RESUMO
Interaction of transforming growth factor-ß (TGFß)-induced canonical signaling with the noncanonical kinase cascades regulates glomerular hypertrophy and matrix protein deposition, which are early features of glomerulosclerosis. However, the specific target downstream of the TGFß receptor involved in the noncanonical signaling is unknown. Here, we show that TGFß increased the catalytic loop phosphorylation of platelet-derived growth factor receptor ß (PDGFRß), a receptor tyrosine kinase expressed abundantly in glomerular mesangial cells. TGFß increased phosphorylation of the PI 3-kinase-interacting Tyr-751 residue of PDGFRß, thus activating Akt. Inhibition of PDGFRß using a pharmacological inhibitor and siRNAs blocked TGFß-stimulated phosphorylation of proline-rich Akt substrate of 40 kDa (PRAS40), an intrinsic inhibitory component of mTORC1, and prevented activation of mTORC1 in the absence of any effect on Smad 2/3 phosphorylation. Expression of constitutively active myristoylated Akt reversed the siPDGFRß-mediated inhibition of mTORC1 activity; however, co-expression of the phospho-deficient mutant of PRAS40 inhibited the effect of myristoylated Akt, suggesting a definitive role of PRAS40 phosphorylation in mTORC1 activation downstream of PDGFRß in mesangial cells. Additionally, we demonstrate that PDGFRß-initiated phosphorylation of PRAS40 is required for TGFß-induced mesangial cell hypertrophy and fibronectin and collagen I (α2) production. Increased activating phosphorylation of PDGFRß is also associated with enhanced TGFß expression and mTORC1 activation in the kidney cortex and glomeruli of diabetic mice and rats, respectively. Thus, pursuing TGFß noncanonical signaling, we identified how TGFß receptor I achieves mTORC1 activation through PDGFRß-mediated Akt/PRAS40 phosphorylation to spur mesangial cell hypertrophy and matrix protein accumulation. These findings provide support for targeting PDGFRß in TGFß-driven renal fibrosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Fibronectinas/metabolismo , Humanos , Córtex Renal/metabolismo , Células Mesangiais/citologia , Células Mesangiais/metabolismo , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Nonalcoholic fatty liver disease (NAFLD) is a spectrum of disorders ranging from hepatic steatosis [excessive accumulation of triglycerides (TG)] to nonalcoholic steatohepatitis, which can progress to cirrhosis and hepatocellular carcinoma. The molecular pathogenesis of steatosis and progression to more severe NAFLD remains unclear. Obesity and aging, two principal risk factors for NAFLD, are associated with a hyperadrenergic state. ß-Adrenergic responsiveness in liver increases in animal models of obesity and aging, and in both is linked to increased hepatic expression of ß2-adrenergic receptors (ß2-ARs). We previously showed that in aging rodents intracellular signaling from elevated hepatic levels of ß2-ARs may contribute to liver steatosis. In this study we demonstrate that injection of formoterol, a highly selective ß2-AR agonist, to mice acutely results in hepatic TG accumulation. Further, we have sought to define the intrahepatic mechanisms underlying ß2-AR mediated steatosis by investigating changes in hepatic expression and cellular localization of enzymes, transcription factors, and coactivators involved in processes of lipid accrual and disposition-and also functional aspects thereof-in livers of formoterol-treated animals. Our results suggest that ß2-AR activation by formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, increased but incomplete ß-oxidation of fatty acids with accumulation of potentially toxic long-chain acylcarnitine intermediates, and reduced TG secretion-all previously invoked as contributors to fatty liver disease. Experiments are ongoing to determine whether sustained activation of hepatic ß2-AR signaling by formoterol might be utilized to model fatty liver changes occurring in hyperadrenergic states of obesity and aging, and thereby identify novel molecular targets for the prevention or treatment of NAFLD.NEW & NOTEWORTHY Results of our study suggest that ß2-adrenergic receptor (ß2-AR) activation by agonist formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, incomplete ß-oxidation of fatty acids with accumulation of long-chain acylcarnitine intermediates, and reduced TG secretion. These findings may, for the first time, implicate a role for ß2-AR responsive dysregulation of hepatic lipid metabolism in the pathogenetic processes underlying NAFLD in hyperadrenergic states such as obesity and aging.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Fígado Gorduroso/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Receptores Adrenérgicos beta 2/fisiologia , Animais , Carnitina/análogos & derivados , Carnitina/análise , Fumarato de Formoterol/farmacologia , Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/fisiologia , Lipogênese/genética , Fígado/química , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Fosfatidato Fosfatase/análise , Triglicerídeos/biossínteseRESUMO
S6 kinase acts as a driver for renal hypertrophy and matrix accumulation, two key pathologic signatures of diabetic nephropathy. As a post-translational modification, S6 kinase undergoes acetylation at the C terminus. The role of this acetylation to regulate kidney glomerular cell hypertrophy and matrix expansion is not known. In mesangial cells, high glucose decreased the acetylation and enhanced phosphorylation of S6 kinase and its substrates rps6 and eEF2 kinase that lead to dephosphorylation of eEF2. To determine the mechanism of S6 kinase deacetylation, we found that trichostatin A, a pan-histone deacetylase (HDAC) inhibitor, blocked all high glucose-induced effects. Furthermore, high glucose increased the expression and association of HDAC1 with S6 kinase. HDAC1 decreased the acetylation of S6 kinase and mimicked the effects of high glucose, resulting in mesangial cell hypertrophy and expression of fibronectin and collagen I (α2). In contrast, siRNA against HDAC1 inhibited these effects by high glucose. A C-terminal acetylation-mimetic mutant of S6 kinase suppressed high glucose-stimulated phosphorylation of S6 kinase, rps6 and eEF2 kinase, and inhibited the dephosphorylation of eEF2. Also, the acetylation mimetic attenuated the mesangial cell hypertrophy and fibronectin and collagen I (α2) expression. Conversely, an S6 kinase acetylation-deficient mutant induced all the above effects of high glucose. Finally, in the renal glomeruli of diabetic rats, the acetylation of S6 kinase was significantly reduced concomitant with increased HDAC1 and S6 kinase activity. In aggregate, our data uncovered a previously unrecognized role of S6 kinase deacetylation in high glucose-induced mesangial cell hypertrophy and matrix protein expression.
Assuntos
Diabetes Mellitus Experimental/patologia , Fibronectinas/metabolismo , Glucose/farmacologia , Hipertrofia/patologia , Glomérulos Renais/patologia , Células Mesangiais/patologia , Proteínas Quinases S6 Ribossômicas/metabolismo , Acetilação , Animais , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Humanos , Hipertrofia/etiologia , Hipertrofia/metabolismo , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas/genética , Transdução de Sinais , Edulcorantes/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
High-glucose increases NADPH oxidase 4 (NOX4) expression, reactive oxygen species generation, and matrix protein synthesis by inhibiting AMP-activated protein kinase (AMPK) in renal cells. Because hydrogen sulfide (H2S) inhibits high glucose-induced matrix protein increase by activating AMPK in renal cells, we examined whether H2S inhibits high glucose-induced expression of NOX4 and matrix protein and whether H2S and NO pathways are integrated. High glucose increased NOX4 expression and activity at 24 h in renal proximal tubular epithelial cells, which was inhibited by sodium hydrosulfide (NaHS), a source of H2S. High glucose decreased AMPK phosphorylation and activity, which was restored by NaHS. Compound C, an AMPK inhibitor, prevented NaHS inhibition of high glucose-induced NOX4 expression. NaHS inhibition of high glucose-induced NOX4 expression was abrogated by N(ω)-nitro-l-arginine methyl ester, an inhibitor of NOS. NaHS unexpectedly augmented the expression of inducible NOS (iNOS) but not endothelial NOS. iNOS siRNA and 1400W, a selective iNOS inhibitor, abolished the ameliorative effects of NaHS on high glucose-induced NOX4 expression, reactive oxygen species generation, and, matrix laminin expression. Thus, H2S recruits iNOS to generate NO to inhibit high glucose-induced NOX4 expression, oxidative stress, and matrix protein accumulation in renal epithelial cells; the two gasotransmitters H2S and NO and their interaction may serve as therapeutic targets in diabetic kidney disease.
Assuntos
Células Epiteliais/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Sulfeto de Hidrogênio/farmacologia , Túbulos Renais Proximais/enzimologia , NADPH Oxidases/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Amidinas/farmacologia , Animais , Benzilaminas/farmacologia , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/terapia , Células Epiteliais/patologia , Proteínas da Matriz Extracelular/metabolismo , Túbulos Renais Proximais/patologia , Camundongos , NADPH Oxidase 4 , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacosRESUMO
Aberrant expression of microRNAs (miRs) contributes to diabetic renal complications, including renal hypertrophy and matrix protein accumulation. Reduced expression of phosphatase and tensin homolog (PTEN) by hyperglycemia contributes to these processes. We considered involvement of miR in the downregulation of PTEN. In the renal cortex of type 1 diabetic mice, we detected increased expression of miR-214 in association with decreased levels of PTEN and enhanced Akt phosphorylation and fibronectin expression. Mesangial and proximal tubular epithelial cells exposed to high glucose showed augmented expression of miR-214. Mutagenesis studies using 3'-UTR of PTEN in a reporter construct revealed PTEN as a direct target of miR-214, which controls its expression in both of these cells. Overexpression of miR-214 decreased the levels of PTEN and increased Akt activity similar to high glucose and lead to phosphorylation of its substrates glycogen synthase kinase-3ß, PRAS40, and tuberin. In contrast, quenching of miR-214 inhibited high-glucose-induced Akt activation and its substrate phosphorylation; these changes were reversed by small interfering RNAs against PTEN. Importantly, respective expression of miR-214 or anti-miR-214 increased or decreased the mammalian target of rapamycin complex 1 (mTORC1) activity induced by high glucose. Furthermore, mTORC1 activity was controlled by miR-214-targeted PTEN via Akt activation. In addition, neutralization of high-glucose-stimulated miR-214 expression significantly inhibited cell hypertrophy and expression of the matrix protein fibronectin. Finally, the anti-miR-214-induced inhibition of these processes was reversed by the expression of constitutively active Akt kinase and hyperactive mTORC1. These results uncover a significant role of miR-214 in the activation of mTORC1 that contributes to high-glucose-induced mesangial and proximal tubular cell hypertrophy and fibronectin expression.
Assuntos
Glicemia/metabolismo , Proliferação de Células , Diabetes Mellitus Tipo 1/enzimologia , Nefropatias Diabéticas/enzimologia , Células Epiteliais/enzimologia , Glomérulos Renais/enzimologia , Túbulos Renais Proximais/enzimologia , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Regiões 3' não Traduzidas , Animais , Células Cultivadas , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Células Epiteliais/patologia , Fibronectinas/metabolismo , Regulação Enzimológica da Expressão Gênica , Mesângio Glomerular/metabolismo , Mesângio Glomerular/patologia , Hipertrofia , Glomérulos Renais/patologia , Túbulos Renais Proximais/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , MicroRNAs/genética , Complexos Multiproteicos/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Ratos , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Transfecção , Fator de Crescimento Transformador beta/metabolismoRESUMO
Increased expression of PDGF receptor-ß (PDGFRß) has been shown in renal proximal tubules in mice with diabetes. The core molecular network used by high glucose to induce proximal tubular epithelial cell collagen I (α2) expression is poorly understood. We hypothesized that activation of PDGFRß by high glucose increases collagen I (α2) production via the Akt/mTORC1 signaling pathway in proximal tubular epithelial cells. Using biochemical and molecular biological techniques, we investigated this hypothesis. We show that high glucose increases activating phosphorylation of the PDGFRß, resulting in phosphorylation of phosphatidylinositol 3-kinase. A specific inhibitor, JNJ-10198409, and small interfering RNAs targeting PDGFRß blocked this phosphorylation without having any effect on MEK/Erk1/2 activation. We also found that PDGFRß regulates high glucose-induced Akt activation, its targets tuberin and PRAS40 phosphorylation, and finally, mTORC1 activation. Furthermore, inhibition of PDGFRß suppressed high glucose-induced expression of collagen I (α2) in proximal tubular cells. Importantly, expression of constitutively active Akt or mTORC1 reversed these processes. As a mechanism, we found that JNJ and PDGFRß knockdown inhibited high glucose-stimulated Hif1α expression. Furthermore, overexpression of Hif1α restored expression of collagen I (α2) that was inhibited by PDGFRß knockdown in high glucose-stimulated cells. Finally, we show increased phosphorylation of PDGFRß and its association with Akt/mTORC1 activation, Hif1α expression, and elevated collagen I (α2) levels in the renal cortex of mice with diabetes. Our results identify PDGFRß as a driver in activating Akt/mTORC1 nexus for high glucose-mediated expression of collagen I (α2) in proximal tubular epithelial cells, which contributes to tubulointerstitial fibrosis in diabetic nephropathy.
Assuntos
Glicemia/metabolismo , Colágeno Tipo I/metabolismo , Nefropatias Diabéticas/enzimologia , Túbulos Renais Proximais/enzimologia , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/complicações , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Fibrose , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Transgênicos , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Interferência de RNA , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
High glucose-induced Akt acts as a signaling hub for mesangial cell hypertrophy and matrix expansion, which are recognized as cardinal signatures for the development of diabetic nephropathy. How mesangial cells sustain the activated state of Akt is not clearly understood. Here we show Akt-dependent phosphorylation of the transcription factor FoxO1 by high glucose. Phosphorylation-deficient, constitutively active FoxO1 inhibited the high glucose-induced phosphorylation of Akt to suppress the phosphorylation/inactivation of PRAS40 and mTORC1 activity. In contrast, dominant negative FoxO1 increased the phosphorylation of Akt, resulting in increased mTORC1 activity similar to high glucose treatment. Notably, FoxO1 regulates high glucose-induced protein synthesis, hypertrophy, and expression of fibronectin and PAI-1. High glucose paves the way for complications of diabetic nephropathy through the production of reactive oxygen species (ROS). We considered whether the FoxO1 target antioxidant enzyme catalase contributes to sustained activation of Akt. High glucose-inactivated FoxO1 decreases the expression of catalase to increase the production of ROS. Moreover, we show that catalase blocks high glucose-stimulated Akt phosphorylation to attenuate the inactivation of FoxO1 and PRAS40, resulting in the inhibition of mTORC1 and mesangial cell hypertrophy and fibronectin and PAI-1 expression. Finally, using kidney cortices from type 1 diabetic OVE26 mice, we show that increased FoxO1 phosphorylation is associated with decreased catalase expression and increased fibronectin and PAI-1 expression. Together, our results provide the first evidence for the presence of a positive feedback loop for the sustained activation of Akt involving inactivated FoxO1 and a decrease in catalase expression, leading to increased ROS and mesangial cell hypertrophy and matrix protein expression.
Assuntos
Retroalimentação Fisiológica/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Glucose/farmacologia , Células Mesangiais/efeitos dos fármacos , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Catalase/genética , Catalase/metabolismo , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Fibronectinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Immunoblotting , Córtex Renal/metabolismo , Córtex Renal/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Células Mesangiais/metabolismo , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Platelet-derived growth factor BB and its receptor (PDGFRß) play a pivotal role in the development of renal glomerular mesangial cells. Their roles in increased mesangial cell proliferation during mesangioproliferative glomerulonephritis have long been noted, but the operating logic of signaling mechanisms regulating these changes remains poorly understood. We examined the role of a recently identified MAPK, Erk5, in this process. PDGF increased the activating phosphorylation of Erk5 and tyrosine phosphorylation of proteins in a time-dependent manner. A pharmacologic inhibitor of Erk5, XMD8-92, abrogated PDGF-induced DNA synthesis and mesangial cell proliferation. Similarly, expression of dominant negative Erk5 or siRNAs against Erk5 blocked PDGF-stimulated DNA synthesis and proliferation. Inhibition of Erk5 attenuated expression of cyclin D1 mRNA and protein, resulting in suppression of CDK4-mediated phosphorylation of the tumor suppressor protein pRb. Expression of cyclin D1 or CDK4 prevented the dominant negative Erk5- or siErk5-mediated inhibition of DNA synthesis and mesangial cell proliferation induced by PDGF. We have previously shown that phosphatidylinositol 3-kinase (PI3-kinase) contributes to PDGF-induced proliferation of mesangial cells. Inhibition of PI3-kinase blocked PDGF-induced phosphorylation of Erk5. Since PI3-kinase acts through Akt, we determined the role of Erk5 on Akt phosphorylation. XMD8-92, dominant negative Erk5, and siErk5 inhibited phosphorylation of Akt by PDGF. Interestingly, we found inhibition of PDGF-induced Erk5 phosphorylation by a pharmacological inhibitor of Akt kinase and kinase dead Akt in mesangial cells. Thus our data unfold the presence of a positive feedback microcircuit between Erk5 and Akt downstream of PI3-kinase nodal point for PDGF-induced mesangial cell proliferation.
Assuntos
Proliferação de Células , Retroalimentação Fisiológica/fisiologia , Células Mesangiais/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/biossíntese , Proteína Oncogênica v-akt/biossíntese , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Retroalimentação Fisiológica/efeitos dos fármacos , Células Mesangiais/efeitos dos fármacos , RatosRESUMO
Skeletal remodeling consists of timely formation and resorption of bone by osteoblasts and osteoclasts in a quantitative manner. Patients with chronic myeloid leukemia receiving inhibitors of c-Abl tyrosine kinase often show reduced bone remodeling due to impaired osteoblast and osteoclast function. BMP-2 plays a significant role in bone generation and resorption by contributing to the formation of mature osteoblasts and osteoclasts. The effects of c-Abl on BMP-2-induced bone remodeling and the underlying mechanisms are not well studied. Using a pharmacological inhibitor and expression of a dominant negative mutant of c-Abl, we show an essential role of this tyrosine kinase in the development of bone nodules containing mature osteoblasts and formation of multinucleated osteoclasts in response to BMP-2. Calvarial osteoblasts prepared from c-Abl null mice showed the absolute requirement of this tyrosine kinase in maturation of osteoblasts and osteoclasts. Activation of phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling by BMP-2 leads to osteoblast differentiation. Remarkably, inhibition of c-Abl significantly suppressed BMP-2-stimulated PI 3-kinase activity and its downstream Akt phosphorylation. Interestingly, c-Abl regulated BMP-2-induced osteoclastogenic CSF-1 expression. More importantly, we identified the requirements of c-Abl in BMP-2 autoregulation and the expressions of alkaline phosphatase and osterix that are necessary for osteoblast differentiation. c-Abl contributed to BMP receptor-specific Smad-dependent transcription of CSF-1, osterix, and BMP-2. Finally, c-Abl associates with BMP receptor IA and regulates phosphorylation of Smad in response to BMP-2. We propose that activation of c-Abl is an important step, which induces into two signaling pathways involving noncanonical PI 3-kinase and canonical Smads to integrate BMP-2-induced osteogenesis.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteína Smad5/metabolismo , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Animais , Proteína Morfogenética Óssea 2/genética , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Fator Estimulador de Colônias de Macrófagos/biossíntese , Fator Estimulador de Colônias de Macrófagos/genética , Camundongos , Osteoblastos/citologia , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Crânio/citologia , Crânio/metabolismo , Proteína Smad5/genética , Fator de Transcrição Sp7 , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces endothelial nitric-oxide synthase (eNOS) uncoupling with enhanced generation of reactive oxygen species (ROS) and decreased production of NO. Ang II promotes a rapid increase in 3-nitrotyrosine formation, and uric acid attenuates Ang II-induced decrease in NO bioavailability, demonstrating that peroxynitrite mediates the effects of Ang II on eNOS dysfunction. Ang II rapidly up-regulates Nox4 protein. Inhibition of Nox4 abolishes the increase in ROS and peroxynitrite generation as well as eNOS uncoupling triggered by Ang II, indicating that Nox4 is upstream of eNOS. This pathway contributes to Ang II-mediated fibronectin accumulation in MCs. Ang II also elicits an increase in mitochondrial abundance of Nox4 protein, and the oxidase contributes to ROS production in mitochondria. Overexpression of mitochondrial manganese superoxide dismutase prevents the stimulatory effects of Ang II on mitochondrial ROS production, loss of NO availability, and MC fibronectin accumulation, whereas manganese superoxide dismutase depletion increases mitochondrial ROS, NO deficiency, and fibronectin synthesis basally and in cells exposed to Ang II. This work provides the first evidence that uncoupled eNOS is responsible for Ang II-induced MC fibronectin accumulation and identifies Nox4 and mitochondrial ROS as mediators of eNOS dysfunction. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal fibrosis.
Assuntos
Angiotensina II/farmacologia , Fibronectinas/metabolismo , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ácido Peroxinitroso/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Disponibilidade Biológica , Fibrose , Inativação Gênica/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/enzimologia , Células Mesangiais/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Modelos Biológicos , NADPH Oxidase 4 , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Ratos , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The plasticity of proximal tubular epithelial cells in response to TGFß contributes to the expression of TWIST1 to drive renal fibrosis. The mechanism of TWIST1 expression is not known. We show that both PI3 kinase and its target mTORC2 increase TGFß-induced TWIST1 expression. TGFß enhances phosphorylation on Ser-660 in the protein kinase C ßII (PKCßII) hydrophobic motif site. Remarkably, phosphorylation-deficient PKCßIIS660A, kinase-dead PKCßII, and PKCßII knockdown blocked TWIST1 expression by TGFß. Inhibition of TWIST1 arrested TGFß-induced tubular cell hypertrophy and the expression of fibronectin, collagen I (α2), and α-smooth muscle actin. By contrast, TWIST1 overexpression induced these pathologies. Interestingly, the inhibition of PKCßII reduced these phenomena, which were countered by the expression of TWIST1. These results provide the first evidence for the involvement of the mTORC2-PKCßII axis in TWIST1 expression to promote tubular cell pathology.
Assuntos
Serina-Treonina Quinases TOR , Fator de Crescimento Transformador beta , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transdução de Sinais , Proteína Quinase C beta , Células Epiteliais/metabolismoRESUMO
BMP-2 (bone morphogenetic protein-2) promotes differentiation of osteoblast precursor cells to mature osteoblasts that form healthy bone. In the present study, we demonstrate a novel mechanism of BMP-2-induced osteoblast differentiation. The antioxidant NAC (N-acetyl-L-cysteine) and the flavoprotein enzyme NAD(P)H oxidase inhibitor DPI (diphenyleneiodonium) prevented BMP-2-stimulated alkaline phosphatase expression and mineralized bone nodule formation in mouse 2T3 pre-osteoblasts. BMP-2 elicited a rapid generation of ROS (reactive oxygen species) concomitant with increased activation of NAD(P)H oxidase. NAC and DPI inhibited BMP-2-induced ROS production and NAD(P)H oxidase activity respectively. NAD(P)H oxidases display structurally similar catalytic subunits (Nox1-5) with differential expression in various cells. We demonstrate that 2T3 pre-osteoblasts predominantly express the Nox4 isotype of NAD(P)H oxidase. To extend this finding, we tested the functional effects of Nox4. Adenovirus-mediated expression of dominant-negative Nox4 inhibited BMP-2-induced alkaline phosphatase expression. BMP-2 promotes expression of BMP-2 for maintenance of the osteoblast phenotype. NAC and DPI significantly blocked BMP-2-stimulated expression of BMP2 mRNA and protein due to a decrease in BMP2 gene transcription. Dominant-negative Nox4 also mimicked this effect of NAC and DPI. Our results provide the first evidence for a new signalling pathway linking BMP-2-stimulated Nox4-derived physiological ROS to BMP-2 expression and osteoblast differentiation.
Assuntos
Proteína Morfogenética Óssea 2/genética , Diferenciação Celular , NADPH Oxidases/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Camundongos , NADPH Oxidase 4 , RNA Mensageiro/genéticaRESUMO
Diabetes and high glucose (HG) increase the generation of NADPH oxidase-derived reactive oxygen species and induce apoptosis of glomerular epithelial cells (podocytes). Loss of podocytes contributes to albuminuria, a major risk factor for progression of kidney disease. Here, we show that HG inactivates AMP-activated protein kinase (AMPK), up-regulates Nox4, enhances NADPH oxidase activity, and induces podocyte apoptosis. Activation of AMPK blocked HG-induced expression of Nox4, NADPH oxidase activity, and apoptosis. We also identified the tumor suppressor protein p53 as a mediator of podocyte apoptosis in cells exposed to HG. Inactivation of AMPK by HG up-regulated the expression and phosphorylation of p53, and p53 acted downstream of Nox4. To investigate the mechanism of podocyte apoptosis in vivo, we used OVE26 mice, a model of type 1 diabetes. Glomeruli isolated from these mice showed decreased phosphorylation of AMPK and enhanced expression of Nox4 and p53. Pharmacologic activation of AMPK by 5-aminoimidazole-4-carboxamide-1-riboside in OVE26 mice attenuated Nox4 and p53 expression. Administration of 5-aminoimidazole-4-carboxamide-1-riboside also prevented renal hypertrophy, glomerular basement thickening, foot process effacement, and podocyte loss, resulting in marked reduction in albuminuria. Our results uncover a novel function of AMPK that integrates metabolic input to Nox4 and provide new insight for activation of p53 to induce podocyte apoptosis. The data indicate the potential therapeutic utility of AMPK activators to block Nox4 and reactive oxygen species generation and to reduce urinary albumin excretion in type 1 diabetes.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose , Diabetes Mellitus/genética , Regulação para Baixo , Podócitos/citologia , Proteína Supressora de Tumor p53/genética , Proteínas Quinases Ativadas por AMP/genética , Animais , Diabetes Mellitus/enzimologia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Glucose/genética , Humanos , Masculino , Camundongos , NADPH Oxidase 4 , NADPH Oxidases , Fosforilação , Podócitos/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The role of insulin receptor (IR) activated by hyperinsulinemia in obesity-induced kidney injury is not well understood. We hypothesized that activation of kidney proximal tubule epithelial IR contributes to obesity-induced kidney injury. We administered normal-fat diet (NFD) or high-fat diet (HFD) to control and kidney proximal tubule IR-knockout (KPTIRKO) mice for 4 months. Renal cortical IR expression was decreased by 60% in male and female KPTIRKO mice. Baseline serum glucose, serum creatinine, and the ratio of urinary albumin to creatinine (ACR) were similar in KPTIRKO mice compared to those of controls. On HFD, weight gain and increase in serum cholesterol were similar in control and KPTIRKO mice; blood glucose did not change. HFD increased the following parameters in the male control mice: renal cortical contents of phosphorylated IR and Akt, matrix proteins, urinary ACR, urinary kidney injury molecule-1-to-creatinine ratio, and systolic blood pressure. Renal cortical generation of hydrogen sulfide was reduced in HFD-fed male control mice. All of these parameters were ameliorated in male KPTIRKO mice. Interestingly, female mice were resistant to HFD-induced kidney injury in both genotypes. We conclude that HFD-induced kidney injury requires renal proximal tubule IR activation in male mice.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Túbulos Renais Proximais/metabolismo , Receptor de Insulina/metabolismo , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/metabolismo , Animais , Epitélio/metabolismo , Feminino , Sulfeto de Hidrogênio/metabolismo , Resistência à Insulina , Córtex Renal/metabolismo , Masculino , Camundongos , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Receptor de Insulina/deficiência , Receptor de Insulina/genética , Fatores Sexuais , Transdução de SinaisRESUMO
Substantial evidence suggests roles of TSC2 and PTEN in the development of cancer predisposition syndromes. Loss of TSC2 results in benign tumors, neurological disorders, and angiomyolipomas. We found that PTEN mRNA and protein levels are elevated in Tsc2(-/-) mouse embryo fibroblasts with concomitant reduction in Akt phosphorylation. Reconstitution of TSC2 in Tsc2(-/-) mouse embryo fibroblasts decreases PTEN levels. Interestingly, increased HIF1alpha activity present in Tsc2 null cells is required for PTEN transcription and protein expression. We identified a canonical hypoxia-responsive element in the PTEN promoter, which regulates the transcription of this tumor suppressor protein in a TSC2-dependent manner. Finally, we demonstrate a positive correlation between expression of HIF1alpha and PTEN in renal angiomyolipomas from TSC patients. Our results reveal a unique function of HIF1alpha in up-regulation of PTEN and provide a new mechanism of reduced Akt phosphorylation in Tsc2 null cells. These data suggest that PTEN may safeguard against developing malignant tumors in patients with TSC deficiency.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Angiomiolipoma/genética , Angiomiolipoma/metabolismo , Angiomiolipoma/patologia , Animais , Ativação Enzimática , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Esclerose Tuberosa/genética , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologia , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
Diabetic nephropathy manifests aberrant activation of TORC1, which senses key signals to modulate protein synthesis and renal hypertrophy. PRAS40 has recently been identified as a raptor-interacting protein and is a component and a constitutive inhibitor of TORC1. The mechanism by which high glucose stimulates TORC1 activity is not known. PRAS40 was identified in the mesangial cells in renal glomeruli and in tubulointerstitium of rat kidney. Streptozotocin-induced diabetic renal hypertrophy was associated with phosphorylation of PRAS40 in the cortex and glomeruli. In vitro, high glucose concentration increased PRAS40 phosphorylation in a PI 3 kinase- and Akt-dependent manner, resulting in dissociation of raptor-PRAS40 complex in mesangial cells. High glucose augmented the inactivating and activating phosphorylation of 4EBP-1 and S6 kinase, respectively, with concomitant induction of protein synthesis and hypertrophy. Expression of TORC1-nonphosphorylatable mutant of 4EBP-1 and dominant-negative S6 kinase significantly inhibited high glucose-induced protein synthesis and hypertrophy. PRAS40 knockdown mimicked the effect of high glucose on phosphorylation of 4EBP-1 and S6 kinase, protein synthesis, and hypertrophy. To elucidate the role of PRAS40 phosphorylation, we used phosphorylation-deficient mutant of PRAS40, which in contrast to PRAS40 knockdown inhibited phosphorylation of 4EBP-1 and S6 kinase, leading to reduced mesangial cell hypertrophy. Thus, our data identify high glucose-induced phosphorylation and inactivation of PRAS40 as a central node for mesangial cell hypertrophy in diabetic nephropathy.
Assuntos
Glucose/metabolismo , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Ativação Enzimática , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Células Mesangiais/citologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Fatores de Transcrição/genéticaRESUMO
Translation, a process of generating a peptide from the codons present in messenger RNA, can be a site of independent regulation of protein synthesis; it has not been well studied in the kidney. Translation occurs in three stages (initiation, elongation, and termination), each with its own set of regulatory factors. Mechanisms controlling translation include small inhibitory RNAs such as microRNAs, binding proteins, and signaling reactions. Role of translation in renal injury in diabetes, endoplasmic reticulum stress, acute kidney injury, and, in physiological adaptation to loss of nephrons is reviewed here. Contribution of mRNA translation to physiology and disease is not well understood. Because it is involved in such diverse areas as development and cancer, it should prove a fertile field for investigation in renal science.
Assuntos
Nefropatias/fisiopatologia , Rim/fisiologia , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/biossíntese , Animais , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/fisiopatologia , Regulação da Expressão Gênica/fisiologia , Humanos , Rim/metabolismo , Nefropatias/metabolismo , Transdução de SinaisRESUMO
The mechanism of PTEN repression by high glucose in diabetic nephropathy is not known. Using proximal tubular cells, we show that inhibition of PI3 kinase/Akt and their inactive enzymes prevents high glucose-induced PTEN downregulation. Similarly, rapamycin (Rapa) and shRaptor block suppression of PTEN by high glucose. In contrast, the constitutive activation of Akt and mechanistic target of rapamycin (mTOR)C1 decrease the expression of PTEN, similarly to high glucose. Remarkably, PI3 kinase/Akt/mTORC1 inhibition significantly attenuates high glucose-stimulated increase in miR-214, which targets PTEN, while constitutively active Akt/mTORC1 increases miR-214. Furthermore, anti-miR-214 and mTORC1 inhibition block high glucose-induced hypertrophy and fibronectin expression. These results reveal the first evidence for the presence of a high glucose-forced positive feedback conduit between the three-layered kinase cascade and miR-214/ PTEN in tubular cell injury.
Assuntos
Glucose/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Retroalimentação Fisiológica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Here, we demonstrate a mechanism of TGFbeta-mediated inhibition of PDGF-induced DNA synthesis in mesangial cells. TGFbeta significantly inhibited nuclear Akt phosphorylation without any effect on PDGF-stimulated phosphorylation of PDGFR at PI 3 kinase binding site (Tyr-751). Remarkably, TGFbeta inhibited cyclin D1 and cyclin E expression with concomitant decrease in CDK2 activity induced by PDGF. More importantly, we demonstrate that TGFbeta significantly abolished Akt-mediated serine-9 phosphorylation of glycogen synthase kinase 3beta (GSK3beta), thus prevented its inactivation. Expression of inactive GSK3betaK85R mutant increased cyclin D1 expression and DNA synthesis similar to PDGF. These results provide the first evidence that TGFbeta intercepts Akt kinase activity in the nucleus to block inactivation of GSK3beta, leading to attenuation of PDGF-induced CDK2 activity and DNA synthesis.
Assuntos
DNA/biossíntese , Quinase 3 da Glicogênio Sintase/metabolismo , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Núcleo Celular/metabolismo , Ciclina D1/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
We examined the effect of rapamycin on the life span of a mouse model of type 2 diabetes, db/db mice. At 4 months of age, male and female C57BLKSJ-lepr (db/db) mice (db/db) were placed on either a control diet, lacking rapamycin or a diet containing rapamycin and maintained on these diets over their life span. Rapamycin was found to reduce the life span of the db/db mice. The median survival of male db/db mice fed the control and rapamycin diets was 349 and 302 days, respectively, and the median survival of female db/db mice fed the control and rapamycin diets was 487 and 411 days, respectively. Adjusting for gender differences, rapamycin increased the mortality risk 1.7-fold in both male and female db/db mice. End-of-life pathological data showed that suppurative inflammation was the main cause of death in the db/db mice, which is enhanced slightly by rapamycin treatment.