Cell Surface Expression Of C-kit Research Articles

Tracheal Smooth Muscle Cells Stimulated by Stem Cell Factor-c-Kit Coordinate the Production of Transforming Growth Factor-β1 and Fibroblast Growth Factor-2 Mediated by Chemokine (C-C Motif) Ligand 3.

The aim of this study was to evaluate the mechanism involved in the stem cell factor (SCF)-induced production of fibroblast growth factor-2 (FGF-2), transforming growth factor-β1 (TGF-β1), and chemokine (C-C motif) ligand 3 (CCL3) in tracheal smooth muscle cells (tSMCs) and the signaling pathway involved in the process. tSMC primary cultures were stimulated with SCF and evaluated at 24 h. Cells treated with specific antibodies did not show any immunolabeling for cytokeratin or fibroblast activation protein, but were positive for α-smooth muscle actin, indicating the purity of the primary cell line. Western blot analysis showed constitutive phosphorylation of c-Kit, as well as increased total protein and phosphorylated c-Kit levels in tSMCs after SCF stimulation. Flow cytometry analysis also showed an increase in cell-surface c-Kit expression in the presence of SCF. SCF induced TGF-β mRNA expression in tSMCs, as well as the production of TGF-β1, CCL3, and FGF-2. Pretreatment with anti-CCL3 antibody blocked TGF-β1 expression and partially inhibited FGF-2 production. On the other hand, anti-c-Kit antibody blocked TGF-β1 expression and FGF-2 production. Thus, TGF-β1 and FGF-2 production were mediated by CCL3 production through c-Kit. Pretreatment with mitogen-activated protein kinase kinase 1, p38, and Jun N-terminal kinase inhibitors showed that the effects mediated by SCF were involved with the modulation of mitogen-activated protein kinase (MAPK) pathways. Development of inhibitors targeting CCL3 through MAPK activation could thus be an attractive strategy to inhibit tSMC activation during asthma.

Antileukemic Activity of 2-Deoxy-d-Glucose through Inhibition of N-Linked Glycosylation in Acute Myeloid Leukemia with FLT3-ITD or c-KIT Mutations.

We assessed the antileukemic activity of 2-deoxy-d-glucose (2-DG) through the modulation of expression of receptor tyrosine kinases (RTK) commonly mutated in acute myeloid leukemia (AML). We used human leukemic cell lines cells, both in vitro and in vivo, as well as leukemic samples from AML patients to demonstrate the role of 2-DG in tumor cell growth inhibition. 2-DG, through N-linked glycosylation inhibition, affected the cell-surface expression and cellular signaling of both FTL3-ITD and mutated c-KIT and induced apoptotic cell death. Leukemic cells harboring these mutated RTKs (MV4-11, MOLM-14, Kasumi-1, and TF-1 c-KIT D816V) were the most sensitive to 2-DG treatment in vitro as compared with nonmutated cells. 2-DG activity was also demonstrated in leukemic cells harboring FLT3-TKD mutations resistant to the tyrosine kinase inhibitor (TKI) quizartinib. Moreover, the antileukemic activity of 2-DG was particularly marked in c-KIT-mutated cell lines and cell samples from core binding factor-AML patients. In these cells, 2-DG inhibited the cell-surface expression of c-KIT, abrogated STAT3 and MAPK-ERK pathways, and strongly downregulated the expression of the receptor resulting in a strong in vivo effect in NOD/SCID mice xenografted with Kasumi-1 cells. Finally, we showed that 2-DG decreases Mcl-1 protein expression in AML cells and induces sensitization to both the BH3 mimetic inhibitor of Bcl-xL, Bcl-2 and Bcl-w, ABT-737, and cytarabine. In conclusion, 2-DG displays a significant antileukemic activity in AML with FLT3-ITD or KIT mutations, opening a new therapeutic window in a subset of AML with mutated RTKs.

Transmyocardial Revascularization Enhances Mesenchymal Stem Cell Engraftment in Infarcted Hearts through SCF–c-Kit and SDF-1–CXCR4 Signaling Axes

Purpose We investigated the roles of stem cell factor(SCF)–c-kit and stromal derived factor-1(SDF-1)–chemokine receptor type 4(CXCR4) stem cell signaling axes in transmyocardial revascularization (TMR)-enhanced engraftment of transplanted mesenchymal stem cells (MSC) in infarcted hearts. Methods and Materials 3 weeks after LAD ligation, female Lewis rats underwent 10-channel needle TMR, followed by daily IV injections of 1 million male donor MSC for 5 days, either wild type (WT) or with knockdown (K/D) of c-kit or CXCR4, accomplished via a shRNA+plasmid in a lentiviral vector. Experimental groups included: WT MSC with or without TMR, c-kit K/D MSC with or without TMR, and CXCR4 K/D MSC with or without TMR (N=6/group). Results In vitro cell surface expression of c-kit (N=3) was reduced from 14±0.7% of WT MSC to 1.6±0.4% c-kit K/D MSC, and CXCR4 (N=3) was reduced from 39±10% WT MSCs to 3.7±0.7% CXCR4 K/D MSC after 1 week (p Conclusions Downregulation of either c-kit or CXCR4 in MSC decreased engraftment of circulating MSC and inhibited the reparative effects of TMR. Hence, both SCF–c-kit and SDF-1–CXCR4 signaling axes are required for TMR-augmented repair of the infarcted heart.

Rab27b regulates c-kit expression by controlling the secretion of stem cell factor

Rab27b, a subfamily of Rab27 small GTPases, was originally identified in platelets. However, the role of Rab27b in megakaryocytic lineage cells remains unknown. Here, using a human megakaryoblastic cell line, CMK, we show that Rab27b negatively regulates c-kit-expression. We found that transfection of shRNA-Rab27b into CMK cells led to specific increase in the amount of the receptor-type tyrosine kinase c-kit. To elucidate the molecular mechanisms by which Rab27b regulates c-kit expression, we analyzed the dynamics of c-kit by the stimulation with its ligand, stem cell factor (SCF). We found that cell surface expression of c-kit was promptly reduced and rapidly degraded in both CMK and Rab27b-knockdown CMK cells. Pretreatment with a lysosome inhibitor bafilomycin suppressed the degradation of c-kit, indicating that c-kit expression is controlled by SCF-induced endolysosomal degradation system. We therefore focused on the potential involvement of SCF in Rab27b-mediated effects on c-kit expression levels. We found that autocrine secretion of SCF was downregulated in Rab27b-knockdown cells as compared with parental CMK cells. These results suggest that Rab27b negatively regulates the cell surface expression of c-kit via secretion of SCF and that ligation of SCF leads to the endolysosomal degradation system of c-kit.

Direct and differential effects of stem cell factor on the neovascularization activity of endothelial progenitor cells

Previous studies on the role of stem cell factor (SCF) in endothelial progenitor cell (EPC)-mediated neovascularization have focused on the EPC mobilization and homing process. However, the direct effects of SCF on neovascularization activity of EPCs have not been characterized. We sought to determine whether SCF regulates the neovascularization ability of EPCs by comparing its roles in mature endothelial cells. In vitro and in vivo assays revealed that SCF substantially increased the neovascularization activity of human EPCs through the c-Kit receptor. Notably, the SCF-induced increase in neovascularization activity was substantially greater in EPCs than that in human umbilical vein endothelial cells (HUVECs). SCF-induced phosphorylation of c-Kit and downstream signalling molecules was consistently found to be more potent and longer-lasting in EPCs than in HUVECs. This high responsiveness of EPCs to SCF was explained by the finding that the cell-surface expression of c-Kit is far higher in EPCs than in HUVECs. A c-Kit promoter assay revealed that the increased expression of c-Kit in EPCs could be attributed to the greater expression of stem cell leukaemia, LIM-only 2, and GATA-binding protein 2. In addition to its documented role in the mobilization and recruitment of EPCs, our findings show that SCF directly enhances the neovascularization activity of EPCs. Furthermore, the present study provides further evidence that EPCs exhibit differentially greater responsiveness to hypoxia-inducible cytokines, including SCF, than mature endothelial cells, suggesting that EPCs in ischaemic tissues function differently from mature endothelial cells, although they exhibit very similar phenotypes.

GATA2 and Sp1 Positively Regulate the c-kit Promoter in Mast Cells

The c-kit gene is expressed in hematopoietic stem cells and lineage progenitor cells but is downregulated during cell development in most lineages, except for mast cells. In mast cells, high expression of c-kit is maintained during development, and c-Kit signaling is essential for mast cell development. To analyze the mechanisms by which c-kit gene expression are regulated in mast cells, we examined mast cell type-specific regulation of the c-kit promoter region. We observed that a GC-box in the c-kit promoter was critical for transcriptional activity and was bound to the transcription factor Sp1 as assessed using reporter assay and electrophoretic mobility assay. Chromatin immunoprecipitation assay and coexpression analyses showed that the transcription factor GATA2, which was recruited to the c-kit promoter in a mast cell-specific manner, in addition to Sp1, transactivated the c-kit promoter via the GC-box. Electrophoretic mobility assay and rechromatin immunoprecipitation assay indicated that GATA2 binds to the GC-box by forming a complex with Sp1. Introduction of Sp1 small interfering RNA significantly reduced the amount not only of Sp1 but also of GATA2 binding to the c-kit promoter in mast cells, resulting in suppression of c-kit transcription. Knockdown of GATA2 suppressed the recruitment of GATA2 toward the c-kit promoter, subsequently suppressing cell surface expression of c-Kit. These findings indicate that GATA2 and Sp1 play crucial roles in expression of the c-kit gene in mast cells.

A critical role for c-kit on dendritic cells in regulating in T helper cell differentiation and allergic asthma (79.3)

Abstract Dendritic cells are integral in instructing the differentiation of T helper cells into Th1, Th2 and Th17 subsets and IL-6 is a key regulator of CD4 T cell differentiation .In this study, we investigated pathways in DCs that promote IL-6 production. We show exposure of DCs to house dust mite (HDM) or the mucosal adjuvant cholera toxin (CT) promotes cell surface expression of c-Kit and its ligand, stem cell factor (SCF), on DCs. This dual upregulation of c-Kit and SCF results in sustained signaling downstream of c-Kit, thereby promoting IL-6 secretion. Intranasal administration of antigen into c-Kit-mutant mice or neutralization of IL-6 in cultures from the lung-draining lymph nodes of immunized wild-type mice blunted the Th2 and Th7 responses. DCs lacking functional c-Kit or those unable to express membrane-bound SCF secreted lower amounts of IL-6 in response to HDM or CT. Expression of the Th2-Notch ligand Jagged-2 was also reduced in DCs from c-Kit-mutant mice in response to CT or HDM and the Th1-inducing adjuvant, CpG oligodeoxynucleotide, did not promote either c-Kit or Jagged-2 expression. DCs expressing nonfunctional c-Kit were unable to induce a robust Th2 or Th17 response and elicited diminished allergic airway inflammation when adoptively transferred into WT mice. DCs generated from mice expressing a catalytically inactive form of the p110delta subunit of phosphatidylinositol-3 (PI3) kinase secreted lower amounts of IL-6 upon stimulation with cholera toxin. Taken together, these results reveal the importance of the c-Kit-PI3 kinase-IL-6 signaling axis in DCs in the regulation of T cell responses.

Ectopic Expression of ERG and FLI1 Enforce a Megakaryocyte Phenotype Independent of GATA-1

Specific genes that contribute to Transient Myeloproliferative Disorder (TMD) and DS Acute Megakaryocytic Leukemia (AMKL) remain largely undefined. To date, only the X-linked transcription factor GATA1, which is mutated in these disorders, has been implicated in contributing to DS hematopoietic malignancies. We recently reported that overexpression of either of the chromosome 21 ETS family members ERG or ETS2 led to a dramatic increase in megakaryopoeisis from wild-type or Gata1-knockdown fetal liver hematopoietic progenitors. Furthermore, we demonstrated that expression of ETS2 or ERG caused a significant increase in megakaryocyte colony formation and led to immortalization of Gata1-knockdown fetal liver hematopoietic progenitors in vitro. In new studies to define the mechanisms by which these ETS proteins promote megakaryopoiesis, we discovered that the ETS family member FLI1, which shares significant homology with ERG, significantly increased CD41 and CD42 expression and the degree of polyploidization of megakaryocytes in vitro in a manner that was nearly identical to that of ectopic ERG. Furthermore, FLI1 also immortalized Gata1-knockdown fetal liver progenitors. To characterize the effects of ectopic ETS family members on transcription, we performed genome wide expression profiling using RNA isolated from fourth generation immortalized Gata1-knockdown cells expressing ETS2, ERG, FLI1 or GFP alone. Principal component analysis showed that the ERG and FLI1 gene expression patterns clustered tightly with one another and were well separated from both the MIGR1 and ETS2 signatures. Unsupervised hierarchical clustering further revealed that 726 genes were differentially expressed in FLI1, ERG, ETS2 immortalized cells compared to MIGR1 control cells. Of these, genes associated with the erythroid lineage, including Klf1, Lmo2, glycophorin A, Egr1, Ank1 and Eraf were strongly repressed. These findings are consistent with recent publications showing that FLI1 promotes the development of megakaryocytes at the expense of red blood cells and suggest that ERG and ETS2 mimic FLI1 in our assays. Notably, we did not observe significant increases in the expression of Hox genes or Meis1, genes whose expression is often associated with transformed hematopoietic cells. However, we did detect increased expression of Bmi1, which has an essential role in regulating the proliferative capacity of both normal and malignant hematopoietic progenitors, as well as increased expression of Jak2 and Stat5, two genes whose activation is associated with myeloproliferative disease and megakaryocytic transformation, in ETS-family transduced cells. Next, to determine whether ETS-family expression affected the immunophenotype of Gata1-knockdown fetal liver progenitors, immortalized cells were assessed for cell surface expression of c-kit and CD41. ERG and FLI1 expressing colonies included a substantial proportion of CD41+/c-kit- cells, indicative of megakaryocytic maturation. Furthermore, colonies formed from progenitors transduced with ERG were comprised of greater than 90% CD41+ cells, confirming that nearly all of the cells within the immortalized colonies are of the megakaryocyte lineage. Finally, because our gene array data showed increased Jak2 and Stat5 mRNA levels in ETS-family transduced cells, and activating mutations in JAK3 are associated with AMKL, we assessed whether JAK/STAT signaling was altered in the immortalized cells. Using flow cytometry, we found that cells within the ERG and FLI1 cultures consistently showed significant increases in phospho-STAT3 staining in comparison to the MIGR1 control. The increases in STAT3 phosphorylation suggest that ERG and FLI1 promote immortalization of Gata1-knockdown fetal liver progenitors in part by activation of the JAK/STAT pathway. Together our data support the hypothesis that an increased dosage of ETS factors, including ETS2 and ERG, contribute to a pro-megakaryocytic phenotype and to the leukemogenic process in DS TMD and AMKL.

Activation of c-Kit in dendritic cells regulates T helper cell differentiation and allergic asthma

Dendritic cells (DCs) are integral to the differentiation of T helper cells into T helper type 1 T(H)1, T(H)2 and T(H)17 subsets. Interleukin-6 (IL-6) plays an important part in regulating these three arms of the immune response by limiting the T(H)1 response and promoting the T(H)2 and T(H)17 responses. In this study, we investigated pathways in DCs that promote IL-6 production. We show that the allergen house dust mite (HDM) or the mucosal adjuvant cholera toxin promotes cell surface expression of c-Kit and its ligand, stem cell factor (SCF), on DCs. This dual upregulation of c-Kit and SCF results in sustained signaling downstream of c-Kit, promoting IL-6 secretion. Intranasal administration of antigen into c-Kit-mutant mice or neutralization of IL-6 in cultures established from the lung-draining lymph nodes of immunized wild-type mice blunted the T(H)2 and T(H)17 responses. DCs lacking functional c-Kit or those unable to express membrane-bound SCF secreted lower amounts of IL-6 in response to HDM or cholera toxin. DCs expressing nonfunctional c-Kit were unable to induce a robust T(H)2 or T(H)17 response and elicited diminished allergic airway inflammation when adoptively transferred into mice. Expression of the Notch ligand Jagged-2, which has been associated with T(H)2 differentiation, was blunted in DCs from c-Kit-mutant mice. c-Kit upregulation was specifically induced by T(H)2- and T(H)17-skewing stimuli, as the T(H)1-inducing adjuvant, CpG oligodeoxynucleotide, did not promote either c-Kit or Jagged-2 expression. DCs generated from mice expressing a catalytically inactive form of the p110delta subunit of phosphatidylinositol-3 (PI3) kinase (p110(D910A)) secreted lower amounts of IL-6 upon stimulation with cholera toxin. Collectively, these results highlight the importance of the c-Kit-PI3 kinase-IL-6 signaling axis in DCs in regulating T cell responses.

Cardiac stem cells: isolation, expansion and experimental use for myocardial regeneration

Cellular cardiomyoplasty (myogenic cell grafting) is actively being explored as a novel method to regenerate damaged myocardium. The adult human heart contains small populations of indigenous committed cardiac stem cells or multipotent cardiac progenitor cells, identified by their cell-surface expression of c-kit (the receptor for stem cell factor), P-glycoprotein (a member of the multidrug resistance protein family), and Sca-1 (stem cell antigen 1, a mouse hematopoietic stem cell marker) or a Sca-1-like protein. Cardiac stem cells represent a logical source to exploit in cardiac regeneration therapy because, unlike other adult stem cells, they are likely to be intrinsically programmed to generate cardiac tissue in vitro and to increase cardiac tissue viability in vitro. Cardiac stem cell therapy could, therefore, change the fundamental approach to the treatment of heart disease.

Effect of STI-571 (imatinib mesylate) in combination with retinoic acid and γ-irradiation on viability of neuroblastoma cells

Neuroblastoma (NB) expresses the tyrosine kinase receptors c-Kit, PDGFR-α and -β—targets for STI-571.We investigated a possible combination therapy of STI-571 with retinoic acid (RA) and γ-irradiation on NB cell viability in vitro. Expression of tyrosine kinase receptors and their ligands was examined in 6 NB cell lines by RT-PCR and FACS. The effect on cell viability was determined by MTT assay. Cell viability of all 6 NB cell lines was significantly inhibited after treatment with 20 μM STI-571 for 72 h, two cell lines responding already to 10 μM. Cell lines responded irrespective of their mRNA status or cell surface expression of c-Kit, PDGFR-α and -β. Co-incubation with 9- cis RA sensitized cells to the inhibitory effects of STI-571. However, pre-treatment with 9- cis RA resulted in resistance of NB cell lines to STI-571 and γ-irradiation. Treatment of NB with STI-571 in combination with 9- cis RA might be a therapeutic strategy for patients in consolidation therapy who have completed γ-irradiation therapy.

Changes in c‐Kit expression and effects of SCF during differentiation of human erythroid progenitor cells

We analysed c-Kit expression during erythroid differentiation using immunocytochemical staining and flow cytometric analysis. Burst-forming units-erythroid (BFU-E)-derived cell aggregates were identified in methylcellulose cultures containing human umbilical cord blood CD34+ cells and were stained by the indirect immunoalkaline phosphatase method. To investigate the changes in levels of cell-surface c-Kit expression, we subjected progenitor cells in liquid culture to flow cytometric analysis. In addition, the effects of stem cell factor (SCF) on cell-surface c-Kit expression were analysed in these two culture systems and the effects of SCF on erythroid colony formation were studied in a methylcellulose culture. c-Kit was expressed on the cell surface from BFU-E to erythroid precursors recognized morphologically as basophilic erythroblasts. Flow cytometric analysis showed that c-Kit expression increased until 6 d in liquid culture, and that decreased expression of c-Kit was associated with the increased expression of glycophorin A. Moreover, SCF increased the size of erythroid colonies when added at days 0, 4 and 8 in methylcellulose cultures. These results indicate that the c-Kit/SCF system still plays in proliferation of erythroid progenitor cells at the colony-forming units-erythroid stage. Finally, expression of c-Kit in erythroid progenitor cells cultured without SCF showed a diffuse pattern on the cell surface, whereas we observed positive c-Kit immunoreactivity in the region of the Golgi apparatus of these cells cultured with SCF. Flow cytometric analysis also showed that the levels of cell-surface c-Kit expression decreased in the presence of SCF. These results suggest that SCF induced down-modulation of cell-surface c-Kit expression, despite continuous synthesis of c-Kit protein.

Membrane-Bound Steel Factor Induces More Persistent Tyrosine Kinase Activation and Longer Life Span of c-kit Gene-Encoded Protein Than Its Soluble Form

Alternative splicing of exon 6 results in the production of two isoforms of Steel factor (SLF): the membrane-bound and soluble forms. To investigate differences in the kinetics of c-kit tyrosine kinase activated by these two isoforms, we used a stromal cell line (SI/SI4) established from SI/SI homozygous murine embryo fetal liver and its stable transfectants containing either hSCF248 cDNA (including exon 6; secreted form) or hSCF220 cDNA (lacking exon 6; membrane-bound form) as the source of each isoform. Interaction of factor dependent myeloid cell line MO7e with stromal cells producing either isoform resulted in activated c-kit tyrosine kinase and induction of the same series of tyrosine phosphorylated cellular proteins in MO7e cells. However, SI4-h220 (membrane-bound form) induced more persistent activation of c-kit kinase than SI4-h248 (soluble form) did. Flow cytometric analysis and pulse-chase studies using [35S]methionine showed that SI4-h248 induced rapid downmodulation of cell-surface c-kit expression and its protein degradation in MO7e cells, whereas SI4-h220 induced more prolonged life span of c-kit protein. Addition of soluble recombinant human SLF to SI4-h220 cultures enhanced reduction of cell-surface c-kit expression and its protein degradation. Because the kinetics of c-kit inactivation strikingly fits with the protein degradation rates of c-kit under the conditions described above, rapid proteolysis of c-kit protein induced by soluble SLF stimulation may function as a "turn-off switch" for activated c-kit kinase.

Transforming growth factor-beta regulates c-kit message stability and cell-surface protein expression in hematopoietic progenitors

The cell-surface receptor c-kit and its cognate ligand stem-cell factor (SCF) or steel factor (SLF) are important for the maintenance of hematopoiesis both in vitro and in vivo. Transforming growth factor- beta (TGF-beta) has been shown to be a potent inhibitor of SLF-mediated synergistic growth of murine Lin-Sca-1+ progenitor cells, as well as more committed progenitors. In the present study, we examined the regulation of c-kit mRNA and cell-surface expression by TGF-beta. Among the murine hematopoietic progenitor cells tested, the myeloid cell line FDC-P1 and the mast-cell line MC-6, as well as progenitor-enriched bone marrow cells, constitutively expressed functional cell-surface c-kit. Treatment of these progenitor cell lines and primary progenitor cells with TGF-beta resulted in downregulation of cell-surface c-kit expression. This effect was not a secondary event of cell-cycle status. TGF-beta inhibition was dose- and time-dependent, with 50% inhibition seen between 0.3 to 3 ng/mL TGF-beta and maximal inhibition at 30 ng/mL. Using the FDC-P1 cell line, we observed that the inhibition of cell-surface c-kit expression by TGF-beta is preceded by a marked reduction in c-kit mRNA levels starting 2 hours after TGF-beta treatment, and reaches a maximum by 6 hours. The inhibition in steady- state c-kit mRNA levels is explained, in part, by a decrease in the half-life of c-kit transcripts (2 to 4 hours for control cells v 0.5 to 1.5 hours for TGF-beta-treated cells). These findings suggest that TGF- beta regulates the responsiveness of murine hematopoietic progenitors to SLF through a decrease in c-kit message stability leading to decreased cell-surface expression.

Transforming Growth Factor-β Regulates c-kit Message Stability and Cell-Surface Protein Expression in Hematopoietic Progenitors

The cell-surface receptor c-kit and its cognate ligand stem-cell factor (SCF) or steel factor (SLF) are important for the maintenance of hematopoiesis both in vitro and in vivo. Transforming growth factor-beta (TGF-beta) has been shown to be a potent inhibitor of SLF-mediated synergistic growth of murine Lin-Sca-1+ progenitor cells, as well as more committed progenitors. In the present study, we examined the regulation of c-kit mRNA and cell-surface expression by TGF-beta. Among the murine hematopoietic progenitor cells tested, the myeloid cell line FDC-P1 and the mast-cell line MC-6, as well as progenitor-enriched bone marrow cells, constitutively expressed functional cell-surface c-kit. Treatment of these progenitor cell lines and primary progenitor cells with TGF-beta resulted in downregulation of cell-surface c-kit expression. This effect was not a secondary event of cell-cycle status. TGF-beta inhibition was dose- and time-dependent, with 50% inhibition seen between 0.3 to 3 ng/mL TGF-beta and maximal inhibition at 30 ng/mL. Using the FDC-P1 cell line, we observed that the inhibition of cell-surface c-kit expression by TGF-beta is preceded by a marked reduction in c-kit mRNA levels starting 2 hours after TGF-beta treatment, and reaches a maximum by 6 hours. The inhibition in steady-state c-kit mRNA levels is explained, in part, by a decrease in the half-life of c-kit transcripts (2 to 4 hours for control cells v 0.5 to 1.5 hours for TGF-beta-treated cells). These findings suggest that TGF-beta regulates the responsiveness of murine hematopoietic progenitors to SLF through a decrease in c-kit message stability leading to decreased cell-surface expression.