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1.
J Biol Chem ; 288(51): 36202-14, 2013 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-24163368

RESUMO

Accumulating evidence suggests that microRNAs (miRNAs) contribute to a myriad of kidney diseases. However, the regulatory role of miRNAs on the key molecules implicated in kidney fibrosis remains poorly understood. Bone morphogenetic protein-7 (BMP-7) and its related BMP-6 have recently emerged as key regulators of kidney fibrosis. Using the established unilateral ureteral obstruction (UUO) model of kidney fibrosis as our experimental model, we examined the regulatory role of miRNAs on BMP-7/6 signaling. By analyzing the potential miRNAs that target BMP-7/6 in silica, we identified miR-22 as a potent miRNA targeting BMP-7/6. We found that expression levels of BMP-7/6 were significantly elevated in the kidneys of the miR-22 null mouse. Importantly, mice with targeted deletion of miR-22 exhibited attenuated renal fibrosis in the UUO model. Consistent with these in vivo observations, primary renal fibroblast isolated from miR-22-deficient UUO mice demonstrated a significant increase in BMP-7/6 expression and their downstream targets. This phenotype could be rescued when cells were transfected with miR-22 mimics. Interestingly, we found that miR-22 and BMP-7/6 are in a regulatory feedback circuit, whereby not only miR-22 inhibits BMP-7/6, but miR-22 by itself is induced by BMP-7/6. Finally, we identified two BMP-responsive elements in the proximal region of miR-22 promoter. These findings identify miR-22 as a critical miRNA that contributes to renal fibrosis on the basis of its pivotal role on BMP signaling cascade.


Assuntos
Proteína Morfogenética Óssea 6/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Rim/metabolismo , MicroRNAs/metabolismo , Animais , Sequência de Bases , Proteína Morfogenética Óssea 6/genética , Proteína Morfogenética Óssea 7/genética , Fibrose/metabolismo , Homeostase , Rim/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , MicroRNAs/genética , Dados de Sequência Molecular , Elementos de Resposta , Transdução de Sinais , Transcrição Gênica
2.
J Biol Chem ; 286(13): 11837-48, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21310958

RESUMO

Although several recent publications have suggested that microRNAs contribute to the pathogenesis of diabetic nephropathy, the role of miRNAs in vivo still remains poorly understood. Using an integrated in vitro and in vivo comparative miRNA expression array, we identified miR-29c as a signature miRNA in the diabetic environment. We validated our profiling array data by examining miR-29c expression in the kidney glomeruli obtained from db/db mice in vivo and in kidney microvascular endothelial cells and podocytes treated with high glucose in vitro. Functionally, we found that miR-29c induces cell apoptosis and increases extracellular matrix protein accumulation. Indeed, forced expression of miR-29c strongly induced podocyte apoptosis. Conversely, knockdown of miR-29c prevented high glucose-induced cell apoptosis. We also identified Sprouty homolog 1 (Spry1) as a direct target of miR-29c with a nearly perfect complementarity between miR-29c and the 3'-untranslated region (UTR) of mouse Spry1. Expression of miR-29c decreased the luciferase activity of Spry1 when co-transfected with the mouse Spry1 3'-UTR reporter construct. Overexpression of miR-29c decreased the levels of Spry1 protein and promoted activation of Rho kinase. Importantly, knockdown of miR-29c by a specific antisense oligonucleotide significantly reduced albuminuria and kidney mesangial matrix accumulation in the db/db mice model in vivo. These findings identify miR-29c as a novel target in diabetic nephropathy and provide new insights into the role of miR-29c in a previously unrecognized signaling cascade involving Spry1 and Rho kinase activation.


Assuntos
Nefropatias Diabéticas/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/biossíntese , Fosfoproteínas/metabolismo , Podócitos/metabolismo , Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Transformada , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/terapia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Técnicas de Silenciamento de Genes , Proteínas de Membrana/genética , Camundongos , MicroRNAs/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Fosfoproteínas/genética , Podócitos/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
3.
J Am Soc Nephrol ; 22(4): 704-17, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21415151

RESUMO

Stem cells may contribute to renal recovery following acute kidney injury, and this may occur through their secretion of cytokines, chemokines, and growth factors. Here, we developed an acellular, nanofiber-based preparation of self-assembled peptides to deliver the secretome of embryonic stem cells (ESCs). Using an integrated in vitro and in vivo approach, we found that nanofibers preconditioned with ESCs could reverse cell hyperpermeability and apoptosis in vitro and protect against lipopolysaccharide-induced acute kidney injury in vivo. The renoprotective effect of preconditioned nanofibers associated with an attenuation of Rho kinase activation. We also observed that the combined presence of follistatin, adiponectin, and secretory leukoprotease during preconditioning was essential to the renoprotective properties of the nanofibers. In summary, we developed a designer-peptide nanofiber that can serve as a delivery platform for the beneficial effects of stem cells without the problems of teratoma formation or limited cell engraftment and viability.


Assuntos
Injúria Renal Aguda/terapia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Embrionárias/transplante , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Células Cultivadas , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Nanofibras/uso terapêutico , Proteinúria/prevenção & controle , Quinases Associadas a rho/fisiologia
4.
J Biol Chem ; 285(30): 23457-65, 2010 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-20501654

RESUMO

Vascular endothelial growth factor (VEGF) is a dimeric glycoprotein that plays a crucial role in microvascular complications of diabetes, including diabetic nephropathy. However, the precise regulatory mechanisms governing VEGF expression in the diabetic milieu are still poorly understood. Here, we provide evidence that microRNA-93 (miR-93) regulates VEGF expression in experimental models of diabetes both in vitro and in vivo. Comparative microRNA expression profile arrays identified miR-93 as a signature microRNA in hyperglycemic conditions. We identified VEGF-A as a putative target of miR-93 in the kidney with a perfect complementarity between miR-93 and the 3'-untranslated region of vegfa in several species. When cotransfected with a luciferase reporter construct containing the mouse vegfa 3'-untranslated region, expression of miR-93 markedly decreased the luciferase activity. We showed that forced expression of miR-93 in cells abrogated VEGF protein secretion. Conversely, anti-miR-93 inhibitors increased VEGF release. Transfection of miR-93 also prevented the effect of high glucose on VEGF downstream targets. Using transgenic mice containing VEGF-LacZ bicistronic transcripts, we found that inhibition of glomerular miR-93 by peptide-conjugated morpholino oligomers elicited increased expression of VEGF. Our findings also indicate that high glucose decreases miR-93 expression by down-regulating the promoter of the host MCM7 gene. Taken together, our findings provide new insights into the role of miR-93 in VEGF signaling pathway and offer a potentially novel target in preventing the progression of diabetic nephropathy.


Assuntos
Hiperglicemia/genética , Hiperglicemia/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Diabetes Mellitus/fisiopatologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Glucose/farmacologia , Células HeLa , Humanos , Hiperglicemia/patologia , Hiperglicemia/fisiopatologia , Camundongos , Microvasos/citologia , Componente 7 do Complexo de Manutenção de Minicromossomo , Dados de Sequência Molecular , Morfolinas/química , Morfolinas/farmacologia , Proteínas Nucleares/genética , Podócitos/citologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/patologia , Polímeros/química , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Genesis ; 48(7): 446-51, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20641128

RESUMO

We report the generation and initial characterization of a mouse line expressing tamoxifen-inducible improved Cre (iCre) recombinase (iCre-ER(T2)) under the regulation of NPHS2 (podocin) gene promoter. The resulting transgenic mouse line was named podocin-iCreER(T2) mice. The efficiency of iCre activity was confirmed by crossing podocin-iCreER(T2) with the ROSA26 reporter mouse. By using the floxed ROSA reporter mice, we found that tamoxifen specifically induced recombination in the kidneys. In the absence of tamoxifen, recombination was undetectable in podocin-iCreER(T2);ROSA26 mice. However, following intraperitoneal injection of tamoxifen, selective recombination was observed in the podocytes of adult animals. We further examined the efficiency of recombination by assessing various tamoxifen exposure regimens in adult mice. These results suggest that podocin-iCre-ER(T2) mouse provides an excellent genetic tool to examine the function of candidate genes in podocytes in a spatially and temporally-restricted manner.


Assuntos
Integrases/fisiologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Tamoxifeno/farmacologia , Fatores Etários , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
6.
J Cell Biol ; 157(2): 303-14, 2002 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-11956231

RESUMO

The low-density lipoprotein receptor-related protein (Lrp)-5 functions as a Wnt coreceptor. Here we show that mice with a targeted disruption of Lrp5 develop a low bone mass phenotype. In vivo and in vitro analyses indicate that this phenotype becomes evident postnatally, and demonstrate that it is secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner. Lrp5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts. In addition, Lrp5-deficient mice display persistent embryonic eye vascularization due to a failure of macrophage-induced endothelial cell apoptosis. These results implicate Wnt proteins in the postnatal control of vascular regression and bone formation, two functions affected in many diseases. Moreover, these features recapitulate human osteoporosis-pseudoglioma syndrome, caused by LRP5 inactivation.


Assuntos
Doenças Ósseas Metabólicas/patologia , Anormalidades do Olho/metabolismo , Proteínas de Neoplasias , Osteoblastos/metabolismo , Osteoblastos/patologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores de LDL/deficiência , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra , Animais , Apoptose , Desenvolvimento Ósseo , Doenças Ósseas Metabólicas/metabolismo , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Cálcio/sangue , Cálcio/urina , Divisão Celular , Subunidade alfa 1 de Fator de Ligação ao Core , Olho/citologia , Olho/patologia , Anormalidades do Olho/patologia , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Proteínas Relacionadas a Receptor de LDL , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Tamanho do Órgão , Fenótipo , Proteínas Proto-Oncogênicas/genética , Receptores de LDL/genética , Receptores de LDL/imunologia , Receptores de LDL/metabolismo , Transdução de Sinais , Proteínas Wnt
7.
Sci Signal ; 6(257): ra2, 2013 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-23300339

RESUMO

Phosphorylation and activation of Akt1 is a crucial signaling event that promotes adipogenesis. However, neither the complex multistep process that leads to activation of Akt1 through phosphorylation at Thr³°8 and Ser47³ nor the mechanism by which Akt1 stimulates adipogenesis is fully understood. We found that the BSD domain-containing signal transducer and Akt interactor (BSTA) promoted phosphorylation of Akt1 at Ser47³ in various human and murine cells, and we uncovered a function for the BSD domain in BSTA-Akt1 complex formation. The mammalian target of rapamycin complex 2 (mTORC2) facilitated the phosphorylation of BSTA and its association with Akt1, and the BSTA-Akt1 interaction promoted the association of mTORC2 with Akt1 and phosphorylation of Akt1 at Ser47³ in response to growth factor stimulation. Furthermore, analyses of bsta gene-trap murine embryonic stem cells revealed an essential function for BSTA and phosphorylation of Akt1 at Ser47³ in promoting adipocyte differentiation, which required suppression of the expression of the gene encoding the transcription factor FoxC2. These findings indicate that BSTA is a molecular switch that promotes phosphorylation of Akt1 at Ser47³ and reveal an mTORC2-BSTA-Akt1-FoxC2-mediated signaling mechanism that is critical for adipocyte differentiation.


Assuntos
Adipócitos/fisiologia , Adipogenia/fisiologia , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Proteínas , Técnicas do Sistema de Duplo-Híbrido
8.
Cell Metab ; 15(2): 186-200, 2012 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-22326220

RESUMO

Several lines of evidence suggest that mitochondrial dysfunction plays a critical role in the pathogenesis of microvascular complications of diabetes, including diabetic nephropathy. However, the signaling pathways by which hyperglycemia leads to mitochondrial dysfunction are not fully understood. Here we examined the role of Rho-associated coiled coil-containing protein kinase 1 (ROCK1) on mitochondrial dynamics by generating two diabetic mouse models with targeted deletions of ROCK1 and an inducible podocyte-specific knockin mouse expressing a constitutively active (cA) mutant of ROCK1. Our findings suggest that ROCK1 mediates hyperglycemia-induced mitochondrial fission by promoting dynamin-related protein-1 (Drp1) recruitment to the mitochondria. Deletion of ROCK1 in diabetic mice prevented mitochondrial fission, whereas podocyte-specific cA-ROCK1 mice exhibited increased mitochondrial fission. Importantly, we found that ROCK1 triggers mitochondrial fission by phosphorylating Drp1 at serine 600 residue. These findings provide insights into the unexpected role of ROCK1 in a signaling cascade that regulates mitochondrial dynamics.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Células Endoteliais/metabolismo , Hiperglicemia/metabolismo , Doenças Mitocondriais/metabolismo , Modelos Animais , Podócitos/metabolismo , Quinases Associadas a rho/metabolismo , Sequência de Aminoácidos , Análise de Variância , Animais , Primers do DNA/genética , Dinaminas/genética , Dinaminas/metabolismo , Ativação Enzimática/fisiologia , Citometria de Fluxo , Deleção de Genes , Técnicas de Introdução de Genes , Membrana Basal Glomerular/patologia , Hiperglicemia/complicações , Camundongos , Camundongos Transgênicos , Doenças Mitocondriais/etiologia , Dados de Sequência Molecular , Mutagênese , Fosforilação , RNA Interferente Pequeno/genética , Alinhamento de Sequência , Quinases Associadas a rho/genética
9.
Aging Cell ; 10(6): 996-1010, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21895961

RESUMO

Aging is associated with increased adiposity in white adipose tissues and impaired thermogenesis in brown adipose tissues; both contribute to increased incidences of obesity and type 2 diabetes. Ghrelin is the only known circulating orexigenic hormone that promotes adiposity. In this study, we show that ablation of the ghrelin receptor (growth hormone secretagogue receptor, GHS-R) improves insulin sensitivity during aging. Compared to wild-type (WT) mice, old Ghsr(-/-) mice have reduced fat and preserve a healthier lipid profile. Old Ghsr(-/-) mice also exhibit elevated energy expenditure and resting metabolic rate, yet have similar food intake and locomotor activity. While GHS-R expression in white and brown adipose tissues was below the detectable level in the young mice, GHS-R expression was readily detectable in visceral white fat and interscapular brown fat of the old mice. Gene expression profiles reveal that Ghsr ablation reduced glucose/lipid uptake and lipogenesis in white adipose tissues but increased thermogenic capacity in brown adipose tissues. Ghsr ablation prevents age-associated decline in thermogenic gene expression of uncoupling protein 1 (UCP1). Cell culture studies in brown adipocytes further demonstrate that ghrelin suppresses the expression of adipogenic and thermogenic genes, while GHS-R antagonist abolishes ghrelin's effects and increases UCP1 expression. Hence, GHS-R plays an important role in thermogenic impairment during aging. Ghsr ablation improves aging-associated obesity and insulin resistance by reducing adiposity and increasing thermogenesis. Growth hormone secretagogue receptor antagonists may be a new means of combating obesity by shifting the energy balance from obesogenesis to thermogenesis.


Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Envelhecimento/genética , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica , Obesidade/metabolismo , Receptores de Grelina/deficiência , Transdução de Sinais/genética , Adiposidade/genética , Animais , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/prevenção & controle , Ingestão de Alimentos/fisiologia , Metabolismo Energético/fisiologia , Grelina/genética , Grelina/metabolismo , Humanos , Resistência à Insulina/genética , Canais Iônicos/genética , Canais Iônicos/metabolismo , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Obesidade/complicações , Obesidade/genética , Obesidade/prevenção & controle , Receptores de Grelina/antagonistas & inibidores , Receptores de Grelina/genética , Termogênese/fisiologia , Proteína Desacopladora 1
10.
J Biol Chem ; 281(45): 34341-8, 2006 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-16968708

RESUMO

Ectopic accumulation of lipid droplets in non-adipose tissues correlates with the degree of insulin resistance in these tissues. Emerging evidence indicates that lipid droplets are specialized organelles that participate in lipid metabolism and intracellular trafficking. These properties are thought to derive from the lipid droplet-associated PAT protein family (perilipin, ADFP, and Tip47). The functions of the ubiquitously distributed adipose differentiation-related protein (ADFP) and Tip47 remain unknown. To evaluate the roles of ADFP and Tip47 in lipid biogenesis and metabolism, ADFP null and wild type (wt) clonal cell lines were established from ADFP null and wt mice, respectively. In ADFP null cells, Tip47 was identified as the sole lipid droplet-associated protein from the PAT family by mass spectroscopy, which was further confirmed by immunoblotting and immunocytochemistry. Following incubation with oleic acid, ADFP null cells were able to form lipid droplets to the same extent as wt cells. No statistical differences between the two cell types were observed in NEFA uptake or lipolysis. Small interference RNAs (siRNAs) against Tip47 were found to down-regulate protein levels for Tip47 by 85%. ADFP null cells treated with Tip47 siRNA retained the ability to form lipid droplets but to a lesser extent and shunted the utilization of exogenously added NEFA from triglycerides to phospholipids. These data support the hypothesis that Tip47 plays an important role in lipid metabolism. Tip47 and ADFP in peripheral tissues may play a critical role in regulating the formation and turnover, and hence metabolic consequences, of ectopic fat.


Assuntos
Adipócitos/citologia , Tecido Adiposo/metabolismo , Diferenciação Celular , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Proteínas de Membrana/fisiologia , Proteínas da Gravidez/fisiologia , Adipócitos/metabolismo , Tecido Adiposo/citologia , Animais , Linhagem Celular , Cromatografia em Camada Fina , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Immunoblotting , Técnicas Imunoenzimáticas , Metabolismo dos Lipídeos , Lipólise , Espectrometria de Massas , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Perilipina-2 , Fosfolipídeos/metabolismo , Proteínas da Gravidez/genética , RNA Interferente Pequeno/farmacologia , Triglicerídeos/metabolismo
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