RESUMO
Abnormal expression and dysfunction of adiponectin and the cognate receptors are involved in diabetes and diabetic kidney disease (DKD), whereas angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) alleviate diabetic albuminuria and prevent development of DKD through upregulation of adiponectin expression. Here we report that high glucose stimulates expression of angiotensin II (AngII) receptors (AT1 and AT2) in renal proximal tubular epithelial cells (NRK-52E). These receptors underwent hetero-dimerization with adiponectin receptor AdipoR1 and AdipoR2, respectively. High glucose inhibited the dimerization between AT1 and AT2. Interestingly, these hetero-dimers instigated tubulointerstitial injury by inhibiting the cytoprotective action of the adiponectin receptors. These modes of receptor-receptor hetero-dimerization may contribute to high glucose-induced renal tubulointerstitial injury and could be potential therapeutic targets.
Assuntos
Nefropatias Diabéticas/metabolismo , Glucose/metabolismo , Nefrite Intersticial/metabolismo , Receptores de Adiponectina/metabolismo , Receptores de Angiotensina/metabolismo , Animais , Linhagem Celular , Dimerização , Ligação Proteica , RatosRESUMO
RATIONALE: Nitro-oleic acid (OA-NO(2)) is a bioactive, nitric-oxide derived fatty acid with physiologically relevant vasculoprotective properties in vivo. OA-NO(2) exerts cell signaling actions as a result of its strong electrophilic nature and mediates pleiotropic cell responses in the vasculature. OBJECTIVE: The present study sought to investigate the protective role of OA-NO(2) in angiotensin (Ang) II-induced hypertension. METHODS AND RESULTS: We show that systemic administration of OA-NO(2) results in a sustained reduction of Ang II-induced hypertension in mice and exerts a significant blood pressure lowering effect on preexisting hypertension established by Ang II infusion. OA-NO(2) significantly inhibits Ang II contractile response as compared to oleic acid (OA) in mesenteric vessels. The improved vasoconstriction is specific for the Ang II type 1 receptor (AT(1)R)-mediated signaling because vascular contraction by other G-protein-coupled receptors is not altered in response to OA-NO(2) treatment. From the mechanistic viewpoint, OA-NO(2) lowers Ang II-induced hypertension independently of peroxisome proliferation-activated receptor (PPAR)gamma activation. Rather, OA-NO(2), but not OA, specifically binds to the AT(1)R, reduces heterotrimeric G-protein coupling, and inhibits IP(3) (inositol-1,4,5-trisphosphate) and calcium mobilization, without inhibiting Ang II binding to the receptor. CONCLUSIONS: These results demonstrate that OA-NO(2) diminishes the pressor response to Ang II and inhibits AT(1)R-dependent vasoconstriction, revealing OA-NO(2) as a novel antagonist of Ang II-induced hypertension.
Assuntos
Angiotensina II/antagonistas & inibidores , Angiotensina II/toxicidade , Hipertensão/induzido quimicamente , Hipertensão/prevenção & controle , Nitrocompostos/uso terapêutico , Ácido Oleico/uso terapêutico , Angiotensina II/fisiologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Hipertensão/fisiopatologia , Ácidos Linoleicos/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Oleicos/uso terapêutico , Ratos , Ratos Sprague-DawleyRESUMO
At the cellular level, 5'-AMP-activated protein kinase (AMPK) serves as a critical link between energy homeostasis and the regulation of fundamental biological activities, including apoptosis. Angiotensin (Ang) II plays a key role in fibrotic lung remodeling. We recently demonstrated that Ang II induces apoptosis in pulmonary artery endothelial cells (PAEC) through the Ang type 2 receptor (AT(2)). AT(2) activates Src-homology two-domain-containing phosphatase-2 (SHP-2) in a signaling cascade leading to Bcl-x(L) mRNA destabilization and initiation of intrinsic apoptosis. We investigated the requirement of AMPK and ATP generation for Ang II-induced apoptosis in PAEC. Ang II activated AMPK, which was required for ATP generation. Inhibition of ATP production by compound C, an AMPK inhibitor, or by oligomycin suppressed Ang II-induced apoptosis. Experiments in Chinese hamster ovary-K1 cells expressing ectopic AT(2) (wild-type, mutant D90A, or carboxy terminal truncated mutant tC319) demonstrated that AT(2) activation of AMPK required the active conformation of the receptor and the carboxy terminal 44 amino acids. AMPK associated with and activated SHP-2 and was required for Bcl-x(L) mRNA destabilization. These are the first findings demonstrating that AMPK is activated by Ang II to produce ATP required for apoptosis. Our data also indicate that AMPK plays an energy-independent role by mediating SHP-2 activation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/biossíntese , Angiotensina II/farmacologia , Células Endoteliais/metabolismo , Ativação Enzimática/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/genética , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Bovinos , Cricetinae , Cricetulus , Células Endoteliais/citologia , Mutação , Plasmídeos , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Artéria Pulmonar/citologia , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Transdução de Sinais/fisiologia , Transfecção , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Domínios de Homologia de srcRESUMO
Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in the synthesis of steroid hormones, essential to fetal development. We have reported that the StAR expression in fetal adrenal is inhibited in a rat model of nicotine-induced intrauterine growth retardation (IUGR). Here using primary human fetal adrenal cortex (pHFAC) cells and a human fetal adrenal cell line NCI-H295A, we show that nicotine inhibits StAR expression and cortisol production in a dose- and time-dependent manner, and prolongs the inhibitory effect on cells proliferating over 5 passages after termination of nicotine treatment. Methylation detection within the StAR promoter region uncovers a single site CpG methylation at nt -377 that is sensitive to nicotine treatment. Nicotine-induced alterations in frequency of this point methylation correlates well with the levels of StAR expression, suggesting an important role of the single site in regulating StAR expression. Further studies using bioinformatics analysis and siRNA approach reveal that the single CpG site is part of the Pax6 binding motif (CGCCTGA) in the StAR promoter. The luciferase activity assays validate that Pax6 increases StAR gene expression by binding to the glucagon G3-like motif (CGCCTGA) and methylation of this site blocks Pax6 binding and thus suppresses StAR expression. These data identify a nicotine-sensitive CpG site at the Pax6 binding motif in the StAR promoter that may play a central role in regulating StAR expression. The results suggest an epigenetic mechanism that may explain how nicotine contributes to onset of adult diseases or disorders such as metabolic syndrome via fetal programming.
Assuntos
Ilhas de CpG/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Fatores de Transcrição Box Pareados/genética , Fosfoproteínas/genética , Proteínas Repressoras/genética , Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/embriologia , Córtex Suprarrenal/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glucagon/genética , Humanos , Hidrocortisona/metabolismo , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , Fator de Transcrição PAX6 , Regiões Promotoras Genéticas , Fatores de TempoRESUMO
Recently, a series of exciting reports have revealed that terminally differentiated somatic cells can be reprogrammed to generate induced pluripotent stem (iPS) cells via overexpression of a cocktail of transcription factors such as Oct3, Sox2, Klf4, and c-Myc or Oct3, Sox2, Nanog, and Lin28. Most recently, these iPS cells has been used to generate viable, live-born progeny by tetraploid complementation. Reprogramming of iPS cells inaugurates a new era of biology and medicine, it inevitably brings new challenges, e.g., how these factors induce reprogramming and how their expression is regulated. To facilitate iPS cell research, this review focuses on how expression and activation of these transcription factors are regulated.
Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Epigênese Genética , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/biossíntese , Animais , Humanos , Fator 4 Semelhante a Kruppel , CamundongosRESUMO
The physiological function of angiotensin II (Ang II) is mediated through the Ang II type 1 (AT1R) and type 2 (AT2R) receptors. Our previous studies have demonstrated that cell surface targeting of AT1R is regulated by Rab and Sar1 GTPases and the F(x)(6)LL motif in the membrane-proximal C terminus. However, the molecular mechanisms underlying the export of nascent AT2R remain poorly defined. In this report, we determined the role of Rab1 GTPase, which specifically controls protein transport from the endoplasmic reticulum (ER) to the Golgi, and receptor dimerization in the biosynthesis of AT2R. Cell surface expression of AT2R was augmented by transient expression of Rab1 and attenuated by dominant-negative Rab1 mutants and small interfering RNA-mediated knockdown of Rab1. Consistently, AT2R inhibition of epidermal growth factor-activated extracellular signal-regulated kinase 1/2 was significantly reduced by the Rab1 mutants, indicating that endogenous Rab1 modulates the cell surface targeting and signaling of AT2R. It is of interest to note that Rab1 augmented the overall expression of AT2R and its mRNA, whereas the Rab1 mutants attenuated the total AT2R expression and enhanced ubiquitin-dependent AT2R degradation. Furthermore, our previously characterized ER export-deficient AT1R mutant in which the F(x)(6)LL motif was mutated formed both homodimers and heterodimers with AT2R. Dimerization of the AT1R mutant with AT2R blocked AT2R trafficking to the cell surface, suggesting constitutive dimerization of both receptors in the ER and an important role of dimerization in ER export of the receptors. These data demonstrate for the first time that Rab1 GTPase and dimerization modulate export traffic from the ER to the cell surface of newly synthesized AT2R.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Membrana/fisiologia , Multimerização Proteica/fisiologia , Receptor Tipo 2 de Angiotensina/biossíntese , Proteínas rab1 de Ligação ao GTP/fisiologia , Linhagem Celular , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Multimerização Proteica/genética , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/fisiologia , Proteínas rab1 de Ligação ao GTP/biossíntese , Proteínas rab1 de Ligação ao GTP/genéticaRESUMO
Poor homing of cells after transplantation is an unresolved common issue in cardiac cell therapies. To enhance stem cell homing, the ligand CXC motif chemokine 12 (CXCL12) and its specific receptor CXC receptor type 4 (CXCR4) have been employed as a system in this study to show that induced expression of the endogenous CXCR4 gene in mouse-induced pluripotent stem cells (iPSCs) improved the cell migration. Loci-specific epigenome editing in the form of CpG demethylation at CXCR4 promoter region of the mouse iPSCs was accomplished with CXCR4b-TAL-Tet1c, chimeric fusion proteins of the catalytic domain of ten-eleven translocation 1 (TET1) to the C-terminal end of the DNA binding domains of predesigned synthetic transcription activator-like effectors (TALEs) that recognize specific DNA sequences within the mouse CXCR4 promoter region. Infection of the mouse iPSCs with the engineered CXCR4b-TAL-Tet1c in the form of lentiviral particles induced the loci-specific CpG demethylation and subsequent activation of CXCR4 expression in mouse iPSCs. As expected, the CXCR4-overexpressing iPSCs exhibited 3.9-fold greater migration than the control iPSCs did without alteration of the stemness and activated phosphorylation of AKT significantly. These results set a sound foundation for subsequent in vivo iPSCs transplantation studies in rodent models of acute myocardial infarction and heart failure. We show that TALEs can enhance the expression of CXCR4 by CpG methylation, and may retain the stemness. Migration of iPSCs activated by CXCL12 is associated with significant phosphorylation of AKT, not ERK1/2.
Assuntos
Movimento Celular , Quimiocina CXCL12/metabolismo , Desmetilação do DNA , Células-Tronco Pluripotentes Induzidas/citologia , Receptores CXCR4/metabolismo , Animais , Camundongos , Fosforilação , Regiões Promotoras Genéticas , Engenharia de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR4/genéticaRESUMO
Overexposure to glucocorticoid during fetal development can result in intrauterine growth retardation (IUGR) as well as other diseases after birth. The purpose of this study is to investigate the possibility of glucocorticoid disturbance-mediated nicotine-induced IUGR after chronic prenatal exposure. Nicotine at 1.0mg/kg twice a day was administered subcutaneously to pregnant rats from gestational day (GD) 8 to GD 15 (mid-gestation) or GD 21 (late-gestation). Placental weights and fetal developmental parameters were recorded. Corticosterone levels were determined by radioimmunoassay. The mRNA expressions of adrenal steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage (P450scc) and placental 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD-2) were determined using real-time quantitative RT-PCR. The results showed that prenatal chronic nicotine exposure causes IUGR in rats (P<0.01); in response to nicotine exposure, maternal serum corticosterone levels were elevated at mid- and late-gestations (P<0.05); mRNA expressions of StAR and P450scc increased in maternal adrenals (P<0.05 or 0.01) but decreased in fetal adrenals (P=0.16 or 0.11). Furthermore, the mRNA levels of placental 11 beta-HSD-2 were reduced at mid- and late-gestations (P<0.05). These results suggest that nicotine-induced IUGR is associated with the disturbances of glucocorticoid homeostasis in maternal and fetal rats. A possible underlying mechanism is that long term nicotine administration leads to fetal overexposure to maternal glucocorticoid by the combined effect of increased maternal glucocorticoid level and impaired placental barrier to it, all of which eventually leads to the fetal adrenocortical dysfunction and IUGR.
Assuntos
Corticosterona/sangue , Retardo do Crescimento Fetal/induzido quimicamente , Exposição Materna , Troca Materno-Fetal/efeitos dos fármacos , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/embriologia , Glândulas Suprarrenais/metabolismo , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Modelos Animais de Doenças , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Retardo do Crescimento Fetal/patologia , Peso Fetal/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Injeções Subcutâneas , Troca Materno-Fetal/fisiologia , Tamanho do Órgão/efeitos dos fármacos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Placenta/efeitos dos fármacos , Placenta/enzimologia , Placenta/patologia , Gravidez , Ratos , Ratos Wistar , Organismos Livres de Patógenos EspecíficosRESUMO
OBJECTIVE: To determine expression of the P2X(7) receptor in normal and in cancer uterine tissues. The rationale was that the receptor P2X(7) regulates constitutive apoptosis in uterine epithelial cells, and previous studies showed diminished P2X(7)-mediated apoptosis in cancer uterine cells compared with normal cells. METHODS: A clinical, experimental feasibility study. Normal (n = 42) and cancer uterine tissues (n = 47) were obtained from a total of 72 women ages 25 to 75. End points for P2X(7) mRNA were quantitative PCR and in situ hybridization, and end points for P2X(7) protein were Western blots and immunostaining using anti-P2X(7) antibody. RESULTS: (a) In normal uteri, P2X(7) mRNA and protein were expressed predominantly in the epithelial (endometrial, endocervical, and ectocervical) cells. (b) Expression of the P2X(7) mRNA and protein was absent from endometrial and endocervical adenocarcinoma tissues and from cervical squamous cell carcinoma tissues. (c) In cervical dysplasia, P2X(7) protein was absent in the dysplastic lesions. (d) Semiquantitative analysis using P2X(7) mRNA (normalized in each tissue to the constitutive glyceraldehyde-3-phosphate dehydrogenase) and P2X(7) protein levels (normalized in each tissue to the constitutive tubulin) revealed that P2X(7) mRNA and/or protein levels can distinguish uterine normal from cancer tissues at high degrees of sensitivity (92%, 100%) and specificity (100%, 90%). SUMMARY AND CONCLUSIONS: (a) Levels of the P2X(7) are lower in uterine epithelial cancer tissues than in the corresponding normal tissues. (b) The data suggest that tissue P2X(7) mRNA and protein levels could be used as a novel biomarker to differentiate normal and cancer uterine epithelial tissues.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Epiteliais e Glandulares/química , Receptores Purinérgicos P2/análise , Neoplasias Uterinas/química , Neoplasias Uterinas/metabolismo , Adenocarcinoma/química , Adenoma/química , Adulto , Idoso , Western Blotting , Carcinoma de Células Escamosas/química , Estudos de Casos e Controles , Estudos de Viabilidade , Feminino , Humanos , Hibridização In Situ , Pessoa de Meia-Idade , Tumor Mulleriano Misto/química , Neoplasias Epiteliais e Glandulares/patologia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Receptores Purinérgicos P2X7 , Sensibilidade e Especificidade , Displasia do Colo do Útero/química , Neoplasias do Colo do Útero/química , Neoplasias Uterinas/patologiaRESUMO
Epidermal growth factor (EGF), epinephrine, and the P2X7 receptor system regulate growth of human uterine cervical epithelial cells, but little is known about how these systems intercommunicate in exerting their actions. The objective of this study was to understand the mechanisms of EGF and epinephrine regulation of growth of cervical cells. Treatment of cultured CaSki cells with 0.2 nM EGF increased cell number via a PD98059-sensitive pathway. Treatment with 2 nM epinephrine increased cell number, and the effect was facilitated by cotreatment with EGF. Whereas the effect of EGF alone involved up-regulation of [3H]-thymidine incorporation and an increase in cell proliferation, the effect of epinephrine was mediated by inhibition of apoptosis. Epinephrine inhibited apoptosis induced by the P2X7 receptor ligand 2',3'-0-(4-benzoylbenzoyl)-ATP, by attenuation of P2X7 receptor plasma membrane pore formation. Cotreatment with EGF facilitated epinephrine effect via a phosphoinositide 3-kinase-dependent mechanism. CaSki cells express the beta2-adrenoceptor, and the epinephrine antiapoptotic effect could be mimicked by beta2-adrenoceptor agonists and by activators of adenylyl cyclase. Likewise, the effect could be blocked by beta2-adrenoceptor blockers and by the inhibitor of protein kinase-A H-89. Western immunoblot analysis revealed that epinephrine decreased the levels of the glycosylated 85-kDa form of the P2X7 receptor and increased receptor degradation, and that EGF potentiated these effects of epinephrine. EGF did not affect cellular levels of the beta2-adrenoceptor. In contrast, EGF, acting via the EGF receptor, augmented beta2-adrenoceptor recycling, and it inhibited beta2-adrenoceptor internalization via a phosphoinositide 3-kinase-dependent mechanism. We conclude that, in cervical epithelial cells, EGF has a dual role: as mitogen, acting via the MAPK/MAPK kinase pathway, and as an antiapoptotic factor by facilitating epinephrine effect and resulting in greater expression of beta2-adrenoceptors in the plasma membrane. These findings underscore a novel signaling network of communication between the receptor tyrosine kinases, the G protein-coupled receptors, and the purinergic P2X7 receptor.
Assuntos
Apoptose/fisiologia , Membrana Celular/fisiologia , Colo do Útero/citologia , Fator de Crescimento Epidérmico/fisiologia , Epinefrina/fisiologia , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/fisiologia , Transdução de Sinais/fisiologia , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Colo do Útero/fisiologia , Sinergismo Farmacológico , Fator de Crescimento Epidérmico/farmacologia , Epinefrina/farmacologia , Feminino , Humanos , Mitocôndrias/fisiologia , Receptores Adrenérgicos beta 2/fisiologia , Receptores Purinérgicos P2X7RESUMO
Prostate cancer is the most commonly diagnosed non-cutaneous cancer and one of the leading causes of cancer death for North American men. Whereas localized prostate cancer can be cured, there is currently no cure for metastatic prostate cancer. Here we report a novel approach that utilizes designed chimeric transcription activator-like effectors (dTALEs) to control prostate cancer metastasis. Transfection of dTALEs of DNA methyltransferase or demethylase induced artificial, yet active locus-specific CpG and subsequent histone modifications. These manipulations markedly altered expression of endogenous CRMP4, a metastasis suppressor gene. Remarkably, locus-specific CpG demethylation of the CRMP4 promoter in metastatic PC3 cells abolished metastasis, whereas locus-specific CpG methylation of the promoter in non-metastatic 22Rv1 cells induced metastasis. CRMP4-mediated metastasis suppression was found to require activation of Akt/Rac1 signaling and down-regulation of MMP-9 expression. This proof-of-concept study with dTALEs for locus-specific epigenomic manipulation validates the selected CpG methylation of CRMP4 gene as an independent biomarker for diagnosis and prognosis of prostate cancer metastasis and opens up a novel avenue for mechanistic research on cancer biology.
Assuntos
Metilases de Modificação do DNA/genética , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Metilação de DNA , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Prognóstico , Regiões Promotoras Genéticas , Neoplasias de Próstata Resistentes à Castração/patologia , TransfecçãoRESUMO
The present study investigated the antiapoptotic effects of estrogen in normal and cancer human cervical cells and the mechanisms involved. Baseline apoptosis in human cervical epithelial cells is mediated predominantly by P2X7-receptor-induced, Ca(2+)-dependent activation of the mitochondrial (caspase-9) pathway. Treatment with 10 nM 17beta-estradiol blocked apoptosis induced by the P2X7-receptor ligands ATP and 2',3'-0-(4-benzoylbenzoyl)-ATP in normal human cervical epithelial cells (hECEs) and attenuated the effect in hECEs immortalized with human papillomavirus-16 (ECE16-1) and the cancer cervical cells HT3 and CaSki. Diethylstilbestrol and to a lesser degree estrone could mimic the effects of 17beta-estradiol, whereas actinomycin-D and cycloheximide attenuated the response. The antiapoptotic effect of estrogen did not depend on cell cycle phase, and in both normal and cancer cervical cells, it involved attenuation of activation of caspase-9 and the terminal caspase-3. However, involvement of cascades upstream to the caspase-9 differed in normal vs. cancer cervical cells. In the normal hECEs estrogen blocked P2X7-receptor-induced calcium influx. In contrast, in the cancer CaSki cells, estrogen up-regulated expression of Bcl-2 and attenuated Ca(2+)-induced mitochondrial swelling (i.e. formation of mitochondrial permeability transition pores). Estrogen had no effect on P2X7-receptor-induced apoptosis in the anaplastic SiHa and Hela cells. These results point to a novel antiapoptotic effect of estrogen in the cervix that is independent of its mitogenic function. The results also suggest that cancer cervical cells evolved antiapoptotic mechanisms that enable the cells to evade apoptosis and could therefore promote tumor progression.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Apoptose/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Estrogênios/farmacologia , Neoplasias do Colo do Útero/patologia , Trifosfato de Adenosina/farmacologia , Adulto , Marcadores de Afinidade/farmacologia , Cálcio/metabolismo , Caspase 3 , Caspase 9 , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Feminino , Humanos , Pessoa de Meia-Idade , Dilatação Mitocondrial/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7 , Células Tumorais CultivadasRESUMO
Caffeine is the most widely consumed psychoactive substance in the world. It can elevate the level of glucocorticoid which is involved in metabolism regulation, stress response, and immune function. However, the specific mechanism has yet to be elucidated. Glucocorticoid is steroid hormone synthesized in adrenal cortex and the key rate-limiting step in its biosynthesis is mediated by steroidogenic acute regulatory protein (StAR). This study was designed to investigate the direct effects and inheritable epigenetic mechanisms of caffeine on cortisol production and StAR expression in human adrenocortical cells. The human adrenocortical cell line NCI-H295A was cultured with 0.4-40µM caffeine. There was a significant increase of the cortisol production in cells. In both acutely and chronically caffeine-treated cell groups, mRNA and protein expressions of StAR were stimulated in a dose-dependent manner. DNA methylation detection via bisulfite-sequencing PCR (BSP) uncovered a single site CpG demethylation at nt -682 within the StAR promoter region. Then we investigated how long the increased StAR expression and the single CpG demethylation could last. The caffeine was withdrawn after 48h of treatment and then the cells were continually subcultured for up to 5 and 10 passages, respectively. The results showed that the StAR expression at post-caffeine passage 10 still increased, as compared with that in the control. The caffeine-induced demethylation at nt -682 in StAR promoter underwent a similar time course as StAR expression does. The present study reveals the direct effect and possible inheritable epigenetic mechanism of caffeine on steroidogenesis in human adrenocortical cells and has implications for our understanding of the consumption of caffeine.
Assuntos
Cafeína/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hidrocortisona/metabolismo , Fosfoproteínas/metabolismo , Córtex Suprarrenal/efeitos dos fármacos , Linhagem Celular , Metilação de DNA/efeitos dos fármacos , Epigênese Genética , Humanos , Fosfoproteínas/genética , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismoRESUMO
A truncated naturally occurring variant of the human receptor P2X7 was identified in cancer cervical cells. The novel protein (P2X7-j), a polypeptide of 258 amino acids, lacks the entire intracellular carboxyl terminus, the second transmembrane domain, and the distal third of the extracellular loop of the full-length P2X7 receptor. The P2X7-j was expressed in the plasma membrane; it showed diminished ligand-binding and channel function capacities and failed to form pores and mediate apoptosis in response to treatment with the P2X7 receptor agonist benzoyl-ATP. The P2X7-j interacted with the full-length P2X7 in a manner suggesting heterooligomerization and blocked the P2X7-mediated actions. Interestingly, P2X7-j immunoreactivity and mRNA expression were similar in lysates of human cancer and normal cervical tissues, but full-length P2X7 immunoreactivity and mRNA expression were higher in normal than in cancer tissues, and cancer tissues lacked 205-kDa P2X7 immunoreactivity suggesting lack of P2X7 homo(tri)-oligomerization. These results identify a novel P2X7 variant with apoptosis-inhibitory actions, and demonstrate a distinct regulatory property for a truncated variant to antagonize its full-length counterpart through hetero-oligomerization. This may represent a general paradigm for regulation of a protein function by its variant.
Assuntos
Receptores Purinérgicos P2/biossíntese , Neoplasias do Colo do Útero/metabolismo , Processamento Alternativo , Animais , Apoptose , Sequência de Bases , Cães , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Receptores Purinérgicos P2X7 , Homologia de Sequência do Ácido NucleicoRESUMO
AIM: To investigate the alterations of placental P-glycoprotein (P-gp) and cytochrome P450 1A1 (CYP1A1) at different gestational days (GD), and to explore the possible significance of placental P-gp and CYP1A1 in tobacco smoke-induced intrauterine growth retardation (IUGR) in rats. METHODS: An IUGR model was produced by passive tobacco smoking from GD7 to parturition (GD21) and predicted using fetal development parameters. Placental structure and function were monitored by observing pathological alteration and antioxidative function, including the content of malondialdehyde and the activities of superoxide dismutase and catalase (CAT). The expressions of CYP1A1 and P-gp (mdr 1a and mdr 1b) were detected using a reverse transcription polymerase chain reaction and immunohistochemistry. RESULTS: Placental pathological changes occurred and the malondialdehyde content increased, whereas the activities of superoxide dismutase and CAT lowered, when compared to their controls. In the rat placenta of the tobacco group, the level of CYP1A1 mRNA increased significantly; the level of mdr1a mRNA increased significantly at GD21 but not at GD14, whereas the level of mdr1b mRNA in different term remained stable; the expression of P-gp increased significantly only in full-term placenta. CONCLUSION: The expression of placental CYP1A1 and P-gp increased in tobacco-induced IUGR. Overexpression of placental CYP1A1 can attribute to the metabolism of tobacco and the generation of reactive metabolites, which can trigger IUGR. As a compulsory mechanism, upregulation of P-gp might decrease tobacco exposure to a developing fetus with IUGR.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Citocromo P-450 CYP1A1/biossíntese , Retardo do Crescimento Fetal/metabolismo , Placenta/patologia , Poluição por Fumaça de Tabaco/efeitos adversos , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Citocromo P-450 CYP1A1/genética , Feminino , Retardo do Crescimento Fetal/etiologia , Retardo do Crescimento Fetal/patologia , Placenta/metabolismo , Gravidez , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
The most striking feature of a G protein-coupled receptor (GPCR) is its highly exclusive agonist specificity. This feature guarantees that a GPCR recognizes only its specific native agonist(s). In this study, we showed that two point mutations of N295S and L305Q enabled the AT(1) receptors to recognize multiple Ang II fragments. Similar to the well established constitutively active AT(1) mutant receptor N111G, the mutations of N295S and L305Q induced an increased production of basal inositol 1,4,5-phosphates in the absence of exogenous Ang II when expressed in HEK293 cells. Distinct from the N111G, however, is the fact that the increased basal activity disappeared in COS-7 cells because of the lack of endogenous Ang II fragments produced by the cells-a pseudo-constitutive activity. It is surprising that the Ang II analog [Sar(1),Ile(4),Ile(8)]Ang II and the native angiotensin II fragments Ang 1-7, Ang IV, and Ang 5-8, which are inactive in activating the wild-type receptor, activated N295S and L305Q. Results generated by lowering the Na(+) concentration suggest that the mutant N295S and L305Q may be trapped in neutral conformational states (R(N)). These data allow us to identify for the first time a novel pattern of GPCR mutations with a broad spectrum of agonist specificity, suggesting possible existence of functional GPCRs in nature that are activated through conformational "selection" rather than "induction" mechanisms.
Assuntos
Angiotensina II/genética , Asparagina/genética , Leucina/genética , Proteínas de Membrana/genética , Mutação , Fragmentos de Peptídeos/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Angiotensina II/metabolismo , Animais , Asparagina/metabolismo , Células COS , Chlorocebus aethiops , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Leucina/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/genética , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Ratos , Receptor Tipo 1 de Angiotensina/metabolismoRESUMO
The objective of this study was to understand the mechanisms involved in P2X(7) receptor activation. Treatments with ATP or with the P2X(7) receptor-specific ligand 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) induced pore formation, but the effect was slower in CaSki cells expressing endogenous P2X(7) receptor than in human embryonic kidney (HEK)-293 cells expressing exogenous P2X(7) receptor (HEK-293-hP2X(7)-R). In both types of cells Western blots revealed expression of three forms of the receptor: the functional 85-kDa form present mainly in the membrane and 65- and 18-kDa forms expressed in both the plasma membrane and the cytosol. Treatments with ATP transiently decreased the 85-kDa form and increased the 18-kDa form in the membrane, suggesting internalization, degradation, and recycling of the receptor. In CaSki cells ATP stimulated phosphorylation of the 85-kDa form on tyrosine and serine residues. Phosphorylation on threonine residues increased with added ATP, and it increased ATP requirements for phosphorylation on tyrosine and serine residues, suggesting a dominant-negative effect. In both CaSki and in HEK-293-hP2X(7)-R cells ATP also increased binding of the 85-kDa form to G protein-coupled receptor kinase (GRK)-3, beta-arrestin-2, and dynamin, and it stimulated beta-arrestin-2 redistribution into submembranous regions of the cell. These results suggest a novel mechanism for P2X(7) receptor action, whereby activation involves a GRK-3-, beta-arrestin-2-, and dynamin-dependent internalization of the receptor into clathrin domains, followed in part by receptor degradation as well as receptor recycling into the plasma membrane.
Assuntos
Trifosfato de Adenosina/fisiologia , Arrestinas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Purinérgicos P2/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Clatrina/fisiologia , Citosol/fisiologia , Dinaminas/fisiologia , Quinase 3 de Receptor Acoplado a Proteína G , Expressão Gênica , Humanos , Fosforilação , Receptores Purinérgicos P2X7 , beta-Arrestina 2 , beta-ArrestinasRESUMO
BACKGROUND: The most striking feature of life is biodiversity. However, mechanisms of biodiversity remain poorly understood, as most protein orthologues of different species are highly homologous in sequence and identical in function. Interestingly, recent evidence has demonstrated heterogeneity for a G protein-coupled angiotensin II (Ang II) type 2 (AT(2)) receptor in both ligand binding and induction of arachidonic acid (AA) release. The present study investigated the properties of AT(2) receptors in closely related species. METHODS: AT(2) receptors cloned from human, rabbit, rat, and mouse were expressed in Chinese hamster ovary cells (CHO-K1), African green monkey kidney cells (COS-1), and human embryonic kidney (HEK)-293 cells and characterized in ligand binding and signal transductions. Critical residues in rabbit AT(2) receptor attributable to heterogeneity were examined using both gain-of-function and loss-of-function approaches with mutagenesis. RESULTS: The newly cloned rabbit AT(2) receptor exhibits distinct biochemical and biologic properties compared to its highly homologous orthologues (91% in overall amino acid sequence) of rat, mouse, and human. All these orthologues activate SH2 domain-containing phosphatase-1 (SHP-1) and show similar binding affinities for Ang II and AT(2)-specific ligands CGP42112A and PD123319. However, reducing agent dithiothreitol (DTT) inactivates the rabbit orthologue but potentiates the others in ligand binding, a hallmark of AT(2) versus AT(1) receptor subtypes. Most interestingly, rabbit AT(2) receptor, but not the other orthologues, induces AA release in various cell systems when stimulated by both Ang II and CGP42112A, the peptide antagonist. Mutagenesis studies and sequence analyses further indicate that residues His(106), Asp(188), and Thr(293) are responsible for the DTT inactivation and residues Val(209) and Val(249) are partially responsible for AA release. CONCLUSION: These results deny the coexistence of an additional AT(2) subtype in rabbit proximal tubule cells and demonstrate for the first time the presence of functional diversity for closely related Eutherian orthologues of a G protein-coupled receptor (GPCR) that are more than 90% homologous in the amino acid sequence.
Assuntos
Receptor Tipo 2 de Angiotensina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Ácido Araquidônico/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Cricetinae , DNA Complementar/genética , Ditiotreitol/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Ligantes , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Coelhos , Ratos , Receptor Tipo 2 de Angiotensina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Internalization of a G-protein-coupled receptor (GPCR) is essential to the desensitization, endocytosis, and signal transduction of the receptor. It has been the general view that conventional homologous internalization of a GPCR requires activation of the G-protein(s) coupled to the receptor. However, whether and how GPCR-mediated G-protein-independent signals trigger receptor internalization remains unknown, although G-protein-independent internalization has been reported. Here we show that an angiotensin II (Ang II) type-1 (AT1) receptor mutant incapable of activating any G-protein still undergoes normal internalization. Substitution of Asp125 with Ala and Arg126 with Leu at the highly conserved DRY motif of the AT1 receptor disabled the ability of the receptor to activate G-proteins, as shown by various Ang II binding studies, GDP-GTP exchange, and inositol phosphate production assays. Surprisingly, the mutant internalized normally in the presence of Ang II and transactivated the epidermal growth factor receptor (EGFR). Similar to the wild-type receptor, overexpression of a dominant-negative K220R mutant GRK2 diminished the internalization of D125A-R126L but not the transactivation of EGFR. These data indicate that G-protein-independent specific signals may also trigger homologous internalizations of the AT1 receptor through beta-arrestin-dependent and -independent pathways, suggesting a possible mechanism for G-protein-independent activation of G-protein-coupled receptor kinases (GRKs). This may represent a general mechanism for triggering GPCR internalization.
Assuntos
Endocitose/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de Sinais/fisiologia , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Receptores ErbB/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ligantes , Mutação , Receptor Tipo 1 de Angiotensina/genética , Receptores Acoplados a Proteínas G/metabolismo , Ativação TranscricionalRESUMO
Conventional mode of activation of SH2 domain-containing phosphatase 1 (SHP-1) by a single transmembrane (TM) inhibitory receptor such as killer cell inhibitory receptor, Fcgamma receptor type IIb1, and paired Ig-like receptors of inhibitory types requires tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory (ITIM) motifs in the cytoplasmic domains of the inhibitory receptors. Contrary to this paradigm, AT(2), a G protein-coupled 7TM receptor that does not undergo tyrosine phosphorylation in response to angiotensin II (Ang II) stimulation, also activates SHP-1. Here we show that SHP-1 constitutively and physically associates with AT(2) receptor in transfected COS-7 cells. On stimulation by Ang II, SHP-1 becomes activated and dissociated from AT(2) receptor, independent of pertussis toxin. Cotransfection of transducin G(betagamma) inhibits SHP-1/AT(2) association and the SHP-1 activation, whereas cotransfection of C-terminal of beta-adrenergic receptor kinase, which abrogates G(betagamma) signaling, facilitates SHP-1 activation. Surprisingly, SHP-1/AT(2) association and the SHP-1 activation requires the presence of G(alphas) as shown by differential coimmunoprecipitation, dominant negative G(alphas), constitutively active G(alphas), and G(alpha) peptides. A mutant AT(2) receptor D141A-R142L that is inactive in G(alpha) protein activation constitutively associates with SHP-1 and activates it. Together, these results indicate that G(alphas) alone, rather than exclusively in the form of G(alphabetagamma) heterotrimer may facilitate signal transduction for G protein-coupled receptors, suggesting a novel mechanism distinct from the classic paradigm of heterotrimeric G proteins. The AT(2)-mediated ITIM-independent activation of SHP-1 that is distinct from the conventional mode of activation, may represent a general paradigm for activation of SHP-1/2-class tyrosine phosphatases by G protein-coupled receptors.