RESUMO
Experimental inhibition of the (pro)renin receptor [(P)RR] is a promising therapeutic strategy in different disease models ranging from cardiorenal to oncological entities. Here, we briefly review the direct protein-protein interaction partners of the (P)RR and the plethora of distinct diseases in which the (P)RR is involved. The first structural work on the (P)RR using AlphaFold, which was recently published by Ebihara et al., is the center of this mini-review since it can mechanistically link the protein-protein interaction level with the pathophysiological level. More detailed insights into the 3D structure of the (P)RR and its interaction domains might guide drug discovery on this novel target. Finally, antibody- and small molecule-based approaches to inhibit the (P)RR are shortly discussed.
Assuntos
Receptor de Pró-Renina , Receptores de Superfície Celular , Humanos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Renina/química , Renina/metabolismo , Comunicação CelularRESUMO
The (pro)renin receptor ((P)RR) is not only a member of the renin-angiotensin system (RAS) but also exerts several RAS-independent functions due to its multiple signal transductions pathways. In this mini-review, we shortly discuss the molecular functions of this receptor and its pathophysiological significance with a focus on cardiorenal diseases. Finally, we provide a short summary regarding a drug discovery and drug development program on small molecule-based renin/ prorenin receptor blockers (RERBs).
Assuntos
Receptor de Pró-Renina , ATPases Vacuolares Próton-Translocadoras , Renina/metabolismo , Sistema Renina-Angiotensina/fisiologia , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The (pro)renin receptor [(P)RR] is crucial for cardio-renal pathophysiology. The distinct molecular mechanisms of this receptor are still incompletely understood. The (P)RR is able to interact with different signalling proteins such as promyelocytic leukemia zinc finger protein (PLZF) and Wnt receptors. Moreover, domains of the (P)RR are essential for V-ATPase activity. V-ATPase- and Wnt-mediated effects imply constitutive, i.e., (pro)renin-independent functions of the (P)RR. Regarding ligand-dependent (P)RR signalling, the role of prorenin glycosylation is currently unknown. Therefore, the aim of this study was to analyse the contribution of constitutive (P)RR activity to its cellular effects and the relevance of prorenin glycosylation on its ligand activity. We were able to demonstrate that high glucose induces (P)RR signal transduction whereas deglycosylation of prorenin abolishes its intrinsic activity in neuronal and epithelial cells. By using siRNA against (P)RR or PLZF as well as the PLZF translocation blocker genistein and the specific V-ATPase inhibitor bafilomycin, we were able to dissect three distinct sub-pathways downstream of the (P)RR. The V-ATPase function is ligand-independently associated with strong pro-proliferative effects whereas prorenin causes moderate proliferation in vitro. In contrast, PLZF per se [i.e., in the absence of (pro)renin] does not interfere with cell number.
Assuntos
Genisteína/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Macrolídeos/farmacologia , Receptores de Superfície Celular/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Glucose/farmacologia , Glicosilação/efeitos dos fármacos , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/genética , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Modelos Biológicos , Peroxissomos/efeitos dos fármacos , Peroxissomos/metabolismo , Regiões Promotoras Genéticas/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Receptores de Superfície Celular/genética , Transdução de Sinais/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
The (pro)renin receptor ((P)RR) signaling is involved in different pathophysiologies ranging from cardiorenal end-organ damage via diabetic retinopathy to tumorigenesis. We have previously shown that the transcription factor promyelocytic leukemia zinc finger (PLZF) is an adaptor protein of the (P)RR. Furthermore, recent publications suggest that major functions of the (P)RR are mediated ligand-independently by its transmembrane and intracellular part, which acts as an accessory protein of V-ATPases. The transcriptome and recruitmentome downstream of the V-ATPase function and PLZF in the context of the (P)RR are currently unknown. Therefore, we performed a set of microarray and chromatin-immunoprecipitation (ChIP)-chip experiments using siRNA against the (P)RR, stable overexpression of PLZF, the PLZF translocation inhibitor genistein and the specific V-ATPase inhibitor bafilomycin to dissect transcriptional pathways downstream of the (P)RR. We were able to identify distinct and overlapping genetic signatures as well as novel real-time PCR-validated target genes of the different molecular functions of the (P)RR. Moreover, bioinformatic analyses of our data confirm the role of (P)RRs signal transduction pathways in cardiovascular disease and tumorigenesis.
Assuntos
Doenças Cardiovasculares/genética , Transformação Celular Neoplásica/genética , Fatores de Transcrição Kruppel-Like/genética , Receptores de Superfície Celular/genética , Transcriptoma , ATPases Vacuolares Próton-Translocadoras/genética , Doenças Cardiovasculares/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica/efeitos dos fármacos , Genisteína/farmacologia , Células HEK293 , Humanos , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/metabolismo , Macrolídeos/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteína com Dedos de Zinco da Leucemia Promielocítica , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismo , Receptor de Pró-ReninaRESUMO
Genetic factors strongly contribute to the pathogenesis of sporadic Alzheimer's disease (AD). Nevertheless, genome-wide association studies only yielded single nucleotide polymorphism loci of moderate importance. In contrast, microsatellite repeats are functionally less characterized structures within our genomes. Previous work has shown that endothelin-converting enzyme-1 (ECE-1) is able to reduce amyloid ß content. Here we demonstrate that a CpG-CA repeat within the human ECE-1c promoter is highly polymorphic, harbors transcriptional start sites, is able to recruit the transcription factors poly(ADP-ribose) polymerase-1 and splicing factor proline and glutamine-rich, and is functional regarding haplotype-specific promoter activity. Furthermore, genotyping of 403 AD patients and 444 controls for CpG-CA repeat length indicated shifted allelic frequency distributions. Sequencing of 245 haplotype clones demonstrated that the overall CpG-CA repeat composition of AD patients and controls is distinct. Finally, we show that human and chimpanzee [CpG](m)-[CA](n) ECE-1c promoter repeats are genetically and functionally distinct. Our data indicate that a short genomic repeat structure constitutes a novel core promoter element, coincides with human evolution, and contributes to the pathogenesis of AD.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Ácido Aspártico Endopeptidases/genética , Evolução Biológica , Metaloendopeptidases/genética , Repetições de Microssatélites/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Animais , Western Blotting , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/fisiopatologia , Cromatografia em Gel , DNA/genética , DNA/isolamento & purificação , Ensaio de Desvio de Mobilidade Eletroforética , Enzimas Conversoras de Endotelina , Genótipo , Humanos , Ensaios de Proteção de Nucleases , Pan troglodytes , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND/AIMS: Putative in vitro-in vivo correlations of pharmacokinetic (PK) parameters are regarded as a prerequisite to filter hits derived from high-throughput screening (HTS) approaches for subsequent murine in vivo PK studies. METHODS: In this study, we assessed stabilities in rat and human microsomes of 121 compounds from an early, academic drug discovery programme targeting the (pro)renin receptor and correlated the respective data with single-dose, in vivo PK parameters of 22 hits administered intravenously in rats. RESULTS: After transformation of in vitro half-lives to predicted in vivo hepatic clearances, r(2) regarding in vitro-in vivo clearance correlations were 0.31 and 0.27 for the rat and human species, respectively. CONCLUSIONS: Our data concerning structurally diverse real-world compounds indicate that microsomal stability testing is not a tool to triage early compounds for in vivo PK testing.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Microssomos Hepáticos/metabolismo , Preparações Farmacêuticas/metabolismo , Farmacocinética , Alternativas aos Testes com Animais/métodos , Animais , Células Cultivadas , Meia-Vida , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos TestesRESUMO
The (pro)renin receptor ((P)RR) and Wnt signalling are both involved in different diseases ranging from cardiac and renal end-organ damage to cancer. (P)RR function involves signalling via the transcription factor promyelocytic leukemia zinc finger protein (PLZF) as well as the furin-mediated generation of vacuolar proton-translocating ATPase (V-ATPase)-associated and soluble (P)RR isoforms. Recently, the (P)RR was described as adaptor protein of Wnt (co)receptors. The aim of this study was to analyse the contribution of these distinct (P)RR functions to Wnt signalling. Using Tcf/Lef reporter gene systems in HEK293T and HepG2 cells and quantification of endogenous axin2 mRNA and protein levels in HEK293T cells we were able to demonstrate that full-length (P)RR acts as a repressor of Wnt signalling in a system preactivated either by Wnt3a stimulation or by constitutively active ß-catenin. These repressive effects are mediated by Dvl but are independent of the mutation status of ß-catenin. Furthermore, the V-ATPase complex, but not PLZF translocation or renin enzymatic activity, is necessary for the induction of Tcf/Lef-responsive genes by Wnt3a. Our data indicate interference of (P)RR and Wnt cascades, a fact that has to be considered concerning pathophysiology of cardio-renal and oncological entities as well as in drug development programs targeting (P)RR or Wnt pathways.
Assuntos
Receptores de Superfície Celular/fisiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Genisteína/farmacologia , Células HEK293 , Células Hep G2 , Humanos , Macrolídeos/farmacologia , Camundongos , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Pró-ReninaRESUMO
Stroke is one of the major medical burdens in industrialized countries. Animal experiments indicate that blockade of the angiotensin AT1 receptor (AT1R) improves neurological outcome after cerebral ischemia. These protective effects are partially mediated by the angiotensin AT2 receptor (AT2R). The transcription factor promyelocytic leukemia zinc finger (PLZF) was identified as a direct adapter protein of the AT2R. Furthermore, our group was able to demonstrate that PLZF also directly binds and mediates the effects of the human (pro)renin receptor [(P)RR] which is involved in brain development. Therefore, we hypothesized that PLZF is involved in neuroprotection. Here we show that PLZF and its receptors (P)RR and AT2R exhibited an ubiquitous expression pattern in different brain regions. Furthermore, stable PLZF overexpression in human neuronal cells was able to mediate neuroprotection in a glutamate toxicity model in vitro. Consistently, PLZF mRNA and protein were downregulated on the ipsilateral side in a stroke model in vivo, whereas the neurodetrimental PLZF target genes cyclin A2 and BID were upregulated under this condition. Further analyses indicated that the neuroprotective AT2R is upregulated upon stable PLZF overexpression in cultured neuronal cells. Finally, reporter gene assays demonstrated the functionality of (P)RR promoter polymorphisms regarding basal and PLZF-induced activity.
Assuntos
Córtex Cerebral/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Neurônios/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Dedos de Zinco/genética , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Córtex Cerebral/patologia , Ciclina A2/genética , Ciclina A2/metabolismo , Regulação para Baixo/genética , Humanos , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Fatores de Transcrição Kruppel-Like/genética , Imageamento por Ressonância Magnética , Masculino , Neurônios/patologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor de Pró-ReninaRESUMO
Molecular mechanisms accompanying ethanol-induced cytotoxicity remain to be defined. The renin-angiotensin system with its respective receptors, the angiotensin AT1 and AT2 receptor (AT1R and AT2R), has been implicated in these processes. The AT2R seems to counteract the pro-inflammatory, pro-hypertrophic, and pro-fibrotic actions of the AT1R and is involved in cellular differentiation and tissue repair. Recently, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel negative transcriptional regulator of the AT2R. However, the complex interactions between ethanol, PARP-1, and the AT2R are largely unknown. In this in vitro study, we aimed to clarify whether acute ethanol treatment modifies AT2R promoter activity or AT2R mRNA and protein levels and whether PARP-1 is involved in ethanol-mediated regulation of the AT2R. Murine fibroblasts of the R3T3 and MEF line (murine embryonic fibroblasts) were exposed to ethanol for 24h. AT2R promoter activity, mRNA and protein levels were analyzed with and without PARP-1 inhibition and in PARP-1 knockout MEF cells. Expression of PARP-1 was analyzed over course of time, and cell viability and DNA fragmentation were measured on single-cell level by flow cytometry. Ethanol exposition induced substantial downregulation of the AT2R on promoter, mRNA and protein levels in a dose-dependent manner. Pharmacological inhibition or ablation of PARP-1 completely abolished this effect. Ethanol treatment did not have any effect on AT1R mRNA and protein levels in MEF cells. Further, acute ethanol treatment promoted DNA fragmentation and caused transcriptional induction of PARP-1. Our findings reveal that PARP-1 is an upstream transcriptional regulator of the AT2 receptor in the context of ethanol exposure and represses the AT2R gene in fibroblasts in vitro. Variations in expression of the potentially tissue-protective AT2R might contribute to ethanol-mediated pathology.
Assuntos
Regulação para Baixo/genética , Etanol/farmacologia , Fibroblastos/metabolismo , Poli(ADP-Ribose) Polimerases/fisiologia , Receptor Tipo 2 de Angiotensina/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Fragmentação do DNA , Fibroblastos/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Humanos , Camundongos , Camundongos Knockout , Neuroblastoma , Células PC12 , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , Ratos , Receptor Tipo 2 de Angiotensina/análiseRESUMO
Angiotensin II type 2 (AT(2)) receptors can be regarded as an endogenous repair system, because the AT(2) receptor is upregulated in tissue damage and mediates tissue protection. A potential therapeutic use of this system has only recently come within reach through synthesis of the first selective, orally active, nonpeptide AT(2) receptor agonist, compound 21 (C21; dissociation constant for AT(2) receptor: 0.4 nM; dissociation constant for angiotensin II type 1 receptor: >10,000 nM). This study tested AT(2) receptor stimulation with C21 as a potential future therapeutic approach for the inhibition of proinflammatory cytokines and of nuclear factor kappaB. C21 dose-dependently (1 nM to 1 micromol/L) reduced tumor necrosis factor-alpha-induced interleukin 6 levels in primary human and murine dermal fibroblasts. AT(2) receptor specificity was controlled for by inhibition with the AT(2) receptor antagonist PD123319 and by the absence of effects in AT(2) receptor-deficient cells. AT(2) receptor-coupled signaling leading to reduced interleukin 6 levels involved inhibition of nuclear factor kappaB, activation of protein phosphatases, and synthesis of epoxyeicosatrienoic acid. Inhibition of interleukin 6 promoter activity by C21 was comparable in strength to inhibition by hydrocortisone. C21 also reduced monocyte chemoattractant protein 1 and tumor necrosis factor-alpha in vitro and in bleomycin-induced toxic cutaneous inflammation in vivo. This study is the first to show the anti-inflammatory effects of direct AT(2) receptor stimulation in vitro and in vivo by the orally active, nonpeptide AT(2) receptor agonist C21. These data suggest that pharmacological AT(2) receptor stimulation may be an orally applicable future therapeutic approach in pathological settings requiring the reduction of interleukin 6 or inhibition of nuclear factor kappaB.
Assuntos
Ácido 8,11,14-Eicosatrienoico/metabolismo , Inflamação/metabolismo , NF-kappa B/metabolismo , Receptor Tipo 2 de Angiotensina/agonistas , Receptor Tipo 2 de Angiotensina/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos/citologia , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The (pro)renin receptor ((P)RR) not only represents a novel component of the renin-angiotensin system but is also a promising novel drug target because of its crucial involvement in the pathogenesis of renal and cardiac end-organ damage. This review discusses the signal transduction of the (P)RR with its adapter protein promyelocytic zinc-finger protein, the impact of this receptor, especially on cardiovascular disease, and its putative interaction with renin inhibitors such as aliskiren. Furthermore, the increasing complexity regarding the cellular function of the (P)RR is addressed, which arises by the intimate link with proton pumps and the phosphatase PRL-1, as well as by the presence of different subcellular localizations and of a soluble isoform of the (P)RR. Finally, the rationale and strategy for the development of small-molecule antagonists of the (P)RR, called renin/prorenin receptor blockers, are presented.
Assuntos
Receptores de Superfície Celular/antagonistas & inibidores , Transdução de Sinais/fisiologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Animais , Doenças Cardiovasculares/etiologia , Humanos , Receptores de Superfície Celular/fisiologia , ATPases Vacuolares Próton-Translocadoras/fisiologia , Receptor de Pró-ReninaRESUMO
The angiotensin AT( 2) receptor (AT(2)R) represents an important component of the renin-angiotensin system since it is involved in the (patho) physiology of different cardiovascular and neuronal diseases. Furthermore, AT(2) receptors can partly mediate beneficial effects of angiotensin AT( 1) receptor (AT(1)R) blockers, and direct pharmacological AT( 2) receptor agonism emerges as a novel therapeutic strategy. This review discusses the constitutive and ligand-mediated activity as well as the signal transduction of the AT(2) receptor, focusing on adapter proteins which directly bind to this receptor. Direct protein-protein interaction partners of the AT(2) receptor described so far include the transcription factor promyelocytic zinc finger protein, AT(2) receptor binding protein and the AT(1) receptor. In addition, the putative crosstalk of the AT(2) receptor with the renin/ prorenin receptor (RER) via the promyelocytic zinc finger protein (PLZF) and the role of oestrogens on the regulation of the AT(2) receptor are presented. Conceiving the coupling of the AT(2) receptor to different adapter proteins with distinct and partly opposing cellular effects and the implications of its constitutive activity might help to overcome the current controversies on the (patho)physiological role of the AT(2) receptor.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica , Miocárdio/metabolismo , Miocárdio/patologia , Regiões Promotoras Genéticas/genética , Receptor Tipo 2 de Angiotensina/genética , Animais , Humanos , Receptor Cross-Talk , Receptor Tipo 2 de Angiotensina/metabolismoRESUMO
Studying the angiotensin type 2 receptor (AT(2)) has been problematic in the past because a pharmacological tool for direct, specific in vitro and in vivo stimulation of the receptor has been lacking. Consequently, current knowledge about AT(2) receptor signalling and function had to be obtained by indirect approaches, like studying animals or cells with genetically altered AT(2) receptor expression levels, inhibitory experiments using specific AT(2) receptor antagonists, stimulation with angiotensin II under concomitant angiotensin II type 1 receptor blockade or stimulation with the peptide agonist CGP42112A, which has additional AT(2) receptor antagonistic properties. The recently developed non-peptide AT(2) receptor agonist Compound 21 now, for the first time, allows direct, selective and specific AT(2) receptor stimulation in vitro and in vivo. This new tool will certainly revolutionize AT(2) receptor research, enable many new insights into AT(2) receptor function and may also have the potential to become a future medical drug. This article reviews milestone findings about AT(2) receptor functional properties obtained by 'conventional' experimental approaches within the last 20 years. Moreover, it provides an overview of the first results obtained by direct AT(2) receptor stimulation with Compound 21, comprising effects on alkaline secretion, neurite outgrowth, blood pressure and post-infarct cardiac function.
Assuntos
Receptor Tipo 2 de Angiotensina/agonistas , Receptor Tipo 2 de Angiotensina/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Humanos , Peptídeos/farmacologiaRESUMO
The renin-angiotensin system (RAS) plays a crucial role in cardiovascular and neuronal (patho-)physiology. The angiotensin AT2 receptor (AT2R) seems to counteract the proinflammatory, prohypertrophic and profibrotic actions of the AT1 receptor. Recently, we identified a novel protein, termed "AT2R binding protein" (ATBP/ATIP) which seems essential for AT2R-mediated growth inhibition. Poly(ADP-ribose) polymerase-1 (PARP-1) can act as a nuclear integrator of angiotensin II-mediated cell signalling, and has been implicated in the pathogenesis of cardiovascular and neuronal disease. In this study, promoters of human AT2R and ATIP1 were cloned and two transcriptional start sites in the ATIP1 promoter were identified whereas only one was detected in the AT2R promoter. Promoter assays indicated that the exon 1-intron 1 region of AT2R is necessary and sufficient for AT2R promoter activity. Inverse cloning experiments indicated that this regulatory region is a promoter but not an enhancer element implicating (a) further start site(s) in this region. Consistently, the exon 1-intron 1 region of AT2R was shown to tether the basal transcriptional machinery. Overexpression, pharmacological inhibition and ablation of PARP demonstrated that PARP-1 activates the ATIP1 gene but represses the AT2R on promoter and mRNA levels in vitro, and in brain tissue in vivo. Additional experiments indicated that AT2R activation does not modulate PARP-1 transcript levels but increases AT2R promoter activity, thereby creating a positive feedback mechanism. Our results demonstrate that PARP-1 acts as novel node within the RAS network based on its ability to regulate downstream targets such as AT2R and its adapter protein ATBP.
Assuntos
Regulação da Expressão Gênica , Poli(ADP-Ribose) Polimerases/fisiologia , Receptor Tipo 2 de Angiotensina/genética , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana Transportadoras , RNA Mensageiro , Transcrição GênicaRESUMO
OBJECTIVE: Ceftriaxone has been reported to reduce neuronal damage in amyotrophic lateral sclerosis and in an in-vitro model of neuronal ischaemia through increased expression and activity of the glutamate transporter, GLT1. We tested the effects of ceftriaxone on mortality, neurological outcome, and infarct size in experimental stroke in rats and looked for underlying mechanisms. METHODS: Male normotensive Wistar rats received ceftriaxone (200 mg/kg intraperitoneal) as a single injection 90 min after middle cerebral artery occlusion (90 min with reperfusion). Forty-eight hours after middle cerebral artery occlusion, infarct size (MRI) and neurological deficits were estimated. GLT1 expression was determined by real time RT-PCR, immunoblotting and promoter reporter assay, astrocyte GLT1 activity by measuring glutamate uptake. Bacterial load in various organs was measured by real time RT-PCR, neurotrophins and IL-6 by immunoblotting. RESULTS: Ceftriaxone dramatically reduced early (24-h) mortality from 34.5% (vehicle treatment, n = 29) to 0% (P < 0.01, n = 19). In a subgroup, followed up for 4 weeks, mortality persisted at 0%. Ceftriaxone strongly tended to reduce infarct size, it significantly improved neuronal survival within the penumbra, reduced neurological deficits (P < 0.001) and led to an upregulation of neurotrophins (P < 0.01) in the peri-infarct zone. Ceftriaxone did not increase GLT1 expression, but increased GLT1 activity (P < 0.05). CONCLUSION: Ceftriaxone causes a significant reduction in acute stroke mortality in a poststroke treatment regimen in animal studies. Improved neurological performance and survival may be due to neuroprotection by activation of GLT1 and a stimulation of neurotrophins resulting in an increased number of surviving neurons in the penumbra.
Assuntos
Antibacterianos/farmacologia , Ceftriaxona/farmacologia , Ácido Glutâmico/metabolismo , Fatores de Crescimento Neural/metabolismo , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/mortalidade , Animais , Temperatura Corporal/fisiologia , Encéfalo/irrigação sanguínea , Infarto Encefálico/patologia , Transtornos Cerebrovasculares/complicações , Modelos Animais de Doenças , Transportador 2 de Aminoácido Excitatório/metabolismo , Interleucina-6/metabolismo , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Acidente Vascular Cerebral/etiologia , Taxa de SobrevidaRESUMO
OBJECTIVES: Inhibition of (pro)renin receptor activation was demonstrated to inhibit or even abolish the development of end-organ damage in animal models. The new renin inhibitor, aliskiren, markedly increases the plasma concentration of the (pro)renin receptor ligands prorenin and renin in patients. The effects of prorenin and of renin inhibitors on the signal transduction cascade of the (pro)renin receptor are currently unknown. RESULTS: Our results indicate that renin and prorenin were equally potent in (pro)renin receptor activation by decreasing (pro)renin receptor mRNA, increasing phosphatidylinositol-3 kinase p85alpha mRNA and augmenting viable cell number, respectively. These effects of renin and prorenin are both abolished using small-interfering RNA against the (pro)renin receptor or its adaptor promyelocytic zinc finger protein. The renin inhibitor aliskiren did not inhibit the renin-induced or prorenin-induced activation of the (pro)renin receptor. CONCLUSION: This is the first report demonstrating equal ligand activities of both, renin and prorenin, on the (pro)renin receptor - promyelocytic zinc finger protein-phosphatidylinositol-3 kinase-p85alpha pathway. The failure of aliskiren to inhibit the noncatalytic effects of renin and prorenin may be of clinical relevance considering the increase in plasma concentrations of (pro)renin under aliskiren treatment.
Assuntos
Amidas/farmacologia , Anti-Hipertensivos/farmacologia , Fumaratos/farmacologia , Receptores de Superfície Celular/metabolismo , Renina/antagonistas & inibidores , Renina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Rim/citologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Ligantes , Fosfatidilinositol 3-Quinases/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Receptores de Superfície Celular/genética , Renina/farmacologia , Receptor de Pró-ReninaRESUMO
For many years, angiotensin II with its respective receptors was considered to be the only effector molecule within the renin-angiotensin system. Nevertheless, several studies indicated that renin (the enzyme catalyzing the generation of angiotensin I) and its enzymatically inactive precursor prorenin may have an angiotensin-II-independent (patho)physiological significance. In 2002, a specific (pro)renin receptor ((P)RR)) which increases the enzymatic activity of its ligands and induces an intrinsic activity upon ligand binding has been published. Recently, our group has demonstrated a novel (P)RR signal transduction pathway involving direct protein-protein interaction between the (P)RR and the transcription factor promyelocytic zinc finger protein (PLZF) and the nuclear translocation of PLZF upon renin stimulation. Downstream effects of (P)RR activation by renin included repression of the (P)RR itself and induction of the p85alpha subunit of the phosphatidylinositol-3 kinase (PI3K-p85alpha) as well as an increase in proliferation and a decrease in apoptotic activity. Various animal models demonstrated that inhibition of prorenin binding to the (P)RR can prevent or even abolish the development of cardiac fibrosis and diabetic nephropathy via angiotensin-II-independent mechanisms. Additional studies that verify these remarkable findings are needed. Moreover, the potency of aliskiren (the first orally active renin inhibitor in the market) to interfere with a putatively detrimental binding of (pro)renin to the (P)RR is of particular interest and has to be elucidated.
Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Humanos , Renina/antagonistas & inibidores , Transdução de Sinais , Receptor de Pró-ReninaRESUMO
Nitric oxide (NO) and endothelin-1 (ET-1) are known to play a major role in renal and vascular pathophysiology and exhibit a close interaction with ET-1, stimulating NO production; NO in turn inhibits ET-1 expression. Our objectives were (1) to establish a novel transgenic mouse model facilitating ET-1 expression assessment in vivo, (2) to validate this model by assessing prepro-ET-1 promoter activity in mice embryos by means of our novel model and comparing expression sites to well-established data on ET-1 in fetal development and (3) to investigate renal ET-NO interaction by assessing prepro-ET-1 promoter activity in different structures of the renal cortex in the setting of blocked NO synthases via L-NAME administration. We established transgenic mice carrying a lacZ reporter gene under control of the human prepro-ET-1 gene promoter sequence (8 kb of 5' sequences). Bluo-Gal staining of tissue sections revealed intracellular blue particles as indicators of prepro-ET-1 promoter activity. In mouse embryos, we detected high prepro-ET-1 promoter activity in the craniofacial region, as well as in bone and cartilage consistent with the literature. In order to investigate the interaction of ET-1 and NO in the kidney in vivo, transgenic mice at the age of 3-4 months were treated with a single dose of the NO synthase inhibitor L-NAME (25 mg (kg bw)(-1) i.p.) 12 h before kidney removal. Bluo-Gal staining of kidney sections revealed intracellular blue particles as indicators of prepro-ET-1 promoter activity in tubular and vascular endothelium and glomerular cells. Particle count was closely correlated to kidney tissue ET-1 content (R=0.918, P<0.001). Comparison of counts revealed an increase by 135+/-53% in L-NAME treated (n=12) compared to non-treated mice (n=10, P=0.001). Cell-type specific evaluation revealed an increase of 136+/-51% in tubular (P=0.001) and 105+/-41% in glomerular cells (P=0.046), but no significant increase in vascular endothelium. In conclusion, our study revealed a close interaction of renal endothelin and the NO system in a cell-type specific manner. Our new transgenic model provides a unique opportunity to analyse regulation of the ET system on a cellular level in vivo.
Assuntos
Endotelina-1/metabolismo , Inibidores Enzimáticos/farmacologia , Rim/efeitos dos fármacos , Óperon Lac , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico/metabolismo , Regiões Promotoras Genéticas , Animais , Pressão Sanguínea/efeitos dos fármacos , Cartilagem/embriologia , Cartilagem/metabolismo , Endotelina-1/genética , Ossos Faciais/embriologia , Ossos Faciais/metabolismo , Humanos , Rim/citologia , Rim/enzimologia , Rim/metabolismo , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Camundongos , Camundongos Transgênicos , Óxido Nítrico Sintase/metabolismo , Fenótipo , Reprodutibilidade dos Testes , Crânio/embriologia , Crânio/metabolismoRESUMO
A human renin/prorenin receptor (RER) has recently been cloned. To gain insight into the molecular function of the RER, we studied its signal transduction mechanisms. Initially, we found a ubiquitous and intracellular expression pattern of the human RER. Consistently, we observed several transcriptional start sites and a high promoter activity of the human RER. We could identify the transcription factor promyelocytic zinc finger (PLZF) protein as a direct protein interaction partner of the C-terminal domain of the RER by yeast 2-hybrid screening and coimmunoprecipitation. Coimmunoprecipitation experiments also indicated homodimerization of the RER. On activation of the RER by renin, PLZF is translocated into the nucleus and represses transcription of the RER itself, thereby creating a very short negative feedback loop, but activates transcription of the p85alpha subunit of the phosphatidylinositol-3 kinase (PI3K-p85alpha). Small interfering RNA against the RER abolished these effects. A PLZF cis-element in the RER promoter was identified by site-directed mutagenesis and electrophoretic mobility-shift assay. Renin stimulation caused a 6-fold recruitment of PLZF to this promoter region as shown by chromatin immunoprecipitation. Moreover, renin stimulation of rat H9c2 cardiomyoblasts induced an increase of cell number and a decrease of apoptosis. These effects were partly abolished by PI3K inhibition and completely abrogated by small interfering RNA against PLZF. Finally, experiments in PLZF knockout mice confirmed the role of PLZF as an upstream regulator of RER and PI3K-p85alpha. Our data demonstrate the existence of a novel signal transduction pathway involving the ligand renin, RER, and the transcription factor PLZF, which is of physiological and putative pathophysiological relevance.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Sistema Renina-Angiotensina/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/fisiologia , Rim/citologia , Fatores de Transcrição Kruppel-Like , Camundongos , Camundongos Knockout , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas/fisiologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , RNA Mensageiro/metabolismo , Renina/metabolismo , Fatores de Transcrição/genéticaRESUMO
For quantification of gene-specific mRNA, quantitative real-time RT-PCR has become one of the most frequently used methods over the last few years. This article focuses on the issue of real-time PCR data analysis and its mathematical background, offering a general concept for efficient, fast and precise data analysis superior to the commonly used comparative CT (DeltaDeltaCT) and the standard curve method, as it considers individual amplification efficiencies for every PCR. This concept is based on a novel formula for the calculation of relative gene expression ratios, termed GED (Gene Expression's CT Difference) formula. Prerequisites for this formula, such as real-time PCR kinetics, the concept of PCR efficiency and its determination, are discussed. Additionally, this article offers some technical considerations and information on statistical analysis of real-time PCR data.