RESUMEN
Steroid and thyroid hormone receptors are members of the superfamily of nuclear receptors (NR) that participate in developmental and homeostatic mechanisms by changes in the transcription of specific genes. These activities are governed by the receptors' cognate ligands and through interaction with the components of the transcriptional machinery. A number of coactivator molecules of the steroid receptor coactivator (SRC)/nuclear receptor coactivator (NCoA) family interact with activation functions within NRs through a conserved region containing helical domains of a core LXXLL sequence and, thereby, participate in transcriptional regulation. Using a mammalian-two-hybrid assay, we show that the thyroid hormone receptor beta (TRbeta) and estrogen receptor beta (ERbeta) have different LXXLL motif preferences for interactions with SRC-1. Using large random and focused (centered on the LXXLL motif) recombinant peptide diversity libraries, we have obtained novel peptide sequences that interact specifically with ERbeta or with TRbeta in a ligand-dependent manner. Random sequence libraries yielded LXXLL-containing peptides, and sequence analysis of selected clones revealed that the preferred residues within and around the LXXLL motif vary significantly between these two receptors. We compared the receptor binding of library-selected peptides to that of peptides derived from natural coactivators. The affinities of selected peptides for the ligand binding domains of ERbeta and TRbeta were similar to the best natural LXXLL motifs tested, but showed a higher degree of receptor selectivity. These selected peptides also display receptor-selective dominant inhibitory activities when introduced into mammalian cells. Finally, by directed mutations in specific residues, we were able to alter the receptor binding preference of these peptides.
Asunto(s)
Péptidos/farmacología , Receptores de Estrógenos/agonistas , Receptores de Hormona Tiroidea/agonistas , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sinergismo Farmacológico , Estradiol/farmacología , Receptor beta de Estrógeno , Histona Acetiltransferasas , Humanos , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Coactivador 1 de Receptor Nuclear , Biblioteca de Péptidos , Péptidos/química , Péptidos/aislamiento & purificación , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Estrógenos/química , Receptores de Hormona Tiroidea/química , Proteínas Recombinantes de Fusión/farmacología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Técnicas del Sistema de Dos HíbridosRESUMEN
Reporter cell lines are often used for high throughput screening of chemical libraries to identify new receptor ligands. Here we show how Cre recombinase can be used in mammalian cells to screen for steroid receptor ligands. A translational fusion of Cre recombinase and the ligand binding domain of the human glucocorticoid receptor was transfected into mammalian cells with a loxP/luciferase reporter gene. The recombinase function of the fusion is dependent on ligand binding to the receptor, and Cre-mediated recombination results in constitutive expression of luciferase from the reporter gene. A stable transfected clone was isolated and used to characterize the kinetics, ligand specificity, and dose response to various receptor ligands. The Cre fusion system, unlike a transcriptional reporter using the mouse mammary tumor virus promoter, can detect binding of the receptor antagonist RU486. We also studied the Cre reporter in a sensitive, miniaturized, assay format using an 864-well plate and show that as few as 560 cells per assay well was sufficient to measure a dose response to ligand.
Asunto(s)
Genes Reporteros , Receptores de Esteroides/agonistas , Receptores de Esteroides/antagonistas & inhibidores , Recombinación Genética , Proteínas Virales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Cricetinae , ADN Recombinante/genética , Antagonistas de Hormonas/metabolismo , Humanos , Integrasas/genética , Cinética , Ligandos , Luciferasas/genética , Ratones , Mifepristona/metabolismo , Datos de Secuencia Molecular , Plásmidos/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , TransfecciónRESUMEN
The lymphokine IL-4 is a potent enhancer of anti-IgM-induced B cell proliferation. Although the mechanism of this enhancement is not known, a commonly held view suggests that IL-4 acts together with anti-IgM as a costimulating factor for the activation of a subpopulation of B cells. To evaluate this hypothesis we examined the effect of IL-4 on the proportion of B cells stimulated to divide by different doses of anti-IgM using flow cytometry in combination with measurements of tritiated-thymidine incorporation. The results suggest a novel and surprisingly simple model for the mode of action of IL-4. Our analysis revealed that at high saturating anti-IgM concentrations, the proportion of live B cells which enter into S phase of the cell cycle is the same (approximately 65%) for cells cultured with or without IL-4. Cultures containing IL-4, however, exhibit a twofold increase in thymidine uptake over cultures without IL-4. This increase can be explained completely by the ability of IL-4 to enhance the viability of small dense B cells over the first 24 hr from approximately 50 to 90% of the starting cell number. Normalizing the maximum response levels obtained with and without IL-4 reveals that B cells incubated with IL-4 exhibit a 10-fold decrease in the concentration of anti-IgM required to stimulate the half-maximum proliferation level. Furthermore, evaluation of the number of cells in S phase by flow cytometry and analysis of the kinetics of cell proliferation revealed that the increased response effected by IL-4 at lower anti-IgM concentrations was due to a greater number of proliferating B cells rather than the same number of cells undergoing a faster division rate. We also found a highly nonlinear relationship between B cell number and proliferative response, implying a requirement for an additional, cell cooperation-mediated, activating signal for maximum B cell proliferation. IL-4 enhanced proliferation by the same proportion at all cell concentrations indicating that it does not replace or alter this requirement for cell cooperation. Taken together these results suggest that anti-IgM in combination with a second unidentified cell-cooperation-dependent signal leads to proliferation of up to 65% of small resting B cells. IL-4 does not provide an essential activation signal but serves to raise the sensitivity of B cells to the anti-IgM-generated signal.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Linfocitos B/inmunología , Interleucina-4/farmacología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Anticuerpos Antiidiotipos/inmunología , Ciclo Celular , Supervivencia Celular , Demecolcina/farmacología , Relación Dosis-Respuesta Inmunológica , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Factores de TiempoRESUMEN
Highly purified, small dense splenic B cells from unstimulated mice showed increased expression of class II major histocompatibility complex (MHC) antigens and enhanced viability when cultured with affinity-purified recombinant interleukin 10 (rIL-10), compared with B cells cultured in medium alone. These responses were blocked by a monoclonal antibody (mAb) specific for IL-10, but not by an isotype-matched control antibody. IL-10 did not upregulate the expression of Fc epsilon receptors (CD23) or class I MHC antigens on small dense B cells or induce their replication as monitored by [3H]thymidine incorporation. While these B cell-stimulatory properties of IL-10 are also mediated by IL-4, the two cytokines appear to act independently in these assays; anti-IL-10 antibodies blocked IL-10 but not IL-4-mediated B cell viability enhancement, and vice versa. Similarly, since IL-4 upregulates CD23 on small dense B cells, the inability of IL-10 to do so argues against its acting via endogenously generated IL-4. Finally, IL-10 did not upregulate class II MHC antigens on B cells from X chromosome-linked immunodeficiency (XID) mice, while the same cells showed normal upregulation of class II antigens in response to IL-4. This report also extends our understanding of the relationship between IL-10 and the highly homologous Epstein-Barr virus (EBV)-encoded Bam HI fragment C rightward reading frame no. 1 (BCRFI) protein. It has previously been shown that BCRFI protein exhibits the cytokine synthesis inhibitory activity of IL-10. This report indicates that BCRFI protein also enhances in vitro B cell viability, but does not upregulate class II MHC antigens on B cells. One explanation for these data is that IL-10 contains at least two functional epitopes, only one of which has been conserved by EBV.
Asunto(s)
Linfocitos B/inmunología , Interleucinas/farmacología , Cromosoma X , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cruzamientos Genéticos , Femenino , Expresión Génica/efectos de los fármacos , Genes MHC Clase I/efectos de los fármacos , Interleucina-10 , Interleucina-4/farmacología , Interleucinas/genética , Cinética , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Proteínas Recombinantes/farmacologíaRESUMEN
Three strains of mice (NZB/W F1 X NZW (NZB/W), BXSB, and MRL/Mp-lpr/lpr (MRL/lpr] develop an autoimmune disease that is clinically and immunologically similar to human SLE. A characteristic of these mice is polyclonal B cell hyperactivity. To explore whether this may be related to hyper-responsiveness to B cell stimulatory factors, we investigated the proliferative and secretory responses of B cells from these mice to semi-purified natural and rIL-5, a major regulator of B cell development in the mouse. As this lymphokine stimulates growth and differentiation of activated B cells, attention was focused on in vivo-activated B cell populations, obtained from the interface of 50/65% Percoll density gradients, from normal or autoimmune mice. This cell population from NZB/W mice secreted IgM and incorporated [3H]TdR at significantly higher levels in response to IL-5, and was more sensitive to IL-5, than a comparable population from several normal murine strains. NZB/W female and male mice displayed heightened responses to IL-5, indicating that this is characteristic of the strain in general and is not associated with the accelerated severe disease of the females. Small resting B cells from NZB/W and normal mice were insensitive to IL-5 stimulation. In contrast to NZB/W mice, no difference was observed in the magnitude of either proliferative or Ig secretory responses between in vivo-activated B cell populations from autoimmune BXSB and MRL/lpr or normal mice. Thus, B cell hyper-responsiveness to IL-5 is a characteristic of NZB/W mice but not of two other lupus-prone murine strains. As one unique feature of NZB/W mouse B cells compared to normal and other autoimmune B cells is an elevated proportion of Ly-1+ B cells, the possibility of IL-5 hyper-responsiveness being associated with this B cell subpopulation was investigated. Fluorescence-activated cell sorter sorted Ly-1+ and Ly-1- B cells both responded to IL-5, however Ly-1+ B cells consistently showed a higher stimulation index in both proliferative and Ig secretory responses to this lymphokine.