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2.
J Biol Chem ; 294(2): 576-592, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30409912

RESUMO

Faithful chromosome segregation during mitosis is critical for maintaining genome integrity in cell progeny and relies on accurate and robust kinetochore-microtubule attachments. The NDC80 complex, a tetramer comprising kinetochore protein HEC1 (HEC1), NDC80 kinetochore complex component NUF2 (NUF2), NDC80 kinetochore complex component SPC24 (SPC24), and SPC25, plays a critical role in kinetochore-microtubule attachment. Mounting evidence indicates that phosphorylation of HEC1 is important for regulating the binding of the NDC80 complex to microtubules. However, it remains unclear whether other post-translational modifications, such as acetylation, regulate NDC80-microtubule attachment during mitosis. Here, using pulldown assays with HeLa cell lysates and site-directed mutagenesis, we show that HEC1 is a bona fide substrate of the lysine acetyltransferase Tat-interacting protein, 60 kDa (TIP60) and that TIP60-mediated acetylation of HEC1 is essential for accurate chromosome segregation in mitosis. We demonstrate that TIP60 regulates the dynamic interactions between NDC80 and spindle microtubules during mitosis and observed that TIP60 acetylates HEC1 at two evolutionarily conserved residues, Lys-53 and Lys-59. Importantly, this acetylation weakened the phosphorylation of the N-terminal HEC1(1-80) region at Ser-55 and Ser-62, which is governed by Aurora B and regulates NDC80-microtubule dynamics, indicating functional cross-talk between these two post-translation modifications of HEC1. Moreover, the TIP60-mediated acetylation was specifically reversed by sirtuin 1 (SIRT1). Taken together, our results define a conserved signaling hierarchy, involving HEC1, TIP60, Aurora B, and SIRT1, that integrates dynamic HEC1 acetylation and phosphorylation for accurate kinetochore-microtubule attachment in the maintenance of genomic stability during mitosis.


Assuntos
Cinetocoros/metabolismo , Lisina Acetiltransferase 5/metabolismo , Microtúbulos/metabolismo , Mitose , Proteínas Nucleares/metabolismo , Acetilação , Segregação de Cromossomos , Proteínas do Citoesqueleto , Células HEK293 , Células HeLa , Humanos , Lisina Acetiltransferase 5/análise , Modelos Moleculares , Proteínas Nucleares/análise , Mapas de Interação de Proteínas , Sirtuína 1/análise , Sirtuína 1/metabolismo
3.
Int J Biochem Cell Biol ; 79: 139-150, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27590851

RESUMO

Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin's actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin's actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin's effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis.


Assuntos
Células Endoteliais da Veia Umbilical Humana/citologia , Leptina/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Receptores Notch/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Notch/genética , Suínos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
4.
Am J Physiol Cell Physiol ; 311(2): C179-89, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27170637

RESUMO

The Iroquois homeobox (Irx5) gene is essential in embryonic development and cardiac electrophysiology. Although recent studies have reported that IRX5 protein is involved in regulation of the cell cycle and apoptosis in prostate cancer cells, little is known about the role of IRX5 in the adult vasculature. Here we report novel observations on the role of IRX5 in adult vascular smooth muscle cells (VSMCs) during proliferation in vitro and in vivo. Comparative studies using primary human endothelial cells, VSMCs, and intact carotid arteries to determine relative expression of Irx5 in the peripheral vasculature demonstrate significantly higher expression in VSMCs. Sprague-Dawley rat carotid arteries were subjected to balloon catherization, and the presence of IRX5 was examined by immunohistochemistry after 2 wk. Results indicate markedly elevated IRX5 signal at 14 days compared with uninjured controls. Total RNA was isolated from injured and uninjured arteries, and Irx5 expression was measured by RT-PCR. Results demonstrate a significant increase in Irx5 expression at 3-14 days postinjury compared with controls. Irx5 genetic gain- and loss-of-function studies using thymidine and 5-bromo-2'-deoxyuridine incorporation assays resulted in modulation of DNA synthesis in primary rat aortic VSMCs. Quantitative RT-PCR results revealed modulation of cyclin-dependent kinase inhibitor 1B (p27(kip1)), E2F transcription factor 1 (E2f1), and proliferating cell nuclear antigen (Pcna) expression in Irx5-transduced VSMCs compared with controls. Subsequently, apoptosis was observed and confirmed by morphological observation, caspase-3 cleavage, and enzymatic activation compared with control conditions. Taken together, these results indicate that Irx5 plays an important role in VSMC G1/S-phase cell cycle checkpoint control and apoptosis.


Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Fase G1/fisiologia , Proteínas de Homeodomínio/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fase S/fisiologia , Fatores de Transcrição/metabolismo , Animais , Apoptose/fisiologia , Artérias Carótidas/metabolismo , Caspase 3/metabolismo , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27 , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley
5.
J Cardiovasc Pharmacol ; 67(5): 433-41, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26841069

RESUMO

Leukocyte infiltration of adventitial and perivascular tissues is an early event in the development of vascular remodeling after injury. We investigated whether Slit/Robo-an axonal chemorepellent system in vertebrate and invertebrate development-is activated during the inflammatory phase that follows endothelial denudation. Using the rat carotid artery model of angioplasty, we conducted a time course analysis of mRNAs encoding Slit ligands (Slit2 and Slit3) and Robo receptors (Robo1, Robo2, and Robo4), as well as proinflammatory cell adhesion molecule (CAM) genes. Adventitial inflammatory cells were counted in immunostained arterial sections. E-selectin, vascular CAM-1, and intercellular CAM-1 were upregulated 2-3 hours after injury, followed by infiltration of neutrophils and monocytes as evidenced by real-time polymerase chain reaction, in situ hybridization, and immunohistochemistry. Slit2, Slit3, and Robo genes exhibited no expression changes at 3 hours; however, they were markedly upregulated 1 day after angioplasty. Intercellular CAM-1 expression was reduced by 50%, and the number of adventitial neutrophils decreased by >75% 1 day after angioplasty. Slit2 has been shown to be a potent chemorepelent of leukocytes, endothelial cells, and smooth muscle cells. Thus, we decided to further investigate the localization of Slit2 in injured vessels. Immunohistochemical stainings revealed the presence of Slit2 within the vessel wall and in the perivascular vasa vasorum of naive and injured arteries. Double immunohistochemical analyses showed that infiltrating monocytes expressed Slit2 in the perivascular and adventitial tissues of injured arteries 1 and 3 days postangioplasty. In addition, recombinant full-length Slit2 and Slit2-N/1118, an N-terminal fragment of Slit2, inhibited stromal cell-derived factor 1-mediated migration of circulating rat peripheral blood mononuclear cells. In summary, adventitial activation of CAM genes and neutrophil infiltration preceded upregulation of Slit/Robo genes. Sli2 expression colocalized with infiltrating inflammatory cells in the adventitial layer. This temporospatial association suggests that leukocyte chemorepellent Slit2 may be involved in halting the adventitial accumulation of inflammatory cells in injured vessels.


Assuntos
Artérias Carótidas/fisiopatologia , Células Endoteliais/metabolismo , Mediadores da Inflamação/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Animais , Moléculas de Adesão Celular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Proteínas de Membrana/biossíntese , RNA Mensageiro , Ratos , Receptores Imunológicos/biossíntese , Regulação para Cima , Proteínas Roundabout
6.
J Biol Chem ; 290(10): 6303-15, 2015 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-25512384

RESUMO

Angiogenesis is a dynamic process required for embryonic development. However, postnatal vascular growth is characteristic of multiple disease states. Despite insights into the multistep process in which adhesion molecules, extracellular matrix proteins, growth factors, and their receptors work in concert to form new vessels from the preexisting vasculature, there remains a lack of insight of the nuclear transcriptional mechanisms that occur within endothelial cells (ECs) in response to VEGF. Iroquois homeobox gene 3 (Irx3) is a transcription factor of the Iroquois family of homeobox genes. Irx homeodomain transcription factors are involved in the patterning and development of several tissues. Irx3 is known for its role during embryogenesis in multiple organisms. However, the expression and function of Irx3 in human postnatal vasculature remains to be investigated. Here we show that Irx3 is expressed in human microvascular endothelial cells, and expression is elevated by VEGF stimulation. Genetic Irx3 gain and loss of function studies in human microvascular endothelial cells resulted in the modulation of EC migration during wound healing, chemotaxis and invasion, and tubulogenesis. Additionally, we observed increased delta-like ligand 4 (Dll4) expression, which suggests an increase in EC tip cell population. Finally, siRNA screening studies revealed that transient knockdown of Hey1, a downstream Notch signaling mediator, resulted in increased Irx3 expression in response to VEGF treatment. Strategies to pharmacologically regulate Irx3 function in adult endothelial cells may provide new therapies for angiogenesis.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Proteínas de Homeodomínio/metabolismo , Neovascularização Patológica/genética , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ligação ao Cálcio , Movimento Celular/genética , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Células Endoteliais/patologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Receptores Notch/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
7.
J Neuroinflammation ; 11: 9, 2014 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-24433482

RESUMO

BACKGROUND: Cerebral Malaria (CM) is a diffuse encephalopathy caused by Plasmodium falciparum infection. Despite availability of antimalarial drugs, CM-associated mortality remains high at approximately 30% and a subset of survivors develop neurological and cognitive disabilities. While antimalarials are effective at clearing Plasmodium parasites they do little to protect against CM pathophysiology and parasite-induced brain inflammation that leads to seizures, coma and long-term neurological sequelae in CM patients. Thus, there is urgent need to explore therapeutics that can reduce or prevent CM pathogenesis and associated brain inflammation to improve survival. Neuregulin-1 (NRG-1) is a neurotrophic growth factor shown to protect against brain injury associated with acute ischemic stroke (AIS) and neurotoxin exposure. However, this drug has not been tested against CM-associated brain injury. Since CM-associated brain injuries and AIS share similar pathophysiological features, we hypothesized that NRG-1 will reduce or prevent neuroinflammation and brain damage as well as improve survival in mice with late-stage experimental cerebral malaria (ECM). METHODS: We tested the effects of NRG-1 on ECM-associated brain inflammation and mortality in P. berghei ANKA (PbA)-infected mice and compared to artemether (ARM) treatment; an antimalarial currently used in various combination therapies against malaria. RESULTS: Treatment with ARM (25 mg/kg/day) effectively cleared parasites and reduced mortality in PbA-infected mice by 82%. Remarkably, NRG-1 therapy (1.25 ng/kg/day) significantly improved survival against ECM by 73% despite increase in parasite burden within NRG-1-treated mice. Additionally, NRG-1 therapy reduced systemic and brain pro-inflammatory factors TNFalpha, IL-6, IL-1alpha and CXCL10 and enhanced anti-inflammatory factors, IL-5 and IL-13 while decreasing leukocyte accumulation in brain microvessels. CONCLUSIONS: This study suggests that NRG-1 attenuates ECM-associated brain inflammation and injuries and may represent a novel supportive therapy for the management of CM.


Assuntos
Antimaláricos/uso terapêutico , Encefalite/tratamento farmacológico , Malária Cerebral/tratamento farmacológico , Malária Cerebral/mortalidade , Neuregulina-1/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Animais , Artemeter , Artemisininas/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Encéfalo/parasitologia , Encéfalo/patologia , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalite/etiologia , Encefalite/patologia , Endotélio/efeitos dos fármacos , Endotélio/patologia , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Malária Cerebral/complicações , Camundongos , Camundongos Endogâmicos C57BL , Neuregulina-1/metabolismo , Plasmodium berghei/fisiologia
8.
J Biol Chem ; 288(48): 34394-402, 2013 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-24133204

RESUMO

Prohibitin (PHB) has been reported to play a crucial role in adipocyte differentiation and mitochondrial function. However, the regulative mechanism of PHB during adipogenesis remains unclear. In this study, we determined that the levels of both microRNA (miR)-27a and miR-27b were down-regulated following adipogenic induction of human adipose-derived stem cells, whereas the mRNA level of PHB was up-regulated. Overexpression of miR-27a or miR-27b inhibited PHB expression and adipocyte differentiation. Using PHB 3'-UTR luciferase reporter assay, we observed that miR-27a and miR-27b directly targeted PHB in human adipose-derived stem cells. A compensation of PHB partially restored the adipogenesis inhibited by miR-27. Moreover, we demonstrated the novel finding that ectopic expression of miR-27a or miR-27b impaired mitochondrial biogenesis, structure integrity, and complex I activity accompanied by excessive reactive oxygen species production. Our data suggest that miR-27 is an anti-adipogenic microRNA partly by targeting PHB and impairing mitochondrial function. Pharmacological modulation of miR-27 function may provide a new therapeutic strategy for the treatment of obesity.


Assuntos
Adipogenia/genética , MicroRNAs/metabolismo , Obesidade/genética , Proteínas Repressoras/metabolismo , Adipócitos/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , MicroRNAs/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Obesidade/metabolismo , Obesidade/patologia , Proibitinas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Células-Tronco , Regulação para Cima
9.
PLoS One ; 8(4): e60898, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23630573

RESUMO

Despite appropriate anti-malarial treatment, cerebral malaria (CM)-associated mortalities remain as high as 30%. Thus, adjunctive therapies are urgently needed to prevent or reduce such mortalities. Overproduction of CXCL10 in a subset of CM patients has been shown to be tightly associated with fatal human CM. Mice with deleted CXCL10 gene are partially protected against experimental cerebral malaria (ECM) mortality indicating the importance of CXCL10 in the pathogenesis of CM. However, the direct effect of increased CXCL10 production on brain cells is unknown. We assessed apoptotic effects of CXCL10 on human brain microvascular endothelial cells (HBVECs) and neuroglia cells in vitro. We tested the hypothesis that reducing overexpression of CXCL10 with a synthetic drug during CM pathogenesis will increase survival and reduce mortality. We utilized atorvastatin, a widely used synthetic blood cholesterol-lowering drug that specifically targets and reduces plasma CXCL10 levels in humans, to determine the effects of atorvastatin and artemether combination therapy on murine ECM outcome. We assessed effects of atorvastatin treatment on immune determinants of severity, survival, and parasitemia in ECM mice receiving a combination therapy from onset of ECM (day 6 through 9 post-infection) and compared results with controls. The results indicate that CXCL10 induces apoptosis in HBVECs and neuroglia cells in a dose-dependent manner suggesting that increased levels of CXCL10 in CM patients may play a role in vasculopathy, neuropathogenesis, and brain injury during CM pathogenesis. Treatment of ECM in mice with atorvastatin significantly reduced systemic and brain inflammation by reducing the levels of the anti-angiogenic and apoptotic factor (CXCL10) and increasing angiogenic factor (VEGF) production. Treatment with a combination of atorvastatin and artemether improved survival (100%) when compared with artemether monotherapy (70%), p<0.05. Thus, adjunctively reducing CXCL10 levels and inflammation by atorvastatin treatment during anti-malarial therapy may represent a novel approach to treating CM patients.


Assuntos
Antimaláricos/farmacologia , Quimiocina CXCL10/antagonistas & inibidores , Malária Cerebral/tratamento farmacológico , Malária Cerebral/metabolismo , Animais , Antimaláricos/uso terapêutico , Apoptose/efeitos dos fármacos , Artemeter , Artemisininas/farmacologia , Atorvastatina , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Caspases/metabolismo , Contagem de Células , Quimiocina CXCL10/sangue , Quimiocina CXCL10/genética , Quimiocina CXCL10/farmacologia , Modelos Animais de Doenças , Interações Medicamentosas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Ácidos Heptanoicos/farmacologia , Ácidos Heptanoicos/uso terapêutico , Humanos , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Malária Cerebral/imunologia , Malária Cerebral/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Conformação de Ácido Nucleico , Pirróis/farmacologia , Pirróis/uso terapêutico , Análise de Sobrevida
10.
Obesity (Silver Spring) ; 20(7): 1481-90, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22262158

RESUMO

Progenitor cells (PCs) are key components of vasculogenic remodeling and hematopoietic development. Decreases in the number and function of angiogenic progenitors have been observed in coronary artery disease, hypertension, and diabetic vasculopathy. Several recent studies have also demonstrated a close relationship between increased visceral fat and cardiovascular disease, implying an association between obesity and vascular dysfunction. However, very little is known about the role of PCs in obesity. We generated whole genome expression profiles of cultured PCs from 18 obese and 6 lean African-American women on Agilent microarrays and analyzed the data through bioinformatic pathway analysis using multiple databases and analytic tools. False-discovery rates (FDR) were calculated to assess statistical significance while controlling for multiple testing. We identified 1,145 upregulated and 2,257 downregulated genes associated with obesity (1.5-fold or greater absolute fold-change). Pathway analysis further identified a statistically significant downregulation of immune-response pathways in the obese subjects, including T-cell receptor signaling, natural killer cell signaling, and chemokine-signaling pathways (FDR <5%). Chemokine gene-expression patterns were consistent with an angiogenic-angiostatic imbalance and a downregulation of CXCR3 receptor-mediated signaling in the PCs from obese subjects. Overall, these findings reveal a novel transcriptional signature in cultured PCs from obese African-American women and further suggest that obesity-associated immune-compromise may originate much earlier in cellular development than currently appreciated. Clinically, this may translate into a lengthier period of immune dysregulation in obese subjects exposing them to greater risks of infection and other morbidities.


Assuntos
Negro ou Afro-Americano/genética , Gordura Intra-Abdominal/metabolismo , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/genética , Obesidade/genética , Transdução de Sinais/genética , Transcriptoma/genética , Adolescente , Adulto , Células Cultivadas , Feminino , Humanos , Células Matadoras Naturais/imunologia , Pessoa de Meia-Idade , Obesidade/imunologia , Obesidade/metabolismo , Receptores CXCR3/genética , Adulto Jovem
11.
J Clin Invest ; 117(2): 299-302, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17273550

RESUMO

The way in which multiple cell types organize themselves into a carefully sculpted, 3D labyrinth of vessels that regulate blood flow throughout the body has been a longstanding mystery. Clinicians familiar with congenital cardiovascular disease recognize how genetic variants and modest perturbations in this complex set of spatiotemporal interactions and stochastic processes can result in life-threatening anomalies. Although the mystery is not yet fully solved, we are poised at an exciting juncture, as insights from murine disease models are converging with advances in human genetics to shed new light on puzzling clinical phenotypes of vascular disease. The study by High et al. in this issue of the JCI establishes a model system that mimics clinical features of congenital cardiovascular disease and further defines the role of the Notch signaling pathway in the neural crest as an essential determinant of cardiovascular structure (see the related article beginning on page 353).


Assuntos
Vasos Sanguíneos/embriologia , Receptores Notch/fisiologia , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Genômica , Humanos , Camundongos , Morfogênese , Músculo Liso Vascular/citologia , Crista Neural/embriologia , Transdução de Sinais , Doenças Vasculares/etiologia
12.
J Clin Invest ; 115(7): 1913-22, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16007255

RESUMO

The renin-angiotensin-aldosterone system controls blood pressure and salt-volume homeostasis. Renin, which is the first enzymatic step of the cascade, is critically regulated at the transcriptional level. In the present study, we investigated the role of liver X receptor alpha (LXR(alpha)) and LXR(beta) in the regulation of renin. In vitro, both LXRs could bind to a noncanonical responsive element in the renin promoter and regulated renin transcription. While LXR(alpha) functioned as a cAMP-activated factor, LXR(beta) was inversely affected by cAMP. In vivo, LXRs colocalized in juxtaglomerular cells, in which LXR(alpha) was specifically enriched, and interacted with the renin promoter. In mouse models, renin-angiotensin activation was associated with increased binding of LXR(alpha) to the responsive element. Moreover, acute administration of LXR agonists was followed by upregulation of renin transcription. In LXR(alpha) mice, the elevation of renin triggered by adrenergic stimulation was abolished. Untreated LXR(beta) mice exhibited reduced kidney renin mRNA levels compared with controls. LXR(alpha)LXR(beta) mice showed a combined phenotype of lower basal renin and blunted adrenergic response. In conclusion, we show herein that LXR(alpha) and LXR(beta) regulate renin expression in vivo by directly interacting with the renin promoter and that the cAMP/LXR(alpha) signaling pathway is required for the adrenergic control of the renin-angiotensin system.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Renina/genética , Animais , Sequência de Bases , Linhagem Celular , DNA Complementar/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores Nucleares Órfãos , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Sistema Renina-Angiotensina/genética , Sistema Renina-Angiotensina/fisiologia , Transdução de Sinais
13.
J Biol Chem ; 278(17): 15252-60, 2003 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-12551904

RESUMO

We have reported previously that liver X receptor-alpha (LXRalpha) can mediate a novel cAMP-dependent increase in renin and c-myc gene transcription by binding as a monomer to a unique regulatory element termed the cAMP-negative response element (CNRE). To determine whether this novel action of LXRalpha has global implications on gene regulation, we employed expression profiling to identify other genes regulated by this unique mechanism. Here we report the existence of a set of known and unknown transcripts regulated in parallel with renin. Querying the Celera Mouse Genome Assembly revealed that a majority of these genes contained the consensus CNRE. We have confirmed the functionality of these CNREs by competition for LXRalpha binding via electrophoretic mobility shift assays (EMSA) and by the use of CNRE decoy molecules documenting the abolishment of the cAMP-mediated gene induction. Taken together, these results demonstrate that the interaction between cAMP-activated LXRalpha and the CNRE enhancer element is responsible for widespread changes in gene expression and identify a set of LXRalpha/cAMP-regulated genes that may have important biological implications.


Assuntos
Regulação da Expressão Gênica , Receptores Citoplasmáticos e Nucleares/genética , Transcrição Gênica , Animais , Sítios de Ligação , Análise por Conglomerados , Sequência Consenso , AMP Cíclico/farmacologia , Proteínas de Ligação a DNA , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores X do Fígado , Camundongos , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Renina/genética , Elementos de Resposta , Ativação Transcricional
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