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Originally published In Press as doi:10.1074/jbc.M307182200 on August 9, 2003

J. Biol. Chem., Vol. 278, Issue 42, 40702-40709, October 17, 2003
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Roles for an Epo Receptor Tyr-343 Stat5 Pathway in Proliferative Co-signaling with Kit*

Ke Li, Chris Miller, Shailajia Hegde and Don Wojchowski {ddagger}

From the Immunobiology Program and Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802

Received for publication, July 5, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Erythroid progenitor cell expansion depends upon co-signaling by Epo receptor (EpoR) and Kit, but underlying mechanisms are incompletely understood. To quantitatively analyze EpoR contributions to co-signaling, phosphotyrosine (Tyr(P)) mutants were expressed as human epidermal growth factor (hEGF) receptor-mEpoR EE chimeras at matched and physiological levels in FDCW2 hematopoietic progenitor cells and were assayed for proliferative activities in the absence or presence of endogenous Kit stimulation. Two Tyr(P)-null (but Jak2-coupled) EpoR forms each retained <=25% of the wild-type activity, whereas the add-back of single Tyr(P) sites in the EpoR forms EE-T-Y343 (Stat5 binding site), EE-Y479 (p85/phosphatidylinositol 3-kinase binding site), or EE-Y464 (Src kinase binding site) significantly enhanced activities (to 100, 95, and 50% of EE-WT (wild type) levels, respectively). EE-Y343&Y401 and EEF343&F401 double add-back and deletion constructs were also prepared and were shown to possess 90 and <=50% of wild-type activity. In contrast, efficient Kit co-signaling activity was retained only by EE-T-Y343 and EE-Y343&Y401 EpoR forms. EE-T-Y343 together with EE-T-Y343F and EE-WT EpoR forms were also analyzed in embryonic stem cell-derived erythroid G1E-2 cells with highly comparable outcomes, including the ability of EE-T-Y343 (but not EE-T-Y-343F) to synergize with Kit. Despite specific connection of EE-T-Y343 to Stat5, the contributions of Kit to EpoR-dependent proliferation did not involve Kit effects on Stat5 activation (but was limited by the mutation of Kit Tyr(P)-567 and Tyr(P)-569 Src kinase recruitment sites). Instead, co-signaling appears to depend upon the downstream integration of Kit signals with the targets of an EpoR/Jak2/Y343/Stat 5 response axis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Erythropoiesis depends sharply upon combined sets of signals that are relayed by the single transmembrane receptors for erythropoietin (EpoR),1 and stem cell factor (Kit). The EpoR is expressed and functions primarily in erythroid and neuronal cells (1, 2), and Epo or EpoR gene disruption blocks erythroid development at the CFUe stage (3, 4). Inactivating mutations in Kit also lead to overt anemia (5, 6), but consistent with the broader expression of Kit (7, 8) such mutations are associated with defects in mast cell, melanocyte, germ cell, and early hematopoietic progenitor cell development (5, 6). In addition, EpoR cytoplasmic deletion mutations have been linked to erythropolycythemias (9), and mutations in Kit have been correlated with acute myeloid leukemia and chronic myeloid leukemia (10) as well as the tumorogenesis of mast (11) and gastrointestinal stromal cells (12).

With regards to EpoR signaling, primary roles for the associated Janus kinase, Jak2, have been defined through studies of EpoR membrane-proximal domain mutants (13), dominant-negative forms of Jak2 (14), and Jak2-/- mice (15). One set of Jak2 targets is eight cytoplasmic tyrosine sites within the EpoR, sites that are conserved in mice, man, and zebra fish (16). Within activated EpoR complexes, these phosphotyrosine (Tyr(P)) sites engage a complex set of SH2 domain-encoding signal transduction factors, including those that regulate Stat transcription factors (17), phosphatidyl inositol metabolism (especially PI3 kinase and SH2 inositol phosphatase) (18, 19), Ras/Raf pathways (20), phosphatase recruitment (e.g. SHPTP1&2, Syp (SH2 domain containing tyrosine phosphatase)) (21, 22), calcium flux (23), protein kinase C (24), CIS (25), and Socs3 (26), NF-{kappa}B (27), and Src (28) or Lyn (29) signaling. Nonetheless, EpoR forms that lack all cytoplasmic Tyr(P) sites for SH2 domain factor binding recently been shown in transgenic (30) and EpoR knock-in mouse models (31) to support red cell development, but at somewhat less than wild-type efficiencies. For efficient EpoR signaling, the relative importance of positively acting Tyr(P) sites therefore remains a basic and somewhat controversial issue, and in vivo models may be complicated by compensatory erythropoietic mechanisms (32).

In an aim to more quantitatively define the relative importance of select Tyr(P) sites in EpoR function, the following approach was presently applied. First, a series of EpoR Tyr(P) mutants were prepared as hEGFR-EpoR "EE" chimeras and were expressed using MSCV-based vectors in IL-3-dependent FDC cells (33), as well as ES cell-derived erythroid G1E-2 cells (34). In cells expressing these Epo receptor forms at matched physiological levels (as isolated by FACS), the abilities of these Tyr(P) mutant EE receptors to support progenitor cell proliferation were then assayed in the absence or presence of endogenous Kit co-activation. This approach was prompted by signaling studies in several laboratories and systems that underline the importance of attention paid to EpoR expression levels (33, 35) and by the limited availability of useful antibodies to the mEpoR (36). In the present studies, activities of EpoR Tyr(P)-null forms were observed to be significantly enhanced by Tyr(P)-343, Tyr(P)-464, or Tyr(P)-479 sites for Stat5, p85/PI3 kinase, and Lyn (or Src) kinase, respectively. However, efficient co-signaling with Kit interestingly proved to be supported in each of two model systems only by EpoR forms containing a Tyr(P)-343 Stat5 binding site. Mechanistically this co-signaling did not involve detectable effects of Kit on Stat5 activity and instead is proposed to involve specific interactions between EpoR/Jak2/Stat5 target gene products and select Kit-directed routes. Findings are discussed in the contexts of core EpoR and Kit co-signaling axes and Epo- and SCF-dependent erythropoiesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines—FDCW2 cells (a myeloid murine marrow IL-3-dependent FDC subline) (33) were maintained in OptiMEM I medium (Invitrogen), 8% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 µg/ml), and amphotericin B (0.25 µg/ml) (PSF) plus 3.5% WEHI3 cell conditioned medium as a source of IL-3. G1E-2 cells are an Epo-plus SCF-dependent erythroid line derived from murine ES cells with a disrupted endogenous Gata1 gene (34) and were maintained in IMDM, 13% FBS, 100 µM monothioglycerol, 50 ng/ml SCF, 2 units/ml Epo plus PSF. GP2-293 cells were maintained on collagen-coated dishes in Dulbecco's modified Eagle's medium, 8% FBS plus PSF.

Epo Receptor and Kit Mutants—cDNAs encoding EpoR mutants (as EE chimera) were cloned to the retrovirus vector pMSCV-Neo-N/X. pMSCV-Neo-N/X was prepared from pMSCV-Neo (Clontech) by inserting NotI and SfiI sites into a polylinker region using the following cassette: 5'-NotI, SfiI, XhoI, HpaI, EcoRI-3' (5'-GGCCGGGCCNNNNGGCCTCGAGGTTAACGAATTC-3'). To generate pMSCV-Neo-N/X-EET-343F, the NotI fragment of pCINeo-EE372-Y343F (37) was cloned to pMSCV-Neo-N/X. For pMSCV-Neo-N/X-EECA and pMSCV-Neo-N/XEE-T-Y343, the BglII to SalI and BglII to XhoI fragments of pCI-Neo-{Delta}BglII-EECA (37) and pSPER396-new stop (37), respectively, were cloned to BglII and XhoI sites in pMSCV-Neo-N/X-EE-T-Y343F. For pMSCV-Neo-N/X-EE-Y343Y401 and pMSCV-Neo-N/X-EE-F343F401, 0.7-kb BglII to XhoI fragments of pcDNA3-EpoR-Y343Y401 and -F343F401 (38) were cloned to pMSCV-Neo-N/X-EE-T-Y343F at BglII and XhoI sites. For pMSCV-Neo-N/X-Y479 and Tyr-464, 890-bp BglII to EcoRV fragments from pcDNA3-mEpoR-F7Y479 and F7Y464 (38) were cloned to pMSCV-Neo-N/X-EE-F343 at BglII to HpaI sites. For construction of pMSCV-Neo-N/X-EE-F8, a full-length cDNA pointed-mutated (to Phe) at each of eight conserved cytoplasmic (P)Y codons (38) was cloned to pMSCV-Neo-N/X at BglII and HpaI sites. The Kit Tyr(P) mutants MK-WT and MK-F567F569 were prepared as hMCSF receptor-murine Kit chimeras (39) and were cloned to a MIEG3 retrovirus vector (39) at 5'NotI and 3'XhoI sites.

Retroviruses—Retroviruses were prepared using GP2-293 cells and a VSVenv-G protein encoding plasmid (pVSVenv-G) (Clontech) (40). Cells at 30% confluency (100-mm dish) were transfected with 8 µg of pMSCVNeo or pMIEG3 EpoR (EE) or Kit (MK) constructs plus 8 µg of pVSVenv-G using 40 µl of FuGENE 6 liposomal reagent (Roche Applied Science). At 14 h after transfection, PSF was added. At 60 h, recombinant retroviruses (30 ml from triplicate transfections) were recovered at 4 °C, filtered (0.45 µm), and concentrated by centrifugation for 90 min at 50,000 x g. Viruses were resuspended in 0.5 ml of 0.1% Hanks' balanced salt solution, incubated for 4 h at 4 °C, and preserved at -80 °C.

Transductions—Exponentially growing FDC cells or G1E-2 cells (4 x 105 cells/1 ml in 12-well plates) were combined with polybrene (8 µg/ml) plus 50 µl of concentrated recombinant retrovirus. At 24 h, transduced cells were cultured in complete growth medium (with 0.8 mg/ml G418 for MSCV-Neo transduced FDCW2 cells).

FACS and Flow Cytometry—To match receptor expression levels among transduced cell lines, cells were gated and sorted by FACS (Coulter Elite ESP System). For EpoR EE chimeras, 2 x 106 cells were collected, washed, and resuspended in 0.2 ml of phosphate-buffered saline, 0.1% bovine serum albumin, and incubated at 4 °C with 5 µg of Fc fragment (Pierce)(15 min) with 2.5 µg of anti-human EGF receptor (Pharmingen) (45 min) and with 1 µg of phycoerythrin-conjugated anti-mouse IgG secondary antibody (Jackson Immunoresearch, catalog number 115-116-072) (30 min). Cells were then washed, resuspended in 1 ml of phosphate-buffered saline, 0.1% bovine serum albumin, and sorted. Sorted cells were collected, washed in IMDM, and cultured initially in 2 ml of culture medium. For MK chimeras, sorting was based on green fluorescent protein fluorescence, and matched MK expression levels were confirmed using 1 µg of rat anti-human M-CSF receptor (Santa Cruz Biotechnology, catalog number F181) and 1 µg of phosphatidylethanolamine-conjugated anti-rat IgG (PharMingen, catalog number 550767). Levels of receptor expression were determined using phosphatidylethanolamine microsphere fluorescence standards (30).

Cytokines and Proliferation Assays—Cytokines used were hEGF (R&D System, Minneapolis, MN), hMCSF (PeproTech, Rocky Hill, NJ), murine SCF (PeproTech), and hEpo (Epoetin Alpha, Amgen, Thousand Oaks, CA). In proliferation assays, exponentially growing FDC and G1E-2 cells were collected, washed twice in 10 ml of Dulbecco's modified Eagle's medium, and adjusted to 5 x 105/ml in culture medium lacking cytokines. To these cells (50 µl/well in 96-well plate), test cytokines (2x in culture medium) were then added. At 44 h of culture, [3H]thymidine (2.0 mCi/mmol, catalog number 2407005, ICN) was added (1 µCi in 10 µl of phosphate-buffered saline plus 0.5% bovine serum albumin), and incorporation over a 2-h interval was assayed (1205 Betaplate counter, KBL Pharmacia).

Stat5 Activation Assay—Exponentially growing FDC cells were washed twice with OptiMEM I medium and adjusted to 3 x 105/ml in OptiMEM I plus 8% FBS (2 ml/well, 6-well plates) in the presence of hEGF (10 ng/ml) and/or SCF (50 ng/ml). For analyses in G1E-2 cells, exponentially growing G1E-2 cells were washed twice with OptiMEM I medium and adjusted to 3 x 105/ml in IMDM, 13% FBS, 100 µM monothioglycerol in the presence of hEGF (10 ng/ml) and/or mCSF (100 ng/ml). At 2 h of culture, cells were transfected using 1.8 µg of pSPIGLE1-Luc (41), 0.2 µg of pSEAP (secreted alkaline phosphatase) (Tropix), and 12 µl of FuGENE 6. At 48 h, cells were washed, and extracts were prepared using reporter lysis buffer (Promega, catalog number E3971). Extracts were assayed for protein content (BCA system, Pierce) and for luciferase activity (luciferin substrate, Promega, catalog number E1483).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Contributions of Epo Receptor Tyr(P)-343, Tyr(P)-464, and Tyr(P)-479 Sites to Progenitor Cell Proliferation—An initial goal of investigations was to quantitatively compare contributions of positively acting EpoR cytoplasmic Tyr(P) sites with hematopoietic progenitor cell proliferation. A central focus was on EpoR Tyr(P)-343, Tyr(P)-464, and Tyr(P)-479 sites due to their respective coupling to Stat5, Src kinase, and p85/PI3K pathways. To allow for the reliable assay (and adjustment) of receptor expression levels (and to bypass endogenous EpoRs) a chimeric receptor approach was used in which the extracellular dimerization domain of the murine EpoR was replaced by that of the human EGF receptor (hEGFR) (42). Cytoplasmic Tyr(P) sites within resulting EE chimeras next were mutated to phenylalanine, recombinant MSCV retroviruses encoding these EpoR Tyr(P) mutants were prepared, and FDCW2 and ES cell-derived erythroid G1E-2 cells were transduced with these vectors. FACS then was used to retrieve transduced lines expressing EE Epo receptor forms at physiological levels (~1000 receptors/cell), and their proliferative signaling capacities were assayed. Compared first in FDCW2 cells were the activities of wild-type (EE-WT), Tyr(P)-343-retaining (EE-T-Y343), and two Tyr(P)-null EpoR forms (EE-F8 and EE-T-Y343F). These EE receptor forms and their expression levels in transduced and FACS-analyzed FDCW2 cells are shown in Fig. 1. When activated by EGF, EE-WT and EE-T-Y343 each efficiently supported high level proliferation (Fig. 2). By direct comparison and for each of two Tyr(P)-null forms studied (EE-F8 and EE-T-Y343F), proliferative activities were uniformly lower (~25% of EE-WT activity). This outcome is in keeping with the non-essential nature of EpoR Tyr(P) sites, yet also underlines apparent significant contributions of at least certain sites (including Tyr(P)-343) to efficient EpoR function.



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  		FIG. 1.
Wild-type, Tyr(P)-null, and Tyr(P)-343 EpoR chimeras and their expression in FDCW2 cells. FDCW2 cells were transduced with MSCV-neo retroviruses encoding the illustrated chimeric receptor forms and were selected in G418. FACS (with an antibody to the hEGFR extracellular domain) was then used to prepare lines expressing matched levels of chimeric EE receptors. Phosphatidylethanolamine fluorescence profiles (i.e. EE receptor expression levels) for each cell line are shown versus parental FDCW2 cells as a negative control.

 


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  		FIG. 2.
Tyr(P)-null EE receptor forms support progenitor cell proliferation, but at limited efficiencies. FDCW2-derived cell lines expressing the diagrammed EpoR forms (as EE chimeras) were cultured for 48 h in the presence of hEGF at increasing concentrations. Rates of hEGF-dependent proliferation were then determined based on [3H]dT incorporation. Graphs are means of triplicate assays ± S.D.

 

Roles for three additional EpoR Tyr(P) sites were next considered: a Tyr(P)-479 site for PI3K recruitment (18, 43); a Tyr(P)-464 site that has been mapped as an Src family kinase docking site (28); and a Tyr(P)-401 site that also has been implicated in Stat5 (17) as well as inhibitory SOCS3 and CIS signaling (25, 26). Contributions of these select Tyr(P) sites to EpoR proliferative signaling were analyzed quantitatively as above in FDCW2 cells transduced with these EE constructs. For Tyr(P)-479 and Tyr(P)-464, this involved their selective reconstitution within EE-F8 (see above). For Tyr(P)-343-plus-Tyr(P)-401, this involved the construction of double add-back and EE-Y343F&Y401F double-deletion forms. These Tyr(P) mutant EpoR forms and their expression levels in transduced and FACS-selected FDC lines are shown in Fig. 3. In proliferative response assays, EE-Y343&Y401 as well as EE-Y479 proved to retain essentially wild-type activities (Fig. 4). By comparison, EpoR forms that either lacked Tyr-343 and Tyr-401 (EE-F343F401), or retained only Tyr-464, possessed limited activity (although each/all Tyr(P)-retaining EpoR forms were significantly more active than Tyr(P)-null EpoR forms). In these direct comparisons, EpoR Tyr(P)-479 p85/PI3K as well as Tyr(P)-343 Stat5 routes each therefore are shown to independently support EpoR proliferative activity at highly comparable (and near wild-type) efficiencies. In addition, positive roles exerted via Tyr(P)-343 plus Tyr(P)-401 in EE-Y343&Y401 appear to overcome of the effects of negatively acting SOCS3 and CIS factors.



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  		FIG. 3.
EpoR (EE) Tyr(P) add-back constructs, and their expression in FDCW2 cells. FDCW2 cells were transduced with MSCV-neo retroviruses encoding the indicated EE receptor forms and were selected in G418. Lines expressing matched levels of EE receptors were then isolated by FACS. PE, phycoerythrin.

 


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  		FIG. 4.
Proliferative activities of EE receptor forms retaining Tyr(P) sites Tyr-479, Tyr-464, Y343&Y401, or Tyr-429/431/443/460/464/479 (i.e. Phe-343, Phe-401). FDCW2 cells expressing these EE EpoR receptor forms were prepared and were assayed for proliferative responsiveness based on rates of hEGF-induced [3H]dT incorporation. Means of triplicate assays ± S.D. are graphed.

 

EpoR Tyr(P)-343-mediated Kit Co-signaling—FDCW2 cells express endogenous and functional Kit (34), and opportunities therefore were provided to investigate Kit and EpoR co-function. Specifically, cells expressing the above EE EpoR forms at matched levels were exposed to hEGF at increasing concentrations in the presence or absence of a limiting dose of SCF (15 ng/ml, ~15% maximal response to SCF only), and effects of EE and Kit co-stimulation on proliferation were determined. This first was studied for EE-WT, EE-T-Y343, EE-F8, and EE-TY343F EpoR forms (Fig. 5). For EE-T-Y343 (and EE-WT), co-function was obvious, and each supported Kit co-signaling (lower panels). Thus, Tyr(P)-343 alone (together with Jak2) can efficiently support this integrated aspect of EpoR biofunction. Whether Kit co-signaling might also be supported by other positively acting EpoR Tyr(P) sites next was investigated. In particular, EE-Y479, EE-Y464, EE-Y343&Y401, and EEF343F401 forms each were assayed for co-activity in the presence or absence of SCF as above. Among these EpoR forms, only the Tyr(P)-343-retaining construct EE-Y343Y401 retained the ability to efficiently integrate this Kit proliferative co-response (Fig. 6). Interestingly, these outcomes therefore implicate the EpoR Tyr(P)-343- (and Tyr(P)-401-) coupled factor Stat5 (and not EpoR-coupled PI3K nor SRC kinases) as a selective positive integrator Kit co-signaling.



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  		FIG. 5.
The Jak2/STAT5-coupled EpoR form EE-T-Y343 efficiently co-signals with Kit. In the upper panels, FDCW2 cells expressing matched levels of EE-T-Y343 or EE-T-Y343F EpoR forms were stimulated with EGF in the absence or presence of mSCF at a low constant concentration (15 µg/ml). EE-WT and EE-F8 forms were included as controls. At 48 h of culture, rates of cytokine-induced [3H]dT incorporation were determined, and are graphed as means of triplicate assays ± S.D. In the lower panel, the overall abilities of EE-WT and EE-T-Y343 EpoR forms to act in synergy with Kit also are summarized as levels of hEGF-plus-SCF-induced growth above calculated additive effects.

 


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  		FIG. 6.
EpoR form EE-Y343Y401, but not EE-Y464, EE-Y479 or EE-F343F401, efficiently co-signals with Kit. In the upper panels, FDCW2 cells expressing matched levels of EE-Y343Y401, EEF343F401, EE-Y464, or EE-Y479 receptor forms were stimulated with EGF, SCF (closed bars, 15 ng/ml), or EGF-plus-SCF. FDCW2-EE-WT cells were included as a control. At 48 h of culture, rates of cytokine-induced [3H]dT incorporation were measured and are graphed as means ± S.D. of triplicate assays. In the lower panel, the ability of the EpoR form EE-Y343Y401 to act in synergy with Kit is illustrated as levels of hEGF-plus-SCF-induced growth above calculated additive effects.

 

Based on the above observed roles for EpoR Tyr(P)-343 in Kit and EpoR co-function, possible effects of Kit on Stat5 activity were tested using a sensitive and Stat5-specific transcriptional reporter, pSpi-GLE1-Luc (41). For EpoR signaling per se, only in cells expressing Tyr-343-containing receptor forms (EE-WT, EE-T-Y343, EE-Y343&Y401) was Stat5 activity strongly stimulated (Fig. 7, upper panel) (although low level Stat5 activation was supported by EpoR EE-F343F401). Moreover, no significant effects of Kit stimulation on Stat5 activation were observed in either the presence or the absence of EE receptor co-stimulation in FDC-EE-TY343F, EE-T-Y343, or EE-WT cells (Fig. 7, lower panel). These outcomes argue that Kit does not directly stimulate Stat5 and that it also fails to significantly modulate EpoR-stimulated Stat5 activity in this system. Instead, EpoR and Kit co-action are suggested to depend upon interactions of Kit signal transduction factors with downstream EpoR-plus-Stat5-regulated target genes.



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  		FIG. 7.
Kit does not significantly activate Stat5 or substantially modulate Stat5 activation by EpoR forms. FDCW2-derived cell lines expressing the indexed EpoR forms (as EE chimeras) were transfected with the STAT5-specific reporter construct pSPI-GLE1-STAT5-Luc and cultured for 48 h in the presence of hEGF (10 ng/ml), SCF (50 ng/ml), or hEGF-plus-SCF. Luciferase activities in cell extracts were then determined. Mean luciferase values ± S.D. for triplicate assays are shown. Upper panel, hEGF- (and EE EpoR form) dependent Stat5 transcriptional activity. Lower panel, hEGF, SCF- and hEGF-plus-SCF-dependent Stat5 activities in FDC-EE-T-Y343, EE-T-Y343F, and EE-WT cells.

 

Key Roles for EpoR Tyr-343 in Proliferative Signaling and Kit Co-function in ES Cell-derived G1E-2 Cells—The above implicated roles of Tyr-343 in EpoR proliferative signaling and Kit co-function next were tested further in an erythroid Epoand SCF-dependent ES cell-derived model, G1E-2 cells (34). Specifically, green fluorescent protein dicistronic MIEG3 retroviruses encoding EpoR forms EE-WT, EE-T-Y343, and EET-Y343F were prepared and used to transduce G1E-2 cells. As above, FACS then was used to isolate G1E-2-EEwt, G1E-2-EET-Y343F lines expressing ~1000 receptors/cell (Fig. 8). Activities of EE EpoR forms first were assessed in hEGF-dependent proliferation assays (Fig. 9, open symbols). As observed in FDCW2 cells, EE-T-Y343 in G1E-2 cells displayed >=100% of wild-type activity (EE-WT), whereas activity of a derived Tyr(P)-null EpoR form EE-T-Y343F was decreased ~4-fold. The abilities of these EpoR forms to co-signal with endogenous Kit were assessed next (Fig. 9, closed symbols). EE-WT and EE-T-Y343 forms, but again not EE-T-Y343F, proved to co-function efficiently with Kit to promote erythroid progenitor cell proliferation (with effects as great as 5-fold above additive effects observed for EE-T-Y343 and EE-WT EpoR forms). Whether this might involve Kit effects on Stat5-specific activation was tested using pSpi-GLE1-Luc, but again revealed no such effects (Table I). Therefore, results in G1E-2 cells likewise point to a key role in Kit co-signaling for Tyr(P)-343 of the EpoR, but again via a secondary action mechanism.



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  		FIG. 8.
EE-WT, EE-T-Y343, and EE-T-Y343F receptor expression levels in G1E-2 cells. G1E-2 cells were transduced with MIEG3 retroviruses encoding the indexed EE receptor forms, and lines expressing matched receptor levels (~1000/cell) were isolated by FACS.

 


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  		FIG. 9.
The EpoR form EE-T-Y343 (but not EE-T-Y343F) co-signals proliferation with Kit in G1E-2 cells. MIEG3 retroviruses encoding EE-WT EE-T-Y343 and EE-T-Y343F were prepared and used to transduce ES cell-derived G1E-2 cells. G1E-2-wtEE, G1E-2-EE-TY343, and G1E-2-EE-T-Y343F cell lines expressing matched EE EpoR levels were then isolated by FACS, and their proliferative responses to hEGF, SCF, or hEGF-plus-SCF were determined (based on stimulated rates of [3H]dT incorporation). Mean rates ±S.D. are graphed and are representative of three independent experiments.

 

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  		TABLE I
Stat5 activation via EE-WT and MK-WT Epo and Kit receptor forms in G1E2 cells

 

Finally, to at least initially consider the converse question of which Kit-linked events might be important for EpoR co-signaling, two Kit juxtamembrane Tyr(P) sites implicated in Src kinase activation (Tyr-567 and Tyr-569) were mutated, and effects on Kit proliferative signaling were tested in G1E-2 cells transduced with wild-type or F567&569 forms. To bypass endogenous Kit, the extracellular dimerization domain of Kit was replaced with that of the human MCSF receptor (to yield MK-WT and MK-F567&569 receptor forms). These MK receptors and their expression levels in transduced and FACS-selected G1E-2 cells are shown in Fig. 10. As compared directly with the wild-type control chimera (MK-WT), MK-F567&569 was inhibited ~2-fold in its independent and EpoR-dependent co-signaling activities (Fig. 11). This outcome suggests that these sites (and linked Src kinase pathways) at least in part contribute in significant ways to Kit and Kit-plus-EpoR co-signaling routes.



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  		FIG. 10.
Construction and expression of hMCSF-mKit chimeras MK-WT and MK-F567F569. MIEG3 retroviruses encoding the diagrammed MK receptor forms were prepared and were used to transduce G1E-2 cells expressing matched levels of EE-WT, EE-T-Y343, or EE-T-Y343F Epo receptor forms. Cells expressing essentially equivalent numbers of MK receptors were then isolated by FACS and were analyzed by flow cytometry using an antibody specific to the hMCSF receptor extracellular domain. PE, phycoerythrin.

 


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  		FIG. 11.
Kit and Epo receptor co-signaling depends in part upon Kit Tyr-567 and Tyr-569 sites (and EpoR Tyr-343). In G1E-2 cells expressing matched levels of the indexed EE (EpoR) MK and (Kit) forms, the abilities of MK-WT and MK-F567F569 Kit forms to co-function with EE-WT, EE-T-Y343, or EE-T-Y343F receptors were assayed. Specifically, MK and EE receptor-dependent levels of proliferation were assayed in cells exposed to increasing concentrations of hEGF in the presence or absence of hMCSF at 15 ng/ml (~15% maximal dose). Data are the means ± S.D. of n = 3 replicates and are expressed as percent maximal response.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present investigations sought to quantitatively assess relative contributions of select EpoR Tyr(P) sites (and linked pathways) to proliferative signaling independently and in the presence of Kit co-activation. Aspects of these studies selected for discussion include: 1) the importance of attention paid to matched receptor expression levels; 2) the significant bolstering of Tyr(P)-null EpoR form activity by Tyr(P)-343, Tyr(P)-479, and to an extent, Tyr(P)-464-coupled pathways; 3) apparent selective roles for an EpoR Tyr(P)-343 Stat5 binding site in Kit co-signaling; and 4) possible molecular mechanisms that underlie EpoR and Kit co-signaling. With regards first to effects of EpoR expression levels, the EpoR is normally expressed at only hundreds of receptors/cell (44), and previously, it has been proposed that quite limited numbers of receptors may efficiently relay quantum signals (45). However, several studies have challenged this notion and have underlined the importance paid in signaling studies to EpoR expression levels. This includes studies by our laboratory in which Tyr(P)-null EpoR forms were expressed in FDCW2 cells at levels similar to Tyr(P)-343-retaining forms (as determined by Northern blotting) yet previously were observed to possess nominal proliferative activity (33); studies by Quelle et al. (46) in which clonal variability in Epo responses was described among Tyr(P)-null EpoR form transfected DA3 cells; and studies in EpoR+/- mice in which a predicted 2-fold decrease expression has been shown to perturb erythroid cell production (35). Assays of receptor expression levels are complicated by the limited availability and utility of antibodies for flow cytometric or Western blot analyses (36) and the variability of equilibrium binding analyses typically performed with biologically compromised 125IEpo preparations (45). Such considerations prompted the present use of hEGFR-mEpoR chimeras plus FACS to prepare FDCW2 and G1E-2 cell lines expressing matched physiological levels of EpoR forms.

With regards to EpoR Tyr(P) sites, although eight cytoplasmic tyrosines are conserved within the EpoR in humans, mice, and zebrafish (16), their overall functional significance recently has been brought into question by the perhaps unexpected ability of a Tyr(P)-null EpoR form ("HM") to support in vivo erythropoiesis as a targeted (knock-in) transgene in EpoR-HM mice (31). In particular, these mice have somewhat decreased hematocrits and hemoglobin levels but appear to be only mildly perturbed in overall erythropoietic capacities. More recently, however, evidence has been provided that EpoR-HM activity in these knock-in mice may depend in part on compensatory mechanisms (32). In addition, defects appear to exist during EpoR-HM-supported stress erythropoiesis, as well as Epo dose-dependent EpoR-HM progenitor cell proliferation and survival in vitro (32). Thus, compensatory mechanisms may significantly complicate interpretations of Tyr(P) mutant function in such primary cell models, and this prompted our return to cell line models for the present studies. For these studies, one basic outcome is a case that EpoR Tyr(P) sites appear to contribute overall positive roles to EpoR-dependent progenitor cell proliferation. This differs from the outcomes of analyses in which a Tyr(P)-null but Jak2-coupled EpoR form was observed in certain transfected 32D sublines (for example) to efficiently support Epo-dependent proliferation (46) but is in agreement with the majority of other studies in DA-3 (47), FDCW2 (48), and SKT6 (37) cells that have described up to 10-fold defects in signaling by Tyr(P)-null EpoR forms. This case for EpoR Tyr(P) function is also supported by studies of EpoR Tyr(P) add-back forms in primary fetal liver cells (50, 51) (which can be efficiently transduced and subsequently retain a sustained proliferative potential). Using transduced fetal liver cells from EpoR-/- mice, Tyr(P)-479 and Tyr(P)-343 each have been reported to be able to restore >=50% of transduced wild-type EpoR activity (51). In this transduced fetal liver system, however, Tyr(P)-479 was somewhat more active, whereas Tyr(P)-464 interestingly supported Epo-dependent proliferation but not CFUe formation (51). This partial activity for the putatively Src or Lyn kinase-linked EpoR Tyr(P)-464 site compares well with the activity presently observed for EE-Y464 in FDC cells (as compared with the more robust effects exerted by EpoR Tyr(P)-343 and Tyr(P)-479 sites).

The proliferative potential of EpoR-expressing erythroid progenitor cells also depends sharply upon co-activation of Kit receptor tyrosine kinase (5, 6, 52), and studies in several laboratories have described important synergistic features of EpoR and Kit co-signaling (51–53). FDCW2 cells (and G1E-2 cells) express endogenous Kit, and FACS-isolated lines expressing matched physiological levels of EE EpoR Tyr(P) forms presently were also assessed for their Kit co-signaling capacities. In each system, co-activation of EpoR plus Kit stimulated proliferation at levels significantly above additive effects. Among EpoR Tyr(P) forms studied, however, Kit synergy interestingly was supported efficiently only by Tyr(P)-343 retaining forms (and not by Tyr(P)-464 or Tyr(P)-479 add-back forms). EpoR EE-T-Y343 and control EE-T-F343 and EE-WTEpoR forms were also studied in this co-signaling context in erythroid G1E-2 cells (34), and high activity for EpoR Tyr(P)-343 (but not EE-T-F343) in Kit co-signaling of proliferation likewise was observed. Tests of the ability of Kit to modulate Stat5, however, discounted a simple possible upstream action mechanism whereby Kit might enhance Stat5 activation. Findings instead point to a more complex secondary mechanism of EpoR/Jak2/Tyr(P)-343/Stat5 plus Kit integrative signaling (see Fig. 12), and results are interesting to consider in the contexts of several recent investigations of Kit and EpoR co-function. One co-signaling model is a direct one in which the function of Kit has been proposed to be dependent upon its ability to bind, phosphorylate, and transactivate the EpoR (51). Support for this model comes from studies of co-signaling in 32D cells (51) and from studies in which SCF was shown to be important for CFUe formation in EpoR construct-transduced EpoR-/- fetal liver cells (but interestingly, not for CFUe formation from WT-EpoR control preparations) (51). As studied in HCD57 cells, however, SCF-plus-Kit-induced tyrosine phosphorylation of the EpoR apparently fails to stimulate EpoR-dependent signaling events (53), and for primary cells from mice with an inactivating Kit W/W mutation, Epo-dependent early stage erythropoiesis does not appear to be markedly perturbed (54). These findings do not discount observed physical interactions between Kit and the EpoR (51, 55) but do suggest that alternate and/or additional co-signaling mechanisms likely exist.



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  		FIG. 12.
A model for EpoR and Kit co-signaling. Synergistic co-signaling by the EpoR and Kit is proposed to depend upon EpoR/Jak2/EpoR-Y343/Stat5-modulated genes. Kit may either co-modulate these genes or integrate its signals with products of select EpoR/Stat5 gene products.

 

The presently proposed model for EpoR and Kit co-signaling implicates EpoR-activated Stat5 as one important integrator, and this model is supported by several recent independent reports. First, although certain studies suggest that Kit might contribute to Jak2 and Stat5 signaling (56), this was not detectable in studies in HCD57 cells by Jacobs-Helber et al. (53) or in previous studies by our laboratory using FDCW2 as well as primary erythroid progenitor cells (33). However, we have shown previously that a Tyr(P)-343-retaining EpoR form enhances the Kit-induced Bcl-x gene expression (33), and Kapur and Zhange (57) have proposed that Kit signaling contributes to the sustained expression of Stat5 protein (which can then be activated by Epo). In the present studies, a sensitive and functional reporter assay system was used to determine that Kit essentially fails to contribute to Stat5 activation in the absence or presence of EpoR stimulation in either FDCW2 or G1E-2 cells. The apparent requirement of EpoR Tyr(P)-343 for Kit co-signaling therefore again invokes a model in which the products of Stat5 target genes act to enhance Kit signaling. In studies of EpoR and Kit co-stimulation of Bcl-x gene expression, synergistic effects interestingly were delayed and were not realized until ~4 h of cytokine co-exposure (33), and this finding is consistent with this proposed secondary action mechanism. Finally, in expressing an AP20187-inducible LV'V-Jak2 fusion protein in primary hematopoietic progenitor cells, Zhao et al. (49) recently discovered that strong proliferative effects are exerted by this dimerizing Jak2 construct only upon the activation of Kit or a related Flt3 receptor tyrosine kinase. In these studies, Stat5a,b-/- mice were used to show that this example of Kit and Jak2 proliferative co-signaling also depended upon Stat5. In future experiments, it should be interesting to discover the extent to which the ability of Kit to co-function with additional Jak2- and/or Stat5-linked type-1 cytokine receptors might likewise involve integrations with Jak/Stat5-activated events, as well as the specific nature of underlining molecular mechanisms.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants RO1s HL44491 and DK40242 (to D. M. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: 115 Henning Bldg., The Pennsylvania State University, University Park, PA 16802. Tel.: 814-863-8329; Fax: 814-863-6140; E-mail: dmw1{at}psu.edu.

1 The abbreviations used are: EpoR, erythropoetin receptor; Jak, Janus kinase; Stat, signal transducer and activator of transcription; CIS, cytokine-inducible SH2 protein; PSF, penicillin streptomycin and fungizone (amphotericin B); FBS, fetal bovine serum; EGF, epidermal growth factor; EGFR, EGF receptor; MCSF, macrophage colony-stimulating factor; SCF, stem cell factor; IL-3, interleukin-3; MSCV, murine stem cell virus; WT, wild type; ES, embryonic stem; FACS, fluorescence-activated cell sorter; PI3K, phosphatidylinositol 3-kinase; IMDM, Iscove's modified Dulbecco's medium; CFUe, colony-forming unit erythroid; h, human; m, murine. Back


   ACKNOWLEDGMENTS
 
The authors thank Dr. Ruben Kapur (Indiana University) for the generous provision of pMIEG3 and a chimeric MK cDNA construct; Dr. Dwayne Barber (University of Toronto, Ontario) for select Epo receptor PY mutant cDNAs; and Dr. Mitchell Weis (University of Pennsylvania) for G1E-2 cells.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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