RESUMEN
Aptamers are widely used as binders that interact with targets with high affinity or as inhibitors of the function of target molecules. However, they have also been used to modulate target protein function, which they achieve by activating the target or stabilizing its conformation. Here, we report a unique aptamer modulator of the insulin receptor (IR), IR-A62. Alone, IR-A62 acts as a biased agonist that preferentially induces Y1150 monophosphorylation of IR. However, when administered alongside insulin, IR-A62 shows variable binding cooperativity depending on the ligand concentration. At low concentrations, IR-A62 acts as a positive allosteric modulator (PAM) agonist that enhances insulin binding, but at high concentrations, it acts as a negative allosteric modulator (NAM) agonist that competes with insulin for IR. Moreover, the concentration of insulin affects the binding of IR-A62 to IR. Finally, the subcutaneous administration of IR-A62 to diabetic mice reduces blood glucose levels with a longer-lasting effect than insulin administration. These findings imply that aptamers can elicit various responses from receptors beyond those of a simple agonist or inhibitor. We expect further studies of IR-A62 to help reveal the mechanism of IR activation and greatly expand the range of therapeutic applications of aptamers.
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Diabetes Mellitus Experimental , Receptor de Insulina , Regulación Alostérica , Animales , Insulina/metabolismo , Ligandos , Ratones , Receptor de Insulina/metabolismoRESUMEN
BACKGROUND: Accumulating evidences indicate that AXL overexpression or activation is associated with cancer progression and acquired resistance to targeted anti-cancer drugs such as epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Despite recent development of several drugs that target multiple receptor tyrosine kinases (RTKs), drugs that selectively target AXL signaling are extremely rare. Short nucleic acid aptamers are non-immunogenic molecules with high binding affinity and specificity to their target molecules that could potentially be used as a novel cancer treatment. METHODS: Modified-DNA aptamers were selected on the basis of its ability to bind recombinant human AXL. AXL aptamers were selected for their inhibition of AXL and then selected aptamers were tested for their use to overcome acquired resistant to EGFR-TKI on a lung cancer cell with acquired resistance to erlotinib. RESULTS: These new AXL aptamers inhibited cell viability to an extent of 30-40% in HCC827/ER cells with acquired resistance to erlotinib. The possible mechanism of overcoming the acquired resistance may be by inhibiting the activation of Akt and Erk. Although, aptamers effectively decreased cell viability of erlotinib-resistant cell line, the combination of aptamers and erlotinib did not synergistically decrease the survival of the resistant cell line. CONCLUSIONS: We developed newly modified DNA aptamers that selectively bind to AXL receptors, and assessed their efficacy in a human lung cancer cell with acquired resistance to EGFR-TKI.
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Hepatocellular carcinoma (HCC) is the most common malignancy of the liver, which can progress rapidly and has a poor prognosis. Glypican-3 (GPC3) has been proposed to be an important diagnostic biomarker and therapeutic target for HCC. Aptamers have emerged as promising drug delivery vehicles because of their high binding affinity for target molecules. Herein, we developed G12msi, a gemcitabine-incorporated DNA aptamer, targeting GPC3, and evaluated its binding specificity and anti-tumor efficacy in GPC3-overexpressing HCC cell lines and murine xenograft models. GPC3-targeted aptamers were selected by using the SELEX process and the chemotherapy drug gemcitabine was internally incorporated into the aptamer. To determine the binding affinity and internalization of the G12msi, flow cytometry and confocal microscopy were performed on GPC3-positive HepG2, Hep3B, and Huh7 cells, as well as a GPC3-negative A431 cell. The anti-tumor activities of G12msi were evaluated with in vitro and in vivo models. We found that G12msi binds to GPC3-overexpressing HCC tumor cells with high specificity and is effectively internalized. Moreover, G12msi treatment inhibited the cell proliferation of GPC3-positive HCC cell lines with minimal cytotoxicity in control A431 cells. In vivo systemic administration of G12msi significantly inhibited tumor growth of HCC HepG2 cells in xenograft models without causing toxicity. These results suggest that gemcitabine-incorporated GPC3 aptamer-based drug delivery may be a promising strategy for the treatment of HCC.
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We validated the diagnostic performance of a previously developed blood-based 7-protein biomarker panel, AptoDetect™-Lung (Aptamer Sciences Inc., Pohang, Korea) using modified aptamer-based proteomic technology for lung cancer detection. Non-small cell lung cancer (NSCLC), 200 patients and benign nodule controls, 200 participants were enrolled. In a high-risk population corresponding to ≥ 55 years of age and ≥ 30 pack-years, the diagnostic performance was improved, showing 73.3% sensitivity and 90.5% specificity with an area under the curve of 0.88. AptoDetect™-Lung (Aptamer Sciences Inc.) offers the best validated performance to discriminate NSCLC from benign nodule controls in a high-risk population and could play a complementary role in lung cancer screening.
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Biomarcadores de Tumor/sangre , Proteínas Sanguíneas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Adulto , Anciano , Área Bajo la Curva , Carcinoma de Pulmón de Células no Pequeñas/patología , Femenino , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Curva ROC , República de Corea , Sensibilidad y Especificidad , FumarRESUMEN
The insulin receptor is an important regulator of metabolic processes in the body, and in particular of glucose homeostasis, including glucose uptake into peripheral tissues. Thus, insulin administration is an effective treatment for diabetes, which is characterized by chronic elevation of blood glucose. However, insulin is not only a metabolic regulator, but also functions as a growth hormone. Accordingly, studies of long-term insulin administration and of the hyperinsulinemia associated with type 2 diabetes have raised concerns about possible increases in the risks of cancer and atherosclerosis, due to excessive stimulation of cell proliferation. Interestingly, some insulin receptor ligands that have been developed based on a peptide, an antibody, and an aptamer selectively have metabolic effects exerted through the insulin receptor but do not cause significant cellular proliferation. Although these ligands therefore have potential as anti-diabetic drugs for advanced diabetes care, the mechanism whereby they specifically activate the insulin receptor is still unclear. Recently, studies of the structure of the insulin receptor have progressed considerably, and have provided further mechanistic understanding of insulin receptor activation. Based on this progress, we propose a mechanistic model of this specificity and discuss the potential for the development of novel anti-diabetic drugs that would not have the adverse effects caused by excessive mitogenic action.
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Diabetes Mellitus/metabolismo , Hipoglucemiantes/farmacología , Receptor de Insulina/metabolismo , Animales , Diabetes Mellitus/tratamiento farmacológico , Humanos , Hipoglucemiantes/uso terapéutico , Insulina/farmacología , Insulina/uso terapéuticoRESUMEN
Diabetic nephropathy, the major cause of chronic kidney disease, is associated with progressive renal fibrosis. Recently, accumulation of periostin, an extracellular matrix protein, was shown to augment renal fibrosis. Aptamers have higher binding affinities without developing the common side effects of antibodies. Thus, we evaluated the effect of periostin inhibition by an aptamer-based inhibitor on renal fibrosis under diabetic conditions. In vitro, transforming growth factor-ß1 (TGF-ß1) treatment significantly upregulated periostin, fibronectin, and type I collagen mRNA and protein expressions in inner medullary collecting duct (IMCD) cells. These increases were attenuated significantly in periostin-binding DNA aptamer (PA)-treated IMCD cells exposed to TGF-ß1. In vivo, PA treatment attenuated the increased blood urea nitrogen levels in the diabetic mice significantly. Fibronectin and type I collagen mRNA and protein expressions increased significantly in the kidneys of diabetic mice: PA administration abrogated these increases significantly. Immunohistochemistry and Sirius Red staining also revealed that fibronectin expression was significantly higher and tubulointersititial fibrosis was significantly worse in diabetic mice kidneys compared with control mice. These changes were ameliorated by PA treatment. These findings suggested that inhibition of periostin using a DNA aptamer could be a potential therapeutic strategy against renal fibrosis in diabetic nephropathy.
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Aptámeros de Nucleótidos/administración & dosificación , Moléculas de Adhesión Celular/antagonistas & inhibidores , Nefropatías Diabéticas/tratamiento farmacológico , Fibrosis/tratamiento farmacológico , Animales , Aptámeros de Nucleótidos/metabolismo , Nitrógeno de la Urea Sanguínea , Moléculas de Adhesión Celular/metabolismo , Colágeno Tipo I/antagonistas & inhibidores , Nefropatías Diabéticas/patología , Modelos Animales de Enfermedad , Fibronectinas/antagonistas & inhibidores , Fibrosis/patología , Ratones , Unión Proteica , Resultado del TratamientoRESUMEN
Peritoneal fibrosis is a major complication in peritoneal dialysis (PD) patients, which leads to dialysis discontinuation. Periostin, increased by transforming growth factor ß1 (TGF-ß1) stimulation, induces the expression of extracellular matrix (ECM) genes. Aberrant periostin expression has been demonstrated to be associated with PD-related peritoneal fibrosis. Therefore, the effect of periostin inhibition by an aptamer-based inhibitor on peritoneal fibrosis was evaluated. In vitro, TGF-ß1 treatment upregulated periostin, fibronectin, α-smooth muscle actin (α-SMA), and Snail expression and reduced E-cadherin expression in human peritoneal mesothelial cells (HPMCs). Periostin small interfering RNA (siRNA) treatment ameliorated the TGF-ß1-induced periostin, fibronectin, α-SMA, and Snail expression and restored E-cadherin expression in HPMCs. Similarly, the periostin-binding DNA aptamer (PA) also attenuated fibronectin, α-SMA, and Snail upregulation and E-cadherin downregulation in TGF-ß1-stimulated HPMCs. In mice treated with PD solution for 4 weeks, the expression of periostin, fibronectin, α-SMA, and Snail was significantly increased in the peritoneum, whereas E-cadherin expression was significantly decreased. The thickness of the submesothelial layer and the intensity of Masson's trichrome staining in the PD group were significantly increased compared to the untreated group. These changes were significantly abrogated by the intraperitoneal administration of PA. These findings suggest that PA can be a potential therapeutic strategy for peritoneal fibrosis in PD patients.
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BACKGROUND: Lung cancer screening using low-dose computed tomography reduces lung cancer mortality. However, the high false-positive rate, cost, and potential harms highlight the need for complementary biomarkers. We compared the diagnostic performance of modified aptamer-based protein biomarkers with Cyfra 21-1. PATIENTS AND METHODS: Participants included 100 patients diagnosed with lung cancer, and 100 control subjects from Asan Medical Center (Seoul, Korea). We investigated candidate biomarkers with new modified aptamer-based proteomic technology and developed a 7-protein panel that discriminates lung cancer from controls. A naive Bayesian classifier was trained using sera from 75 lung cancers and 75 controls. An independent set of 25 cases and 25 controls was used to verify performance of this classifier. The panel results were compared with Cyfra 21-1 to evaluate the diagnostic accuracy for lung nodules detected by computed tomography. RESULTS: We derived a 7-protein biomarker classifier from the initial train set comprising: EGFR1, MMP7, CA6, KIT, CRP, C9, and SERPINA3. This classifier distinguished lung cancer cases from controls with an area under the curve (AUC) of 0.82 in the train set and an AUC of 0.77 in the verification set. The 7-marker naive Bayesian classifier resulted in 91.7% specificity with 75.0% sensitivity for the subset of individuals with lung nodules. The AUC of the classifier for lung nodules was 0.88, whereas Cyfra 21-1 had an AUC of 0.72. CONCLUSION: We have developed a protein biomarker panel to identify lung cancers from controls with a high accuracy. This integrated noninvasive approach to the evaluation of lung nodules deserves further prospective validation among larger cohorts of patients with lung nodules in screening strategy.
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Adenocarcinoma/diagnóstico , Biomarcadores de Tumor/sangre , Carcinoma de Células Grandes/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Proteómica/métodos , Adenocarcinoma/sangre , Antígenos de Neoplasias/sangre , Aptámeros de Péptidos/metabolismo , Área Bajo la Curva , Teorema de Bayes , Carcinoma de Células Grandes/sangre , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Células Escamosas/sangre , Estudios de Casos y Controles , Detección Precoz del Cáncer , Femenino , Humanos , Queratina-19/sangre , Neoplasias Pulmonares/sangre , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , PronósticoRESUMEN
Periostin is an extracellular matrix (ECM) protein that is overexpressed in a variety of human cancers, and its functions appear to be linked to tumor growth, metastasis, and angiogenesis. Recent clinical evidence suggests that aberrant periostin expression is correlated with poor outcome in patients with breast cancer. To identify novel tools to regulate the functional role of periostin, we generated benzyl-d(U)TP-modified DNA aptamers that were directed against human periostin (PNDAs) and characterized their functional roles in breast cancer progression. PNDA-3 selectively bound to the FAS-1 domain of periostin with nanomolar affinity and disrupted the interaction between periostin and its cell surface receptors, αvß3 and αvß5 integrins. PNDA-3 markedly antagonized the periostin-induced adhesion, migration, and invasion of breast cancer cells and blocked the activation of various components of the αvß3 and αvß5 integrin signal transduction pathways. In a 4T1 orthotopic mouse model, PNDA-3 administration significantly reduced primary tumor growth and distant metastasis. Thus, our results demonstrated that periostin-integrin signaling regulates breast cancer progression at multiple levels in tumor cells and the tumor microenvironment. DNA aptamers targeting periostin may potentially be used to inhibit breast cancer progression.
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Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Moléculas de Adhesión Celular/metabolismo , Animales , Aptámeros de Nucleótidos/química , Secuencia de Bases , Neoplasias de la Mama/patología , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Integrinas/metabolismo , Ratones , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Unión Proteica , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We introduce a new methodology named ligand profiling and identification for effective discovery of bioactive ligands such as peptide hormones. This technology was developed from a new concept of parallel column chromatography and active fraction profiling by nano-LC MS. Traditional methods use sequential column chromatography, and thus are inevitably limited by the low abundance of the peptide of interest and by a low yield due to the many column steps. Using this new technology, insulin was successfully identified and diarginylinsulin, a minor intermediate form of insulin, was unexpectedly also identified simultaneously from 100 mg of porcine pancreatic tissue. This integrative technology could be used to search for various low-abundance peptides (or bioactive molecules) rapidly and simultaneously, by applying this to the later stages of traditional sequential purification.
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Cromatografía Liquida/métodos , Insulina/análisis , Espectrometría de Masas/métodos , Péptidos/análisis , Animales , Western Blotting , Células Cultivadas , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Densitometría , Fibroblastos/citología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Insulina/análogos & derivados , Ligandos , Nanotecnología , Páncreas/química , Péptido Hidrolasas/farmacología , Ratas , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , PorcinosRESUMEN
Although the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in the regulation of several immune responses, its target receptors and signaling mechanisms have yet to be fully elucidated in immune cells. In this study, we found that PACAP27, but not PACAP38, specifically stimulated intracellular calcium mobilization and ERK phosphorylation in human neutrophils. Moreover, formyl peptide receptor-like 1 (FPRL1) was identified as a PACAP27 receptor, and PACAP27 was found to selectively stimulate intracellular calcium increase in FPRL1-transfected rat basophil leukocytes-2H3 cell lines. In addition, PACAP27-induced calcium increase and ERK phosphorylation were specifically inhibited by an FPRL1 antagonist, Trp-Arg-Trp-Trp-Trp-Trp (WRW4), thus supporting the notion that PACAP27 acts on FPRL1. In terms of the functional role of PACAP27, we found that the peptide stimulated CD11b surface up-regulation and neutrophil chemotactic migration, and that these responses were completely inhibited by WRW4. The interaction between PACAP27 and FPRL1 was analyzed further using truncated PACAPs and chimeric PACAPs using vasoactive intestinal peptide, and the C-terminal region of PACAP27 was found to perform a vital function in the activation of FPRL1. Taken together, our study suggests that PACAP27 activates phagocytes via FPRL1 activation, and that this results in proinflammatory behavior, involving chemotaxis and the up-regulation of CD11b.
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Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Secuencia de Aminoácidos , Animales , Antígeno CD11b/biosíntesis , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Ligandos , Datos de Secuencia Molecular , Activación Neutrófila/inmunología , Neutrófilos/enzimología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Isoformas de Proteínas/metabolismo , Ratas , Transducción de Señal/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Phospholipase A(2) plays a key role in phagocytic cell functions. By screening a synthetic hexapeptide combinatorial library, we identified 24 novel peptides based on their ability to stimulate arachidonic acid release associated with cytosolic phospholipase A(2) activity in differentiated HL60 cells. The identified peptides, that contain the consensus sequence (K/R/M)KYY(P/V/Y)M, also induce intracellular calcium release in a pertussis toxin-sensitive manner showing specific action on phagocytic leukocytes, but not on other cells. Functionally, the peptides stimulate superoxide generation and chemotactic migration in human neutrophils and monocytes. Four of the tested active peptides were ligands for formyl peptide receptor like 1. Among these, two peptides with the consensus sequence (R/M)KYYYM can induce intracellular calcium release in undifferentiated HL60 cells that do not express formyl peptide receptor like 1, indicating usage of other receptor(s). A study of intracellular signaling in differentiated HL60 cells induced by the peptides has revealed that four of the novel peptides can induce extracellular signal-regulated protein kinase activation via shared and distinct signaling pathways, based on their dependence of phospatidylinositol-3-kinase, protein kinase C, and MEK. These peptides provide previously unavailable tools for study of differential signaling in leukocytes.
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Factores Quimiotácticos/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Leucocitos/efectos de los fármacos , Fosfolipasas A/fisiología , Ácido Araquidónico/metabolismo , Calcio/metabolismo , Quimiotaxis de Leucocito/fisiología , Células HL-60 , Humanos , Leucocitos/fisiología , Péptidos/farmacología , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Transducción de Señal/efectos de los fármacos , Superóxidos/metabolismoRESUMEN
Formyl peptide receptor (FPR) and formyl peptide receptor like 1 (FPRL1) play important roles in inflammation and immunity. Stimulation of FPR and FPRL1 initiates a cascade of signaling events, leading to activation of various phagocyte responses, including chemotaxis, superoxide generation, and exocytosis. Trp-Lys-Tyr-Met-Val-d-Met-NH2 (WKYMVm) is a synthetic peptide that binds to and activates FPR and FPRL1. To develop agonists that selectively activate phagocyte functions and therefore protect host from unwanted tissue damage, we generated various WKYMVm analogs and examined their effects on cellular responses in FPR- or FPRL1-expressing RBL-2H3 cells. Analogs with substitution at the third position such as WKGMVm, WKRMVm, as well as analogs with substitution at the sixth d-Met, selectively altered calcium mobilization in cells expressing FPRL1 but not in cells expressing FPR. Whereas binding of WKYMVm to FPR activates a broad spectrum of cellular signaling events, including phospholipase C-mediated intracellular calcium concentration ([Ca2+]i) mobilization and activation of extracellular signal-regulated kinase (ERK) and Akt, WKGMVm and WKRMVm could only activate ERK and Akt but did not induce [Ca2+]i mobilization. With respect to phagocyte functions, WKYMVm could induce both chemotaxis and exocytosis, but the two analogs WKGMVm and WKRMVm could only induce chemotaxis but not exocytosis. This study demonstrates that a major phagocyte chemoattractant receptor FPR may be activated differentially by distinct peptide ligands. Our results suggest that WKGMVm and WKRMVm may be useful model for further development of pharmacological agents that selectively activate FPR-mediated functions.
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Quimiotaxis/efectos de los fármacos , Oligopéptidos/farmacología , Proteínas Serina-Treonina Quinasas , Receptores Inmunológicos/metabolismo , Receptores de Lipoxina , Receptores de Péptidos/metabolismo , Línea Celular , Exocitosis/efectos de los fármacos , Humanos , Ligandos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores de Formil Péptido , Receptores Inmunológicos/efectos de los fármacos , Receptores de Péptidos/efectos de los fármacosRESUMEN
Phosphoinositide-specific phospholipase C (PLC) plays a pivotal role in the signal transduction of various cellular responses. However, although it is undeniably important that modulators of PLC activity be identified, no direct PLC activity modulator has been identified until now. In this study, by screening more than 10,000 different compounds in human neutrophils, we identified a compound that strongly enhances superoxide-generating activity, which is well known to be PLC-dependent. The active compound 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS) stimulated a transient intracellular calcium concentration ([Ca(2+)](i)) increase in neutrophils. Moreover, m-3M3FBS stimulated the formation of inositol phosphates in U937 cells, indicating that it stimulates PLC activity. The compound showed no cell-type specificity in terms of [Ca(2+)](i) increase in the various cell lines including leukocytes, fibroblasts, and neuronal cells. We also ruled out the possible involvement of heterotrimeric G proteins in m-3M3FBS-stimulated signaling by confirming the following: 1) pertussis toxin does not inhibit m-3M3FBS-induced [Ca(2+)](i) increase; 2) m-3M3FBS does not stimulate cyclic AMP generation; and 3) the inhibition of G(q) by the regulator of G protein-signaling 2 does not affect the m-3M3FBS-induced [Ca(2+)](i) increase. We also observed that m-3M3FBS stimulated PLC activity in vitro. The purified isoforms of PLC that were tested (i.e., beta2, beta3, gamma1, gamma2, and delta1) were activated by m-3M3FBS and showed no isoform specificity. Taken together, these results demonstrate that m-3M3FBS modulates neutrophil functions by directly activating PLC. Because m-3M3FBS is the first compound known to directly activate PLC, it should prove useful in the study of the basic molecular mechanisms of PLC activation and PLC-mediated cell signaling.
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Activadores de Enzimas/farmacología , Neutrófilos/efectos de los fármacos , Sulfonamidas/farmacología , Fosfolipasas de Tipo C/metabolismo , Células 3T3 , Animales , Calcio/metabolismo , Activación Enzimática/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Células HL-60 , Células HeLa , Humanos , Fosfatos de Inositol/biosíntesis , Ratones , Neutrófilos/metabolismo , Células PC12 , Ratas , Transducción de Señal/efectos de los fármacos , Superóxidos/metabolismo , Células U937RESUMEN
Previously, we showed that Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) stimulates superoxide generation and chemotactic migration in monocytes and neutrophils. In this study, we examined the effect of WKYMVm on monocyte survival. Serum starvation-induced monocyte death was attenuated in the presence of WKYMVm, which was abated when the cells were preincubated with LY294002, suggesting the involvement of phosphoinositide-3-kinase (PI 3-kinase) in the peptide-induced monocyte survival. WKYMVm stimulated ERK and Akt activity via PI 3-kinase activation in monocytes. We also investigated the signaling pathway of WKYMVm-induced ERK and Akt activation. The WKYMVm-induced ERK activation was PI 3-kinase-dependent but PKC-independent. However, Akt activation by WKYMVm was dependent not only on PI 3-kinase but also on the PKC pathway. When monocytes were incubated with WKYMVm, caspase-3 activity, which is important for cell death, was inhibited. Pretreatment of the cells with LY294002, GF109203X, and Go 6976 but not PD98059 blocked WKYMVm-induced monocyte survival and caspase-3 inhibition. In summary, the novel chemoattractant WKYMVm enhances monocyte survival via Akt-mediated pathways, and in this process, PKC and PI 3-kinase act upstream of Akt.
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Proteínas Quimioatrayentes de Monocitos/farmacología , Monocitos/citología , Monocitos/efectos de los fármacos , Oligopéptidos/farmacología , Proteínas Serina-Treonina Quinasas , Transducción de Señal/efectos de los fármacos , Adulto , Supervivencia Celular/efectos de los fármacos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Monocitos/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal/fisiologíaRESUMEN
Although the functional significance of neuronal phospholipase D (PLD) is being recognized, little is known about its regulatory role in neuronal cells. To elucidate the regulatory mechanism of neuronal PLD, we investigated PLD(2)-binding neuronal protein from rat brain cytosol. During the fractionation of rat brain cytosol by four-column chromatography, a 62-kDa PLD(2)-interacting protein was detected by PLD(2) overlay assay and identified as collapsin response mediator protein-2 (CRMP-2), which controls neuronal axon guidance and outgrowth. Using bacterially expressed glutathione S-transferase fusion proteins, we found that two regions (amino acids 65-192 (the phagocytic oxidase domain) and 724-825) of PLD(2) and a single region (amino acids 243-300) of CRMP-2 are required for the direct binding of both proteins. A co-immunoprecipitation study in COS-7 cells also showed an in vivo interaction between CRMP-2 and PLD(2). Interestingly, CRMP-2 was found to potently inhibit PLD(2) activity in a concentration-dependent manner (IC(50) = 30 nm). Overexpression studies also showed that CRMP-2 is an in vivo inhibitor of PLD(2) in PC12 cells. Moreover, increasing the concentration of semaphorin 3A, one of the repulsive axon guidance cues, showed that PLD(2) activity can be inhibited in PC12 cells. Immunocytochemistry further revealed that PLD(2) is co-localized with CRMP-2 in the distal tips of neurites, its possible action site, in differentiated PC12 cells. Taken together, our results indicate that CRMP-2 may interact directly with and inhibit neuronal PLD(2), suggesting that this inhibitory mode of regulation may play a role in neuronal pathfinding during the developmental stage.