RESUMO
Cannabinoids modulate intestinal permeability through cannabinoid receptor 1 (CB1). The endocannabinoid-like compounds oleoylethanolamine (OEA) and palmitoylethanolamine (PEA) play an important role in digestive regulation, and we hypothesized they would also modulate intestinal permeability. Transepithelial electrical resistance (TEER) was measured in human Caco-2 cells to assess permeability after application of OEA and PEA and relevant antagonists. Cells treated with OEA and PEA were stained for cytoskeletal F-actin changes and lysed for immunoassay. OEA and PEA were measured by liquid chromatography-tandem mass spectrometry. OEA (applied apically, logEC50 -5.4) and PEA (basolaterally, logEC50 -4.9; apically logEC50 -5.3) increased Caco-2 resistance by 20-30% via transient receptor potential vanilloid (TRPV)-1 and peroxisome proliferator-activated receptor (PPAR)-α. Preventing their degradation (by inhibiting fatty acid amide hydrolase) enhanced the effects of OEA and PEA. OEA and PEA induced cytoskeletal changes and activated focal adhesion kinase and ERKs 1/2, and decreased Src kinases and aquaporins 3 and 4. In Caco-2 cells treated with IFNγ and TNFα, OEA (via TRPV1) and PEA (via PPARα) prevented or reversed the cytokine-induced increased permeability compared to vehicle (0.1% ethanol). PEA (basolateral) also reversed increased permeability when added 48 or 72 h after cytokines (P < 0.001, via PPARα). Cellular and secreted levels of OEA and PEA (P < 0.001-0.001) were increased in response to inflammatory mediators. OEA and PEA have endogenous roles and potential therapeutic applications in conditions of intestinal hyperpermeability and inflammation.-Karwad, M. A., Macpherson, T., Wang, B., Theophilidou, E., Sarmad, S., Barrett, D. A., Larvin, M., Wright, K. L., Lund, J. N., O'Sullivan, S. E. Oleoylethanolamine and palmitoylethanolamine modulate intestinal permeability in vitro via TRPV1 and PPARα.
Assuntos
Etanolaminas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ácidos Oleicos/farmacologia , PPAR alfa/metabolismo , Ácidos Palmíticos/farmacologia , Canais de Cátion TRPV/metabolismo , Amidas , Células CACO-2 , Citocinas , Citoesqueleto , Humanos , Intestinos/efeitos dos fármacos , PPAR alfa/genética , Permeabilidade/efeitos dos fármacos , Transdução de Sinais , Canais de Cátion TRPV/genéticaRESUMO
The endocannabinoid system has previously been shown to play a role in the permeability and inflammatory response of the human gut. The goal of our study was to determine the effects of endogenous anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) on the permeability and inflammatory response of intestinal epithelium under normal, inflammatory, and hypoxic conditions. Human intestinal mucosa was modeled using Caco-2 cells. Human tissue was collected from planned colorectal resections. Accumulation of AEA and 2-AG was achieved by inhibiting their metabolizing enzymes URB597 (a fatty acid amide hydrolase inhibitor) and JZL184 (a monoacylglycerol lipase inhibitor). Inflammation and ischemia were simulated with TNF-α and IFN-γ and oxygen deprivation. Permeability changes were measured by transepithelial electrical resistance. The role of the CB1 receptor was explored using CB1-knockdown (CB1Kd) intestinal epithelial cells. Endocannabinoid levels were measured using liquid chromatography-mass spectrometry. Cytokine secretion was measured using multiplex and ELISA. URB597 and JZL184 caused a concentration-dependent increase in permeability via CB1 (P < 0.0001) and decreased cytokine production. Basolateral application of JZL184 decreased permeability via CB1 (P < 0.0001). URB597 and JZL184 increased the enhanced (worsened) permeability caused by inflammation and hypoxia (P < 0.0001 and P < 0.05). CB1Kd cells showed reduced permeability response to inflammation (P < 0.01) but not hypoxia. 2-AG levels were increased in response to inflammation and hypoxia in Caco-2 cells. In human mucosal tissue, inflammation increased the secretion of granulocyte macrophage-colony stimulating factor, IL-12, -13, and -15, which was prevented with ex vivo treatment with URB597 and JZL184, and was inhibited by a CB1 antagonist. The results of this study show that endogenous AEA and 2-AG production and CB1 activation play a key modulatory roles in normal intestinal mucosa permeability and in inflammatory and hypoxic conditions.-Karwad, M. A., Couch, D. G., Theophilidou, E., Sarmad, S., Barrett, D. A., Larvin, M., Wright, K. L., Lund, J. N., O'Sullivan, S. E. The role of CB1 in intestinal permeability and inflammation.
Assuntos
Ácidos Araquidônicos/metabolismo , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Intestinos/fisiologia , Alcamidas Poli-Insaturadas/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Benzamidas/farmacologia , Benzodioxóis/farmacologia , Células CACO-2 , Carbamatos/farmacologia , Neoplasias Colorretais/metabolismo , Citocinas/genética , Citocinas/metabolismo , Impedância Elétrica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestinos/patologia , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismo , Consumo de Oxigênio , Permeabilidade , Piperidinas/farmacologia , Receptor CB1 de Canabinoide/genética , Técnicas de Cultura de TecidosRESUMO
One of the most exciting developments in Raman spectroscopy in the last decade has been its application to cells and tissues for diagnostic and pharmaceutical applications, and in particular its use in the analysis of cellular dynamics. Raman spectroscopy is rapidly advancing as a cell imaging method that overcomes many of the limitations of current techniques and is earning its place as a routine tool in cell biology. In this review we focus on important developments in Raman spectroscopy that have evolved into the exciting technique of live-cell Raman microscopy and highlight some of the most recent and significant applications to cell biology.
Assuntos
Microscopia , Análise Espectral Raman , Humanos , Análise de Célula ÚnicaRESUMO
Autophagy is a catabolic process involved in homeostatic and regulated cellular protein recycling and degradation via the lysosomal degradation pathway. Emerging data associate impaired autophagy, increased activity in the endocannabinoid system, and upregulation of suppressor of cytokine signaling-3 (SOCS3) protein expression during intestinal inflammation. We have investigated whether these three processes are linked. By assessing the impact of the phytocannabinoid cannabidiol (CBD), the synthetic cannabinoid arachidonyl-2'-chloroethylamide (ACEA), and the endocannabinoid N-arachidonoylethanolamine (AEA) on autophagosome formation, we explored whether these actions were responsible for cyclic SOCS3 protein levels. Our findings show that all three cannabinoids induce autophagy in a dose-dependent manner in fully differentiated Caco-2 cells, a model of mature intestinal epithelium. ACEA and AEA induced canonical autophagy, which was cannabinoid type 1 receptor-mediated. In contrast, CBD was able to bypass the cannabinoid type 1 receptor and the canonical pathway to induce autophagy, albeit to a lesser extent. Functionally, all three cannabinoids reduced SOCS3 protein expression, which was reversed by blocking early and late autophagy. In conclusion, the regulatory protein SOCS3 is regulated by autophagy, and cannabinoids play a role in this process, which could be important when therapeutic applications for the cannabinoids in inflammatory conditions are considered.
Assuntos
Autofagia/efeitos dos fármacos , Agonistas de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Receptor CB1 de Canabinoide/agonistas , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Ácidos Araquidônicos/farmacologia , Western Blotting , Células CACO-2 , Canabidiol/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo , Endocanabinoides/farmacologia , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Microscopia Confocal , Alcamidas Poli-Insaturadas , Interferência de RNA , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Fatores de Tempo , TransfecçãoRESUMO
Electrical stimulation (ES) can serve as a therapeutic modality accelerating the healing of wounds, particularly chronic wounds which have impaired healing due to complications from underlying pathology. This review explores how ES affects the cellular mechanisms of wound healing, and its effectiveness in treating acute and chronic wounds. Literature searches with no publication date restrictions were conducted using the Cochrane Library, Medline, Web of Science, Google Scholar and PubMed databases, and 30 full-text articles met the inclusion criteria. In vitro and in vivo experiments investigating the effect of ES on the general mechanisms of healing demonstrated increased epithelialization, fibroblast migration, and vascularity around wounds. Six in vitro studies demonstrated bactericidal effects upon exposure to alternating and pulsed current. Twelve randomized controlled trials (RCTs) investigated the effect of pulsed current on chronic wound healing. All reviewed RCTs demonstrated a larger reduction in wound size and increased healing rate when compared to control groups. In conclusion, ES therapy can contribute to improved chronic wound healing and potentially reduce the financial burden associated with wound management. However, the variations in the wound characteristics, patient demographics, and ES parameters used across studies present opportunities for systematic RCT studies in the future.
RESUMO
In situ forming hydrogels are a class of biomaterials that can fulfil a variety of important biomedically relevant functions and hold promise for the emerging field of patient-specific treatments (e.g., cell therapy, drug delivery). Here we report the results of our investigations on the generation of in situ forming hydrogels with potential for wound healing applications (e.g., complex blast injuries). The combination of polysaccharides that were oxidized to display aldehydes, amine displaying chitosan and nanostructured ZnO yields in situ forming bionanocomposite hydrogels. The physicochemical properties of the components, their cytotoxicity towards HaCat cells and the in vitro release of zinc ions on synthetic skin were studied. The in situ gel formation process was complete within minutes, the components were non-toxic towards HaCat cells at functional levels, Zn2+ was released from the gels, and such materials may facilitate wound healing.
RESUMO
Cannabinoids have long been proposed to affect the immune system, especially as one of the cannabinoid receptors, the cannabinoid receptor-2 (CB(2)R) has been found almost exclusively on immune cells. Here, using human in vitro activated peripheral blood-derived T lymphocytes we investigated the long-term changes in cannabinoid receptor protein expression following cellular activation and the effects of cannabinoids on migration. We report that resting T lymphocytes do not detectably express either the cannabinoid receptor-1 (CB(1)R) or CB(2)R at the protein level. However, CB(2)R protein expression is upregulated in a biphasic manner in T lymphocytes following activation by superantigen. The cannabinoids 2-AG and JWH-133 were found to elicit activation of downstream biochemical effectors (as assessed by the phosphorylation of the ERK1/2 MAP kinases). Neither 2-AG nor JWH-133 induced chemotaxis in day 5 activated T lymphocytes, when receptor expression was at its highest. Interestingly, both 2-AG and JWH-133 inhibited CXCL12-induced chemotaxis, suggesting a modulatory role for cannabinoids in activated T lymphocytes.
Assuntos
Canabinoides/farmacologia , Quimiocinas CXC/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Receptor CB2 de Canabinoide/análise , Linfócitos T/efeitos dos fármacos , Adulto , Amidoidrolases/genética , Ácidos Araquidônicos/farmacologia , Quimiocina CXCL12 , Endocanabinoides , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glicerídeos/farmacologia , Células HT29 , Humanos , Monoacilglicerol Lipases/genética , Fosforilação , Alcamidas Poli-Insaturadas/farmacologia , RNA Mensageiro/análise , Receptor CB2 de Canabinoide/genética , Linfócitos T/química , Linfócitos T/imunologiaRESUMO
The anti-cancer effect of the plant-derived cannabinoid, cannabidiol, has been widely demonstrated both in vivo and in vitro. However, this body of preclinical work has not been translated into clinical use. Key issues around this failure can be related to narrow dose effects, the cell model used and incomplete efficacy. A model of acute lymphoblastic disease, the Jurkat T cell line, has been used extensively to study the cannabinoid system in the immune system and cannabinoid-induced apoptosis. Using these cells, this study sought to investigate the outcome of those remaining viable cells post-treatment with cannabidiol, both in terms of cell size and tracking any subsequent recovery. The phosphorylation status of the mammalian Target of Rapamycin (mTOR) signaling pathway and the downstream target ribosomal protein S6, were measured. The ability of cannabidiol to exert its effect on cell viability was also evaluated in physiological oxygen conditions. Cannabidiol reduced cell viability incompletely, and slowed the cell cycle with fewer cells in the G2/M phase of the cell cycle. Cannabidiol reduced phosphorylation of mTOR, PKB and S6 pathways related to survival and cell size. The remaining population of viable cells that were cultured in nutrient rich conditions post-treatment were able to proliferate, but did not recover to control cell numbers. However, the proportion of viable cells that were gated as small, increased in response to cannabidiol and normally sized cells decreased. This proportion of small cells persisted in the recovery period and did not return to basal levels. Finally, cells grown in 12% oxygen (physiological normoxia) were more resistant to cannabidiol. In conclusion, these results indicate that cannabidiol causes a reduction in cell size, which persists post-treatment. However, resistance to cannabidiol under physiological normoxia for these cells would imply that cannabidiol may not be useful in the clinic as an anti-leukemic agent.
RESUMO
Ligands of peroxisome proliferator-activated receptor-gamma (PPAR(gamma)) are thought to possess anti-inflammatory properties mediated via both PPAR(gamma) dependent and independent mechanisms. This work investigates the effects of PPAR(gamma) ligands on the regulation of cyclooxygenase-2 (COX-2) in the human lung epithelial cell line, A549. The synthetic ligand troglitazone activated the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase pathway (MAPK), whereas the endogenous ligand, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), only activated the PI3K pathway. 15d-PGJ2 had no detectable effects on COX-2, mPGES expression, or PGE2 production. However, troglitazone induced time-dependent COX-2 expression, which was insensitive to PPAR(gamma) antagonists, but was abrogated by inhibitors of PI3K and the ERK MAP kinase pathway. Furthermore, troglitazone induced mPGES expression and PGE2 production. Neither troglitazone nor 15d-PGJ2 was able to convincingly activate NF-kappaB in A549 cells. Further heterogeneity in the responses to troglitazone and 15d-PGJ2 was observed in the regulation of gene expression as assessed by microarray analysis. In summary, this study provides compelling evidence that troglitazone (like 15d-PGJ2) can exert functional effects independently of actions via PPAR(gamma). Moreover, we have identified unique biochemical and functional actions of troglitazone that are not shared by 15d-PGJ2, which may influence the therapeutic potential of this compound in inflammatory settings.
Assuntos
Cromanos/farmacologia , PPAR gama/agonistas , Prostaglandina D2/análogos & derivados , Prostaglandina-Endoperóxido Sintases/biossíntese , Mucosa Respiratória/enzimologia , Tiazolidinedionas/farmacologia , Linhagem Celular , Ciclo-Oxigenase 2 , Dinoprostona/biossíntese , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas I-kappa B/metabolismo , Proteínas de Membrana , NF-kappa B/metabolismo , PPAR gama/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Prostaglandina D2/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Troglitazona , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
1. Cyclooxygenase (COX)-2 expression and activity in response to pro-inflammatory cytokines TNF alpha and IFN gamma was evaluated in the colonic epithelial cell line HT29 and the airway epithelial cell line A549. 2. TNF alpha induced concentration- and time-dependent upregulation of COX-2 mRNA, protein and prostaglandin (PG)E(2) synthesis. 3. Co-stimulation of TNF alpha with IFN gamma resulted in reduced COX-2 mRNA and protein expression. 4. IFN gamma had no effect on the stability of TNF alpha-induced COX-2 mRNA. 5. TNF alpha-induced PGE(2) biosynthesis was significantly enhanced by the simultaneous addition of IFN gamma and was COX-2 dependent. 6. The combination of IFN gamma and TNF alpha induced the microsomal prostaglandin E synthase (mPGES), comensurate with the enhanced PGE(2) synthesis. 7. These results suggest that, in terms of PGE(2) biosynthesis, IFN gamma plays a negative regulatory role at the level of COX-2 expression and a positive regulatory role at the level of mPGES expression. This may have important implications for the clinical use of IFN gamma in inflammatory diseases.
Assuntos
Colo/metabolismo , Células Epiteliais/metabolismo , Interferon gama/farmacologia , Prostaglandinas E/biossíntese , Animais , Células CACO-2 , Colo/efeitos dos fármacos , Colo/patologia , Inibidores de Ciclo-Oxigenase/metabolismo , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Estudos de Avaliação como Assunto , Células HT29 , Humanos , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Interferon gama/antagonistas & inibidores , Interferon gama/metabolismo , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Camundongos , Microssomos/enzimologia , Prostaglandina-E Sintases , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Regulação para CimaRESUMO
The Caco-2 cell model is widely used as a model of colon cancer and small intestinal epithelium but, like most cell models, is cultured in atmospheric oxygen conditions (â¼21%). This does not reflect the physiological oxygen range found in the colon. In this study, we investigated the effect of adapting the Caco-2 cell line to routine culturing in a physiological oxygen (5%) environment. Under these conditions, cells maintain a number of key characteristics of the Caco-2 model, such as increased formation of tight junctions and alkaline phosphatase expression over the differentiation period and maintenance of barrier function. However, these cells exhibit differential oxidative metabolism, proliferate less and become larger during differentiation. In addition, these cells were more sensitive to cannabidiol-induced antiproliferative actions through changes in cellular energetics: from a drop of oxygen consumption rate and loss of mitochondrial membrane integrity in cells treated under atmospheric conditions to an increase in reactive oxygen species in intact mitochondria in cells treated under low-oxygen conditions. Inclusion of an additional physiological parameter, sodium butyrate, into the medium revealed a cannabidiol-induced proliferative response at low doses. These effects could impact on its development as an anticancer therapeutic, but overall, the data supports the principle that culturing cells in microenvironments that more closely mimic the in vivo conditions is important for drug screening and mechanism of action studies.
Assuntos
Canabidiol/farmacologia , Neoplasias do Colo/patologia , Mucosa Intestinal/efeitos dos fármacos , Oxigênio/metabolismo , Fosfatase Alcalina/biossíntese , Células CACO-2 , Diferenciação Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismoAssuntos
Fármacos Antiobesidade/efeitos adversos , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/prevenção & controle , Mucosa Intestinal/efeitos dos fármacos , Obesidade/tratamento farmacológico , Piperidinas/efeitos adversos , Pirazóis/efeitos adversos , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Adenoma/prevenção & controle , Fármacos Antiobesidade/administração & dosagem , Fármacos Antiobesidade/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma/prevenção & controle , Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/metabolismo , Dronabinol/farmacologia , Agonismo Inverso de Drogas , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Obesidade/complicações , Obesidade/metabolismo , Piperidinas/administração & dosagem , Piperidinas/farmacologia , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , RimonabantoRESUMO
Intestinal myofibroblasts have been implicated in the pathogenesis of chronic inflammatory conditions such as Crohn's disease via interactions with an elaborate network of cytokines, growth factors, and other inflammatory mediators. CXCR3 is a Galpha(i) protein-coupled receptor that binds the proinflammatory chemokines CXCL9, CXCL10, and CXCL11, which are released from the intestinal epithelium. The three CXCR3 ligands shared the ability to activate biochemical (e.g., PI3K and MAPK activation) and functional events (actin reorganization) in intestinal myofibroblasts. However, CXCL11 is unique in its ability to elevate intracellular calcium. Surprisingly, although CXCR3 mRNA is detectable in these myofibroblasts, there is no detectable surface expression of CXCR3. Furthermore, the biochemical responses and actin reorganization stimulated by the CXCR3 ligands in intestinal myofibroblasts are insensitive to the Galpha(i) inhibitor, pertussis toxin. This suggests either the existence of differential receptor coupling mechanisms in myofibroblasts for CXCR3 that are distinct from those observed in PBLs and/or that these cells express a modified or variant CXCR3 compared with the CXCR3 expressed on PBLs.
Assuntos
Actinas/metabolismo , Quimiocinas CXC/fisiologia , Fibroblastos/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Mucosa Intestinal/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Cálcio/metabolismo , Células Cultivadas , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL9 , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR3 , Receptores de Quimiocinas/metabolismo , Receptores de Quimiocinas/fisiologia , Quinases Associadas a rhoRESUMO
Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 are a multifunctional chemokine/receptor system with essential roles in the development of the immune system and other aspects of embryogenesis, including vascularization and organ development. SDF-1 is also a potent chemoattractant for T cells and has roles in both inflammation and immune homeostasis. Our group has previously demonstrated that phosphoinositide 3-kinase (PI 3-kinase) is activated in SDF-1-stimulated T cells and is indeed required for SDF-1-mediated chemotaxis. In this study Jurkat clones were established, stably expressing dominant negative constructs of class IA and class IB PI 3-kinases under the control of the tetracycline off inducible gene system, to determine the relative roles of these PI 3-kinases in SDF-1 signaling. Our results show that expression of either kinase-dead PI3Kgamma (KD-PI3Kgamma) or Deltap85 (a construct unable to bind class I(A) p110alpha, -beta, or -delta) leads to a partial inhibition of SDF-1-stimulated protein kinase B phosphorylation, but had no effect on SDF-1-induced phosphorylation of the mitogen-activated protein kinase ERK1/2. Functional studies demonstrated that expression of KD-PI3Kgamma markedly inhibited SDF-1-mediated chemotaxis, typically eliciting 40-60% inhibition. Interestingly, the expression of Deltap85 also leads to inhibition of the SDF-1-mediated chemotactic response, albeit to a much lesser extent than achieved with the KD-PI3Kgamma mutant, typically in the range of 20-40% inhibition. Furthermore, the inhibition of chemotaxis by the expression of dominant negative class IA or class IB PI 3-kinases could be enhanced by the presence of the PI 3-kinase inhibitor LY294002. Together, these results demonstrate that optimal chemotactic response of leukemic T cells to SDF-1 requires the activation of both class IA and class IB PI 3-kinases.
Assuntos
Quimiocinas CXC/fisiologia , Quimiotaxia de Leucócito/imunologia , Isoenzimas/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Animais , Bovinos , Inibição de Migração Celular , Quimiocina CXCL12 , Quimiocinas CXC/antagonistas & inibidores , Cromonas/farmacologia , Classe Ib de Fosfatidilinositol 3-Quinase , Células Clonais , Sinergismo Farmacológico , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/imunologia , Vetores Genéticos , Humanos , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/fisiologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/biossíntese , Isoenzimas/genética , Células Jurkat , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/biossíntese , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Suínos , Subpopulações de Linfócitos T/metabolismo , Tetraciclina/metabolismoRESUMO
The leukemic T cell line Jurkat is deficient in protein expression of the lipid phosphatases Src homology 2 domain containing inositol polyphosphate phosphatase (SHIP) and phosphatase and tensin homolog deleted on chromosome ten (PTEN). We examined whether the lack of expression of SHIP-1 and PTEN is shared by other leukemic T cell lines and PBLs. Analysis of a range of cell lines and PBLs revealed that unlike Jurkat cells, two other well-characterized T cell lines, namely CEM and MOLT-4 cells, expressed the 5'-phosphatase SHIP at the protein level. However, the 3-phosphatase PTEN was not expressed by CEM or MOLT-4 cells or Jurkat cells. The HUT78 cell line and PBLs expressed both SHIP and PTEN. Jurkat cells exhibited high basal levels of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3); the lipid substrate for both SHIP and PTEN) as well as saturated protein kinase B (PKB) phosphorylation. Lower levels of PI(3,4,5)P(3) and higher levels of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) as well as unsaturated constitutive phosphorylation of PKB were observed in CEM and MOLT-4 cells compared with Jurkat cells. In PBLs and HUT78 cells which express both PTEN and SHIP-1, there was no constitutive PI(3,4,5)P(3) or PKB phosphorylation, and receptor stimuli were able to elicit robust phosphorylation of PKB. Expression of a constitutively active SHIP-1 protein in Jurkat cells was sufficient to reduce both constitutive PKB membrane localization and PKB phosphorylation. Together, these data indicate important differences between T leukemic cells as well as PBLs, regarding expression of key lipid phosphatases. This study provides the first evidence that SHIP-1 can influence the constitutive levels of PI(3,4,5)P(3) and the activity of downstream phosphoinositide 3-kinase effectors in T lymphocytes.