CRL4 dissociation to assemble the CRL4COP1 E3 ligase, which targets p53 to derepress glycolytic enzymes. A genetic or pharmacologic disruption of the O-GlcNAc-CK2-CSN2-CRL4COP1 axis abrogates glucose-induced p53 degradation and cancer cell proliferation. Diet-induced overnutrition upregulates the CRL4COP1-p53 axis to promote PyMT-induced mammary tumorigenesis in wild type but not in mammary-gland-specific p53 knockout mice. These effects of overnutrition are reversed by P28, an investigational peptide inhibitor of COP1-p53 interaction. Thus, glycometabolism self-amplifies via a glucose-induced post-translational modification cascade culminating in CRL4COP1-mediated p53 degradation. Such mutation-independent p53 checkpoint bypass may represent the carcinogenic origin and targetable vulnerability of hyperglycemia-driven cancer.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Animais , Camundongos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Glucose , Ubiquitina-Proteína Ligases/metabolismo , Carcinogênese/genética , Transformação Celular Neoplásica/genéticaRESUMO
Kisspeptin receptor (Kiss1R) is an important receptor that plays central regulatory roles in reproduction by regulating hormone release in the hypothalamus. We hypothesize that the formation of heterocomplexes between Kiss1R and other hypothalamus G protein-coupled receptors (GPCRs) affects their cellular signaling. Through screening of potential interactions between Kiss1R and hypothalamus GPCRs, we identified G protein-coupled estrogen receptor (GPER) as one interaction partner of Kiss1R. Based on the recognised function of kisspeptin and estrogen in regulating the reproductive system, we investigated the Kiss1R/GPER heterocomplex in more detail and revealed that complex formation significantly reduced Kiss1R-mediated signaling. GPER did not directly antagonize Kiss1R conformational changes upon ligand binding, but it rather reduced the cell surface expression of Kiss1R. These results therefore demonstrate a regulatory mechanism of hypothalamic hormone receptors via receptor cooperation in the reproductive system and modulation of receptor sensitivity.
Assuntos
Hipotálamo/metabolismo , Complexos Multiproteicos/genética , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Kisspeptina-1/genética , Animais , Hormônios/biossíntese , Hormônios/genética , Humanos , Complexos Multiproteicos/ultraestrutura , Ligação Proteica/genética , Receptores de Superfície Celular/genética , Receptores de Estrogênio/ultraestrutura , Receptores Acoplados a Proteínas G/ultraestrutura , Receptores de Kisspeptina-1/ultraestrutura , Transdução de Sinais/genéticaRESUMO
Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.
Assuntos
Aorta Torácica/cirurgia , Constrição Patológica , Modelos Animais de Doenças , Procedimentos Cirúrgicos Vasculares , Animais , Aorta Torácica/fisiopatologia , Artérias Carótidas/fisiopatologia , Ecocardiografia , Ecocardiografia Doppler , Coração/fisiopatologia , Insuficiência Cardíaca/patologia , Ventrículos do Coração/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Procedimentos Cirúrgicos Minimamente Invasivos , Ultrassonografia Doppler em CoresRESUMO
Ovarian cancer is the seventh most common cancer in women and the most lethal gynecological cancer, causing over 151,000 deaths worldwide each year. Dysregulated production of endocrine hormones, known to have pluripotent effects on cell function through the activation of receptor signaling pathways, is believed to be a high-risk factor for ovarian cancer. An increasing body of evidence suggests that endocrine G protein-coupled receptors (GPCRs) are involved in the progression and metastasis of ovarian neoplasms. GPCRs are attractive drug targets because their activities are regulated by more than 25% of all drugs approved by the Food and Drug Administration. Therefore, understanding the role of endocrine GPCRs during ovarian cancer progression and metastasis will allow for the development of novel strategies to design effective chemotherapeutic drugs against malignant ovarian tumors. In this review, we address the signaling pathways and functional roles of several key endocrine GPCRs that are related to the cause, progression, and metastasis of ovarian cancer.
RESUMO
Secretin (SCT) was first considered to be a gut hormone regulating gastrointestinal functions when discovered. Recently, however, central actions of SCT have drawn intense research interest and are supported by the broad distribution of SCT in specific neuronal populations and by in vivo physiological studies regarding its role in water homeostasis and food intake. The direct action of SCT on a central neuron was first discovered in cerebellar Purkinje cells in which SCT from cerebellar Purkinje cells was found to potentiate GABAergic inhibitory transmission from presynaptic basket cells. Because Purkinje neurons have a major role in motor coordination and learning functions, we hypothesize a behavioral modulatory function for SCT. In this study, we successfully generated a mouse model in which the SCT gene was deleted specifically in Purkinje cells. This mouse line was tested together with SCT knockout and SCT receptor knockout mice in a full battery of behavioral tasks. We found that the knockout of SCT in Purkinje neurons did not affect general motor ability or the anxiety level in open field tests. However, knockout mice did exhibit impairments in neuromuscular strength, motor coordination, and motor learning abilities, as shown by wire hanging, vertical climbing, and rotarod tests. In addition, SCT knockout in Purkinje cells possibly led to the delayed development of motor neurons, as supported by the later occurrence of key neural reflexes. In summary, our data suggest a role in motor coordination and motor learning for SCT expressed in cerebellar Purkinje cells.
Assuntos
Aprendizagem/fisiologia , Atividade Motora/fisiologia , Células de Purkinje/metabolismo , Secretina/metabolismo , Animais , Ansiedade/metabolismo , Comportamento Exploratório/fisiologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Força Muscular/fisiologia , Testes Neuropsicológicos , Receptores Acoplados a Proteínas G/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Teste de Desempenho do Rota-Rod , Secretina/genéticaRESUMO
In several vertebrates, it has been demonstrated that alternative splicing of PAC1 receptor (PAC1-R) transcripts can generate a number of functional receptor variants which utilize different signal transduction pathways to mediate their activities. As PACAP is a physiological growth hormone-releasing factor in fish, and PACAP and the PAC1-R are highly conserved in vertebrate evolution, it would be of interest to investigate the structure and cellular distribution, particularly in the pituitary, of PAC1-R splice variants in a fish model. Our laboratory has previously cloned a receptor cDNA corresponding to the goldfish PAC1-R-s (goldfish PAC1-R-short). In the present study, a goldfish PAC1-R-hop1 variant was characterized. Functional expression of goldfish PAC1-R-s and PAC1-R-hop1 in Chinese Hamster Ovary cells revealed that, upon stimulation by ovine PACAP38, these receptor variants exhibited similar EC50 values (8.7+/-1.5 and 8.8+/-1.9 nM, respectively) and maximal responses in activating intracellular cAMP production. The presence and expression levels of these transcripts were measured by quantitative real-time PCR in the brain, heart, pituitary and male gonad, and goldfish PAC1-R-s were found to be the predominant form. In situ hybridization of goldfish PAC1-R in the pituitary revealed its prevalent presence in the pars distalis. In summary, the present study provides information to confirm the role of PACAP in the pituitary and to elucidate the pleiotropic effects of PACAP in fish.
Assuntos
Processamento Alternativo , Proteínas de Peixes/genética , Carpa Dourada/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , DNA Complementar/metabolismo , Proteínas de Peixes/fisiologia , Carpa Dourada/metabolismo , Carpa Dourada/fisiologia , Hibridização In Situ , Dados de Sequência Molecular , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Hipófise/citologia , Hipófise/metabolismo , Estrutura Terciária de Proteína , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Distribuição TecidualRESUMO
Previous studies have revealed distribution of histaminergic fibers and presence of histamine receptors in globus pallidus (GP). In this study, the brain slice preparation of adult rats was used to examine the effect of histamine on the spontaneous unitary discharge of GP neurons and the underlying receptor mechanism. Ninety-five GP neurons were extracellularly recorded from 42 slices containing the GP, of which 87 (91.6%) were excited by the stimulation of histamine. The histamine-induced excitation was concentration-dependent and persisted in low Ca2+/high Mg2+ medium (n = 9), demonstrating that the action of histamine on the GP neurons was postsynaptic. The excitatory effect of histamine on the GP neurons was not blocked by selective histamine H1 receptor antagonist triprolidine (n = 16) or chlorpheniramine (n = 6), but was effectively suppressed by ranitidine, a highly selective histamine H2 receptor antagonist (n = 21). On the other hand, highly selective histamine H2 receptor agonist dimaprit mimicked the excitatory effect of histamine on the GP neurons (n = 23), while histamine H1 receptor agonists, including 2-pyridylethylamine (n = 22), 2-thiazolyethylamine (n = 9) and betahistine (n = 9), did not cause GP neurons any response. The dimaprit-induced GP neuronal excitation was effectively antagonized by selective histamine H2 receptor antagonist ranitidine (n = 14) but not influenced by selective histamine H1 receptor antagonist triprolidine (n = 12). Moreover, adenylate cyclase (AC) activator forskolin (n = 7) was observed to evoke GP neurons an excitatory response, whereas the histamine-induced excitation was effectively reduced by H-89 (n = 9), a selective and potent inhibitor of protein kinase A (PK(A)). Finally, it was noted that neurons of both subdivisions of the GP, the internal (GPi, n = 35) and external (GPe, n = 60) segment, showed no differences in their responses to stimulations of the tested histaminergic reagents. These results demonstrated that histamine excited GP (including GPi and GPe) neurons via histamine H2 receptors and H2 receptors linked intracellular G-protein-AC-PK(A) signaling pathway, suggesting that the hypothalamic histaminergic afferent fibers innervating GP may play an important modulatory role in motor control through its excitatory effect on GP neurons.
Assuntos
Potenciais de Ação/fisiologia , Globo Pálido/fisiologia , Histamina/metabolismo , Neurônios/fisiologia , Receptores Histamínicos H2/fisiologia , Transmissão Sináptica/fisiologia , Potenciais de Ação/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Doenças dos Gânglios da Base/metabolismo , Doenças dos Gânglios da Base/fisiopatologia , Cálcio/metabolismo , Cálcio/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Feminino , Globo Pálido/efeitos dos fármacos , Histamina/farmacologia , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Magnésio/metabolismo , Magnésio/farmacologia , Masculino , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Receptores Histamínicos H2/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
Secretin is a neuropeptide that is expressed in distinct central neurones. As there is no information on how the secretin gene is regulated in neuronal cells, a well established neuronal differentiation cell model, SH-SY5Y, was used to study transcriptional regulation of the human secretin gene. High secretin transcript and peptide levels were found in this cell, and secretin gene expression and promoter activity were up-regulated upon all-trans retinoic acid (RA) treatment. Within the promoter, a functional GC-box 1 (-131 from ATG, relative to the ATG initiation codon) was found to be regulated by a brain-specific Sp protein, Sp4, and ubiquitous factors Sp1 and Sp3. The human secretin gene in SH-SY5Y cells is controlled by the (Sp1 + Sp4)/Sp3 ratio and the RA-induced activation is a partial result of a decrease in Sp3 levels. In addition to the GC-box 1, an N1 motif in close proximity was also responsible for RA-induced secretin gene activation. Competitive gel mobility shift and southwestern blot studies revealed binding of Nuclear Factor I (NFI) with the N1 motif. Overexpression of NFI-C increased promoter activity upon RA treatment. Consistent with this observation, NFI-C transcript levels were augmented after RA treatment. We conclude that RA induction of the secretin gene in neuronal cells is regulated by the combined actions of reducing Sp3 and increasing NFI-C expression.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Proteínas de Ligação a DNA/fisiologia , Secretina/biossíntese , Secretina/genética , Fator de Transcrição Sp1/fisiologia , Fatores de Transcrição/fisiologia , Tretinoína/farmacologia , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Dados de Sequência Molecular , Fatores de Transcrição NFI , Elementos de Resposta/efeitos dos fármacos , Fator de Transcrição Sp3 , Fator de Transcrição Sp4 , Ativação TranscricionalRESUMO
To unravel the mechanisms that regulate the human secretin gene expression, in this study, we have used secretin-expressing (HuTu-80 cells, human duodenal adenocarcinoma) and non-secretin-expressing [PANC-1 (human pancreatic ductile carcinoma) and HepG2 (human hepatocellular carcinoma) cells] cell models for in vitro and in vivo analyses. By transient transfection assays, within the promoter region (-11 to -341 from ATG, relative to the ATG initiation codon), we have initially identified several functional motifs including an E-box and 2 GC-boxes. Results from gel mobility shift and chromatin immunoprecipitation assays confirmed further that NeuroD, E2A, Sp1, and Sp3 bind to these E- and GC-boxes in HuTu-80 cells in vitro and in vivo, whereas only high levels of Sp3 is observed to bind the promoter in HepG2 cells. In addition, overexpression of Sp3 resulted in a dose-dependent repression of the Sp1-mediated transactivation. Collectively, these data suggest that the Sp1/Sp3 ratio is instrumental to controlling secretin gene expression in secretin-producing and non-secretin-producing cells. The functions of GC-box and Sp proteins prompted us to investigate the possible involvement of DNA methylation in regulating this gene. Consistent with this idea, we found a putative CpG island (-336 to 262 from ATG) that overlaps with the human secretin gene promoter. By methylation-specific PCR, all the CpG dinucleo-tides (26 of them) within the CpG island in HuTu-80 cells are unmethylated, whereas all these sites are methylated in PANC-1 and HepG2 cells. The expressions of secretin in PANC-1 and HepG2 cells were subsequently found to be significantly activated by a demethylation agent, 5'-Aza-2' deoxycytidine. Taken together, our data indicate that the human secretin gene is controlled by the in vivo Sp1/Sp3 ratio and the methylation status of the promoter.
Assuntos
Ilhas de CpG , Proteínas de Ligação a DNA/metabolismo , Elementos E-Box , Secretina/genética , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Região 5'-Flanqueadora , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Metilação de DNA , Proteínas de Ligação a DNA/genética , Desoxicitidina/farmacologia , Drosophila/citologia , Drosophila/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Plicamicina/farmacologia , Regiões Promotoras Genéticas , Secretina/efeitos dos fármacos , Secretina/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3 , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
The human secretin receptor (hSR) is an important glycoprotein receptor for regulating the secretion of pancreatic bicarbonate, water, and electrolytes. In this study we investigated the transcriptional regulation of the hSR gene. A minimal 106-bp promoter was identified, and it contains two GC boxes (GC box-A, -240 to -226; and GC box-B, -203 to -194, from the translation start site). EMSA and supershift analyses showed that both GC boxes interact with Sp1 and Sp3 transcription factors. Transient transfection in pancreas-derived human pancreatic ductule carcinoma (PANC)-1 and bovine pancreatic duct-1 cells showed that mutation of either GC box-A or -B reduced the promoter strength by 56-67%, whereas mutation of both GC boxes caused more than 90% reduction of promoter activity. Cotransfections of the hSR promoter with Sp1 and Sp3 expression vectors in Sp-deficient Drosophila SL-2 Schneider cells further demonstrated that the ratio of Sp1 to Sp3 is the key mechanism to modulate hSR gene expression. The methylation statuses of 27 CpG sites within the promoter region (-400 to -151 bp) were assessed in various human pancreas and liver cell lines. The hSR promoter is unmethylated (CAPAN-1, human pancreatic adenocarcinoma) or partially methylated (PANC-1 and HPAC, human pancreatic adenocarcinoma) in hSR-expressing cell lines but is completely methylated in hSR nonexpressing HepG2 cells. Methyltransferase inhibitor 5-aza-2'deoxycytidine increased hSR gene expression level in PANC-1 cells and induced hSR gene expression in HepG2 cells. Together, our study shows that, in addition to Sp1 and Sp3, promoter methylation also plays a role in the regulation of hSR gene expression.
Assuntos
Ilhas de CpG , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Receptores dos Hormônios Gastrointestinais/genética , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Animais , Azacitidina/farmacologia , Composição de Bases , Sequência de Bases , Bovinos , Células Cultivadas , Citosina/metabolismo , Metilases de Modificação do DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Drosophila/citologia , Drosophila/genética , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Regiões Promotoras Genéticas/genética , Receptores Acoplados a Proteínas G , Receptores dos Hormônios Gastrointestinais/efeitos dos fármacos , Receptores dos Hormônios Gastrointestinais/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3 , Fatores de Transcrição/genéticaRESUMO
Peptide histidine isoleucine (PHI), peptide histidine valine (PHV), and vasoactive intestinal polypeptide (VIP) are cosynthesized from the same precursor and share high levels of structural similarities with overlapping biological functions. In this study, the first PHI/PHV receptor was isolated and characterized in goldfish. To study this receptor using homologous peptides, we have also characterized the goldfish prepro-PHI/VIP, and, surprisingly, a shorter transcript lacking the VIP coding region was isolated. A PHI/VIP precursor without the VIP coding sequence has never before been reported. Initial functional expression of the PHI/PHV receptor in Chinese hamster ovary cells revealed that it could be activated by human PHV [50% effective concentration (EC(50)): 43 nM] and to a lesser extent human PHI (EC(50): 133 nM) and helodermin (EC(50): 166 nM) but not fish and mammalian pituitary adenylate cyclase-activating polypeptides and VIPs. Subsequent studies indicated that, similar to the pituitary adenylate cyclase-activating polypeptide receptors (PAC1-R, VPAC1-R, and VPAC2-R), the receptor isolated in this study is able to interact with goldfish PHI and its C-terminally extended form, PHV with EC(50) values 93 and 43 nM, respectively. Northern blot and RT-PCR/Southern blot analyses revealed that the PHI/VIP gene is expressed in the intestine, brain, and gall bladder and the PHI/PHV receptor gene is primarily expressed in the pituitary and to a lesser extend in the intestine and gall bladder, suggesting that PHI/PHV may play a role, notably in the regulation of pituitary function. In conclusion, our results demonstrate for the first time the existence of a PHI/PHV receptor, indicating that the functions of PHI and PHV could be mediated by their own receptor in addition to VIP receptors.