The distal cytoplasmic domain of the erythropoietin receptor induces granulocytic differentiation in 32D cells

The role of hematopoietic growth factors in lineage commitment and differentiation is unclear. We present evidence that heterologous expression of an erythroid specific receptor allows granulocytic differentiation of a myeloid cell line. We have previously characterized a truncation mutant of the erythropoietin receptor (EpoR), which is associated with familial erythrocytosis (Blood 89:4628, 1997). This truncated EpoR lacks the distal 70 amino acids of the cytoplasmic domain. To study the functional role of this distal receptor domain, 32D cells, a murine interleukin-3 (IL-3)-dependent myeloid line, were transfected with the wild-type EpoR (32D/EpoR WT) or the truncated EpoR (32D/EpoR FE). 32D cells expressing either the full-length or truncated EpoR display equivalent proliferative rates in saturating concentrations of Epo. There is a dramatic difference in maturational phenotype between the two cell lines, however. The 32D/EpoR FE cells and mock transfected 32D cells have an immature, monoblastic morphology and do not express the primary granule protein myeloperoxidase. The 32D/EpoR WT cells, on the other hand, demonstrate granulocytic differentiation with profuse granulation, mature, clumped chromatin, and myeloperoxidase expression. There is no evidence of erythroid differentiation in 32D cells transfected with either the full-length or truncated EpoR. Treatment of the cells with the specific Jak2 inhibitor tyrphostin AG 490 inhibits myeloid differentiation driven by the distal EpoR. We conclude that: (1) the distal cytoplasmic domain of the EpoR is able to induce a specific myeloid differentiation signal distinct from mitogenic signaling, and (2) these data extend to myelopoiesis the growing body of evidence that the cellular milieu, not the specific cytokine receptor, determines the specificity of differentiation after cytokine receptor activation.     

The prolactin receptor rescues EpoR-/- erythroid progenitors and replaces EpoR in a synergistic interaction with c-kit

We recently showed that a retrovirally transduced prolactin receptor (PrlR) efficiently supports the differentiation of wild-type burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors in response to prolactin and in the absence of erythropoietin (Epo). To examine directly whether the Epo receptor (EpoR) expressed by wild-type erythroid progenitors was essential for their terminal differentiation, we infected EpoR-/- progenitors with retroviral constructs encoding either the PrlR or a chimeric receptor containing the extracellular domain of the PrlR and intracellular domain of EpoR. In response to prolactin, both receptors were equally efficient in supporting full differentiation of the EpoR-/- progenitors into erythroid colonies in vitro. Therefore, there is no requirement for an EpoR-unique signal in erythroid differentiation; EpoR signaling has no instructive role in red blood cell differentiation. A synergistic interaction between EpoR and c-kit is essential for the production of normal numbers of red blood cells, as demonstrated by the severe anemia of mice mutant for either c-kit or its ligand, stem cell factor. We show that the addition of stem cell factor potentiates the ability of the PrlR to support differentiation of both EpoR-/- and wild-type CFU-e progenitors. This synergism is quantitatively equivalent to that observed between c-kit and EpoR. Therefore, there is no requirement for an EpoR-unique signal in the synergistic interaction between c-kit and EpoR.     

The response of IL-3 dependent B6SUtA bone marrow cells to both erythropoietin and chemical inducers of differentiation

To develop cell lines which respond to both a physiological cytokine and chemical agents by the induction of differentiation pathway, factor dependent B6SUtA murine bone marrow cells were transfected with the erythropoietin receptor (EpoR). Clones were obtained that exhibited different sensitivities to erythopoietin (Epo), with one clone exhibiting erythroid differentiation in response to Epo, while in another Epo acted as a proliferation stimulus. Moreover, parental B6SUtA cells were sensitive to the initiation of differentiation by butyrate, diazepam and hemin. Thus, B6SUtA cells appear to represent a unique model to dissect the signaling molecules involved in the growth and differentiation pathways employed by Epo and non-physiological chemicals.     

Deletions in one domain of the Friend virus-encoded membrane glycoprotein overcome host range restrictions for erythroleukemia

Although the Friend virus-encoded membrane glycoprotein (gp55) activates erythropoietin receptors (EpoR) to cause erythroblastosis only in certain inbred strains of mice but not in other species, mutant viruses can overcome aspects of mouse resistance. Thus, mice homozygous for the resistance allele of the Fv-2 gene are unaffected by gp55 but are susceptible to mutant glycoproteins that have partial deletions in their ecotropic domains. These and other results have suggested that proteins coded for by polymorphic Fv-2 alleles might directly or indirectly interact with EpoR and that changes in gp55 can overcome this defense. A new viral mutant with an exceptionally large deletion in its ecotropic domain is now also shown to overcome Fv-2rr resistance. In all cases, the glycoproteins that activate EpoR are processed to cell surfaces as disulfide-bonded dimers. To initiate analysis of nonmurine resistances, we expressed human EpoR and mouse EpoR in the interleukin 3-dependent mouse cell line BaF3 and compared the abilities of Friend virus-encoded glycoproteins to convert these cells to growth factor independence. Human EpoR was activated in these cells by erythropoietin but was resistant to gp55. However, human EpoR was efficiently activated in these cells by the same viral mutants that overcome Fv-2rr resistance in mice. By construction and analysis of human-mouse EpoR chimeras, we obtained evidence that the cytosolic domain of human EpoR contributes to its resistance to gp55 and that this resistance is mediated by accessory cellular factors. Aspects of host resistance in both murine and nonmurine species are targeted specifically against the ecotropic domain of gp55.     

Physical and functional interaction between p72(syk) and erythropoietin receptor

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid cells through interaction with a cell surface receptor (EpoR) that belongs to the cytokine receptor family. The Jak2 tyrosine kinase was previously shown to bind to the EpoR, to be activated upon Epo stimulation, and to play a critical role in Epo-induced proliferation. However, little is known about the role of other tyrosine kinases in Epo signaling. In this paper, we examined whether Syk was involved in EpoR activation. Coimmunoprecipitation experiments showed that the phosphorylated EpoR was associated with the Syk kinase in activated UT7 cells. The interaction of Epo with its receptor led to an increased kinase activity. The use of recombinant Syk Src homology 2 (SH2) domains expressed in tandem or individually revealed that both N- and C-SH2 domains of Syk participated in EpoR binding with a major contribution of the C-terminal SH2 domain. Far Western blotting further indicated that Syk directly binds to the EpoR and that the interaction of Syk with EpoR only occurred after Epo activation. These data suggest that phosphorylation of EpoR on tyrosine residues may mediate Syk binding to the receptor through interaction between the two SH2 domains of Syk and tyrosines of the receptor. We propose that in addition to Jak2, Syk protein kinase may be a component of EpoR signaling.     

Participation of IHF and a distant UP element in the stimulation of the phage lambda PL promoter

We examined signaling by erythropoietin in highly purified human colony forming unit-erythroid cells, generated in vitro from CD34(+) cells. We found that erythropoietin induces tyrosine phosphorylation of Jak2, STAT5A, and STAT5B. Tyrosine phosphorylation of Jak2 reaches a peak around 10 minutes after stimulation and is maximum at 5 U/mL of erythropoietin. Tyrosine phosphorylation of STAT5 is accompanied by the translocation of activated STAT5 to the nucleus as shown by electrophoretic mobility shift assay (EMSA) using 32Pi-labeled STAT5 binding site in the beta-casein promoter. Tyrosine phosphorylation STAT1 or STAT3 was not detected in human erythroid precursors after stimulation with erythropoietin. Crkl, an SH2/SH3 adapter protein, becomes coimmunoprecipitated specifically with STAT5 from erythropoietin-stimulated erythroid cells; although it was shown to become associated with c-Cbl in the studies using cell lines. Thus, human erythroid precursors can be expanded in vitro in sufficient numbers and purity to allow its usage in signal transduction studies. This report sets a basis for further studies on signaling in primary cultured human erythroid precursors, which in turn contribute to our better understanding in the differentiation processes of erythrocytes and their precursors.     

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The erythropoietin receptor (EpoR) has been previously shown to contain a cytoplasmic C-terminal negative regulatory domain, experimental deletion or mutation of which leads to increased sensitivity of expressing cells to the effects erythropoietin (Epo). We have studied a naturally occurring C-terminal truncation mutant of the human EpoR by stably transfecting the growth factor-dependent hematopoietic tissue culture cell line 32D with expression plasmids containing either the wildtype or mutant human EpoR cDNA, thus rendering the cells dependent on Epo for viability and proliferation. In Epo dose-response assays, cells expressing the mutant EpoR displayed hyperresponsiveness to Epo compared with cells expressing comparable numbers of the wild-type EpoR cultured in the presence of fetal bovine serum. We investigated whether enhanced Epo sensitivity of cells expressing the truncated EpoR is associated with alteration in Epo receptor-mediated activation of Stat5, which could have a role in Epo-induced proliferation. Although maximal Stat5 activation in response to a given concentration of Epo was comparable in 32D cells expressing the wild-type or truncated EpoRs, the time course of Epo-induced Stat5 activation was very different. Gel-mobility shift studies revealed the presence of Stat5 DNA-binding activity in nuclear and cytoplasmic extracts of cells expressing the truncated EpoR for a significantly longer time than that observed in similar extracts of cells expressing the wild-type EpoR consistent with decreased rate of inactivation of Stat5 in cells expressing the mutant EpoR. Epo-dependent tyrosine phosphorylation of both Stat5 and Jak2 was also substantially prolonged in cells expressing the truncated EpoR. These results suggest a role for Stat5 in regulation of Epo-mediated cell growth and implicate altered kinetics of Epo-induced Jak2 and Stat5 activation in the pathogenesis of familial erythrocytosis associated with this naturally occurring EpoR gene mutation.     

Angiotensin II stimulates proliferation of normal early erythroid progenitors

Angiotensin II exerts a mitogenic effect in several in vitro models, but a direct effect on erythroid progenitors has not been documented. Angiotensin-converting enzyme inhibitors and losartan, an angiotensin II type 1 receptor (AT1) antagonist, ameliorate posttransplant erythrocytosis, without altering serum erythropoietin levels. We studied erythroid differentiation and the effect of angiotensin II on proliferation of erythroid progenitors by culturing CD34+ hematopoietic progenitor cells in liquid serum-free medium favoring growth of erythroid precursors. Aliquots of cells were collected every third day, and were used for RNA preparation. AT1 mRNA was detected after 6 d. In these same samples, erythroid-specific mRNA (erythropoietin receptor) was also detected. AT1 protein was detected in 7-d-old burst-forming units-erythroid colonies by Western blotting. The CD34+ cell liquid cultures were used to incubate erythroid precursors with angiotensin II from days 6-9. After incubation, cells were transferred to semisolid medium and cultured with erythropoietin. Angiotensin II increased proliferation of early erythroid progenitors, defined as increased numbers of burst-forming units-erythroid colonies. Losartan completely abolished this stimulatory effect of angiotensin II. Moreover, we observed increased numbers of erythroid progenitors in the peripheral blood of posttransplant erythrocytosis patients. Thus, activation of AT1 with angiotensin II enhances erythropoietin-stimulated erythroid proliferation in vitro. A putative defect in the angiotensin II/AT1 pathway may contribute to the pathogenesis of posttransplant erythrocytosis.     

Coinduction of embryonic and adult-type globin mRNAs by sodium butyrate and trichostatin A in two murine interleukin-3-dependent bone marrow-derived cell lines

Using an RNase protection assay, globin mRNA species expressed in clones derived from Ba/F3 and B6SUtA cells transfected with the erythropoietin receptor (EpoR) and selected with erythropoietin (Epo) were compared with globin mRNA species induced in corresponding parental cells by sodium butyrate (SB) and trichostatin A (TSA). betaMajor/betaminor- and -1/-2-globin mRNAs were the major species, with trace amounts of epsilon-globin mRNA, formed in Epo-stimulated EpoR+ Ba/F3 clones, whereas SB and TSA allowed expression of all species of globin mRNAs, ie, epsilon, betah1, betamajor/betaminor, zeta, and -1/-2, in parental Ba/F3 cells. In contrast, epsilon- and -1/-2-globin mRNAs were the major species present in Epo-stimulated EpoR+ B6SUtA clones, whereas SB and TSA activated epsilon-, betah1-, betaS/betaT-, and -1/-2-globin genes in parental B6SUtA cells; zeta-globin mRNA was not detected in SB- and TSA-treated B6SUtA cells. Because TSA is a specific inhibitor of histone deacetylase, the mimicry of action exhibited by SB and TSA suggests that the effects of SB are mediated through its ability to inhibit histone deacetylase and that histone deacetylase is an integral part of the repression of globin genes in these interleukin-3-dependent cells. Efficient coinduction of embryonic and adult types of globin mRNA in bone marrow cell lines derived from adult mice indicates that adult hematopoietic precursors possess an embryonic nature. These cell lines are useful models to study the mechanism(s) of developmental globin gene switching.     

SHP-1 phosphatase C-terminus interacts with novel substrates p32/p30 during erythropoietin and interleukin-3 mitogenic responses

SHP-1 protein tyrosine phosphatase is a critical negative regulator of mitogenic signaling, as demonstrated by the heightened growth responses to hematopoietic growth factors in hematopoietic cells of motheaten mice, which lack functional SHP-1 expression due to mutations in the SHP-1 gene. The mitogenic signaling molecules dephosphorylated by SHP-1 have not been fully identified. We detected two proteins (p32/p30) that are hyperphosphorylated in a DA3/erythropoietin receptor (EpoR) cell line that expresses a mutant containing the SHP-1 C-terminus that suppresses the function of the endogenous phosphatase and induces hyperproliferative responses to interleukin-3 (IL-3) and Epo. Hyperphosphorylated p32/p30 are also detected in motheaten hematopoietic cells, demonstrating an association of p32/p30 hyperphosphorylation with SHP-1-deficiency and growth factor-hyperresponsiveness. The hyperphosphorylated p32/30 associate with SHP-1 via its C-terminus, because they coimmunoprecipitate with the phosphatase and the C-terminal mutant and they bind in vitro to a synthetic peptide of the mutant but not the GST fusion proteins of SHP-1 SH2 domains. Induction of p32/p30 phosphorylation by IL-3 or Epo occurs mainly at 2 to 18 hours poststimulation in the DA3/EpoR cell line, indicating p32/p30 as novel signaling molecules during cell cycle progression. These data demonstrate a function for the SHP-1 C-terminus in recruiting potential substrates p32/p30 and suggest that SHP-1 may regulates mitogenic signaling by dephosphorylating p32/p30.     

Distinct actions of interleukin-9 and interleukin-4 on a hematopoietic stem cell line, EMLC1

EMLC1 is a hematopoietic stem cell line that depends on stem cell factor (SCF) for growth and generates lymphoid, erythroid and myeloid progenitors in the presence of different cytokines. We have studied signaling events leading to cell proliferation and differentiation of EMLC1 mediated by interleukin (IL)-4 and IL-9. It was found that IL-9 enhances SCF-induced cell proliferation and promotes erythropoietin (EPO)-dependent erythroid differentiation of EMLC1 cells. However, IL-9 alone cannot support the growth of this cell line. In contrast, IL-4 by itself is sufficient to promote the growth of EMLC1 cells, even in the absence of SCF. Antiphosphotyrosine immunoblots of total cell lysates demonstrated that IL-4 and IL-9 induce tyrosine phosphorylation of different cellular substrates. Both IL-4 and IL-9 stimulated tyrosine phosphorylation of SHP-2, whereas the 90-kD tyrosine phosphorylated protein induced by IL-9 stimulation is Stat3. We have also shown that IL-4 is much more potent than IL-9 in inducing the expression of primary response gene c-myc. It was further determined that c-myc antisense oligodeoxynucleotide blocked IL-4 supported cell growth. Taken together, these results indicate that IL-4 may serve as a growth-promoting factor for hematopoietic stem cells, and IL-9 enhances both growth and erythroid differentiation of primitive hematopoietic progenitors. The results also suggest that differences in tyrosine phosphorylation induced by IL-4 and IL-9 may in part determine their distinct biological functions.     

Positive selection of apoptosis-resistant cells correlates with activation of dominant-negative STAT5

The STAT5 activation has important roles in cell differentiation, cell cycle control, and development. However, the potential implications of STAT5 in the control of apoptosis remain unexplored. To evaluate any possible link between the erythropoietin receptor (EpoR) JAK2/STAT5 transduction pathway and apoptosis, we have investigated apoptosis-resistant cells (ApoR) that arose from positive selection of the erythroid-committed Ba/F3EpoR cells triggered to apoptosis by ectopic expression of the HOX-B8 homeotic gene. We show that JAK2 is normally activated by Epo in both Ba/F3EpoR and ApoR cells. In contrast, both STAT5a and STAT5b isoforms are uniquely activated in a C-truncated form (86 kDa) only in ApoR cells. Analysis of ApoR and Ba/F3EpoR subclones confirmed that the switch to the truncated STAT5 isoform coincides with apoptosis survival and that ApoR do not derive from preexisting cells with a shortened STAT5. In addition, ApoR cells die in the absence of Epo. This indicates that resistance to apoptosis is not because of a general defect in the apoptotic pathway of ApoR cells. Furthermore, we show that the 86-kDa STAT5 protein presents a dominant-negative (DN) character. We hypothesize that the switch to a DN STAT5 may be part of a mechanism that allows ApoR cells to be selectively advantaged during apoptosis. In conclusion, we provide evidence for a functional correlation between a naturally occurring DN STAT5 and a biological response.     

Characterization of the domains of the erythropoietin receptor necessary for induction of cell growth and differentiation

To define the cytoplasmic region(s) of the erythropoietin receptor (EpoR) necessary for promotion of growth and induction of differentiation, mutated EpoR cDNAs containing truncations and conversions of tyrosine residues to phenylalanines were generated. Constructs were introduced into IL-3 dependent Ba/F3 cells by electroporation, and individual transfectants were propagated in methylcellulose-containing medium in the presence of erythropoietin (Epo). Truncated receptor at amino acid 350 was partially deficient in promoting cellular growth and the receptor lacking the box 2 region was not capable of inducing cellular growth. When a large number of cells were screened in Epo-containing liquid medium, clones arose in which Epo-dependent growth was due to activation of the endogenous EpoR gene. Analysis of the degree of Epo-dependent differentiation of the transfectants, based on the steady-state levels of beta major-globin mRNA, showed that the carboxyl terminal 133 amino acids and tyrosyl residues located at positions 429/431 and 460/464 were not necessary for the induction of differentiation. Examination of clones from diverse origins demonstrated that an inverse relationship existed between the rate of Epo-induced cellular replication and the degree of Epo-induced differentiation.     

Prognostic value of alpha 6 beta 4 integrin expression in breast carcinomas is affected by laminin production from tumor cells

Familial erythrocytosis, associated with high haemoglobin levels and low serum erythropoietin (Epo), has been shown to co-segregate with a sequence repeat polymorphism at the 5' region of the erythropoietin receptor (EpoR) in a large Finnish family. We have investigated the cause of erythrocytosis in an English boy. Sequencing of the cytoplasmic region of the EpoR detected a de novo transition mutation of G to A at nucleotide 6002. This mutation resulted in the formation of a stop codon at amino acid 439 with the loss of 70 amino acids from the carboxy terminus. The mutation (G6002A) has arisen independently in a Finnish family and de novo in this English boy. Patients with unexplained erythrocytosis and low serum Epo levels should be investigated for EpoR mutations.     

Establishment and characterization of a new erythropoietin-dependent acute myeloid leukemia cell line, AS-E2

We have established an erythropoietin-dependent human leukemia cell line, AS-E2, from a patient with acute myeloid leukemia. These cells have many characteristics of late erythroid progenitor cells, they are positive for CD36, Glycophorin A, and CD71 but negative for CD41, and positive for benzidine and PAS staining. These cells express GATA-1 and have low affinity erythropoietin (EPO) receptor on their surface. Interestingly, AS-E2 cells are strictly dependent on EPO for their growth and survival; other cytokines including GM-CSF, stem cell factor, or IL-3 cannot support the growth of this cell line. These features are similar to late erythroid lineage cells, like normal BFU-E or CFU-E, and we have demonstrated that EPO stimulation induces the tyrosine phosphorylation of several proteins in AS-E2 cells including the EPO receptor and JAK2 kinase. This new cell line is a useful reagent to study biological and molecular events during the late stages of erythropoiesis, and to understand transforming events in human erythroid cells.     

CD4 regulation of TCR signaling and T cell differentiation following stimulation with peptides of different affinities for the TCR

To define the role of CD4 in modulating T cell signaling pathways and regulating Th1 and Th2 differentiation, we have examined the activation and differentiation characteristics of naive T cells from CD4 mutant mice. Using peptides with differing affinities for the moth cytochrome c-specific TCR, we test the hypothesis that differences in coreceptor recruitment and signaling explain the qualitatively distinct signaling pathways seen in CD4 T cells following high affinity agonist and low affinity altered peptide ligand (APL) ligation. We find that the absence of CD4 signaling during stimulation with a strong agonist peptide does not qualitatively change the pattern of early TCR-mediated biochemical signaling events into a pattern resembling the response of CD4+ T cells to APLs. In contrast, the response to APL stimulation, by T cells bearing the same TCR, does require a component of CD4 signaling. The proliferative response and calcium signals normally seen following APL stimulation are markedly diminished in the absence of CD4. In addition, we find that naive T cell differentiation into Th2 effector cells is impaired in the absence of CD4. These data suggest that the altered pattern of biochemical signals generated by APLs require CD4 coreceptor function and that some of these signals may be required to initiate Th2 differentiation.