 |
INTRODUCTION |
Survival, proliferation, and differentiation of hematopoietic
cells are regulated by multiple cytokines (1), and erythropoietin (Epo)1 is essential for
erythropoiesis. By binding to the erythropoietin receptor (EpoR), a
member of the hematopoietic cytokine receptor superfamily, Epo prevents
apoptosis of erythroid progenitor cells and promotes their
proliferation and erythroid maturation (2). The formation of
erythrocytes is tightly controlled by the coordinated activation of
several signal-promoting and signal-terminating cascades activated by
the EpoR (3). The receptor-associated tyrosine kinase JAK2 is
essential for surface appearance of the EpoR (4) and becomes activated
after Epo binding. JAK2 in turn phosphorylates several tyrosine
residues on the EpoR-cytosolic domain and probably on JAK2 itself that
serve as docking sites for SH2 or protein tyrosine binding
domains of downstream signal transduction proteins such as STAT5,
phosphatidylinositol 3-kinase, Shc, and tyrosine phosphatases
SHP1 and SHP2 (5-10).
EpoR (phospho)tyrosine residues 343 and 401 serve as docking sites for
the latent transcription factor STAT5 (5-7). STAT5 becomes
tyrosine-phosphorylated upon receptor recruitment, homodimerizes, and
migrates to the nucleus where it promotes the activation of target
genes. STAT5 activates transcription of the Bcl-xL gene (11, 12). Bcl-xL has an essential role in
preventing apoptosis of primitive and definitive erythrocytes at the
end of maturation (13), and the STAT5 Bcl-xL signaling pathway
has been suggested to protect cells from apoptosis and to promote cell
proliferation (11, 12). A STAT5a/b null mutation
in fetal and neonatal mice leads to defects in erythroid maturation
accompanied by increased apoptosis of erythroid progenitors (14),
supporting the notion that the STAT5 Bcl-xL pathway
mediates anti-apoptotic effects in erythroid cells.
For the controlled production of erythroid cells, it is critical that
the activation of signal-promoting cascades is counter-balanced by
terminating events. In erythroid cells, these include activation of the
tyrosine phosphatase SHP1 that dephosphorylates JAK2 (9) and induction
of the negative regulatory protein CIS (cytokine-inducible SH2
domain-containing protein) (15). CIS lacks enzymatic activity and
belongs to the family of suppressor of cytokine signaling (SOCS)
proteins. These proteins possess a SH2 domain in the middle that
mediates binding to phosphotyrosine residues and a SOCS Box at the C
terminus that has been implicated in proteasomal degradation. By the
use of a limited set of mutated EpoRs, tyrosine 401 in the cytoplasmic
domain has been identified as the binding site for CIS (16). However,
Tyr-401 is a multi-task residue that binds (besides CIS) other SOCS
family members such as SOCS2 (17) and SOCS3 (18, 19) as well as the
phosphatidylinositol phosphatase SHIP, the tyrosine
phosphatase SHP2 (10), and STAT5 (5-7).
Forced expression of CIS inhibits proliferation of cell lines in
response to Epo or interleukin-3 (IL-3) and results in reduced activation of STAT5 (20, 21). The molecular mechanism of this effect is
poorly understood because CIS lacks the kinase inhibitory region that
mediates JAK2 inactivation by SOCS1 and SOCS3 (18). It has been
suggested that the CIS SH2 domain competes with STAT5 for binding to
the EpoR (18), and the CIS SOCS Box has been proposed to mediate
proteasome-dependent degradation of the EpoR (16). The
physiological role of CIS for the regulation of erythropoiesis remains
to be determined since analyses of the two transgenic animal models
harboring CIS under the control of the
-casein promoter (22) or the
CD4 promoter (23) were focused on development of the mammary gland and
on T-cell signaling, respectively.
During embryonic development, CIS is first expressed in the fetal liver
at embryonic day 12.5, the same stage as the EpoR (24). The role of CIS
in fetal erythropoiesis has not been addressed yet since most studies
on growth and survival have been undertaken in cell lines. Here we show
that CIS overexpression mimics STAT5 loss-of-function in erythroid
progenitor cells, and we determine which CIS domains are essential for
this effect. To this end, we expressed GFP-tagged versions of the CIS
protein in erythroid progenitor cells under the control of a retroviral
promoter, which uncoupled CIS synthesis from activation of the
JAK/STAT pathway. We show that elevated levels of CIS inhibit
proliferation of erythroid progenitor cells. Although erythroid
differentiation is unaffected, an intact CIS SH2 domain is essential
for inhibition of proliferation. Similar to bone marrow-derived
erythroid progenitor cells in STAT5 knock-out mice (14), erythroid
progenitor cells from fetal liver overexpressing CIS show an increased
tendency to undergo apoptosis, which requires both the CIS SH2 domain
and the SOCS box. Thus, we propose that CIS negatively regulates
signaling through the EpoR by two mechanisms. 1) Recruitment of CIS to
the EpoR via its SH2 domain is sufficient to repress proliferative
responses. 2) In addition, the SOCS Box mediates apoptotic effects of
CIS expression in fetal liver erythroid progenitor cells.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Single or double Tyr to Phe mutant EpoRs were
generated by PCR mutagenesis and inserted into the eukaryotic
expression vector pXM as described previously (5, 9). EpoR Y401F
was subcloned in frame as a ApaI- and
EcoRI-digested fragment into the appropriate restriction
sites of the retroviral expression vector pMX (puro)-EpoR. GFP-CIS
fusion proteins were established by introducing an in-frame BglII restriction site at the 5'-end and a EcoRI
restriction site at the 3'-end of the CIS cDNA and subcloning the
DNA fragment in frame into the BamHI and EcoRI
restriction sites of the retroviral expression vector pMX-enhanced GFP
or pOSdSV (25). The SOCS Box from CIS was deleted at amino acid 182, and the fragment was cloned via BglII and EcoRI
into the BamHI and EcoRI restriction sites of
pMOWS-GFP (25). GFP-SOCS3 was generated by introducing BamHI
and NotI restriction sites at the 5' and 3' ends and
subcloned in-frame into pMOWS-GFP. Arginine 107 localized within the
SH2 domain of CIS was replaced by lysine using overlap extension PCR and subcloned into the BamHI and EcoRI
restriction sites of pMX-enhanced GFP.
Cell Lines and Cultures--
BaF3 cell lines expressing single
or double Tyr to Phe mutant EpoRs in pXM were generated as described
previously (5, 9). By retroviral transduction, pMX-EpoR or pMX-EpoR
Y401F was introduced into the IL-3-dependent pro-B cell
line BaF3. BaF3 cells were maintained in RPMI 1640 medium
(Invitrogen) supplemented with 10% fetal calf serum
(Invitrogen) and 10% WEHI-conditioned medium. Pools of stable
transfectants were selected in 1.5 µg/ml puromycin (Sigma). The
retroviral packaging cell line Phoenix-eco was maintained in
Dulbecco's modified Eagle's medium (Invitrogen) supplemented with
10% fetal calf serum.
Fetal Liver Cell Preparation and Transduction--
Fetal livers
from 13.5-day-old BALB/c mouse embryos were prepared as
described elsewhere (25). For Ter119 expression kinetics, the fetal
liver cell preparation was incubated with anti-Ter119 (provided by Dr.
Albrecht Müller) and subjected to AutoMACS depletion (Miltenyi
Biotech, Bergisch-Gladbach, Germany). For TUNEL assay, the cells were
depleted for hematopoietic lineages with an antibody mixture as
described previously (25). Retroviral expression vectors were
transiently transfected into Phoenix-eco cells using the
Calcium-phosphate method (25). Twenty-four hours after transfection of
Phoenix cells, the medium was changed to Iscove's modified Eagle's
medium containing 50 µM
-mercaptoethanol and
30% fetal calf serum. Twenty-four hours later, 1 ml of
virus-containing supernatant was harvested and filtered through a
0.45-µm filter. For spin infection, the supernatant was mixed with
1 × 104 (for colony assays) or 5 × 104 (for liquid culture) freshly prepared fetal
liver cells and spun for 2 h in an Eppendorf centrifuge at 1800 rpm at room temperature. The cells were supplemented with 0.4 unit/ml
Epo (Cilag-Jansen, Bad Hamburg, Germany) and plated in 0.8%
methylcellulose (StemCell Technologies, Vancouver, Canada) or seeded in
Iscove's modified Eagles's medium, 30% fetal calf serum, 50 µM
-mercaptoethanol supplemented with 0.4 unit/ml Epo.
GFP-positive and hemoglobinized erythroid colonies were identified by
benzidine staining.
Growth Assay--
BaF3 cells expressing the indicated EpoRs in
the context of pMX were washed three times in medium and plated at a
density of 5 × 104 cells/well in 24-well plates.
After 3 days in culture in the indicated concentrations of Epo or in
10% WEHI-conditioned medium, cell numbers were determined using a
Coulter counter.
Immunoprecipitation and Immunoblotting--
For BaF3 cells
expressing the wild-type EpoR or EpoR, Y401F 1 × 107
cells were used per immunoprecipitation. Lysis and immunoprecipitation experiments were carried out as described previously (9) using the
following antibodies: crude rabbit antiserum raised against the
extracellular domain of the EpoR (9), rabbit antiserum raised against
CIS (kindly provided by A. Yoshimura), and rabbit antiserum raised
against a GST-GFP fusion protein. Proteins were detected by
immunoblotting with anti-phosphotyrosine antiserum 4G10 (Upstate
Biotechnology, Palo Alto, CA) followed by enhanced chemiluminescence
(Amersham Biosciences). Membranes were denatured with
-mercaptoethanol/SDS before reprobing for control of equal protein loading.
FACScan Analysis--
To determine surface expression of
HA-tagged EpoR (26), BaF3 cells were transduced with retroviral
supernatants and sorted for GFP-expression using MoFlo (Cytomation,
Colorado). The sorted cells were incubated with rat anti-HA (Roche
Molecular Biochemicals) as primary antibody and anti-rat IgG coupled to
Cy5 (Dianova) as secondary antibody and analyzed for green and red
fluorescence by flow cytometry. For apoptosis assay, the cells were
stained with 5 µl of VIAprobe 7-AAD (BD Biosciences) and 5 µl of annexin V coupled to phycoerythrin (PE) or Cy5 (BD Biosciences)
according to the manufacturer's instructions. For TUNEL assay, lineage
negative fetal liver cells were cultivated for 24 h in serum-free
medium PANserin401 (PANbiotech) supplemented with 0.05 unit/ml Epo. The cells were washed with phosphate-buffered saline, fixed with 2% paraformaldehyde, and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline. The TUNEL assay was performed using the
TMR-Red in situ cell death kit (Roche
Molecular Biochemicals) according to the manufacturer's instructions.
Ter119 staining of fetal liver cells as a marker for erythroid
differentiation (27) was performed with a rat anti-Ter119 antiserum
(kindly provided by Dr. Albrecht Müller, Julius-Maximilian
University, Würzburg, Germany) and a secondary anti-rat IgG
coupled to Cy5. GFP, PE, Cy5, and TMR-Red fluorescence were detected
with a Becton Dickinson FACScan (BD Biosciences) using the CellQuest software.
 |
RESULTS |
Importance of EpoR Tyrosine 401 for Proliferative Signaling and
Survival--
To confirm that Tyr-401 in the EpoR cytoplasmic domain
represents the major CIS binding site, we expressed the wild type EpoR or a panel of EpoRs containing specific single or double tyrosine to
phenylalanine mutations in the IL-3-dependent pro-B cell
line BaF3 and tested in co-immunoprecipitation experiments for their ability to bind CIS. Despite lacking one or two tyrosine residues, the
mutant EpoRs were tyrosine-phosphorylated upon Epo stimulation to the
same extent as the wild type receptor as judged by immunoprecipitation with antiserum recognizing the EpoR followed by immunoblotting with an
anti-phosphotyrosine antibody (Fig.
1A). In agreement with the
observation of others, tyrosine-phosphorylated mutant EpoR Y401F was
greatly impaired in its ability to associate with CIS (Fig.
1A, lower panel, lane 3). Because a
faint band representing the tyrosine-phosphorylated EpoR Y401F was
consistently observed in these co-immunoprecipitation experiments, we
conclude that Tyr-401 represents the major binding site for CIS on the
EpoR but that other minor binding sites exist.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
Proliferation and survival of BaF3 cells
stably expressing the wild-type EpoR or the mutant EpoR Y401F.
A, BaF3 cells or BaF3 cells stably expressing the wild type
EpoR or the various tyrosine to phenylalanine point mutant EpoRs were
stimulated with 100 units/ml Epo for 5 min, lysed, and subjected to
immunoprecipitation (IP) using antiserum directed against
the EpoR ( EpoR) or CIS ( CIS). The
immunoprecipitates were analyzed by SDS-PAGE and visualized by
anti-phosphotyrosine immunoblotting (IB: PTyr)
followed by ECL. B, pools of BaF3-EpoR or BaF3-EpoR Y401F
cells were seeded at a density of 5 × 104 cells/ml in
the presence of either 0.1-10 units/ml Epo or in the presence of 10%
WEHI-conditioned medium as a source of IL-3. After 3 days in culture,
the cell number was determined using a Coulter counter. The cell
numbers obtained were plotted as the percentage of growth obtained in
the presence of WEHI-conditioned medium. For BaF3 cells stably
expressing the mutant EpoR Y401F, three independent cell pools were
analyzed and the results were reproduced at least three times.
C, cell surface expression of HA-tagged EpoR and EpoR.
Parental BaF3 cells (dark gray) or BaF3 cells stably
expressing HA-EpoR (medium gray), HA-EpoR Y401F
(white) were incubated with rat anti-HA antiserum followed
by Cy5-labled anti-rat IgG and analyzed by flow cytometry. The mean
fluorescence intensity (MFI) detected in three independent
experiments ± S.D. is indicated. D, BaF3 cells stably
expressing EpoR (solid line) or EpoR Y401F (dashed
line) were stained with 7-AAD to determine by flow cytometry the
number of dead cells after 24 h in culture in the indicated
concentrations of Epo or IL-3.
|
|
Since Tyr-401 has been identified as the binding site for multiple
signal-promoting and signal-terminating molecules including CIS, we
asked whether the absence of Tyr-401 increased or decreased biological
responsiveness of the EpoR. To examine proliferative signaling, BaF3
cells stably expressing the wild type EpoR or the mutant EpoR Y401F
were cultivated in the presence of increasing concentrations of Epo
ranging from 0.1 to 10 units/ml. After 3 days, the cell number was
determined and normalized to the cell growth obtained in the presence
of WEHI-conditioned medium. All cell pools analyzed showed comparable
growth rates in WEHI-conditioned medium. When grown in Epo,
half-maximal proliferation of BaF3 cells expressing the wild type EpoR
was achieved at 1 unit/ml Epo and equaled 17% of the growth obtained
in the presence of WEHI-conditioned medium (Fig. 1B). At 10 units/ml Epo, these cells grew to 45% of the number achieved in
WEHI-conditioned medium. In contrast, although proliferation of BaF3
cells expressing EpoR Y401F remained Epo-dependent, these
cells grew better at all Epo concentrations. At the highest Epo
concentration employed, 76% of the growth observed in WEHI-conditioned
medium was achieved compared with 45% obtained upon expression of the
wild type EpoR. The expression of mutant EpoR Y401F allows cells to
proliferate in Epo 2-4-fold less than normal and enables cells
to grow to higher density. To confirm that the growth-promoting effects
of EpoR Y401F are not caused by increased surface prevalence, we expressed HA-tagged forms of the wild type EpoR and EpoR Y401F in BaF3
cells and analyzed the extent of surface expression by flow cytometry
(Fig. 1C). This analysis revealed that in comparison to the
HA-tagged wild type receptor, the amount of HA-EpoR Y401F detectable on
the cell surface is not enhanced but rather slightly reduced.
To determine whether the increased yield of BaF3 cells expressing EpoR
Y401F correlates with increased cell survival, we performed viability
staining of BaF3-EpoR and BaF3 EpoR Y401F cells. The cells were
cultivated in the absence of growth factor or in the presence of 0.1 or
1 unit/ml Epo or, as a positive control, 2 ng/ml IL-3. The cells were
stained with 7-AAD, a dye that stains dead cells. (Fig. 1D).
As expected, in the absence of cytokine, the majority of cells (90.7%
of BaF3-EpoR and 85.9% of BaF3-EpoR Y401F cells) underwent death
within 24 h, whereas in the presence of IL-3, death was reduced to
~13% of the cells. Similarly, in 1 unit/ml Epo, only 20.5%
BaF3-EpoR cells and 10.8% BaF3-EpoR Tyr-401 cells were
7-AAD-positive. At a lower Epo concentration (0.1 unit/ml), the number
of 7-AAD-positive BaF3-EpoR cells increased to 50.0% but only 16.5%
BaF3-EpoR Y401F cells were dead. Thus, the absence of Tyr-401 from the
EpoR decreases sensitivity to undergo cell death and increases the
proliferative yield, suggesting that EpoR Tyr-401 is important for
activating a down-modulating signal.
Role of the CIS SH2 Domain for Binding to the EpoR--
To
determine the domains in CIS required for binding to the EpoR and for
exerting biological functions, we generated GFP-tagged versions of wild
type CIS and a mutant CIS harboring an inactivated SH2 domain (CIS
R107K). To examine binding to (phospho)tyrosine 401 of the EpoR, GFP,
GFP-CIS, and GFP-CIS R107K were stably expressed in parental BaF3,
BaF3-EpoR, and BaF3-EpoR Y401F cells. The cells were stimulated
with Epo, lysed, and subjected to immunoprecipitation using
antisera against the EpoR or GFP. Tyrosine-phosphorylated EpoR
complexed with GFP fusion proteins were subsequently identified by
immunoblotting with anti-phosphotyrosine antiserum (Fig.
2). As expected, GFP alone was unable to
bind either one of tyrosine-phosphorylated receptors (Fig. 2,
lanes 5 and 6). However, compared with the total
amount of phosphorylated EpoR (Fig. 2, lanes 2 and
3), GFP-CIS preferentially bound the tyrosine-phosphorylated
wild type EpoR and to a lesser extent tyrosine-phosphorylated EpoR
Y401F (Fig. 2, lanes 8 and 9). By testing a panel
of tyrosine-to-phenylalanine mutant EpoRs (Fig. 1A), we
could demonstrate that, despite the residual association of GFP-CIS
with EpoR Y401F, Tyr-401 represents the major binding site for CIS in
the EpoR (Fig. 2, lane 8). Conversely, mutating the critical
arginine 107 in the CIS SH2 domain completely abrogated the ability
of CIS to bind the tyrosine-phosphorylated EpoR (Fig. 2, lanes
11 and 12). This finding confirms the importance of the
SH2 domain for receptor recruitment of CIS.

View larger version (87K):
[in this window]
[in a new window]
|
Fig. 2.
Association of a green fluorescent
protein-tagged CIS with tyrosine phosphorylated EpoR. Parental
BaF3 cells or BaF3 cells stably expressing the wild type EpoR or the
mutant EpoR Y401F were transduced with retroviral expression vectors
pMX-GFP, pMX-GFP-CIS, or pMX-GFP-CIS R107K. In this retroviral vector,
the transcription of the GFP-CIS gene is promoted by the long terminal
repeats and transcription of the puromycin resistance gene is from an
internal SV40 promoter. Cell pools expressing GFP or the GFP fusion
proteins were selected in the presence of puromycin and grown in the
presence of WEHI. After immunoprecipitation with anti-EpoR or anti-GFP
antiserum, tyrosine-phosphorylated EpoR was detected by immunoblotting
with anti-phosphotyrosine and anti-GFP antiserum. The positions of the
tyrosine-phosphorylated EpoR (pY-EpoR), GFP-CIS, and GFP are
indicated with arrows.
|
|
CIS Reduces Growth of Fetal Liver Erythroid Progenitor
Cells--
To elucidate whether binding of CIS to the EpoR negatively
regulates erythropoiesis, GFP-CIS and GFP-CIS R107K were transduced by
retroviral infection into fetal liver cells and tested for their effect
on CFU-E colony formation. The transduction efficiency ranged from 30 to 50% and was the same for transduction of GFP-CIS and GFP-CIS R107K.
As shown in Fig. 3A, the total
number of CFU-E colonies expressing GFP-CIS was not significantly
reduced compared with colonies expressing GFP-CIS R107K or GFP.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of GFP-CIS fusion proteins on CFU-E
colony formation and erythroid differentiation. A, GFP,
GFP-CIS, and GFP-CIS R107K were transduced into Ter119-depleted day
13.5 fetal liver cells and grown in methylcellulose for 3 days in 0.5 units/ml Epo. The colonies were stained with benzidine and
benzidine-positive colonies with >8 cells/colony considered as CFU-E.
B, after 23 h, GFP-positive erythroid colonies were
identified by fluorescence microscopy (middle panel) and
bright field microscopy (left panel) using a Zeiss Axiovert
100 microscope. After 72 h, hemoglobinization of the colonies was
visualized by benzidine staining (right panel). Pictures of
representative colonies are shown. C, after 23 h,
GFP-positive erythroid colonies at the 4-, 8-, and 16-cell stage were
identified by fluorescence microscopy and the number of cells within
each GFP-positive colony was counted. The white bars
represent values obtained for erythroid progenitors expressing GFP-CIS,
whereas the gray bars indicate the values for GFP-CIS R107K.
The numbers presented are the mean of three independent
experiments ± S.D. The overall infection rate of erythroid
colonies was 41 and 31% for GFP-CIS and GFP-CIS R107K, respectively.
Single cells and two-cell colonies were not considered. Significances
were calculated according to a two-sided paired Student's t
test.
|
|
Colonies grown in methylcellulose supplemented with 0.4 unit/ml Epo, thus favoring growth of CFU-E colonies, were inspected by
fluorescence microscopy to monitor GFP-positive CFU-E colonies. As
shown in Fig. 3B, the expression of GFP-CIS and GFP-CIS
R107K in erythroid progenitors is comparable with each other and is predominantly localized to the cytoplasm.
To determine the effect of unregulated CIS expression on proliferation
of erythroid progenitor cells, we investigated the cell proliferation
rate within each GFP-positive colony. After 20-25 h of culture in Epo,
colonies containing 4-16 cells predominated. To detect alterations in
colony size, the formed erythroid colonies were classified into three
categories: 4 cell, 4-8 cell, and 9-16 cell. After 23 h in
culture, the percentage of the largest (9-16 cell) colonies expressing
GFP-CIS (25%) was reduced compared with the percentage of the 9-16
cell colonies expressing GFP-CIS R107K (71%) (Fig. 3C). The
latter result was identical to the distribution of colony sizes
after transduction of the control GFP protein (data not shown). Thus,
the expression of GFP-CIS but not GFP-CIS R107K reduces proliferation
of erythroid progenitor cells, indicating the importance of the CIS SH2
domain in this process.
To determine whether these erythroid colonies were defective in
differentiation, we directly examined all GFP-positive colonies after
24 h of cultivation for the extent of benzidine staining and
detected no difference among the fusion proteins (data not shown). We
conclude that the observed colonies are of erythroid origin and that
maturation of erythroid progenitors is normal even though proliferation
is impaired as a consequence of CIS expression. Accordingly, benzidine
staining of CFU-E colonies after 3 days in culture with Epo did not
show a difference between cells expressing GFP-CIS, GFP-CIS R107K, or
GFP (Fig. 3B, right panel). Similarly, as judged
by fluorescence-activated cell sorter analysis, Ter119 expression
occurred at normal rates in GFP-CIS, GFP-CIS R107K, and GFP-expressing
cells, reaching a plateau of >80% positive cells after 12-18 h of
culture (data not shown). Ter119 is closely associated with
glycophorin, an erythroid-specific integral membrane protein (27).
Thus, the recruitment of CIS via its SH2 domain to the EpoR negatively
regulates proliferative responses in erythroid progenitor cells,
whereas erythroid differentiation is not affected.
SH2 Domain-mediated Receptor Recruitment of CIS Accelerates
Apoptosis of BaF3 Cells--
To further dissect the role of the
domains present in CIS, we generated, in addition to the GFP-tagged CIS
protein harboring an inactivated SH2 domain (GFP-CIS R107K), a GFP-CIS
fusion protein lacking the C-terminal region including the SOCS Box
that has been implicated in proteasomal degradation (GFP-CIS
Box). To
gauge the effectiveness of CIS, we constructed a GFP-tagged SOCS3
protein since SOCS3 has been previously demonstrated to significantly reduce growth of Epo-dependent BaF3 cells (18). As in
erythroid progenitor cells, the subcellular localization of GFP-CIS and GFP-CIS R107K (data not shown) in the adherent retroviral packaging cell line Phoenix-eco was comparable and exclusively cytosolic (Fig.
4A), whereas GFP and
remarkably GFP-SOCS3 and GFP-CIS
Box were detected throughout the
cell.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of GFP-CIS on growth in BaF3
cells. A, GFP, GFP-CIS, GFP-CIS Box, or GFP-SOCS3 was
transfected into the retroviral packaging cell line Phoenix-eco.
Representative cells were photographed 48 h after transfection
using a Zeiss Axiovert 100 fluorescence microscope. B,
GFP-CIS, GFP-CIS R107K, GFP-SOCS3, and GFP as a control were transduced
into BaF3 cells stably expressing the wild type EpoR and were
cultivated in the presence of 1 unit/ml Epo. At the indicated time
points, the expression of GFP or the GFP fusion proteins was evaluated
by flow cytometry and used to calculate the number of transduced
GFP-positive cells.
|
|
To determine the effect of the various GFP-tagged fusion proteins on
proliferative responses, we used retroviral vectors to generate stable
populations of BaF3-EpoR cells expressing GFP-CIS, GFP-CIS R107K,
GFP-CIS
Box, and GFP and monitored the percentage of GFP-positive
cells during 1-13 days in the presence of 1 unit/ml Epo. By applying
comparable viral titers for each fusion protein, we routinely achieved
transduction rates of 23-27% GFP-positive BaF3-EpoR cells. During the
observation period, the fraction of GFP expressing BaF3-EpoR steadily
increased from ~27 to 40%, showing that GFP expression had no
general toxic effect on cell proliferation. The percentage of
GFP-SOCS3-expressing cells was reduced to 15% just after 1 day of
culture and reached 2.1% after 3 days (Fig. 4B). Thus, the
expression of this SOCS3 fusion protein blocked cell proliferation. The
expression of GFP-CIS was less inhibitory. After 3 days, the fraction
of GFP-CIS-positive cells decreased to 29.2%, and this percentage
decreased with time in culture to 13% after 9 days. Similar inhibition
of proliferation was achieved by expression of GFP-CIS
Box in
BaF3-EpoR cells (data not shown), confirming the notion that the SOCS
Box is not essential for inhibition of cell proliferation. In contrast,
the GFP-CIS R107K mutant was constantly expressed in 26-29% of the
cells between days 3 and 13, indicating that it does not lead to
inhibition of cell proliferation. Thus, the anti-proliferative effect
of CIS in BaF3 cells requires receptor binding of CIS via the SH2
domain, whereas proteasomal degradation facilitated by the CIS SOCS Box
is of lesser importance.
To elucidate whether CIS binding to tyrosine 401 is involved in
apoptosis, we transduced GFP, GFP-CIS, and GFP-CIS R107K into BaF3-EpoR
and BaF3-EpoR Y401F cells and cultured the cells for 24 h in 0.1 unit/ml Epo (Fig. 5A). By flow
cytometry, we quantified the amount of apoptotic cells in these
cultures as annexin V-positive viable cells. The shown panels were
gated on GFP-positive cells, and in both cell lines, ~53-65% of the
cells expressed GFP-CIS and GFP-CIS R107K. Strikingly, the
overexpression of GFP-CIS led to an increased number of annexin
V-positive BaF3-EpoR cells (33.0%) compared with GFP-CIS R107K
(11.9%) or GFP alone (10.4%). In contrast, in BaF3-EpoR Y401F cells,
GFP-CIS had no effect on the number of apoptotic cells. Furthermore,
BaF3-EpoR cells expressing GFP-CIS
Box showed an overall reduced GFP
fluorescence compared with BaF3-EpoR cells expressing GFP-CIS but a
similar annexin V-staining pattern that differed from BaF3-EpoR cells
harboring GFP alone (Fig. 5B). Thus, we conclude that
through the binding of its SH2 domain to tyrosine 401 of the EpoR,
GFP-CIS promotes apoptotic cell death in BaF3-EpoR cells at low Epo
concentrations.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of GFP-CIS on apoptosis in BaF3
cells. A, BaF3 cells stably expressing EpoR or
EpoR Y401F were transduced with GFP-CIS (blue line), GFP-CIS
R107K (red line), and GFP (green line) and grown
in 1 unit/ml Epo. After 24 h, the cells were stained with annexin
V coupled to PE to determine the number of apoptotic cells and analyzed
by flow cytometry. The panels are gated on GFP-positive cells.
B, BaF3 cells stably expressing the wild type EpoR were
transduced with GFP, GFP-CIS, or GFP-CIS Box, were sorted for GFP
expression, and cultivated in 0.01 unit/ml Epo. After 28 h, the
cells were stained with annexin V coupled to Cy5 and analyzed by flow
cytometry.
|
|
Receptor Recruitment of CIS Decreases STAT5 Activation by the EpoR
in BaF3 Cells--
To elucidate mechanisms mediating pro-apoptotic
effects of GFP-CIS in BaF3 cells, we asked whether the effect of GFP,
GFP-CIS, and GFP-CIS R107K expression on apoptosis in BaF3 cells
paralleled inhibition of Epo-induced STAT5 activation. To test this
possibility, GFP, GFP-CIS, or GFP-CIS R107K were expressed in BaF3
cells stably expressing the wild type EpoR or the mutant EpoR Y401F.
The ability of the obtained cell lines to activate STAT5 in response to
increasing concentrations of Epo ranging from 0.5 to 10 units/ml Epo
was tested by immunoprecipitation of STAT5 from the cellular lysates and subsequent analysis by immunoblotting with anti-phosphotyrosine (Fig. 6). At low Epo
concentrations (0.5 unit/ml Epo), STAT5 tyrosine phosphorylation was
reduced by the expression of GFP-CIS to ~50% of the level achieved
in BaF3 EpoR cells co-expressing GFP or GFP-CIS R107K. At higher Epo
concentrations, the extent of STAT5 tyrosine phosphorylation was
identical in cells expressing GFP, GFP-CIS, or GFP-CIS R107K. In
contrast, in BaF3-EpoR Y401F cells, tyrosine phosphorylation of STAT5
was elevated ~2-fold at lower Epo concentrations and was not
influenced in a concentration-dependent manner by the
co-expression of GFP-CIS. Therefore, in comparison with the
apoptosis-promoting effects, CIS-mediated inhibition of STAT5
phosphorylation depends upon the presence of an intact SH2 domain
within CIS and the presence of (phospho)tyrosine 401 within the EpoR.
This finding suggests that in BaF3 cells the recruitment of CIS to the
EpoR can impair STAT5 activation and thereby accelerate the induction
of apoptosis.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
CIS reduces STAT5 activation in BaF3
cells. BaF3 cells stably expressing the wild type EpoR (BaF3 EpoR)
or the mutant EpoR Y401F (BaF3 EpoR Y401F) in conjunction with GFP or
GFP-CIS or GFP-CIS R107K were left unstimulated or were stimulated for
5 min with 0.5, 1, 5, or 10 units/ml Epo, lysed, and subjected to
immunoprecipitation with an anti-STAT5 antiserum. The analysis by
immunoblotting was performed using an anti-phosphotyrosine monoclonal
antibody ( PTyr). To verify equal loading, the blots were
stripped and reprobed with anti-STAT5 antiserum. As control, parental
BaF3 cells were stimulated with 10 units/ml Epo.
|
|
CIS SH2 Domain and SOCS Box Are Required to Promote Apoptosis in
Erythroid Progenitor Cells--
To investigate whether CIS promotes
apoptosis in erythroid progenitor cells, we performed TUNEL staining of
retroviral transduced fetal liver erythroid progenitor cells grown in
serum-free medium supplemented with 0.05 unit/ml Epo. The flow
cytometry analysis shown in Fig. 7
revealed that under these conditions ~3% untransduced or
GFP-expressing erythroid progenitor cells were undergoing apoptosis. In
contrast, of the green fluorescent cells expressing GFP-SOCS3, 12%
were undergoing apoptosis. Similarly, the expression of GFP-CIS increased the proportion of apoptotic cells to 9%. This increase in
apoptotic cells upon the expression of GFP-CIS was significant (p < 0.05) and reproducibly observed also with
bicistronic expression using the pOS-IRES-GFP vector (data not shown).
Importantly, CIS mutants defective in the SH2 domain (4.7%) or missing
the SOCS Box (3.6%) did not promote apoptosis, thus indicating that
both domains are essential to mediate the pro-apoptotic effect of CIS in erythroid progenitor cells.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 7.
CIS enhances apoptosis in erythroid
progenitor cells. Fetal liver cells were depleted by MACS beads
with antibodies raised against hematopoietic lineages, transduced with
empty vector, GFP-CIS, GFP-CIS R107K, GFP-CIS Box, and GFP-SOCS3, and
cultured for 24 h in serum-free medium PANserin401 supplemented
with 0.05 unit/ml Epo. Fixed and permeabilized cells were stained with
the TMR-Red TUNEL assay kit, and apoptotic cells were analyzed
concomitantly in GFP-positive cells by flow cytometry. The
boxes indicate GFP and TMR-Red positive cells gated on
viable cells according to forward scatter/side scatter discrimination
given as percentage of all viable cells.
|
|
 |
DISCUSSION |
At the molecular level, developmental processes such
as maturation of erythrocytes are controlled by precise orchestration of activating and inhibiting intracellular signal transduction cascades. Our principal result is that the overexpression of the negative regulatory protein CIS reduces signaling through the EpoR both
in cell lines and in primary erythroid progenitors. This results in
reduced proliferation and increased apoptosis of erythroid progenitor
cells, suggesting an important role for CIS in setting a threshold for
erythrocyte production triggered by Epo.
SH2 Domain-mediated Receptor Recruitment of CIS and Inhibition of
Cell Proliferation--
Our results showed that the recruitment to the
EpoR is essential for the negative regulatory function of CIS. In
agreement with the studies of Verdier et al. (16), we
identified (phospho)tyrosine residue 401 in the EpoR cytoplasmic domain
as the principal binding site for the CIS SH2 domain (16). By analyzing
an extended panel of Tyr-to-Phe mutant EpoRs, we confirmed that,
despite a residual association of GFP-CIS to a mutant EpoR lacking
Tyr-401 (EpoR Y401F), this residue represents the major CIS binding
site. The negative regulatory effect of CIS binding to
(phospho)tyrosine 401 is supported by our observation that the
expression of EpoR Y401F in BaF3 cells confers hyper-responsiveness to
Epo and decreases the sensitivity of cells to undergo apoptosis at low
Epo concentrations.
It is possible that in other cell lines, this negative regulatory role
of CIS is less important for signaling through the EpoR since Tauchi
et al. (10) observed that expression in DA-3 cells of an
EpoR lacking Tyr-401 resulted in reduced rather than enhanced
proliferative responses (10). This observation supports the notion that
the efficiency of signaling pathways strongly depends on the cellular
background used for analysis. However, we showed that CIS proteins
mutant in the SH2 domain and thus unable to bind to the EpoR also could
not inhibit proliferation of primary erythroid progenitor cells. This
finding supports the role of the CIS SH2 domain in negatively
regulating signaling through the EpoR in primary cells.
Role of CIS SOCS Box for Apoptosis-promoting Effects--
It was
previously shown that CIS is ubiquitinated and that treatment of cells
with the proteasome inhibitor LLnL protects the CIS·EpoR
complex from degradation (16). This result suggested that CIS
negatively regulates EpoR signaling by targeting the receptor for
degradation. However, we showed that deletion of the CIS SOCS Box, the
segment mediating the association of CIS with the ubiquitin degradation
cascade, does not affect the ability of CIS to inhibit proliferation of
BaF3 cells. Thus, our results show that whereas SH2 domain-mediated
receptor recruitment of CIS is critical for its anti-proliferative
effects in BaF3 cells, the SOCS Box is not required. However, in
primary erythroid progenitors, the SOCS Box as well as the SH2 domain
is essential for the ability of the CIS protein to promote apoptosis.
This finding suggests that CIS utilizes different mechanisms to repress
growth and to increase sensitivity for entering apoptosis.
Interaction of CIS with the STAT5 Bcl-xL
Pathway--
In contrast to other members of the SOCS family of
proteins, CIS does not directly interact with or inhibit the activity
of Janus kinases (28). CIS binds via its single SH2 domain,
specifically, to (phospho)tyrosine 401 in the activated EpoR and
thereby could compete binding of other signal-transducing proteins
including STAT5. Accordingly, it was observed that forced
overexpression of CIS reduces the activation of STAT5 by the EpoR (20,
21). We show here that reduced activation of STAT5 depends on SH2
domain-mediated receptor recruitment of CIS. However, because the EpoR
contains a second STAT5 binding site (phospho-Tyr-343) that is equally efficient in activating STAT5, it was proposed that the negative regulatory effect of CIS was amplified by targeting the activated EpoR
for degradation (16). We show that cell surface expression of the
mutant EpoR lacking the CIS binding site (EpoR Y401F) is slightly
reduced compared with the wild type EpoR, suggesting that CIS binding
to (phospho)tyrosine 401 does not down-regulate cell surface expression
of the EpoR. In addition, our results show that a mutant CIS missing
the SOCS Box is as efficient as wild type CIS in repressing the
proliferative response of BaF3 cells, supporting the hypothesis that by
binding to (phospho) tyrosine 401 CIS either sterically shields
(phospho)tyrosine 343 or induces conformational changes in the receptor
that impair STAT5 binding to (phospho)tyrosine 343.
CIS itself is transcriptionally induced by the STAT5-signaling cascade
but not mitogen-activated protein kinase or phosphatidylinositol 3-kinase (21), and therefore CIS has been suggested to be part of a
negative feedback loop controlling the activity of the pathway (15).
Numerous reports show a role of STAT5 in proliferation and
differentiation mediated by the EpoR (29). Furthermore, STAT5 has been
implicated in protection from apoptosis by directly inducing
the expression of the anti-apoptotic gene
Bcl-xL (12). Adult
STAT5a
/
/STAT5b
/
mice have persistent anemia despite a marked compensatory expansion in
their erythropoietic tissues. Importantly, decreased expression of
Bcl-xL and increased apoptosis in adult and neonatal STAT5a
/
/STAT5b
/
early
erythroblasts correlated with the degree of anemia (14). Similarly, in
fetal erythropoiesis, the absence of Bcl-xL could be sufficient
to render STAT5a
/
/STAT5b
/
cells more sensitive to apoptosis. Mice deficient in
Bcl-xL have demonstrated an essential role for Bcl-xL
in preventing apoptosis at the end of maturation of both primitive and
definitive erythrocytes (13).
In addition to the activation of Bcl-xL, a role for NF
B has
been proposed in EpoR signaling to prevent apoptosis (30). Epo-mediated
activation of NF
B was induced by the same membrane-proximal tyrosines that serve as binding sites for STAT5 (30). An EpoR lacking
all of the tyrosine residues in the cytoplasmic domain and an EpoR with
a mutation of the JAK2 binding site have been demonstrated to induce
apoptosis in BaF3 cells (30), supporting the idea that signals involved
in protection from apoptosis emanate from the EpoR. We propose that
forced expression of CIS impairs activation of the STAT5-signaling
cascade and potentially the NF
B pathway and thereby increases the
sensitivity of cells to undergo apoptosis. It has been shown that GATA1
cooperates with erythropoietin to regulate Bcl-xL expression
(31) and GATA1 can enhance Bcl-xL expression by the EpoR/STAT5
pathway (32). The interplay between GATA1, NF
B, STAT5, and CIS
clearly demands further investigations.
Two Functions of CIS in Primary Erythroid Cells--
Previous
studies investigating the role of CIS in erythropoiesis have been
limited to analysis of proliferation in cell lines. Here, we have used
retroviral expression to analyze the role of CIS in proliferation,
differentiation, and apoptosis of primary erythroid progenitor cells.
Upon expression of CIS in fetal liver cells, erythroid progenitors form
smaller colonies and show a reduced growth rate and an increased
frequency to undergo apoptosis. We propose that CIS exerts its negative
regulatory function by two interconnected mechanisms. CIS
represses recruitment of other signaling molecules to the EpoR, which
is sufficient to suppress proliferative responses. However, the ability
of CIS to enhance apoptosis in erythroid progenitor cells depends on
the SOCS Box, and hence, the enhancement of apoptosis may require
degradation of activated receptors by the proteasome. Thus, the amount
of CIS induced in cells sets a threshold to EpoR signaling and
therefore tightly controls the elicited response.