Erythropoietin-stimulated Raf-1 tyrosine phosphorylation is associated with the tyrosine kinase Lyn in J2E erythroleukemic cells.

The serine/threonine kinase Raf-1 is crucial for transducing intracellular signals emanating from numerous growth factors. Here we used the J2E erythroid cell line transformed by the nu-raf/nu-myc oncogenes to examine the effects of erythropoietin on endogenous Raf-1 activity. Despite the presence of constitutively active v-raf in these cells, Raf-1 exokinase activity increased after erythropoietin stimulation. This increase in enzymatic activity coincided with tyrosine phosphorylation of Raf-1 on residue Y341. Significantly, the tyrosine kinase Lyn coimmunoprecipitated with Raf-1, and Raf-1 was not tyrosine-phosphorylated in a J2E subclone lacking Lyn. Therefore, it was concluded that Lyn may be the kinase responsible for tyrosine phosphorylating Raf-1 and increasing its exokinase activity in response to erythropoietin.

Thyroid hormone receptor-interacting protein 1 modulates cytokine and nuclear hormone signaling in erythroid cells.

Erythropoietin (Epo) and thyroid hormone (T(3)) are key molecules in the development of red blood cells. We have shown previously that the tyrosine kinase Lyn is involved in differentiation signals emanating from an activated erythropoietin receptor. Here we demonstrate that Lyn interacts with thyroid hormone receptor-interacting protein 1 (Trip-1), a transcriptional regulator associated with the T(3) receptor, providing a link between the Epo and T(3) signaling pathways. Trip-1 co-localized with Lyn and the T(3) receptor alpha in the cytoplasm/plasma membrane of erythroid cells but translocated to discrete nuclear foci shortly after Epo-induced differentiation. Our data reveal that T(3) stimulated the proliferation of immature erythroid cells, and inhibited maturation promoted by erythropoietin. Removal of T(3) reduced cell division and enhanced terminal differentiation. This was accompanied by large increases in the cell cycle inhibitor p27(Kip1) and by increasing expression of erythroid transcription factors GATA-1, EKLF, and NF-E2. Strikingly, a truncated Trip-1 inhibited both erythropoietin-induced maturation and T(3)-initiated cell division. This mutant Trip-1 acted in a dominant negative fashion by eliminating endogenous Lyn, elevating p27(Kip1), and blocking T(3) response elements. These data demonstrate that Trip-1 can simultaneously modulate responses involving both cytokine and nuclear receptors.

Maturation of erythroid cells and erythroleukemia development are affected by the kinase activity of Lyn.

This study examined the impact of the tyrosine kinase Lyn on erythropoietin-induced intracellular signaling in erythroid cells. In J2E erythroleukemic cells, Lyn coimmunoprecipitated with numerous proteins, including SHP-1, SHP-2, ras-GTPase-activating protein, signal transducers and activators of transcription (STAT) 5a, STAT5b, and mitogen-activated protein kinase; however, introduction of a dominant-negative Lyn (Y397F Lyn) inhibited the interaction of Lyn with all of these molecules except SHP-1. Cells containing the dominant-negative Lyn displayed altered intracellular phosphorylation patterns, including mitogen-actiated protein kinase, but not erythropoietin receptor, Janus-activated kinase (JAK) 2, or STAT5. As a consequence, erythropoietin-initiated differentiation and basal proliferation were severely impaired. Y397F Lyn reduced the protein levels of erythroid transcription factors erythroid Kruppel-like factor and GATA-1 up to 90%, which accounts for the inability of J2E cells expressing Y397F Lyn to synthesize hemoglobin. Although Lyn was shown to bind several sites on the cytoplasmic domain of the erythropoietin receptor, it was not activated when a receptor mutated at the JAK2 binding site was ectopically expressed in J2E cells indicating that JAK2 is the primary kinase in erythropoietin signaling and that Lyn is a secondary kinase. In normal erythroid progenitors, erythropoietin enhanced phosphorylation of Lyn; moreover, exogenous Lyn increased colony forming unit-erythroid, but not burst forming uniterythroid, colonies from normal progenitors, demonstrating a stage-specific effect of the kinase. Significantly, altering Lyn activity in J2E cells had a profound effect on the development of erythroleukemias in vivo: the mortality rate was markedly reduced and latent period extended when either wild-type Lyn or Y397F Lyn was introduced into these cells. Taken together, these data show that Lyn plays an important role in intracellular signaling in nontransformed and leukemic erythroid cells.

Ectopic expression of transcription factor NF-E2 alters the phenotype of erythroid and monoblastoid cells.

In this study, regulation of transcription factor NF-E2 was examined in differentiating erythroid and myeloid cells, and the impact of raising NF-E2 concentrations within these cell types was assessed. NF-E2 was expressed in the J2E erythroid cell line, but the levels increased only marginally during erythropoietin-induced differentiation. In contrast, rare myeloid variants of J2E cells did not express NF-E2. Although NF-E2 was present in M1 monoblastoid cells, it was undetectable as these cells matured into macrophages. Compared with erythroid cells, transcription of the NF-E2 gene was reduced, and the half-life of the mRNA was significantly shorter in monocytoid cells. Ectopic expression of NF-E2 had a profound impact upon the J2E cells; morphologically mature erythroid cells spontaneously emerged in culture, but the cells failed to synthesize hemoglobin, even in the presence of erythropoietin. Although proliferation and viability increased in the NF-E2-transfected J2E cells, their responsiveness to erythropoietin was severely diminished. Strikingly, increasing the expression of NF-E2 in M1 cells produced sublines that contained erythroid or immature megakaryocytic cells. Finally, overexpression of NF-E2 in primary hemopoietic progenitors from fetal liver increased erythroid colony formation in the absence of erythropoietin. These data demonstrate that elevated NF-E2 (i) had a dominant effect on the phenotype and maturation of J2E erythroid cells, (ii) was able to reprogram the M1 monocytoid line, and (iii) promoted the development of erythroid colonies by normal progenitors.

Dominant action of mutated erythropoietin receptors on differentiation in vitro and erythroleukemia development in vivo.

J2E cells produce rapid, fatal erythroleukemias in vivo but still respond to erythropoietin (epo) in vitro by differentiating, proliferating and remaining viable in the absence of serum. Mutant epo receptors were introduced into these cells to determine whether they could influence the different biological responses to epo in vitro and the development of erythroleukemias. Three mutant receptors were used as cytoplasmic truncation mutants Delta257 and Delta321 (above box 1 and below box 2 respectively), and the cytoplasmic point mutant W282R (defective for JAK2 activation). Strikingly, the Delta321 mutation produced a hyper-sensitive response in vitro to epo-induced differentiation and viability, but not to proliferation. In contrast with the Delta321 receptor, the Delta257 and W282R mutants inhibited all biological responses to epo due to impaired JAK2 phosphorylation. Significantly, erythroleukemias took almost twice as long to develop with cells containing the W282R mutation, indicating that JAK2 plays an important role in the emergence of these leukemias. These data demonstrate that mutant epo receptors dominantly altered responses of J2E cells to epo in culture and the development of erythroleukemias. Oncogene (2000) 19, 953 - 960.

HS1 interacts with Lyn and is critical for erythropoietin-induced differentiation of erythroid cells.

Erythroid cells terminally differentiate in response to erythropoietin binding its cognate receptor. Previously we have shown that the tyrosine kinase Lyn associates with the erythropoietin receptor and is essential for hemoglobin synthesis in three erythroleukemic cell lines. To understand Lyn signaling events in erythroid cells, the yeast two-hybrid system was used to analyze interactions with other proteins. Here we show that the hemopoietic-specific protein HS1 interacted directly with the SH3 domain of Lyn, via its proline-rich region. A truncated HS1, bearing the Lyn-binding domain, was introduced into J2E erythroleukemic cells to determine the impact upon responsiveness to erythropoietin. Truncated HS1 had a striking effect on the phenotype of the J2E line-the cells were smaller, more basophilic than the parental proerythoblastoid cells and had fewer surface erythropoietin receptors. Moreover, basal and erythropoietin-induced proliferation and differentiation were markedly suppressed. The inability of cells containing the truncated HS1 to differentiate may be a consequence of markedly reduced levels of Lyn and GATA-1. In addition, erythropoietin stimulation of these cells resulted in rapid, endosome-mediated degradation of endogenous HS1. The truncated HS1 also suppressed the development of erythroid colonies from fetal liver cells. These data show that disrupting HS1 has profoundly influenced the ability of erythroid cells to terminally differentiate.

The erythropoietin receptor.

The erythropoietin (epo) receptor is a member of the cytokine receptor family. It is expressed almost exclusively on erythroid precursor cells and controls the development of red blood cells. The epo receptor has no intrinsic kinase activity, but binds intracellular tyrosine kinases to elicit its signals. Alterations in the transmission of the signalling cascade lead to clinically abnormal red blood cell production.

A novel ADP-ribosylation like factor (ARL-6), interacts with the protein-conducting channel SEC61beta subunit.

We report here the isolation of a new member of the ADP-ribosylation factor (ARF)-like family (ARL-6) present in the J2E erythroleukemic cell line, but not its myeloid variants. Consistent with this lineage-restricted expression, ARL-6 mRNA increased with erythropoietin-induced maturation of J2E cells, and decreased with interleukin 6-induced differentiation of M1 monoblastoid cells. In tissues, ARL-6 mRNA was most abundant in brain and kidney. While ARL-6 protein was predominantly cytosolic, its membrane association increased following exposure to GTP-gammaS, like many members of the ARF/ARL family. Using the yeast two-hybrid system, six molecules which interact with ARL-6 were identified including SEC61beta, a subunit of the heterotrimeric protein conducting channel SEC61p. Co-immunoprecipitation of ARL-6 confirmed a stable association between ARL-6 and SEC61beta in COS cells. These results demonstrate that ARL-6, a novel member of the ADP-ribosylation factor-like family, interacts with the SEC61beta subunit.

HLS7, a hemopoietic lineage switch gene homologous to the leukemia-inducing gene MLF1.

Hemopoietic lineage switching occurs when leukemic cells, apparently committed to one lineage, change and display the phenotype of another pathway. cDNA representational difference analysis was used to identify myeloid-specific genes that may be associated with an erythroid to myeloid lineage switch involving the murine J2E erythroleukemic cell line. One of the genes isolated (HLS7) is homologous to the novel human oncogene myeloid leukemia factor 1 (MLF1) involved in the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia. Enforced expression of HLS7 in J2E cells induced a monoblastoid phenotype, thereby recapitulating the spontaneous erythroid to myeloid lineage switch. HLS7 also inhibited erythropoietin- or chemically-induced differentiation of erythroleukemic cell lines and suppressed development of erythropoietin-responsive colonies in semi-solid culture. However, intracellular signaling activated by erythropoietin was not impeded by ectopic expression of HLS7. In contrast, HLS7 promoted maturation of M1 monoblastoid cells and increased myeloid colony formation in vitro. These data show that HLS7 can influence erythroid/myeloid lineage switching and the development of normal hemopoietic cells.

Transcription factor erythroid Krüppel-like factor (EKLF) is essential for the erythropoietin-induced hemoglobin production but not for proliferation, viability, or morphological maturation.

The erythroid Krüppel-like factor (EKLF) is essential for the transcription of betamaj globin in erythroid cells. We show here that RNA for this transcription factor did not alter during erythropoietin-induced differentiation of J2E cells; however, EKLF protein content decreased and was inversely related to globin production. This unexpected result was also observed during chemically induced maturation of two murine erythroleukemia cell lines. To explore the role of EKLF in erythroid terminal differentiation, an antisense EKLF construct was introduced into J2E cells. As a consequence EKLF RNA and protein levels fell by approximately 80%, and the cells were unable to manufacture hemoglobin in response to erythropoietin. The failure to produce hemoglobin was due to reduced transcription of not only globin genes but also key heme enzyme genes. However, numerous other genes, including several erythroid transcription factors, were unaffected by the decrease in EKLF. Although hemoglobin synthesis was severely impaired with depleted EKLF levels, morphological maturation in response to erythropoietin continued normally. Moreover, erythropoietin-induced proliferation and viability were unaffected by the decrease in EKLF levels. We conclude that EKLF affects a specific set of genes, which regulates hemoglobin production and has no obvious effect on morphological changes, cell division, or viability in response to erythropoietin.

Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway.

Nitric oxide (NO) has been invoked as an important pathogenic factor in a wide range of immunologically mediated diseases. The present study demonstrates that macrophage-derived NO may conversely function to fine tune T cell-mediated inflammation via reversible dephosphorylation of intracellular signaling molecules, which are involved in the control of T cell proliferation. Thus, T cells activated in the presence of alveolar macrophages are unable to proliferate despite expression of IL-2R and secretion of IL-2. This process is reproduced by the NO generator S-nitroso-N-acetylpenicillamine and is inhibitable by the NO synthase inhibitor N(G)-methyl-L-arginine. Analysis of T cell lysates by immunoprecipitation with specific Abs and subsequent immunoblotting indicated marked reduction of tyrosine phosphorylation of Jak3 and STAT5 mediated by NO. Further studies indicated that NO-mediated T cell suppression was reversible by the guanylate cyclase inhibitors methylene blue and LY-83583 and was reproduced by a cell-permeable analogue of cyclic GMP, implicating guanylate cyclase activation as a key step in the inhibition of T cell activation by NO.

Complex regulation of transferrin receptors during erythropoietin-induced differentiation of J2E erythroid cells--elevated transcription and mRNA stabilisation produce only a modest rise in protein content.

The regulation of transferrin-receptor synthesis was studied in J2E erythroid cells induced to differentiate with erythropoietin. Nuclear run-on assays demonstrated that transcription of the transferrin-receptor gene rose markedly after erythropoietin treatment. In addition, transferrin-receptor mRNA was stabilised and this was associated with an increase in the activity of the RNA-binding protein IRP (iron regulatory protein). As a result of increased transcription and mRNA stabilisation, steady-state RNA levels increased 10-20-fold. However, despite these large increases in mRNA, translation only doubled; consequently, modest increases in total protein and surface transferrin receptors were observed. Moreover, this rise in transferrin receptors was transient, and correlated with a burst of proliferation shortly after erythropoietin treatment. The expected inverse relationship between transferrin receptors and ferritin did not occur during J2E maturation as translation of both ferritin subunits increased when transferrin-receptor mRNA levels rose. Analysis of mutant J2E clones incapable of synthesising haemoglobin revealed that surface transferrin-receptor levels were only 15-25% that of the parental erythroid line. We propose that the surface expression of transferrin receptors in J2E cells is governed by three factors: basal levels essential for normal growth in culture; elevated levels needed for haemoglobin synthesis; and a transient erythropoietin-induced increase that is required for the final burst of proliferation. It was concluded that the regulation of transferrin-receptor production in erythropoietin-stimulated J2E cells is complex and that there are several sites of control.

Increased transcription of the E mu-myc transgene and mRNA stabilisation produce only a modest elevation in Myc protein.

Mice bearing the E mu-myc transgene, which links the immunoglobulin heavy chain enhancer (E mu) with c-myc, are predisposed to developing B cell lymphomas. Several B lineage cell lines have been isolated from these animals, and some have been converted to macrophages following infection with v-raf. In this study we compared the regulation of myc expression in E mu-myc B lymphoma lines, their macrophage counterparts and other non-myc transformed B cell lines. Nuclear run-on analyses demonstrated that transcription of the transgene was elevated in E mu-myc B cell lines. Moreover, the presence of a 600 bp phiX174 marker in the 3' end of the transgene produced a marked stabilisation of this RNA species. Consequently, steady state myc mRNA levels in the E mu-myc B lymphoma cells were tenfold higher than the macrophage derivatives and non-myc transformed B lineage lines. Despite the considerable difference in myc RNA levels, the E mu-myc B cell lines contained only 30-50% more Myc protein than the other cell lines. This discrepancy between RNA and protein content was not due to increased degradation of the protein as the half life was normal in the transgenic cell lines. These results indicate that both E mu and phiX174 sequences influence transgenic myc expression and that protein levels do not correlate with RNA content in E mu-myc cell lines.

Lyn tyrosine kinase is essential for erythropoietin-induced differentiation of J2E erythroid cells.

Erythropoietin stimulates the immature erythroid J2E cell line to terminally differentiate and maintains the viability of the cells in the absence of serum. In contrast, a mutant J2E clone (J2E-NR) fails to mature in response to erythropoietin; however, it remains viable in the presence of the hormone. We have shown previously that intracellular signalling is disrupted in the J2E-NR cell line and that tyrosine phosphorylation is dramatically reduced after erythropoietin stimulation. In this study we investigated the defect in J2E-NR cells that is responsible for their inability to differentiate. Screening of numerous signalling molecules revealed that the lyn tyrosine kinase appeared to be absent from J2E-NR cells. On closer examination, both lyn mRNA and protein content were reduced >500-fold. Consistent with a defect in lyn, amphotropic retroviral infection of J2E-NR cells with lyn restored the ability of the cells to synthesize haemoglobin and enabled the cells to mature morphologically. Conversely, the ability of J2E cells to differentiate in response to epo was severely curtailed when antisense lyn oligonucleotides or a dominant negative lyn were introduced into the cells. However, erythropoietin-supported viability was unaffected by reducing lyn activity. The ability of two other erythropoietin-responsive cell lines (R11 and R24) to differentiate in response to the hormone was also reduced by dominant negative lyn. Finally, co-immunoprecipitation and yeast two-hybrid analyses indicated that lyn directly associated with the erythropoietin receptor complex. These data indicate for the first time an essential role for lyn in erythropoietin-initiated differentiation of J2E cells but not in the maintenance of cell viability.

Prevention of apoptosis in J2E erythroid cells by erythropoietin: involvement of JAK2 but not MAP kinases.

The J2E erythroid cell line, transformed by retroviral v-raf/v-myc oncogenes, proliferates and differentiates in response to erythropoietin. Here we show that J2E cells undergo apoptosis rapidly after serum withdrawal and that only erythropoietin of seven growth factors tested, could protect the cells from death. The role of JAK2 and MAP kinases in transmitting viability signals initiated by erythropoietin was examined in these cells. Despite constitutive raf kinase activity, phosphorylation of MAP kinases fell after serum withdrawal. However, an antisense oligonucleotide strategy revealed that JAK2, but not the MAP kinases, was involved in transmitting signals to maintain the viability of J2E cells. Several cell cycle proteins and transcription factors were also studied; although c-jun rose sharply during apoptosis, erythropoietin could not suppress this increase. It was concluded that erythropoietin-induced protection from apoptosis involved JAK2, but not MAP kinases or c-jun.

Haemoglobin synthesis in erythropoietin-stimulated J2E cells does not require increased numbers of transferrin receptors.

Changes in transferrin-receptor numbers and iron utilisation were monitored during erythropoietin-induced maturation of J2E erythroid cells. Uptake of transferrin and iron doubled 24 h after exposure to erythropoietin, due to a twofold rise in surface transferrin receptors. In addition, a tenfold increase in iron incorporation into haem was observed after erythropoietin stimulation, as iron taken up from transferrin was directed towards haem biosynthesis and away from storage in ferritin. The rise in iron chelation into haem correlated extremely well with haemoglobin synthesis. However, the increase in numbers of transferrin receptors was not essential for haemoglobin synthesis; rather, it was linked with a burst in proliferation stimulated by erythropoietin. We have shown previously that amiloride blocks erythropoietin-enhanced proliferation of J2E cells, but potentiates maturation [Callus, B. A., Tilbrook, P. A., Busfield, S. J. & Klinken, S. P. (1995) Exp. Cell Res. 219, 39-46]. Here we demonstrate that amiloride suppressed the hormone-induced increase in transferrin receptors, whereas the enhanced incorporation of iron into haem was not inhibited. Similarly, when sodium butyrate was used to induce differentiation of J2E cells, proliferation ceased and surface transferrin receptors remained unaltered, while haemoglobin production was accelerated. It was concluded from these experiments that the erythropoietin-stimulated rise in transferrin receptors during the final stages of J2E cell maturation is linked to cell division, and is not essential for haemoglobin synthesis.

Regulation of the erythropoietin receptor and involvement of JAK2 in differentiation of J2E erythroid cells.

In response to erythropoietin, J2E cells proliferate and differentiate into mature hemoglobin-producing erythroid cells. Here we show that following hormonal stimulation, between 10 and 17 proteins, including the erythropoietin receptor and JAK2, were tyrosine phosphorylated immediately after exposure to the hormone. Although the receptor was only phosphorylated to 15% of its maximum with 0.1 unit/ml erythropoietin, this was sufficient to induce peak hemoglobin synthesis. The importance of JAK2 to J2E cell maturation was demonstrated by inhibiting JAK2 protein synthesis with antisense oligonucleotides; not only was erythropoietin-stimulated mitogenesis inhibited by this procedure, but differentiation was also suppressed. In addition, the activation of STAT5 paralleled the kinetics of receptor phosphorylation. During differentiation, 94% decrease in surface erythropoietin receptors was detected 48 h after ligand binding, but transcription of the receptor gene, mRNA steady-state levels, protein content, and translation rates did not alter with hormonal stimulation. We concluded from these experiments that (a) sub-maximal receptor phosphorylation is sufficient for differentiation to proceed; (b) JAK2 is required for erythropoietin-induced cell division and maturation; and (c) post-translational processing, or translocation, play important roles in controlling surface erythropoietin receptor numbers.

Truncated erythropoietin receptor in a murine erythroleukemia cell line.

The Friend spleen focus forming virus produces a 55 kDa envelope glycoprotein which associates with the erythropoietin receptor. We compared the erythropoietin receptor in Friend virus transformed murine erythroleukemic F4N and 707 cell lines with the J2E erythroid line generated by the J2 retrovirus. Reverse transcriptase PCR was used to determine transcript size. Erythropoietin receptor cDNAs were then sequenced and protein products analysed by Western blotting and immunoprecipitation. We show here that the F4N murine erythroleukemic cell line had an enlarged erythropoietin receptor mRNA. In contrast, the 707 and J2E cell line had normal sized transcripts for the receptor. Sequence analysis of the receptor in F4N cells revealed that introns which separate the exons coding for the cytoplasmic domain of the receptor were retained in these transcripts. As a consequence, a premature stop codon had been introduced, leaving only four amino acids in the intracellular portion of the receptor molecule. The normal erythropoietin receptor is approx. 66-70 kDa, but immunoprecipitation of [35S]methionine/cysteine labelled cell lysates with an antibody to the amino-terminus of the erythropoietin receptor identified a truncated 37 kDa protein in F4N cells. Despite the severe carboxy-terminal truncation of the erythropoietin receptor, F4N cells continued to proliferate like the other murine erythroleukemia cell lines. This study shows that failure to remove introns from the erythropoietin receptor mRNA in F4N cells has resulted in the production of a smaller protein with virtually no cytoplasmic domain.

Disrupted signaling in a mutant J2E cell line that shows enhanced viability, but does not proliferate or differentiate, with erythropoietin.

The immature erythroid J2E cell line proliferates and terminally differentiates following erythropoietin stimulation. In contrast, the mutant J2E-NR clone does not respond to erythropoietin by either proliferating or differentiating. Here we show that erythropoietin can act as a viability factor for both the J2E and J2E-NR lines, indicating that erythropoietin-initiated maturation is separable from the prevention of cell death. The inability of J2E-NR cells to mature in response to erythropoietin was not due to a defect in the erythropoietin receptor sequence, although surface receptor numbers were reduced. Both the receptor and Janus kinase 2 were phosphorylated after erythropoietin stimulation of J2E-NR cells. However, protein interactions with the erythropoietin receptor and Grb2 were restricted in the mutant cells. Subsequent investigation of several other signaling molecules exposed numerous alterations in J2E-NR cells; phosphorylation changes to phosphatidylinositol 3-kinase, phospholipase Cgamma, p120 GAP, and mitogen-activated protein kinases (p42 and p44) observed in erythropoietin-stimulated J2E cells were not seen in the J2E-NR line. These data indicate that some pathways activated during erythropoietin-induced differentiation may not be essential for the prevention of apoptosis.