Synergistic Effect Of Stem Cell Factor Research Articles

Synergistic effect of SCF and G-CSF on stem-like properties in prostate cancer cell lines

Bone marrow metastases are formed in the late phases of prostate cancer disease. Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) are present in the microenvironment of the bone marrow and play a vital role in cell biology therein. The present study was to investigate the influence of SCF and G-CSF on stem-like properties in prostate cancer cell lines. Upon stimulation with SCF or G-CSF, higher levels of CD117, ABCG2, and CD44 were observed in PC-3 and DU145 cells examined by flow cytometry. Simultaneously, the expressions of Oct3/4 and Nanog were upregulated. Moreover, quantitative real-time PCR verified that the increased Nanog under the stimulations was mostly derived from NANOGP8. In parallel with the increasing expressions of these proteins, higher colony and sphere formation efficiencies were seen in these cells in response to the cytokine stimulations. Furthermore, a synergistic effect of SCF and G-CSF on colony and sphere formations and ABCG2 expression was disclosed. Our results indicate a favorable bone marrow niche for prostate cancer cells where higher levels of cell stemness are maintained at least partly by the cytokines SCF and G-CSF.

Socs3 maintains the specificity of biological responses to cytokine signals during granulocyte and macrophage differentiation

Granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) play key roles in regulating emergency granulopoiesis and inflammation, and are both negatively regulated by the inducible intracellular protein suppressor of cytokine signaling-3 (Socs3). Mice with Socs3 deleted specifically in hematopoietic cells succumb to severe neutrophil and macrophage-driven inflammation by 1 year of age, and responses to G-CSF are grossly exacerbated. In order to determine which elements of cellular responses to cytokines require Socs3, we have examined the differentiative and proliferative capacity of hematopoietic progenitor cells stimulated by G-CSF and IL-6. The differentiation of Socs3-deficient progenitor cells is skewed toward macrophage production in response to G-CSF or IL-6, whereas wild-type progenitor cells produce mainly neutrophils. The proliferative capacity of Socs3-deficient progenitor cells is greatly enhanced in response to G-CSF at all concentrations, but only at low concentrations for IL-6. Strikingly, synergistic responses to costimulation with stem cell factor and IL-6 (but not G-CSF) are lost at higher concentrations in Socs3-deficient progenitor cells. Cytokine-induced expression of transcriptional regulators including cebpb, Ets2, Bcl3, c-Myc, Jun, and Fosl2 are differentially regulated in Socs3-deficient cells. The tight regulation by Socs3 of signal transducer and activator of transcription 3 phosphorylation and gene transcription after cytokine receptor ligation significantly influences the fate of myeloid progenitor cells.

Design of Recombinant Stem Cell Factor–macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity In Vitro

Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF-M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF-M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1-165aa) and M-CSF (1-149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of approximately 84 KD under non-reducing conditions and a monomer of approximately 42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.

Synergistic effect of SCF and TNF-α on the up-regulation of cell-surface expression of ICAM-1 on human leukemic mast cell line (HMC)-1 cells

Intercellular adhesion molecule-1 (ICAM-1) has been shown to play crucial roles in mast cell interaction with other inflammatory cells and recruitment into the inflamed tissue. In the present study, human mast cell line-1 (HMC-1) was stimulated with different cytokines including stem cell factor (SCF), tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-13, IL-18, and IL-25. Cell-surface expression of ICAM-1 was assessed by flow cytometry. To elucidate the intracellular signal transduction regulating the ICAM-1 expression, phosphorylated extracellular signal-regulated kinase (ERK), phosphorylated p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-kappaB translocation were assessed by enzyme-linked immunosorbent assay. Results showed that SCF, TNF-alpha, and IL-13 but not IL-18 and IL-25 could up-regulate the surface expression of ICAM-1 on HMC-1 cells. A synergistic effect of SCF and TNF-alpha on ICAM-1 expression was demonstrated. This synergistic effect was shown to be dose-dependently enhanced by SCF but not TNF-alpha. Results indicated that SCF activated ERK, and TNF-alpha activated the p38 MAPK and NF-kappaB pathway. Selective inhibitor of ERK, PD098059, and c-kit inhibitors, STI571 and PP1, suppressed the combined SCF and TNF-alpha-induced ICAM-1 expression. BAY117082 but not SB203580, which are the inhibitors of NF-kappaB and p38 MAPK, respectively, suppressed the TNF-alpha-induced ICAM-1 expression. Therefore, SCF and TNF-alpha acted through ERK and the NF-kappaB pathway to regulate the ICAM-1 expression and elicited the synergistic effect. In conclusion, our results provide insight for cross-talk between different signaling pathways that can help in understanding the fine control of adhesion molecule expression under the concerted effects of cytokines.

SCF and G-CSF lead to the synergistic induction of proliferation and gene expression through complementary signaling pathways

Stem cell factor (SCF) is a potent costimulatory molecule for many cytokines. Its synergy with granulocyte colony-stimulating factor (G-CSF) results in important biologic and clinical effects, although the mechanism by which this occurs remains poorly understood. To investigate this interaction, this study used a retroviral vector to transduce the G-CSF receptor into MO7e cells, which are known to express the SCF receptor. The transduced G-CSF receptor is functionally active, and the resultant MO7e-G cells recapitulate the proliferative synergy between SCF and G-CSF. When treated with both cytokines, a marked shortening of the G(0)/G(1) phase of the cell cycle occurs, associated with a suppression of the cyclin-dependent kinase inhibitor p27(kip-1). In addition, SCF and G-CSF induce the synergistic activation of c-fos, a proto-oncogene involved in propagation of mitogenic signals in hematopoietic cells. G-CSF, but not SCF, induces the tyrosine phosphorylation of STAT1 and STAT3, transcription factors that can mediate the induction of c-fos. However, SCF induces phosphorylation of STAT3 on serine727 (ser727), which is necessary for maximal STAT transcriptional activity, and the combination of SCF and G-CSF leads to complete STAT3 phosphorylation on ser727. The pathways by which SCF and G-CSF lead to serine phosphorylation of STAT3 are distinct and are partially dependent on phosphatidylinositol-3 kinase and ERKs, pathways that are also necessary for the synergistic effects of SCF and G-CSF on proliferation and c-fos induction. Thus, MO7e-G cells provide a powerful system in which the molecular basis of the synergy between SCF and G-CSF can be dissected.

Mcl-1 is a common target of stem cell factor and interleukin-5 for apoptosis prevention activity via MEK/MAPK and PI-3K/Akt pathways

Stem cell factor (SCF) has been suggested as essential for optimal production of various hematopoietic lineages mainly because of its apoptosis prevention function when it costimulates with other cytokines. However, the underlying mechanism of this synergism of apoptosis prevention is largely unknown. The present study examined the expression of some Bcl-2 family members, including Bcl-2, Bcl-XL, Mcl-1, and Bax, in response to cytokine stimulation in TF-1 and JYTF-1 cells in which SCF costimulation is differentially required for optimal proliferation. The results revealed that only the expression of Mcl-1 highly correlated with the antiapoptotic activity of interleukin-5 (IL-5) and the synergistic effect of SCF. In TF-1 cells, the defect of IL-5 in apoptosis suppression and Mcl-1 induction was associated with the incapability to highly phosphorylate Janus kinases (JAK1, JAK2), signal transducer and activator of transcription-5 (STAT5), mitogen-activated protein kinase (MAPK), and Akt/PKB, whereas SCF costimulation restored the potent phosphorylation of MAPK and Akt/PKB, but not STAT5. The importance of MAPK and Akt/PKB signaling pathways in regulating the expression of Mcl-1 and cell survival was further supported by the observation that inhibition of MEK by PD98059 or phosphatidylinositol-3 kinase (PI-3K) by LY294002 independently resulted in the reduction of Mcl-1 expression and loss of cell viability. Therefore, the data suggest that Mcl-1 is a common antiapoptotic target of both early-stage cytokine SCF and late-stage cytokine IL-5. Both MEK/MAPK and PI-3K/Akt signaling pathways are essential in the regulation of Mcl-1 expression and apoptosis prevention.

Optimal Proliferation of a Hematopoietic Progenitor Cell Line Requires Either Costimulation With Stem Cell Factor or Increase of Receptor Expression That Can Be Replaced by Overexpression of Bcl-2

In vitro proliferation of hematopoietic stem cells requires costimulation by multiple regulatory factors whereas expansion of lineage-committed progenitor cells generated by stem cells usually requires only a single factor. The distinct requirement of factors for proliferation coincides with the differential temporal expression of the subunits of cytokine receptors during early stem cell differentiation. In this study, we explored the underlying mechanism of the requirement of costimulation in a hematopoietic progenitor cell line TF-1. We found that granulocyte-macrophage colony-stimulating factor (GM-CSF) optimally activated proliferation of TF-1 cells regardless of the presence or absence of stem cell factor (SCF). However, interleukin-5 (IL-5) alone sustained survival of TF-1 cells and required costimulation of SCF for optimal proliferation. The synergistic effect of SCF was partly due to its anti-apoptosis activity. Overexpression of the IL-5 receptor  subunit (IL5R) in TF-1 cells by genetic selection or retroviral infection also resumed optimal proliferation due to correction of the defect in apoptosis suppression. Exogenous expression of an oncogenic anti-apoptosis protein, Bcl-2, conferred on TF-1 cells an IL-5–dependent phenotype. In summary, our data suggested SCF costimulation is only necessary when the expression level of IL5R is low and apoptosis suppression is defective in the signal transduction of IL-5. Expression of Bcl-2 proteins released the growth restriction of the progenitor cells and may be implicated in leukemia formation.

Optimal Proliferation of a Hematopoietic Progenitor Cell Line Requires Either Costimulation With Stem Cell Factor or Increase of Receptor Expression That Can Be Replaced by Overexpression of Bcl-2

AbstractIn vitro proliferation of hematopoietic stem cells requires costimulation by multiple regulatory factors whereas expansion of lineage-committed progenitor cells generated by stem cells usually requires only a single factor. The distinct requirement of factors for proliferation coincides with the differential temporal expression of the subunits of cytokine receptors during early stem cell differentiation. In this study, we explored the underlying mechanism of the requirement of costimulation in a hematopoietic progenitor cell line TF-1. We found that granulocyte-macrophage colony-stimulating factor (GM-CSF) optimally activated proliferation of TF-1 cells regardless of the presence or absence of stem cell factor (SCF). However, interleukin-5 (IL-5) alone sustained survival of TF-1 cells and required costimulation of SCF for optimal proliferation. The synergistic effect of SCF was partly due to its anti-apoptosis activity. Overexpression of the IL-5 receptor α subunit (IL5Rα) in TF-1 cells by genetic selection or retroviral infection also resumed optimal proliferation due to correction of the defect in apoptosis suppression. Exogenous expression of an oncogenic anti-apoptosis protein, Bcl-2, conferred on TF-1 cells an IL-5–dependent phenotype. In summary, our data suggested SCF costimulation is only necessary when the expression level of IL5Rα is low and apoptosis suppression is defective in the signal transduction of IL-5. Expression of Bcl-2 proteins released the growth restriction of the progenitor cells and may be implicated in leukemia formation.

Stem Cell Factor Enhances Interleukin-3-Dependent Induction of 68-kD Calmodulin-Binding Protein and Thymidine Kinase Activity in NFS-60 Cells

Stem cell factor (SCF) is known to act synergistically with other hematopoietic factors in increasing the colony formation of hematopoietic progenitor cells. We have shown that interleukin-3 (IL-3)-dependent proliferation of NFS-60 cells is associated with the induction of a specific calmodulin-binding protein of about 68 kD (CaM-BP68). To evaluate the relationship between proliferative stimulation and the induction of CaM-BP68 by cytokines, we examined whether the increased proliferative potential of NFS-60 cells in response to SCF is reflected in an increased induction of the CaM-BP68. We observed that SCF alone has a limited effect on proliferative stimulation and on the induction of CaM-BP68 in factor-deprived NFS-60 cells. However, when combined with IL-3, granulocyte colony-stimulating factor (G-CSF), or IL-6, it caused a significant increase in cytokine-dependent proliferative stimulation, as well as in the induction of CaM-BP68. Furthermore, an increase in IL-3-dependent induction of CaM-BP68 in the presence of SCF coincided with a corresponding increase in thymidine kinase activity, whose expression is linked to G1/S transition of the cells. At low concentrations SCF caused a synergistic increase in IL-3-dependent induction of both CaM-BP68 and thymidine kinase activity. In contrast to the changes in CaM-BP68 and thymidine kinase activity, no significant changes in DNA polymerase alpha were observed in factor-deprived NFS-60 cells in response to IL-3 and/or SCF. These observations suggest an increased expression of CaM-BP68 and thymidine kinase are associated with the synergistic effect of SCF on factor-dependent proliferation of hematopoietic progenitor cells.