From the
Apoptosis, or programmed cell death (PCD), recently has emerged
as an important homeostatic mechanism within several hematopoietic
lineages. This process is subject to both positive and negative
modulation by cytokines and within the erythroid lineage is inhibited
by interleukin-3, stem cell factor, and erythropoietin (Epo). Through
the expression of carboxyl-truncated Epo receptor mutants in FDC-P1
cells, a receptor form possessing 80 membrane-proximal cytoplasmic
residues is shown to efficiently mediate Epo-dependent inhibition of
PCD. This is in contrast to previous studies that attributed this
activity to a distal carboxyl-terminal receptor subdomain (and/or
heterodimerization of wild type Epo receptors with a truncated
non-functional receptor form). Epo-dependent inhibition of PCD also is
shown to be blocked by ectopic expression of kinase-deficient
dominant-negative forms of Jak2 (Jak2
During the development of hematopoietic cells, an apparently
constitutively poised program of apoptosis (or programmed cell death
(PCD))
With regard to
molecular effectors of PCD, several classes of factors have been
defined. These include Bcl-2 and related factors (Bcl-x, Bcl-x-s, Bax,
Mcl-1, A1, and Bhrf-1; see Ref. 22 for review), Myc (23, 24, 25) and Myb (26) as immediate
early gene products that enforce apoptosis when expressed at elevated
levels, the tumor suppresser gene p53(27, 28) ,
and several families of proteins whose expression has been associated
specifically with apoptosis (i.e. Myd, Gadd, and Gas gene
products)(22) . In hematopoietic systems, the ability of Bcl-2
to block PCD upon withdrawal of IL-3 has been demonstrated in both
lymphoid (29) and myeloid cells(7) . The forced
expression of Myc and Myb has been shown to advance
PCD(25, 26) , and apoptosis has been shown in
Epo-dependent murine FVA cells to proceed from both G1 and S phases of
the cell cycle(30) . However, little is presently understood
concerning how these effectors of PCD might be modulated upon cytokine
exposure.
An important advance toward understanding cytokine action
is constituted by the recently defined association between cytokine
receptors of the type I/II superfamilies and members of the Janus
family of protein-tyrosine kinases (i.e. Jak1, Jak2, Jak3, and
Tyk-2)(31, 32, 33, 34, 35, 36, 37, 38) .
In the Epo receptor system, for example, Epo activates Jak2, and this
cytosolic protein-tyrosine kinase has been evidenced to associate with
the Epo receptor within a conserved box 1/box 2-encoding
membrane-proximal receptor subdomain (39). Recently, through the
development of a dominant-negative form of Jak2, we have shown that
activation of this receptor-associated kinase is required for
Epo-induced mitogenesis(40, 41) . In the present study
we have used factor-dependent murine FDC-ER and DAER cell lines to
assess whether Jak2 (as a receptor-associated effector) might also be
involved in the effects of Epo on PCD. First, experiments using Epo
receptor cytoplasmic domain mutants delineate a membrane-proximal
receptor domain that supports Epo-inhibited PCD. Notably, this is in
contrast to recent studies by Nakamura et al.(42) , who
have implicated a role for a carboxyl-terminal domain of the Epo
receptor in inhibited PCD. Second, evidence that Epo-induced activation
of Jak2 is essential for Epo-dependent inhibition of PCD is provided by
studies using ectopically expressed dominant-negative forms of this
Janus kinase. To our knowledge, this is the first direct evidence among
type I cytokine receptor systems for a critical role for a specific
protein-tyrosine kinase in this signaling pathway.
In primary studies aimed at defining the possible involvement
of Jak2 in Epo-dependent inhibition of PCD, PCD-associated DNA
fragmentation was analyzed in FDC-P1 cells expressing a series of
carboxyl-terminally truncated Epo receptor mutants (Fig. 1). This
includes receptor forms that efficiently mediate Jak2 activation and
proliferation (wild type Epo receptor and ER-372), efficiently support
Jak2 activation but require high levels of Epo to support proliferation
(ER-329), or fail to mediate either Jak2 activation or proliferation
(ER-256). Levels of cell surface Epo receptor expression in these cell
lines are highly comparable and have been assessed by equilibrium
binding analyses, [
To further affirm the present findings,
analyses of apoptosis of FDC-wtER versus FDCER-329 cells also
were performed in the presence of Epo at decreasing concentrations (Fig. 3). Interestingly, higher concentrations of Epo were
required to inhibit the apoptosis of FDCER-329 cells. This is also the
case for Epo-dependent mitogenesis of FDCER-329 versus FDC-wtER cells as shown by He et al.(46) and
Quelle et al.(43) . Thus, it is interesting to at least
speculate that Epo-dependent mitogenesis and inhibition of PCD may, in
fact, depend upon common upstream effectors.
In several systems, bcl-2 has been implicated as a mediator of
IL-3-dependent inhibition of PCD. This includes studies in lymphoid
cells derived from IL-3-dependent pro-B lymphocyte line FL5.12 (29) as well as murine myeloid 32D cells(50) . This
implied role for Bcl-2 depends largely upon its observed capacity to
attenuate PCD upon forced expression. In studies by Baffy et
al.(50) , evidence is also provided for an
IL-3/Bcl-2-dependent redistribution of cellular calcium, whereas
studies by May et al.(51, 52) suggest a
possible role for the protein kinase C-dependent, IL-3- and Epo-induced
phosphorylation of Bcl-2. However, these events presently are only
associated with Bcl-2 action, and further studies are required to
establish possible direct roles for Bcl-2 and related factors (Bcl-x,
Bcl-x-s, Bax, Mcl-1, and A1) in cytokine-inhibited PCD. Notably, this
includes the delineation of downstream effectors that mediate the
presently established role for Jak2 in this process.
In the context
of pathways to inhibition of PCD versus mitogenesis, positive
roles for both Myc and Myb in PCD have been evidenced. Specifically,
PCD is activated by over-expression of either
factor(25, 26) . For Myc, this apparently depends upon
cooperativity with Max (53) and is associated with a specific
increase in cyclin A (54). Also, in transgenic mice harboring a c-Fos
promoter/
Finally, studies by Otani et al.(7) in IL-3/IL-2-dependent 32D-derived b53 cells have
provided indirect evidence (using herbimycin A) for an essential role
for tyrosine phosphorylation events in IL-3-inhibited PCD. The present
study extends these analyses by defining Jak2 kinase as at least one
requisite factor in this pathway. To our knowledge, this is the first
identification of a role for a specific, cytokine-regulated
protein-tyrosine kinase in PCD. The nature of additional factors that
transduce Jak2-dependent signals to PCD effectors comprises a presently
unresolved problem of significant interest.
We thank Dr. Steven Elliot of Amgen, Inc. for the
generous provision of the recombinant human Epo used in these studies.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
VIII and Jak2-829),
further underlining a role of this membrane-proximal subdomain of the
Epo receptor in the inhibition of PCD. To our knowledge, this comprises
the first direct evidence for an essential role for a Jak tyrosine
kinase (Jak2) in this apoptotic response pathway.
(
)has been shown to significantly affect
progenitor cell survival and homeostasis(1, 2) . Among
at least certain lineages, commitment to this program is prevented
through exposure to select cytokines including IL-2, IL-3, IL-6,
granulocyte colony-stimulating factor, granulocyte-macrophage
colony-stimulating factor, and cillary neurotropic
factor(3, 4, 5, 6, 7, 8, 9, 10) .
Within the erythroid lineage, PCD has been shown to be attenuated by
IL-3, stem cell factor, insulin-like growth factor-1(11) , and
erythropoietin (Epo)(12, 13, 14) . Notably, this
inhibited PCD can be contrasted with and is perhaps balanced by factors
that induce PCD, including IL-4(15) , tumor necrosis
factor(16) , corticosteroids versus early thymic
lymphocytes(17, 18) , and the Fas
antigen(19, 20, 21) .
Cell Lines
The FDC-P1-derived murine myeloid
cell lines (FDC-ER, FDCER-372, FDCER-329, and FDCER-256) (43) and DA-1-derived cell lines (DAER, DAERJK2VIII, and
DAERJak2-829) (40, 41) used in this study have
been described previously (also see below). Each was
maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 10
mol/liter
2-mercaptoethanol, and 3% conditioned medium from WEHI-3B cells.
DNA Fragmentation Assays
In assays of apoptosis,
cells were incubated in Dulbecco's modified Eagle's medium
with 1% fetal calf serum in the presence or the absence of Epo
8-9 h prior to the assay of PCD-associated DNA fragmentation. The
method of genomic DNA extraction was adapted from that described by
Wang et al. (44). Briefly, 8 10
cells were
collected and washed twice in phosphate-buffered saline. Washed cells
were lysed by vortexing (1 min) in 1% Triton X-100, 0.32 M
sucrose, 5 mM MgCl
, 10 mM Tris-HCl, pH
7.5. Nuclei were then isolated by centrifugation (600
g for 5 min) and were suspended in 0.2 ml of 1% SDS, 5 mM Na
EDTA, 10 mM Tris-HCl, pH 8.0. Sequential
incubations with RNase A (10 mg/ml for 10 min at 37 °C) and
proteinase K (1 mg/ml for 1 h at 37 °C) were performed to liberate
DNA. DNA was then precipitated by the addition of 0.3 ml of 7.6 M NaI, 20 mM Na
EDTA, 40 mM Tris-HCl, pH 8.0, 0.5 ml of isopropanol. Precipitates were
collected by centrifugation and were washed sequentially in 40%
isopropanol and 70% ethanol. Air-dried samples were resuspended in 1
mM Na
EDTA, 10 mM Tris, pH 8.0, and were
analyzed by electrophoresis (1.2% agarose gel, ethidium bromide
staining).
I]Epo cross-linking studies,
and assays of sensitivity of receptor forms to endoglycosidase H to
assess intracellular processing and transport(43, 45) .
Properties of these receptor forms and corresponding FDC cell lines are
summarized in .
Figure 1:
Epo receptor truncation mutants and
derived FDC-ER cell lines used in PCD
analyses.
In assays of Epo-inhibited PCD,
FDC-derived cell lines expressing the wild type receptor (FDC-ER
cells), the truncation mutant ER-372 (FDCER-372 cells), and the
truncation mutant ER-329 (FDCER-329 cells) were observed to efficiently
support inhibited PCD (Fig. 2). In this and subsequent studies,
PCD was assayed based on levels of cytokine-inhibited DNA
fragmentation. In contrast, in cells expressing the highly truncated
Epo receptor mutant ER-256 (FDCER-256 cells), DNA fragmentation was
observed in both the absence and the presence of Epo. Among these
receptor forms, each either supported Epo-inhibited apoptosis (wild
type ER, ER-372, ER-329) or was apparently fully deficient in this
activity (ER-256). This result, together with the consideration that
each receptor form is expressed at comparable levels ()
argues that the observed activities in inhibited PCD are attributable
to form rather than to possible general differences in expression. In
addition, each of the receptor forms wild type ER, ER-372, and ER-329
has been shown to efficiently mediate Epo-induced activation of Jak2 at
comparable efficiencies(46) , and mitogenic properties have been
shown to be essentially equivalent among at least 5 independent clones
for each cell line(45) .
Figure 2:
Epo-dependent inhibition of apoptosis as
mediated by carboxyl-truncated Epo receptor mutants in FDC-ER cell
lines. Epo-dependent inhibition of PCD-associated DNA fragmentation was
assessed in FDC-ER, FDCER-374, FDCER-329, and FDCER-256 cell lines as
described under ``Materials and
Methods.''
The above data show that a
membrane-proximal cytoplasmic subdomain of the Epo receptor, which is
essential for Jak2 binding and activation (36), likewise is necessary
and sufficient for supporting Epo-dependent inhibition of
PCD-associated DNA fragmentation. Notably, this finding is in contrast
to studies of the human Epo receptor by Nakamura et al.(42) in which Epo-inhibited apoptosis was suggested to be
mediated by a carboxyl-terminal receptor subdomain. These latter
studies aimed to assess Epo-inhibited PCD activity for a truncated Epo
receptor form (EpoR-T), which derives from the alternate splicing of
transcripts in early erythroid progenitor cells. Stably transfected
murine BaF3 cells were used as a model to reconstitute activity for a
corresponding Epo receptor cDNA. However, it recently has been resolved
that the BaF3 cell line used in these analyses expresses significant
levels of endogenous full-length Epo receptors and that the receptor
form studied apparently is mitogenically inactive. To explain observed
apparent effects on apoptosis, an alternate model more recently has
been proposed in which heterodimerization of this truncated form with
full-length receptors might act to down-regulate mitogenesis and cell
survival(47) . However, as noted by Schwall(48) , the
further consideration that the apparent failure of EpoR-T to inhibit
apoptosis depends upon Epo concentration is inconsistent with the
conclusion that this receptor form lacks the domains required to
inhibit PCD. Finally, it is relevant to consider that, although BaF3
cells were originally described as pro-B cells, this cell line recently
has been shown to express several erythroid-specific factors (including
Gata-1 and Eklf) and to support Epo-induced induction of globin gene transcription (but not hemoglobinization) following
stable transfection with the wild type Epo receptor(49) . Due to
this potential for erythroid differentiation, the use of BaF3 cells
possibly complicates interpretations of effects of Epo on cell
survival. In contrast, the models used in the present studies (FDC-P1-
and DA-1-derived cell lines) do not express erythroid-specific factors
at detectable levels (43)
(
)and are not subject
to this complication.
Figure 3:
Concentration-dependence of Epo-inhibited
PCD in FDC-ER versus FDCER-329 cells. Exponentially growing
washed cells were cultured in the presence (or the absence (-))
of Epo at the indexed concentrations for 7 h. PCD-associated DNA
fragmentation was then assayed as described under ``Materials and
Methods.''
The above experiments
using Epo receptor carboxyl-terminal truncation mutants implicate a
role for Jak2 in Epo-inhibited PCD yet do not directly test whether
Jak2 activation is necessary to support the observed Epo-dependent
inhibition of DNA fragmentation. This possibility was addressed using
DA-1-derived cell lines, which ectopically express both the cloned
murine Epo receptor and one of two kinase-deficient, dominant-negative
forms of Jak2 (i.e. Jk2VIII or Jak2-829
(DAERJak2
VIII and DAERJak2-829 cell lines, respectively)).
Jk2
VIII is a construct mutated within the type VIII
phosphotransferase motif of the carboxyl-terminal protein-tyrosine
kinase domain, and Jak2-829 is a carboxyl-terminal truncation
mutant lacking this protein-tyrosine kinase domain (Fig. 4). Each
mutant has recently been shown to efficiently inhibit Epo-induced
activation of endogenous Jak2 and mitogenesis(40, 41) .
The ability of DAERJak2
VIII and DAERJak2-829 cells to
support Epo-inhibited PCD-associated DNA fragmentation was assessed
through comparison with control parental DAER cells (Fig. 4). For
cells expressing each dominant-negative form of Jak2, Epo exerted
little, if any, detectable protection against PCD yet efficiently
inhibited DNA fragmentation in control DAER cells. Thus, these latter
studies using cells specifically inhibited in Epo-induced activation of
endogenous Jak2 provide direct evidence for a requisite role for Jak2
in Epo inhibition of PCD.
Figure 4:
Epo-dependent inhibition of PCD-associated
DNA fragmentation is blocked in DAER cell lines expressing
dominant-negative Jak2 mutants Jk2VIII and Jak2-829. A, kinase-deficient dominant-negative forms of Jak2 expressed
ectopically in DAER cells. B, PCD-associated DNA fragmentation
in DAER versus DAERJk2
VIII (left) and
DAERJak2-829 cells (right) in the absence (-) or
the presence of Epo (12 units/ml).
As indicated above, the conclusion that
the Epo-dependent activation of Jak2 is required for inhibition of PCD
by this cytokine is also supported indirectly by the observed ability
of the receptor form ER-329 to protect against PCD-associated DNA
fragmentation in FDCER-329 cells. Specifically, this receptor form,
although somewhat deficient in mitogenesis, contains a cytoplasmic
region that is necessary and sufficient for Epo-induced Jak2
activation(43, 46) . Whereas this conclusion regarding
the apparent requirement for Jak2 in Epo-inhibited PCD derives from
studies of two independent sets of cell lines that cannot be equated
directly with regard to molecular mechanisms of cytokine-mediated
inhibition of apoptosis, these findings are of interest in several
contexts. This includes potential roles of known apoptotic effectors in
cytokine-inhibited PCD, effects of mitogenic factors on apoptosis, and
the nature of downstream effectors of Epo- and Jak2-inhibited PCD.
-galactosidase receptor construct, sustained
transcriptional activation at the Fos promoter has been observed
penultimate to commitment to PCD(55) . Together, these
observations raise important questions regarding the extent to which
overlap might exist in sets of effectors of mitogenesis versus inhibition of PCD. In this context, the receptor form ER-329 (Fig. 1) lacks carboxyl-terminal sites recently suggested by
Miura et al. (56) to recruit p85/p110 phosphatidylinositol
3-kinase to receptor complexes. Thus, Epo-inhibited PCD may occur
independently of this effector.
Table: Properties of Epo receptor forms and derived FDC
cell lines
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.