Kinetics of reduction of tyrosine phenoxyl radicals by glutathione

Erythropoietin (Epo) is crucial for promoting the survival, proliferation, and differentiation of mammalian erythroid progenitors. The central role played by tyrosine phosphorylation of erythropoietin receptor (EpoR) in Epo-cell activation has focused attention on protein tyrosine phosphatases (PTPs) as candidates implicated in the pathogenesis of the resistance to therapy with human recombinant Epo. Prototypic member of the PTP family is PTP1B, which has been implicated in the regulation of EpoR signaling pathways. In previous reports we have shown that PTP1B is reciprocally modulated by Epo in undifferentiated UT-7 cell line. However, no information is available with respect to the modulation of this phosphatase in non-Epo depending cells or at late stages of erythroid differentiation. In order to investigate these issues we induced UT-7 cells to differentiate and studied their PTP1B expression pattern. Simultaneous observations were performed in TF-1 cells which can be cultured either with GM-CSF, IL-3 or Epo. We found that Epo induced PTP1B cleaveage in TF-1 and differentiated UT-7 cells. This pattern of PTP1B modulation may be due to an increased TRPC3/TRPC6 expression ratio which could explain the larger and sustained calcium response to Epo and calpain activation in Epo treated TF-1 and differentiated UT-7 cells.     

GM-CSF rescues TF-1 cells from growth factor withdrawal-induced, but not differentiation-induced apoptosis: the role of BCL-2 and MCL-1.

Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 promote the survival and stimulate the proliferation of haematopoietic cells. Using the GM-CSF-dependent TF-1 myeloid leukaemia cell line, the authors show that the endogenous levels of BCL-2 and MCL-1 are downregulated upon GM-CSF withdrawal, whereas the levels of BCL-x(L)and Bax are unchanged. Re-exposure of growth factor deprived cells to GM-CSF resulted in an early and transient increase in MCL-1 expression, and prolonged induction of BCL-2, which prevented apoptosis. In contrast, the expression of BCL-2 and MCL-1 were not modulated during TPA-induced differentiation of TF-1 cells, which was followed by apoptosis despite the presence of GM-CSF. TF-1 cells overexpressing BCL-2 or MCL-1 underwent delayed apoptosis upon growth factor withdrawal, but displayed no impaired apoptosis in response to TPA. Erythropoietin (Epo) induced the expression of BCL-2 and MCL-1 protein in TF-1 cells, however it did not support their long term proliferation, further demonstrating that upregulation of these anti-apoptotic genes is insufficient for the long term proliferation of TF-1 cells.     

Growth factor-independent proliferation of erythroid cells infected with Friend spleen focus-forming virus is protein kinase C dependent but does not require Ras-GTP

Interaction of erythropoietin (Epo) with its cell surface receptor activates signal transduction pathways which result in the proliferation and differentiation of erythroid cells. Infection of erythroid cells with the Friend spleen focus-forming virus (SFFV) leads to the interaction of the viral envelope glycoprotein with the Epo receptor and renders these cells Epo independent. We previously reported that SFFV induces Epo independence by constitutively activating components of several Epo signal transduction pathways, including the Jak-Stat and the Raf-1/mitogen-activated protein kinase (MAPK) pathways. To further evaluate the mechanism by which SFFV activates the Raf-1/MAPK pathway, we investigated the effects of SFFV on upstream components of this pathway, and our results indicate that SFFV activates Shc and Grb2 and that this leads to Ras activation. While studies with a dominant-negative Ras indicated that Ras was required for Epo-induced proliferation of normal erythroid cells, the Epo-independent growth of SFFV-infected cells can still occur in the absence of Ras, although at reduced levels. In contrast, protein kinase C (PKC) was shown to be required for the Epo-independent proliferation of SFFV-infected cells. Further studies indicated that PKC, which is thought to be involved in the activation of both Raf-1 and MAPK, was required only for the activation of MAPK, not Raf-1, in SFFV-infected cells. Our results indicate that Ras and PKC define two distinct signals converging on MAPK in both Epo-stimulated and SFFV-infected erythroid cells and that activation of only PKC is sufficient for the Epo-independent proliferation of SFFV-infected cells.     

Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin

We have established a novel cell line, designated as TF-1, from a patient with erythroleukemia, which showed complete growth dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or on interleukin-3 (IL-3) and carried a homogeneous chromosomal abnormality (54X). Erythropoietin (EPO) also sustained the short-term growth of TF-1, but did not induce erythroid differentiation. These three hematopoietic growth factors acted on TF-1 synergistically. Transforming growth factor-beta and interferons inhibited the factor-dependent growth of TF-1 cells in a dose-dependent fashion, and monocyte-colony stimulating factor and interkeukin-1 enhanced the GM-CSF-dependent growth of TF-1. Ultrastructural studies revealed some very immature features in this cell line. Although TF-1 cells do not express glycophorin A or carbonyl anhydrase I, the morphological and cytochemical features, and the constitutive expression of globin genes, indicate the commitment of TF-1 to erythroid lineage. When induced to differentiate, TF-1 entered two different pathways. Specifically, hemin and delta-aminolevulinic acid induced hemoglobin synthesis, whereas TPA induced dramatic differentiation of TF-1 into macrophage-like cells. In summary, TF-1 is a cell line of immature erythroid origin that requires GM-CSF, IL-3, or EPO for its growth and that has the ability to undergo differentiation into either more mature erythroid cells or into macrophage-like cells. TF-1 is a useful tool for analyzing the human receptors for IL-3, GM-CSF, and EPO or the signal transduction of these hemopoietic growth factors.     

TGFβ inhibits GM-CSF-induced phosphorylation of ERK and MEK in human myeloid leukaemia cell lines via inhibition of phosphatidylinositol 3-kinase (PI3-k)

Objectives:  Activation of SMAD-independent p44/42 MAPK (ERK1/2) signalling by TGFβ has been recently reported in various cell types. However, the mechanisms for the linkage between the SMAD-dependent and -independent pathways are poorly understood. In this study, we investigated whether TGF-β activates the ERK pathway and how TGFβ communicates with the MAP kinase signals induced by a mitogen, in human myeloid leukaemia cells.
Materials and methods and results:  TGFβ dramatically suppressed proliferation of MV4–11 and TF-1 cells without detectable phosphorylation of ERK1/2 and MEK1/2 for the duration of 48 h, as detected by MTT assay and Western blot analysis, respectively. In contrast, GM-CSF induced rapid and transient phosphorylation of MEK1/2 and ERK1/2 and up-regulated cell proliferation. Both GM-CSF-induced ERK1/2 activation and cell proliferation were significantly inhibited by TGFβ. GM-CSF also induced transient phosphorylation of the p85 subunit of PI3-kinase. Corresponding to this change, phosphorylated p85 was found to bind to the GM-CSF receptor-α subunit, as detected by immunoprecipitation and Western blot analysis. PD98059, a selective inhibitor of MEK, blocked GM-CSF-induced phosphorylation of MEK and ERK but not p85. However, TGFβ and LY294002, a potent inhibitor of PI3-kinase, significantly inhibited phosphorylation of both p85 and ERK1/2.
Conclusions:  These studies thus indicate that TGFβ does not activate the ERK pathway but turns off the GM-CSF-induced ERK signal via inhibition of the PI3-kinase-Akt pathway, in these human laeukemia cells.     

Growth signal erythropoietin activates the same tyrosine kinases as interleukin 3, but activates only one tyrosine kinase as differentiation signal.

By Western blotting with anti-phosphotyrosine-specific antibody, we demonstrated that both erythropoietin (Epo) and interleukin 3 (IL3) induce rapid and transient tyrosine phosphorylation of a common set of proteins of 45, 55, 69, 87, 90, 95 and 160 KDa as a growth signal in Epo- and IL3-dependent FD-M6 cells. In contrast, only two proteins of 87 and 90 KDa were transiently phosphorylated in Epo-induced erythroid differentiation of SKT6 cells. Furthermore, no tyrosine phosphorylation was observed in dimethyl sulfoxide-induced differentiation of SKT6 cells. Taken together with other observations, these results indicate that Epo, IL3 and GM-CSF activate the same tyrosine protein kinases as growth signal and that Epo-induced differentiation signal uses only a part of the tyrosine kinase pathway.     

Activin A induces erythroid gene expressions and inhibits mitogenic cytokine-mediated K562 colony formation by activating p38 MAPK

Activin A, a member of the transforming growth factor (TGF)-beta superfamily, is involved in the regulation of erythroid differentiation. Previous studies have shown that activin A inhibited the colony-forming activity of mouse Friend erythroleukemia cells, however, the mechanism remains unknown. First, we show herein that activin A induced the expression and activated the promoters of alpha-globin and zeta-globin in K562 cells, confirming that activin A induces erythroid differentiation in K562 cells. The p38 mitogen activated protein kinase (MAPK) inhibitor, SB203580, inhibited and the extracellular signal regulated kinase (ERK) inhibitor, PD98059, enhanced the expression and promoter activities of alpha-globin and zeta-globin by activin A, indicating that p38 MAPK and ERK are crucial for activin A-induced erythroid genes expression. Second, SB203580 inhibited the inhibitory effect of activin A on the colony-forming activity of K562 cells using the methylcellulose colony assay, indicating that activin A inhibits K562 colony formation by activating p38 MAPK. In addition, mitogenic cytokines SCF, IL-3, and GM-CSF induced colony formation of K562 cells that could be inhibited by PD98059 or enhanced by SB203580, respectively, indicating that these mitogenic cytokines induce K562 colony formation by activating ERK and inactivating p38 MAPK. Furthermore, activin A reduced the induction effect of these mitogenic cytokines on K562 colony formation in a dose-dependent manner. The inhibition of p38 MAPK reverted the inhibitory effect of activin A on mitogenic cytokine-mediated K562 colony formation. We conclude that activin A can regulate the same pathway via p38 MAPK to coordinate cell proliferation and differentiation of K562 cells.     

Stromal cell-derived factor 1alpha (SDF-1alpha) induces gene-expression of early growth response-1 (Egr-1) and VEGF in human arterial endothelial cells and enhances VEGF induced cell proliferation

Abstract. Stromal cell-derived factor-1 (SDF-1), mainly known as a chemotactic factor for haematopoietic progenitor cells, also provides angiogenetic potency. Since the intracellular signalling of SDF-1-induced neovascularization remains unclear, we studied in human umbilical arterial endothelial cells (HUAEC) the influence of SDF-1α on induction of the genes of early growth response-1 (Egr-1) and VEGF, as well as the activation of extracellular regulated kinases (ERK) 1/2, which are all known to be involved in endothelial cell proliferation. We found a time-dependent induction of Egr-1 and VEGF mRNA expression and phosphorylation of ERK1/2 by SDF-1α. Furthermore, we demonstrated that Egr-1 expression is dependent on ERK 1/2 activation. Finally, we tried to confirm the relevance of the induced gene expression by detecting the [3H]thymidine incorporation as a marker for cell proliferation in HUAEC after stimulation with SDF-1α alone or together with VEGF. This particular test showed, that SDF-1α alone has no effect, but is able to significantly enhance VEGF induced DNA synthesis. In summary, SDF-1α is involved in different steps of endothelial cell proliferation, but, since Egr-1 and VEGF offer different functions, it may also play a so far undefined role on other conditions of the endothelium.     

Protein kinase C alpha controls erythropoietin receptor signaling

Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors with distinct modes of action on EpoR signaling in primary human erythroblasts and in a recently established murine erythroid cell line. Active PKC appeared essential for Epo-induced phosphorylation of the Epo receptor itself, STAT5, Gab1, Erk1/2, AKT, and other downstream targets. Under the same conditions, stem cell factor-induced signal transduction was not impaired. LY294002, a specific inhibitor of phosphoinositol 3-kinase, also suppressed Epo-induced signal transduction, which could be partially relieved by activators of PKC. PKC inhibitors or LY294002 did not affect membrane expression of the EpoR, the association of JAK2 with the EpoR, or the in vitro kinase activity of JAK2. The data suggest that PKC controls EpoR signaling instead of being a downstream effector. PKC and phosphoinositol 3-kinase may act in concert to regulate association of the EpoR complex such that it is responsive to ligand stimulation. Reduced PKC-activity inhibited Epo-dependent differentiation, although it did not effect Epo-dependent "renewal divisions" induced in the presence of Epo, stem cell factor, and dexamethasone.