RESUMO
Apurinic/apyrimidinic endonuclease/redox factor-1 (Ref-1), a multifunctional protein secreted from stimulated cells, has been identified as a new serological biomarker. Despite recent reports on the role of Ref-1 in inflammation, the biological function of secreted Ref-1 remains unknown, especially in vivo. This study aimed to evaluate the possible roles of secreted Ref-1 in lipopolysaccharide-induced systemic inflammation in vivo. We generated a secretory Ref-1 adenoviral vector system, AdPPT-LS-Ref-1, by conjugation of preprotrypsin leading sequence (PPT-LS) with full-length Ref-1 sequences. Expression of tumor necrosis factor-α (TNF-α)-induced vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells and lipopolysaccharide (LPS)-induced cyclooxygenase-2 in Raw264.7â¯cells was inhibited by secretory Ref-1, and this inhibitory effect was abrogated following neutralization of Ref-1 with anti-Ref-1 antibody. Plasma Ref-1 levels following administration of AdPPT-LS-Ref-1 (2â¯×â¯109 ifu, i.p.) for 24â¯h were substantially higher than those recorded following administration of Adßgal (84.6⯱â¯7.2â¯ng/ml vs. 4.4⯱â¯1.5â¯ng/ml). Treatment with LPS (10â¯mg/kg, i.v. for 6â¯h) markedly increased VCAM-1 expression, cathepsin or myeloperoxidase activity, which were significantly suppressed by treatment with AdPPT-LS-Ref-1. Furthermore, LPS-induced cytokines, such as TNF-α, interleukin (IL)-1ß, IL-6, and monocyte chemoattractant protein 1, were significantly inhibited in AdPPT-LS-Ref-1-treated mice. However, LPS-induced myeloperoxidase activities were not suppressed by treatment with the redox mutant of secretory Ref-1, AdPPT-LS-Ref-1(C65A/C93A), or wild-type AdRef-1. Collectively, these results suggest that secreted Ref-1 has anti-inflammatory properties and that its redox cysteine residue is associated with the anti-inflammatory activity in vivo. Furthermore, our findings indicate that secretory Ref-1 may be useful as a therapeutic biomolecule against systemic inflammation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , Sepse/terapia , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Catepsinas/genética , Catepsinas/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos ICR , Peroxidase/genética , Peroxidase/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Células RAW 264.7 , Sepse/induzido quimicamente , Sepse/genética , Sepse/patologia , Tripsina/genética , Tripsina/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
OBJECTIVE: To investigate the function of REF1 in the proliferation and collagen synthesis of neonatal rat cardiac fibroblasts, and the underlying mechanisms. METHODS: Neonatal rat cardiac fibroblasts were transfected with the adenoviral vector containing rat wild type Ref1 (Ad-Ref1) or mutated Ref1 (Ad-mutRef1). The mutations resulted in Cys to Ala at amino acids 65 and 93, which eliminated the redox function of the REF1 protein. MTT was used to check the cell viability and flow cytometry was used to analyze the cell proliferation with the count of cell numbers and the percentage of cells in S phase of the cell cycle. The expressions of Ref1, collagen I (Col I) and collagen III (Col III) were determined by RT-PCR and Western blot. The translocation of REF1 was examined by fluorescence staining and revealed under fluorescence microscope. Electrophoretic mobility shift assay (EMSA) was used to check the effect of REF1 on AP1 DNA binding ability. The high glucose medium (25 mmol/L) was applied to culture cardiac fibroblasts. The effect of high glucose on AP1 DNA binding activity, the expression and translocation of REF1 were examined. RESULTS: MTT analysis showed that Ad-Ref1 promoted the relative viability of cardiac fibroblasts (0.671+/-0.044 vs control 0.364+/-0.007, n=6, P<0.01). The percentage of cells in S phase of the cell cycle was increased significantly in the Ad-Ref1 transfected cells (16.8%+/-0.62% vs control 9.04%+/-0.43%, n=3, P<0.05), as demonstrated by flow cytometry analysis. The expressions of Col I and Col III at mRNA level were increased when cells transfected with Ad-Ref1, while Ad-mutRef1 did not show such effects. Compared with the redox-deficient mutant Ad-mutRef1 (C65/93A), EMSA results demonstrated that Ad-Ref1 resulted in a marked increase in AP1 DNA binding. We also found that the cardiac fibroblasts cultured in high glucose (25 mmol/L) medium resulted in an increase in AP1 DNA binding activity, which was similar as seen in Ad-Ref1 transfected cells. There was also an increased accumulation of nuclear REF1 protein when cells were cultured in high glucose medium, although the expressions of REF1 at both mRNA and protein levels were not affected. CONCLUSION: REF1 can increase proliferation and collagen synthesis of cardiac fibroblasts, which may be related to its ability to up-regulate AP1 DNA binding.
Assuntos
Proliferação de Células/efeitos dos fármacos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/farmacologia , Fibroblastos/citologia , Miocárdio/citologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Colágeno/biossíntese , Fibroblastos/metabolismo , Miocárdio/metabolismo , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
Previous scientific research has elucidated the correlation between changes in levels of the DNA base excision repair protein, apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1), and ischemic neuronal DNA damage. However, to date, no studies have addressed the question of whether treatment involving this protein's repair function may prevent ischemic neuron death in vivo. Therefore, we aimed to investigate whether treatment with APE peptide is sufficient to prevent neuron death after ischemia/reperfusion (I/R) in mice. Mice were subjected to intraluminal suture occlusion of the middle cerebral artery for 1h followed by reperfusion. Post-ischemic treatment with the peptide containing only the APE repair functional domain was introduced intracerebroventricularly. Endonuclease activity assay and immunohistochemistry were performed. Assays of apurinic/apyrimidinic (AP) sites, single-strand DNA breaks, caspase-3 activity, and cell death were examined and quantified. We found that post-ischemic administration of the APE peptide up to 4h after reperfusion significantly inhibited the induction of cell death and subsequent infarct volume, measured 24h after I/R.
Assuntos
Isquemia Encefálica/patologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/farmacologia , Peptídeos/farmacologia , Traumatismo por Reperfusão/patologia , Análise de Variância , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/enzimologia , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Simples , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/administração & dosagem , Modelos Animais de Doenças , Endodesoxirribonucleases/metabolismo , Endorribonucleases/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/administração & dosagem , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/enzimologia , Sais de Tetrazólio , Fatores de TempoRESUMO
Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/Ref-1) is a multifunctional protein involved in DNA base excision repair and redox regulation of many transcription factors. In different melanoma cell lines, we found that both nucleus and cytoplasm exhibited higher levels of Ref-1 compared with normal melanocytes. Similar increases of Ref-1 expression, detected by immunohistofluorescence, were also evident in nevi and malignant melanoma biopsies compared with normal skin, which were predominantly localized in the nucleus. Using recombinant adenovirus Adref-1, encoding full-length Ref-1, we transiently overexpressed APE/Ref-1 in human melanocytes, which protected these cells from UVB-induced apoptosis and increased foci formation in culture. Ref-1 overexpression also protected melanoma cells from cisplatin- or H2O2-induced apoptosis, whereas increased apoptosis was observed with Ref-1 antisense construct infection. These observations suggested that intracellular Ref-1 levels played an important role in sensitization of melanoma cells to apoptosis. Electrophoretic mobility shift assay results showed that in both cultured primary and metastatic melanomas DNA-binding activities of activator protein-1 and nuclear factor-kappaB were significantly diminished or shifted when anti-APE/Ref-1 antibody was added to deplete APE/Ref-1 from the binding complexes. Induced nuclear factor-kappaB transcriptional activities were also evident after Ref-1 overexpression. Furthermore, using three-dimensional molecular structure modeling and virtual screening, we found that resveratrol, a natural compound found in fruits and vegetables, docks into a druggable pocket of Ref-1 protein. In vitro studies revealed that resveratrol inhibited, in a dose-dependent manner, Ref-1-activated activator protein-1 DNA-binding activities as well as Ref-1 endonuclease activities and rendered melanoma cells more sensitive to dacarbazine treatment.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Inibidores Enzimáticos/farmacologia , Melanoma/enzimologia , Estilbenos/farmacologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Imunofluorescência , Humanos , Melanócitos/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , ResveratrolRESUMO
Many environmental metals are co-carcinogens, eliciting their effects via inhibition of DNA repair. Apurinic/apyrimidinic (AP) endonuclease 1 (Ape1) is the major mammalian abasic endonuclease and initiates repair of this cytotoxic/mutagenic lesion by incising the DNA backbone via a Mg(2+)-dependent reaction. In this study we examined the effects of arsenite [As(III)], cadmium [Cd(II)], cobalt [Co(II)], iron [Fe(II)], nickel [Ni(II)], and lead [Pb(II)] at concentrations ranging from 0.3 to 100 microM on the incision activity of Ape1 in the presence of 1 mM MgCl(subscript)2(/subscript). Pb(II) and Fe(II) inhibited Ape1 activity at each of the concentrations tested, with an IC(subscript)50(/subscript) (half-maximal inhibitory concentration) of 0.61 and 1.0 microM, respectively. Cd(II) also inhibited Ape1 activity but only at concentrations > 10 microM. No inhibition was seen with As(III), Co(II), or Ni(II). A similar inhibition pattern was observed with the homologous Escherichia coli protein, exonuclease III, but no inhibition was seen with the structurally distinct AP endonuclease E. coli endonuclease IV, indicating a targeted effect of Pb(II), Fe(II), and Cd(II) on the Ape1-like repair enzymes. Excess nonspecific DNA did not abrogate the metal inactivation, suggesting a protein-specific effect. Notably, Cd(II), Fe(II), and Pb(II) [but not As(III), Co(II), or Ni(II)] inhibited AP endonuclease activity in whole-cell extracts but had no significant effect on single nucleotide gap filling, 5'-flap endonuclease, and nick ligation activities, supporting the idea of selective inactivation of Ape1 in cells. Our results are the first to identify a potential DNA repair enzyme target for lead and suggest a means by which these prevalent environmental metals may elicit their deleterious effects.
Assuntos
Cádmio/toxicidade , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/farmacologia , Ferro/toxicidade , Chumbo/toxicidade , Metais Pesados/toxicidade , Técnicas de Cultura de Células , Dano ao DNA , Reparo do DNA , Relação Dose-Resposta a Droga , Escherichia coli/enzimologia , Perfilação da Expressão Gênica , HumanosRESUMO
Redox factor-1 (Ref-1) is a ubiquitously expressed protein with proven roles as a modulator of redox-sensitive transcription, and as an endonuclease in the base excision repair pathway of oxidatively damaged DNA. Although Ref-1 is induced by a variety of oxidative stress and protects cells against oxidative stress, the function of Ref-1 in regulating nitric oxide (NO) synthesis has not been elucidated to date. We investigated the role of Ref-1 in regulating NO synthesis and NO-mediated apoptosis employing adenoviral-mediated overexpression of Ref-1 in lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells. LPS treatment produced NO synthesis and NO-mediated apoptosis. Forced overexpression of Ref-1 suppressed LPS-stimulated NO synthesis. In parallel with this, Ref-1 also mitigated alteration of inducible NO synthase expression and NO-mediated apoptosis. Our findings suggest that Ref-1 is implicated in protection against cell death resulting from oxidative stimuli containing NO.
Assuntos
Apoptose/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/biossíntese , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Óxido Nítrico/biossíntese , Adenoviridae/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Nitritos/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , TransgenesRESUMO
Telomerase is an important ribonucleoprotein enzyme involved in cellular proliferation and senescence. Activation of telomerase has been detected in a vast majority of human cancer cells. In this article, we demonstrated that Interleukin-2 (IL-2) which is the pivotal cytokine in the immune system could stimulate the activity of telomerase in the cultured BA/F3beta cells. It was also found that the level of IL-2-induced telomerase activity was decreased by the treatment with chemical oxidant in vitro. Since IL-2 stimulation produces a oxidative shift of the intracellular environment, the activation and maintenance of telomerase in this oxidative circumstance requires particular protection. Here we proved the redox factor-1 (Ref-1) protein was involved in this process. The addition of GST-Ref-1 protein increased the level of IL-2-induced telomerase activity in the TRAP assay, while elimination of the endogenous Ref-1 protein by immunodepletion decreased it. Consistent with these in vitro results, IL-2-induced telomerase activity could be enhanced by transient overexpression of Ref-1 protein in BA/F3beta cells. Taken together, these findings proved that Ref-1 protein benefits the activation of telomerase activity in the oxidative microenvironment of the BA/F3beta cells stimulated by IL-2.