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The distal region and receptor tyrosines of the Epo receptor are non‐essential for in vivo erythropoiesis

The distal region and receptor tyrosines of the Epo receptor are non‐essential for in vivo... The EMBO Journal Vol. 20 No. 12 pp. 3156-3166, 2001 The distal region and receptor tyrosines of the Epo receptor are non-essential for in viva erythropoiesis 1 2 1 used to date. One approach has utilized mutant stains of Heesuk Zang • , Ken Sato , 1 3 mice that lack components of various signaling pathways. Hideaki Nakajima 1, Catriona McKay , , 1 2 1 2 3 4 However, this is limited by the potential involvement of Paul A.Ney • and James N.lhle , , , the signaling pathways in other receptor systems. The 1 3 Department of Biochemistry and Howard Hughes Medical Institute, other approach has utilized receptor mutations that have St Jude Children's Research Hospital, Memphis, TN 38105 and lost or retained the ability to activate signaling pathways Department of Biochemistry, University of Tennessee Health Science when expressed in cell lines. Center, Memphis, TN 38105, USA The first studies of EpoR mutants came to the Corresponding author at: Howard Hughes Medical Institute, St Jude unexpected conclusion that the distal half of the cytoplas­ Children's Research Hospital, Memphis, TN 38105, USA mic domain of the EpoR was not essential for receptor e-mail: [email protected] function in cell lines and, indeed, negatively influenced H.Zang and K.Sato contributed equally to this work receptor functions (D' Andrea et al., 1991). The concept of a negative role for the distal domain of the receptor gained The erythropoietin receptor (EpoR) is required for support from the observation that there were mutations of the proliferation and survival of committed erythroid the human EpoR that deleted 70-- 79 amino acids of the lineage cells. Previous studies have utilized receptor distal region of the receptor and were associated with a mutations to show the requirement for the distal half phenotype of erythrocytosis (De La Chapelle et al., 1993; of the cytoplasmic domain of the EpoR and receptor Sokol et al., 1995; Arcasoy et al., 1997). Interestingly, all tyrosines for activation of signaling pathways poten­ the cases reported to date are heterozygous for the tially critical to Epo function. To extend these studies mutation. The biochemical basis for the increased func­ to in vivo erythropoiesis, we have created two mutant tionality of the distally truncated receptor is not known strains of mice. One strain (H) contains a truncation precisely. However, it has been shown that the distal of the distal half of the cytoplasmic domain, while the region of the EpoR contains tyrosines that can be bound second strain (HM) contains the same truncation as by the SH2 domains of the protein tyrosine specific well as the mutation of the residual tyrosine (Y ) to a phosphatase SHP-1 (Yi et al., 1995), a phosphatase that, phenylalanine. Strikingly, both strains of mice are when deleted in mice, results in the hyperproliferation of viable, with only slight alterations in constitutive many cell lineages (Shultz et al., 1993). In one report, it erythropoiesis or in in vitro assays of red cell lineage was suggested that recruitment to the receptor complex is function. Challenging H mutant mice with continuous critical to bring SHP-1 in proximity to Jak2 (Klingmuller injections of Epo results in an erythrocytosis that is et al., 1995). However, subsequent studies have demon­ not seen in HM mice. The results demonstrate that strated that SHP-1 can interact directly with Jak2 and, neither the distal region nor receptor tyrosines are consequently, the requirement for receptor tyrosine for essential for in vivo EpoR function, but contribute to recruitment is unclear (Jiao et al., 1996). receptor function in a subtle manner. A variety of studies have been directed at the identi­ Keywords: cytokine signaling/erythropoiesis/ fication of phosphorylated tyrosine residues in the EpoR erythropoietin/J ak2 kinase/receptor that are recognized by proteins that may be important in transduction of a proliferative or differentiative response. For example, the signal transducers and activators of Introduction transcription Sa and Sb (Stat5a/b) can bind to multiple sites The production of erythrocytes is tightly regulated through of tyrosine phosphorylation within the receptor, including the production of erythropoietin (Epo) and its ability to Y . In the context of a truncated receptor that only 343 343 function through its receptor to support the survival and contains Y , mutation of Y to a phenylalanine expansion of erythroid progenitors (Krantz, 1991). The eliminates the ability of the receptor to recruit and activate Epo receptor (EpoR) is a member of the cytokine receptor Stat5a/b (Gobert et al., 1996; Quelle et al., 1996). The family and functions to couple Epo binding to the consequences of this mutation have been variably reported activation of the Janus protein tyrosine kinase Jak2 to have no consequence (Quelle et al., 1996), to reduce the (Witthuhn et al., 1993). Following the activation of Jak2, proliferative function of the receptor (Damen et al., 1995a; multiple sites on the receptor are tyrosine phosphorylated, Gobert et al., 1996) and/or to promote differentiation as as well as on substrates that are recruited to the receptor well as hemoglobinization (Iwatsuki et al., 1997; Gregory complex. One of the challenges of studying signal et al., 1998). transduction by the EpoR, as with all members of the Other sites of tyrosine phosphorylation within the cytokine receptor superfamily, has been to define the receptor have been shown to be critical for recruit­ physiological consequences of the activation of individual ment of the p85 regulatory subunit of phosphatidylinositol signaling pathways. Two general approaches have been 3-kinase (PI-3K) (Damen et al., 1995b). Gabl and Gab2 3156 © European Molecular Biology Organization The distal region and receptor tyrosines of EpoR are non-essential have been implicated in Epo signaling, and shown to require the distal region of the receptor, with Gab2 425 3 7 specifically requiring Y and/or Y 6 for recruitment to the receptor complex (Wickrema et al., 1999). Epo­ induced calcium channel activation has been shown to require Y 60 in the distal region of the receptor (Miller et al., 1999). Activation of AP-1 transcription factors has been reported to require the presence of at least one of -fft +f- + multiple tyrosines, including Y (Bergelson et al., 1998). B C Lyn has been reported to physically associate with the IJUl'WT 4 4 479 EpoR through Y 6 and/or Y in the distal region (Chin et al., 1998), while Syp has been reported to bind to Y , - - - .. _ --- 4 1 which corresponds to Y 0 in the commonly used nomen­ �---- -- clature. (Tauchi et al., 1996). Using retrovirally trans­ ....... duced mutants of a constitutive active form of the EpoR, �-· recent studies concluded that efficient red blood cell "' it • .� ,;,j.l"./ 4".Jl>;. 7 development was dependent upon receptor cytoplasmic domain tyrosine residues. However, the functionality of a tyrosine-less receptor could be restored by multiple individual tyrosines, although Y and Y uniquely supported immature burst-forming progenitors (Longmore ... et al., 1998). Transfection of mutant receptors into EpoR-deficient fetal liver cells has identified a novel, Stat5a/b-independent pathway that requires Y and is necessary and sufficient for Epo responses (Klingmuller et al., 1997). However, the role of the recruitment of the above proteins is unclear since the sites required for their recruitment and activation are in the distal half of the 'i.Ml VJ") cytoplasmic domain. The above experiments with transfected EpoR mutants ""'' have several limitations. In many cases, the possibility ,,, .. QI exists that the cell lines used are transformed to the extent y;u that a signaling pathway that is essential in normal v_. ,, ... hematopoiesis is obviated in the cell line by transforming Y'1't events that allowed its establishment. The other difficulty WT 4Qat is to obtain receptor expression levels that are comparable Fig. 1. Generation of EpoR-H and EpoR-HM mice. (A) Targeting to that occurring during normal erythroid lineage differ­ strategy. Solid boxes with numbers 1-8 indicate ex ons of the EpoR entiation. Because of these limitations, we have generated gene. Arrows labeled a and b indicate PCR primers used for geno­ typing. E, EcoRI. (H/HM) indicates H and HM mutation of exon 8. mutant strains of mice by modification of the endogenous The H mutation was produced by truncation of the C-terminal 108 receptor gene to contain the most commonly studied EpoR amino acids. The HM mutation was produced by the same truncation mutations. Quite strikingly, the deletion of the distal half 343 343 and a mutation of Y to F on exon 8. Homologous recombination of the cytoplasmic domain had very little consequence on replaced exon 8 with the H or HM mutant DNA, resulting in a in vivo erythropoiesis, although it provided some support truncated receptor with only one tyrosine residue on its cytoplasmic domain (EpoR-H) or a truncated receptor with Phe substitution for to the concept that the distal region negatively influences residue Y (EpoR-HM). (B) PCR analysis of tail clippings from wild­ receptor function to a small degree. Removal of the last type and mutant mice: +/+, wild type; +/-, heterozygous for wild type tyrosine (Y343) in the truncated receptor similarly had and H allele; -/-, homozygous for H allele. Genomic DNA from tail only a very minor effect on receptor function and tended to clippings was used for PCR with primers a and b. The same PCR eliminate the increase in receptor function seen with the analysis was used to identify EpoR-HM mice (data not shown). (C) Southern blot analysis of DNA from offspring derived from truncation and returned the receptor function to a level heterozygous matings of wild-type and H mice. Genomic DNA was comparable to that of the wild-type receptor. digested with EcoRI and probed with the 0.8 kb genomic DNA fragment indicated in (A). (D) Semi-quantitative RT-PCR analysis of EpoR, EpoR-H and EpoR-HM mRNA in adult bone marrow cells. The Results PCR products are 667 bp for EpoR wild-type allele, 349 bp for EpoR-H or EpoR-HM mutant allele and 900 bp for �-actin. The The roles of the distal region of the EpoR and receptor numbers indicate the number of PCR cycles performed. (E) Schematic tyrosines have been extensively studied in transfected cell diagram of wild-type, H and HM EpoR. The relative positions of lines. To address the role of the distal region under more tyrosine residues are marked. physiological conditions, two mouse strains were created with the gene-targeting strategies illustrated in Figure lA. The H strain of mice was obtained by using homologous was mutated to phenylalanine. ES cells contammg the recombination to delete the distal 108 amino acids of the appropriately modified receptor loci were injected into EpoR in ES cells. Since the resulting receptor would blastocysts to obtain chimeric mice, which were then bred contain one tyrosine (Y ), a second strain of mice (HM) was created in which the distal region was deleted and Y to obtain gerrnline transmission. Heterozygous mice for 3157 H.Zang et al. WT H/Jl HMlDM Table I. Genotype distribution of heterozygote breeding -1L:L m -1.1..l.. w:i � w T1me(h} 0 0 ,5 3 0 . S 3 0 0 . S J 0,5 3 0 0.5 J 0,5 3 Strain +/+ -/- EpoR-H 73 (27%) 127 (47%) 70 (26%) EpoR-HM 37 (27%) 69 (50%) 31 (23%) Stat5a/b 103 (25%) 219 (52%) 97 (23%) Fig. 2. Effect of EpoR-H or EpoR-HM on the activation of Stat5a/b in For each strain, offspring from heterozygous matings of the mutant bone marrow cells. Bone marrow cells from femurs of adult (;e.4 weeks) alleles on a background of C57B1/6 X 129Sv were genotyped as mice were isolated and suspended in a.-MEM medium. After 4 h of described in Materials and methods. The expected Mendelian growth arrest, cells were stimulated with either 10 ng/ml recombinant distribution of offspring is 25% +/+, 50% +/- and 25% -/-. The mIL-3 or 50 U/ml recombinant hEpo for 30 min or 3 h. Whole-cell 32 observed frequencies are not statistically different from those predicted extracts were prepared and analyzed by EMSA. y- P-labeled DNA based on the use of a x goodness of fit test. from the Stat5a/b binding site in the �-casein promoter was used as binding sequence for the assay. the activation of Stat5a/b was not detected following Epo stimulation of bone marrow cells from HM mutant mice, the modified loci were interbred to obtain mice that were again consistent with studies with this receptor mutant homozygous for each locus. Mice homozygous for the H transfected in cell lines, although IL-3-induced levels of and HM mutations were obtained, and were grossly Stat5a/b activation were comparable to wild-type cells. normal; they were subjected to a more detailed analysis, as Together, the results demonstrate that the mutant mouse described below. strains contain receptor mutations with properties pre­ To ensure that the H and HM strains contained the dicted from the studies of these receptors in cell lines. appropriately modified receptor loci, PCR and RT-PCR As indicated above, viable mice were obtained that approaches were used. PCR analysis and Southern blot contained either the H or the HM mutation, suggesting that analysis of tail tissues revealed the segregation of the these mutations did not grossly affect erythropoiesis, and anticipated genomic DNA fragments (Figure 1B and C). consequently more detailed studies were carried out. To To examine expressed sequences, RNA from bone marrow initially assess the consequences on embryonic erythro­ cells of control and mutant strains was used to prepare poiesis, the distributions of offspring from crosses of cDNA, which was used for PCR (Figure lD). As indicated heterozygous mice were examined. As indicated in Table I, in Figure 1, the upstream primer was in coding sequences the distribution of H and HM genotypes was that predicted in exon 7, and therefore the cDNA derived from expressed from Mendelian segregation, suggesting the absence of sequences could be distinguished from genomic DNA by any embryonic lethality among the homozygous mutant the absence of the intronic sequences between ex ons 7 and mice. Since the HM mutation reduces or eliminates the 8 (93 bp ). As illustrated, bone marrow cells from the H or ability to activate Stat5a/b, the distribution of genotypes HM strains contained RNA that yielded products of the from a cross of mice heterozygous for Stat5a/b deficiency size expected for deletion of the distal region of the is also presented. Similar to the H and HM strains, the receptor. Based on the similarities of the yields of the PCR distribution of genotypes was that predicted by Mendelian products with various numbers of cycles of amplification, segregation, demonstrating the absence of any embryonic the levels of expression of the mutated receptors are lethality associated with Stat5a/b deficiency. comparable to those of the wild-type receptor. Sequencing Analysis of adult mice also indicated the lack of any of the PCR products further substantiated the presence of unexpected lethality or pathology. In particular, HM mice the predicted receptor truncation, as well as the presence of did not develop splenomegaly that is consistently seen in the Y F mutation in the HM strain of mice ( data not Stat5a/b-deficient mice. This splenomegaly is due to shown). extramedullary hematopoiesis and includes a striking Since the mice had no overt phenotype, we wanted to expansion of Ter119-positive erythroid lineage progeni­ establish that the mutant receptors were functionally tors (Moriggl et al., 1999). Since splenomegaly was not altered. Previous studies with cell lines have demonstrated observed in the HM mutant mice, the results suggest that that truncated receptors retaining Y mediate a stronger the splenomegaly seen in Stat5a/b-deficient mice is not activation of Stat5a/b tyrosine phosphorylation and DNA due to Stat5a/b deficiency in the erythroid lineage. binding activity than the wild-type receptor (Quelle et al., Consistent with this conclusion, crossing the Stat5a/b 1996). However, the mutation of Y to phenylalanine deficiency onto a Rag2-deficient background to eliminate eliminates activation of Stat5a/b by the truncated receptor. the T-cell alterations associated with Stat5a/b deficiency Therefore, we stimulated bone marrow cells with Epo and eliminates the splenomegaly (our unpublished results). examined the extent of activation of Stat5a/b. As illus­ A previous study reported that Stat5a/b deficiency is trated (Figure 2), stimulation of bone marrow cells from associated with visually evident severe embryonic anemia wild-type mice resulted in the activation of Stat5a/b DNA (Socolovsky et al., 1999). Therefore, it would be antici­ binding activity, as assessed by electrophoretic mobility pated that a comparable phenotype would be observed shift assay (EMSA). Epo stimulation of bone marrow cells in HM embryos, since they lack the ability to activate from the H mutant strain resulted in a reproducibly Stat5a/b. Photographs of El2.5 embryos of the various stronger activation of Stat5a/b, consistent with studies of genotypes are shown in Figure 3. As illustrated, H and this receptor mutant in transfected cell lines. The activa­ HM homozygous embryos were visually indistinguish­ tion of Stat5a/b by interleukin-3 (IL-3) is provided as an able from wild-type embryos. Stat5a/b-deficient embryos internal control, and is comparable in bone marrow are also depicted in Figure 3 for comparison and are preparations from wild-type and H mutant mice. Lastly, similarly indistinguishable from wild-type embryos. For +/- The distal region and receptor tyrosines of EpoR are non-essential Fig. 3. Morphology of EpoR-H and EpoR-HM embryos. Representative photographs of embryos at El2.5 from wild type (A), EpoR-H (B) and --1 --1 --1 EpoR-HM (C). Photographs of Jak2 - (D), EpoR - (E) and Stat5afb - (F) at E12.5 are shown for comparison. comparison, Jak2- or EpoR-deficient embryos are pre­ and CFU-E responses to Epo were also compared with sented to illustrate the readily observable anemia that is decreasing doses of Epo, and the mutant strains had associated with these mutants. comparable dose-response curves (data not shown). To assess the effects of the mutations on erythropoiesis, Embryonic and adult hematocrits provide another colony assays were performed with either feta! liver cells potential means of assessing the consequences of the from El2.S embryos or bone marrow cells of adult mice receptor mutations on embryonic erythropoiesis. We therefore compared the embryonic hematocrits of H and (Figure 4). As illustrated, the numbers of CFU-E per 10 feta! liver cells from H and HM mutant embryos were HM mutant embryos with those of wild-type or Stat5a / b­ comparable to those of wild-type embryos, and also deficient embryos as controls. The hematocrits of wild­ comparable to those obtained with feta! liver cells from type and StatSa/b-deficient heterozygous and homozygous Stat5a / b-deficient mice. However, the numbers of BFU-E embryos or adults are also shown in Figure S for were slightly reduced in H mutant feta! liver cell comparison. Also for comparison are shown the white preparations relative to wild type, and further reduced in and red cell blood counts (Figure SA and B) for wild-type, HM mutant feta! liver cell preparations. As a control, and H and HM mice. The hematocrit values for individual mice or embryos are illustrated in Figure SC and D, for comparison, the colony-forming response to IL-3 is also shown and is comparable in all strains. In the bone respectively. The mean values for these and additional marrow of adult mice, the numbers of CFU-E were embryos and animals are shown in Figure SE. As increased ~2-fold in H mutant mice, while the numbers in illustrated by the mean values (Figure SE), StatSa/b­ HM mutant mice were comparable to those in wild-type or deficient embryos had consistently lowered hematocrits StatSa / b-deficient mice. Lastly, BFU-E from bone marrow from the onset of feta! liver erythropoiesis, throughout the from adult mice were only marginally increased in last half of embryonic development and as adults. H mutant mice and marginally decreased in HM mutant The values ranged from 70 to 90% of those of control embryos or adult mice. The values at E13.5 are compar­ mice to a level comparable to the BFU-E response of StatSa / b mice. As a control, and for comparison, the able to recently reported hematocrits for StatSa / b-deficient colony-forming response to IL-3 is also shown and was embryos at E13.S (Socolovsky et al., 1999), with the comparable among the various strains of mice. The BFU-E exception that we rarely find individual embryos with 3159 H.Zang et al. •wr EEPOR-H .EPOR-HM 60 240 □ STAT5afb CFU-E <40 160 20 60 300 60 ,. BFU-E L4 CFU-E ,. 200 <40 100 20 15wn 0 0 --1 Fig. 4. In vitro colony formation of hematopoietic progenitors from wild-type, EpoR-H, EpoR-HM and StatSafb - mice in response to various cytokines. The numbers of colonies/10 cells formed from E12.5 fetal liver cell cultures (A) or from bone marrow cell cultures (B) are plotted. The mean and standard deviation are shown from six independent assays. The two-tailed P values for comparison of the various mutant strains with wild­ type mice or embryos are >0.01, with the exception of the BFU-E colonies for bone marrow from HM mice (P = 0.004) and of CFU-E colonies for fetal liver cells from H mice (P = 0.00006). Cells from E12.5 embryos or femurs of adult mice were prepared in a-MEM medium containing 2% FBS and counted in the presence of 3% acetic acid to lyse erythrocytes. Diluted cell suspensions and cytokines were mixed with Methocult 3230 to a final concentration of 0.9% methylcellulose. For CFU-E assay, cells were cultured in 0.2 U/ml recombinant hEpo. For BFU-E assay, cells were cultured in 3 U/ml recombinant hEpo and 10 ng/ml recombinant murine IL-3. IL-3 colony assays were performed in the presence of 10 ng/ml recombinant mIL-3. The plating conditions, cell concentration for each assay, culture conditions and colony scoring methods were exactly as described previously (Parganas et al., 1998; Teglund et al., 1998). severely decreased hematocrits. As illustrated (Figure 5D increased sensitivity to apoptosis in the absence of Epo. and E), H mutant embryos at E14.5, El5.5 and E18.5 Since no significant apoptosis was seen in fetal liver cells consistently had hematocrits that were slightly higher than of any of the mutants when examined directly from the those of wild-type embryos. Moreover, the hematocrits of embryos (data not shown), the cells were cultured for 18 h HM embryos at these ages were consistently lower than in the presence or absence of Epo. The cells were then those of wild-type embryos. In adult mice, the hematocrits examined by fluorescence-activated cell sorting (FACS) of H mutant mice were slightly higher than those of wild­ analysis for the expression of the erythroid lineage marker type mice, while the hematocrits of HM mutant mice were Ter119, and for apoptosis by TUNEL staining. As consistently lower. For comparison and for an indication illustrated (Figure 6), fetal liver cells from H or HM of the significance of the differences seen, the normal mutants cultured in the presence of 10 U/ml Epo showed hematocrits of two strains of mice are also shown no consistent differences in the levels of apoptosis, (Figure 5E; BL, C57Bl/6 and C3H/HeJ). comparable to the fetal liver cells from wild-type embyros. The above results consistently demonstrated increased When cultured in the absence of Epo, significant fractions hematocrits associated with the H mutation, whereas the of apoptotic cells were evident in cultures of wild-type HM mutation is associated with reduced levels. Since Y cells (7.1 %), slightly fewer apoptotic cells in cell cultures has been shown to be important for the activation of from H mutant embryos (5.83%) and significantly several signaling pathways, as well as Stat5a/b, we wished increased numbers of apoptotic cells in cultures from to determine whether the primary difference between the HM mutant embryos (25.5%). For comparison, typical H and HM mutations was associated with the absence of results obtained with cells from Stat5a/b-deficient Stat5a/b activation. To assess this, we crossed the H embryos are also illustrated. As above, no differences in mutation onto the Stat5a/b-deficient background, thus the numbers of apoptotic cells were evident when fetal eliminating the contribution of Stat5a/b while preserving liver cells were examined directly from E13.5 embryos. other functions requiring Y . As illustrated (Figure 5C), However, culture of the cells in the absence of Epo this resulted in reduced hematocrits that were comparable resulted in an increased degree of apoptosis in cultures of to those seen in Stat5a/b-deficient mice containing a wild­ Stat5a/b-deficient fetal liver cells comparable to that seen type receptor. The results would, therefore, suggest that in the HM mutant mice (Figure 6B). As an approach to establish additional in vivo biological the change in the hematocrits that occurs when Y is consequences of the receptor mutations, H and HM mice mutated in the context of the truncated receptor is due to the loss of the ability to activate Stat5a/b. were challenged with repeated doses of Epo, and the The distal region of the EpoR and Y have been hematocrits assessed (Figure 7). Comparable experiments implicated in the regulation of pathways that affect were not carried out with Stat5a/b-deficient mice because apoptosis. We therefore examined fetal liver cells from of the splenomegaly and extramedullary hematopoiesis mutant embryos of E13.5 for increased apoptosis or associated with the altered T-cell functions in these mice. 3160 70 The distal region and receptor tyrosines of EpoR are non-essential 21 11 N=143 N=143 ,. --- --- 12 ']: .... .... D IOI ._, IOI • 7 B . • · _J_ • t-- •. - ::I 5 ---· · D WT 6E POR-H OEPOR-HM ■ H xSTATSallr' ·- .. ··--- D .. �-------- � <>S TATSallr' N=208 N=185 60 so+- ---� .-------------< ·· -- ..... ····- .: el --- .. +- --- -------�-----, --- t 4 E-- -- E-- so+<>- � +=----��---___, i I X 20 +- ...... .__.._�+- --=- ----, - - E1 8.5 E1 4.5 E1 5.5 EM �------------------------ N=749 eo+-- ----------------------------< D STAT5alb ./-. ./+, +/+ fllli1 EPOR-H IIIE POR-HM -S TAT5alb"· xEPOR-H - C57B� and C3H/HeJ 13.5 14.5 15.5 16.5 17.5 Ul.5 19.5 N.,.bora Ad11lt Fig. 5. Hematocrits and blood cell counts of embryos or adult mice with the indicated genotypes. For adult mice (;a.4 weeks old), white blood cell numbers (A), red blood cell numbers (B) and hematocrits (C and E) were machine scored by a Hemavet 3700R counter. For embryos (D and E), blood samples were acquired using 2 µl glass micropipettes and then briefly centrifuged to measure hematocrits. N, total number of mice used for the -1 experiment. H X StatSafb - indicates mice cross-breeding from StatSa/b null mice with EpoR-H mice. (E) Numbers indicate days of gestation. The P values for all the comparisons support statistically significant differences in the hematocrits. To indicate the types of P values associated with the 3 12 results, in (C) the two-tailed P values for the means of the various mutants relative to wild type are: H, 1.3 X 10-; HM, 8.0 X 10- ; StatSa/b, 6.4 X lo-4; H/StatSa/b, 9.2 X 10-. Repeated injections of Epo (three times weekly) into wild­ essential for embryonic and adult erythropoiesis in vivo. type mice over 30 days resulted in an increasing Numerous studies have examined the properties of hematocrit during the period of injections. As illustrated, mutated EpoRs in cell lines, particularly receptors lacking this increase was enhanced in H mutant mice, consistent tyrosine residues, which would have suggested that such with the concept that the receptor functions slightly better mutations would have a dramatic phenotype in vivo. This than the wild-type receptor. In contrast, there was a lower was not the case and specifically neither the H nor HM rate of increase in hematocrits in HM mice. The results are mutations were associated with any evidence of embry­ generally consistent with the concept that the H truncation onic or adult lethality. This contrasts with deficiencies of results in a moderately more efficient receptor, while the the receptor-associated kinase Jak2 (Neubauer et al., 1998; mutation of the last tyrosine in the truncated receptor Parganas et al., 1998) or the EpoR-regulated genes Bcl-X reduces the efficiency of the receptor. (Motoyama et al., 1995) and N-myc (Moens et al., 1993; Sawai et al., 1993), all of which exhibit, like deficiency of the EpoR (Wu et al., 1995), embryonic lethal phenotypes Discussion associated with profound deficiencies in expansion of The results demonstrate the striking finding that neither the erythroid lineage progenitors with dramatically increased distal region of the EpoR nor receptor tyrosines are apoptosis in vivo. The lack of consistency with the data 3161 ) 0 . 5 2 H.Zang et al. POR- HM EPOR-H "o "o I') .., ..,o g g ;:::i "b 5.83 c... -o -0 -g U-1 0.72 0.0 1 0.87 0.0 0.0 0 0 g g 0 3 "' ° 1 2 3 4 o 1 2 3 4 0 0 0 0 0 1 1 ,ii- 10 1 1 0 1 1 0 0 1 1 10 0 o 1 1 10 0 "o "o "'o ---I .., 8 £ ..._ ,, ,I l' "' • a "' ,..... "!=! 0.3 0 . 6 2.7 1 c.., 0 -0 -� '-1,l 0 .0 0.0 0 . 84 0.0 0.36 0� 0 ° 3 4 1 4 0 1 2 3 1 0 10 !0 ° 2 0 0 0 0 10 1 103 1 1 1 0 I0 10� 1 0 ./. ... --, 5' --- 0� c., 22.6 I .:? 1 7 U. 2 0.03 2 --- - * 4 c 1 , 0 .. !< �L TUN EL ., M e 5' ..._ ... 1.72 u .u z ,ii2 I0 Fig. 6. FACS analysis of feta! liver cells at E13.5 with combined TUNEL- Terl 19 staining. Numbers in plots indicate the percentage of sorted cell population in each quadrant. The upper right quadrant in each plot represents Te rll 9-positive erythrocytes with TUNEL-positive apoptotic cells. (A) Fetal liver cells from EpoR wild-type, EpoR-H or EpoR-HM embryos were isolated at E13.5 and cultured in a.-MEM with 2% FBS for 18 h in the presence or absence of 10 U/ml recombinant hEpo. Cells were then fixed with formalin and double stained with TUNEL reagent and Ter i 19 antibody. The cell suspension was analyzed by FACScan. (B) Feta! livers from StatSa/b wild-type embryos or StatSa/b null embryos were used for TUNEL­ Terl 19 double staining. Note that the EpoR mutants were performed independently of the StatSa/b mutant studies and, consequently, should not be compared across the two experiments, but rather the mutants should be compared with their respective wild-type controls. with cell lines may reflect the need for additional were the first to suggest that the distal half of the pathways, perhaps in a cell line-dependent manner, for cytoplasmic domain negatively influenced EpoR signal­ the maintenance of the cell lines as opposed to the ing. Our results with modification of the receptor by gene transient requirement for Epo to mediate the expansion of targeting provide in vivo support to the concept that the erythroid lineage cells. distal region negatively affects receptor signaling. The first studies of mutant EpoRs indicated the exist­ Consistent with the experiments in cell lines, the activation ence of non-overlapping positive and negative regulatory of Stat5a/b was increased and prolonged in bone domains (D' Andrea et al., 1991). In particular, the studies marrow cells from H mutant mice stimulated with Epo. 3162 The distal region and receptor tyrosines of EpoR are non-essential which any differences observed with the H mutation were only evident in the homozygous state. The basis for the differences is not known, but may well reside in the �60 differences that exist between the human and murine !;.., owr receptors, and the extent of the truncations, as noted above. CH The other possibility might be mouse strain differences, 6HM although we have not seen any significant differences in H �: :: .7- 6. � .. or HM mutant mice on a C57Bl/6 or Balb/c background. Numerous studies have used receptor mutants to identify the domains and/or tyrosine in the EpoR that are 20 ---- required for the activation of signal transduction pathways. days 0 6 12 18 24 The potential role for many of these pathways was inferred Fig. 7. Response of the EpoR mutant mice to continual injections of from the activity of transfected mutated receptors or from Epo. Mice were injected three times during a week, as described in the use of dominant-negative proteins, inhibitors or Materials and methods, with recombinant hEpo. To prevent anomalous antisense reduction of protein levels. Because of this results from frequent bleeding, the mice were separated into groups, which were bled only every 14 days. The average hematocrits for two extensive body of research, it is particularly striking that a mice are indicated by the individual symbols. The deviations among distally truncated receptor containing no tyrosines was the samples for each symbol were between 2 and 5%; consequently, able to sustain erythropoiesis at the levels observed. It error bars are not included. The line presents the linear regression of must be concluded that either the activation of the the data points for each of the mutant strains. pathways studied is not essential for receptor function or that there is some type of selection for compensating functions that is occurring in vivo and that is sufficiently Unfortunately, due to the quantity of cells required and the efficient to completely obviate the role of the receptor heterogeneous nature of bone marrow cells, we have been tyrosines. Unfortunately, in most cases, it is not possible to unable to assess the Jak2 activation. Also consistent with a determine whether signal transduction pathways are still negative role of the distal region in regulating signaling, activated in cells from HM mutant mice because of the the hematocrits of H mutant mice were modestly increased requirement for large quantities of homogeneous cells. We and there was an increased response to repeated injections are currently breeding the HM mutation on a background of Epo. However, the effects were not as striking as might that is susceptible to Friend virus disease, as a way of have been predicted based on the mutant receptor studies obtaining large quantities of erythroid lineage cells for or based on the mutations of the distal region of the EpoR further biochemical studies (Koury et al., 1984). Using in humans (De La Chapelle et al., 1993; Sokol et al., 1995; these mice, we will also be able to evaluate the cell surface Arcasoy et al., 1997). In particular, individuals who are expression patterns of the receptor mutants, which has not heterozygous for a truncation of 70 amino acids were been possible with bone marrow cells. However, we would reported to have an erythrocytosis in which hemoglobin not expect that the expression of the mutants would be levels were 1.33-fold higher than controls. In contrast, different since the gene modifications are quite distant mice homozygous for a truncation of 108 amino acids from the promoter of the gene. Moreover, extensive have hemoglobin levels that are only 1.08-fold those of studies with these mutants in transduced cell lines have controls (data not shown). Lastly, it should be noted that consistently shown that, when expressed comparably, they whereas the erythrocytosis associated with the human are processed and expressed at the cell surface in receptor mutations has been observed with individuals comparable patterns. heterozygous for the receptor mutations, mice hetero­ In contrast to most of the signaling pathways, the zygous for the H mutation display no increase in sensitivity of EMSA allowed us to assess the ability of H hematocrits (data not shown). One possible basis for the and HM mice to recruit and activate Stat5a/b, for which difference is the extent of the truncations. In particular, the Y is known to be necessary and sufficient for its human truncations retain an additional tyrosine and this activation. The results demonstrate that the strong activa­ may provide an additional positive signal that contributes tion of Stat5a/b that is seen in H mutant mice is completely to the overall larger positive response. eliminated in HM mutant mice. Thus, HM mice have not In a recent study (Divoky et al., 2001), mutant mouse activated a compensating pathway that either allows Epo strains were created in which the endogenous murine to activate Stat5a/b or results in a constitutive level of EpoR gene was replaced with either the wild-type human Stat5a/b activation comparable to that seen in Epo­ EpoR gene or a carboxyl-truncated human EpoR gene as a stimulated cells. The HM mutation thus renders the mice model for the human primary familial congenital poly­ specifically deficient in Stat5a/b function within the cythemia associated with receptor truncations. Consistent erythroid lineage, and allows a potentially more precise with our studies with the H strain of mice, no loss of analysis of Stat5a/b function in erythroid cells than viability was observed. Also consistent with our results complete deletion of the gene because of the critical role was the lack of striking changes in BFU-E or CFU-E. that Stat5a/b play in other lineages of cells and particularly However, the extent of erythrocytosis, as assessed with in T cells (Teglund et al., 1998; Moriggl et al., 1999). hematocrits, was considerably greater than that seen in our Based on the published properties of erythropoiesis in H strain of mice. Moreover, erythrocytosis was evident in Stat5a/b-deficient embryos from the strain of mice we heterozygous animals for the truncation in combination produced (Socolovsky et al., 1999), we had anticipated a with either the wild-type murine endogenous gene or the dramatic phenotype of severe fetal anemia in HM mice. wild-type human gene. This is in contrast to our results in However, many of the phenotypes that were reported were 3163 H.Zang et al. and Hindlll digestion utilizing a Hindlll site on exon 8 and a newly not evident in our studies of Stat5a/b-deficient embryos. In created Hindlll site was followed. For Y F mutation, a 0. 8 kb Hindlll particular, HM mutant embryos, like Stat5a/b-deficient fragment utilizing Hindlll site on intron 6 and exon 8 was subcloned into embryos, were morphologically indistinguishable from pBluescript. Site-directed mutagenesis was performed as described in the wild-type embryos. Moreover, no embryonic lethality was manufactu re r's protocol (Qiagen) and the plasmid containing the correct mutation was used for the construction of HM targeting vector. For evident in either HM mutant or Stat5a/b-deficient negative selection, a diphtheria toxin A gene (DTA) driven by the embryos. The frequencies of BFU-E and CFU-E were polyoma virus enhancer cassette (Adachi et al., 1995) was inserted in the comparable among wild-type, HM mutant and Stat5a/b­ 5' end of the targeting construct. A thymidine kinase promoter-driven neo deficient embryos. Lastly, there was no apoptosis in fetal cassette was inserted after the stop codon of exon 8 to confer positive livers of HM mutant or Stat5a/b-deficient embryos, selection. consistent with the absence of apoptosis in fetal livers of Transfection of ES cells and generation of EpoR-H or wild-type embryos. The basis for the differences in the EpoR-HM mice results is yet to be resolved. W9.5 ES cells and El4 (129/0la mouse strain) ES cells were cultured as Consistent with the previous studies, however previously described (Parganas et al., 1998). W9.5 ES cells were used for the generation of EpoR-H mice and El 4 cells were used for the generation (Socolovsky et al., 1999), Stat5a/b-deficient embryos of EpoR-HM mice. Twenty-five micrograms of Sall-linearized EpoR-H had hematocrits that were reduced relative to those of or EpoR-HM plasmid construct were electroporated into the W9.5 or El 4 wild-type mice throughout embryonic development and as ES cells. Twenty-four hours after electroporation, selection was adults. The differences are comparable to that seen performed in 350 µg/ml geneticin (G418; Gibco-BRL). Conditions for between different strains of mice. Importantly, a compar­ blastocyst injection of correctly targeted and karyotypically normal ES clones, and breeding to generate mice homozygous for the mutated EpoR able reduction in hematocrits was seen in HM mutant gene, were performed essentially as described (van Deursen et al., 1993). embryos and adults. The possibility that the reduction seen ES clones were injected into C57Bl/6 blastocysts, and five clones from in HM mutants is due to the loss of Stat5a/b function is EpoR-H targeting and three clones from EpoR-HM targeting gave supported by the observation that the increased hemato­ germline transmission. For PCR analysis, mice tails were clipped and digested overnight in lysis buffer (500 mM KCI, 100 mM Tris pH 8.3, 0.1 crits seen in H mutant embryos are reduced to that of mg/ml gelatin, 1 % NP-40, 1 % Tween-20, 500 µg/ml proteinase K). PCR Stat5a/b-deficient or HM mutant embryos when crossed was carried out with primer a (5'-GAGTTTGAGGGTCTCTTCACC-3') onto the Stat5a/b-deficient background. The Stat5a/b in exon 7 and primer b (5' -TAGGCTGGAGTCCTAGGAGC-3') in exon 8 functions that mediate the 10--30% reduction in hemato­ of the EpoR gene. Since primer b corresponds to a DNA sequence at the crits are unclear from studies of erythroid progenitors beginning of the stop codon in exon 8 and extends into the following non­ coding 3' sequences that are retained in the truncation, the 442 bp in vivo. However, fetal liver cells from both HM and fragment is characteristically derived from the mutant containing the Stat5a/b-deficient embryos are more susceptible to apop­ truncation, while the wild-type allele gives rise to a 760 bp fragment. For tosis when cultured in the absence of Epo than are wild­ EpoR-HM mice, the PCR product was sequenced to confirm HM type or H mutant fetal liver cells. It has been proposed that mutation. For Southern blot analysis, EcoRI- Xhol digestion was performed on isolated EpoR genomic DNA, and the 0.8 kbp fragment Stat5a/b is required for the Epo-induced expression of comprising exon 2 and an intron between exon 1 and exon 2 was used as a Bcl-X (Socolovsky et al., 1999); however, when exam­ probe. Genomic DNA from mouse tail was digested by EcoRI and, after ined, the levels of Bcl-X protein in fetal liver erythroid probing, the mutant allele shows a 4.5 kb fragment and the wild-type cells are not detectably altered in Stat5a/b-deficient allele shows an ~12 kb fragment. For RT-PCR analysis, total cellular embryos (Marine et al., 1999). RNA was extracted from bone marrow cells of adult mice (;a,,4 weeks) using RNAzol B (Tel-Test). First-strand cDNA synthesis using oligo(dn In summary, while it might have been anticipated that primer was carried out as described in the manufactur er's protocol the creation of mouse strains containing a severely (Gibco-BRL Superscript reverse transcriptase). PCR was performed truncated EpoR, which lacks any tyrosine residues, using PCR primers a and b for the EpoR gene, and �-actin primers might have resulted in striking consequences to erythro­ (5'-GTGACGAGGCCCAGAGCAAGAG-3', 5'-AGGGGCCGGACT­ CATCGTACTC-3') for the �-actin gene. poiesis, this was not the case. The results support the concept that many of the signal transducing events that EMSA have been well characterized with the use of mutant Bone marrow cells (8.0 X 10 ) were collected from femurs of wild-type, receptors in cell lines may have limited significance in EpoR-H and EpoR-HM mice. Cells were suspended in a-MEM medium defining the critical events in EpoR signaling. The with 2% fetal calf serum, 1 % L-glutarnine and appropriate amounts of antibiotics. Cells were then starved for 4 h and stimulated with 50 U/ml availability of these mutant strains of mice will be recombinant human Epo (hEpo; Amgen) or 10 ng/ml recombinant murine particularly useful in allowing studies to focus on a more IL-3 (mIL-3; R & D systems) for 30 min or 3 h. Whole-cell extract was restricted set of signal transducing pathways, retained by prepared by incubation in extraction buffer [50 mM Tris pH 8.0, 50 mM the mutant receptors, in more physiologically relevant NaCl, 0.1 mM EDTA, 0.5 mM NaV0 , 0.5% NP-40, 50 mM NaF, 10% 3 4 glycerol, 20 mM �-mercaptoethanol, 1 mM dithiothreitol (DTn, 0.4 mM in vivo studies. The availability of these mutant strains will phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail] for also allow examination of the possibility that the distal 30 min at 4C. For Stat5 DNA binding sequence, sense and antisense region of the receptor is required in very specific situations oligonucleotides representing the Stat5a/b site in �-casein promoter to modify or amplify a 'c ore ' signal. (5'-AGATTTCTAGGAATTCAATCC-3') were synthesized, annealed to make double-stranded DNA and labeled with [y- P]ATP. Ten micro­ grams of cell extract were incubated in binding buffer [10 mM Tris, 50 mM NaCl, 0.1 mM EDTA, 0.2 mM PMSF, 1 mM DTT, 0.1 % NP-40, Materials and methods 5% glycerol, 2 µg poly(dl-dC), 30 µg/ml bovine serum albumin] for Construction of targeting vector 30 min at room temperature, and the reaction mixtures were separated on The EpoR gene was isolated from the El4 (129/0la mouse strain) 4% native TBE gel. genomic library in EMBL3 using EpoR full-length cDNA as a probe. Positive clones were restriction mapped and sequenced. Briefly, a Xhol Colony assay fragment including intron 2-exon 8 of the EpoR gene was subcloned into Petal liver cells were prepared from livers of E12.5 embryos, and bone pBluescript. For truncation of the receptor, a Hindlll site was introduced marrow cells from femurs of wild-type, EpoR-H, EpoR-HM and --1- just upstream of the stop codon by site-directed mutagenesis (5'­ StatSafb mice. The cells were plated and the colonies scored exactly TATGTGGCCTGCTCCTAG-3' to 5'-TATGAAGCTTGCTCCTAG-3'), as described previously (Parganas et al., 19 98; Teglund et al., 1998). 3164 The distal region and receptor tyrosines of EpoR are non-essential Blood cell counts and hematocrits D' Andrea,A.D., Yoshimura,A., Youssoufian,H., Zon,L.I., Koo,J.W. and For adult mice, blood samples were taken from orbital sinus using a Lodish,H.F. (1991) The cytoplasmic region of the erythropoietin microcapillary tube. Twenty microliters of sample blood were used for receptor contains nonoverlapping positive and negative growth­ analysis in a MASCOT Hemavet 3700R counter (CDC Technologies) and regulatory domains. Mol. Cell. Biol., 11, 1980-1987. scored for white blood cells and red blood cells. Hematocrits were De La Chapelle,A., Traskelin,A.-L. and Juvonen,E. (1993) Truncated calculated by Hemavet counter based on red blood cell counts and mean erythropoietin receptor causes dominantly inherited benigu human corpuscular volume. For embryos, blood samples were taken from the erythrocytosis. Proc. Natl Ac ad. Sci. USA , 90, 4495-4499. carotid artery using heparinized 2 µ1 microcapillary tubes. Tubes were Divoky,V., Liu,Z., Ryan,T.M., Prchal,J.F., Townes,T.M. and Prchal,J.T. sealed at one end and centrifuged for 5 min at 3000 r.p.m. to measure (2001) Mouse model of congenital polycythemia: homologous hematocrits. replacement of murine gene by mutant human erythropoietin receptor gene. Proc. Natl Acad. Sci. USA, 98, 986-991. Flow cytometry and TUNEL staining Gobert,S. et al. (1996) Identification of tyrosine residues within the E13.5 fetal livers were isolated from embryos, and made into single-cell intracellular domain of the erythropoietin receptor crucial for ST AT5 suspension by passing them through a G25 needle. Cells were cultured in activation. EMB O J. , 15, 2434-2441. cx-MEM medium with 2% fetal bovine serum (FBS) for 18 h. For Gregory,R.C., Jiang,N., Todokoro,K., Crouse,J., Pacifici,R.E. and Epo stimulation, recombinant hEpo was added to a final concentration of Wojchowski,D.M. (1998) Erythropoietin receptor and STAT5- 10 U/ml in culture medium. After culture, cells were centrifuged and specific pathways promote SKT6 cell hemoglobinization. Blood, 92, washed with phosphate-buffered saline (PBS) containing 1 % FBS. Cells 1104-1118. were then centrifuged for 5 min at 1000 g and PBS decanted, followed by Iwatsuki,K., Endo,T., Misawa,H., Yokouchi,M., Matsumoto,A., fixation with formalin for 20 min at room temperature and subsequent Ohtsubo,M., Mori,K.J. and Yoshimura,A. (1997) STAT5 activation treatment with 0.1% Triton X-100 and 0.1% sodium citrate. Cells were correlates with erythropoietin receptor-mediated erythroid placed on ice for 2 min and washed with PBS. TUNEL reaction was differentiation of an erythroleukemia cell line. J. Biol. Chem, 272, performed as described in the manufacturer's protocol (ln situ cell death 8149-8152. detection kit, Fluorescein; Boehringer Manheim) and, after washing with Jiao,H., Berrada,K., Yang,W., Tabrizi,M., Platanias,L.C. and Yi,T. PBS, Ter119 antibody was added, followed by incubation on ice for an (1996) Direct association and dephosphorylation of Jak2 kinase by hour and several washing with PBS. The cell suspension was then SH2 domain-containing protein tyrosine phosphatase SHP-1. Mol. analyzed in a Becton-Dickinson FACScan using CellQuest software. Cell. Biol., 16, 6985--6992. Klingmuller,U., Lorenz,U., Cantley,L.C., Neel,B.G. and Lodish,H.F. Epo challenge analysis (1995) Specific recruitment of the hematopoietic protein tyrosine A total of 30 mice were selected for the experiment. The selection was phosphatase SH-PTPl to the erythropoietin receptor causes gender balanced and mice of various ages were included. Epo (500 U/kg inactivation of JAK2 and termination of proliferative signals. Cell, body weight) was injected into all 30 mice three times a week. Blood 80, 729- 738. samples were drawn between injections and complete blood counts were Klingmuller,U., Wu,H., Hsiao,J.G., Toker,A., Duckworth,B.C., Cantley, performed. Hematocrits were monitored for 4 weeks. To prevent any L.C. and Lodish,H.F. (1997) Identification of a novel pathway anomaly in the experimental results due to frequent bleeding, mice were important for proliferation and differentiation of primary erythroid separated into five groups and scheduled to take turns for bleeding. progenitors. Proc. Natl Acad. Sci. USA, 94, 3016-3021. A group of six mice consisted of two mice from each genotype: wild type, Koury,M.J., Sawyer,S.T. and Bondurant,M.C. (1984) Splenic EpoR-H and EpoR-HM. erythroblasts in anemia-inducing Friend disease: a source of cells for studies of erythropoietin-mediated differentiation. J. Cell Physiol., 121, 526-532. Ackn owledgements Krantz,S.B. (1991) Erythropoietin. Blood, 77, 419-434. Longmore,G.D., You,Y., Molden,J., Liu,K.D., Mikami,A., Lai,S.Y., We would like to thank Linda Snyder, Kristen Rothammer and Melanie Pharr,P. and Goldsmith,M.A. (1998) Redundant and selective roles for Loyd for technical support; Drs John Cleveland, Gerry Zambetti and erythropoietin receptor tyrosines in erythropoiesis in vivo. Blood, 91, Nick Carpino for helpful discussion; and Richard Morrigl for helping 870-878. with HCT analysis of Stat5a/b embryos. This work was supported by the Marine,J.C. et al. (1999) SOCS3 is essential in the regulation of fetal Cancer Center CORE grant CA21765, by grants ROl DK42932 and POl liver erythropoiesis. Cell, 98, 617--627. HL53749, and by the American Lebanese Syrian Associated Charities Miller,B.A., Barber,D.L., Bell,L.L., Beattie,B.K., Zhang,M.Y., Neel, (ALSAC). B.G., Yoakim,M., Rothblum,L.I. and Cheung,J.Y. (1999) Identification of the erythropoietin receptor domain required for calcium channel activation. J. Biol. Chem. , 274, 20465-20472. Referen ces Moens,C.B., Stanton,B.R., Parada,L.F. and Rossant,J. (1993) Defects in heart and lung development in compound heterozygotes for two Adachi,M., Suematsu,S., Kondo,T., Ogasawara,J., Tanaka,T., Yoshida,N. different targeted mutations at the N-myc locus. Development, 119, and Nagata,S. (1995) Targeted mutation in the Fas gene causes 485-499. hyperplasia in peripheral lymphoid organs and liver. Natu re Genet., 11, Moriggl,R. et al. (1999) Stat5 is required for IL-2 induced cell cycle 294-300. progression of peripheral T cells. Imm unity, 10, 249-259. Arcasoy,M.O., Degar,B.A., Harris,K.W. and Forget,B.G. (1997) Familial Motoyama,N. et al. (1995) Massive cell death of immature erythrocytosis associated with a short deletion in the erythropoietin hematopoietic cells and neurons in Bcl-x-deficient mice. Science, receptor gene. Blood, 89, 4628-4635. 267, 1506-1510. Bergelson,S., Klingmuller,U., Socolovsky,M., Hsiao,J.G. and Lodish, Neubauer,H., Cumano,A., Muller,M., Wu,H., Huffstadt,U. and H.F. (1998) Tyrosine residues within the intracellular domain of the Pfeff er,K. (1998) Jak2 deficiency defines an essential developmental erythropoietin receptor mediate activation of AP-1 transcription checkpoint in definitive hematopoiesis. Cell, 93, 397-409. factors. J. Biol. Chem, 273, 2396-2401. Parganas,E. et al. (1998) Jak2 is essential for signaling through a variety Chin,H., Arai,A., Wakao,H., Kamiyama,R., Miyasaka,N. and Miura,O. of cytokine receptors. Cell, 93, 385-395. (1998) Lyn physically associates with the erythropoietin receptor and Quelle,F.W., Wang,D., Nosaka,T., Thierfelder,W.E., Stravopodis ,D., may play a role in activation of the Stat5 pathway. Blood, 91, Weinstein,Y. and Ihle,J.N. (1996) Erythropoietin induces activation of 3734-3745. Stat5 through association with specific tyrosines on the receptor that Damen,J.E., Wakao,H., Miyajima,A., Krosl,J., Humphries,R.K., Cutler, are not required for a mitogenic response. Mol. Cell. Biol., 16, R.L. and Krystal,G. (1995a) Tyrosine 343 in the erythropoietin 1622-1631. receptor positively regulates erythropoietin-induced cell proliferation Sawai,S., Shimono,A., Wakamatsu,Y., Palmes,C., Hanaoka,K. and and Stat5 activation. EMBO J. , 14, 5557-5568. Damen,J.E., Cutler,R.L., Jiao,H., Yi,T. and Krystal,G. (1995b) Kondoh,H. (1993) Defects of embryonic organogenesis resulting Phosphorylation of Y in the erythropoietin receptor (EpR) is from targeted disruption of the N-myc gene in the mouse. essential for binding the p85 subunit of phosphatidylinositol (PI) 3- Development, 117, 1445-1455. kinase and for EpR associated PI 3-kinase activity. J. Biol. Chem, Shultz,L.D., Schweitzer,P.A., Rajan,T.V., Yi,T., Ihle,J.N., Matthews, 270, 23402-23408. R.J., Thomas,M.L. and Beier,D.R. (1993) Mutations at the murine 3165 H.Zang et al. motheaten locus are within the hematopoietic cell protein tyrosine phosphatase (Hcph) gene. Cell, 73, 1445-1454. Socolovsk:y,M., Fallon,A.E., Wang,S., Brugnara,C. and Lodish,H.F. (19 99) Fetal anemia and apoptosis of red cell progenitors in --f- 1 Stat5a 5b-- mice: a direct role for Stat5 in Bcl-X(L) induction. Cell, 98, 181 -191. Sokol,L., Luhovy,M., Guan,Y., Prchal,J.F., Semenza,G.L. and Prchal,J.T. (19 95) Primary familial polycythemia: a frameshift mutation in the erythropoietin receptor gene and increased sensitivity of erythroid progenitors to erythropoietin. Blood, 86, 15 -22. Tauch i,T., Damen,J.E., Toyarna,K., Feng,G.S., Broxmeyer,H.E. and Krystal,G. (1 996) Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis. Blood, 87, 4495-4501. Teglun d,S. et al. (19 98) Stat5a and Stat5b proteins have essential and non-essential, or redundant, roles in cytokine responses. Cell, 93, 841-850. van Deursen,J. , Heerschap,A., Oerlemans,F., Ruitenbeek,W., Jap,P., ter Laak,H. and Wieringa,B . (19 93) Skeletal muscles of mice deficient in muscle creatine kinase lack burst activity. Cell, 74, 621- 63 1. Wickrema,A. et al. (19 99) Engagement of Gabl and Gab2 in erythropoietin signaling. J. Biol. Chem, 274, 24469-24474. Witthuhn,B ., Quelle,F.W., Silvennoinen,O., Yi,T ., Tan g,B ., Miura,O. and Thle,J.N. (19 93) JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following EPO stimulation. Cell, 74, 227-236. Wu,H ., Liu,X., Jaenisch,R. and Lodish,H.F. (19 95) Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell, 83, 59-67. Yi,T ., Zhang,J., Miura,O. and Ihle,J.N. (19 95) Hematopoietic cell phosphatase (HCP) associates with the erythropoietin receptor following Epo induced receptor tyrosine phosphorylation: identification of potential binding sites. Blood, 85, 87-95. Received February 23, 2001; revised and acce pted April 26, 2001 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

The distal region and receptor tyrosines of the Epo receptor are non‐essential for in vivo erythropoiesis

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Springer Journals
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Copyright © European Molecular Biology Organization 2001
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0261-4189
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1460-2075
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10.1093/emboj/20.12.3156
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Abstract

The EMBO Journal Vol. 20 No. 12 pp. 3156-3166, 2001 The distal region and receptor tyrosines of the Epo receptor are non-essential for in viva erythropoiesis 1 2 1 used to date. One approach has utilized mutant stains of Heesuk Zang • , Ken Sato , 1 3 mice that lack components of various signaling pathways. Hideaki Nakajima 1, Catriona McKay , , 1 2 1 2 3 4 However, this is limited by the potential involvement of Paul A.Ney • and James N.lhle , , , the signaling pathways in other receptor systems. The 1 3 Department of Biochemistry and Howard Hughes Medical Institute, other approach has utilized receptor mutations that have St Jude Children's Research Hospital, Memphis, TN 38105 and lost or retained the ability to activate signaling pathways Department of Biochemistry, University of Tennessee Health Science when expressed in cell lines. Center, Memphis, TN 38105, USA The first studies of EpoR mutants came to the Corresponding author at: Howard Hughes Medical Institute, St Jude unexpected conclusion that the distal half of the cytoplas­ Children's Research Hospital, Memphis, TN 38105, USA mic domain of the EpoR was not essential for receptor e-mail: [email protected] function in cell lines and, indeed, negatively influenced H.Zang and K.Sato contributed equally to this work receptor functions (D' Andrea et al., 1991). The concept of a negative role for the distal domain of the receptor gained The erythropoietin receptor (EpoR) is required for support from the observation that there were mutations of the proliferation and survival of committed erythroid the human EpoR that deleted 70-- 79 amino acids of the lineage cells. Previous studies have utilized receptor distal region of the receptor and were associated with a mutations to show the requirement for the distal half phenotype of erythrocytosis (De La Chapelle et al., 1993; of the cytoplasmic domain of the EpoR and receptor Sokol et al., 1995; Arcasoy et al., 1997). Interestingly, all tyrosines for activation of signaling pathways poten­ the cases reported to date are heterozygous for the tially critical to Epo function. To extend these studies mutation. The biochemical basis for the increased func­ to in vivo erythropoiesis, we have created two mutant tionality of the distally truncated receptor is not known strains of mice. One strain (H) contains a truncation precisely. However, it has been shown that the distal of the distal half of the cytoplasmic domain, while the region of the EpoR contains tyrosines that can be bound second strain (HM) contains the same truncation as by the SH2 domains of the protein tyrosine specific well as the mutation of the residual tyrosine (Y ) to a phosphatase SHP-1 (Yi et al., 1995), a phosphatase that, phenylalanine. Strikingly, both strains of mice are when deleted in mice, results in the hyperproliferation of viable, with only slight alterations in constitutive many cell lineages (Shultz et al., 1993). In one report, it erythropoiesis or in in vitro assays of red cell lineage was suggested that recruitment to the receptor complex is function. Challenging H mutant mice with continuous critical to bring SHP-1 in proximity to Jak2 (Klingmuller injections of Epo results in an erythrocytosis that is et al., 1995). However, subsequent studies have demon­ not seen in HM mice. The results demonstrate that strated that SHP-1 can interact directly with Jak2 and, neither the distal region nor receptor tyrosines are consequently, the requirement for receptor tyrosine for essential for in vivo EpoR function, but contribute to recruitment is unclear (Jiao et al., 1996). receptor function in a subtle manner. A variety of studies have been directed at the identi­ Keywords: cytokine signaling/erythropoiesis/ fication of phosphorylated tyrosine residues in the EpoR erythropoietin/J ak2 kinase/receptor that are recognized by proteins that may be important in transduction of a proliferative or differentiative response. For example, the signal transducers and activators of Introduction transcription Sa and Sb (Stat5a/b) can bind to multiple sites The production of erythrocytes is tightly regulated through of tyrosine phosphorylation within the receptor, including the production of erythropoietin (Epo) and its ability to Y . In the context of a truncated receptor that only 343 343 function through its receptor to support the survival and contains Y , mutation of Y to a phenylalanine expansion of erythroid progenitors (Krantz, 1991). The eliminates the ability of the receptor to recruit and activate Epo receptor (EpoR) is a member of the cytokine receptor Stat5a/b (Gobert et al., 1996; Quelle et al., 1996). The family and functions to couple Epo binding to the consequences of this mutation have been variably reported activation of the Janus protein tyrosine kinase Jak2 to have no consequence (Quelle et al., 1996), to reduce the (Witthuhn et al., 1993). Following the activation of Jak2, proliferative function of the receptor (Damen et al., 1995a; multiple sites on the receptor are tyrosine phosphorylated, Gobert et al., 1996) and/or to promote differentiation as as well as on substrates that are recruited to the receptor well as hemoglobinization (Iwatsuki et al., 1997; Gregory complex. One of the challenges of studying signal et al., 1998). transduction by the EpoR, as with all members of the Other sites of tyrosine phosphorylation within the cytokine receptor superfamily, has been to define the receptor have been shown to be critical for recruit­ physiological consequences of the activation of individual ment of the p85 regulatory subunit of phosphatidylinositol signaling pathways. Two general approaches have been 3-kinase (PI-3K) (Damen et al., 1995b). Gabl and Gab2 3156 © European Molecular Biology Organization The distal region and receptor tyrosines of EpoR are non-essential have been implicated in Epo signaling, and shown to require the distal region of the receptor, with Gab2 425 3 7 specifically requiring Y and/or Y 6 for recruitment to the receptor complex (Wickrema et al., 1999). Epo­ induced calcium channel activation has been shown to require Y 60 in the distal region of the receptor (Miller et al., 1999). Activation of AP-1 transcription factors has been reported to require the presence of at least one of -fft +f- + multiple tyrosines, including Y (Bergelson et al., 1998). B C Lyn has been reported to physically associate with the IJUl'WT 4 4 479 EpoR through Y 6 and/or Y in the distal region (Chin et al., 1998), while Syp has been reported to bind to Y , - - - .. _ --- 4 1 which corresponds to Y 0 in the commonly used nomen­ �---- -- clature. (Tauchi et al., 1996). Using retrovirally trans­ ....... duced mutants of a constitutive active form of the EpoR, �-· recent studies concluded that efficient red blood cell "' it • .� ,;,j.l"./ 4".Jl>;. 7 development was dependent upon receptor cytoplasmic domain tyrosine residues. However, the functionality of a tyrosine-less receptor could be restored by multiple individual tyrosines, although Y and Y uniquely supported immature burst-forming progenitors (Longmore ... et al., 1998). Transfection of mutant receptors into EpoR-deficient fetal liver cells has identified a novel, Stat5a/b-independent pathway that requires Y and is necessary and sufficient for Epo responses (Klingmuller et al., 1997). However, the role of the recruitment of the above proteins is unclear since the sites required for their recruitment and activation are in the distal half of the 'i.Ml VJ") cytoplasmic domain. The above experiments with transfected EpoR mutants ""'' have several limitations. In many cases, the possibility ,,, .. QI exists that the cell lines used are transformed to the extent y;u that a signaling pathway that is essential in normal v_. ,, ... hematopoiesis is obviated in the cell line by transforming Y'1't events that allowed its establishment. The other difficulty WT 4Qat is to obtain receptor expression levels that are comparable Fig. 1. Generation of EpoR-H and EpoR-HM mice. (A) Targeting to that occurring during normal erythroid lineage differ­ strategy. Solid boxes with numbers 1-8 indicate ex ons of the EpoR entiation. Because of these limitations, we have generated gene. Arrows labeled a and b indicate PCR primers used for geno­ typing. E, EcoRI. (H/HM) indicates H and HM mutation of exon 8. mutant strains of mice by modification of the endogenous The H mutation was produced by truncation of the C-terminal 108 receptor gene to contain the most commonly studied EpoR amino acids. The HM mutation was produced by the same truncation mutations. Quite strikingly, the deletion of the distal half 343 343 and a mutation of Y to F on exon 8. Homologous recombination of the cytoplasmic domain had very little consequence on replaced exon 8 with the H or HM mutant DNA, resulting in a in vivo erythropoiesis, although it provided some support truncated receptor with only one tyrosine residue on its cytoplasmic domain (EpoR-H) or a truncated receptor with Phe substitution for to the concept that the distal region negatively influences residue Y (EpoR-HM). (B) PCR analysis of tail clippings from wild­ receptor function to a small degree. Removal of the last type and mutant mice: +/+, wild type; +/-, heterozygous for wild type tyrosine (Y343) in the truncated receptor similarly had and H allele; -/-, homozygous for H allele. Genomic DNA from tail only a very minor effect on receptor function and tended to clippings was used for PCR with primers a and b. The same PCR eliminate the increase in receptor function seen with the analysis was used to identify EpoR-HM mice (data not shown). (C) Southern blot analysis of DNA from offspring derived from truncation and returned the receptor function to a level heterozygous matings of wild-type and H mice. Genomic DNA was comparable to that of the wild-type receptor. digested with EcoRI and probed with the 0.8 kb genomic DNA fragment indicated in (A). (D) Semi-quantitative RT-PCR analysis of EpoR, EpoR-H and EpoR-HM mRNA in adult bone marrow cells. The Results PCR products are 667 bp for EpoR wild-type allele, 349 bp for EpoR-H or EpoR-HM mutant allele and 900 bp for �-actin. The The roles of the distal region of the EpoR and receptor numbers indicate the number of PCR cycles performed. (E) Schematic tyrosines have been extensively studied in transfected cell diagram of wild-type, H and HM EpoR. The relative positions of lines. To address the role of the distal region under more tyrosine residues are marked. physiological conditions, two mouse strains were created with the gene-targeting strategies illustrated in Figure lA. The H strain of mice was obtained by using homologous was mutated to phenylalanine. ES cells contammg the recombination to delete the distal 108 amino acids of the appropriately modified receptor loci were injected into EpoR in ES cells. Since the resulting receptor would blastocysts to obtain chimeric mice, which were then bred contain one tyrosine (Y ), a second strain of mice (HM) was created in which the distal region was deleted and Y to obtain gerrnline transmission. Heterozygous mice for 3157 H.Zang et al. WT H/Jl HMlDM Table I. Genotype distribution of heterozygote breeding -1L:L m -1.1..l.. w:i � w T1me(h} 0 0 ,5 3 0 . S 3 0 0 . S J 0,5 3 0 0.5 J 0,5 3 Strain +/+ -/- EpoR-H 73 (27%) 127 (47%) 70 (26%) EpoR-HM 37 (27%) 69 (50%) 31 (23%) Stat5a/b 103 (25%) 219 (52%) 97 (23%) Fig. 2. Effect of EpoR-H or EpoR-HM on the activation of Stat5a/b in For each strain, offspring from heterozygous matings of the mutant bone marrow cells. Bone marrow cells from femurs of adult (;e.4 weeks) alleles on a background of C57B1/6 X 129Sv were genotyped as mice were isolated and suspended in a.-MEM medium. After 4 h of described in Materials and methods. The expected Mendelian growth arrest, cells were stimulated with either 10 ng/ml recombinant distribution of offspring is 25% +/+, 50% +/- and 25% -/-. The mIL-3 or 50 U/ml recombinant hEpo for 30 min or 3 h. Whole-cell 32 observed frequencies are not statistically different from those predicted extracts were prepared and analyzed by EMSA. y- P-labeled DNA based on the use of a x goodness of fit test. from the Stat5a/b binding site in the �-casein promoter was used as binding sequence for the assay. the activation of Stat5a/b was not detected following Epo stimulation of bone marrow cells from HM mutant mice, the modified loci were interbred to obtain mice that were again consistent with studies with this receptor mutant homozygous for each locus. Mice homozygous for the H transfected in cell lines, although IL-3-induced levels of and HM mutations were obtained, and were grossly Stat5a/b activation were comparable to wild-type cells. normal; they were subjected to a more detailed analysis, as Together, the results demonstrate that the mutant mouse described below. strains contain receptor mutations with properties pre­ To ensure that the H and HM strains contained the dicted from the studies of these receptors in cell lines. appropriately modified receptor loci, PCR and RT-PCR As indicated above, viable mice were obtained that approaches were used. PCR analysis and Southern blot contained either the H or the HM mutation, suggesting that analysis of tail tissues revealed the segregation of the these mutations did not grossly affect erythropoiesis, and anticipated genomic DNA fragments (Figure 1B and C). consequently more detailed studies were carried out. To To examine expressed sequences, RNA from bone marrow initially assess the consequences on embryonic erythro­ cells of control and mutant strains was used to prepare poiesis, the distributions of offspring from crosses of cDNA, which was used for PCR (Figure lD). As indicated heterozygous mice were examined. As indicated in Table I, in Figure 1, the upstream primer was in coding sequences the distribution of H and HM genotypes was that predicted in exon 7, and therefore the cDNA derived from expressed from Mendelian segregation, suggesting the absence of sequences could be distinguished from genomic DNA by any embryonic lethality among the homozygous mutant the absence of the intronic sequences between ex ons 7 and mice. Since the HM mutation reduces or eliminates the 8 (93 bp ). As illustrated, bone marrow cells from the H or ability to activate Stat5a/b, the distribution of genotypes HM strains contained RNA that yielded products of the from a cross of mice heterozygous for Stat5a/b deficiency size expected for deletion of the distal region of the is also presented. Similar to the H and HM strains, the receptor. Based on the similarities of the yields of the PCR distribution of genotypes was that predicted by Mendelian products with various numbers of cycles of amplification, segregation, demonstrating the absence of any embryonic the levels of expression of the mutated receptors are lethality associated with Stat5a/b deficiency. comparable to those of the wild-type receptor. Sequencing Analysis of adult mice also indicated the lack of any of the PCR products further substantiated the presence of unexpected lethality or pathology. In particular, HM mice the predicted receptor truncation, as well as the presence of did not develop splenomegaly that is consistently seen in the Y F mutation in the HM strain of mice ( data not Stat5a/b-deficient mice. This splenomegaly is due to shown). extramedullary hematopoiesis and includes a striking Since the mice had no overt phenotype, we wanted to expansion of Ter119-positive erythroid lineage progeni­ establish that the mutant receptors were functionally tors (Moriggl et al., 1999). Since splenomegaly was not altered. Previous studies with cell lines have demonstrated observed in the HM mutant mice, the results suggest that that truncated receptors retaining Y mediate a stronger the splenomegaly seen in Stat5a/b-deficient mice is not activation of Stat5a/b tyrosine phosphorylation and DNA due to Stat5a/b deficiency in the erythroid lineage. binding activity than the wild-type receptor (Quelle et al., Consistent with this conclusion, crossing the Stat5a/b 1996). However, the mutation of Y to phenylalanine deficiency onto a Rag2-deficient background to eliminate eliminates activation of Stat5a/b by the truncated receptor. the T-cell alterations associated with Stat5a/b deficiency Therefore, we stimulated bone marrow cells with Epo and eliminates the splenomegaly (our unpublished results). examined the extent of activation of Stat5a/b. As illus­ A previous study reported that Stat5a/b deficiency is trated (Figure 2), stimulation of bone marrow cells from associated with visually evident severe embryonic anemia wild-type mice resulted in the activation of Stat5a/b DNA (Socolovsky et al., 1999). Therefore, it would be antici­ binding activity, as assessed by electrophoretic mobility pated that a comparable phenotype would be observed shift assay (EMSA). Epo stimulation of bone marrow cells in HM embryos, since they lack the ability to activate from the H mutant strain resulted in a reproducibly Stat5a/b. Photographs of El2.5 embryos of the various stronger activation of Stat5a/b, consistent with studies of genotypes are shown in Figure 3. As illustrated, H and this receptor mutant in transfected cell lines. The activa­ HM homozygous embryos were visually indistinguish­ tion of Stat5a/b by interleukin-3 (IL-3) is provided as an able from wild-type embryos. Stat5a/b-deficient embryos internal control, and is comparable in bone marrow are also depicted in Figure 3 for comparison and are preparations from wild-type and H mutant mice. Lastly, similarly indistinguishable from wild-type embryos. For +/- The distal region and receptor tyrosines of EpoR are non-essential Fig. 3. Morphology of EpoR-H and EpoR-HM embryos. Representative photographs of embryos at El2.5 from wild type (A), EpoR-H (B) and --1 --1 --1 EpoR-HM (C). Photographs of Jak2 - (D), EpoR - (E) and Stat5afb - (F) at E12.5 are shown for comparison. comparison, Jak2- or EpoR-deficient embryos are pre­ and CFU-E responses to Epo were also compared with sented to illustrate the readily observable anemia that is decreasing doses of Epo, and the mutant strains had associated with these mutants. comparable dose-response curves (data not shown). To assess the effects of the mutations on erythropoiesis, Embryonic and adult hematocrits provide another colony assays were performed with either feta! liver cells potential means of assessing the consequences of the from El2.S embryos or bone marrow cells of adult mice receptor mutations on embryonic erythropoiesis. We therefore compared the embryonic hematocrits of H and (Figure 4). As illustrated, the numbers of CFU-E per 10 feta! liver cells from H and HM mutant embryos were HM mutant embryos with those of wild-type or Stat5a / b­ comparable to those of wild-type embryos, and also deficient embryos as controls. The hematocrits of wild­ comparable to those obtained with feta! liver cells from type and StatSa/b-deficient heterozygous and homozygous Stat5a / b-deficient mice. However, the numbers of BFU-E embryos or adults are also shown in Figure S for were slightly reduced in H mutant feta! liver cell comparison. Also for comparison are shown the white preparations relative to wild type, and further reduced in and red cell blood counts (Figure SA and B) for wild-type, HM mutant feta! liver cell preparations. As a control, and H and HM mice. The hematocrit values for individual mice or embryos are illustrated in Figure SC and D, for comparison, the colony-forming response to IL-3 is also shown and is comparable in all strains. In the bone respectively. The mean values for these and additional marrow of adult mice, the numbers of CFU-E were embryos and animals are shown in Figure SE. As increased ~2-fold in H mutant mice, while the numbers in illustrated by the mean values (Figure SE), StatSa/b­ HM mutant mice were comparable to those in wild-type or deficient embryos had consistently lowered hematocrits StatSa / b-deficient mice. Lastly, BFU-E from bone marrow from the onset of feta! liver erythropoiesis, throughout the from adult mice were only marginally increased in last half of embryonic development and as adults. H mutant mice and marginally decreased in HM mutant The values ranged from 70 to 90% of those of control embryos or adult mice. The values at E13.5 are compar­ mice to a level comparable to the BFU-E response of StatSa / b mice. As a control, and for comparison, the able to recently reported hematocrits for StatSa / b-deficient colony-forming response to IL-3 is also shown and was embryos at E13.S (Socolovsky et al., 1999), with the comparable among the various strains of mice. The BFU-E exception that we rarely find individual embryos with 3159 H.Zang et al. •wr EEPOR-H .EPOR-HM 60 240 □ STAT5afb CFU-E <40 160 20 60 300 60 ,. BFU-E L4 CFU-E ,. 200 <40 100 20 15wn 0 0 --1 Fig. 4. In vitro colony formation of hematopoietic progenitors from wild-type, EpoR-H, EpoR-HM and StatSafb - mice in response to various cytokines. The numbers of colonies/10 cells formed from E12.5 fetal liver cell cultures (A) or from bone marrow cell cultures (B) are plotted. The mean and standard deviation are shown from six independent assays. The two-tailed P values for comparison of the various mutant strains with wild­ type mice or embryos are >0.01, with the exception of the BFU-E colonies for bone marrow from HM mice (P = 0.004) and of CFU-E colonies for fetal liver cells from H mice (P = 0.00006). Cells from E12.5 embryos or femurs of adult mice were prepared in a-MEM medium containing 2% FBS and counted in the presence of 3% acetic acid to lyse erythrocytes. Diluted cell suspensions and cytokines were mixed with Methocult 3230 to a final concentration of 0.9% methylcellulose. For CFU-E assay, cells were cultured in 0.2 U/ml recombinant hEpo. For BFU-E assay, cells were cultured in 3 U/ml recombinant hEpo and 10 ng/ml recombinant murine IL-3. IL-3 colony assays were performed in the presence of 10 ng/ml recombinant mIL-3. The plating conditions, cell concentration for each assay, culture conditions and colony scoring methods were exactly as described previously (Parganas et al., 1998; Teglund et al., 1998). severely decreased hematocrits. As illustrated (Figure 5D increased sensitivity to apoptosis in the absence of Epo. and E), H mutant embryos at E14.5, El5.5 and E18.5 Since no significant apoptosis was seen in fetal liver cells consistently had hematocrits that were slightly higher than of any of the mutants when examined directly from the those of wild-type embryos. Moreover, the hematocrits of embryos (data not shown), the cells were cultured for 18 h HM embryos at these ages were consistently lower than in the presence or absence of Epo. The cells were then those of wild-type embryos. In adult mice, the hematocrits examined by fluorescence-activated cell sorting (FACS) of H mutant mice were slightly higher than those of wild­ analysis for the expression of the erythroid lineage marker type mice, while the hematocrits of HM mutant mice were Ter119, and for apoptosis by TUNEL staining. As consistently lower. For comparison and for an indication illustrated (Figure 6), fetal liver cells from H or HM of the significance of the differences seen, the normal mutants cultured in the presence of 10 U/ml Epo showed hematocrits of two strains of mice are also shown no consistent differences in the levels of apoptosis, (Figure 5E; BL, C57Bl/6 and C3H/HeJ). comparable to the fetal liver cells from wild-type embyros. The above results consistently demonstrated increased When cultured in the absence of Epo, significant fractions hematocrits associated with the H mutation, whereas the of apoptotic cells were evident in cultures of wild-type HM mutation is associated with reduced levels. Since Y cells (7.1 %), slightly fewer apoptotic cells in cell cultures has been shown to be important for the activation of from H mutant embryos (5.83%) and significantly several signaling pathways, as well as Stat5a/b, we wished increased numbers of apoptotic cells in cultures from to determine whether the primary difference between the HM mutant embryos (25.5%). For comparison, typical H and HM mutations was associated with the absence of results obtained with cells from Stat5a/b-deficient Stat5a/b activation. To assess this, we crossed the H embryos are also illustrated. As above, no differences in mutation onto the Stat5a/b-deficient background, thus the numbers of apoptotic cells were evident when fetal eliminating the contribution of Stat5a/b while preserving liver cells were examined directly from E13.5 embryos. other functions requiring Y . As illustrated (Figure 5C), However, culture of the cells in the absence of Epo this resulted in reduced hematocrits that were comparable resulted in an increased degree of apoptosis in cultures of to those seen in Stat5a/b-deficient mice containing a wild­ Stat5a/b-deficient fetal liver cells comparable to that seen type receptor. The results would, therefore, suggest that in the HM mutant mice (Figure 6B). As an approach to establish additional in vivo biological the change in the hematocrits that occurs when Y is consequences of the receptor mutations, H and HM mice mutated in the context of the truncated receptor is due to the loss of the ability to activate Stat5a/b. were challenged with repeated doses of Epo, and the The distal region of the EpoR and Y have been hematocrits assessed (Figure 7). Comparable experiments implicated in the regulation of pathways that affect were not carried out with Stat5a/b-deficient mice because apoptosis. We therefore examined fetal liver cells from of the splenomegaly and extramedullary hematopoiesis mutant embryos of E13.5 for increased apoptosis or associated with the altered T-cell functions in these mice. 3160 70 The distal region and receptor tyrosines of EpoR are non-essential 21 11 N=143 N=143 ,. --- --- 12 ']: .... .... D IOI ._, IOI • 7 B . • · _J_ • t-- •. - ::I 5 ---· · D WT 6E POR-H OEPOR-HM ■ H xSTATSallr' ·- .. ··--- D .. �-------- � <>S TATSallr' N=208 N=185 60 so+- ---� .-------------< ·· -- ..... ····- .: el --- .. +- --- -------�-----, --- t 4 E-- -- E-- so+<>- � +=----��---___, i I X 20 +- ...... .__.._�+- --=- ----, - - E1 8.5 E1 4.5 E1 5.5 EM �------------------------ N=749 eo+-- ----------------------------< D STAT5alb ./-. ./+, +/+ fllli1 EPOR-H IIIE POR-HM -S TAT5alb"· xEPOR-H - C57B� and C3H/HeJ 13.5 14.5 15.5 16.5 17.5 Ul.5 19.5 N.,.bora Ad11lt Fig. 5. Hematocrits and blood cell counts of embryos or adult mice with the indicated genotypes. For adult mice (;a.4 weeks old), white blood cell numbers (A), red blood cell numbers (B) and hematocrits (C and E) were machine scored by a Hemavet 3700R counter. For embryos (D and E), blood samples were acquired using 2 µl glass micropipettes and then briefly centrifuged to measure hematocrits. N, total number of mice used for the -1 experiment. H X StatSafb - indicates mice cross-breeding from StatSa/b null mice with EpoR-H mice. (E) Numbers indicate days of gestation. The P values for all the comparisons support statistically significant differences in the hematocrits. To indicate the types of P values associated with the 3 12 results, in (C) the two-tailed P values for the means of the various mutants relative to wild type are: H, 1.3 X 10-; HM, 8.0 X 10- ; StatSa/b, 6.4 X lo-4; H/StatSa/b, 9.2 X 10-. Repeated injections of Epo (three times weekly) into wild­ essential for embryonic and adult erythropoiesis in vivo. type mice over 30 days resulted in an increasing Numerous studies have examined the properties of hematocrit during the period of injections. As illustrated, mutated EpoRs in cell lines, particularly receptors lacking this increase was enhanced in H mutant mice, consistent tyrosine residues, which would have suggested that such with the concept that the receptor functions slightly better mutations would have a dramatic phenotype in vivo. This than the wild-type receptor. In contrast, there was a lower was not the case and specifically neither the H nor HM rate of increase in hematocrits in HM mice. The results are mutations were associated with any evidence of embry­ generally consistent with the concept that the H truncation onic or adult lethality. This contrasts with deficiencies of results in a moderately more efficient receptor, while the the receptor-associated kinase Jak2 (Neubauer et al., 1998; mutation of the last tyrosine in the truncated receptor Parganas et al., 1998) or the EpoR-regulated genes Bcl-X reduces the efficiency of the receptor. (Motoyama et al., 1995) and N-myc (Moens et al., 1993; Sawai et al., 1993), all of which exhibit, like deficiency of the EpoR (Wu et al., 1995), embryonic lethal phenotypes Discussion associated with profound deficiencies in expansion of The results demonstrate the striking finding that neither the erythroid lineage progenitors with dramatically increased distal region of the EpoR nor receptor tyrosines are apoptosis in vivo. The lack of consistency with the data 3161 ) 0 . 5 2 H.Zang et al. POR- HM EPOR-H "o "o I') .., ..,o g g ;:::i "b 5.83 c... -o -0 -g U-1 0.72 0.0 1 0.87 0.0 0.0 0 0 g g 0 3 "' ° 1 2 3 4 o 1 2 3 4 0 0 0 0 0 1 1 ,ii- 10 1 1 0 1 1 0 0 1 1 10 0 o 1 1 10 0 "o "o "'o ---I .., 8 £ ..._ ,, ,I l' "' • a "' ,..... "!=! 0.3 0 . 6 2.7 1 c.., 0 -0 -� '-1,l 0 .0 0.0 0 . 84 0.0 0.36 0� 0 ° 3 4 1 4 0 1 2 3 1 0 10 !0 ° 2 0 0 0 0 10 1 103 1 1 1 0 I0 10� 1 0 ./. ... --, 5' --- 0� c., 22.6 I .:? 1 7 U. 2 0.03 2 --- - * 4 c 1 , 0 .. !< �L TUN EL ., M e 5' ..._ ... 1.72 u .u z ,ii2 I0 Fig. 6. FACS analysis of feta! liver cells at E13.5 with combined TUNEL- Terl 19 staining. Numbers in plots indicate the percentage of sorted cell population in each quadrant. The upper right quadrant in each plot represents Te rll 9-positive erythrocytes with TUNEL-positive apoptotic cells. (A) Fetal liver cells from EpoR wild-type, EpoR-H or EpoR-HM embryos were isolated at E13.5 and cultured in a.-MEM with 2% FBS for 18 h in the presence or absence of 10 U/ml recombinant hEpo. Cells were then fixed with formalin and double stained with TUNEL reagent and Ter i 19 antibody. The cell suspension was analyzed by FACScan. (B) Feta! livers from StatSa/b wild-type embryos or StatSa/b null embryos were used for TUNEL­ Terl 19 double staining. Note that the EpoR mutants were performed independently of the StatSa/b mutant studies and, consequently, should not be compared across the two experiments, but rather the mutants should be compared with their respective wild-type controls. with cell lines may reflect the need for additional were the first to suggest that the distal half of the pathways, perhaps in a cell line-dependent manner, for cytoplasmic domain negatively influenced EpoR signal­ the maintenance of the cell lines as opposed to the ing. Our results with modification of the receptor by gene transient requirement for Epo to mediate the expansion of targeting provide in vivo support to the concept that the erythroid lineage cells. distal region negatively affects receptor signaling. The first studies of mutant EpoRs indicated the exist­ Consistent with the experiments in cell lines, the activation ence of non-overlapping positive and negative regulatory of Stat5a/b was increased and prolonged in bone domains (D' Andrea et al., 1991). In particular, the studies marrow cells from H mutant mice stimulated with Epo. 3162 The distal region and receptor tyrosines of EpoR are non-essential which any differences observed with the H mutation were only evident in the homozygous state. The basis for the differences is not known, but may well reside in the �60 differences that exist between the human and murine !;.., owr receptors, and the extent of the truncations, as noted above. CH The other possibility might be mouse strain differences, 6HM although we have not seen any significant differences in H �: :: .7- 6. � .. or HM mutant mice on a C57Bl/6 or Balb/c background. Numerous studies have used receptor mutants to identify the domains and/or tyrosine in the EpoR that are 20 ---- required for the activation of signal transduction pathways. days 0 6 12 18 24 The potential role for many of these pathways was inferred Fig. 7. Response of the EpoR mutant mice to continual injections of from the activity of transfected mutated receptors or from Epo. Mice were injected three times during a week, as described in the use of dominant-negative proteins, inhibitors or Materials and methods, with recombinant hEpo. To prevent anomalous antisense reduction of protein levels. Because of this results from frequent bleeding, the mice were separated into groups, which were bled only every 14 days. The average hematocrits for two extensive body of research, it is particularly striking that a mice are indicated by the individual symbols. The deviations among distally truncated receptor containing no tyrosines was the samples for each symbol were between 2 and 5%; consequently, able to sustain erythropoiesis at the levels observed. It error bars are not included. The line presents the linear regression of must be concluded that either the activation of the the data points for each of the mutant strains. pathways studied is not essential for receptor function or that there is some type of selection for compensating functions that is occurring in vivo and that is sufficiently Unfortunately, due to the quantity of cells required and the efficient to completely obviate the role of the receptor heterogeneous nature of bone marrow cells, we have been tyrosines. Unfortunately, in most cases, it is not possible to unable to assess the Jak2 activation. Also consistent with a determine whether signal transduction pathways are still negative role of the distal region in regulating signaling, activated in cells from HM mutant mice because of the the hematocrits of H mutant mice were modestly increased requirement for large quantities of homogeneous cells. We and there was an increased response to repeated injections are currently breeding the HM mutation on a background of Epo. However, the effects were not as striking as might that is susceptible to Friend virus disease, as a way of have been predicted based on the mutant receptor studies obtaining large quantities of erythroid lineage cells for or based on the mutations of the distal region of the EpoR further biochemical studies (Koury et al., 1984). Using in humans (De La Chapelle et al., 1993; Sokol et al., 1995; these mice, we will also be able to evaluate the cell surface Arcasoy et al., 1997). In particular, individuals who are expression patterns of the receptor mutants, which has not heterozygous for a truncation of 70 amino acids were been possible with bone marrow cells. However, we would reported to have an erythrocytosis in which hemoglobin not expect that the expression of the mutants would be levels were 1.33-fold higher than controls. In contrast, different since the gene modifications are quite distant mice homozygous for a truncation of 108 amino acids from the promoter of the gene. Moreover, extensive have hemoglobin levels that are only 1.08-fold those of studies with these mutants in transduced cell lines have controls (data not shown). Lastly, it should be noted that consistently shown that, when expressed comparably, they whereas the erythrocytosis associated with the human are processed and expressed at the cell surface in receptor mutations has been observed with individuals comparable patterns. heterozygous for the receptor mutations, mice hetero­ In contrast to most of the signaling pathways, the zygous for the H mutation display no increase in sensitivity of EMSA allowed us to assess the ability of H hematocrits (data not shown). One possible basis for the and HM mice to recruit and activate Stat5a/b, for which difference is the extent of the truncations. In particular, the Y is known to be necessary and sufficient for its human truncations retain an additional tyrosine and this activation. The results demonstrate that the strong activa­ may provide an additional positive signal that contributes tion of Stat5a/b that is seen in H mutant mice is completely to the overall larger positive response. eliminated in HM mutant mice. Thus, HM mice have not In a recent study (Divoky et al., 2001), mutant mouse activated a compensating pathway that either allows Epo strains were created in which the endogenous murine to activate Stat5a/b or results in a constitutive level of EpoR gene was replaced with either the wild-type human Stat5a/b activation comparable to that seen in Epo­ EpoR gene or a carboxyl-truncated human EpoR gene as a stimulated cells. The HM mutation thus renders the mice model for the human primary familial congenital poly­ specifically deficient in Stat5a/b function within the cythemia associated with receptor truncations. Consistent erythroid lineage, and allows a potentially more precise with our studies with the H strain of mice, no loss of analysis of Stat5a/b function in erythroid cells than viability was observed. Also consistent with our results complete deletion of the gene because of the critical role was the lack of striking changes in BFU-E or CFU-E. that Stat5a/b play in other lineages of cells and particularly However, the extent of erythrocytosis, as assessed with in T cells (Teglund et al., 1998; Moriggl et al., 1999). hematocrits, was considerably greater than that seen in our Based on the published properties of erythropoiesis in H strain of mice. Moreover, erythrocytosis was evident in Stat5a/b-deficient embryos from the strain of mice we heterozygous animals for the truncation in combination produced (Socolovsky et al., 1999), we had anticipated a with either the wild-type murine endogenous gene or the dramatic phenotype of severe fetal anemia in HM mice. wild-type human gene. This is in contrast to our results in However, many of the phenotypes that were reported were 3163 H.Zang et al. and Hindlll digestion utilizing a Hindlll site on exon 8 and a newly not evident in our studies of Stat5a/b-deficient embryos. In created Hindlll site was followed. For Y F mutation, a 0. 8 kb Hindlll particular, HM mutant embryos, like Stat5a/b-deficient fragment utilizing Hindlll site on intron 6 and exon 8 was subcloned into embryos, were morphologically indistinguishable from pBluescript. Site-directed mutagenesis was performed as described in the wild-type embryos. Moreover, no embryonic lethality was manufactu re r's protocol (Qiagen) and the plasmid containing the correct mutation was used for the construction of HM targeting vector. For evident in either HM mutant or Stat5a/b-deficient negative selection, a diphtheria toxin A gene (DTA) driven by the embryos. The frequencies of BFU-E and CFU-E were polyoma virus enhancer cassette (Adachi et al., 1995) was inserted in the comparable among wild-type, HM mutant and Stat5a/b­ 5' end of the targeting construct. A thymidine kinase promoter-driven neo deficient embryos. Lastly, there was no apoptosis in fetal cassette was inserted after the stop codon of exon 8 to confer positive livers of HM mutant or Stat5a/b-deficient embryos, selection. consistent with the absence of apoptosis in fetal livers of Transfection of ES cells and generation of EpoR-H or wild-type embryos. The basis for the differences in the EpoR-HM mice results is yet to be resolved. W9.5 ES cells and El4 (129/0la mouse strain) ES cells were cultured as Consistent with the previous studies, however previously described (Parganas et al., 1998). W9.5 ES cells were used for the generation of EpoR-H mice and El 4 cells were used for the generation (Socolovsky et al., 1999), Stat5a/b-deficient embryos of EpoR-HM mice. Twenty-five micrograms of Sall-linearized EpoR-H had hematocrits that were reduced relative to those of or EpoR-HM plasmid construct were electroporated into the W9.5 or El 4 wild-type mice throughout embryonic development and as ES cells. Twenty-four hours after electroporation, selection was adults. The differences are comparable to that seen performed in 350 µg/ml geneticin (G418; Gibco-BRL). Conditions for between different strains of mice. Importantly, a compar­ blastocyst injection of correctly targeted and karyotypically normal ES clones, and breeding to generate mice homozygous for the mutated EpoR able reduction in hematocrits was seen in HM mutant gene, were performed essentially as described (van Deursen et al., 1993). embryos and adults. The possibility that the reduction seen ES clones were injected into C57Bl/6 blastocysts, and five clones from in HM mutants is due to the loss of Stat5a/b function is EpoR-H targeting and three clones from EpoR-HM targeting gave supported by the observation that the increased hemato­ germline transmission. For PCR analysis, mice tails were clipped and digested overnight in lysis buffer (500 mM KCI, 100 mM Tris pH 8.3, 0.1 crits seen in H mutant embryos are reduced to that of mg/ml gelatin, 1 % NP-40, 1 % Tween-20, 500 µg/ml proteinase K). PCR Stat5a/b-deficient or HM mutant embryos when crossed was carried out with primer a (5'-GAGTTTGAGGGTCTCTTCACC-3') onto the Stat5a/b-deficient background. The Stat5a/b in exon 7 and primer b (5' -TAGGCTGGAGTCCTAGGAGC-3') in exon 8 functions that mediate the 10--30% reduction in hemato­ of the EpoR gene. Since primer b corresponds to a DNA sequence at the crits are unclear from studies of erythroid progenitors beginning of the stop codon in exon 8 and extends into the following non­ coding 3' sequences that are retained in the truncation, the 442 bp in vivo. However, fetal liver cells from both HM and fragment is characteristically derived from the mutant containing the Stat5a/b-deficient embryos are more susceptible to apop­ truncation, while the wild-type allele gives rise to a 760 bp fragment. For tosis when cultured in the absence of Epo than are wild­ EpoR-HM mice, the PCR product was sequenced to confirm HM type or H mutant fetal liver cells. It has been proposed that mutation. For Southern blot analysis, EcoRI- Xhol digestion was performed on isolated EpoR genomic DNA, and the 0.8 kbp fragment Stat5a/b is required for the Epo-induced expression of comprising exon 2 and an intron between exon 1 and exon 2 was used as a Bcl-X (Socolovsky et al., 1999); however, when exam­ probe. Genomic DNA from mouse tail was digested by EcoRI and, after ined, the levels of Bcl-X protein in fetal liver erythroid probing, the mutant allele shows a 4.5 kb fragment and the wild-type cells are not detectably altered in Stat5a/b-deficient allele shows an ~12 kb fragment. For RT-PCR analysis, total cellular embryos (Marine et al., 1999). RNA was extracted from bone marrow cells of adult mice (;a,,4 weeks) using RNAzol B (Tel-Test). First-strand cDNA synthesis using oligo(dn In summary, while it might have been anticipated that primer was carried out as described in the manufactur er's protocol the creation of mouse strains containing a severely (Gibco-BRL Superscript reverse transcriptase). PCR was performed truncated EpoR, which lacks any tyrosine residues, using PCR primers a and b for the EpoR gene, and �-actin primers might have resulted in striking consequences to erythro­ (5'-GTGACGAGGCCCAGAGCAAGAG-3', 5'-AGGGGCCGGACT­ CATCGTACTC-3') for the �-actin gene. poiesis, this was not the case. The results support the concept that many of the signal transducing events that EMSA have been well characterized with the use of mutant Bone marrow cells (8.0 X 10 ) were collected from femurs of wild-type, receptors in cell lines may have limited significance in EpoR-H and EpoR-HM mice. Cells were suspended in a-MEM medium defining the critical events in EpoR signaling. The with 2% fetal calf serum, 1 % L-glutarnine and appropriate amounts of antibiotics. Cells were then starved for 4 h and stimulated with 50 U/ml availability of these mutant strains of mice will be recombinant human Epo (hEpo; Amgen) or 10 ng/ml recombinant murine particularly useful in allowing studies to focus on a more IL-3 (mIL-3; R & D systems) for 30 min or 3 h. Whole-cell extract was restricted set of signal transducing pathways, retained by prepared by incubation in extraction buffer [50 mM Tris pH 8.0, 50 mM the mutant receptors, in more physiologically relevant NaCl, 0.1 mM EDTA, 0.5 mM NaV0 , 0.5% NP-40, 50 mM NaF, 10% 3 4 glycerol, 20 mM �-mercaptoethanol, 1 mM dithiothreitol (DTn, 0.4 mM in vivo studies. The availability of these mutant strains will phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail] for also allow examination of the possibility that the distal 30 min at 4C. For Stat5 DNA binding sequence, sense and antisense region of the receptor is required in very specific situations oligonucleotides representing the Stat5a/b site in �-casein promoter to modify or amplify a 'c ore ' signal. (5'-AGATTTCTAGGAATTCAATCC-3') were synthesized, annealed to make double-stranded DNA and labeled with [y- P]ATP. Ten micro­ grams of cell extract were incubated in binding buffer [10 mM Tris, 50 mM NaCl, 0.1 mM EDTA, 0.2 mM PMSF, 1 mM DTT, 0.1 % NP-40, Materials and methods 5% glycerol, 2 µg poly(dl-dC), 30 µg/ml bovine serum albumin] for Construction of targeting vector 30 min at room temperature, and the reaction mixtures were separated on The EpoR gene was isolated from the El4 (129/0la mouse strain) 4% native TBE gel. genomic library in EMBL3 using EpoR full-length cDNA as a probe. Positive clones were restriction mapped and sequenced. Briefly, a Xhol Colony assay fragment including intron 2-exon 8 of the EpoR gene was subcloned into Petal liver cells were prepared from livers of E12.5 embryos, and bone pBluescript. For truncation of the receptor, a Hindlll site was introduced marrow cells from femurs of wild-type, EpoR-H, EpoR-HM and --1- just upstream of the stop codon by site-directed mutagenesis (5'­ StatSafb mice. The cells were plated and the colonies scored exactly TATGTGGCCTGCTCCTAG-3' to 5'-TATGAAGCTTGCTCCTAG-3'), as described previously (Parganas et al., 19 98; Teglund et al., 1998). 3164 The distal region and receptor tyrosines of EpoR are non-essential Blood cell counts and hematocrits D' Andrea,A.D., Yoshimura,A., Youssoufian,H., Zon,L.I., Koo,J.W. and For adult mice, blood samples were taken from orbital sinus using a Lodish,H.F. (1991) The cytoplasmic region of the erythropoietin microcapillary tube. Twenty microliters of sample blood were used for receptor contains nonoverlapping positive and negative growth­ analysis in a MASCOT Hemavet 3700R counter (CDC Technologies) and regulatory domains. Mol. Cell. Biol., 11, 1980-1987. scored for white blood cells and red blood cells. Hematocrits were De La Chapelle,A., Traskelin,A.-L. and Juvonen,E. (1993) Truncated calculated by Hemavet counter based on red blood cell counts and mean erythropoietin receptor causes dominantly inherited benigu human corpuscular volume. For embryos, blood samples were taken from the erythrocytosis. Proc. Natl Ac ad. Sci. USA , 90, 4495-4499. carotid artery using heparinized 2 µ1 microcapillary tubes. Tubes were Divoky,V., Liu,Z., Ryan,T.M., Prchal,J.F., Townes,T.M. and Prchal,J.T. sealed at one end and centrifuged for 5 min at 3000 r.p.m. to measure (2001) Mouse model of congenital polycythemia: homologous hematocrits. replacement of murine gene by mutant human erythropoietin receptor gene. Proc. Natl Acad. Sci. USA, 98, 986-991. Flow cytometry and TUNEL staining Gobert,S. et al. (1996) Identification of tyrosine residues within the E13.5 fetal livers were isolated from embryos, and made into single-cell intracellular domain of the erythropoietin receptor crucial for ST AT5 suspension by passing them through a G25 needle. Cells were cultured in activation. EMB O J. , 15, 2434-2441. cx-MEM medium with 2% fetal bovine serum (FBS) for 18 h. For Gregory,R.C., Jiang,N., Todokoro,K., Crouse,J., Pacifici,R.E. and Epo stimulation, recombinant hEpo was added to a final concentration of Wojchowski,D.M. (1998) Erythropoietin receptor and STAT5- 10 U/ml in culture medium. After culture, cells were centrifuged and specific pathways promote SKT6 cell hemoglobinization. Blood, 92, washed with phosphate-buffered saline (PBS) containing 1 % FBS. Cells 1104-1118. were then centrifuged for 5 min at 1000 g and PBS decanted, followed by Iwatsuki,K., Endo,T., Misawa,H., Yokouchi,M., Matsumoto,A., fixation with formalin for 20 min at room temperature and subsequent Ohtsubo,M., Mori,K.J. and Yoshimura,A. (1997) STAT5 activation treatment with 0.1% Triton X-100 and 0.1% sodium citrate. Cells were correlates with erythropoietin receptor-mediated erythroid placed on ice for 2 min and washed with PBS. TUNEL reaction was differentiation of an erythroleukemia cell line. J. Biol. Chem, 272, performed as described in the manufacturer's protocol (ln situ cell death 8149-8152. detection kit, Fluorescein; Boehringer Manheim) and, after washing with Jiao,H., Berrada,K., Yang,W., Tabrizi,M., Platanias,L.C. and Yi,T. PBS, Ter119 antibody was added, followed by incubation on ice for an (1996) Direct association and dephosphorylation of Jak2 kinase by hour and several washing with PBS. The cell suspension was then SH2 domain-containing protein tyrosine phosphatase SHP-1. Mol. analyzed in a Becton-Dickinson FACScan using CellQuest software. Cell. Biol., 16, 6985--6992. Klingmuller,U., Lorenz,U., Cantley,L.C., Neel,B.G. and Lodish,H.F. Epo challenge analysis (1995) Specific recruitment of the hematopoietic protein tyrosine A total of 30 mice were selected for the experiment. The selection was phosphatase SH-PTPl to the erythropoietin receptor causes gender balanced and mice of various ages were included. Epo (500 U/kg inactivation of JAK2 and termination of proliferative signals. Cell, body weight) was injected into all 30 mice three times a week. Blood 80, 729- 738. samples were drawn between injections and complete blood counts were Klingmuller,U., Wu,H., Hsiao,J.G., Toker,A., Duckworth,B.C., Cantley, performed. Hematocrits were monitored for 4 weeks. To prevent any L.C. and Lodish,H.F. (1997) Identification of a novel pathway anomaly in the experimental results due to frequent bleeding, mice were important for proliferation and differentiation of primary erythroid separated into five groups and scheduled to take turns for bleeding. progenitors. Proc. Natl Acad. Sci. USA, 94, 3016-3021. A group of six mice consisted of two mice from each genotype: wild type, Koury,M.J., Sawyer,S.T. and Bondurant,M.C. (1984) Splenic EpoR-H and EpoR-HM. erythroblasts in anemia-inducing Friend disease: a source of cells for studies of erythropoietin-mediated differentiation. J. Cell Physiol., 121, 526-532. Ackn owledgements Krantz,S.B. (1991) Erythropoietin. Blood, 77, 419-434. Longmore,G.D., You,Y., Molden,J., Liu,K.D., Mikami,A., Lai,S.Y., We would like to thank Linda Snyder, Kristen Rothammer and Melanie Pharr,P. and Goldsmith,M.A. (1998) Redundant and selective roles for Loyd for technical support; Drs John Cleveland, Gerry Zambetti and erythropoietin receptor tyrosines in erythropoiesis in vivo. Blood, 91, Nick Carpino for helpful discussion; and Richard Morrigl for helping 870-878. with HCT analysis of Stat5a/b embryos. This work was supported by the Marine,J.C. et al. (1999) SOCS3 is essential in the regulation of fetal Cancer Center CORE grant CA21765, by grants ROl DK42932 and POl liver erythropoiesis. Cell, 98, 617--627. HL53749, and by the American Lebanese Syrian Associated Charities Miller,B.A., Barber,D.L., Bell,L.L., Beattie,B.K., Zhang,M.Y., Neel, (ALSAC). B.G., Yoakim,M., Rothblum,L.I. and Cheung,J.Y. (1999) Identification of the erythropoietin receptor domain required for calcium channel activation. J. Biol. Chem. , 274, 20465-20472. Referen ces Moens,C.B., Stanton,B.R., Parada,L.F. and Rossant,J. (1993) Defects in heart and lung development in compound heterozygotes for two Adachi,M., Suematsu,S., Kondo,T., Ogasawara,J., Tanaka,T., Yoshida,N. different targeted mutations at the N-myc locus. Development, 119, and Nagata,S. (1995) Targeted mutation in the Fas gene causes 485-499. hyperplasia in peripheral lymphoid organs and liver. Natu re Genet., 11, Moriggl,R. et al. (1999) Stat5 is required for IL-2 induced cell cycle 294-300. progression of peripheral T cells. Imm unity, 10, 249-259. Arcasoy,M.O., Degar,B.A., Harris,K.W. and Forget,B.G. (1997) Familial Motoyama,N. et al. (1995) Massive cell death of immature erythrocytosis associated with a short deletion in the erythropoietin hematopoietic cells and neurons in Bcl-x-deficient mice. Science, receptor gene. Blood, 89, 4628-4635. 267, 1506-1510. Bergelson,S., Klingmuller,U., Socolovsky,M., Hsiao,J.G. and Lodish, Neubauer,H., Cumano,A., Muller,M., Wu,H., Huffstadt,U. and H.F. (1998) Tyrosine residues within the intracellular domain of the Pfeff er,K. (1998) Jak2 deficiency defines an essential developmental erythropoietin receptor mediate activation of AP-1 transcription checkpoint in definitive hematopoiesis. Cell, 93, 397-409. factors. J. Biol. Chem, 273, 2396-2401. Parganas,E. et al. (1998) Jak2 is essential for signaling through a variety Chin,H., Arai,A., Wakao,H., Kamiyama,R., Miyasaka,N. and Miura,O. of cytokine receptors. Cell, 93, 385-395. (1998) Lyn physically associates with the erythropoietin receptor and Quelle,F.W., Wang,D., Nosaka,T., Thierfelder,W.E., Stravopodis ,D., may play a role in activation of the Stat5 pathway. Blood, 91, Weinstein,Y. and Ihle,J.N. (1996) Erythropoietin induces activation of 3734-3745. Stat5 through association with specific tyrosines on the receptor that Damen,J.E., Wakao,H., Miyajima,A., Krosl,J., Humphries,R.K., Cutler, are not required for a mitogenic response. Mol. Cell. Biol., 16, R.L. and Krystal,G. (1995a) Tyrosine 343 in the erythropoietin 1622-1631. receptor positively regulates erythropoietin-induced cell proliferation Sawai,S., Shimono,A., Wakamatsu,Y., Palmes,C., Hanaoka,K. and and Stat5 activation. EMBO J. , 14, 5557-5568. Damen,J.E., Cutler,R.L., Jiao,H., Yi,T. and Krystal,G. (1995b) Kondoh,H. (1993) Defects of embryonic organogenesis resulting Phosphorylation of Y in the erythropoietin receptor (EpR) is from targeted disruption of the N-myc gene in the mouse. essential for binding the p85 subunit of phosphatidylinositol (PI) 3- Development, 117, 1445-1455. kinase and for EpR associated PI 3-kinase activity. J. Biol. Chem, Shultz,L.D., Schweitzer,P.A., Rajan,T.V., Yi,T., Ihle,J.N., Matthews, 270, 23402-23408. R.J., Thomas,M.L. and Beier,D.R. (1993) Mutations at the murine 3165 H.Zang et al. motheaten locus are within the hematopoietic cell protein tyrosine phosphatase (Hcph) gene. Cell, 73, 1445-1454. Socolovsk:y,M., Fallon,A.E., Wang,S., Brugnara,C. and Lodish,H.F. (19 99) Fetal anemia and apoptosis of red cell progenitors in --f- 1 Stat5a 5b-- mice: a direct role for Stat5 in Bcl-X(L) induction. Cell, 98, 181 -191. Sokol,L., Luhovy,M., Guan,Y., Prchal,J.F., Semenza,G.L. and Prchal,J.T. (19 95) Primary familial polycythemia: a frameshift mutation in the erythropoietin receptor gene and increased sensitivity of erythroid progenitors to erythropoietin. Blood, 86, 15 -22. Tauch i,T., Damen,J.E., Toyarna,K., Feng,G.S., Broxmeyer,H.E. and Krystal,G. (1 996) Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis. Blood, 87, 4495-4501. Teglun d,S. et al. (19 98) Stat5a and Stat5b proteins have essential and non-essential, or redundant, roles in cytokine responses. Cell, 93, 841-850. van Deursen,J. , Heerschap,A., Oerlemans,F., Ruitenbeek,W., Jap,P., ter Laak,H. and Wieringa,B . (19 93) Skeletal muscles of mice deficient in muscle creatine kinase lack burst activity. Cell, 74, 621- 63 1. Wickrema,A. et al. (19 99) Engagement of Gabl and Gab2 in erythropoietin signaling. J. Biol. Chem, 274, 24469-24474. Witthuhn,B ., Quelle,F.W., Silvennoinen,O., Yi,T ., Tan g,B ., Miura,O. and Thle,J.N. (19 93) JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following EPO stimulation. Cell, 74, 227-236. Wu,H ., Liu,X., Jaenisch,R. and Lodish,H.F. (19 95) Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell, 83, 59-67. Yi,T ., Zhang,J., Miura,O. and Ihle,J.N. (19 95) Hematopoietic cell phosphatase (HCP) associates with the erythropoietin receptor following Epo induced receptor tyrosine phosphorylation: identification of potential binding sites. Blood, 85, 87-95. Received February 23, 2001; revised and acce pted April 26, 2001

Journal

The EMBO Journal – Springer Journals

Published: Jun 15, 2001

Keywords: cytokine signaling; erythropoiesis; erythropoietin; Jak2 kinase; receptor

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