RESUMO
In 1957, cyclic adenosine monophosphate (cAMP) was identified as the first secondary messenger, and the first signaling cascade discovered was the cAMP-protein kinase A (PKA) pathway. Since then, cAMP has received increasing attention given its multitude of actions. Not long ago, a new cAMP effector named exchange protein directly activated by cAMP (Epac) emerged as a critical mediator of cAMP's actions. Epac mediates a plethora of pathophysiologic processes and contributes to the pathogenesis of several diseases such as cancer, cardiovascular disease, diabetes, lung fibrosis, neurological disorders, and others. These findings strongly underscore the potential of Epac as a tractable therapeutic target. In this context, Epac modulators seem to possess unique characteristics and advantages and hold the promise of providing more efficacious treatments for a wide array of diseases. This paper provides an in-depth dissection and analysis of Epac structure, distribution, subcellular compartmentalization, and signaling mechanisms. We elaborate on how these characteristics can be utilized to design specific, efficient, and safe Epac agonists and antagonists that can be incorporated into future pharmacotherapeutics. In addition, we provide a detailed portfolio for specific Epac modulators highlighting their discovery, advantages, potential concerns, and utilization in the context of clinical disease entities.
Assuntos
Fatores de Troca do Nucleotídeo Guanina , Transdução de Sinais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transdução de Sinais/fisiologia , Descoberta de DrogasRESUMO
Agonists for glucagon-like-peptide-1 receptor (GLP-1R) are currently used for the treatment of type 2 diabetes and obesity. Their benefits have been centered on pancreas and hypothalamus, but their roles in other organ systems are not well understood. We studied the action of GLP-1R on secretions of adrenal medulla. Exendin-4, a synthetic analog of GLP-1, increases the synthesis and the release of catecholamines (CAs) by increasing cyclic AMP (cAMP) production, without apparent participation of cAMP-regulated guanine nucleotide exchange factor (Epac). Exendin-4, when incubated for 24 h, increases CA synthesis by promoting the activation of tyrosine hydroxylase. Short incubation (20 min) increases the quantum size of exocytotic events by switching exocytosis from partial to full fusion. Our results give a strong support to the role of GLP-1 in the fine control of exocytosis.
Assuntos
Células Cromafins/metabolismo , Exenatida/farmacologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/efeitos dos fármacos , Animais , Células Cromafins/efeitos dos fármacos , AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Exenatida/metabolismo , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ratos , Receptores de Glucagon/efeitos dos fármacos , Receptores de Glucagon/metabolismoRESUMO
Previous studies reported the critical role of the brefeldin A-inhibited guanine nucleotide exchange protein 3-prohibitin 2 (BIG3-PHB2) complex in modulating estrogen signaling activation in breast cancer cells, yet its pathophysiological roles in osteosarcoma (OS) cells remain elusive. Here, we report a novel function of BIG3-PHB2 in OS malignancy. BIG3-PHB2 complexes were localized mainly in mitochondria in OS cells, unlike in estrogen-dependent breast cancer cells. Depletion of endogenous BIG3 expression by small interfering RNA (siRNA) treatment led to significant inhibition of OS cell growth. Disruption of BIG3-PHB2 complex formation by treatment with specific peptide inhibitor also resulted in significant dose-dependent suppression of OS cell growth, migration, and invasion resulting from G2/M-phase arrest and in PARP cleavage, ultimately leading to PARP-1/apoptosis-inducing factor (AIF) pathway activation-dependent apoptosis in OS cells. Subsequent proteomic and bioinformatic pathway analyses revealed that disruption of the BIG3-PHB2 complex might lead to downregulation of inner mitochondrial membrane protein complex activity. Our findings indicate that the mitochondrial BIG3-PHB2 complex might regulate PARP-1/AIF pathway-dependent apoptosis during OS cell proliferation and progression and that disruption of this complex may be a promising therapeutic strategy for OS.
Assuntos
Neoplasias Ósseas/patologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Mitocôndrias/metabolismo , Osteossarcoma/patologia , Proteínas Repressoras/fisiologia , Animais , Apoptose/fisiologia , Fator de Indução de Apoptose/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/terapia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Bases de Dados Factuais , Regulação para Baixo , Pontos de Checagem da Fase G2 do Ciclo Celular , Inativação Gênica , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Membranas Mitocondriais/metabolismo , Invasividade Neoplásica , Transplante de Neoplasias , Osteossarcoma/metabolismo , Osteossarcoma/terapia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proibitinas , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/metabolismoRESUMO
Despite the renal expression of P2Y12, the purinergic receptor for adenosine diphosphate, few data are available to discuss the renotherapeutic potential of ticagrelor, one of its reversible blockers. Indeed, the tonic inhibitory effect of this receptor has been linked to the activation of exchange protein activated by cyclic adenosine monophosphate-1 (Epac-1) protein through the cyclic adenosine monophosphate cascade. Epac-1 is considered a crossroad protein, where its activation has been documented to manage renal injury models. Hence, the current study aimed to investigate the possible therapeutic effectiveness of ticagrelor, against renal ischemia/reperfusion (I/R) model with emphasis on the involvement of Epac-1 signaling pathway using R-CE3F4, a selective Epac-1 blocker. Accordingly, rats were randomized into four groups; viz., sham-operated, renal I/R, I/R post-treated with ticagrelor for 3 days, and ticagrelor + R-CE3F4. Treatment with ticagrelor ameliorated the I/R-mediated structural alterations and improved renal function manifested by the reduction in serum BUN and creatinine. On the molecular level, ticagrelor enhanced renal Epac-1 mRNA expression, Rap-1 activation (Rap-1-GTP) and SOCS-3 level. On the contrary, it inhibited the protein expression of JAK-2/STAT-3 hub, TNF-α and MDA contents, as well as caspase-3 activity. Additionally, ticagrelor enhanced the protein expression/content of AKT/Nrf-2/HO-1 axis. All these beneficial effects were obviously antagonized upon using R-CE3F4. In conclusion, ticagrelor reno-therapeutic effect is partly mediated through modulating the Epac-1/Rap-1-GTP, AKT/Nrf-2/HO-1 and JAK-2/STAT-3/SOCS-3 trajectories, pathways that integrate to afford novel explanations to its anti-inflammatory, anti-oxidant, and anti-apoptotic potentials.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Ticagrelor/uso terapêutico , Proteínas rap1 de Ligação ao GTP/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Isquemia , Testes de Função Renal , Masculino , Ratos , Ratos Wistar , Circulação Renal/efeitos dos fármacos , Proteínas rap1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
Elucidation of the mechanism of dopamine signaling to ERK that underlies plasticity in dopamine D1 receptor-expressing neurons leading to acquired cocaine preference is incomplete. NCS-Rapgef2 is a novel cAMP effector, expressed in neuronal and endocrine cells in adult mammals, that is required for D1 dopamine receptor-dependent ERK phosphorylation in mouse brain. In this report, we studied the effects of abrogating NCS-Rapgef2 expression on cAMP-dependent ERKâEgr-1/Zif268 signaling in cultured neuroendocrine cells; in D1 medium spiny neurons of NAc slices; and in either male or female mouse brain in a region-specific manner. NCS-Rapgef2 gene deletion in the NAc in adult mice, using adeno-associated virus-mediated expression of cre recombinase, eliminated cocaine-induced ERK phosphorylation and Egr-1/Zif268 upregulation in D1-medium spiny neurons and cocaine-induced behaviors, including locomotor sensitization and conditioned place preference. Abrogation of NCS-Rapgef2 gene expression in mPFC and BLA, by crossing mice bearing a floxed Rapgef2 allele with a cre mouse line driven by calcium/calmodulin-dependent kinase IIα promoter also eliminated cocaine-induced phospho-ERK activation and Egr-1/Zif268 induction, but without effect on the cocaine-induced behaviors. Our results indicate that NCS-Rapgef2 signaling to ERK in dopamine D1 receptor-expressing neurons in the NAc, but not in corticolimbic areas, contributes to cocaine-induced locomotor sensitization and conditioned place preference. Ablation of cocaine-dependent ERK activation by elimination of NCS-Rapgef2 occurred with no effect on phosphorylation of CREB in D1 dopaminoceptive neurons of NAc. This study reveals a new cAMP-dependent signaling pathway for cocaine-induced behavioral adaptations, mediated through NCS-Rapgef2/phospho-ERK activation, independently of PKA/CREB signaling.SIGNIFICANCE STATEMENT ERK phosphorylation in dopamine D1 receptor-expressing neurons exerts a pivotal role in psychostimulant-induced neuronal gene regulation and behavioral adaptation, including locomotor sensitization and drug preference in rodents. In this study, we examined the role of dopamine signaling through the D1 receptor via a novel pathway initiated through the cAMP-activated guanine nucleotide exchange factor NCS-Rapgef2 in mice. NCS-Rapgef2 in the NAc is required for activation of ERK and Egr-1/Zif268 in D1 dopaminoceptive neurons after acute cocaine administration, and subsequent enhanced locomotor response and drug seeking behavior after repeated cocaine administration. This novel component in dopamine signaling provides a potential new target for intervention in psychostimulant-shaped behaviors, and new understanding of how D1-medium spiny neurons encode the experience of psychomotor stimulant exposure.
Assuntos
Cocaína/farmacologia , Condicionamento Operante/efeitos dos fármacos , Inibidores da Captação de Dopamina/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Receptores de Dopamina D1/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , AMP Cíclico/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína 1 de Resposta de Crescimento Precoce/efeitos dos fármacos , Feminino , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Accumbens/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Estriado Ventral/efeitos dos fármacosRESUMO
BACKGROUND: Total intravenous anesthesia with propofol has been shown to reduce postoperative pain in some clinical studies, but knowledge of its underlying analgesic mechanism remains limited. In this study, we compared the analgesic effects of propofol versus isoflurane in an animal model of postoperative pain and evaluated its underlying molecular mechanisms. METHODS: Plantar incision was made in the hind paws of rats under general anesthesia with 2.5% of inhalational isoflurane (isoflurane group) or intravenous infusion of propofol (1.5 mg kg-1 min-1 , propofol group). Mechanical allodynia was assessed by paw withdrawal threshold before and after incision. Spinal dorsal horns (L3-L5) were harvested 1 hr after incision to assess the level of phosphorylated GluN2B, p38MAPK, ERK, JNK, and EPAC using Western blot and immunofluorescence. RESULTS: Mechanical allodynia induced by plantar incision peaked at 1 hr and lasted for 3 days after incision. It was significantly less in the propofol group compared with the isoflurane group in the first 2 hr following incision. The incision-induced increases in phosphorylated GluN2B, p38MAPK, and EPAC1 were significantly reduced in the propofol group. The number of spinal dorsal neurons co-expressed with EPAC1 and c-Fos after the incision was significantly lower in the propofol group. CONCLUSION: Propofol reduced pain responses in an animal model of postoperative pain and suppressed the spinal GluN2B-p38MAPK/EPAC1 signaling pathway. Since the p38MAPK/EPAC pathway plays a critical role in the development of postoperative hyperalgesia, our results provide evidence-based behavioral, molecular, and cellular mechanisms for the analgesic effects of propofol when used for general anesthesia. SIGNIFICANCE: These findings may provide a new mechanism for the postsurgical analgesic effect of propofol, which is particularly interesting during the subacute period after surgery as it is the critical period for the development of persistent postsurgical pain.
Assuntos
Anestésicos Inalatórios/farmacologia , Anestésicos Intravenosos/farmacologia , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Isoflurano/farmacologia , Dor Pós-Operatória/metabolismo , Propofol/farmacologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Corno Dorsal da Medula Espinal/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Hiperalgesia/metabolismo , Masculino , Período Pós-Operatório , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Rat pancreatic acinar cells possess only the p21-activated kinase (PAKs), PAK4 of the group II PAK, and it is activated by gastrointestinal hormones/neurotransmitters stimulating PLC and by a number of growth factors. However, little is known generally of cAMP agents causing PAK4 activation, and there are no studies with gastrointestinal hormones/neurotransmitters activating cAMP cascades. In the present study, we examined the ability of VIP and secretin, which stimulate cAMP generation in pancreatic acini, to stimulate PAK4 activation, the signaling cascades involved, and their possible role in activating sodium-potassium adenosine triphosphatase (Na+,K+-ATPase). PAK4 activation was compared with activation of the well-established cAMP target, cyclic AMP response element binding protein (CREB). Secretin-stimulated PAK4 activation was inhibited by KT-5720 and PKA Type II inhibitor (PKI), protein kinase A (PKA) inhibitors, whereas VIP activation was inhibited by ESI-09 and HJC0197, exchange protein directly activated by cAMP (EPAC) inhibitors. In contrast, both VIP/secretin-stimulated phosphorylation of CREB (pCREB) via EPAC activation; however, it was inhibited by the p44/42 inhibitor PD98059 and the p38 inhibitor SB202190. The specific EPAC agonist 8-CPT-2- O-Me-cAMP as well 8-Br-cAMP and forskolin stimulated PAK4 activation. Secretin/VIP activation of Na+,K+-ATPase, was inhibited by PAK4 inhibitors (PF-3758309, LCH-7749944). These results demonstrate PAK4 is activated in pancreatic acini by stimulation of both VIP-/secretin-preferring receptors, as is CREB. However, they differ in their signaling cascades. Furthermore, PAK4 activation is needed for Na+,K+ATPase activation, which mediates pancreatic fluid secretion. These results, coupled with recent studies reporting PAKs are involved in both pancreatitis/pancreatic cancer growth/enzyme secretion, show that PAK4, similar to PAK2, likely plays an important role in both pancreatic physiological/pathological responses. NEW & NOTEWORTHY Pancreatic acini possess only the group II p21-activated kinase, PAK4, which is activated by PLC-stimulating agents/growth factors and is important in enzyme-secretion/growth/pancreatitis. Little information exists on cAMP-activating agents stimulating group II PAKs. We studied ability/effect of cyclic AMP-stimulating agents [vasoactive intestinal polypeptide (VIP), secretin] on PAK4 activity in rat pancreatic-acini. Both VIP/secretin activated PAK4/CREB, but the cAMP signaling cascades differed for EPAC, MAPK, and PKA pathways. Both hormones require PAK4 activation to stimulate sodium-potassium adenosine triphosphatase activity. This study shows PAK4 plays an important role in VIP-/secretin-stimulated pancreatic fluid secretion and suggests it plays important roles in pancreatic acinar physiological/pathophysiological responses mediated by cAMP-activating agents.
Assuntos
Células Acinares/efeitos dos fármacos , Antineoplásicos/farmacologia , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Células Acinares/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Masculino , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Pirazóis/farmacologia , Pirróis/farmacologia , Quinazolinas/farmacologia , Ratos Sprague-Dawley , Secretina/efeitos dos fármacosRESUMO
PURPOSE: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. METHODS: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. RESULTS: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). CONCLUSION: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.
Assuntos
Ácido Ascórbico/farmacologia , Queimaduras/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Adulto , Araquidonato 12-Lipoxigenase/análise , Araquidonato 12-Lipoxigenase/efeitos dos fármacos , Queimaduras/tratamento farmacológico , Células Cultivadas , Estudos Transversais , Ciclo-Oxigenase 1/análise , Ciclo-Oxigenase 1/efeitos dos fármacos , Oxidases Duais/análise , Oxidases Duais/efeitos dos fármacos , Feminino , Glutationa Peroxidase/análise , Glutationa Peroxidase/efeitos dos fármacos , Glutationa Transferase/análise , Glutationa Transferase/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Humanos , Masculino , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/análise , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/efeitos dos fármacos , Peroxirredoxinas/análise , Peroxirredoxinas/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Reprodutibilidade dos Testes , Pele/efeitos dos fármacos , Pele/patologia , Estatísticas não Paramétricas , Ubiquitina-Proteína Ligases/análise , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Adulto JovemRESUMO
Activation of Rap1 by exchange protein activated by cAMP (Epac) promotes cell adhesion and actin cytoskeletal polarization. Pharmacologic activation of Epac-Rap signaling by the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP during ischemia-reperfusion (IR) injury reduces renal failure and application of 8-pCPT-2'-O-Me-cAMP promotes renal cell survival during exposure to the nephrotoxicant cisplatin. Here, we found that activation of Epac by 8-pCPT-2'-O-Me-cAMP reduced production of reactive oxygen species during reoxygenation after hypoxia by decreasing mitochondrial superoxide production. Epac activation prevented disruption of tubular morphology during diethyl maleate-induced oxidative stress in an organotypic three-dimensional culture assay. In vivo renal targeting of 8-pCPT-2'-O-Me-cAMP to proximal tubules using a kidney-selective drug carrier approach resulted in prolonged activation of Rap1 compared with nonconjugated 8-pCPT-2'-O-Me-cAMP. Activation of Epac reduced antioxidant signaling during IR injury and prevented tubular epithelial injury, apoptosis, and renal failure. Our data suggest that Epac1 decreases reactive oxygen species production by preventing mitochondrial superoxide formation during IR injury, thus limiting the degree of oxidative stress. These findings indicate a new role for activation of Epac as a therapeutic application in renal injury associated with oxidative stress.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/fisiologia , Túbulos Renais Proximais/metabolismo , Estresse Oxidativo , Urotélio/metabolismo , Animais , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Urotélio/efeitos dos fármacosRESUMO
3',5'-Cyclic adenosine monophosphate (cAMP) is a pivotal second messenger that regulates numerous biological processes under physiological and pathological conditions, including cancer, diabetes, heart failure, inflammation, and neurological disorders. In the past, all effects of cAMP were initially believed to be mediated by protein kinase A (PKA) and cyclic nucleotide-regulated ion channels. Since the discovery of exchange proteins directly activated by cyclic adenosine 5'-monophosphate (EPACs) in 1998, accumulating evidence has demonstrated that the net cellular effects of cAMP are also regulated by EPAC. The pursuit of the biological functions of EPAC has benefited from the development and applications of a growing number of pharmacological probes targeting EPACs. In this review, we seek to provide a concise update on recent advances in the development of chemical entities including various membrane-permeable analogues of cAMP and newly discovered EPAC-specific ligands from high throughput assays and hit-to-lead optimizations.
Assuntos
Descoberta de Drogas , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Cristalografia por Raios X , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Modelos Moleculares , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
GPR119 is a G protein-coupled receptor expressed on enteroendocrine L-cells that synthesize and secrete the incretin hormone glucagon-like peptide-1 (GLP-1). Although GPR119 agonists stimulate L-cell GLP-1 secretion, there is uncertainty concerning whether GLP-1 biosynthesis is under the control of GPR119. Here we report that GPR119 is functionally coupled to increased proglucagon (PG) gene expression that constitutes an essential first step in GLP-1 biosynthesis. Using a mouse L-cell line (GLUTag) that expresses endogenous GPR119, we demonstrate that PG gene promoter activity is stimulated by GPR119 agonist AS1269574. Surprisingly, transfection of GLUTag cells with recombinant human GPR119 (hGPR119) results in a constitutive and apparently ligand-independent increase of PG gene promoter activity and PG mRNA content. These constitutive actions of hGPR119 are mediated by cAMP-dependent protein kinase (PKA) but not cAMP sensor Epac2. Thus, the constitutive action of hGPR119 to stimulate PG gene promoter activity is diminished by: 1) a dominant-negative Gαs protein, 2) a dominant-negative PKA regulatory subunit, and 3) a dominant-negative A-CREB. Interestingly, PG gene promoter activity is stimulated by 6-Bn-cAMP-AM, a cAMP analog that selectively activates α and ß isoforms of type II, but not type I PKA regulatory subunits expressed in GLUTag cells. Finally, our analysis reveals that a specific inhibitor of Epac2 activation (ESI-05) fails to block the stimulatory action of 6-Bn-cAMP-AM at the PG gene promoter, nor is PG gene promoter activity stimulated by: 1) a constitutively active Epac2, or 2) cAMP analogs that selectively activate Epac proteins. Such findings are discussed within the context of ongoing controversies concerning the relative contributions of PKA and Epac2 to the control of PG gene expression.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proglucagon/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Enteroendócrinas , Etanolaminas/farmacologia , Regulação da Expressão Gênica , Peptídeo 1 Semelhante ao Glucagon/biossíntese , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Incretinas/biossíntese , Incretinas/metabolismo , Células L , Camundongos , Proglucagon/biossíntese , Proglucagon/genética , Regiões Promotoras Genéticas , Pirimidinas/farmacologia , RNA Mensageiro/biossíntese , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Neuropeptide Y (NPY) administration into the basolateral amygdala (BLA) decreases anxiety-like behavior, mediated in part through the Y1 receptor (Y1R) isoform. Activation of Y1Rs results in G-protein-mediated reduction of cAMP levels, which results in reduced excitability of amygdala projection neurons. Understanding the mechanisms linking decreased cAMP levels to reduced excitability in amygdala neurons is important for identifying novel anxiolytic targets. We studied the intracellular mechanisms of activation of Y1Rs on synaptic transmission in the BLA. Activating Y1Rs by [Leu(31),Pro(34)]-NPY (L-P NPY) reduced the amplitude of evoked NMDA-mediated excitatory postsynaptic currents (eEPSCs), without affecting AMPA-mediated eEPSCs, but conversely increased the amplitude of GABAA-mediated evoked inhibitory postsynaptic currents (eIPSCs). Both effects were abolished by the Y1R antagonist, PD160170. Intracellular GDP-ß-S, or pre-treatment with either forskolin or 8Br-cAMP, eliminated the effects of L-P NPY on both NMDA- and GABAA-mediated currents. Thus, both the NMDA and GABAA effects of Y1R activation in the BLA are G-protein-mediated and cAMP-dependent. Pipette inclusion of protein kinase A (PKA) catalytic subunit blocked the effect of L-P NPY on GABAA-mediated eIPSCs, but not on NMDA-mediated eEPSCs. Conversely, activating the exchange protein activated by cAMP (Epac) with 8CPT-2Me-cAMP blocked the effect of L-P NPY on NMDA-mediated eEPSCs, but not on GABAA-mediated eIPSCs. Thus, NPY regulates amygdala excitability via two signal-transduction events, with reduced PKA activity enhancing GABAA-mediated eIPSCs and Epac deactivation reducing NMDA-mediated eEPSCs. This multipathway regulation of NMDA- and GABAA-mediated currents may be important for NPY plasticity and stress resilience in the amygdala.
Assuntos
Tonsila do Cerebelo/fisiologia , Neuropeptídeo Y/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Neuropeptídeos/fisiologia , Transdução de Sinais/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Aminoquinolinas/farmacologia , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Ansiolíticos/farmacologia , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neuropeptídeo Y/administração & dosagem , Neuropeptídeo Y/análogos & derivados , Neuropeptídeo Y/antagonistas & inibidores , Neuropeptídeo Y/farmacologia , Ratos , Receptores de AMPA/efeitos dos fármacos , Receptores de AMPA/fisiologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/fisiologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/fisiologia , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Tionucleotídeos/farmacologiaRESUMO
It is well known that sulphonylureas (SUs), commonly used in the treatment of type 2 diabetes mellitus, stimulate insulin secretion by closing ATP-sensitive K(+) (K(ATP) ) channels in pancreatic ß-cells by binding to the SU receptor SUR1. SUs are now known also to activate cAMP sensor Epac2 (cAMP-GEFII) to Rap1 signalling, which promotes insulin granule exocytosis. For SUs to exert their full effects in insulin secretion, they are required to activate Epac2 as well as to inhibit the ß-cell K(ATP) channels. As Epac2 is also necessary for potentiation of glucose-induced insulin secretion by cAMP-increasing agents, such as incretin, Epac2 is a target of both cAMP and SUs. The distinct effects of various SUs appear to be because of their different actions on Epac2/Rap1 signalling as well as K(ATP) channels. Differently from other SUs, gliclazide is unique in that it is specific for ß-cell K(ATP) channel and does not activate Epac2.
Assuntos
Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hipoglicemiantes/uso terapêutico , Células Secretoras de Insulina/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Receptores de Droga/efeitos dos fármacos , Compostos de Sulfonilureia/uso terapêutico , Transportadores de Cassetes de Ligação de ATP/metabolismo , AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Exocitose , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Canais KATP/efeitos dos fármacos , Canais KATP/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Droga/metabolismo , Complexo Shelterina , Transdução de Sinais/efeitos dos fármacos , Receptores de Sulfonilureias , Proteínas de Ligação a Telômeros/efeitos dos fármacos , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Endogenous noradrenaline presumably prohibits neuroinflammation by stimulation of ß-adrenergic receptor-dependent suppression of the production of inflammatory mediators. Using the microglial cell line, BV-2, as well as primary murine microglial cells, we show here that the ß-adrenergic agonist, isoproterenol, suppresses uptake of hydrophobic polystyrene microspheres. The number of cells showing a specific number of engulfed microspheres followed a Poisson distribution. Isoproterenol decreased the number of engulfed particles per cell and the number of cells showing at least one incorporated particle. Elevation of intracellular cAMP by activation of adenylyl cyclase activity with forskolin, suppression of phosphodiesterase activity with 3-isobutyl-1-methylxanthine (IBMX), or application of the membrane-permeable cAMP analog, 8-bromo-cAMP, suppressed particle uptake. The protein kinase A inhibitor, H-89, did not prevent isoproterenol-dependent suppression of particle engulfment. However, activation of exchange protein activated by cAMP (Epac), specific guanine nucleotide exchange factors for the Ras GTPase homologues, Rap1 and Rap2, with the Epac1-specific cAMP analog, 8-pCPT-2'-O-Me-cAMP, mimicked the suppressive effect of isoproterenol on particle uptake. Our results suggest that ß-adrenergic receptor stimulation suppresses particle uptake in microglia by cAMP-dependent activation of Epac.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Microglia/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Isoproterenol/farmacologia , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microesferas , Norepinefrina/fisiologia , Diester Fosfórico Hidrolases/metabolismo , Poliestirenos , Transdução de Sinais/efeitos dos fármacos , Estimulação QuímicaRESUMO
Mechanisms of brain metastatic melanoma (BMM) remain largely unknown. Understanding the modulation of signaling pathways that alter BMM cell invasion and metastasis is critical to develop new therapies for BMM. Heparanase has been widely implicated in cancer and is the dominant mammalian endoglycosidase which degrades heparan sulfate chains of proteoglycans (HSPG) including syndecans (SDCs). Recent findings also indicate that heparanase possesses non-enzymatic functions in its latent form. We hypothesized that extracellular heparanase modulates BMM cell signaling by involving SDC1/4 carboxy terminal-associated proteins and downstream targets. We digested BMM cell surface HS with human recombinant active or latent heparanase to delineate their effects on cytoskeletal dynamics and cell invasiveness. We identified the small GTPase guanine nucleotide exchange factor-H1 (GEF-H1) as a new component of a SDC signaling complex that is differentially expressed in BMM cells compared to corresponding non-metastatic counterparts. Second, knockdown of GEF-H1, SDC1, or SDC4 decreased BMM cell invasiveness and GEF-H1 modulated small GTPase activity of Rac1 and RhoA in conjunction with heparanase treatment. Third, both active and latent forms of heparanase affected Rac1 and RhoA activity; notably increasing RhoA activity. Both forms of heparanase were found to mediate the expression and subcellular localization of GEF-H1, and treatment of BMM with latent heparanase modulated SDC1/4 gene expression. Finally, treatment with exogenous heparanase downregulated BMM cell invasion. These studies indicate the relevance of heparanase signaling pathways in BMM progression, and provide insights into the molecular mechanisms regulating HSPG signaling in response to exogenous heparanase.
Assuntos
Neoplasias Encefálicas/secundário , Glucuronidase/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Melanoma/patologia , Transdução de Sinais , Citoesqueleto/metabolismo , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Proteoglicanas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Sindecanas/metabolismoRESUMO
It has been recently shown that beta-adrenergic receptors are able to activate phospholipase C via the cyclic adenosine monophosphate-binding protein Epac. This new interconnection may participate in isoproterenol (Iso)-induced preconditioning. We evaluated here whether Epac could induce PKCepsilon activation and could play a role in ischemic preconditioning through the phosphorylation of connexin43 (Cx43) and changes in gap junctional intercellular communication (GJIC). In cultured rat neonatal cardiomyocytes, we showed that in response to Iso and 8-CPT, a specific Epac activator, PKCepsilon content was increased in particulate fractions of cell lysates independently of protein kinase A (PKA). This was associated with an increased Cx43 phosphorylation. Both Iso and 8-CPT induced an increase in GJIC that was blocked by the PKC inhibitor bisindolylmaleimide. Interestingly, inhibition of PKA partly suppressed both Iso-induced increases in Cx43 phosphorylation and in GJIC. The same PKCepsilon-dependent Cx43 phosphorylation by beta-adrenergic stimulation via Epac was found in adult rat hearts. However, in contrast with Iso that induced a preconditioning effect, perfusion of isolated hearts with 8-CPT prior to ischemia failed to improve the post-ischemia functional recovery. In conclusion, Epac stimulation induces PKCepsilon activation and Cx43 phosphorylation with an increase in GJIC, but Epac activation does not induce preconditioning to ischemia in contrast with beta-adrenergic stimulation.
Assuntos
Conexina 43/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Precondicionamento Isquêmico Miocárdico , Miócitos Cardíacos/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Western Blotting , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/fisiologia , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Isoproterenol/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Fosforilação , Proteína Quinase C-épsilon/metabolismo , Ratos , Teofilina/análogos & derivados , Teofilina/farmacologiaRESUMO
BACKGROUND: Long-lasting increases in dendritic spine density and gene expression in the nucleus accumbens and in the ambulatory response to cocaine occur following chronic cocaine treatment. Despite numerous reports of these findings, the molecular mechanisms leading to these morphological, biochemical, and behavioral changes remain unclear. METHODS: We used mice genetically lacking Kalirin7 (Kal7(KO)), a Rho guanine nucleotide exchange factor that regulates dendritic spine formation and function. Both wild-type (Wt) and Kal7(KO) mice were given high-dose cocaine (20 mg/kg) for 4 or 8 consecutive days. Locomotor sensitization and conditioned place preference elicited by cocaine were evaluated. The nucleus accumbens core was diolistically labeled and spine density and morphology were quantified using confocal microscopy. RESULTS: Cocaine increased Kalirin7 messenger RNA and protein expression in the nucleus accumbens of Wt mice. The Kal7(KO) animals showed greater locomotor sensitization to cocaine than Wt mice. In contrast, Kal7(KO) mice exhibited decreased place preference for cocaine, despite displaying a normal place preference for food. While Wt mice showed a robust increase in dendritic spine density after 4 and 8 days of cocaine treatment, dendritic spine density failed to increase in cocaine-exposed Kal7(KO) mice. Wild-type mice treated with cocaine for 8 days exhibited larger dendritic spines than cocaine-treated Kal7(KO) mice. CONCLUSIONS: Kalirin7 is an essential determinant of dendritic spine formation following cocaine treatment. The absence of this single isoform of one of the many Rho guanine nucleotide exchange factors expressed in the nucleus accumbens results in enhanced locomotor sensitization and diminished place preference in response to cocaine.
Assuntos
Comportamento Animal/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Cocaína/farmacologia , Espinhas Dendríticas/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Núcleo Accumbens/efeitos dos fármacos , Animais , Aprendizagem por Associação/efeitos dos fármacos , Condicionamento Clássico/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Núcleo Accumbens/citologia , Núcleo Accumbens/metabolismo , RNA Mensageiro/análiseRESUMO
Mantle cell lymphoma (MCL) is characterized by translocation t(11;14)(q13;q32), aggressive clinical behaviour, and poor patient outcomes following conventional chemotherapy. New treatment approaches are needed that target novel biological pathways. All trans retinoic acid (ATRA) is a key retinoid that acts through nuclear receptors that function as ligand-inducible transcription factors. The present study evaluated cell killing effects of ATRA-enriched nanoscale delivery particles, termed nanodisks (ND), on MCL cell lines. Results show that ATRA-ND induced cell death more effectively than naked ATRA (dimethyl sulphoxide) or empty ND. ATRA-ND induced reactive oxygen species (ROS) generation to a greater extent than naked ATRA. The antioxidant, N-acetylcysteine, inhibited ATRA-ND induced apoptosis. Compared to naked ATRA, ATRA-ND enhanced G1 growth arrest, up-regulated p21and p27, and down regulated cyclin D1. At ATRA concentrations that induced apoptosis, expression levels of retinoic acid receptor-alpha (RARalpha) and retinoid X receptor-gamma (RXRgamma) were increased. Compared to naked ATRA, ATRA-ND significantly stimulated transcriptional activity of RARA in a model carcinoma cell line. Furthermore, the RAR antagonist, Ro 41-5253, inhibited ATRA-ND induced ROS generation and prevented ATRA-ND induced cell growth arrest and apoptosis. In summary, incorporation of ATRA into ND enhanced the biological activity of this retinoid in cell culture models of MCL.
Assuntos
Antineoplásicos/farmacologia , Linfoma de Célula do Manto/patologia , Receptores do Ácido Retinoico/efeitos dos fármacos , Tretinoína/farmacologia , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Benzoatos/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/efeitos dos fármacos , Cromanos/farmacologia , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Humanos , Linfoma de Célula do Manto/metabolismo , Nanopartículas , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas Nucleares/biossíntese , Proteínas Nucleares/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides/efeitos dos fármacos , Receptores X de Retinoides/metabolismo , Transcrição Gênica/efeitos dos fármacos , Tretinoína/administração & dosagem , Células Tumorais CultivadasRESUMO
In infected cells, hepatitis C virus (HCV) induces the formation of membrane alterations referred to as membranous webs, which are sites of RNA replication. In addition, HCV RNA replication also occurs in smaller membrane structures that are associated with the endoplasmic reticulum. However, cellular mechanisms involved in the formation of HCV replication complexes remain largely unknown. Here, we used brefeldin A (BFA) to investigate cellular mechanisms involved in HCV infection. BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARF), which can lead to a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways. Our data show that HCV RNA replication is highly sensitive to BFA. Individual knockdown of the cellular targets of BFA using RNA interference and the use of a specific pharmacological inhibitor identified GBF1, a guanine nucleotide exchange factor for small GTPases of the ARF family, as a host factor critically involved in HCV replication. Furthermore, overexpression of a BFA-resistant GBF1 mutant rescued HCV replication in BFA-treated cells, indicating that GBF1 is the BFA-sensitive factor required for HCV replication. Finally, immunofluorescence and electron microscopy analyses indicated that BFA does not block the formation of membranous web-like structures induced by expression of HCV proteins in a nonreplicative context, suggesting that GBF1 is probably involved not in the formation of HCV replication complexes but, rather, in their activity. Altogether, our results highlight a functional connection between the early secretory pathway and HCV RNA replication.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hepacivirus/fisiologia , RNA Viral/biossíntese , Replicação Viral , Fatores de Ribosilação do ADP/metabolismo , Animais , Brefeldina A/farmacologia , Linhagem Celular , Imunofluorescência , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Humanos , Microscopia Eletrônica , Replicação Viral/efeitos dos fármacosRESUMO
A variety of cardiovascular, neurological, and neoplastic conditions have been associated with oxidative stress, i.e., conditions under which levels of reactive oxygen species (ROS) are elevated over significant periods. Nuclear factor erythroid 2-related factor (Nrf2) regulates the transcription of several gene products involved in the protective response to oxidative stress. The transcriptional regulatory and signaling relationships linking gene products involved in the response to oxidative stress are, currently, only partially resolved. Microarray data constitute RNA abundance measures representing gene expression patterns. In some cases, these patterns can identify the molecular interactions of gene products. They can be, in effect, proxies for protein-protein and protein-DNA interactions. Traditional techniques used for clustering coregulated genes on high-throughput gene arrays are rarely capable of distinguishing between direct transcriptional regulatory interactions and indirect ones. In this study, newly developed information-theoretic algorithms that employ the concept of mutual information were used: the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE), and Context Likelihood of Relatedness (CLR). These algorithms captured dependencies in the gene expression profiles of the mouse lung, allowing the regulatory effect of Nrf2 in response to oxidative stress to be determined more precisely. In addition, a characterization of promoter sequences of Nrf2 regulatory targets was conducted using a Support Vector Machine classification algorithm to corroborate ARACNE and CLR predictions. Inferred networks were analyzed, compared, and integrated using the Collective Analysis of Biological Interaction Networks (CABIN) plug-in of Cytoscape. Using the two network inference algorithms and one machine learning algorithm, a number of both previously known and novel targets of Nrf2 transcriptional activation were identified. Genes predicted as novel Nrf2 targets include Atf1, Srxn1, Prnp, Sod2, Als2, Nfkbib, and Ppp1r15b. Furthermore, microarray and quantitative RT-PCR experiments following cigarette-smoke-induced oxidative stress in Nrf2(+/+) and Nrf2(-/-) mouse lung affirmed many of the predictions made. Several new potential feed-forward regulatory loops involving Nrf2, Nqo1, Srxn1, Prdx1, Als2, Atf1, Sod1, and Park7 were predicted. This work shows the promise of network inference algorithms operating on high-throughput gene expression data in identifying transcriptional regulatory and other signaling relationships implicated in mammalian disease.