ABSTRACT
Previous studies have demonstrated that activation of Kit by stem cell factor (SCF), its natural ligand, or by gain-of-function point mutation in its intracellular domain, confers significant protection against apoptosis induced by growth factor deprivation or radiation. However, the effects of Kit activation on the cellular response to anti-tumor agents have not been so extensively documented. This study shows that daunorubicin-induced apoptosis and cytotoxicity were reduced in the murine Ba/F3 cells transfected with Kit (Ba/F3-Kit) in the presence of SCF and in Ba/F3 cells transfected with a constitutively active Kit variant (Ba/F3-KitDelta27), compared to either parental Ba/F3 (Ba/F3-wt) or unstimulated Ba/F3-Kit cells. In Ba/F3-wt and in Ba/F3-Kit cells, daunorubicin stimulated within 8-15 min neutral sphingomyelinase and ceramide production but not in SCF-stimulated Ba/F3-Kit or in Ba/F3-KitDelta27 whereas all Ba/F3 cells were equally sensitive to exogenous cell-permeant C6-ceramide. In Ba/F3-Kit, SCF-induced Kit activation resulted in a rapid phospholipase Cgamma (PLCgamma) tyrosine phosphorylation followed by diacylglycerol release and protein kinase C (PKC) stimulation. U-73122, a PLCgamma inhibitor, not only blocked diacylglycerol production and PKC stimulation but also restored daunorubicin-induced sphingomyelinase stimulation, ceramide production, and apoptosis. These results suggest that Kit activation results in PLCgamma-mediated PKC-dependent sphingomyelinase inhibition which contributes to drug resistance in Kit-related malignancies.
Subject(s)
Apoptosis , Daunorubicin/pharmacology , Isoenzymes/physiology , Proto-Oncogene Proteins c-kit/physiology , Type C Phospholipases/physiology , Animals , Antibiotics, Antineoplastic/pharmacology , CHO Cells , Cell Line , Cricetinae , DNA Fragmentation , Diglycerides/metabolism , Enzyme Activation , Enzyme Inhibitors/pharmacology , Estrenes/pharmacology , Ligands , Mice , Mutation , Phospholipase C gamma , Phospholipids , Phosphorylation , Protein Binding , Protein Isoforms , Protein Kinase C/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins c-kit/metabolism , Pyrrolidinones/pharmacology , Signal Transduction , Sphingomyelin Phosphodiesterase/metabolism , Stem Cell Factor/metabolism , Time FactorsABSTRACT
We have previously observed that the synthetic immunomodulator Murabutide inhibits human immunodeficiency virus type 1 (HIV-1) replication at multiple levels in macrophages and dendritic cells. The present study was designed to profile the activity of Murabutide on CD8-depleted phytohemagglutinin-activated lymphocytes from HIV-1-infected subjects and on the outcome of HIV-1 infection in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID mice). Maintaining cultures of CD8-depleted blasts from 36 patients in the presence of Murabutide produced dramatically reduced levels of viral p24 protein in the supernatants. This activity correlated with reduced viral transcripts and proviral DNA, was evident in cultures harboring R5, X4-R5, or X4 HIV-1 isolates, was not linked to inhibition of cellular DNA synthesis, and did not correlate with beta-chemokine release. Moreover, c-myc mRNA expression was down-regulated in Murabutide-treated cells, suggesting potential interference of the immunomodulator with the nuclear transport of viral preintegration complexes. On the other hand, daily treatment of HIV-1-infected hu-PBL-SCID mice with Murabutide significantly reduced the viral loads in plasma and the proviral DNA content in human peritoneal cells. These results are the first to demonstrate that a clinically acceptable synthetic immunomodulator with an ability to enhance the host's nonspecific immune defense mechanisms against infections can directly regulate cellular factors in infected lymphocytes, leading to controlled HIV-1 replication.
Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Adjuvants, Immunologic , CD4-Positive T-Lymphocytes/virology , HIV Infections/virology , HIV-1/drug effects , Virus Replication/drug effects , Acetylmuramyl-Alanyl-Isoglutamine/analogs & derivatives , Adult , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/drug effects , Cell Division , Cell Survival , Cells, Cultured , Cytokines/metabolism , DNA, Viral/blood , Down-Regulation , Gene Expression Regulation, Viral , HIV Infections/blood , HIV-1/genetics , HIV-1/physiology , Humans , Leukocytes, Mononuclear/cytology , Mice , Mice, SCID , Proto-Oncogene Proteins c-myc/genetics , RNA, Messenger , RNA, Viral/blood , Receptors, CCR5/biosynthesis , Receptors, CXCR4/biosynthesis , Receptors, Interleukin-2/biosynthesis , Viral LoadABSTRACT
The smallest unit of bacterial peptidoglycans known to be endowed with biological activities is muramyl dipeptide (MDP). A clinically acceptable synthetic derivative of MDP, namely murabutide (MB), has been found to present interesting pharmacological properties and to suppress HIV-1 replication in monocyte-derived macrophages (MDM). We have addressed the signaling events activated in MDM following stimulation with either MB or the potent immunostimulant LPS. We also examined whether signaling by muramyl peptides involves the use of cell surface receptors, including CD14 and Toll-like receptor 2 (TLR2) or TLR4 that are known to be signal-transducing receptors for other bacterial cell wall components. We demonstrate that, unlike LPS, the safe immunomodulator MB selectively activates extracellular signal-regulated kinases (Erk) 1/2, in the absence of detectable Jun N-terminal kinase (JNK) or p38 mitogen-activated kinase activation. Furthermore, STAT1 activation but weak or no activation of STAT3 or STAT5 respectively, could be detected in MB-stimulated MDM. Using MonoMac6 cells, we observed high C/EBPbeta and AP-1 but weaker and transient NF-kappaB activation by MB.Moreover, the truncated form of C/EBPbeta, known to repress HIV-1 transcription, was detected in extracts from MB-treated THP-1 cells. Surprisingly, neither MB nor MDP were able to transduce signals via CD14 and TLR2 or 4. These findings present major differences in the early cell activation process between LPS and muramyl peptides, and strongly argue for the implication of co-receptors other than TLR2 and TLR4 in mediating the signaling events induced by defined subunits of bacterial peptidoglycans.
Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Adjuvants, Immunologic/pharmacology , Anti-HIV Agents/pharmacology , CCAAT-Enhancer-Binding Protein-beta/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins , Macrophages/metabolism , Trans-Activators/metabolism , Acetylmuramyl-Alanyl-Isoglutamine/analogs & derivatives , Animals , CHO Cells , Cell Line , Cells, Cultured , Cricetinae , Enzyme Activation , Humans , Lipopolysaccharide Receptors/physiology , Macrophages/drug effects , Membrane Glycoproteins/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Monocytes/immunology , Phosphorylation , Receptors, Cell Surface/physiology , STAT1 Transcription Factor , Signal Transduction , Toll-Like Receptor 2 , Toll-Like Receptor 4 , Toll-Like Receptors , Transcription Factor AP-1/metabolismABSTRACT
Development of the hematopoietic lineages is partially under the control of hematopoietic receptors with tyrosine kinase activity (RTK). To compare the cellular functions of two of the class III RTK, FLT3 and KIT, a murine chimeric FMS/FLT3 (FF3) receptor was expressed ectopically using retroviral infection, in normal IL3-derived cultured mast cells. Stimulation of the chimeric receptor produced a full mitogenic signal and led to mast cell maturation, as occurs upon activation of the endogenous KIT receptor. When introduced into mast cells derived from KIT-deficient White spotting (W) mutant mice, the FF3 receptor bypassed their mitogenic defect. KIT activation induced a synergistic mitogenic activity in mast cells upon IL3 stimulation, whereas FF3 appeared to down-modulate the IL3 response. Adhesion to fibronectin was specifically associated with KIT signaling.
Subject(s)
Amidohydrolases , Mast Cells/ultrastructure , Proto-Oncogene Proteins c-kit/physiology , Proto-Oncogene Proteins/physiology , Receptor Protein-Tyrosine Kinases/physiology , Aminopeptidases/biosynthesis , Aminopeptidases/genetics , Aminopeptidases/physiology , Animals , Bone Marrow Cells/ultrastructure , Cell Adhesion/physiology , Cells, Cultured , Female , Fibronectins/metabolism , Interleukin-3/pharmacology , Liver/cytology , Male , Mast Cells/physiology , Mice , Pregnancy , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-kit/biosynthesis , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/metabolism , Receptors, Interleukin-3/physiology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/physiology , Sensitivity and Specificity , fms-Like Tyrosine Kinase 3ABSTRACT
Flt3/Flk2 is a receptor tyrosine kinase that is expressed on early hematopoietic progenitor cells. Flt3/Flk2 belongs to a family of receptors, including Kit and colony-stimulating factor-1R, which support growth and differentiation within the hematopoietic system. The Flt3/Flk2 ligand, in combination with other growth factors, stimulates the proliferation of hematopoietic progenitors of both lymphoid and myeloid lineages in vitro. We report that phosphatidylinositol 3'-kinase (PI3K) binds to a unique site in the carboxy tail of murine Flt3/Flk2. In distinction to Kit and colony-stimulating factor-1R, mutant receptors unable to couple to PI3K and expressed in rodent fibroblasts or in the interleukin 3-dependent cell line Ba/F3 provide a mitogenic signal comparable to wild-type receptors. Flt3/Flk2 receptors that do not bind to PI3K also normally down-regulate, a function ascribed to PI3K in the context of other receptor systems. These data point to the existence of other unidentified pathways that, alone or in combination with PI3K, transduce these cellular responses following the activation of Flt3/Flk2.
Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Animals , Binding Sites , Cell Line , Cytoplasm/metabolism , Enzyme Activation , Mitosis , Phosphatidylinositol 3-Kinases , Phosphorylation , Phosphotyrosine/metabolism , Point Mutation , Protein Binding , Proteins/metabolism , Rats , Shc Signaling Adaptor Proteins , Signal Transduction , Src Homology 2 Domain-Containing, Transforming Protein 1 , Structure-Activity Relationship , fms-Like Tyrosine Kinase 3ABSTRACT
Flt3 is a receptor tyrosine kinase (RTK) structurally related to the CSF-1R encoded by the c-fms locus, Kit and the PDGFR which is restricted in its expression to hematopoietic precursor populations and several distinct cell types within the central nervous system. Although the ligand for Flt3 has recently been identified, the developmental function of Flt3 within these tissues has not yet been described. In order to examine the signalling properties of this receptor, we previously constructed a chimeric molecule containing the extracellular domain of CSF-1R fused to the transmembrane and cytoplasmic domain of mouse Flt3 (FF3). The ability of the FF3 to directly associate with or tyrosine phosphorylate specific cytoplasmic signalling molecules in vivo was examined. GAP, Vav, Shc, and to a lesser extent PLC gamma become tyrosine-phosphorylated but no in vivo association with the receptor was detectable. FF3 associates with PI3K activity and the SH2 domains of p85 and Grb-2. Phosphopeptide competition experiments suggest that the PI3K binding site is located outside of the kinase insert in the carboxy tail of the receptor.
Subject(s)
Phosphotransferases (Alcohol Group Acceptor)/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Amino Acid Sequence , Animals , Binding Sites , GTPase-Activating Proteins , Macrophage Colony-Stimulating Factor/pharmacology , Mice , Molecular Sequence Data , Phosphatidylinositol 3-Kinases , Phosphorylation , Proteins/metabolism , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/genetics , Receptor, Macrophage Colony-Stimulating Factor/metabolism , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Type C Phospholipases/metabolism , Tyrosine/metabolism , fms-Like Tyrosine Kinase 3ABSTRACT
The FLT3 receptor tyrosine kinase (RTK) belongs to the class III subfamily which includes PDGF, CSF1 and SLF receptors. The recent cloning of the FLT3 ligand suggesting its important role in the differentiation and proliferation of the hematopoietic stem cells, has confirmed the initial expression analysis showing restricted pattern of receptor expression within the primitive hematopoietic population. To better understand the function of the FLT3 receptor and its relationship with the other hematopoietic RTKs, we analyzed the mitogenic pathway and substrate specificity of this receptor. The construction of a chimeric receptor called FF3, between the extracellular region of the CSF1 receptor fused with the transmembrane and the cytoplasmic regions of FLT3, has allowed an analysis in the absence of FLT3 ligand. We have shown in previous studies that FF3 is able to transduce the signal induced by CSF1, to induce tyrosine phosphorylation and/or association of several cytoplasmic proteins. We show here that this new receptor is fully functional in Ba/F3 hematopoietic cells, inducing a CSF1 dependence when expressed at the surface of this IL3 dependent cell line. The PI3' Kinase interacts with the FF3 receptor through SH2 domains and its binding site is localized on the tyrosine residue 958 in the C terminal part of the receptor.
Subject(s)
Hematopoietic Stem Cells/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Proto-Oncogene Proteins/physiology , Receptor Protein-Tyrosine Kinases/physiology , Recombinant Fusion Proteins/metabolism , Animals , Binding Sites , Cell Division , Cell Line , Cells, Cultured , Chlorocebus aethiops , Hematopoietic Stem Cells/drug effects , Interleukin-3/pharmacology , Macrophage Colony-Stimulating Factor/pharmacology , Mice , Mitosis , Models, Biological , Multigene Family , Mutagenesis, Site-Directed , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Rats , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Macrophage Colony-Stimulating Factor/genetics , Receptor, Macrophage Colony-Stimulating Factor/metabolism , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Receptors, Cell Surface/physiology , Signal Transduction , fms-Like Tyrosine Kinase 3ABSTRACT
We have designed a convenient procedure for the analysis of V beta repertoire expression in polyclonal T-cell populations. In this procedure T-cell RNA is converted to cDNA, polydC-tailed with terminal deoxynucleotidyl transferase and submitted to one-side specificity PCR amplification with a constant region oligonucleotide primer. The amplified material is then analysed by reverse spot-test hybridization: after 32P-labelling, the amplification product is put to hybridize on a membrane where specially designed V beta subfamily-specific probes are immobilized. The radioactivity fixed on each probe can then be easily quantified and the signal obtained is directly proportional to the initial amount of homologous RNA. We applied this technique to the study of V beta gene selection following T-cell stimulation by staphylococcal enterotoxins B and E. We show that with these toxins two almost non-overlapping sets of T-cells are recruited and that this selection is likely to be dependent on specific amino acid residues shaping the fourth complementarity determining region of the TCR-beta chain. These residues constitute two tandemly-conserved tripeptide sequences (Asp39Pro40Gly41)-(Val69Ser70Arg71) and (Arg66Phe67Ser68)-(Asp88Ser89Ala90) in the SEB- and the SEE-responsive V beta gene clusters respectively.