RESUMO
The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A(2A) adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (T(reg)) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4(+) and CD8(+) T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated T(reg). Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4(+) T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of T(reg) but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.
Assuntos
5'-Nucleotidase/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Neoplasias Ovarianas/enzimologia , Receptor A2A de Adenosina/metabolismo , Linfócitos T/imunologia , 5'-Nucleotidase/imunologia , Adenosina/metabolismo , Antígenos CD/imunologia , Apirase/imunologia , Linhagem Celular Tumoral , Separação Celular , Feminino , Citometria de Fluxo , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Imuno-Histoquímica , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Interferência de RNA , Receptor A2A de Adenosina/imunologiaRESUMO
Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: 1) attraction of phagocytes via soluble "find me" signals, 2) recognition and phagocytosis via cell surface-presenting "eat me" signals, and 3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of proinflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the current study, using wild-type and adenosine A(2A) receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, as a result of possible activation of liver X receptor and peroxisome proliferators activated receptor δ. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase/protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether, our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation.
Assuntos
Adenosina/imunologia , Apoptose/imunologia , Inflamação/imunologia , Fagocitose/imunologia , Receptor A2A de Adenosina/imunologia , Animais , Inflamação/patologia , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Peritonite/imunologiaRESUMO
BACKGROUND/AIMS: Elevated plasma homocysteine (Hcy) levels have been identified as a pathogenic factor causing a variety of pathological changes in different cells and tissues. In vertebrates, Hcy is produced solely from S-adenosylhomocysteine (AdoHcy) through the catalysis of AdoHcy-hydrolase. The direction of AdoHcy-hydrolase activity is determined by its cytosolic substrate concentrations, thereby controlling intracellular AdoHcy levels. Most S-adenosylmethionine (AdoMet)-dependent methyltransferases are regulated in vivo by the ratio of AdoMet/AdoHcy, which is termed "methylation potential" (MP). To test whether high rates of erythropoietin (EPO) expression is reduced by a low MP in vivo we choosed the model of increased EPO production following carbon monoxide (CO) exposure in rats in which high transcriptional activity is responsible for renal EPO production. RESULTS: To induce a sustained hyperhomocysteinemia in rats, we infused i.v. a low or high dose of Hcy resulting in Hcy plasma levels of 87.4+/-6.2 and 300.8+/-23.7 mumol/l, respectively. Renal tissue contents of AdoHcy, AdoMet, and adenosine (Ado) were measured after freeze clamp by means of HPLC. Within 4h of CO exposure EPO serum levels increased from 13.6+/-0.4 (control) to 2254.8+/-278.3 mIU/ml. Only high dose of Hcy reduces both, the MP from 40.8+/-2.0 to 8.2+/-1.0 in the kidney as well as EPO serum levels by 40% compared to control rats. CONCLUSION: Our data show that severe hyperhomocysteinemia (HHcy) affects the MP in the renal tissue and lowers EPO expression following CO induced intoxication. This result supports the concept that efficient EPO production requires an unimpaired MP.
Assuntos
Eritropoetina/sangue , Hiper-Homocisteinemia/metabolismo , Adenosil-Homocisteinase/metabolismo , Animais , Monóxido de Carbono/toxicidade , Homocisteína/sangue , Hiper-Homocisteinemia/induzido quimicamente , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
S-Adenosylhomocysteine-hydrolase (AdoHcy-hydrolase) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine (Hcy). Since Ado competes with cAMP at the high affinity-binding site of the enzyme, we determined the effect of cAMP on enzyme activity and its binding characteristics to purified AdoHcy-hydrolase from bovine kidney in its native, in its fully oxidized (NAD(+)), and in its fully reduced (NADH) form. cAMP (10 micromol/l) enhanced the hydrolytic activity of native AdoHcy-hydrolase by 35%, whereas the activity of the enzyme in its NAD(+) form was not stimulated by cAMP. In contrast to azido-Ado, binding of azido-cAMP did not inhibit the enzymatic activity of AdoHcy-hydrolase. Furthermore, cAMP did not prevent the Ado induced inhibition of the AdoHcy hydrolysis. Saturation binding experiments with the three different forms of AdoHcy-hydrolase, native, NAD(+), and NADH showed only one binding site with high affinity. This binding site was identified after photoaffinity labeling of the enzyme with 8-azido-[2-(3)H]-cAMP. One photolabeled peptide was isolated as Trp(310)-Val(325) from each AdoHcy-hydrolase from native, NAD(+), and NADH. The cAMP-labeled peptide is located in the NAD-binding domain of AdoHcy-hydrolase. In conclusion, our data show that the cAMP-binding site at the AdoHcy-hydrolase is independent of the NAD(+)/NADH ratio of the enzyme and is identical with the high affinity-binding site of Ado. Moreover, cAMP did not interact with the catalytic site of AdoHcy-hydrolase and did not act as an allosteric effector for the AdoHcy-hydrolase.
Assuntos
Adenosil-Homocisteinase/metabolismo , AMP Cíclico/metabolismo , S-Adenosil-Homocisteína/metabolismo , Adenosina/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Bovinos , AMP Cíclico/administração & dosagem , Humanos , Hidrólise , Rim/enzimologia , Rim/metabolismo , Oxirredução , Marcadores de Fotoafinidade , Ligação ProteicaRESUMO
BACKGROUND & AIMS: The liver tolerates ischemia/reperfusion (IR) poorly. The discovery of ischemic preconditioning (IP) has raised hopes that natural pathways could be activated to increase hepatic resistance to ischemia. However, mechanisms of hepatic IP remain largely unknown. Extracellular adenosine has been implicated as an innate anti-inflammatory metabolite, particularly during ischemia. We investigated whether ecto-5'-nucleotidase (CD73), the "pacemaker" enzyme of extracellular adenosine production, is critical for hepatic protection by IP. METHODS: Mice were subjected to 4 cycles of portal triad occlusion and reperfusion (3 minutes of ischemia/3 minutes of reperfusion) prior to IR or IR alone. RESULTS: Hepatic IP was associated with a significant induction of CD73 transcript and protein. Targeted gene deletion or pharmacologic inhibition of CD73 abolished hepatic protection by IP as measured by lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase serum levels or histologic injury. Increases in extracellular adenosine with IP were significantly attenuated in cd73-deficient (cd73(-/-)) mice. Reconstitution of cd73(-/-) mice with soluble 5'-nucleotidase resulted in complete restoration of hepatoprotection by IP, and hepatic injury following ischemia was attenuated by treatment of WT mice with soluble 5'-nucleotidase. Mice deficient in CD73 did not demonstrate the same degree of IP-dependent inhibition of acute phase complement gene expression/activation as did wild-type mice suggesting that extracellular adenosine attenuates hepatic IR via complement regulation. CONCLUSIONS: Extracellular adenosine production by CD73 mediates protection during murine hepatic IP. Use of soluble 5'-nucleotidase may be a potential therapeutic for hepatic ischemia.
Assuntos
5'-Nucleotidase/genética , Adenosina/biossíntese , Precondicionamento Isquêmico/métodos , Fígado/irrigação sanguínea , RNA/genética , Traumatismo por Reperfusão/prevenção & controle , Transcrição Gênica , 5'-Nucleotidase/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Líquido Extracelular/enzimologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND/AIMS: S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy), which is a potent product inhibitor of S-adenosylmethionine (AdoMet)-dependent methyltransferases. While previous studies have shown that AdoHcyase inhibition or deficiency lead to a decreased AdoMet/AdoHcy ratio resulting in impaired transmethylation, the effect of enhanced AdoHcyase activity on AdoMet/AdoHcy metabolism and methylation reactions has not been studied in detail. METHODS: To investigate the effect of enhanced AdoHcyase activity, we generated HEK-293 cell lines stably overexpressing AdoHcyase. RESULTS: Initial studies revealed that 2-10-fold AdoHcyase overexpression resulted in decreased intracellular AdoHcy and elevated adenosine levels, whereas 16-fold AdoHcyase overexpression increased adenosine and AdoHcy levels, lowered energy charge, and altered cell morphology. Furthermore, we found a correlation between AdoHcyase activity and cell viability. Caspase-activity assays and DNA fragmentation analysis revealed that the cell death in AdoHcyase overexpressing cells was due to apoptosis. Global DNA methylation was not altered in the different AdoHcyase overexpressing cell lines. CONCLUSION: Taken together, these data show that 2-5-fold enhanced AdoHcyase activity is well tolerated by the cell, while greatly enhanced AdoHcyase activity results in adenosine-induced apoptosis. The fact that enhanced AdoHcyase activity does not increase transmethylation activity suggests that AdoHcyase activity under physiological conditions is not rate limiting for efficient transmethylation.
Assuntos
Adenosina/metabolismo , Adenosil-Homocisteinase/metabolismo , Metilação de DNA , Espaço Intracelular/metabolismo , Adenosina Desaminase/metabolismo , Adenosil-Homocisteinase/genética , Caspases/metabolismo , Morte Celular , Linhagem Celular , Forma Celular , Sobrevivência Celular , Células Clonais , DNA/metabolismo , Fragmentação do DNA , Eletroforese em Gel de Ágar , Metabolismo Energético , Regulação Enzimológica da Expressão Gênica , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Frações Subcelulares , TransfecçãoRESUMO
OBJECTIVE: Adenosine signaling is known to inhibit platelet aggregation. Extracellular adenosine mainly stems from enzymatic phosphohydrolysis of precursor nucleotides via ecto-5'-nucleotidase. Previous studies suggest that soluble 5'-nucleotidase (5'-NT) derived from Crotalus atrox venom may be clinically beneficial in vascular leakage, myocardial, renal, and intestinal ischemia, or acute lung injury. However, the effects of 5'-NT treatment on platelet aggregation remain unknown. We examined the direct effects of 5'-NT treatment on platelet aggregation in vivo and ex vivo using a whole blood aggregation method. METHODS AND RESULTS: Platelet aggregation in whole human blood was completely inhibited by 5'-NT. When 5'-[alphabeta-methylene] diphosphate (APCP), a specific 5'-ecto-nucleotidase inhibitor, was added together with 5'-NT, APCP fully restored collagen- or ADP-induced aggregation. Adenosine levels in whole blood were significantly increased after 5'-NT treatment compared to controls and inhibition of platelet aggregation by 5'-NT was completely reversed by pretreatment with the nonspecific adenosine receptor antagonist 8-(p-sulfophenyl)theophylline hydrate (8-SPT), suggesting that 5'-NT inhibits aggregation via increased adenosine signaling. Administration of 5'-NT to mice in vivo abolished ADP- and collagen-induced platelet aggregation and increased adenosine concentrations and tail bleeding time. CONCLUSIONS: 5'-NT treatment inhibits platelet aggregation via generation of increased levels of extracellular adenosine and subsequent adenosine receptor signaling.
Assuntos
5'-Nucleotidase/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , 5'-Nucleotidase/química , Adenosina/metabolismo , Animais , Venenos de Crotalídeos/química , Humanos , Camundongos , Receptores Purinérgicos P1/metabolismo , Tempo de Coagulação do Sangue TotalRESUMO
The DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) protects cells against the cytotoxic effects of alkylating agents. Therefore, modulation of MGMT expression in tumors is a possible strategy for improving the efficiency of cancer therapy. MGMT expression and activity is lost frequently in association with DNA hypermethylation of the MGMT promoter region. Since DNA and mRNA methylation are controlled by intracellular S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) levels, we hypothesized a role for AdoMet/AdoHcy ratio in the regulation of MGMT promoter methylation and mRNA expression. Our initial studies showed that AdoMet/AdoHcy ratios vary over a wide range (7.0-50) in different glioblastoma and hepatoma cell lines. The studied cell lines exhibit distinct MGMT promoter methylation patterns: MGMT promoter was completely unmethylated in LN-18 and Tu 132 cells, hypermethylated in LN-229, U87-MG, and Tu 113 cells, and partially methylated in HepG2 cells. Furthermore, MGMT promoter methylation patterns and global DNA methylation are not related to intracellular AdoMet/AdoHcy ratio under control conditions. To lower AdoMet/AdoHcy ratio to values <1 we used AdoHcy hydrolase inhibitor adenosine-2',3'-dialdehyde (30 microM) and found that neither short-term (24 h) nor long-term changes (7 weeks) in AdoMet/AdoHcy ratio altered global or MGMT promoter methylation. However, experimentally elevated AdoHcy levels significantly decreased MGMT mRNA levels by >50% in all MGMT-expressing cell lines, which is most likely the result of impaired mRNA methylation. Thus, the present study suggests elevation of AdoHcy levels by AdoHcy hydrolase inhibition as a novel pharmacological approach to modulate MGMT expression and to increase the responsiveness to alkylating agents.
Assuntos
Antineoplásicos/farmacologia , Metilação de DNA , Regulação Enzimológica da Expressão Gênica/fisiologia , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Regiões Promotoras Genéticas/fisiologia , S-Adenosil-Homocisteína/metabolismo , Adenosil-Homocisteinase/antagonistas & inibidores , Adenosil-Homocisteinase/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioma/tratamento farmacológico , Glioma/metabolismo , Hepatoblastoma/tratamento farmacológico , Hepatoblastoma/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/genética , RNA Mensageiro/metabolismoRESUMO
Extracellular adenosine has been implicated as an innate antiinflammatory metabolite, particularly during conditions of limited oxygen availability such as ischemia. Because extracellular adenosine generation is primarily produced via phosphohydrolysis from its precursor molecule adenosine-monophosphate (AMP) through the enzyme ecto-5'-nucleotidase (CD73), we examined the contribution of CD73-dependent adenosine production in modulation of intestinal ischemia-reperfusion (IR) injury. Following transcriptional and translational profiling of intestinal tissue that revealed a prominent induction of murine CD73, we next determined the role of CD73 in protection against intestinal IR injury. Interestingly, pharmacological inhibition or targeted gene deletion of CD73 significantly enhanced not only local intestinal injury, but also secondary organ injury, following IR as measured by intestinal and lung myeloperoxidase, aspartate and alanine aminotransferase, interleukin (IL) -1, IL-6, and histological injury. To confirm the role of CD73 in intestinal adenosine production, we measured adenosine tissue levels and found that they were increased with IR injury. In contrast, CD73-deficient (cd73(-/-)) mice had lower adenosine levels at baseline and no increase with IR injury. Finally, reconstitution of cd73(-/-) mice or treatment of wild-type mice with soluble 5'-nucleotidase was associated with significantly lower levels of injury. These data reveal a previously unrecognized role of CD73 in attenuating intestinal IR-mediated injury.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/lesões , Traumatismo por Reperfusão/metabolismo , 5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/deficiência , 5'-Nucleotidase/genética , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Sequência de Bases , Células CACO-2 , Linhagem Celular , Primers do DNA/genética , Inibidores Enzimáticos/farmacologia , Líquido Extracelular/metabolismo , Feminino , Humanos , Hidrólise , Interleucina-1beta/farmacologia , Interleucina-6/farmacologia , Intestinos/irrigação sanguínea , Intestinos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Purinérgicos P1/metabolismo , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia , Transdução de SinaisRESUMO
Previous studies have yielded conflicting results as to whether extracellular adenosine generation and signaling contributes to hypoxia-induced increases in renal erythropoietin (EPO) secretion. In this study, we combined pharmacological and genetic approaches to elucidate a potential contribution of extracellular adenosine to renal EPO release in mice. To stimulate EPO secretion, we used murine carbon monoxide exposure (400 and 750 parts per million CO, 4 h), ambient hypoxia (8% oxygen, 4 h), or arterial hemodilution. Because the ecto-5-nucleotidase (CD73, conversion of AMP to adenosine) is considered the pacemaker of extracellular adenosine generation, we first tested the effect of blocking extracellular adenosine generation with the specific CD73-inhibitor adenosine 5'-(alpha,beta-methylene) diphosphate (APCP) or by gene-targeted deletion of cd73. These studies showed that neither APCP-treatment nor targeted deletion of cd73 resulted in changes of stimulated EPO mRNA or serum levels, although the increases of adenosine levels in the kidney following CO exposure were attenuated in mice with APCP treatment or in cd73(-/-) mice. Moreover, pharmacological studies using specific inhibitors of individual adenosine receptors (A1 AR, DPCPX; A 2A AR, DMPX; A 2B AR, PSB 1115; A3AR, MRS 1191) showed no effect on stimulated increases of EPO mRNA or serum levels. Finally, stimulated EPO secretion was not attenuated in gene-targeted mice lacking A1A(-/-, A2A AR-/-, A2BAR(-/-), or A3AR-/-. Together, these studies combine genetic and pharmacological in vivo evidence that increases of EPO secretion during limited oxygen availability are not affected by extracellular adenosine generation or signaling.
Assuntos
Adenosina/metabolismo , Eritropoetina/metabolismo , Líquido Extracelular/metabolismo , Hipóxia/metabolismo , Rim/metabolismo , Transdução de Sinais , 5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/deficiência , Adenosina/antagonistas & inibidores , Adenosina/biossíntese , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Artérias/fisiologia , Monóxido de Carbono/farmacologia , Eritropoetina/sangue , Eritropoetina/genética , Hemodiluição , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antagonistas de Receptores Purinérgicos P1 , RNA Mensageiro/metabolismo , Receptores Purinérgicos P1/deficiênciaRESUMO
Acute renal failure from ischemia significantly contributes to cardiovascular morbidity and mortality. Extracellular adenosine has been implicated as an anti-inflammatory metabolite particularly during conditions of limited oxygen availability (e.g., ischemia). Because ecto-5'-nucleotidase (CD73) is rate limiting for extracellular adenosine generation, this study examined the contribution of CD73-dependent adenosine production to ischemic preconditioning (IP) of the kidneys. After the initial observation that murine CD73 transcript, protein, and function are induced by renal IP, its role in IP-mediated kidney protection was studied. In fact, increases in renal adenosine concentration with IP are attenuated in cd73(-/-) mice. Moreover, pharmacologic inhibition of CD73 or its targeted gene deletion abolished renal protection by IP as measured by clearance studies, plasma electrolytes, and renal tubular destruction, and reconstitution of cd73(-/-) mice with soluble 5'-nucleotidase resulted in complete restoration of renal protection by IP. Finally, renal injury after ischemia was attenuated by intraperitoneal treatment of wild-type mice with soluble 5'-nucleotidase to a similar degree as by IP. Taken together, these data reveal what is believed to be a previously unrecognized role of CD73 in renal protection from ischemia and suggest treatment with soluble 5'-nucleotidase as a novel therapeutic approach in the treatment of renal diseases that are precipitated by limited oxygen availability.
Assuntos
5'-Nucleotidase/metabolismo , Injúria Renal Aguda/prevenção & controle , Adenosina/biossíntese , Isquemia/metabolismo , Precondicionamento Isquêmico , Rim/metabolismo , 5'-Nucleotidase/deficiência , 5'-Nucleotidase/genética , Injúria Renal Aguda/etiologia , Animais , Modelos Animais de Doenças , Isquemia/complicações , Rim/irrigação sanguínea , Rim/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND/AIMS: Tissue hypoxia induces a variety of functional changes including enhanced transcriptional activity associated with high transmethylation activity (e.g. mRNA cap methylation) in the nucleus. It is well known that the kidney responds to hypoxia with enhanced transcription of erythropoietin (EPO) in the interstitial cells. Since S-adenosylhomocysteine (AdoHcy)-hydrolase regulates most S-adenosylmethionine (AdoMet) dependent transmethylation reactions by hydrolyzing the potent feedback inhibitor AdoHcy to adenosine and homocysteine we studied the effect of hypoxia by carbon monoxide (CO) inhalation (1200 ppm) on AdoHcy-hydrolase gene expression and its localization in rat kidneys. RESULTS: CO lowered renal AdoHcy-hydrolase mRNA expression by 64% whereas AdoHcy-hydrolase activity was not changed during 4h of CO exposure 0.7+/-0.04 mU/mg (control) vs. 0.75+/-0.06 mU/mg protein. Using two-channel immunofluorescence confocal laser scanning microscope AdoHcy-hydrolase was visualized in different cells of the hypoxic rat kidney. A very bright immunofluorescence of AdoHcy-hydrolase was observed in the nuclei of single interstitial cells of renal cortex and outer medulla which respond to hypoxia with increased EPO secretion indicating translocation of AdoHcy-hydrolase from the cytosol to the nucleus. CONCLUSIONS: These data suggest that AdoHcy-hydrolase accumulation in the nucleus of adult mammalian cells is involved in maintaining efficient transmethylation reactions in transcriptionally active cells by removing the product inhibitor AdoHcy.
Assuntos
Adenosil-Homocisteinase/metabolismo , Monóxido de Carbono/farmacologia , Rim/enzimologia , Animais , Núcleo Celular/metabolismo , Eritropoetina/sangue , Eritropoetina/metabolismo , Hipóxia , Imuno-Histoquímica , Hibridização In Situ , Rim/efeitos dos fármacos , Masculino , Metilação , Ratos , Ratos Sprague-Dawley , Transcrição GênicaRESUMO
Adenosine has been shown to initiate apoptosis through different mechanisms: (i) activation of adenosine receptors, (ii) intracellular conversion to AMP and stimulation of AMP-activated kinase, (iii) conversion to S-adenosylhomocysteine (AdoHcy), which is an inhibitor of S-adenosylmethionine (AdoMet)-dependent methyltransferases. Since the pathways involved are still not completely understood, we further investigated the role of AdoHcy hydrolase in adenosine-induced apoptosis. In HepG2 cells, adenosine induced caspase-like activity and DNA fragmentation, a marker of apoptosis. These effects were potentiated by co-incubation with homocysteine or adenosine deaminase inhibitor, pentostatin, and were mimicked by inhibition of AdoHcy hydrolase by adenosine-2',3'-dialdehyde (Adox). Adenosine-induced effects were significantly inhibited by dipyridamole, an inhibitor of adenosine transporter, whereas inhibitors of adenosine kinase did not affect adenosine-induced changes. Various adenosine receptor agonists and AICAR, an activator of AMP-activated kinase, did not mimic the effect of adenosine. Thus, adenosine-induced apoptosis is likely due to intracellular action of AdoHcy and independent of AMP-activated kinase and adenosine receptors. Because elevated AdoHcy levels are associated with reduced mRNA methylation, we studied mRNA expression in Adox-treated cells by microarray analysis. Since several p53-target genes and other apoptosis-related genes were up-regulated by Adox, we conclude that AdoHcy is involved in adenosine-induced apoptosis by altering gene expression.
Assuntos
Adenosina/farmacologia , Adenosil-Homocisteinase/fisiologia , Apoptose/genética , Caspases/metabolismo , Regulação da Expressão Gênica , Inibidores de Adenosina Desaminase , Adenosina Quinase/antagonistas & inibidores , Adenosina Quinase/metabolismo , Adenosil-Homocisteinase/antagonistas & inibidores , Adenosil-Homocisteinase/genética , Inibidores de Caspase , Caspases/análise , Fragmentação do DNA , Dipiridamol/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Homocisteína/farmacologia , Humanos , Metilação , Proteínas de Transporte de Nucleosídeos/antagonistas & inibidores , Análise de Sequência com Séries de Oligonucleotídeos , Pentostatina/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Mensageiro/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMO
S-adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of AdoHcy to adenosine and homocysteine. Increased levels of AdoHcy may play a role in the development of cardiovascular diseases and numerous other conditions associated with hyperhomocysteinemia. Several polymorphic isoforms named SAHH-1 to 4 may be resolved by horizontal starch gel electrophoresis from red blood cells. We have identified the genetic background of isoforms SAHH-2 and SAHH-3. SAHH-2 represents the previously described polymorphism in exon 2 of the AdoHcyase gene (112 C>T; p.R38W). Isoform SAHH-3 is based on a new polymorphism in exon 3 (377 G>A), leading to the conversion of glycine to arginine at amino-acid position 123. To shed light on the effects of these polymorphisms on the molecular and catalytic properties of AdoHcyase, we made recombinant wild-type and polymorphic R38W and G123R enzymes for a comparative analysis. The amino-acid exchanges did not bring about major changes to the catalytic rates of the recombinant proteins. However, circular dichroism analysis showed that both polymorphisms effect the thermal stability of the recombinant protein in vitro, reducing the unfolding temperature by approximately 2.6 degrees C (R38W) and 1.5 degrees C (G123R) compared to wild-type protein. In view of the altered thermal stability, and slightly decreased enzymatic activity of polymorphic proteins (< or =6%), one may consider the analyzed AdoHcyase isoforms as risk markers for diseases caused by irregular AdoHcyase metabolism.
Assuntos
Adenosil-Homocisteinase/fisiologia , Substituição de Aminoácidos , Adenosil-Homocisteinase/química , Adenosil-Homocisteinase/genética , Dicroísmo Circular , Estabilidade Enzimática/genética , Temperatura Alta , Humanos , Isoenzimas/genéticaRESUMO
Recently, a proven case of human S-adenosylhomocysteine-hydrolase (SAHH) deficiency was reported in a Croatian boy. As molecular analysis of the SAHH gene in this case revealed two different mutant alleles, we investigated the polymorphism of human SAHH in a total of 237 red blood samples from unrelated Croats using starch gel electrophoresis and an enzyme-specific staining procedure. From the relative enzymatic activity of SAHH--determined by densitometric assessment of electrophoretic patterns, and calculated on the basis of the protein concentration of the red blood cells-we detected three individuals as being heterozygous for an SAHH 0-allele. Moreover, a total of four different electromorphic SAHHs have been observed, giving allele frequencies calculated as SAHH 1 = 0.941, SAHH 2 = 0.032, SAHH 3 = 0.006, SAHH 4 = 0.015, and SAHH 0 = 0.006.
Assuntos
Adenosil-Homocisteinase/genética , Polimorfismo Genético , Alelos , Croácia , Frequência do Gene , HumanosRESUMO
BACKGROUND/AIMS: The methylation potential (MP) is defined as the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). It was shown recently that hypoxia increases AdoMet/AdoHcy ratio in HepG2 cells (Hermes et al., Exp Cell Res 294: 325-334, 2004). In the present study, we compared AdoMet/AdoHcy ratio and energy metabolism in HepG2, HEK-293, HeLa, MCF-7 and SK-HEP-1 cell lines under normoxia and hypoxia. METHODS: Metabolite concentrations were measured by HPLC. In addition, AdoHcy hydrolase (AdoHcyase) activity was determined photometrically. RESULTS: Under normoxia HepG2 cells show the highest AdoMet/AdoHcy ratio of 53.4 +/- 3.3 followed by MCF-7 and SK-HEP-1 cells with a AdoMet/AdoHcy ratio of 14.4 +/- 1.1 and 21.1 +/- 1.3, respectively. The lowest AdoMet/AdoHcy ratios are exhibited by HeLa and HEK-293 cells (6.6 +/- 0.7 and 7.1 +/- 0.3). Hypoxia does not significantly change the MP in MCF-7 and HeLa cells, but alters the MP in HepG2, HEK-293 and SK-HEP-1 cells. These alterations are dependent on the cell density. Under normoxia HepG2 cells exhibit AdoHcyase activity of 2.5 +/- 0.2 nmol min(-1) mg(-1) protein. All other cell lines show 3-5 times lower enzyme activity. Interestingly, hypoxia affects AdoHcyase activity only in HepG2 cells. CONCLUSIONS: Our data clearly show that the cell lines are characterized by different MP and different behavior under hypoxia. That implies that a lower MP is not necessarily associated with impaired transmethylation activity and cellular function.
Assuntos
Adenosil-Homocisteinase/metabolismo , S-Adenosil-Homocisteína/metabolismo , Adenosil-Homocisteinase/análise , Anaerobiose/fisiologia , Contagem de Células , Hipóxia Celular , Linhagem Celular , Sobrevivência Celular , Metabolismo Energético/fisiologia , Humanos , Metilação , S-Adenosilmetionina/metabolismoRESUMO
The enzymatic properties of glycine N-methyltransferase from rabbit liver and the effects of endogenous adenosine nucleosides, nucleotides and methyltransferase inhibitors were investigated using a photometrical assay to detect sarcosine with o-dianisidine as a dye. After isolation and purification the denatured enzyme showed a two-banded pattern by SDS-PAGE. The enzyme was highly specific for its substrates with a pH-optimum at pH 8.6. Glycine N-methyltransferase exhibits Michaelis-Menten kinetics for its substrates, S-adenosylmethionine and glycine, respectively. The apparent Km and Vmax values were determined for both the substrates, the other substrate being present at saturating concentrations. The enzyme was strongly inhibited in the presence of S-adenosylhomocysteine, 3-deazaadenosine, and 5'-S-isobutylthio-5'-deoxyadenosine. All other inhibitors investigated, adenosine, 2'-deoxyadenosine, aciclovir, and 5'-N-ethylcarboxamidoadenosine were poor inhibitors of the methylation reaction. Adenine nucleotides and vidarabin were without effect on the enzymatic activity. Based on the kinetic data glycine N-methyltransferase from rabbit liver exhibits appreciable activity at physiological S-adenosylmethionine and S-adenosylhomocysteine levels.
Assuntos
Fígado/enzimologia , Metiltransferases/química , Animais , Inibidores Enzimáticos/química , Glicina N-Metiltransferase , Concentração de Íons de Hidrogênio , Cinética , Metiltransferases/isolamento & purificação , Desnaturação Proteica , Coelhos , Especificidade por SubstratoRESUMO
S-Adenosylhomocysteine hydrolase (AdoHcyase) controls intracellular levels of S-adenosylhomocysteine (AdoHcy). AdoHcy is a potent product inhibitor of some S-adenosylmethionine-dependent methyltransferases. Pharmacological modulation of AdoHcyase to indirectly inhibit methyltransferases can be guided by the fact that adenosine binds with high affinity to AdoHcyase and inhibits enzyme activity. cAMP can compete with adenosine and can counteract the adenosine-induced inhibition of AdoHcyase. Thus, the ratio between adenosine and cAMP, which can vary under different physiological conditions, might result in changes in, for example, DNA promoter methylation and therefore transcription.
Assuntos
Adenosina/farmacologia , Adenosil-Homocisteinase/efeitos dos fármacos , Animais , AMP Cíclico/fisiologia , Humanos , Modelos Moleculares , S-Adenosil-Homocisteína/metabolismoRESUMO
S-adenosylhomocysteine (AdoHcy), a by-product and inhibitor of S-adenosylmethionine (AdoMet)-dependent methylation reactions, is removed by AdoHcy hydrolase. The ratio of AdoMet and AdoHcy, also termed methylation potential (MP), is a metabolic indicator for cellular methylation status. In the present study, we have investigated the influence of hypoxia and inhibition of AdoHcy hydrolase on MP in HepG2 cells. Furthermore, we studied the impact of deviations in MP on mRNA and DNA methylation and the expression of selected genes: erythropoietin, VEGF-A, AdoHcy hydrolase, cyclophilin, and HIF-1alpha. Under hypoxic conditions, the MP raised from 53.4 +/- 3.3 to 239.4 +/- 24.8, which is the result of increased AdoMet and decreased AdoHcy levels. Inhibition of AdoHcy hydrolase by adenosine-2',3'-dialdehyde leads to a 40-fold reduction of the MP under both normoxic and hypoxic conditions. Hypoxia increases erythropoietin (2.7-fold) and VEGF-A (5-fold) mRNA expression. During a reduced MP erythropoietin mRNA expression is lowered under normoxia and hypoxia by 70%, whereas VEGF-A mRNA expression is only reduced under hypoxic conditions by 60%. The mRNA expression of AdoHcy hydrolase, HIF-1alpha, and cyclophilin is insensitive to an altered MP. Furthermore, decreased MP leads to a highly significant decrease in overall mRNA methylation. Our results show that the mRNA levels of the studied genes respond differentially to changes in MP. This implies that genes with a slower transcription rate and mRNAs with a slower turnover are insensitive to short-term changes in MP.
Assuntos
Adenosina/análogos & derivados , Adenosil-Homocisteinase/metabolismo , Regulação da Expressão Gênica/genética , Hipóxia/metabolismo , RNA Mensageiro/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Adenosina/farmacologia , Adenosil-Homocisteinase/antagonistas & inibidores , Linhagem Celular Tumoral , Ciclofilinas/biossíntese , Ciclofilinas/genética , Metilação de DNA , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Eritropoetina/biossíntese , Eritropoetina/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Metilação , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine. On the basis of the kinetics of Ado binding to AdoHcy hydrolase we have shown that AdoHcy hydrolase binds Ado with different affinities [Kidney Blood Press. Res. 19 (1996) 100]. Since AdoHcy hydrolase in its totally reduced form binds Ado with high affinity we determined in the present study the Ado binding characteristics of purified AdoHcy hydrolase from bovine kidney (native form) and of reconstituted forms with defined NAD(+)/NADH ratios. AdoHcy hydrolase in its native form and at a ratio of 50% NAD(+) and 50% NADH exhibits two binding sites for Ado with a K(D1) of 9.2+/-0.6 nmol/L and a K(D2) of 1.4+/-0.1 micromol/L, respectively. Binding of Ado to AdoHcy hydrolase in its NADH form and in its NAD(+) form exhibits only one binding site with high affinity 48.3+/-2.7 nmol/L for the NADH form and with a low affinity of 4.9+/-0.3 micromol/L for the NAD(+) form. To identify these two Ado binding sites, AdoHcy hydrolase was covalently modified with [2-3H]-8-azido-Ado. After irradiation of the native AdoHcy hydrolase two different photolabeled peptides were isolated and identified as Asp(307)-Val(325) and Tyr(379)-Thr(410). When the reconstituted AdoHcy hydrolase in its NADH and in its NAD(+) form was irradiated with [2-3H]-8-azido-Ado only one peptide was identified as Asn(312)-Lys(318) from the NADH form and as Asp(391)-Ala(396) from the NAD(+) form. Based on the crystallographic data, the labeled peptide Asp(391)-Ala(396) (low affinity binding site), appears to belong to the catalytic domain of AdoHcy hydrolase, whereas the labeled peptide, identified as Asn(312)-Lys(318) (high affinity binding site), is located in the NAD domain. In conclusion, our data show that AdoHcy hydrolase has two different Ado binding sites which are dependent upon the enzyme-bound NAD(+)/NADH ratios.