CD8 beta increases CD8 coreceptor function and participation in TCR-ligand binding.

To study the role of CD8 beta in T cell function, we derived a CD8 alpha/beta-(CD8-/-) T cell hybridoma of the H-2Kd-restricted N9 cytotoxic T lymphocyte clone specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260. This hybridoma was transfected either with CD8 alpha alone or together with CD8 beta. All three hybridomas released interleukin 2 upon incubation with L cells expressing Kd-peptide derivative complexes, though CD8 alpha/beta cells did so more efficiently than CD8 alpha/alpha and especially CD8-/- cells. More strikingly, only CD8 alpha/beta cells were able to recognize a weak agonist peptide derivative variant. This recognition was abolished by Fab' fragments of the anti-Kd alpha 3 monoclonal antibody SF1-1.1.1 or substitution of Kd D-227 with K, both conditions known to impair CD8 coreceptor function. T cell receptor (TCR) photoaffinity labeling indicated that TCR-ligand binding on CD8 alpha/beta cells was approximately 5- and 20-fold more avid than on CD8 alpha/a and CD8-/- cells, respectively. SF1-1.1.1 Fab' or Kd mutation D227K reduced the TCR photoaffinity labeling on CD8 alpha/beta cells to approximately the same low levels observed on CD8-/- cells. These results indicate that CD8 alpha/beta is a more efficient coreceptor than CD8alpha/alpha, because it more avidly strengthens TCR-ligand binding.

SOCS-1 protein prevents Janus Kinase/STAT-dependent inhibition of beta cell insulin gene transcription and secretion in response to interferon-gamma.

In the pathogenesis of type I diabetes mellitus, activated leukocytes infiltrate pancreatic islets and induce beta cell dysfunction and destruction. Interferon (IFN)-gamma, tumor necrosis factor-alpha and interleukin (IL)-1 beta play important, although not completely defined, roles in these mechanisms. Here, using the highly differentiated beta Tc-Tet insulin-secreting cell line, we showed that IFN-gamma dose- and time-dependently suppressed insulin synthesis and glucose-stimulated secretion. As described previously IFN-gamma, in combination with IL-1 beta, also induces inducible NO synthase expression and apoptosis (Dupraz, P., Cottet, S., Hamburger, F., Dolci, W., Felley-Bosco, E., and Thorens, B. (2000) J. Biol. Chem. 275, 37672--37678). To assess the role of the Janus kinase/signal transducer and activator of transcription (STAT) pathway in IFN-gamma intracellular signaling, we stably overexpressed SOCS-1 (suppressor of cytokine signaling-1) in the beta cell line. We demonstrated that SOCS-1 suppressed cytokine-induced STAT-1 phosphorylation and increased cellular accumulation. This was accompanied by a suppression of the effect of IFN-gamma on: (i) reduction in insulin promoter-luciferase reporter gene transcription, (ii) decrease in insulin mRNA and peptide content, and (iii) suppression of glucose-stimulated insulin secretion. Furthermore, SOCS-1 also suppressed the cellular effects that require the combined presence of IL-1 beta and IFN-gamma: induction of nitric oxide production and apoptosis. Together our data demonstrate that IFN-gamma is responsible for the cytokine-induced defect in insulin gene expression and secretion and that this effect can be completely blocked by constitutive inhibition of the Janus kinase/STAT pathway.

Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

HLA photoaffinity labeling reveals overlapping binding of homologous melanoma-associated gene peptides by HLA-A1, HLA-A29, and HLA-B44.

Melanoma-associated genes (MAGEs) encode tumor-specific antigens that can be recognized by CD8+ cytotoxic T lymphocytes. To investigate the interaction of the HLA-A1-restricted MAGE-1 peptide 161-169 (EADPT-GHSY) with HLA class I molecules, photoreactive derivatives were prepared by single amino acid substitution with N beta-[iodo-4-azidosalicyloyl]-L-2,3-diaminopropionic acid. These derivatives were tested for their ability to bind to, and to photoaffinity-label, HLA-A1 on C1R.A1 cells. Only the derivatives containing the photoreactive amino acid in position 1 or 7 fulfilled both criteria. Testing the former derivative on 14 lymphoid cell lines expressing over 44 different HLA class I molecules indicated that it efficiently photoaffinity-labeled not only HLA-A1, but possibility also HLA-A29 and HLA-B44. MAGE peptide binding by HLA-A29 and HLA-B44 was confirmed by photoaffinity labeling with photoreactive MAGE-3 peptide derivatives on C1R.A29 and C1R.B44 cells, respectively. The different photoaffinity labeling systems were used to access the ability of the homologous peptides derived from MAGE-1, -2, -3, -4a, -4b, -6, and -12 to bind to HLA-A1, HLA-A29, and HLA-B44. All but the MAGE-2 and MAGE-12 nonapeptides efficiently inhibited photoaffinity labeling of HLA-A1, which is in agreement with the known HLA-A1 peptide-binding motif (acidic residue in P3 and C-terminal tyrosine). In contrast, photoaffinity labeling of HLA-A29 was efficiently inhibited by these as well as by the MAGE-3 and MAGE-6 nonapeptides. Finally, the HLA-B44 photoaffinity labeling, unlike the HLA-A1 and HLA-A29 labeling, was inhibited more efficiently by the corresponding MAGE decapeptides, which is consistent with the reported HLA-B44 peptide-binding motif (glutamic acid in P2, and C-terminal tyrosine or phenylalanine). The overlapping binding of homologous MAGE peptides by HLA-A1, A29, and B44 is based on different binding principles and may have implications for immunotherapy of MAGE-positive tumors.

Cutting edge: influence of the TCR V beta domain on the avidity of CD1d:alpha-galactosylceramide binding by invariant V alpha 14 NKT cells.

CD1d tetramers loaded with alpha-galactosylceramide (alpha-GalCer) bind selectively to mouse invariant Valpha14 (Valpha14i) NKT cells and their human counterparts. Whereas tetramer binding strictly depends on the expression of a Valpha14-Jalpha18 chain in murine NKT cells, the associated beta-chain (typically expressing Vbeta8.2 or Vbeta7) appears not to influence tetramer binding. In this study, we describe novel alpha-GalCer-loaded mouse and human CD1d-IgG1 dimers, which revealed an unexpected influence of the TCR-beta chain on the avidity of CD1d:alpha-GalCer binding. A subset of Valpha14i NKT cells clearly discriminated alpha-GalCer bound to mouse or human CD1d on the basis of avidity differences conferred by the Vbeta domain of the TCR-beta chain, with Vbeta8.2 conferring higher avidity binding than Vbeta7.

Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML1-ETO mouse model.

The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was followed by a second substantial rewiring of transcriptional networks occurring in the trajectory to manifest leukaemia. We also find that both HSC and lineage-restricted granulocyte macrophage progenitors (GMPs) acquired leukaemic stem cell (LSC) potential being capable of initiating and maintaining the disease. Finally, our data demonstrate that long-term expression of AE induces an indolent myeloproliferative disease (MPD)-like myeloid leukaemia phenotype with complete penetrance and that acute inactivation of AE function is a potential novel therapeutic option.

The interaction of human pathogenic arena viruses with the host cell

SummaryResearch projects presented in this thesis aimed to investigate two major aspects of the arenaviruses life cycle in the host cell: viral entry and the biosynthesis of the viral envelope glycoprotein.Old World arenaviruses (OWAV), such as Lassa virus (LASV) and lymphocytic choriomeningitis virus (LCMV), attach to the cell by binding to their receptor, alpha-dystroglycan. Virions are then internalized by a largely unknown pathway of endocytosis and delivered to the late endosome/lysosome where fusion occurs at low pH. In the major project of my thesis, we sought to identify cellular factors involved in OWAV cell entry. Our work indicates that OWAV cell entry requires microtubular transport and a functional multivesicular body (MVB) compartment. Infection indeed depends on phosphatidyl inositol 3-kinase (PI3K) activity and lysobisphosphatidic acid (LBPA), a lipid found in membranes of intraluminal vesicles (ILVs) of the MVB. We further found a requirement of factors that are part of the endosomal sorting complex required for transport (ESCRT), involved in the formation of ILVs. This suggests an ESCRT-mediated sorting of virus- receptor complex during the entry process.During viral replication, biosynthesis of viral glycoprotein takes place in the endoplasmic reticulum (ER) of the host cell. When protein load exceeds the folding capacity of the ER, the accumulation of unfolded proteins is sensed by three ER resident proteins, activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and PKR-like ER kinase (PERK), whose signaling induces the cellular unfolded protein response (UPR). Our results indicate that acute LCMV infection transiently induces the activation of the ATF6 branch of the UPR, whereas the PERK, and IRE1 axis of UPR are neither triggered nor blocked during infection. Our data also demonstrate that activation of ATF6 pathway is required for optimal viral replication during acute infection.The formation of the mature, fusion-active form of arenaviruses glycoproteins requires proteolytic cleavage mediated by the cellular protease subtilisin kexin isozyme-1 (SKI-l)/site-l protease (SIP). We show that targeting the SKI-1/S1P enzymatic activity with specific inhibitors is a powerful strategy to block arenaviruses productive infection. Moreover, characterization of protease function highlights differences in processing between cellular and viral substrates, opening new possibilities in term of drug development against human pathogenic arenaviruses.RésuméLes projets de recherche présentés dans cette thèse visaient à étudier deux aspects du cycle de vie des arenavirus: l'entrée du virus dans la cellule hôte et la biosynthèse de la glycoprotéine durant la réplication virale.Les arenavirus du vieux monde (OWAV), tels que le virus de Lassa (LASV) et le virus de la chorioméningite lymphocytaire (LCMV) s'attachent à la cellule hôte en se liant à leur récepteur, l'alpha-dystroglycane. Les virions sont ensuite intemalisés par une voie d'endocytose inconnue et livrés à l'endosome tardif/lysosome, où le pH acide permet la fusion entre l'enveloppe virale et la membrane du compartiment. Le projet principal de ma thèse consistait à identifier les facteurs cellulaires impliqués dans l'entrée des OWAV dans la cellule hôte. Nos résultats indiquent que l'entrée des OWAV nécessite le transport microtubulaire et la présence d'un corps multivésiculaire (MVB) fonctionnel. L'infection dépend en effet de l'activité de phosphatidyl inositol 3-kinase (PI3K) et de lysobisphosphatidic acid (LBPA), un lipide présent dans les membranes des vésicules intraluminales (ILVs) du MVB. Nous avons également trouvé l'implication de facteurs constituant l'endosomal sorting complex required for sorting (ESCRT) qui joue un rôle dans la formation des ILVs. Ces donnés suggèrent l'incorporation du complexe virus-récepteur dans des ILVs durant le processus d'entrée.Lors de la réplication virale, la biosynthèse de la glycoprotéine virale a lieu dans le réticulum endoplasmique (ER) de la cellule hôte. Lorsque la charge de protéines nouvellement synthétisées excède la capacité de pliage des protéines dans le ER, l'accumulation de protéines mal pliées est détectée par trois facteurs: activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) et PKR-like ER kinase (PERK). Leur signalisation constitue la réponse cellulaire face aux protéines mal pliées (UPR). Nos résultats montrent que l'infection aiguë avec LCMV induit transitoirement l'activation de la voie de signalisation ATF6 alors que les axes PERK et IRE1 de l'UPR ne sont ni induits ni bloqués pendant l'infection. Nos données prouvent également que l'activation de la voie ATF6 est nécessaire à une réplication virale optimale lors de l'infection aiguë avec LCMV.La maturation des glycoprotéines des arenavirus nécessite un clivage protéolytique par la protéase cellulaire subtilisin kexin isozyme-1 (SKI-l)/site-l protease (SIP). Nous avons démontré que le ciblage de l'activité enzymatique de SKI-1/SIΡ avec des inhibiteurs spécifiques est une stratégie prometteuse pour bloquer l'infection par les arenavirus. La caractérisation du mécanisme d'action de la protéase a, par ailleurs, révélé des différences au niveau du clivage entre les substrats cellulaires et viraux, ce qui ouvre de nouvelles perspectives en terme de développement de médicaments contre les arenavirus pathogènes pour l'homme.

Discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (GIP) receptor: the impact on the pancreatic beta cell responses and functional expression studies in Chinese hamster fibroblast cells.

The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are insulinotropic factors released from the small intestine to the blood stream in response to oral glucose ingestion. The insulinotropic effect of GLP-1 is maintained in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas, for unknown reasons, the effect of GIP is diminished or lacking. We defined the exon-intron boundaries of the human GIP receptor, made a mutational analysis of the gene and identified two amino acid substitutions, A207 V and E354Q. In an association study of 227 Caucasian Type II diabetic patients and 224 matched glucose tolerant control subjects, the allelic frequency of the A207 V polymorphism was 1.1% in Type II diabetic patients and 0.7% in control subjects (p = 0.48), whereas the allelic frequency of the codon 354 polymorphism was 24.9% in Type II diabetic patients versus 23.2% in control subjects. Interestingly, the glucose tolerant subjects (6% of the population) who were homozygous for the codon 354 variant had on average a 14% decrease in fasting serum C-peptide concentration (p = 0.01) and an 11% decrease in the same variable 30 min after an oral glucose load (p = 0.03) compared with subjects with the wild-type receptor. Investigation of the function of the two GIP receptor variants in Chinese hamster fibroblasts showed, however, that the GIP-induced cAMP formation and the binding of GIP to cells expressing the variant receptors were not different from the findings in cells expressing the wildtype GIP receptor. In conclusion, amino acid variants in the GIP receptor are not associated with random Type II diabetes in patients of Danish Caucasian origin or with altered GIP binding and GIP-induced cAMP production when stably transfected in Chinese hamster fibroblasts. The finding of an association between homozygosity for the codon 354 variant and reduced fasting and post oral glucose tolerance test (OGTT) serum C-peptide concentrations, however, calls for further investigations and could suggest that GIP even in the fasting state regulates the beta-cell secretory response.

Positive and negative roles of the trans-acting T cell factor-1 for the acquisition of distinct Ly-49 MHC class I receptors by NK cells.

Members of the Ly-49 gene family code for class I MHC-specific receptors that regulate NK cell function. Due to a combinatorial distribution of Ly-49 receptors, NK cells display considerable clonal heterogeneity. The acquisition of one Ly-49 receptor, Ly-49A is strictly dependent on the transcriptional trans-acting factor T cell-specific factor-1 (TCF-1). Indeed, TCF-1 binds to two sites in the Ly-49a promoter and regulates its activity, suggesting that the Ly-49a gene is a direct TCF-1 target. TCF-1 deficiency resulted in the altered usage of additional Ly-49 receptors. We show in this study, using TCF-1 beta(2)-microglobulin double-deficient mice, that these repertoire alterations are not due to Ly-49/MHC class I interactions. Our findings rather suggest a TCF-1-dependent, cell autonomous effect on the acquisition of multiple Ly-49 receptors. Besides reduced receptor usage (Ly-49A and D), we also observed no effect (Ly-49C) and significantly expanded (Ly-49G and I) receptor usage in the absence of TCF-1. These effects did not in all cases correlate with the presence of TCF binding sites in the respective proximal promoter. Therefore, besides TCF-1 binding to the proximal promoter, Ly-49 acquisition may also be regulated by TCF-1 binding to more distant cis-acting elements and/or by regulating the expression of additional trans-acting factors. Consistent with the observed differential, positive or negative role of TCF-1 for Ly-49 receptor acquisition, reporter gene assays revealed the presence of an inducing as well as a repressing TCF site in certain proximal Ly-49 promoters. These findings reveal an important role of TCF-1 for the formation of the NK cell receptor repertoire.

Ezrin-Radixin-Moesin-binding Sequence of PSGL-1 Glycoprotein Regulates Leukocyte Rolling on Selectins and Activation of Extracellular Signal-regulated Kinases.

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the capture (tethering) of free-flowing leukocytes and subsequent rolling on selectins. PSGL-1 interactions with endothelial selectins activate Src kinases and spleen tyrosine kinase (Syk), leading to α(L)β(2) integrin-dependent leukocyte slow rolling, which promotes leukocyte recruitment into tissues. In addition, but through a distinct pathway, PSGL-1 engagement activates ERK. Because ezrin, radixin and moesin proteins (ERMs) link PSGL-1 to actin cytoskeleton and because they serve as adaptor molecules between PSGL-1 and Syk, we examined the role of PSGL-1 ERM-binding sequence (EBS) on cell capture, rolling, and signaling through Syk and MAPK pathways. We carried out mutational analysis and observed that deletion of EBS severely reduced 32D leukocyte tethering and rolling on L-, P-, and E-selectin and slightly increased rolling velocity. Alanine substitution of Arg-337 and Lys-338 showed that these residues play a key role in supporting leukocyte tethering and rolling on selectins. Importantly, EBS deletion or Arg-337 and Lys-338 mutations abrogated PSGL-1-induced ERK activation, whereas they did not prevent Syk phosphorylation or E-selectin-induced leukocyte slow rolling. These studies demonstrate that PSGL-1 EBS plays a critical role in recruiting leukocytes on selectins and in activating the MAPK pathway, whereas it is dispensable to phosphorylate Syk and to lead to α(L)β(2)-dependent leukocyte slow rolling.

Identification of proteoglycans as the APRIL-specific binding partners.

B cell activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) are closely related ligands within the TNF superfamily that play important roles in B lymphocyte biology. Both ligands share two receptors--transmembrane activator and calcium signal--modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA)--that are predominantly expressed on B cells. In addition, BAFF specifically binds BAFF receptor, whereas the nature of a postulated APRIL-specific receptor remains elusive. We show that the TNF homology domain of APRIL binds BCMA and TACI, whereas a basic amino acid sequence (QKQKKQ) close to the NH2 terminus of the mature protein is required for binding to the APRIL-specific "receptor." This interactor was identified as negatively charged sulfated glycosaminoglycan side chains of proteoglycans. Although T cell lines bound little APRIL, the ectopic expression of glycosaminoglycan-rich syndecans or glypicans conferred on these cells a high binding capacity that was completely dependent on APRIL's basic sequence. Moreover, syndecan-1-positive plasma cells and proteoglycan-rich nonhematopoietic cells displayed high specific, heparin-sensitive binding to APRIL. Inhibition of BAFF and APRIL, but not BAFF alone, prevented the survival and/or the migration of newly formed plasma cells to the bone marrow. In addition, costimulation of B cell proliferation by APRIL was only effective upon APRIL oligomerization. Therefore, we propose a model whereby APRIL binding to the extracellular matrix or to proteoglycan-positive cells induces APRIL oligomerization, which is the prerequisite for the triggering of TACI- and/or BCMA-mediated activation, migration, or survival signals.

The C76R transmembrane activator and calcium modulator cyclophilin ligand interactor mutation disrupts antibody production and B-cell homeostasis in heterozygous and homozygous mice.

BackgroundMutations in TNFRSF13B, the gene encoding transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), are found in 10% of patients with common variable immunodeficiency. However, the most commonly detected mutation is the heterozygous change C104R, which is also found in 0.5% to 1% of healthy subjects. The contribution of the C104R mutation to the B-cell defects observed in patients with common variable immunodeficiency therefore remains unclear.ObjectiveWe sought to define the functional consequences of the C104R mutation on B-cell function.MethodsWe performed in vitro studies of TACI C104R expression and signaling. A knock-in mouse with the equivalent mutation murine TACI (mTACI) C76R was generated as a physiologically relevant model of human disease. We examined homozygous and heterozygous C76R mutant mice alongside wild-type littermates and studied specific B-cell lineages and antibody responses to T cell-independent and T cell-dependent challenge.ResultsC104R expression and ligand binding are significantly diminished when the mutant protein is expressed in 293T cells or in patients' cell lines. This leads to defective nuclear factor κB activation, which is proportionally restored by reintroduction of wild-type TACI. Mice heterozygous and homozygous for mTACI C76R exhibit significant B-cell dysfunction with splenomegaly, marginal zone B-cell expansion, diminished immunoglobulin production and serological responses to T cell-independent antigen, and abnormal immunoglobulin synthesis.ConclusionsThese data show that the C104R mutation and its murine equivalent, C76R, can significantly disrupt TACI function, probably through haploinsufficiency. Furthermore, the heterozygous C76R mutation alone is sufficient to disturb B-cell function with lymphoproliferation and immunoglobulin production defects.

Cathepsin X cleavage of the beta2 integrin regulates talin-binding and LFA-1 affinity in T cells.

T cell migration, essential for immune surveillance and response, is mediated by the integrin LFA-1. CatX, a cysteine carboxypeptidase, is involved in the regulation of T cell migration by interaction with LFA-1. We show that sequential cleavage of C-terminal amino acids from the β(2) cytoplasmic tail of LFA-1, by CatX, enhances binding of the adaptor protein talin to LFA-1 and triggers formation of the latter's high-affinity form. As shown by SPR analysis of peptides constituting the truncated β(2) tail, the cleavage of three C-terminal amino acids by CatX resulted in a 1.6-fold increase of talin binding. Removal of one more amino acid resulted in a 2.5-fold increase over the intact tail. CatX cleavage increased talin-binding affinity to the MD but not the MP talin-binding site on the β(2) tail. This was shown by molecular modeling of the β(2) tail/talin F3 complex to be a result of conformational changes affecting primarily the distal-binding site. Analysis of LFA-1 by conformation-specific mAb showed that CatX modulates LFA-1 affinity, promoting formation of high-affinity from intermediate-affinity LFA-1 but not the initial activation of LFA-1 from a bent to extended form. CatX post-translational modifications may thus represent a mechanism of LFA-1 fine-tuning that enables the trafficking of T cells.

Regulation of stem cell factor expression in inflammation and asthma

Stem cell factor (SCF) is a major mast cell growth factor, which could be involved in the local increase of mast cell number in the asthmatic airways. In vivo, SCF expression increases in asthmatic patients and this is reversed after treatment with glucocorticoids. In vitro in human lung fibroblasts in culture, IL-1beta, a pro-inflammatory cytokine, confirms this increased SCF mRNA and protein expression implying the MAP kinases p38 and ERK1/2 very early post-treatment, and glucocorticoids confirm this decrease. Surprisingly, glucocorticoids potentiate the IL-1beta-enhanced SCF expression at short term treatment, implying increased SCF mRNA stability and SCF gene transcription rate. This potentiation involves p38 and ERK1/2. Transfection experiments with the SCF promoter including intron1 also confirm this increase and decrease of SCF expression by IL-1beta and glucocorticoids, and the potentiation by glucocorticoids of the IL-1beta-induced SCF expression. Deletion of the GRE or kappaB sites abolishes this potentiation, and the effect of IL-1beta or glucocorticoids alone. DNA binding of GR and NF-kappaB are also demonstrated for these effects. In conclusion, this review concerns new mechanisms of regulation of SCF expression in inflammation that could lead to potential therapeutic strategy allowing to control mast cell number in the asthmatic airways.