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1.
J Mol Med (Berl) ; 95(3): 257-271, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28054119

RESUMO

Renal mesangial cells are regarded as main players in glomerular inflammatory diseases. To investigate a possible crosstalk between inflammatory and hypoxia-driven signaling processes, we stimulated cultured mouse mesangial cells with different inflammatory agents and analyzed the expression of prolyl hydroxylase domain containing proteins (PHDs), the main regulators of hypoxia-inducible factor (HIF) stability. Administration of IL-1ß (1 nM) and TNF-α (1 nM), a combination further referred to as cytokine mix (CM), resulted in a fivefold increase in PHD3 but not PHD1 and PHD2 mRNA expression compared to untreated controls. In contrast, a combination of IL-1ß, TNF-α with lipopolysaccharide (10 µg/ml), and interferon-γ (20 ng/ml) designated as CM+ showed a high (60-fold) induction of PHD3 and a moderate (twofold) induction of PHD2 mRNA expression. Interestingly, CM+ but not CM induced the expression of inducible NO synthase and endogenously produced NO was responsible for the immense induction of PHD3 in mesangial cells treated with CM+. We found that CM+ affected PHD3 expression mainly via the NO/HIF axis, whereas PHD3 regulation by CM occurred in a NF-κB-dependent manner. In turn, silencing of PHD3 expression resulted in a decrease in the mRNA expression of ICAM-1, MIP-2, MCP-1, and CXCL-10, which are under control of NF-κB. In a rat model of mesangio-proliferative glomerulonephritis, PHD3 mRNA and protein expression was markedly induced and this effect was nearly abolished when rats were treated with the iNOS-specific inhibitor L-NIL, thus confirming our findings also in vivo. KEY MESSAGE: PHD3 expression induced by cytokines is NF-κB dependent in mesangial cells. Endogenously produced NO further augments PHD3 expression via HIF-1α. PHD3 expression is induced by NO in anti-Thy-1 glomerulonephritis.


Assuntos
Glomerulonefrite/genética , Óxido Nítrico/imunologia , Pró-Colágeno-Prolina Dioxigenase/genética , Regulação para Cima , Animais , Células Cultivadas , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Humanos , Interleucina-1beta/imunologia , Células Mesangiais/imunologia , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/imunologia , Pró-Colágeno-Prolina Dioxigenase/imunologia , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/imunologia
2.
Brain Res ; 1624: 380-389, 2015 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-26271715

RESUMO

Accumulating lines of evidence indicate that hydrogen sulfide (H2S) contributes to the processing of chronic pain. However, the sources of H2S production in the nociceptive system are poorly understood. Here we investigated the expression of the H2S releasing enzyme cystathionine γ-lyase (CSE) in the nociceptive system and characterized its role in chronic pain signaling using CSE deficient mice. We show that paw inflammation and peripheral nerve injury led to upregulation of CSE expression in dorsal root ganglia. However, conditional knockout mice lacking CSE in sensory neurons as well as global CSE knockout mice demonstrated normal pain behaviors in inflammatory and neuropathic pain models as compared to WT littermates. Thus, our results suggest that CSE is not critically involved in chronic pain signaling in mice and that sources different from CSE mediate the pain relevant effects of H2S.


Assuntos
Cistationina gama-Liase/metabolismo , Gânglios Espinais/metabolismo , Sulfeto de Hidrogênio/metabolismo , Inflamação/metabolismo , Neuralgia/metabolismo , Animais , Cistationina gama-Liase/genética , Modelos Animais de Doenças , Formaldeído/toxicidade , Regulação da Expressão Gênica/genética , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Inflamação/induzido quimicamente , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/fisiologia , Neuralgia/patologia , Medição da Dor , Medula Espinal/metabolismo , Regulação para Cima , Zimosan/farmacologia
3.
Biochem Pharmacol ; 85(1): 101-8, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23103565

RESUMO

Inflammatory glomerular kidney diseases are often accompanied with a massive production of reactive oxygen species (ROS) that affect the function of the glomerular filtration barrier and contribute to mesangiolysis via the induction of cell death in mesangial cells. Intriguingly, ROS also trigger fine-tuned signalling processes that affect gene expression and cell proliferation or migration. To define such redox-driven signalling devices, a proteomics approach was performed to identify the formation of protein complexes induced by ROS. To this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250 µM). Thereafter cell lysates were subjected to diagonal 2D gel electrophoresis and putative redox-affected proteins were analysed by MS/MS analysis. Among others, the regulatory subunit of protein kinase A (PKA) could be identified that forms homodimers under oxidative conditions. To evaluate whether ROS dependent dimerization of PKA also occurs in a more physiological setting, rat mesangial cells were treated with platelet-derived growth factor-BB (PDGF-BB) to induce ROS formation. This regimen resulted in a redox dependent dimerization of the R-subunits of PKA. To demonstrate whether PDGF-BB induced ROS formation affects PKA dependent pathways, the effects of PDGF-BB on phosphorylation of serine 157 of vasodilator stimulated protein (VASP) a classical target of PKA were analysed. Interestingly PDGF-BB induced VASP phosphorylation in a ROS dependent manner but independent of changes in cAMP levels. Taken together, we demonstrate a redox-mediated activation of PKA by PDGF-BB thus highlighting a physiological role of ROS as regulator of PKA activity in rat mesangial cells.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Mesangiais/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Animais , Becaplermina , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Células Mesangiais/efeitos dos fármacos , Proteínas dos Microfilamentos/metabolismo , Oxirredução , Fosfoproteínas/metabolismo , Fosforilação , Podócitos/metabolismo , Multimerização Proteica , Subunidades Proteicas/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-sis/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Serina/metabolismo , Transdução de Sinais
4.
Br J Pharmacol ; 166(8): 2231-42, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22428706

RESUMO

BACKGROUND AND PURPOSE: So far, there is only limited information about the regulation of the endogenous synthesis of hydrogen sulfide (H(2) S), an important gaseous signalling molecule. This study was done to evaluate the redox-dependent signalling events that regulate the expression of the H(2) S synthesising enzyme cystathionine-γ-lyase (CSE) in rat mesangial cells. EXPERIMENTAL APPROACH: The effects of platelet-derived growth factor (PDGF)-BB and antioxidants on CSE expression and activity in cultured rat renal mesangial cells were assessed. Activity of nuclear factor erythroid-2-related factor-2 (Nrf2) was measured as the binding capacity to a radiolabelled consensus element by electrophoretic mobility shift assay (EMSA). Furthermore, CSE and Nrf2 expression was analysed in a rat model of anti-Thy-1-induced glomerulonephritis by immunohistochemistry. KEY RESULTS: Treatment of mesangial cells with PDGF-BB resulted in a marked time- and dose-dependent up-regulation of CSE mRNA and protein levels, as well as CSE activity accompanied with increased formation of reactive oxygen species. Remarkably, co-administration of antioxidants, such as N-acetylcysteine, ebselen or diphenylene iodonium chloride, drastically reduced PDGF-BB-induced CSE expression. PDGF-BB induced binding of Nrf2 to a corresponding consensus antioxidant element in a redox-dependent manner. Furthermore, PDGF-BB-induced CSE expression in mouse mesangial cells was completely abolished in Nrf2 knockout mice compared with wild-type mice. In a rat model of anti-Thy-1-induced proliferative glomerulonephritis, we observed a marked up-regulation of CSE protein paralleled by a stabilization of Nrf2 protein. CONCLUSIONS AND IMPLICATIONS: PDGF-BB regulated CSE via a redox-mediated activation of Nrf2. Such action would aid the resolution of glomerular inflammatory diseases. LINKED ARTICLE: This article is commented on by Gallyas, pp. 2228-2230 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01976.x.


Assuntos
Cistationina gama-Liase/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células Mesangiais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-sis/farmacologia , Animais , Antioxidantes/farmacologia , Becaplermina , Células Cultivadas , Cistationina gama-Liase/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/metabolismo , Isoanticorpos/farmacologia , Macrófagos , Células Mesangiais/enzimologia , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Baço/citologia
5.
Mol Biol Cell ; 20(18): 4010-20, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19625447

RESUMO

Prostate apoptosis response-4 (Par-4) was initially identified as a gene product up-regulated in prostate cancer cells undergoing apoptosis. In rat fibroblasts, coexpression of Par-4 and its interaction partner DAP-like kinase (Dlk, which is also known as zipper-interacting protein kinase [ZIPK]) induces relocation of the kinase from the nucleus to the actin filament system, followed by extensive myosin light chain (MLC) phosphorylation and induction of apoptosis. Our analyses show that the synergistic proapoptotic effect of Dlk/Par-4 complexes is abrogated when either Dlk/Par-4 interaction or Dlk kinase activity is impaired. In vitro phosphorylation assays employing Dlk and Par-4 phosphorylation mutants carrying alanine substitutions for residues S154, T155, S220, or S249, respectively, identified T155 as the major Par-4 phosphorylation site of Dlk. Coexpression experiments in REF52.2 cells revealed that phosphorylation of Par-4 at T155 by Dlk was essential for apoptosis induction in vivo. In the presence of the Par-4 T155A mutant Dlk was partially recruited to actin filaments but resided mainly in the nucleus. Consequently, apoptosis was not induced in Dlk/Par-4 T155A-expressing cells. In vivo phosphorylation of Par-4 at T155 was demonstrated with a phospho-specific Par-4 antibody. Our results demonstrate that Dlk-mediated phosphorylation of Par-4 at T155 is a crucial event in Dlk/Par-4-induced apoptosis.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Sequência de Aminoácidos , Aminoácidos Acídicos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/química , Biocatálise/efeitos dos fármacos , Proteínas Quinases Associadas com Morte Celular , Lisofosfolipídeos/farmacologia , Mimetismo Molecular/efeitos dos fármacos , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ratos , Serina/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Treonina/metabolismo , Fatores de Tempo
6.
J Am Soc Nephrol ; 20(9): 1963-74, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19578009

RESUMO

Cytokines and nitric oxide (NO) stimulate rat mesangial cells to synthesize and secrete inflammatory mediators. To understand better the signaling pathways that contribute to this response, we exposed rat mesangial cells to the prototypic inflammatory cytokine IL-1beta and analyzed the changes in the pattern of gene expression. IL-1beta downregulated the gene encoding the matricellular glycoprotein secreted modular calcium-binding protein 1 (SMOC-1) in mesangial cells. Inflammatory cytokines attenuated SMOC-1 mRNA and protein expression through endogenous production of NO, which activated the soluble guanylyl cyclase. Silencing SMOC-1 expression with small interfering RNA decreased the formation of TGF-beta, reduced SMAD binding to DNA, and decreased mRNA expression of genes regulated by TGF-beta. In a rat model of anti-Thy-1 glomerulonephritis, glomerular SMOC-1 mRNA and protein decreased and inducible NO synthase expression increased simultaneously. Treatment of nephritic rats with the inducible NO synthase-specific inhibitor l-N(6)-(1-iminoethyl)-lysine prevented SMOC-1 downregulation. In summary, these data suggest that NO attenuates SMOC-1 expression in acute glomerular inflammation, thereby limiting TGF-beta-mediated profibrotic signaling.


Assuntos
Glomerulonefrite/metabolismo , Células Mesangiais/metabolismo , Óxido Nítrico/metabolismo , Osteonectina/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Células Cultivadas , Regulação para Baixo/fisiologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Glomerulonefrite/imunologia , Glomerulonefrite/fisiopatologia , Guanilato Ciclase/metabolismo , Interleucina-1beta/farmacologia , Isoanticorpos/imunologia , Células Mesangiais/citologia , Células Mesangiais/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Osteonectina/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/fisiologia , Guanilil Ciclase Solúvel
7.
Exp Cell Res ; 311(2): 177-91, 2005 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-16229834

RESUMO

Par-4 (prostate apoptosis response-4) sensitizes cells to apoptotic stimuli, but the exact mechanisms are still poorly understood. Using Par-4 as bait in a yeast two-hybrid screen, we identified Amida as a novel interaction partner, a ubiquitously expressed protein which has been suggested to be involved in apoptotic processes. Complex formation of Par-4 and Amida occurs in vitro and in vivo and is mediated via the C-termini of both proteins, involving the leucine zipper of Par-4. Amida resides mainly in the nucleus but displays nucleo-cytoplasmic shuttling in heterokaryons. Upon coexpression with Par-4 in REF52.2 cells, Amida translocates to the cytoplasm and is recruited to actin filaments by Par-4, resulting in enhanced induction of apoptosis. The synergistic effect of Amida/Par-4 complexes on the induction of apoptosis is abrogated when either Amida/Par-4 complex formation or association of these complexes with the actin cytoskeleton is impaired, indicating that the Par-4-mediated relocation of Amida to the actin cytoskeleton is crucial for the pro-apoptotic function of Par-4/Amida complexes in REF52.2 cells. The latter results in enhanced phosphorylation of the regulatory light chain of myosin II (MLC) as has previously been shown for Par-4-mediated recruitment of DAP-like kinase (Dlk), suggesting that the recruitment of nuclear proteins involved in the regulation of apoptotic processes to the actin filament system by Par-4 represents a potent mechanism how Par-4 can trigger apoptosis.


Assuntos
Actinas/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Proteínas Reguladoras de Apoptose/análise , Núcleo Celular/química , Células Cultivadas , Citoplasma/química , Humanos , Camundongos , Cadeias Leves de Miosina/metabolismo , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Fosforilação , Ratos , Técnicas do Sistema de Duplo-Híbrido
8.
Exp Cell Res ; 305(2): 392-408, 2005 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15817164

RESUMO

Prostate apoptosis response-4 (Par-4) is a 38-kDa protein originally identified as a gene product upregulated in prostate cancer cells undergoing apoptosis. Cell death mediated by Par-4 and its interaction partner DAP like kinase (Dlk) is characterized by dramatic changes of the cytoskeleton. To uncover the role of the cytoskeleton in Par-4/Dlk-mediated apoptosis, we analyzed Par-4 for a direct association with cytoskeletal structures. Confocal fluorescence microscopy revealed that endogenous Par-4 is specifically associated with stress fibers in rat fibroblasts. In vitro cosedimentation analyses and in vivo FRET analyses showed that Par-4 directly binds to F-actin. Actin binding is mediated by the N-terminal 266 amino acids, but does not require the C-terminal region of Par-4 containing the leucine zipper and the death domain. Furthermore, the interaction of Par-4 with actin filaments leads to the formation of actin bundles in vitro and in vivo. In rat fibroblasts, this microfilament association is essential for the pro-apoptotic function of Par-4, since both disruption of the actin cytoskeleton by cytochalasin D treatment and overexpression of Par-4 constructs impaired in actin binding result in a significant decrease of apoptosis induction by Par-4 and Dlk. We propose a model, in which Par-4 recruits Dlk to stress fibers, leading to enhanced phosphorylation of the regulatory light chain of myosin II (MLC) and to the induction of apoptosis.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Apoptose/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Citoesqueleto de Actina/química , Actinas/análise , Animais , Proteínas Reguladoras de Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Miosinas Cardíacas/metabolismo , Linhagem Celular Tumoral , Proteínas Quinases Associadas com Morte Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/genética , MAP Quinase Quinase Quinases , Masculino , Camundongos , Mutação/genética , Cadeias Leves de Miosina/metabolismo , Fosforilação , Ratos , Regulação para Cima
9.
Int J Oncol ; 26(1): 159-67, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15586236

RESUMO

Prostate apoptosis response-4 (Par-4) is a pro-apoptotic protein originally identified as a gene product upregulated in prostate tumor cells undergoing apoptosis. Down-regulation of Par-4 has been linked to several cancers. Since Par-4 also plays a crucial role in neuronal apoptosis, we investigated the expression of Par-4 in tumor cell lines derived from representative tumor types of the CNS, including primitive neuroectodermal tumor (PNET), medulloblastoma, neuroblastoma and glioma of human, rat and murine origin. We show that Par-4 is frequently down-regulated, either transcriptionally or post-transcriptionally in the CNS tumor cell lines. Moreover, we demonstrate that ectopic expression of Par-4 is sufficient to directly induce apoptosis in these CNS tumor cells, in contrast to other cancer cells where replenishment of Par-4 levels only sensitizes the cells to apoptotic stimuli. Induction of apoptosis by Par-4 in the neural tumor cell lines is independent of endogenous Bcl-2 levels and PKCzeta activity, although it has been proposed that Par-4 can exert its pro-apoptotic function by down-modulation of Bcl2 expression and inhibition of PKCzeta. Co-expression of Par-4 and a dominant-negative mutant of FADD resulted in a slight reduction of apoptosis in some tumor cell lines, indicating that Par-4 may partially induce apoptosis via the Fas death pathway. Furthermore, these data suggested that the pro-apoptotic function of Par-4 involves (an)other yet unidentified apoptotic pathway(s) in the CNS tumor cell lines. Since Par-4 by itself is not sufficient to induce apoptosis in non-tumor cells, reintroduction of Par-4 into primary CNS tumors or reactivation of the pathways of Par-4-mediated apoptosis represent promising targets in anti-tumor therapy.


Assuntos
Apoptose , Neoplasias do Sistema Nervoso Central/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Neoplasias do Sistema Nervoso Central/genética , Regulação para Baixo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Ratos , Transfecção
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