Mitogenic Signaling and Inhibition of Apoptosis via the Erythropoietin Receptor Box-1 Domain*

(Received for publication, May 25, 1996, and in revised form, February 10, 1997)

Bhavana Joneja and Don M. Wojchowski Dagger

From the Graduate Program in Biochemistry and Molecular Biology, Center for Gene Regulation and the Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Studies of proliferative signaling via type 1 cytokine receptors have revealed a three-step activation mechanism. Cytokine-induced receptor dimerization mediates the trans-phosphorylation of Jak kinases, Jaks phosphorylate receptors at tyrosine sites, and SH2 domain-encoding effectors then are recruited to these sites. Signaling factors that associate with activated erythropoietin (Epo) receptor complexes include phospholipase C-gamma , phosphatidylinositol 3-kinase, SHIP, Shc, Grb2, Cbl, Crk-l, HCP, Syp, and STAT5. While at least certain of these factors modulate proliferative signaling, mutated Epo receptor forms lacking Tyr(P) sites retain substantial mitogenic activity. Presently we show that a highly truncated Epo receptor form that retains box-1, yet lacks the conserved box-2 domain (and all Tyr(P) sites) nonetheless effectively promotes mitogenesis, survival, and Myc and Pim-1 expression. In addition, mitogenesis and Myc expression are shown to be supported by a direct Epo receptor-Jak2 kinase domain chimera. Thus, Epo-dependent mitogenesis and inhibition of apoptosis each depend critically upon only the Epo receptor box-1 domain, with no essential role exerted in these response pathways by the box-2 domain.


INTRODUCTION

Primary regulation over hematopoietic cell growth is exerted through cytokine activation of receptors of the type 1 superfamily. Included in this family are the receptors for Epo,1 interleukins-2, -3, -4, -5, -7, and -9, ciliary neurotrophic factor, oncostatin M, prolactin, growth hormone, G-CSF, granulocyte-macrophage-colony stimulating factor, and thrombopoietin (1, 2). Within extracellular domains of these receptors, co-distributed cysteine residues, predicted paired seven-fold alpha  helical domains, and an intervening WSXWS motif comprise the most highly conserved structural features. In addition, within cytoplasmic domains a membrane-proximal eight-amino acid box-1 motif typically is represented, and in at least certain receptors (Epo, IL-2, IL-3, G-CSF, growth hormone, prolactin receptors, for example) an adjacent 10-amino acid box-2 motif is discernible (3-10). Functionally, type 1 receptors possess no known enzymatic activities. However, delimited receptor domains containing these box motifs have been shown to mediate the recruitment of one or more of the Janus-type protein-tyrosine kinases Jak-1, -2, and -3 and Tyk2 (11, 12). In this receptor family, the catalytic activation of Janus kinases is thought to be driven by trans-phosphorylation as mediated by ligand-induced receptor oligomerization. The activation of primary signaling factors then proceeds from the phosphorylation of receptor tyrosine residues, to the recruitment of an increasingly complex set of SH2 domain-encoding effectors. In our laboratory, work has focused primarily on signaling in the Epo receptor system. This type 1 cytokine receptor comprises an attractive minimal model in that unlike many related receptors the Epo receptor apparently exists as a single transmembrane subunit (13), is activated via simple homodimerization (14), and activates a single member of the Jak kinase family, Jak2 (15, 16). Moreover, disruption of Epo receptor gene expression in chimeric mice recently has provided evidence that the physiological role for the Epo receptor apparently is also strictly defined, and is restricted in its essential activities to supporting the development of late erythroid progenitor cells (17). By comparison, cellular response pathways that are activated by Epo are diverse, and important roles for Epo have been demonstrated in the mitogenesis, survival, and terminal differentiation of erythroid progenitor cells (18). To date, the majority of studies of Epo receptor signaling have focused on mitogenesis. This is largely due to the practical consideration that it is relatively straightforward to reconstitute this signaling pathway in stably transfected IL-3-dependent murine lymphoid and myeloid cell lines. Studies in these systems have revealed that eight cytoplasmic tyrosine motifs in the murine Epo receptor function to recruit and/or facilitate the tyrosine phosphorylation of the following set of SH2 domain-encoding effectors: p95vav (19), phospholipase C-gamma (20), p85 (p110), phosphatidylinositol 3-kinase (21), SHIP (22), Grb2, Shc (23), Cbl (24), Crk-l (24), Cis (25), HCP (26), Syp (27), and STAT5 (and possibly STATs 1 and 3) (28, 29). In addition, activation of a mSos1, Ras, Raf-1 pathway to MAP kinases has been described (30), and Epo stimulation of the transcription of myc, oncostatin-M, cis, and fos genes has been demonstrated (31, 32). Finally, Epo-induced serine phosphorylation of Bcl2 (33) and the induced expression of the cytosolic serine-threonine kinase Pim-1 (19) have been demonstrated.

Despite this above progress in identifying Epo-activated transducing factors, for the majority of these factors the extent to which their activation is important for Epo-stimulated proliferative signaling remains a central, unresolved issue. This point is underlined by studies from several laboratories, which recently have demonstrated mitogenic signaling by truncated and/or (phospho)tyrosine-deficient Epo receptor forms (2, 21, 28, 29). Work from our laboratory, for example, has assisted in mapping essential membrane-proximal cytoplasmic domains (34), has defined the box-1 domain as a specific subdomain for Jak2 activation (2), and has evidenced a requirement for Jak2 kinase activation in Epo-stimulated proliferation (16, 34, 35). Presently, to more directly assess roles in this process for Jak2 kinase activation, the abilities of two novel minimal Epo receptor forms to mediate Epo-dependent mitogenesis and inhibition of apoptosis have been studied. These include a highly truncated form that retains box-1, but lacks the conserved box-2 domain and all distal residues (ER-Bx1), and a form in which the extracellular and trans-membrane domains of the Epo receptor are fused directly to the PTK-like and PTK domains of Jak2 (ER-J2KK). Notably, ER-Bx1 is shown to efficiently support Epo-induced mitogenesis, inhibition of apoptosis, and the expression of Myc and Pim-1. The chimera ER-J2KK likewise was active in supporting Epo-stimulated mitogenesis and myc gene expression, and detectably mediated the induced expression of Pim-1 protein. Findings are discussed in the context of Epo receptor minimal subdomains (and associated effectors), which are essential for the activation of mitogenic and survival response pathways.


MATERIALS AND METHODS

FDCP1 and Derived Cell Lines

IL-3-dependent murine myeloid FDCP1 cells have been characterized previously as a model for studies of Epo receptor function (2, 9, 16, 34, 35). The subline used (FDC-WEHI2, or FDC2) has low potential for transformation to factor-independent growth. FDC2 and derived lines were maintained at 37 °C, 5% CO2 in 8% FBS, 10 µM 2-mercaptoethanol, Opti-MEM medium supplemented with 3% conditioned medium from WEHI-3B cells as a source of IL-3 (36). FDC2 lines ectopically expressing select Epo receptor forms (see below) were prepared by stable co-electrotransfection with pXM expression vector constructs (50 mg) and pCINeo (2.5 mg) as described previously (34). Polyclonal lines were established by selection in G418 (1 mg/ml) and Epo (50 units/ml).

Epo Receptor Constructs

Epo receptor forms studied include: the murine wt Epo receptor (13), ER-396, a carboxyl-terminal deletion mutant truncated at Met396 (34) (i.e. residue 372 of the mature Epo receptor), ER-Bx1, a form truncated to delete the box-2 subdomain (and all distal cytoplasmic residues), and ER-J2KK, a chimeric construct in which the extracellular and transmembrane domains of the Epo receptor (Met1-Lys280) were fused directly to the PTK and kinase-like domains (Glu775-Val1160) of murine Jak2 kinase (15). Expression of wild-type and ER-396 constructs (prepared by Exo III-mediated mutagenesis) has been described previously (34). ER-Bx1 and ER-J2KK receptor forms were constructed as follows. For ER-Bx1, an XbaI site was introduced by PCR (CACCTAGAGCATCTAGAG; His328-Leu329-Glu330) immediately preceding the first leucine of the box-2 domain. A stop codon then was introduced at this position using an XbaI amber stop cassette (Pharmacia Biotech Inc.). For ER-J2KK, a 2000-base pair fragment of a murine Jak2 cDNA (16) was ligated to a natural BglII site within a murine Epo receptor cDNA. Each cDNA construct then was cloned into a modified pXM expression vector. Levels of Epo receptor construct expression in stably transfected FDC2 lines were assayed using 125I-Epo (324 Ci/mmol, Amersham) in equilibrium binding assays as described previously (37).

Assays of Cytokine-induced Proliferation

Cytokine-induced mitogenesis in FDC2 and derived cell lines was assayed based on rates of stimulated reduction of the tetrazolium compound MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-2H-tetrazolium)) to formazan (Promega) (2), or on rates of stimulated incorporation of [3H]thymidine (34). Cells (3 × 105 cells/ml, 50 ml/well, 96-well plate) were exposed to cytokines (50 ml) for 48 h (37 °C, 5% CO2). MTS and phenazine methosulfate were added, and at 2 h of incubation absorbance (A490) was measured (microplate reader, model 550, Bio-Rad). In [3H]dT incorporation assays, cells were plated (2 × 104 cells/well) exposed to cytokines (48 h) and incubated with [3H]thymidine (1 mCi) for 2 h. Scintillation counting of harvested cells was performed using a 1205 Betaplate counter (Pharmacia).

Jak2 Activation Assays

FDC2-derived cell lines (at 8 × 105 cells/ml) were washed in Opti-MEM and incubated for 12 h at 37 °C, 5% CO2 in medium containing 1% fetal calf serum. Na3VO4 (500 mM) then was added for a 20-min period, followed by Epo (± 50 units/ml, 8 min). Cells were chilled to 0 °C, washed once with Opti-MEM, and lysed in 0.2 ml of lysis buffer (1.1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, 1 mM Na2EDTA, 0.02% NaDOC, 1 mM Na3VO4, 2 mM NaF, pH 7.5, 0.5 mg/ml leupeptin, 0.7 mg/ml pepstatin, 50 mg/ml phenylmethylsulfonyl fluoride). Cleared lysates were incubated with Jak2 antiserum (1.5 h, 4 °C) (Upstate Biotechnology, Inc. (UBI), Lake Placid, NY) and with protein A-Sepharose CL4B (90 min, 4 °C). Immune complexes were washed twice with wash buffer (lysis buffer containing Nonidet P-40 at 0.55%) eluted in sample buffer (3.4% SDS, 0.2 M dithiothreitol, 0.05 mM bromphenol blue, 10% glycerol, and 0.12 M Tris-HCl, pH 6.8 (100 °C, 5 min)), electrophoresed (7.5% acrylamide, 0.2% bisacrylamide SDS gels), and transferred to Nitro-Plus membranes (MSI, Westboro, MA). Membranes were blocked (1% powdered milk, 3% BSA, 0.1% Tween 20, in 10 µM Tris base, 150 mM NaCl, 0.05% Tween 20) and probed with phosphotyrosine antibody 4G10 (UBI). Complexes were detected by ECL (Amersham) (38). For reprobing, membranes were stripped in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7 (50 °C, 20 min).

Western Blotting of Bcl2, Pim-1, and Epo Receptor Forms in FDC2-derived Cell Lines

In analyses of Bcl2 and Pim-1 expression, cell lines were allowed to accumulate in G1 phase by incubating exponentially growing cells (8 × 105 cells/ml) in Opti-MEM, 1% FBS, 10 µM 2-mercaptoethanol in the absence of IL-3 or Epo for 9 h. Cells then were exposed to Epo, and at 0, 3, and 9 h of exposure, lysates were prepared for Western blotting. Briefly, cells were collected, washed once in Opti-MEM, and lysed in RIPA buffer (1% Nonidet P-40, 0.5% NaDOC, 0.1% SDS, 0.5 mg/ml leupeptin, 0.7 mg/ml pepstatin, 50 mg/ml phenylmethylsulfonyl fluoride, in 145 mM NaCl, 12 mM Na2HPO4, 2.5 mM NaH2PO4, pH 7.2). Electrophoresis was in 12% acrylamide, 0.3% methylene bisacrylamide SDS gels. Antisera to Bcl2 (Santa Cruz Biotechnology) and Pim-1 (see "Acknowledgments") were used at 0.1 and 2.5 mg/ml, respectively. Antibody-antigen complexes were detected using a horseradish peroxidase-conjugated secondary antibody and ECL (38). Western blotting of Epo receptor constructs was performed as described previously (34).

Apoptosis-associated Nucleosomal Fragmentation of DNA

In assays of apoptosis-associated DNA fragmentation, exponentially growing FDC-WEHI2-derived cell lines (6-9 × 105 cell/ml) were washed twice in Opti-MEM and resuspended in pre-equilibrated Opti-MEM, 1% fetal calf serum, 10 µM 2-mercaptoethanol at 8 × 105 cells/ml. Cytokines (IL-3, Epo, SCF) then were added as indicated. At 7 h, DNA was isolated using a Trevigel (Gaithersburg, MD) extraction system. Inter-nucleosomally fragmented DNA was then labeled using [32P]GATP (800 Ci/mmol) and DNA polymerase I (Klenow fragment) (5-min reaction, 23 °C). Reactions were terminated by the addition of Na2EDTA (5 mM) and heating (15 min at 70 °C). Products were electrophoresed on a 1.4% TrevigelTM 500 agarose gel in TAE (4 mM Tris, 2 mM NaC2H3O2, 1 mM Na2EDTA, pH 7.8) and were transferred to Nytran membranes. Fragmented DNA was analyzed by autoradiography, and quantitatively by phosphorimaging.

Northern Blotting

RNA was isolated from FDC2 and derived cell lines by the method of Chomczynski and Sacchi (39). Total RNA (20 µg/sample) was electrophoresed in denaturing gels (3% formaldehyde) and blotted to nylon membranes. Probes used in hybridizations were labeled by random priming using the Klenow fragment of DNA polymerase I and [32P]alpha -dATP (3000 Ci/mmol). These included full-length cDNAs for murine Pim-1 (40), c-Myc, and glyceraldehyde-3-phosphate dehydrogenase.


RESULTS

Epo Receptor Constructs and Mitogenic Activities

In initial studies, two novel Epo receptor constructs were developed toward advancing an understanding of possible direct contributions of Jak2 kinase to Epo-dependent mitogenesis and inhibition of apoptosis. These included ER-Bx1, a form in which the conserved box-2 subdomain (and all distal carboxyl residues) of the Epo receptor were deleted, and ER-J2KK, a form in which the extracellular and transmembrane domains of the Epo receptor were fused directly to the kinase-like and PTK subdomains of murine Jak2 kinase. These receptor forms are diagramed in Fig. 1, together with the previously described truncation mutant ER-396 (31), the wt Epo receptor, and wt Jak2 kinase.


Fig. 1. Epo receptor ER-Bx1 and Epo receptor/Jak2 chimera ER-J2KK constructs. Panel A, shown schematically are the minimal Epo receptor forms ER-Bx1 and ER-J2KK. Also diagramed are the wt Epo receptor, the previously described carboxyl-terminal truncation mutant ER-396, and wt Jak2 kinase. Panel B, sequence comparison of membrane-proximal regions of selected type 1 cytokine receptors with discernible box-1 and box-2 motifs.
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In proliferative signaling studies, the above Epo receptor constructs were transfected into myeloid FDC2 cells, and lines expressing the above receptor forms were established by selection in G418, and Epo. Receptor expression was confirmed by Western blotting, and in FDCER-Bx1 and FDCER-J2KK cells receptor forms of expected molecular mass were detected (Mr 34,000 and 72,000, respectively) (Fig. 2). For FDCER-Bx1, the occurrence of two Mr forms is thought to be attributable to differential glycosylation as has been observed previously for several alternate, active Epo receptor constructs (13, 37). For the wt Epo receptor and ER-396 receptor forms, expression has been characterized previously (34). In addition, levels of expression of each receptor form were assayed in 125I-Epo equilibrium binding assays. As shown in Table I, receptor densities were highly comparable among cell lines (approximately 400-600 receptors/cell) and approximated normal levels of Epo receptor expression in erythroid progenitor cells (18). In parental FDC-WEHI2 cells, no expression of endogenous Epo receptors was detected by Western blotting, Northern blotting (34), or reverse transcription-PCR (2).


Fig. 2. Expression of Epo receptor forms ER-Bx1 and ER-J2KK in FDC-WEHI2 cells. IL-3-dependent FDC-WEHI2 cells were transfected with pXM expression vectors encoding receptor forms ER-Bx1 and ER-J2KK, and stable lines (FDCER-Bx1 and FDCER-J2KK cells) were isolated by selection in G418 and Epo. Expression of receptor forms was confirmed by ECL Western blotting of Triton X-100 cell lysates (31). FDCwtER cells also were assayed. Arrows indicate positions of ER-Bx1, ER-J2KK, and wtER receptors. Positions of Mr standards are indexed in the left margin.
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Table I.

Densities of ectopically expressed Epo receptor constructs in FDCwtER, FDER-396, FDCER-Bx1, and FDCER-J2KK cell lines

Receptor numbers were assayed under equilibrium conditions using 125I-labeled recombinant human Epo (324 Ci/mmol) as described previously (34).


Cell line Epo receptor no.a

mean ± S.D.
FDCwtER 412  ± 112
FDCER-396 442  ± 112
FDCER-Bx1 489  ± 242
FDCER-J2KK 597  ± 87

a Mean values for n = 3 independent assays.

Experiments next were performed to assess the mitogenic activities of the above minimal receptor forms. In previous studies, our laboratory (2, 16, 34, 35) and others (10, 34, 41, 42) have developed Epo receptor forms with mutations in, or proximal to, the box-2 cytoplasmic subdomain that are either inactive or substantially compromised in mitogenic signaling. Based on these studies, it has been concluded that this receptor subdomain is critical to mitogenic function. However, and as shown in Fig. 3, Epo-induced proliferation proved to be supported efficiently by the box-2-deficient Epo receptor construct, ER-Bx1. Specifically, concentrations of Epo that were required to support proliferation of FDCER-Bx1 cells at 50% of maximal rates (1-2 units/ml) were essentially equivalent to FDCER-396 cells. In accordance with previous studies (34), mitogenic signaling via ER-396 was approximately 50% as efficient as via the wt Epo receptor in FDC-wtER cells. For FDCER-Bx1 and FDCER-396 cells, maximal levels of proliferation also were comparable yet again were somewhat lower than levels supported by the wt Epo receptor (approximately 65% of maximal versus FDCwtER cells; average of three independent experiments). Thus, these primary analyses reveal that the box-2 subdomain of the Epo receptor is nonessential for mitogenic function in FDC cells, and suggest that inactivity of at least certain previously studied Epo receptor forms with mutations in (and/or immediately flanking) this domain may be the trivial consequence of misfolding.


Fig. 3. Mitogenic activity of Epo receptor form ER-Bx1 in FDC-WEHI2 cells. The ability of receptor form ER-Bx1 to mediate Epo-stimulated mitogenesis in FDCER-Bx1 cells (open circle ) was assayed based on stimulated rates of MTS reduction and was compared directly to activities of the wt Epo receptor (FDCwtER cells) (black-square) and ER-396 (FDC-ER396 cells) (bullet ). Proliferative responses were analyzed in linear and log-linear formats. As an additional control, parental FDC-WEHI2 cells (square ) also were assayed.
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Based on the observed efficient mitogenic signaling via the above ER-Bx1 construct (and on the defined role of the box-1 subdomain in Jak kinase recruitment/activation among related type1 receptors) (5, 8, 42), the ability of a direct Epo receptor/Jak2 kinase fusion construct, ER-J2KK, to function mitogenically in FDC2 cells next was tested. In preliminary MTS-based assays, activity reproducibly was indicated for this construct. To better quantitate this response, in FDCER-J2KK cells, rates of Epo-stimulated [3H]thymidine incorporation were assayed and were compared directly to FDCER-Bx1 cells as a control (Fig. 4). Notably, this Epo receptor-Jak2 chimera proved to support Epo-induced [3H]dT incorporation at significant levels, although, maximal mitogenic rates were attenuated (40% versus ERBx1, and 25% versus wtER, see Fig. 3). Whether this restricted activity is attributable to folding events for this ER-J2KK chimera, or to the loss of an ability to activate at least certain downstream mitogenic signaling factors is addressed below. It is clear, however, that this chimera effectively supports Epo-induced proliferation, and evidence therefore is provided that activation of Jak2 (in the absence of Epo receptor cytoplasmic domains) promotes mitogenesis in a ligand-regulated fashion.


Fig. 4. Mitogenic activity of the Epo receptor/Jak2 chimera ER-J2KK in FDC-WEHI2 cells. The ability of the Epo receptor/Jak2 chimera ER-J2KK to mediate Epo-stimulated mitogenesis in FDC(ERJ2KK) cells (black-triangle) was assayed based on stimulated rates of [3H]dT incorporation and was compared directly to activities of the ER-Bx1 (FDC-ERBx1 cells) (open circle ). Proliferative responses were analyzed in linear and log-linear formats. As an additional control, parental FDC-WEHI2 cells (square ) also were assayed.
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Jak2 Kinase Activation via the Minimal Epo Receptor Form ER-Bx1, and Apparent Constitutive Tyrosine Phosphorylation of ER-J2KK

To initially address mechanisms by which activity of the above Epo receptor forms is regulated by Epo, tyrosine phosphorylation of the ER-Bx1 and ER-J2KK receptor forms was studied in FDC cells. As shown in Fig. 5 (panel A), ER-Bx1 mediated Epo-induced Jak2 activation at levels comparable to the wt-Epo receptor. In contrast, for the chimeric construct ER-J2KK, no effect of Epo exposure on tyrosine phosphorylation was detectable in repeated independent experiments performed in parallel with FDCwtER cells. Instead, tyrosine phosphorylation of ER-J2KK appeared to be constitutive (Fig. 5, panel B). This finding suggests that Epo-induced dimerization of Jak2 (in addition to its tyrosine phosphorylation) is probably critical to its activity in proliferative signaling. These experiments, however, were compromised to an extent by the limited reactivity of Jak2 antibodies with ER-J2KK, and by the lack of useful antibodies to residual Epo receptor domains (for use in immunoprecipitation). Therefore, to further confirm the identity of the indicated Tyr(P)-ERJ2KK protein in FDCER-J2KK cells, this chimeric construct also was expressed in COS cells (Fig. 5, panel B). ER-J2KK from COS cells likewise was constitutively tyrosine-phosphorylated, and this result is consistent with the observation by Duhe et al. (43) that catalytic activity of Jak2 increased upon the removal of amino-terminal homology domains.


Fig. 5. Activation of Jak2 kinase in FDCER-Bx1 cells, and ERJ2KK in FDCER-J2KK cells. Panel A, the ability of Epo receptor form ER-Bx1 to mediate the Epo-stimulated activation (induced tyrosine phosphorylation) of Jak2 in FDCER-Bx1 cells was tested by exposing cells to Epo, immunoprecipitating Jak2 from exposed (+) versus untreated (-) cells, and assaying tyrosine phosphorylation by Western blotting with antibody 4G10. For comparison, activation of Jak2 by Epo in FDCwtER cells was co-assayed. Exposure times for blots were as follows: FDCwtER, 1 min, and FDCER-Bx1, 3 min. Panel B, assay of the activation/tyrosine phosphorylation of ER-J2KK in FDCER-J2KK cells (upper panel) was assayed as described above. To confirm the mobility (and constitutive phosphorylation) of ER-J2KK, this chimeric construct also was expressed in COS cells, and lysates were analyzed directly by Western blotting with antibody 4G10 (and Jak2, lower panel). Exposure times for blots were as follows: ER-J2KK in FDCER-J2KK cells, 3 min, ER-J2KK in COS cells, 1 min.
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Activity of ER-Bx1 and ER-J2KK Receptor Forms in the Epo-dependent Inhibition of Apoptosis

Effects of Epo on red cell production have been well evidenced to include primary effects on progenitor cell survival (44). However, little is known regarding Epo-activated upstream effectors of this response pathway. Thus, it was of interest to test whether apoptosis might be inhibited by the above-described minimal Epo receptor forms in FDCER-Bx1, and FDCER-J2KK cells. As controls, FDCwtER cells also were analyzed, and inhibition of apoptosis by IL-3 and SCF was compared in each cell line. Here, apoptosis was assayed based on internucleosomal fragmentation of DNA (35). As shown in Fig. 6, Epo efficiently protected FDCER-Bx1 cells against apoptosis. As compared with levels of apoptotic inhibition supported by the wt Epo receptor (FDCwtER cells), however, protection in FDCER-Bx1 cells was slightly diminished. By comparison, IL-3-inhibition of DNA fragmentation was highly efficient, while inhibition by SCF reproducibly was partial (therefore providing two useful internal controls). Quantitative assays next were performed to more precisely assess the ability of the ER-Bx1 receptor form (as compared with the wt Epo receptor) to inhibit DNA fragmentation (Fig. 7). Here, Epo concentrations were varied and fragmentation was assayed quantitatively via phosphorimaging. These analyses confirmed the high activity of the ER-Bx1 receptor form in mediating Epo-dependent survival. In contrast, the ability of the Epo receptor-Jak2 chimera ER-J2KK to mediate Epo-inhibited apoptosis was marginal, at best. This was observed in three independent experiments and at least suggests that differences may exist in the ability of this latter receptor form to promote mitogenesis versus survival.


Fig. 6. Epo-dependent inhibition of apoptosis as mediated by Epo receptor forms ER-Bx1 and ER-J2KK in FDC-WEHI2 cells. Activities of the Epo receptor forms ER-Bx1 and ER-J2KK in mediating inhibition of apoptosis in FDC cells was assayed based on accumulation of internucleosomally fragmented DNA in the presence versus absence of Epo. As controls FDC-wtER cells also were analyzed, and apoptosis in the presence of IL-3 and SCF was co-assayed. Epo dosage for FDC cell lines was as follows: FDCwtER, 20 units/ml, 7 h; FDC-ERBx1, 20 units/ml, 7 h; FDCER-J2KK, 50 units/ml, 7 h.
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Fig. 7. Epo-dependent cell survival as mediated by Epo receptor form ER-Bx1 at varying Epo concentrations. Panel A, activities of the Epo receptor form ER-Bx1 to support inhibition of apoptosis in FDC cells was assayed based on accumulation of inter-nucleosomally fragmented DNA in the presence of varying concentration of Epo. As controls FDC-wtER cells also were assayed, and apoptosis in the presence of IL-3 was co-assayed. Epo dosage for FDC cell lines was as follows: FDCwtER and FDCER-Bx1, 0.5, 5, and 50 units/ml, 7 h. Panel B, levels of DNA fragmentation in the above experiments (panel A) were analyzed quantitatively by phosphorimaging.
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Activity of ER-Bx1 and ER-J2KK Receptor Forms in Mediating the Epo-induced Expression of Bcl2, Myc, and Pim-1

Possible mechanisms underlying the ability of ER-Bx1 ER-J2KK Epo receptor forms to support Epo-stimulated proliferation next were addressed. In at least certain related type 1 receptor systems, cytokine-induced expression of Bcl2 has been implicated in proliferative signaling (45). In FDCwtER, FDCER-Bx1, or FDCER-J2KK cells, however, little if any effect on levels of Bcl2 was observed over a 9-h time course of Epo exposure (Fig. 8). By comparison, Pim-1 expression recently has been shown to be induced by Epo receptor forms containing both box-1 and -2 subdomains, and Tyr343 (19). Interestingly, analyses of the above samples from Epo-exposed FDCwtER, FDCER-Bx1, and FDCER-J2KK cells revealed the Epo-induced expression of Pim-1 in each cell line (Fig. 9). Levels of Pim-1 expression as stimulated via ER-J2KK, however, were limited. In these studies, two forms of Pim-1 reproducibly were detected (Mr 34,000 and 35,000), and this is consistent with studies from other laboratories (46). Therefore, to further assess these effects (and associated signaling mechanisms), transcriptional activation of pim-1 and myc genes via ER-Bx1 and ER-J2KK receptor forms also were studied (Fig. 10). In FDCER-Bx1 cells, Epo rapidly and efficiently activated the transcription of both c-myc and pim-1 genes. As compared directly to FDC2-wtER cells, rates and levels of transcriptional activation via ER-Bx1 receptors were detectably but not markedly attenuated. In FDCER-J2KK cells, c-myc transcription also was activated upon Epo exposure. pim-1 gene transcription, however, was not supported at significant levels via this chimeric Epo receptor form. pim-1 and c-myc gene transcription were stimulated strongly by IL-3 in FDCER-J2KK cells, and pathways to pim-1 transcriptional activation in this subline therefore are not perturbed. These studies serve to confirm the activity of the ER-Bx1 construct in mediating Pim-1 expression, demonstrate c-myc activation by ER-Bx1 as well as ER-J2KK receptor forms, and at least suggest that effectors of pim-1 transcription might depend upon domains that are represented in ER-Bx1, but lacking in ER-J2KK.


Fig. 8. Bcl2 expression is not modulated by Epo in FDCwtER or FDCER-Bx1 cells. To test whether Bcl2 expression might be modulated by Epo, FDC-WEHI2 cells expressing the wt Epo receptor (FDCwtER) or ER-Bx1 (FDC-ERBx1) were cultured without Epo (or IL-3) for 9 h in 1% FBS, Opti-MEM, and then stimulated with Epo (50 units/ml). At the indicated intervals (0, 0.3, 3, 9 h), cells were lysed and equivalent amounts of lysates were analyzed for Bcl2 levels by direct Western blotting.
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Fig. 9. Epo-induced Pim-1 expression by Epo in FDCwtER, FDCER-Bx1, and FDCER-J2KK cells. The ability of the Epo receptor forms wtER, ER-Bx1, and ER-J2KK to mediate the induced expression of Pim-1 in FDC cells was assayed by culturing cell lines in 1% FBS, Opti-MEM for 9 h in the absence cytokines, stimulating with Epo for the indicated intervals, and assaying for Pim-1 expression in direct cell lysates by Western blotting. Panel A, Epo-induced expression of Pim-1 via the Epo receptor form ER-Bx1 and the wtER in FDC-ERBx1 and FDCwtER cells (exposure time for blot 2 min). Panel B, Epo-induced expression of Pim-1 via the Epo receptor/Jak2 chimera ER-J2KK (exposure time for blot 15 min). In each cell line, the expression of two forms of Pim-1 (arrows) reproducibly was induced. The positions of Mr standards are indexed in the left margin.
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Fig. 10. Epo-induced transcription of c-myc and pim-1 in FDCER-Bx1 and FDCER-J2KK cells. Activities of the minimal Epo receptor forms ER-Bx1 and ER-J2KK in mediating Epo-induced c-myc and pim-1 gene transcription were assayed as follows. FDCwtER, FDCER-Bx1, and FDCER-J2KK cells first were cultured for 8 h in 1% FBS, 10-5 M 2-mercaptoethanol, Opti-MEM. Cells then were stimulated with Epo (50 units/ml) and at the indicated intervals (0, 20, 60, 180 min) total RNA was isolated. Levels of c-myc and pim-1 transcripts were analyzed by Northern blotting (20 µg of RNA/lane). As a positive control, FDC2-ERJ2KK cells also were exposed to IL-3. To control for any variability in loading and/or transfer blots were hybridized to a glyceraldehyde-3-phosphate dehydrogenase cDNA probe.
[View Larger Version of this Image (64K GIF file)]



DISCUSSION

Since the original identification of type 1 cytokine receptors as single-transmembrane, non-enzymatic proteins (1), this family of receptors has been revealed to function in concert with an increasingly diverse set of co-assembled primary signaling factors. In the Epo system, ligand-induced receptor dimerization has been shown to promote the binding, (trans-)phosphorylation, and catalytic activation of Jak2 (12). Jak2-mediated phosphorylation of Epo receptor tyrosine motifs then leads to the SH2 domain-mediated recruitment of the phospholipid-modifying enzymes phospholipase C-gamma , phosphatidylinositol 3-kinase, and SHIP; the tyrosine phosphatases HCP and Syp; the molecular adaptors Grb2, Shc, Cbl, Crk-l, and Cis; and the latent transcription factor STAT5. Accordingly, significant attention has focused on defining mechanisms of factor recruitment, interaction, and function in this and related receptor systems. However, in an increasing number of type 1 cytokine systems the ability of membrane-proximal (phospho)tyrosine-deficient receptor domains to efficiently signal growth has been demonstrated (5, 7, 8, 42). These findings limit the likelihood that distal cytoplasmic receptor domains and associated factors contribute more than modulating roles, and reinforce the apparent essential nature of Jak kinases in cytokine-stimulated proliferation. The present studies of Epo receptor function were performed to further define minimal Epo receptor domains (and effectors), which contribute critically to Epo-dependent mitogenesis and inhibition of apoptosis.

A highly truncated Epo receptor construct ER-Bx1 first was developed that retains the conserved membrane-proximal box-1 domain, but lacks box-2 and all distal (phospho)tyrosine sites for effector recruitment. Notably, this receptor form efficiently mediated both mitogenesis, and Epo-dependent inhibition of apoptosis. These findings underline a necessary and sufficient role for the Epo receptor box-1 domain (and the associated activation of Jak2 kinase) in these proliferative signaling pathways. Mechanistically, this ER-Bx1 receptor form also was shown to mediate the Epo-induced expression of Myc and of the serine-threonine kinase Pim-1. To further test contributions of Jak2 kinase activity in Epo-stimulated growth, a chimeric construct also was developed in which the Epo receptor extracellular and transmembrane domains were fused directly to the kinase and kinase-like domains of Jak2. Interestingly, this chimera likewise effectively mediated mitogenesis and Epo-induced myc transcription. In contrast, Pim-1 expression was only nominally supported, and this chimera did not function detectably in inhibiting apoptosis.

Signaling properties of the Epo receptor forms ER-Bx1 and ER-J2KK are of interest to consider based on comparisons to previously studied, related Epo receptor mutants. As mentioned above, several Epo receptor forms that are mutated within the conserved box-2 domain or immediate carboxyl flanking regions have been described previously, and these typically are either highly compromised or inactive in mitogenic signaling (see Table II). This receptor domain therefore has been concluded to be important, if not essential, to this activity. The high proliferative activity of the presently studied ER-Bx1 mutant, however, demonstrates that this is not the case and suggests that mutations in the box-2 domain and adjacent residues simply may compromise functions of the essential box-1 domain. These conclusions are supported by recent studies by Hilton et al. (47) in which essential mitogenic activity was demonstrated for highly truncated Epo receptor forms expressed in BaF/3 cells. Unfortunately, however, BaF/3 cells recently have been revealed to express endogenous Epo receptors at levels sufficient to confound interpretations of the activities of ectopically expressed Epo receptor forms (48, 49). Presently, the apparent lack of an essential role for the Epo receptor box-2 domain in proliferative signaling raises the obvious question as to the role of this conserved domain among type 1 receptors. One possibility that previously has been suggested is a supporting role in Jak2 binding and activation (12). For the Epo receptor, however, substitution of the box-1 domain into a corresponding box-1 region in the IL-2-beta receptor converts the IL-2 alpha /beta /gamma receptor from a Jak1,3 to a functional Jak2,3 signaling complex (2). Thus, the selective activation of Jak2 apparently depends only upon the box-1 domain. In addition, highly truncated forms of the receptors for G-CSF and growth hormone likewise have been recently shown to efficiently support mitogenic signaling in the absence of the box-2 domain (5-8, 50). Notably, both Epo and G-CSF are known to synergize in mitogenic signaling with the receptor for SCF, c-Kit (51). It therefore is interesting to speculate that this synergy might depend at least in part on features in the box-2 region. In recent work by Wu et al. (52), in fact, the direct binding of c-Kit to a box-2 associated domain of the Epo receptor has been demonstrated. Thus, it should be of interest to test whether this possible role for the Epo receptor box-2 domain (and flanking residues) is evident in functional analyses.

Table II.

Proliferative activity of box-2 Epo receptor mutants


Epo receptor mutant Proliferative activity Reference no.

% wt
A353P/P357A/S360G 0 10
ER352 13 34
L306R 9 41
L307K 18 41
ER337 (S-mutant) 0 42
ER375 (H-mutant) 35 42

To assess whether ligand-mediated activation of Jak2 per se might support proliferative signaling, the kinase (and kinase-like) domain of Jak2 presently was fused directly to the extracellular and trans-membrane domains of the Epo receptor, and signaling activities of this ER-J2KK chimera in FDC2 cells were studied. Interestingly, ligand-dependent mitogenic signaling was supported by ER-J2KK, albeit at reduced rates (40% of levels supported by the ER-Bx1 construct). In contrast, this construct did not exert detectable activity in the Epo-dependent inhibition of apoptosis. With regard to signaling mechanisms, ER-J2KK was shown to efficiently mediate Epo-induced Myc expression, but activated Pim-1 protein expression at only low levels did not appreciably stimulate pim-1 transcription. In keeping with findings in related systems (53), this latter result at least suggests that Pim-1 expression may be regulated in the Epo receptor system at both transcriptional and post-transcriptional levels. These findings also are consistent with the prospect that Epo-induction of pim-1 gene expression might depend upon as yet unidentified effectors that are activated by the ER-Bx1 construct, but not by ER-J2KK. In a broader context, factors that act to regulate Pim-1 expression generally are not well understood, and FDC2-ER-Bx1 cells should provide a tractable system to investigate this Epo signaling pathway. Finally, while the present studies were in progress, properties of two distinct receptor-Jak2 chimeric constructs were reported. First a CD16-Jak2 fusion construct was developed, and the tyrosine phosphorylation of this construct was shown to be ligand regulated (i.e. stimulated by antibody-induced cross-linking) (54). However, this CD16-Jak2 chimera apparently was inactive mitogenically. Second, Jak2 PTK and PTK-like domains were fused to the trans-membrane and extracellular domains of the EGF receptor (55). Interestingly, this EGF receptor-Jak2 chimera efficiently supported EGF-dependent mitogenesis as well as inhibition of apoptosis in 32D cells. Moreover, and despite the absence of canonical sites for the recruitment of either Grb2-mSos complexes or STAT5, MAP kinase, and STAT5 activation was supported in this system. One interpretation of these results is that novel and as yet undefined pathways exist through which Jak2 can stimulate Ras, MAP kinase kinase, MAP, and STAT5 activation. However, the possibility also should be considered that abnormally increased Jak2 activation levels might result in the tyrosine phosphorylation of uncoupled effectors, which then promote growth and survival. In addition, this stimulation would proceed in the absence of inhibitory effects, which normally are exerted on Jak2 by the SH2 tyrosine phosphatase, HCP (26). Increased stimulation of Jak2 might result from the overexpression of chimeric constructs, or overstimulation with ligand. In studies by Nakamura et al. (55), levels of EGF receptor-Jak2 chimera expression in 32D cells were not addressed, and ligand concentrations >= 1 µg/ml were used. In contrast, in the present studies of ER-J2KK signaling, FDC2 cells expressing normal levels of receptors were used, and ligand concentrations were limited (2-3 orders of magnitude lower). This likely explains why tyrosine phosphorylation and activation of SH2 domain-encoding effectors (including STAT5) are not detectable in FDC-ERJ2KK or FDC-ERBx1 cells,2 as would be predicted based on the lack of (phospho)tyrosine recruitment sites.

A final property of the wt Epo receptor and of the minimal construct ER-Bx1 that merits discussion is their ability to efficiently mediate Epo inhibition of apoptosis. Among type 1 cytokine receptors, the Epo receptor and IL-2 beta -receptor structurally are relatively closely related, and their homology in part led to the discovery of this receptor superfamily. In the context of apoptosis, IL-2 has been shown to activate Bcl-2 expression (45), and this observation promoted the present analyses of Epo effects on Bcl-2 expression. However, Bcl-2 levels were not affected by Epo in FDC2 cells, and in recent analyses of Bcl-xL no significant effects of Epo on this Bcl-2 homologue were apparent.3 For Bcl-2, this result is consistent with studies by May et al. (33), in which Epo likewise did not affect Bcl-2 expression (yet interestingly did stimulate Bcl-2 serine phosphorylation). By comparison, in HCD57 cells Epo recently has been shown to stimulate increases in the expression of Bcl-xL (56). Thus, it seems likely that Bcl-2-related factors may act to regulate Epo effects or survival, and this prospect presently is being investigated further in our FDC-ER and FDC-ER-Bx1 models.

Last, and as mentioned above, roles may exist for Pim-1 in Epo-dependent inhibition of programmed cell death. Support for this notion derives from three considerations. First, in the context of erythropoiesis, Pim-1 expression has been associated with erythroleukemias (57) and has been correlated with modulated erythrocyte volume in transgenic models (58). Second, and as studied in lymphocytes from lpr mice, the forced expression of Pim-1 leads to hyperproliferation and interestingly this appears to depend upon effects on cell survival rather than mitogenesis (59). Third, Pim-1 expression commonly is activated by hematopoietic cytokines which promote cell survival, yet little presently is known about its regulated expression or the targets of Pim-1 phosphorylation. In the present study, Epo-induced expression of Pim-1 clearly is shown to depend upon the conserved box-1 domain of the Epo receptor. Future studies that address mechanisms of Pim-1 expression and function in this minimal model should be of significant interest.


FOOTNOTES

*   This work was supported by National Institutes of Health DK 40242 (to D. M. W.), Research Career Development Award Grant HL 03042 (to D. M. W.), and a Sigma Xi grant-in-aid (to B. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: 115 William L. Henning Bldg., The Pennsylvania State University, University Park, PA 16802. Tel.: 814-865-0657; Fax: 814-863-6140; E-mail: dmw1{at}psu.edu.
1   The abbreviations used are: Epo, erythropoietin; ECL, enhanced chemiluminescence; EGF, epidermal growth factor; G-CSF, granulocyte colony-stimulating factor; HCP, hematopoietic cell phosphatase; IL, interleukin; MAP, mitogen-activated protein; STAT, signal transducers and activators of transcription; PCR, polymerase chain reaction; SCF, stem cell factor; SH, Src homology domain; FBS, fetal bovine serum; wt, wild type; PTK, protein-tyrosine kinase; MTS, 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl2Htetrazolium).
2   B. Joneja and D. M. Wojchowski, unpublished data.
3   B. Joneja, unpublished data.

ACKNOWLEDGEMENTS

We thank Amgen for the generous provision of recombinant Epo, Dr. Steven Pelech (University of British Columbia, Vancouver) for antibodies to Pim-1, Ning Jiang (The Pennsylvania State University) for assistance with Jak2 kinase assays, Ron Gmerek for contributions in apoptosis studies, and Loretta Muchinsky for valued typing efforts.


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