RESUMO
Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is an abundant phospholipid that contributes to second messenger formation and has also been shown to contribute to the regulation of cytoskeletal dynamics in all eukaryotic cells. Although the alpha, beta, and gamma isoforms of phosphatidylinositol-4-phosphate-5-kinase I (PIP5KI) all synthesize PIP2, mammalian cells usually contain more than one PIP5KI isoform. This raises the question of whether different isoforms of PIP5KI fulfill different functions. Given the speculated role of PIP(2) in platelet and megakaryocyte actin dynamics, we analyzed murine megakaryocytes lacking individual PIP5KI isoforms. PIP5KIgamma(-/-) megakaryocytes exhibited plasma membrane blebbing accompanied by a decreased association of the membrane with the cytoskeleton. This membrane defect was rescued by adding back wild-type PIP5KIgamma, but not by adding a catalytically inactive mutant or a splice variant lacking the talin-binding motif. Notably, both PIP5KIbeta- and PIP5KIgamma(-/-) cells had impaired PIP(2) synthesis. However, PIP5KIbeta-null cells lacked the membrane-cytoskeleton defect. Furthermore, overexpressing PIP5KIbeta in PIP5KIgamma(-/-) cells failed to revert this defect. Megakaryocytes lacking the PIP5KIgamma-binding partner, talin1, mimicked the membrane-cytoskeleton defect phenotype seen in PIP5KIgamma(-/-) cells. These findings demonstrate a unique role for PIP5KIgamma in the anchoring of the cell membrane to the cytoskeleton in megakaryocytes, probably through a pathway involving talin. These observations further demonstrate that individual PIP5KI isoforms fulfill distinct functions within cells.
Assuntos
Membrana Celular/ultraestrutura , Citoesqueleto/ultraestrutura , Megacariócitos/ultraestrutura , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Talina/metabolismo , Animais , Citoesqueleto/enzimologia , Masculino , Megacariócitos/enzimologia , Camundongos , Camundongos Mutantes , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Chemokines acting through G protein-coupled receptors play an essential role in the immune response. PI3K and phospholipase C (PLC) are distinct signaling molecules that have been proposed in the regulation of chemokine-mediated cell migration. Studies with knockout mice have demonstrated a critical role for PI3K in G(alphai) protein-coupled receptor-mediated neutrophil and lymphocyte chemotaxis. Although PLCbeta is not essential for the chemotactic response of neutrophils, its role in lymphocyte migration has not been clearly defined. We compared the chemotactic response of peripheral T cells derived from wild-type mice with mice containing loss-of-function mutations in both of the two predominant lymphocyte PLCbeta isoforms (PLCbeta2 and PLCbeta3), and demonstrate that loss of PLCbeta2 and PLCbeta3 significantly impaired T cell migration. Because second messengers generated by PLCbeta lead to a rise in intracellular calcium and activation of PKC, we analyzed which of these responses was critical for the PLCbeta-mediated chemotaxis. Intracellular calcium chelation decreased the chemotactic response of wild-type lymphocytes, but pharmacologic inhibition of several PKC isoforms had no effect. Furthermore, calcium efflux induced by stromal cell-derived factor-1alpha was undetectable in PLCbeta2beta3-null lymphocytes, suggesting that the migration defect is due to the impaired ability to increase intracellular calcium. This study demonstrates that, in contrast to neutrophils, phospholipid second messengers generated by PLCbeta play a critical role in T lymphocyte chemotaxis.
Assuntos
Sinalização do Cálcio/imunologia , Quimiotaxia/imunologia , Isoenzimas/imunologia , Linfócitos T/imunologia , Fosfolipases Tipo C/imunologia , Animais , Sinalização do Cálcio/genética , Quimiocina CXCL12 , Quimiocinas/genética , Quimiocinas/imunologia , Quimiocinas CXC/genética , Quimiocinas CXC/imunologia , Quimiotaxia/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/imunologia , Isoenzimas/deficiência , Isoenzimas/genética , Camundongos , Camundongos Knockout , Mutação , Neutrófilos/enzimologia , Neutrófilos/imunologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosfolipase C beta , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologia , Linfócitos T/enzimologia , Fosfolipases Tipo C/deficiênciaRESUMO
The malignant cells in Sezary syndrome express the skin trafficking molecules' cutaneous lymphocyte associated antigen (CLA) and chemokine receptor 4 (CCR4). High levels of the CCR4 ligand, thymus, and activation-regulated chemokine (TARC), have been reported in the blood and skin of patients. The rexinoid X-receptor specific retinoid, bexarotene, has contributed to the resolution of cutaneous disease among patients. To evaluate the effects of bexarotene on skin trafficking molecule expression and chemotaxis, peripheral blood mononuclear cells from Sezary syndrome patients and healthy controls were treated with bexarotene in vitro. CCR4 and CLA expression levels and chemotaxis in response to TARC (6.25 ng/ml) were evaluated among lymphocytes before and after treatment with bexarotene (10 microM). Flow cytometric analysis was performed to evaluate CD4, CD26, CLA, and CCR4 cell surface expression. Transwell migration assays were performed to evaluate chemotaxis to TARC. Prior to treatment, malignant cells exhibited higher CCR4 expression (45-90%) and greater than four times more chemotaxis to TARC compared with healthy controls. After treatment with bexarotene for 36-96 hr, a 28% reduction in CCR4 expression was noted (P < 0.05) among the malignant population with an associated 9% decrease in chemotaxis to TARC (P < 0.05). Our results show that bexarotene may inhibit malignant cell trafficking to the skin through an ability to suppress CCR4 expression among Sezary syndrome lymphocytes.
Assuntos
Anticarcinógenos/farmacologia , Quimiocinas CC/imunologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Receptores de Quimiocinas/imunologia , Síndrome de Sézary/tratamento farmacológico , Tetra-Hidronaftalenos/farmacologia , Idoso , Bexaroteno , Estudos de Casos e Controles , Células Cultivadas , Quimiocina CCL17 , Quimiocinas CC/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Receptores CCR4 , Receptores de Quimiocinas/metabolismo , Síndrome de Sézary/imunologia , Síndrome de Sézary/metabolismo , Fatores de TempoRESUMO
Pleckstrin-2 is composed of 2 pleckstrin homology (PH) domains and a disheveled-Egl-10-pleckstrin (DEP) domain. A lipid-binding assay revealed that pleckstrin-2 binds with greatest affinity to D3 and D5 phosphoinositides. Pleckstrin-2 expressed in Jurkat T cells bound to the cellular membrane and enhanced actin-dependent spreading only after stimulation of the T-cell antigen receptor or the integrin alpha4beta1. A pleckstrin-2 variant containing point mutations in both PH domains failed to associate with the Jurkat membrane and had no effect on spreading under the same conditions. Although still membrane bound, a pleckstrin-2 variant containing point mutations in the DEP domain demonstrated a decreased ability to induce membrane ruffles and spread. Pleckstrin-2 also colocalized with actin at the immune synapse and integrin clusters via its PH domains. Although pleckstrin-2 can bind to purified D3 and D5 phosphoinositides, the intracellular membrane association of pleckstrin-2 and cell spreading are dependent on D3 phosphoinositides, because these effects were disrupted by pharmacologic inhibition of phosphatidylinositol 3-kinase (PI3K). Our results indicate that pleckstrin-2 uses its modular domains to bind to membrane-associated phosphatidylinositols generated by PI3K, whereby it coordinates with the actin cytoskeleton in lymphocyte spreading and immune synapse formation.