RESUMEN
PROBLEM: Qa-2, the product of the Ped (preimplantation development) gene, regulates the rate of cell division of preimplantation mouse embryos by an unknown mechanism. Due to the limited availability of preimplantation embryos, T cells were used as a model system to assess the possible roles of Fyn and Lck, and two downstream effectors, PI-3 kinase and Akt, in Qa-2 induced cell proliferation. METHOD OF STUDY: Resting T cells were stimulated to proliferate by treating with mouse anti-Qa-2 antibody, cross-linking with anti-mouse immunoglobulin, and adding PMA. The effects of kinase inhibitors on this proliferation were studied. Co-immunoprecipitates of T-cell lysates were analyzed for possible associations between Qa-2 and Fyn or Lck. Fyn knockout mice (Fyn-/-) were used to determine whether Fyn is required for T-cell activation induced by cross-linking Qa-2. RESULTS: An inhibitor of Src family kinases and inhibitors of PI-3 kinase and Akt suppressed proliferation of resting T cells induced by cross-linking Qa-2. Fyn, but not Lck, co-immunoprecipitated with Qa-2. Fyn-/- T cells failed to proliferate in response to Qa-2 cross-linking. CONCLUSION: Fyn, PI-3 kinase, and Akt are required for the activation of T cells by cross-linking Qa-2.
Asunto(s)
Proliferación Celular , Antígenos de Histocompatibilidad Clase I/metabolismo , Activación de Linfocitos , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Androstadienos/farmacología , Animales , Anticuerpos , Blastocisto/enzimología , Blastocisto/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cromonas/farmacología , Reactivos de Enlaces Cruzados/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulinas/metabolismo , Inmunoprecipitación , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfolinas/farmacología , Nucleósidos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-fyn/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fyn/deficiencia , Proteínas Proto-Oncogénicas c-fyn/genética , Piridazinas/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Linfocitos T/inmunología , Acetato de Tetradecanoilforbol/farmacología , WortmaninaRESUMEN
Qa-2, a murine class Ib major histocompatibility complex (MHC) molecule, is a possible functional homolog of human leukocyte antigen G (HLA-G). Both molecules have been implicated in immunoregulation and embryonic development and both occur in membrane-bound and soluble isoforms that arise by alternative splicing. Soluble splice variants have been implicated in the reproductive functions of HLA-G. While soluble variants of Qa-2 have been previously detected in T lymphocytes, we now demonstrate the presence of mRNA for one of the two known soluble forms of Qa-2 in eight-cell embryos and in blastocysts. Qa-2 is glycosylphosphatidylinositol (GPI) linked in the outer leaflet of the cell membrane and is found in lipid raft microdomains where other raft-associated proteins transduce signals into the cell. In contrast, HLA-G has a truncated six amino acid cytoplasmic tail. By fluorescence co-localization in JEG-3 cells, using fluorescent cholera toxin beta subunit (a lipid raft marker) and anti-HLA-G antibody, we have demonstrated that membrane-bound HLA-G also localizes to lipid rafts, consistent with functional homology between the two molecules. Finally, our experiments in which we have purified Qa-2 and transferred it via a process known as protein painting to Qa-2 negative cells represent a model for potential therapy involving HLA-G.