RESUMEN
Dissemination of HIV in the host involves transit of the virus and virus-infected cells across the lymphatic endothelium. HIV may alter lymphatic endothelial permeability to foster dissemination, but the mechanism is largely unexplored. Using a primary human lymphatic endothelial cell model, we found that HIV-1 envelope protein gp120 induced lymphatic hyperpermeability by disturbing the normal function of Robo4, a novel regulator of endothelial permeability. HIV-1 gp120 induced fibronectin expression and integrin α5ß1 phosphorylation, which led to the complexing of these three proteins, and their subsequent interaction with Robo4 through its fibronectin type III repeats. Moreover, pretreatment with an active N-terminus fragment of Slit2, a Robo4 agonist, protected lymphatic endothelial cells from HIV-1 gp120-induced hyperpermeability by inhibiting c-Src kinase activation. Our results indicate that targeting Slit2/Robo4 signaling may protect the integrity of the lymphatic barrier and limit the dissemination of HIV in the host.
Asunto(s)
Endotelio Linfático/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Activación Enzimática , Genes src , Células HEK293 , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/genética , Humanos , Integrina alfa5beta1/genética , Integrina alfa5beta1/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas del Tejido Nervioso/genética , Permeabilidad , Receptores de Superficie Celular/genéticaRESUMEN
BACKGROUND: Signaling through vascular endothelial growth factor C (VEGFC) and VEGF receptor 3 (VEGFR-3) plays a central role in lymphangiogenesis and the metastasis of several cancers via the lymphatics. Recently, the Slit2/Robo4 pathway has been recognized as a modulator of vascular permeability and integrity. Signaling via the Robo receptor inhibits VEGF-mediated effects; however, its effects on lymphatic endothelial cell function have not been well characterized. RESULTS: We found that pretreatment with Slit2N, an active fragment of Slit2, inhibited VEGF-C-mediated lung-derived lymphatic endothelial cell (L-LEC) proliferation, migration, and in vitro tube formation. Slit2N induced the internalization of VEGFR-3, which blocked its activation, and inhibited the activation of the PI3K/Akt pathway by VEGF-C in L-LECs. Moreover, we found that inhibition of VEGF-C-induced effects by Slit2N was Robo4-dependent. CONCLUSION: These results indicate that Slit2N/Robo4 modulates several key cellular functions, which contribute to lymphangiogenesis, and identify this ligand-receptor pair as a potential therapeutic target to inhibit lymphatic metastasis of VEGF-C-overexpressing cancers and manage lymphatic dysfunctions characterized by VEGF-C/VEGFR-3 activation.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Linfangiogénesis , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Factor C de Crecimiento Endotelial Vascular/farmacología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Línea Celular , Movimiento Celular , Proliferación Celular , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Vasos Linfáticos/citología , Proteínas del Tejido Nervioso/genética , Fragmentos de Péptidos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Superficie Celular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Endocannabinoids are arachidonic acid derivatives and part of a novel bioactive lipid signaling system, along with their G-coupled cannabinoid receptors (CB1 and CB2) and the enzymes involved in their biosynthesis and degradation. However, their roles in hematopoiesis and hematopoietic stem and progenitor cell (HSPC) functions are not well characterized. Here, we show that bone marrow stromal cells express endocannabinoids (anandamide and 2-arachidonylglycerol), whereas CB2 receptors are expressed in human and murine HSPCs. On ligand stimulation with CB2 agonists, CB2 receptors induced chemotaxis, migration, and enhanced colony formation of bone marrow cells, which were mediated via ERK, PI3-kinase, and Gαi-Rac1 pathways. In vivo, the CB2 agonist AM1241 induced mobilization of murine HSPCs with short- and long-term repopulating abilities. In addition, granulocyte colony-stimulating factor -induced mobilization of HSPCs was significantly decreased by specific CB2 antagonists and was impaired in Cnr2(-/-) cannabinoid type 2 receptor knockout mice. Taken together, these results demonstrate that the endocannabinoid system is involved in hematopoiesis and that CB2/CB2 agonist axis mediates repopulation of hematopoiesis and mobilization of HSPCs. Thus, CB2 agonists may be therapeutically applied in clinical conditions, such as bone marrow transplantation.
Asunto(s)
Hematopoyesis/fisiología , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/fisiología , Receptor Cannabinoide CB2/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Moduladores de Receptores de Cannabinoides/metabolismo , Cannabinoides/farmacología , Movimiento Celular/efectos de los fármacos , Ciclohexanoles/farmacología , Femenino , Citometría de Flujo , Hematopoyesis/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/genética , Células del Estroma/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0046526.].
RESUMEN
Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB(1) and CB(2). The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB(1) receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10(7) cells), and 2-AG (75.2 ng/10(7) cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10(7) cells) and 2-AG (98.8 ng/10(7) cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB(1) agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1(-/-) showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)(-/-) mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1(-/-), as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.
Asunto(s)
Células de la Médula Ósea/metabolismo , Moduladores de Receptores de Cannabinoides/biosíntesis , Endocannabinoides , Células Madre Hematopoyéticas/metabolismo , Células del Estroma/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Animales , Ácidos Araquidónicos/biosíntesis , Western Blotting , Células de la Médula Ósea/citología , Moduladores de Receptores de Cannabinoides/fisiología , Comunicación Celular/fisiología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Ciclohexanoles/farmacología , Femenino , Citometría de Flujo , Glicéridos/biosíntesis , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/citología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Alcamidas Poliinsaturadas , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/genética , Receptor Cannabinoide CB2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Población Lateral/citología , Células de Población Lateral/metabolismo , Nicho de Células Madre/citología , Nicho de Células Madre/metabolismo , Células del Estroma/citologíaRESUMEN
Methamphetamine (Meth) abuse is a worldwide public health problem and contributes to HIV-1 pathobiology and poor adherence to anti-retroviral therapies. Specifically, Meth is posited to alter molecular mechanisms to provide a more conducive environment for HIV-1 replication and spread. Enhanced expression of inflammatory cytokines, such as Interleukin-1ß (IL-1ß), has been shown to be important for HIV-1 pathobiology. In addition, microRNAs (miRNAs) play integral roles in fine-tuning the innate immune response. Notably, the effects of Meth abuse on miRNA expression are largely unknown. We studied the effects of Meth on IL-1ß and miR-146a, a well-characterized member of the innate immune signaling network. We found that Meth induces miR-146a and triggers an IL-1ß auto-regulatory loop to modulate innate immune signaling in CD4+ T-cells. We also found that Meth enhances HIV-1 replication via IL-1 signaling. Our results indicate that Meth activates an IL-1ß feedback loop to alter innate immune pathways and favor HIV-1 replication. These observations offer a framework for designing targeted therapies in HIV-infected, Meth using hosts.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Estimulantes del Sistema Nervioso Central/toxicidad , VIH-1/efectos de los fármacos , Interleucina-1beta/metabolismo , Metanfetamina/toxicidad , Replicación Viral/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , Retroalimentación Fisiológica/efectos de los fármacos , Retroalimentación Fisiológica/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , VIH-1/inmunología , Humanos , MicroARNs/biosíntesis , MicroARNs/efectos de los fármacosRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV; also named human herpesvirus 8) is necessary but not sufficient for the development of Kaposi's sarcoma. A variety of factors may contribute to the pathogenesis of Kaposi's sarcoma in addition to KSHV. Marijuana is a widely used recreational agent, and Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the major active component of marijuana, is prescribed for medicinal use. To evaluate how cannabinoids may affect the pathogenesis of Kaposi's sarcoma, we studied primary human dermal microvascular endothelial cells (HMVEC) exposed to KSHV. There was an increased efficiency of KSHV infection in the presence of low doses of Delta(9)-THC. We also found that Delta(9)-THC increased the viral load in KSHV-infected HMVEC through activation of the KSHV lytic switch gene, the open reading frame 50. Furthermore, we observed that Delta(9)-THC stimulated expression of the KSHV-encoded viral G protein-coupled receptor and Kaposi's sarcoma cell proliferation. Our results indicate that Delta(9)-THC can enhance KSHV infection and replication and foster KSHV-mediated endothelial transformation. Thus, use of cannabinoids may place individuals at greater risk for the development and progression of Kaposi's sarcoma.
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Dronabinol/análogos & derivados , Endotelio Vascular/virología , Herpesvirus Humano 8/fisiología , Western Blotting , Adhesión Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , ADN Viral/análisis , Dronabinol/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Herpesvirus Humano 8/efectos de los fármacos , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Receptores de Quimiocina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/irrigación sanguínea , Transactivadores/metabolismo , Transfección , Carga Viral , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Methamphetamine (Meth) exacerbates HIV-1 pathobiology by increasing virus transmission and replication and accelerating clinical progression to AIDS. Meth has been shown to alter the expression of HIV-1 co-receptors and impair intrinsic resistance mechanisms of immune cells. However, the exact molecular mechanisms involved in augmenting HIV-1 replication in T-cells are still not yet clear. Here, we demonstrate that pretreatment with Meth of CD4+ T-cells enhanced HIV-1 replication. We observed upregulation of CD4+ T-cell activation markers and enhanced expression of miR-34c-5p and miR-155 in these cells. Further, we noted activation of the sigma-1 receptor and enhanced intracellular Ca2+ concentration and cAMP release in CD4+ T-cells upon Meth treatment, which resulted in increased phosphorylation and nuclear translocation of transcription factors NFκB, CREB, and NFAT1. Increased gene expression of IL-4 and IL-10 was also observed in Meth treated CD4+ T-cells. Moreover, proteasomal degradation of Ago1 occurred upon Meth treatment, further substantiating the drug as an activator of T-cells. Taken together, these findings show a previously unreported mechanism whereby Meth functions as a novel T-cell activator via the sigma-1 signaling pathway, enhancing replication of HIV-1 with expression of miR-34c-5p, and transcriptional activation of NFκB, CREB and NFAT1.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Metanfetamina/farmacología , Receptores sigma/metabolismo , Proteínas Argonautas/metabolismo , Biomarcadores/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Calcio/metabolismo , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , VIH-1/efectos de los fármacos , Humanos , MicroARNs/genética , MicroARNs/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Receptores Dopaminérgicos/metabolismo , Replicación Viral/efectos de los fármacos , Receptor Sigma-1RESUMEN
HCV and HIV infections are very common among injection drug users (IDUs). It is well known that 80-90% of HIV-infected IDUs are also infected with HCV. Furthermore, patients with HCV/HIV co-infection are at a higher risk of progressing to end-stage liver disease, namely cirrhosis. Even though there is increasing global awareness of HCV/HIV co-infection and extended therapeutic programs for this infected population, little is known about the HCV/HIV pathophysiology that mediates the rapid progression to hepatic disease. Liver disease caused by HCV/HIV co-infection is characterized by inflammation and cell-death. Recent reports suggest that the HIV and HCV envelope proteins may induce apoptosis and inflammation in hepatocytes via a novel pathway involving collaborative signaling. Moreover, HCV/HIV co-infection may also alter the cytokine production in vivo. Further studies to elucidate the molecular mechanisms of HCV and HIV-mediated pathogenesis will help in the development of therapeutic strategies against HCV/HIV co-infection in these patients.
Asunto(s)
Infecciones por VIH/epidemiología , Hepatitis C Crónica/diagnóstico , Hepatitis C Crónica/fisiopatología , Cirrosis Hepática/epidemiología , Cirrosis Hepática/patología , Abuso de Sustancias por Vía Intravenosa/epidemiología , Antígenos CD4/inmunología , Antígenos CD8/inmunología , Muerte Celular , Progresión de la Enfermedad , Hepatitis C Crónica/epidemiología , Humanos , Inflamación/epidemiología , Inflamación/inmunología , Inflamación/patologíaRESUMEN
Cannabinoids have been shown to influence the immune system. However, their immunomodulatory effects have not been extensively studied. In this investigation, we have observed that both primary and Jurkat T cells express a functional cannabinoid receptor 2 (CB(2)). Furthermore, both the synthetic cannabinoids CP55,940 and WIN55,212-2, as well as the CB(2)-selective agonist JWH-015, caused a significant inhibition of the chemokine CXCL12-induced and CXCR4-mediated chemotaxis of Jurkat T cells, as well as their transendothelial migration. Involvement of the CB(2) receptor was further confirmed by partial reversal of the inhibition using the CB(2)-specific antagonist, AM630. Similarly, CP55,940 and JWH-015 inhibited the CXCL12-induced chemotaxis of primary CD4(+) and CD8(+) T lymphocytes. Further investigation of signaling studies to delineate the mechanism of inhibition revealed that cannabinoids enhance CXCL12-induced p44/42 MAP kinase activity. However, enhanced MAP kinase activity was not responsible for the inhibition of chemotaxis. This suggests that cannabinoids differentially regulate CXCR4-mediated migration and MAP kinase activation in T cells. Cannabinoids were also found to downregulate the PMA-enhanced enzyme activity of matrix metalloproteinase-9, which is known to play an important role in transendothelial migration. This study provides novel information regarding cannabinoid modulation of functional effects in T cells.
Asunto(s)
Cannabinoides/farmacología , Quimiotaxis de Leucocito , Inmunosupresores/farmacología , Receptor Cannabinoide CB2/metabolismo , Linfocitos T/inmunología , Calcio/metabolismo , Cannabinoides/inmunología , Quimiocina CXCL12 , Quimiocinas CXC/inmunología , Quimiocinas CXC/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Ciclohexanos/farmacología , Ciclohexanoles , Endotelio Vascular/fisiología , Humanos , Indoles/farmacología , Células Jurkat , Inhibidores de la Metaloproteinasa de la Matriz , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fenoles/farmacología , Receptor Cannabinoide CB2/genética , Receptores CXCR4/metabolismo , Transducción de Señal , Linfocitos T/efectos de los fármacos , Venas Umbilicales/citologíaRESUMEN
DC-SIGN is a dendritic cell surface structure which participates in binding and transmission of HIV-1. Here, for the first time we demonstrate that cocaine induces over expression of DC-SIGN and significantly enhances virus transfer from DCs to T-cells by increasing the binding and internalization of HIV-1 in DCs. We found that cocaine activates a DC-SIGN mediated 'signalosome' complex by enhancing its association with LARG and LSP1. Further, LARG was observed to participate in DC-SIGN mediated internalization of HIV-1 in DCs. Intracellular trafficking studies of HIV-1 in cocaine treated DCs revealed increased co-localization of HIV-1 with endosomal or multi vesicular body (MVB) markers such as CD81 and VPS4 and decreased co-localization with the phagolysomal marker LAMP1; this signified altered intracellular trafficking and decreased degradation of HIV-1 in cocaine treated DCs. Furthermore, we found that cocaine induced activation of LARG which in turn activated Rho A and the focal adhesion molecules FAK, Pyk2 and paxillin. This signaling cascade enhanced the formation of an infectious synapse between DCs and T-cells. Our study provides insight into the molecular mechanisms of cocaine's contribution to key components in HIV pathogenesis and highlights novel targets for interrupting the virus life cycle in substance using hosts.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Moléculas de Adhesión Celular/metabolismo , Cocaína/metabolismo , Células Dendríticas/metabolismo , Infecciones por VIH/metabolismo , Infecciones por VIH/transmisión , Lectinas Tipo C/metabolismo , Proteínas de Microfilamentos/metabolismo , Receptores de Superficie Celular/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Transporte Biológico , Linfocitos T CD4-Positivos/efectos de los fármacos , Cocaína/farmacología , Células Dendríticas/efectos de los fármacos , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Adhesiones Focales/metabolismo , Expresión Génica , Infecciones por VIH/virología , VIH-1/fisiología , Interacciones Huésped-Patógeno , Humanos , Sinapsis Inmunológicas , Proteínas de Membrana de los Lisosomas/metabolismo , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Tetraspanina 28/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Internalización del Virus , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
We have previously shown that hepatocytes exposed to hepatitis C virus (HCV) and human immunodeficiency virus (HIV) envelope proteins undergo apoptosis. In this article, we further elucidate the signaling mechanisms that mediate this effect. We found that, in human hepatocellular carcinoma (HepG2) cells, HCV E2 protein and HIV glycoprotein (gp) 120 significantly up-regulated the Fas ligand (FasL) and enhanced the formation of the Fas death-inducing signaling complex downstream of Fas receptor activation. Moreover, after stimulation with HCV E2 and HIV gp120, enhanced expression of caspases 2 and 7 and increased caspase 3 activity were observed. In addition, we showed up-regulation of the proapoptotic molecule Bid and its association with caspase 8 after treatment with these envelope proteins. We also found that HCV E2 and HIV gp120 induced a partial translocation of Bid to the mitochondria, which resulted in the release of cytochrome C and the apoptosis-inducing factor. Thus, the results of this study suggest that FasL and Bid play an important role in HCV and HIV envelope protein-induced apoptosis.
Asunto(s)
Infecciones por VIH/patología , VIH/fisiología , Hepacivirus/fisiología , Hepatitis C/patología , Hígado/patología , Apoptosis/efectos de los fármacos , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Proteína Ligando Fas/metabolismo , Proteína gp120 de Envoltorio del VIH/farmacología , Infecciones por VIH/complicaciones , Hepatitis C/complicaciones , Hepatocitos/patología , Humanos , Hígado/enzimología , Mitocondrias Hepáticas/metabolismo , Fragmentos de Péptidos/farmacología , Factores de Tiempo , Regulación hacia Arriba , Proteínas del Envoltorio Viral/farmacología , Receptor fas/metabolismoRESUMEN
Dendritic cells are among the first cells to encounter sexually acquired human immunodeficiency virus (HIV-1), in the mucosa, and they can transmit HIV-1 to CD4(+) T-cells via an infectious synapse. Recent studies reveal that actin-rich membrane extensions establish direct contact between cells at this synapse and facilitate virus transmission. Genesis of these contacts involves signaling through c-Src and Cdc42, which modulate actin polymerization and filopodia formation via the Arp2/3 complex and Diaphanous 2 (Diaph2). We found that Slit2N, a ligand for the Roundabout (Robo) receptors, blocked HIV-1-induced signaling through Arp2/3 and Diaph2, decreased filopodial extensions on dendritic cells, and inhibited cell-to-cell transmission of HIV-1 in a Robo1-dependent manner. Employing proteomic analysis, we identified Flightless-1 as a novel, Robo1-interacting protein. Treatment with shRNAs reduced levels of Flightless-1 and demonstrated its role in efficient cell-to-cell transfer of HIV-1. These results suggest a novel strategy to limit viral infection in the host by targeting the Slit/Robo pathway with modulation of cytoskeletal elements previously unrecognized in HIV-1 transmission.
Asunto(s)
Citoesqueleto/metabolismo , Células Dendríticas/virología , VIH-1/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Proteínas del Tejido Nervioso/farmacología , Linfocitos T/virología , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Proteínas Portadoras/metabolismo , Extensiones de la Superficie Celular/efectos de los fármacos , Extensiones de la Superficie Celular/metabolismo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Células Dendríticas/efectos de los fármacos , Células Dendríticas/ultraestructura , Activación Enzimática/efectos de los fármacos , Forminas , Células HEK293 , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/ultraestructura , Humanos , Modelos Biológicos , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica/efectos de los fármacos , Proteína Proto-Oncogénica c-fli-1/metabolismo , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Receptores Inmunológicos/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Virión/metabolismo , Virión/ultraestructura , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Familia-src Quinasas/metabolismo , Proteínas RoundaboutRESUMEN
Pulmonary complications are common in both AIDS patients and cocaine users. We addressed the cellular and molecular mechanisms by which HIV and cocaine may partner to induce their deleterious effects. Using primary lung lymphatic endothelial cells (L-LECs), we examined how cocaine and HIV-1 gp120, alone and together, modulate signaling and functional properties of L-LECs. We found that brief cocaine exposure activated paxillin and induced cytoskeletal rearrangement, while sustained exposure increased fibronectin (FN) expression, decreased Robo4 expression, and enhanced the permeability of L-LEC monolayers. Moreover, incubating L-LECs with both cocaine and HIV-1 gp120 exacerbated hyperpermeability, significantly enhanced apoptosis, and further impaired in vitro wound healing as compared with cocaine alone. Our studies also suggested that the sigma-1 receptor (Sigma-1R) and the dopamine-4 receptor (D4R) are involved in cocaine-induced pathology in L-LECs. Seeking clinical correlation, we found that FN levels in sera and lung tissue of HIV(+) donors were significantly elevated as compared to HIV(-) donors. Our in vitro data demonstrate that cocaine and HIV-1 gp120 induce dysfunction and damage of lung lymphatics, and suggest that cocaine use may exacerbate pulmonary edema and fibrosis associated with HIV infection. Continued exploration of the interplay between cocaine and HIV should assist the design of therapeutics to ameliorate HIV-induced pulmonary disorders within the drug using population.
RESUMEN
PURPOSE: Mantle cell lymphoma (MCL) is an incurable B-cell lymphoma, and new therapeutic strategies are urgently needed. EXPERIMENTAL DESIGN: The effects of ON 013105, a novel benzylstyryl sulfone kinase inhibitor, alone or with doxorubicin or rituximab, were examined in Granta 519 and Z138C cells. For in vivo studies, CB17/SCID mice were implanted subcutaneously with Z138C cells and treated with various combinations of ON 013105, doxorubicin, and rituximab. Tumor burden and body weight were monitored for 28 days. RESULTS: ON 013105 induced mitochondria-mediated apoptosis in MCL cells. Death was preceded by translocation of tBid to the mitochondria and cytochrome c release. In addition, ON 013105-treated cells exhibited reduced levels of cyclin D1, c-Myc, Mcl-1, and Bcl-xL. Using nuclear magnetic resonance (NMR) spectroscopy, we showed specific binding of ON 013105 to eIF4E, a critical factor for the initiation of protein translation. We proffer that this drug-protein interaction preferentially prevents the translation of the aforementioned proteins and may be the mechanism by which ON 013105 induces apoptosis in MCL cells. Efficacy studies in a mouse xenograft model showed that ON 013105 inhibited MCL tumor growth and that combining ON 013105 with rituximab reduced tumor burden further with negligible unwanted effects. CONCLUSIONS: Our findings suggest that ON 013105, alone or in combination with rituximab, may be a potent therapeutic agent to treat MCLs.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Linfoma de Células del Manto/metabolismo , Linfoma de Células del Manto/patología , Estirenos/administración & dosificación , Sulfonas/administración & dosificación , Carga Tumoral/efectos de los fármacos , Animales , Antibióticos Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Modelos Animales de Enfermedad , Doxorrubicina/administración & dosificación , Factor 4E Eucariótico de Iniciación/metabolismo , Femenino , Expresión Génica , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/genética , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Rituximab , Estirenos/metabolismo , Estirenos/farmacocinética , Sulfonas/metabolismo , Sulfonas/farmacocinéticaRESUMEN
Cell-mediated transmission and dissemination of sexually-acquired human immunodeficiency virus 1 (HIV-1) in the host involves the migration of immature dendritic cells (iDCs). iDCs migrate in response to the HIV-1 envelope protein, gp120, and inhibiting such migration may limit the mucosal transmission of HIV-1. In this study, we elucidated the mechanism of HIV-1-gp120-induced transendothelial migration of iDCs. We found that gp120 enhanced the binding of Wiskott-Aldrich Syndrome protein (WASp) and the Actin-Related Protein 2/3 (Arp2/3) complex with ß-actin, an interaction essential for the proper formation of podosomes, specialized adhesion structures required for the migration of iDCs through different tissues. We further identified Leukocyte-Specific Protein 1 (LSP1) as a novel component of the WASp-Arp2/3-ß-actin complex. Pretreating iDCs with an active fragment of the secretory glycoprotein Slit2 (Slit2N) inhibited HIV-1-gp120-mediated migration and podosome formation, by inducing the cognate receptor Roundabout 1 (Robo1) to bind to and sequester WASp and LSP1 from ß-actin. Slit2N treatment also inhibited Src signaling and the activation of several downstream molecules, including Rac1, Pyk2, paxillin, and CDC42, a major regulator of podosome formation. Taken together, our results support a novel mechanism by which Slit2/Robo1 may inhibit the HIV-1-gp120-induced migration of iDCs, thereby restricting dissemination of HIV-1 from mucosal surfaces in the host.
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Movimiento Celular/inmunología , Células Dendríticas/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Proteínas de Microfilamentos/inmunología , Proteínas del Tejido Nervioso/inmunología , Receptores Inmunológicos/inmunología , Proteína del Síndrome de Wiskott-Aldrich/inmunología , Complejo 2-3 Proteico Relacionado con la Actina/inmunología , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/inmunología , Actinas/metabolismo , Western Blotting , Células Cultivadas , Células Dendríticas/metabolismo , Quinasa 2 de Adhesión Focal/inmunología , Quinasa 2 de Adhesión Focal/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Proteínas del Tejido Nervioso/metabolismo , Paxillin/inmunología , Paxillin/metabolismo , Unión Proteica/inmunología , Proteínas Proto-Oncogénicas pp60(c-src)/inmunología , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Seudópodos/inmunología , Interferencia de ARN , Receptores Inmunológicos/metabolismo , Transducción de Señal/inmunología , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteína de Unión al GTP cdc42/inmunología , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/inmunología , Proteína de Unión al GTP rac1/metabolismo , Proteínas RoundaboutRESUMEN
Kaposi sarcoma is the most common neoplasm caused by Kaposi sarcoma-associated herpesvirus (KSHV). It is prevalent among the elderly in the Mediterranean, inhabitants of sub-Saharan Africa, and immunocompromised individuals such as organ transplant recipients and AIDS patients. Current treatments for Kaposi sarcoma can inhibit tumor growth but are not able to eliminate KSHV from the host. When the host's immune system weakens, KSHV begins to replicate again, and active tumor growth ensues. New therapeutic approaches are needed. Cannabidiol (CBD), a plant-derived cannabinoid, exhibits promising antitumor effects without inducing psychoactive side effects. CBD is emerging as a novel therapeutic for various disorders, including cancer. In this study, we investigated the effects of CBD both on the infection of endothelial cells (ECs) by KSHV and on the growth and apoptosis of KSHV-infected ECs, an in vitro model for the transformation of normal endothelium to Kaposi sarcoma. While CBD did not affect the efficiency with which KSHV infected ECs, it reduced proliferation and induced apoptosis in those infected by the virus. CBD inhibited the expression of KSHV viral G protein-coupled receptor (vGPCR), its agonist, the chemokine growth-regulated protein α (GRO-α), vascular endothelial growth factor receptor 3 (VEGFR-3), and the VEGFR-3 ligand, vascular endothelial growth factor C (VEGF-C). This suggests a potential mechanism by which CBD exerts its effects on KSHV-infected endothelium and supports the further examination of CBD as a novel targeted agent for the treatment of Kaposi sarcoma.
RESUMEN
BACKGROUND: The pro-fibrogenic cytokine connective tissue growth factor (CTGF) plays an important role in the development and progression of fibrosis in many organ systems, including liver. However, its role in the pathogenesis of hepatitis C virus (HCV)-induced liver fibrosis remains unclear. METHODS: In the present study, we assessed CTGF expression in HCV-infected hepatocytes using replicon cells containing full-length HCV genotype 1 and the infectious HCV clone JFH1 (HCV genotype 2) by real-time PCR, Western blot analysis and confocal microscopy. We evaluated transforming growth factor ß1 (TGF-ß1) as a key upstream mediator of CTGF production using neutralizing antibodies and shRNAs. We also determined the signaling molecules involved in CTGF production using various immunological techniques. RESULTS: We demonstrated an enhanced expression of CTGF in two independent models of HCV infection. We also demonstrated that HCV induced CTGF expression in a TGF-ß1-dependent manner. Further dissection of the molecular mechanisms revealed that CTGF production was mediated through sequential activation of MAPkinase and Smad-dependent pathways. Finally, to determine whether CTGF regulates fibrosis, we showed that shRNA-mediated knock-down of CTGF resulted in reduced expression of fibrotic markers in HCV replicon cells. CONCLUSION: Our studies demonstrate a central role for CTGF expression in HCV-induced liver fibrosis and highlight the potential value of developing CTGF-based anti-fibrotic therapies to counter HCV-induced liver damage.
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Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Hepacivirus/metabolismo , Hepatocitos/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Secuencia de Bases , Western Blotting , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Hepacivirus/fisiología , Hepatocitos/virología , Humanos , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Replicación ViralRESUMEN
Cannabidiol (CBD), a major nonpsychoactive constituent of cannabis, is considered an antineoplastic agent on the basis of its in vitro and in vivo activity against tumor cells. However, the exact molecular mechanism through which CBD mediates this activity is yet to be elucidated. Here, we have shown CBD-induced cell death of breast cancer cells, independent of cannabinoid and vallinoid receptor activation. Electron microscopy revealed morphologies consistent with the coexistence of autophagy and apoptosis. Western blot analysis confirmed these findings. We showed that CBD induces endoplasmic reticulum stress and, subsequently, inhibits AKT and mTOR signaling as shown by decreased levels of phosphorylated mTOR and 4EBP1, and cyclin D1. Analyzing further the cross-talk between the autophagic and apoptotic signaling pathways, we found that beclin1 plays a central role in the induction of CBD-mediated apoptosis in MDA-MB-231 breast cancer cells. Although CBD enhances the interaction between beclin1 and Vps34, it inhibits the association between beclin1 and Bcl-2. In addition, we showed that CBD reduces mitochondrial membrane potential, triggers the translocation of BID to the mitochondria, the release of cytochrome c to the cytosol, and, ultimately, the activation of the intrinsic apoptotic pathway in breast cancer cells. CBD increased the generation of reactive oxygen species (ROS), and ROS inhibition blocked the induction of apoptosis and autophagy. Our study revealed an intricate interplay between apoptosis and autophagy in CBD-treated breast cancer cells and highlighted the value of continued investigation into the potential use of CBD as an antineoplastic agent.