RESUMO
The cAMP responsive element-binding protein (CREB) functions in a broad array of biological and pathophysiological processes. We found that salt-inducible kinase 2 (SIK2) was abundantly expressed in neurons and suppressed CREB-mediated gene expression after oxygen-glucose deprivation (OGD). OGD induced the degradation of SIK2 protein concomitantly with the dephosphorylation of the CREB-specific coactivator transducer of regulated CREB activity 1 (TORC1), resulting in the activation of CREB and its downstream gene targets. Ca(2+)/calmodulin-dependent protein kinase I/IV are capable of phosphorylating SIK2 at Thr484, resulting in SIK2 degradation in cortical neurons. Neuronal survival after OGD was significantly increased in neurons isolated from sik2(-/-) mice, and ischemic neuronal injury was significantly reduced in the brains of sik2(-)(/-) mice subjected to transient focal ischemia. These findings suggest that SIK2 plays critical roles in neuronal survival, is modulated by CaMK I/IV, and regulates CREB via TORC1.
Assuntos
Proteína de Ligação a CREB/metabolismo , Hipóxia Celular , Sobrevivência Celular , Neurônios/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Contagem de Células , Células Cultivadas , Córtex Cerebral/citologia , DNA Recombinante , Expressão Gênica , Glucose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs , Oxigênio/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , RatosRESUMO
The (pro)renin receptor ((P)RR), which is a recently discovered molecule of the renin-angiotensin system, plays an important role in the development of cardiovascular diseases. However, the molecular properties and the subcellular distribution of (P)RR remain controversial. In this study, (P)RR-Venus in Chinese hamster ovary (CHO) cells ((P)RR-Venus-CHO) or endogenous (P)RR in human vascular smooth muscle cells (VSMC) were constitutively cleaved without any stimulation, and secretion of the amino-terminal fragment (NTF-(P)RR) into the media was determined using western blot analysis. Immunofluorescent analysis showed robust expression of (P)RR in the endoplasmic reticulum (ER) or the Golgi but not in the plasma membrane. Moreover, we identified ADAM19, which is expressed in the Golgi, as one of cleaving proteases of (P)RR. Transfected ADAM19 evoked the shedding of (P)RR, whereas transfected dominant negative ADAM19 suppressed it. Although (P)RR contains a furin cleavage site, neither the furin-deficient LoVo cells nor furin inhibitor-treated VSMC lost NTF-(P)RR in the media. The secreted NTF-(P)RR induced the renin activity of prorenin in the extracellular space. We describe that (P)RR is mainly localized in the subcellular organelles, such as the ER and Golgi, and (P)RR is cleaved by ADAM19 in the Golgi resulting in two fragments, NTF-(P)RR and CTF-(P)RR. These results may suggest that (P)RR is predominantly secreted into the extracellular space.
Assuntos
Proteínas ADAM/metabolismo , Espaço Extracelular/metabolismo , Complexo de Golgi/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Humanos , Músculo Liso Vascular/metabolismo , Transporte Proteico , Receptor de Pró-ReninaRESUMO
cAMP response element-binding protein (CREB) promotes melanogenesis by inducing microphthalmia-associated transcription factor (Mitfâ) gene expression. We report here that the CREB-specific coactivator TORC and its repressor, salt-inducible kinase 2 (SIK2), are fundamental determinants of the melanogenic program in mice. Exposure of B16 melanoma cells to ultraviolet (UV) light results in the immediate nuclear translocation of TORC1, which is inhibited by SIK2. Overexpression of dominant-negative TORC1 also inhibits UV-induced Mitf gene expression and melanogenesis. α-MSH signaling regulates hair pigmentation, and the decrease in α-MSH activity in hair follicle melanocytes switches the melanin synthesis from eumelanin (black) to pheomelanin (yellow). Mice with the lethal yellow allele of agouti (A(y)) have yellow hair because of impaired activation of the α-MSH receptor. To examine the involvement of SIK2 in the regulation of the melanogenesis switch in vivo, we prepared SIK2-knockout mice, and the Sik2(-/-) genotype was introduced into A(y)/a mice. The resultant Sik2(-/-); A(y)/a mice had brown hair, indicating that SIK2 represses eumelanogenesis in mice.
Assuntos
Regulação para Baixo/genética , Melaninas/biossíntese , Proteínas Serina-Treonina Quinases/genética , Animais , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Regulação para Baixo/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Cabelo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Melanoma/enzimologia , Melanoma/genética , Melanoma/patologia , Camundongos , Pigmentação/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Raios Ultravioleta , alfa-MSH/metabolismoRESUMO
Salusin-alpha and salusin-beta are multifunctional bioactive peptides originally identified using bioinformatics analyses. Salusin-beta has been shown to exert potent hypotensive, bradycardic, and pro-atherosclerotic effects. The form in which it exists in biological fluids remains undetermined due to technical difficulties originating from its unexpected physicochemical properties. Here we show that salusin-beta peptide adheres to polypropylene and polystyrene, so that the aliquoted peptide dissolved in distilled water may rapidly disappear from the solution. By circumventing these features and using an antibody against C-terminal portion of salusin-beta, we have successfully established a specific radioimmunoassay suitable for detection of immunoreactive human salusin-beta. We have characterized the molecular form of salusin-beta in human plasma and urine. The assay detected immunoreactive salusin-beta concentrations as low as 5 fmol/tube and the concentration required for 50% inhibition of binding was 122 fmol/tube. Cross-reactivities with salusin-alpha and other bioactive peptides were negligible. Reverse-phase high performance liquid chromatography coupled with the radioimmunoassay detection after extraction from plasma and urine and using an octyl-silica column, revealed a major immunoreactive component that co-eluted with authentic salusin-beta. Salusin-beta-like immunoreactivity in normal human urine ranged from 0.23 to 2.22 nmol/l (mean+/-SD, 1.16+/-0.84 nmol/l, n=10). These data present the first evidence that salusin-beta circulates and is excreted in its authentic form, thereby verifying the initially predicted processing sites for salusin-beta in humans.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/análise , Adulto , Sequência de Aminoácidos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/urina , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , RadioimunoensaioRESUMO
Ginsenoside Rb1 (GRb1), a major component of the traditional herb ginseng, has been reported to show a neuroprotective effect in a rodent ischemic model. The purpose of this study was to investigate effects of GRb1 on early and delayed brain injuries in a non-human primate thromboembolic stroke model. Thromboembolic stroke was induced by occlusion of the middle cerebral artery by injection of an autologous blood clot into the left internal carotid artery. GRb1 (300 microg/kg per day, i.v.) and vehicle were administered from 7 days before embolization to the day following embolization (total: 8 times). Neurological deficits were observed at 1, 6, and 24 h and at 2, 4, and 7 days after embolization. At 7 days after embolization, neuron damage in the peri-infarct area and core region were assessed by NeuN, TUNEL, and GFAP staining. GRb1 improved the skeletal muscle coordination score of the neurologic deficits (median: GRb1 vs vehicle = 10 vs 12, P<0.05). In the GRb1 group, positive neurons expressed by NeuN staining were noted in the ischemic peri-infarct area, and TUNEL- and GFAP-positive cells significantly decreased, when compared with vehicle. These results demonstrated that GRb1 ameliorated both early and delayed injuries in the thromboembolic stroke model in non-human primates.
Assuntos
Astrócitos/efeitos dos fármacos , Ginsenosídeos/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Degeneração Neural/prevenção & controle , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Tromboembolia/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Astrócitos/patologia , Ataxia/etiologia , Ataxia/prevenção & controle , Edema Encefálico/etiologia , Edema Encefálico/prevenção & controle , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/etiologia , Infarto da Artéria Cerebral Média/patologia , Macaca fascicularis , Masculino , Degeneração Neural/etiologia , Degeneração Neural/patologia , Neurônios/patologia , Tromboembolia/complicações , Tromboembolia/patologia , Fatores de TempoRESUMO
BACKGROUND AND PURPOSE: Arachidonic acid that is released following cerebral ischemia can be metabolized to 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a potent vasoconstrictor that may contribute to ischemic injury. This study examined the effects of blockading the synthesis of 20-HETE with TS-011 on infarct size after transient occlusion of the middle cerebral artery (MCAO) of rats and after thromboembolic stroke in monkeys. METHODS: Rats were treated with TS-011 or vehicle at various times after MCAO. Infarct size was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining and plasma levels of 20-HETE were determined by liquid chromatography mass spectrometry (LC/MS). The effect of TS-011 on infarct size was also studied in monkeys after introduction of a clot into the internal carotid artery. RESULTS: Plasma levels of 20-HETE increased after MCAO in rats. TS-011 (0.01 to 1.0 mg/kg per hour) reduced infarct volume by 40%. Chronic administration of TS-011 for 7 days reduced neurological deficits after MCAO in rats. TS-011 given in combination with tissue plasminogen activator also improved neurological outcome in the stroke model in monkeys. CONCLUSIONS: These results suggest that blockade of the formation of 20-HETE with TS-011 may be useful for the treatment of ischemic stroke.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Formamidas/farmacologia , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Morfolinas/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Isquemia Encefálica/sangue , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Relação Dose-Resposta a Droga , Cromatografia Gasosa-Espectrometria de Massas , Haplorrinos , Ácidos Hidroxieicosatetraenoicos/sangue , Masculino , Ratos , Ratos Wistar , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologiaRESUMO
One of the most important prognostic factors in the thrombolytic treatment of acute ischemic stroke is to re-canalize. The purpose of this study was to evaluate the effectiveness and safety of urokinase in a primate thromboembolic stroke model. Thromboembolic stroke was accomplished via occlusion of the middle cerebral artery (MCA) obtained by injecting an autologous blood clot into the left internal carotid artery in 21 male cynomolgus monkeys. Animals were randomly assigned to the following treatment groups: Group 1: vehicle (saline), Group 2: urokinase (40,000 IU), Group 3: urokinase (120,000 IU,) over 2 or 6 h via intra-internal carotid catheter starting 1 h after embolization, respectively. In the urokinase-treated groups, neurologic deficits were improved in consciousness and skeletal muscle coordination, but not sensory and motor systems. The infarction size in Group 2 (11.9 +/- 3.9% of the hemisphere) and 3 (7.6 +/- 2.5%) were significantly smaller than that (24.7 +/- 3.5%) in Group 1. However, 2 of 5 animals in Group 3 died. In conclusion, urokinase improved neurologic deficits and reduced cerebral infarction on thromboembolic stroke in the cynomolgus monkey.
Assuntos
Fibrinolíticos/uso terapêutico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Doenças do Sistema Nervoso/prevenção & controle , Tromboembolia/tratamento farmacológico , Terapia Trombolítica , Ativador de Plasminogênio Tipo Uroquinase/uso terapêutico , Animais , Modelos Animais de Doenças , Esquema de Medicação , Fibrinolíticos/administração & dosagem , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/patologia , Infusões Intra-Arteriais , Macaca fascicularis , Masculino , Doenças do Sistema Nervoso/etiologia , Tromboembolia/complicações , Tromboembolia/patologia , Ativador de Plasminogênio Tipo Uroquinase/administração & dosagemRESUMO
We determined whether up to 24 h of ischemia could induce the expression of cyclooxygenase-2 (COX-2) in the brain of nonhuman primates. Randomized animals were subjected to either a 2 h ischemia (group II; n=3) or a 24 h ischemia (group III; n=3). Three animals in group I served as controls. In group III, regional cerebral blood flow (CBF) and the cerebral glucose metabolic rate (CMRglc) were evaluated using positron emission tomography. Upregulation of COX-2 mRNA expression was observed after 2 h of ischemia, but disappeared by 24 h in the ischemic temporal cortex, in which both CMRglc and CBF were markedly reduced. In the ischemic parietal cortex, where CMRglc was preserved, COX-2 expression persisted even 24 h after ischemia. This study is the first to demonstrate neuronal COX-2 induction within potentially viable hypoperfused brain areas in nonhuman primates.