Growth Hormone Prevents Apoptosis through Activation of Nuclear Factor-
B in Interleukin-3-Dependent Ba/F3 Cell Line
Sébastien Jeay,
Gail E. Sonenshein,
Marie-Catherine Postel-Vinay and
Elena Baixeras1
INSERM Unité 344, Endocrinologie Moléculaire (S.J.,
M.C.P-V., E.B.) Faculté de Médecine Necker Paris
Cedex 15, France 75730
Department of Biochemistry
(G.E.S.) Boston University School of Medicine Boston,
Massachusetts 02118
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ABSTRACT
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The pro-B Ba/F3 cell line requires
interleukin-3 and serum for growth, and their removal results in
cell apoptosis. Ba/F3 cells transfected with the GH receptor (GHR) cDNA
become able to proliferate in response to GH. To investigate the role
of GH in the control of apoptosis, Ba/F3 cells expressing either the
wild-type rat GHR (Ba/F3 GHR) or a mutated rat GHR (Ba/F3 ILV/T) were
used. We show that Ba/F3 GHR cells, but not parental Ba/F3 or Ba/F3
ILV/T cells, were able to survive in the absence of growth factor.
Furthermore, an autocrine/paracrine mode of GH action was suggested by
the demonstration that Ba/F3 cells produce GH, and that addition of GH
antagonists (B2036 and G120K) promotes apoptosis of Ba/F3 GHR cells.
Consistent with survival, the levels of both antiapoptotic proteins
Bcl-2 and Bag-1 were maintained in Ba/F3 GHR cells, but not in parental
Ba/F3 cells upon growth factor deprivation. Constitutive activation of
the transcription factor nuclear factor-
B (NF-
B), which has been
shown to promote cell survival, was sustained in Ba/F3 GHR cells,
whereas no NF-
B activation was detected in parental Ba/F3 cells in
the absence of growth factor. Furthermore, addition of GH induced
NF-
B DNA binding activity in Ba/F3 GHR cells. Overexpression of the
mutated I
B
(A32/36) protein, known to inhibit NF-
B activity,
resulted in death of growth factor-deprived Ba/F3 GHR cells, and
addition of GH was no longer able to rescue these cells from apoptosis.
Together, our results provide evidence for a new GH-mediated pathway
that initiates a survival signal through activation of the
transcription factor NF-
B and sustained levels of the antiapoptotic
proteins Bcl-2 and Bag-1.
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INTRODUCTION
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The multiple actions of GH are initiated by binding of the hormone
to its receptor (GHR), which belongs to the cytokine receptor
superfamily (1, 2). Binding of GH is followed by receptor dimerization
(3, 4) and activation of the tyrosine kinase Jak2, which then
autophosphorylates itself and phosphorylates tyrosine residues within
the GHR (5). The phosphorylated tyrosines in the receptor and Jak2
provide binding sites for various signaling molecules that contain SH2
(Src homologous 2) or PTB (phosphotyrosine binding) domains.
Recruitment of these molecules initiates a number of signaling pathways
mediating a variety of cellular responses. GH can activate the Stat
(signal transducer and activator of transcription) pathway, including
Stat1, Stat3, and both isoforms of Stat5 (6, 7), and thereby regulate
transcription of GH-responsive genes such as serine protease inhibitor
2.1 (8) and c-fos (9). Shc (Src homologous containing)
proteins that recruit Grb2 (growth factor receptor bound 2) and SOS
(son of sevenless) are also activated by GH and can initiate the
Ras-MAP (mitogen activated protein) kinase pathway, involved in the
regulation of gene transcription and cellular growth and
differentiation (10). The IRS (insulin receptor substrate) proteins,
IRS-1 and -2, which regulate glucose transport and lipid synthesis
(11), and protein kinase C (12) and phosphatase SHP-2 (13) have been
implicated in GH signaling. Other pathways involved in various
physiological actions of GH remain to be identified.
Growth factors and hormones have been shown to be regulators of cell
death. For example, epidermal growth factor, nerve growth factor,
platelet-derived growth factor, and insulin-like growth factor-1 act as
survival factors in hematopoietic cells and neurons (14). Steroid
hormones are also potent regulators of apoptosis in several tissues
such as the mammary gland (14). Similarly, PRL has been reported not
only to trigger proliferation of Nb2 lymphoma cells, but also to
counteract the glucocorticoid-driven apoptosis (15, 16). A recent study
reported a protective action of GH on Fas-mediated apoptosis in
monocytes (17).
Members of the Bcl-2 and NF-
B protein families have been extensively
implicated in cytokine-signaling of cell survival (18, 19). The Bcl-2
family includes pro-survival (e.g. Bcl-2 and
Bcl-XL) and proapoptotic proteins
(e.g. Bax and Bid) (20). Studies have demonstrated that
Bcl-2 and Bcl-XL are differentially regulated
during B cell development (21), and overexpression of Bcl-2 or
Bcl-XL was shown not only to lengthen survival of
cytokine-dependent cells upon cytokine withdrawal (18), but also to
protect B cells from apoptotic signals induced by glucocorticoids and
chemotherapeutic drugs (22). Bag-1, a Bcl-2 binding protein, was
recently found to enhance cellular proliferation and viability during
growth factor deprivation in interleukin-3 (IL-3)-dependent Ba/F3 and
PRL-dependent Nb2 cells (16). Moreover, altered expression of Bcl-2 and
Bax are associated with the antiapoptotic effect of PRL in Nb2 cells
(23). Likewise, the effect of GH on the survival of monocytes exposed
to Fas-mediated cell death is associated with up-regulation of Bcl-2
(17).
NF-
B/Rel factors have also been found to promote cell survival in a
number of cells and growth conditions (24). Classical NF-
B is
composed of p50 and p65 (RelA) subunits. In most cells, other than
mature B lymphocytes, NF-
B/Rel proteins are expressed ubiquitously,
but sequestered in inactive forms in the cytoplasm by association with
inhibitory proteins, termed I
Bs, for which I
B
represents the
paradigm (25). Activation of NF-
B/Rel involves I
B phosphorylation
on serine residues, followed by ubiquitination and rapid degradation of
the inhibitory protein by the proteasome, which allows for nuclear
translocation of the NF-
B/Rel factor (25). Constitutively active
NF-
B/Rel factors have been found in mature B cells and in pro-B
cells, including the Ba/F3 cell line (26, 27, 28). Further, constitutive
activation of NF-
B by the oncogenic TEL/platelet-derived growth
factor receptor ß fusion protein was shown to protect Ba/F3 cells
from apoptosis induced by IL-3 deprivation (28).
The aim of this study was to examine the role of GH in the regulation
of cell death. Stable Ba/F3 transfectants expressing the wild-type rat
GHR (Ba/F3 GHR) or a functionally deficient form of the rat GHR (Ba/F3
ILV/T) were used to study GH signaling pathways involved in cell
survival. We have previously shown that Ba/F3 GHR cells depend on GH
for their growth and can be maintained in culture in the presence of
the hormone (29). Here we show that Ba/F3 GHR cells escape from death
upon cytokine and serum deprivation. We found that locally produced GH
is responsible for such a protective effect, which is associated with
sustained expression of Bcl-2 and Bag-1 proteins. Moreover, we have
identified a novel signaling pathway for GH via activation of NF-
B,
which is crucial in mediating the antiapoptotic effect of GH.
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RESULTS
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Cell Cycle Analyses of Ba/F3 Cells Expressing the GH Receptor
The potential role of GH as a growth and survival factor was
studied using Ba/F3 cells stably transfected with the rat GHR cDNA
(Ba/F3 GHR) (29). As negative controls, parental Ba/F3 cells (Ba/F3
WT), which do not express GHRs, or Ba/F3 cells expressing a mutated rat
GHR (Ba/F3 ILV/T), which has been shown to be inactive, were used (29).
Ba/F3 WT, Ba/F3 ILV/T, and Ba/F3 GHR cells were cultured in serum- and
IL-3-free medium (starvation medium) for 6 h to induce maximal
synchronization. Cell cycle analyses were performed after 48 h of
cell treatment under three culture conditions: 1) normal medium; 2)
starvation medium; 3) starvation medium plus 1 µg/ml bovine GH (bGH).
When cultured in normal medium, the three cell lines showed a typical
cell cycle with 5669% of cells in
G0/G1 phase and 3042% of
cells in S/M phase (Fig. 1
). Under this
condition, almost no cells appeared to be undergoing apoptosis, as
judged by the DNA content (<1%) in
sub-G0/G1 phase. Under
starvation conditions, Ba/F3 WT and Ba/F3 ILV/T cells extensively
underwent apoptosis (84% and 77%, respectively), whereas Ba/F3 GHR
cells were arrested in
G0/G1 phase (88%), and 5%
of cells were undergoing apoptosis (Fig. 1
). Addition of bGH did not
modify the proportion of Ba/F3 WT and Ba/F3 ILV/T cells undergoing
apoptosis, as expected, since these cells do not express a functional
GHR. On the contrary, bGH treatment of Ba/F3 GHR cells promoted cell
cycle progression, with 33% of the cells in S/M phase. Thus, Ba/F3
cells expressing GHR are resistant to apoptosis upon growth factor
deprivation, and addition of exogenous GH induces cell cycle
progression in these cells.

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Figure 1. Cell Cycle Analyses of Ba/F3 Cells under Various
Culture Conditions and GH Treatment
Ba/F3 WT, Ba/F3 ILV/T, and Ba/F3 GHR cell types were starved for 6
h, and then cultured under three conditions: in normal culture medium,
starvation medium, or in starvation medium plus 1 µg/ml bGH.
Cells were harvested 48 h later and cell pellets were
permeabilized and stained with PI, and cell cycle analyses were
performed by flow cytometry. The DNA content vs. cell
number is presented in each histogram. Percentages correspond to cells
in the G0/G1 phase (2n content) or in the S/M
phase (4n content) or in apoptosis phase (hypodiploid content). Results
are representative of four independent experiments.
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Production of GH by Ba/F3 Cells
To explain the survival of Ba/F3 GHR cells cultured in starvation
conditions, the possibility of a local production of hormone by the
cells was addressed. As Ba/F3 WT cells underwent apoptosis upon growth
factor deprivation, we took advantage of the ability of Ba/F3 GHR cells
to survive under starvation conditions (Fig. 1
) to investigate the
presence of GH in cell supernatant. Ba/F3 GHR cells were then
maintained in starvation medium for 24, 48, and 72 h, and presence
of GH was evaluated by RIA in the supernatants of cells. Presence of GH
in starvation medium alone was also evaluated as a control. As shown in
Table 1
, low GH concentrations were
detected in 150-fold concentrated supernatants from Ba/F3 GHR cells,
and the levels increased with longer times of culture. No GH was found
in starvation medium. Thus, Ba/F3 cells produce GH.
We next asked whether this locally produced GH was responsible for cell
survival by using two human GH antagonists, B2036 and G120K, which have
been shown to bind to the rat GHR with a lower affinity than to the
human GHR (30). These two antagonists were tested at concentrations
ranging from 0.5 to 2 µg/ml for their ability to inhibit the
potential effects of endogenous GH on Ba/F3 GHR cell survival.
Starvation of Ba/F3 GHR cells led to apoptosis of a small proportion of
cells (11%). Addition of 0.5 µg/ml of B2036 or G120K was sufficient
to enhance the level of cell death up to 30%. Maximal effect of both
antagonists was reached at 1 to 2 µg/ml where about 40% of cells
underwent apoptosis (Fig. 2
). Taken
together, these results strongly suggest that locally produced GH is
sufficient to rescue Ba/F3 GHR cells from apoptosis.
Expression of Bcl-2 Related Proteins in Ba/F3 GHR Cells
Previous studies have shown that Ba/F3 cells are able to express
the antiapoptotic Bcl-2, Bcl-XL, and Bag-1
proteins under growing conditions, and overexpression of these proteins
was found to enhance cell survival (16, 31). Expression of these
proteins was evaluated by immunoblot analyses of total cell lysates
prepared from Ba/F3 WT and Ba/F3 GHR cells either unstimulated (Fig. 3
, lanes 2 and 6) or stimulated with bGH
(Fig. 3
, lanes 3, 4, 7, and 8). Cells cultured in normal medium for
8 h were used as positive control for expression of Bcl-2-related
proteins in Ba/F3 WT and Ba/F3 GHR cell lysates (Fig. 3
, lanes 1 and
5). Immunoblot analysis of Bcl-2 and Bag-1 showed that expression of
these proteins was down-regulated in unstimulated Ba/F3 WT cells after
6 h of culture in starvation medium (Fig. 3
, lane 2). In contrast,
only a slight decrease was observed in the levels of Bcl-2 and Bag-1 in
lysates from starved Ba/F3 GHR cells (Fig. 3
, lane 6) compared with
those in cells cultured in normal medium (Fig. 3
, lane 5).
Bcl-XL expression was not detectable in lysates
from either Ba/F3 WT or Ba/F3 GHR cells cultured in starvation medium
(Fig. 3
, lanes 2 and 6). As expected, treatment of Ba/F3 WT cells with
bGH for 6 or 8 h (Fig. 3
, lanes 3 and 4) did not affect the
expression of Bcl-2, Bcl-XL, and Bag-1, and
levels were similar to those obtained in starvation conditions (Fig. 3
, lane 2). Also, addition of exogenous bGH to Ba/F3 GHR cell culture for
6 and 8 h caused a modest increase in the expression levels of
Bcl-2 and Bag-1 proteins back to baseline values (Fig. 3
, lanes 7 and
8). In contrast, Bcl-XL expression was induced in
Ba/F3 GHR cells after 6 and 8 h of treatment with bGH (Fig. 3
, lanes 7 and 8) to levels comparable to those obtained in cells grown in
normal medium (Fig. 3
, lane 5). Therefore, constant levels of Bcl-2 and
Bag-1 are associated with survival of starved Ba/F3 GHR cells.
Bcl-XL, however, is not present in starved Ba/F3
GHR cells, although it is inducible by addition of exogenous GH. Thus,
our results suggest that endogenous GH is sufficient to induce
constant levels of Bcl-2 and Bag-1 proteins, which are likely to be
involved in the short-term survival in Ba/F3 GHR cells upon cytokine
deprivation.

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Figure 3. Expression of Bcl-2, Bcl-XL, and Bag-1
Proteins in Ba/F3 Cells
Ba/F3 WT and Ba/F3 GHR cells were starved for 6 h before
incubation in normal medium for 6 h (lanes 1 and 5), or in
starvation medium alone for 6 h (lanes 2 and 6) or in starvation
medium containing 1 µg/ml bGH for 6 h (lanes 3 and 7) or for
8 h (lanes 4 and 8). Then Bcl-2, Bcl-XL, and Bag-1
proteins were detected by immunoblot analysis of total cell lysates, as
detailed in Materials and Methods.
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NF-
B Activation by GH in Ba/F3 GHR Cells
The transcription factor NF-
B can protect a wide variety of
cells from apoptosis (24). The ability of GH to induce NF-
B
activation was then examined. Nuclear extracts of Ba/F3 cells have been
reported to yield two NF-
B complexes, the upper and lower bands
corresponding to classical p65/p50 heterodimers and p50/p50 homodimers,
respectively (28). Indeed, electrophoretic mobility shift assay (EMSA)
of nuclear extracts from Ba/F3 WT and Ba/F3 GHR cells grown overnight
(18 h) in normal medium showed two bands (Fig. 4A
). The specificity of these two
complexes in Ba/F3 GHR nuclear extracts was verified upon successful
competition with 10- and 20-fold excess of the unlabeled wild type
upstream region element (URE) oligonucleotide, whereas addition of a
mutant version, with two internal G to C conversions, was much less
effective (Fig. 4A
). An antibody against the p65 subunit (27), known to
block the binding of p65/p50 complexes (32), was next used to test for
the presence of p65 in the slower migrating complex. Incubation of
Ba/F3 WT and Ba/F3 GHR nuclear extracts with the anti-p65 antibody
specifically eliminated binding of the upper complex (Fig. 4A
). These
data corroborate the specificity of DNA binding and indicate that Ba/F3
GHR cells, like the parental Ba/F3 cells, contain p65/p50 heterodimers
of classical NF-
B.
We next analyzed the effects of starvation conditions, in the absence
and in the presence of bGH, on NF-
B DNA binding ability. Ba/F3 WT
and Ba/F3 GHR cells were incubated overnight under starvation
conditions and then incubated in the absence or in the presence of bGH
(Fig. 4B
). After overnight starvation, p65/p50 heterodimer complexes
were barely detected in nuclear extracts of Ba/F3 WT cells. In
contrast, NF-
B activity was maintained in Ba/F3 GHR cells under
similar starvation conditions. Furthermore, addition of bGH to the
starvation medium appeared to enhance NF-
B levels in Ba/F3 GHR, but
not in Ba/F3 WT cells. Taken together, these results suggest that
maintenance of NF-
B activity may play a role in survival of Ba/F3
GHR cells. Furthermore, the activation of NF-
B could be mediated via
GHR signaling, presumably due to the small quantities of endogenously
secreted GH in the Ba/F3 GHR cells.
To test directly the effects of GH-induced signaling on NF-
B
activation, a time course experiment was performed. Nuclear extracts
were prepared from Ba/F3 WT or Ba/F3 GHR cells incubated under
starvation conditions for 6 h (0 time point) followed by treatment
with bGH for 15 min to 16 h. NF-
B binding was analyzed by EMSA
(Fig. 5
). Very low or undetectable basal
levels of p65/p50 heterodimer were found in starved Ba/F3 WT cells and
they were not altered by bGH. In contrast, exogenous bGH enhanced
NF-
B activation in Ba/F3 GHR cells. An increase in NF-
B
activation was detected by 15 min post-bGH addition. Levels were
maximal at 1 h of stimulation. The presence of p65/p50 heterodimer
was maintained in Ba/F3 GHR cells even after 16 h of bGH
stimulation (Fig. 5
). Thus, stimulation by exogenous bGH increases the
expression of classical NF-
B in Ba/F3 GHR cells.
Involvement of NF-
B in GH-Induced Survival of Ba/F3 GHR
Cells
To investigate the potential role of NF-
B in the GH-induced
survival of Ba/F3 GHR cells, we performed analysis of cell death after
inhibition of NF-
B pathway. The pRCßactin
I
B
(A32/36) vector encoding the dominant negative mutant I
B
(A32/36) was transiently transfected in Ba/F3 GHR cells. Then, the
capacity of GH to induce survival in Ba/F3 GHR cells in these
conditions was examined. As a control, the empty parental
pRCßactin vector DNA was similarly transfected
to assess for effects of the vector sequences. Cell cycle analyses were
performed on Ba/F3 GHR cells expressing I
B
(A32/36) protein after
48 h postelectroporation with pRCßactin
I
B
(A32/36) vector. Cells sham transfected or transfected with
the empty vector were maintained either in normal medium (Fig. 6
, panels A and B) or in medium
containing bGH (Fig. 6
, panels G and H). Under these conditions, no
differences in cell cycle profiles and apoptosis rate were detected as
judged by DNA content. Likewise, cells expressing the mutant I
B
(A32/36) protein and maintained in normal medium showed a cell cycle
similar to that found in cells transfected with the empty vector (Fig. 6
, panels C vs. B). Sham or empty vector transfected cells
maintained in starvation conditions exhibited 19% of cells in
apoptosis. This observation can be attributed to the absence of growth
factor-mediated survival signals that contribute to cell
restoration after electroporation handling (Fig. 6
, panels D and
E). Upon the same starving conditions, cells expressing the mutant
I
B
(A32/36) protein showed 4-fold increase in apoptosis (72%)
(Fig. 6
, panels F vs. D and E). Moreover, apoptosis was no
longer abolished by addition of bGH to I
B
(A32/36) expressing
cells, which exhibited an apoptosis rate of 83%, a similar value to
that found in starvation conditions (Fig. 6
, panels I vs.
F).
From these observations, we can conclude that the protection mediated
by GH was lost upon introduction of the dominant negative I
B
(A32/36), which strongly suggests that NF-
B activation is directly
involved in the GH survival signal. Furthermore, the survival signal
involved in GHR activation pathway seems to differ from that induced by
the normal medium, which contains IL-3 plus serum, a cocktail of
cytokine and growth factors that does not appear to be affected by the
NF-
B inactivation.
Levels of Bcl-2 Related Proteins in Ba/F3 GHR Cells Expressing the
Mutant I
B
(A32/36)
As shown in Fig. 3
, Bcl-2 and Bag-1 proteins were expressed in
lysates from starved Ba/F3 GHR cells, which did not undergo apoptosis,
whereas the expression of Bcl-XL was only
observed in cells in proliferation. In an attempt to establish a
relationship between NF-
B activation and Bcl-2, Bag-1, and
Bcl-XL protein expression, we examined the
presence of these proteins in Ba/F3 GHR cells transfected with the
mutant I
B
(A32/36) vector. Ba/F3 GHR cells were transfected with
the pRCßactin vector or
pRCßactin I
B
(A32/36) vector and then
cultured in either normal or starvation medium or in the presence of
bGH. Nuclear extracts were prepared from these cells to first check the
effect of the mutant I
B
(A32/36) on NF-
B binding activity by
EMSA (Fig. 7A
). In comparison with cells
transfected with control vector (Fig. 7A
, lanes 1, 3, and 5), a marked
decrease in NF-
B activation was detected in Ba/F3 GHR cells
overexpressing I
B
(A32/36) (Fig. 7A
, lanes 2, 4, and 6). This
decrease was independent of culture conditions. As expected, the
results show that overexpression of I
B
(A32/36) strongly inhibits
NF-
B activation in Ba/F3 GHR cells.

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Figure 7. Expression of Bcl-2 Related Proteins in Ba/F3 GHR
Cells Overexpressing Mutant I B (A32/36) Protein
Ba/F3 GHR cells were transfected with either empty
pRCßactin vector (lanes 1, 3, and 5) or with the
pRCßactin I B (A32/36) vector (lanes 2, 4, and 6),
as described in Fig. 6 . After 24 h in normal medium, cells were
starved for 2 h and placed in either normal medium again, or in
starvation medium or in the presence of 1 µg/ml of bGH for an
additional 6 h as indicated. Nuclear extracts were prepared and
subjected to EMSA for NF- B binding (A). Presence of I B , Bcl-2,
Bag-1, and Bcl-XL proteins were sequentially determined in
the same membrane by immunoblot analyses of total cell lysates for each
treatment (B).
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The expression of I
B
, Bcl-2, Bag-1, and
Bcl-XL proteins was next assessed sequentially by
immunoblot analysis of total cell lysates (Fig. 7B
). As expected,
I
B
was clearly detected in lysates from cells transfected with
the pRCßactin I
B
(A32/36) vector (Fig. 7B
, lanes 2, 4, and 6), but was present in greatly reduced amounts in
lysates from parental vector-transfected cells (Fig. 7B
, lanes 1, 3,
and 5). Bcl-2 or Bag-1 protein levels were detected at equivalent
levels independently of culture conditions in lysates from parental
vector-transfected cells. Cells expressing the dominant negative
I
B
showed a clear decrease in the levels of Bcl-2 protein in all
three culture conditions (Fig. 7B
, lanes 2, 4, and 6). In contrast,
while Bag-1 levels were decreased in the presence of the dominant
negative I
B
in cells incubated under normal (Fig. 7
, lanes 1
vs. 2) or starvation conditions (Fig. 7B
, lanes 3
vs. 4), a partial induction was noted upon bGH treatment
(Fig. 7B
, lanes 4 vs. 6). In cells transfected with control
vector, bGH treatment as well as normal culture conditions induced
up-regulation of Bcl-XL levels (Fig. 7B
, lanes 1
and 5) in comparison to the very low expression of
Bcl-XL in deprived cells (Fig. 7B
, lane 3).
Overexpression of the mutant I
B
(A32/36) promoted a 2-fold
decrease of Bcl-XL expression under bGH treatment
(Fig. 7B
, lane 6), whereas no differences were found in cells cultured
in starvation or in normal conditions (Fig. 7B
, lanes 2 and 4). Thus,
NF-
B seems able to regulate expression of Bcl-2, Bag-1, and
Bcl-XL, although the regulation of Bag-1 and
Bcl-XL appears to be more complex, with the
possible involvement of other signaling molecules. Taken together,
these results demonstrate that GH mediates activation of NF-
B and
suggest that NF-
B could, in turn, promote cell survival via
expression of Bcl-2, and potentially Bag-1 and
Bcl-XL.
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DISCUSSION
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In this work, we demonstrate that GH can protect pro-B Ba/F3 cells
expressing the rat GHR against growth factor deprivation through an
autocrine mechanism. We show that Ba/F3 GHR cells produce GH, and the
presence of endogenous GH upon growth factor deprivation results in
sustained Bcl-2 and Bag-1 protein levels as well as constitutive
activation of the transcription factor NF-
B. An essential role of
this pathway in cell survival was indicated when inhibition of NF-
B
activity upon expression of the mutated I
B
(A32/36) protein
resulted in cell death. Our findings provide evidence for a new pathway
involved in signaling for GH antiapoptotic action.
Several lines of evidence indicate that a number of hormones, such as
PRL, insulin-like growth factor-1, and GH, play a role in cell survival
as well as in development and function of the immune system (14, 33).
The GH receptor has been shown to be widely expressed in hematopoietic
cells (34), and GH is able to stimulate the proliferation of several
cell types including activated murine T cells (35, 36), and direct
evidence of the GH proliferative effect on pro-B Ba/F3 cells expressing
the GHR has been reported (29).
Local production of GH in lymphoid tissues has been demonstrated (33, 37). Indeed, we show that Ba/F3 GHR cells produce GH. Interestingly,
the relevance for the locally produced GH is demonstrated since Ba/F3
GHR cells can survive upon serum and cytokine withdrawal from the
culture medium. Further demonstration of the importance of local
production of GH came from the use of B2036 and G120K hGH antagonists
that prevent receptor dimerization (3, 38) and thus GH action. Addition
of both antagonists resulted in enhanced mortality of Ba/F3 GHR cells
cultured in starvation medium. These findings suggest that GH, in
addition to its endocrine mode of action, can act in an
autocrine/paracrine manner in cells of the immune system.
It is known that in a quiescent cell, survival and control of cell
cycle entry predominantly depends on the expression of proteins of the
Bcl-2 family (31, 39, 40). As reported, IL-3 induces cell cycle
progression coupled to the expression of Bcl-XL,
but not of Bcl-2, in Ba/F3 cells (31). On the contrary,
Bcl-XL protein down-regulates rapidly upon serum
and IL-3 deprivation, and then cell death follows (31). This
observation has suggested the existence of two interdependent pathways
leading to apoptosis in IL-3-deprived Ba/F3 cells: while the presence
of Bcl-2 correlated with short-term survival after cytokine
deprivation, de novo expression of
Bcl-XL after addition of IL-3 seemed crucial for
long-term survival of cells (31). Our results show that a large
proportion of starved Ba/F3 GHR cells were arrested at
G0/G1 phase of the cell
cycle and did not undergo apoptosis. At this stage, levels of Bcl-2 and
Bag-1 were sustained, while levels of Bcl-XL
decreased. Addition of bGH, which induces proliferation of Ba/F3 GHR
cells, restored Bcl-XL expression levels while
levels of Bcl-2 and Bag-1 were only modestly increased. Therefore, as
shown for IL-3 (31), Bcl-2 is likely involved in GH signaling for
short-term survival. Bcl-XL, however, appears
involved in GH-induced long-term cell survival coupled to cellular
proliferation process.
Overexpression of the Bcl-2 binding protein, Bag-1, was previously
reported to induce cell survival of cytokine-deprived Ba/F3 and Nb2
cells (16). As shown for Bcl-2, sustained levels of Bag-1 were
associated with short-term survival of Ba/F3 GHR cells. Accordingly,
coordinated expression of Bcl-2 and Bag-1 genes has been shown to be
essential for IL-2-mediated protection from apoptosis of Ba/F3 cells
(41). Altogether, it can be concluded that Bcl-2 and Bag-1 are involved
in pathways for GH signaling of Ba/F3 cell survival.
We present evidence for the involvement of NF-
B activation in GH
survival effects in starved Ba/F3 GHR cells. Locally produced GH
appeared to be sufficient to induce constitutive NF-
B DNA binding
activity, which likely contributes to Ba/F3 GHR cell survival. NF-
B
DNA binding activity was enhanced by addition of bGH to Ba/F3 GHR cell
culture. Cell cycle analyses on cells expressing the mutant I
B
(A32/36) protein demonstrated that the ability of GH to inhibit cell
death is dependent on NF-
B activation pathway in Ba/F3 GHR cells.
Therefore, we can conclude that, as described for IL-3 in Ba/F3 cells
(28), NF-
B is a crucial mediator of the antiapoptotic signal
delivered by GH.
We found that expression of at least Bcl-2 was dependent on the
activation of NF-
B by GH in Ba/F3 GHR cells. The regulation of Bag-1
and Bcl-XL expression appears complex, mediated
via NF-
B-dependent and -independent signaling pathways.
Overexpression of the mutant I
B
(A32/36) provoked a decrease of
NF-
B activation and reduced the expression of the three Bcl-2 family
members to varying degrees, both in starved and bGH-treated Ba/F3 GHR
cells (Fig. 7
, A and B). In these conditions, a marked increase of cell
mortality occurred (Fig. 6
, panels F and I). In contrast, Ba/F3 GHR
cells overexpressing I
B
(A32/36) cultured in the presence of
serum and IL-3 were in cell cycle progression, although expression
levels of Bcl-2 and Bag-1, but not Bcl-XL, were
decreased. These results suggest that cell survival and proliferation
induced by serum could occur through the sustained expression of
Bcl-XL, in a NF-
B-independent manner. These
observations are consistent with the existence of a link between
Bcl-2-related molecules and NF-
B signaling pathways. Indeed,
induction of Bcl-2 and Bcl-XL expression through
NF-
B activation has been implicated in the protective effect of
tumor necrosis factor against neuron-induced injury (42).
Interestingly, the presence of NF-
B DNA binding sites in the
promoters of Bcl-2 and Bcl-X genes has been shown
and more recently the prosurvival Bcl-2 homolog Bfl-1/A1 has
been described as a direct transcriptional target of NF-
B (43, 44).
In addition, activation of NF-
B by Bcl-2 through degradation of
I
B
correlates with the protection of rat myocytes from apoptosis
(45).
Aberrant activation of NF-
B/Rel factors has now been observed in
many tumors. We first reported that breast cancer cells were typified
by aberrant activation of NF-
B (46). Specifically, mammary tumors
induced upon carcinogen treatment of Sprague Dawley rats, human breast
tumor cell lines, and primary human breast tumor tissue samples were
found to constitutively express high levels of nuclear NF-
B/Rel,
whereas normal rat mammary glands and untransformed breast epithelial
cells contained the expected low basal levels. Importantly, inhibition
of this activity in breast cancer cells in culture led to apoptosis.
Other tumors recently shown to display constitutive activation of
NF-
B, include the human cutaneous T cell lymphoma HuT-78 (47),
Hodgkins lymphomas (48), melanoma (49), pancreatic adenocarcinoma
(50), and primary adult T cell leukemias (51). The mechanism whereby
activation of NF-
B occurs remains to be elucidated. Recently, a
secreted factor has been implicated in Hodgkins lymphomas. It is
intriguing to speculate on whether such an autocrine mechanism involves
GH or other hormone or growth factor.
In conclusion, our results demonstrate that: 1) GH exerts an
antiapoptotic effect; 2) GH is able to activate NF-
B; 3) the
antiapoptotic effect of GH is closely mediated through NF-
B
activation; 4) low concentrations of GH induce Bcl-2 and Bag-1 whereas
high concentrations of GH can induce Bcl-XL
expression coupled to cell cycle progression. A possible link between
activation of NF-
B by GH and expression of Bcl-2 and potentially
Bag-1 and Bcl-XL is also suggested.
 |
MATERIALS AND METHODS
|
---|
Antibodies
Mouse monoclonal anti-Bcl-2, rabbit polyclonal anti-Bag-1, and
rabbit polyclonal anti-I
B
antibodies were obtained from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit
polyclonal anti-Bcl-XL antibody was from
Transduction Laboratories, Inc. (Lexington, KY). Anti-p65
antibody (27) was generously provided by Nancy Rice (National Cancer
Institute, Frederick MD).
Cell Culture and Treatment Conditions
The Ba/F3 is a pro-B murine cell line that does not express
endogenous GHR. Stable transfectants expressing either the wild-type
GHR (Ba/F3 GHR) or a mutated GHR (Ba/F3 ILVT) have been prepared as
previously described (29). Ba/F3 ILV/T cells express a mutant form of
the rat GHR: three amino acids (I, L, and V) in the box 1 sequence of
the receptor have been substituted to threonine, which results in a
mutant GHR unable to initiate the phosphorylation of Jak2 and therefore
to promote cell proliferation (29). Cell lines were maintained in RPMI
1640 medium supplemented with 10% heat-inactivated FBS, 50
µM 2-mercaptoethanol, 2 mM
L-glutamine, 10 U/ml penicillin, 10 µg/ml streptomycin,
and 10% WEHI-3B cell supernatant as a source of IL-3 (normal medium).
Under starvation conditions, cells were extensively washed in RPMI, and
then preincubated for 6 h in a serum- and IL-3-free medium
containing 2% BSA (Fraction V, Sigma, St. Louis, MO), 50
µM 2-mercaptoethanol, 2 mM L-glutamine, 10
U/ml penicillin, and 10 µg/ml streptomycin (starvation medium). For
GH stimulation (bGH treatment), cells were starved for 6 h in
starvation medium and then 1 µg/ml of bGH (kindly provided by William
Baumbach, American Cyanamid Co., Princeton, NJ) was added
to the cell culture.
Cell Cycle Analysis and Assessment of Apoptosis
Cell cycle and apoptosis were assessed by DNA content analysis
after staining with the DNA intercalator propidium iodide (PI).
Briefly, cells (106) were harvested by
centrifugation and permeabilized with 30 µl of DNA-Prep LPR reagent,
followed by addition of 0.5 ml of DNA-Prep stain PI solution (DNA-Prep
reagents, Coulter Corp., Hialeah, FL). After vortexing, samples were
incubated at 37 C for 30 min, and then analyzed by flow cytometry on a
FACScan (Becton Dickinson and Co., Mountain View, CA)
using low flow rate. Cell cycle distribution was determined using
CellQuest software (Becton Dickinson and Co.) and manual
gating. Apoptosis was determined as the percentage of DNA localized in
the hypodiploid peak
(sub-G0/G1) of the cell
cycle.
GH Assays
For GH measurement in culture medium, stable clones of Ba/F3
cells (1.5 x 106 cells/ml) expressing
wild-type GHR (Ba/F3 GHR) were incubated in starvation medium without
BSA for 24, 48, and 72 h. Cell-conditioned media were collected
and concentrated 150-fold using Centricon-plus 80 membrane
(Millipore Corp., Bedford, MA). GH measurements were done
using a modified RIA method, as previously described (52). Human GH
antagonists B2036 and G120K were obtained from Sensus Drug Development
Corp. (Austin, TX). Activity of the antagonists was assessed on Ba/F3
GHR cells incubated for 48 h in starvation medium in the absence
or in the presence of increasing concentrations of B2036 or G120K
ranging from 0.5 to 2 µg/ml.
Immunoblotting
Cells (106) were washed in PBS and lysed
either in sample buffer containing dithiothreitol or in lysis buffer
(1% Triton X-100, 50 mM Tris-HCl, pH 7.5, 150
mM NaCl, 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 10 µg/ml trypsin inhibitor). Protein lysate concentration
was measured by Bradford assay using the Bio-Rad reagent (Bio-Rad Laboratories, Inc. Hercules, CA), according to the
manufacturers directions. Samples (50 µg of protein) were resolved
by 10% SDS-PAGE under reducing conditions. Protein lysates were
transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.) and stained with red ponceau to verify the amount of
protein per lane. The membranes were incubated overnight at 4 C in
TBS-T (50 mM Tris-HCl, pH 7.6, 200 mM NaCl,
0.1% Tween 20) with 2% BSA. Proteins were detected by incubation with
the specific antibody in TBS-T with 2% BSA. After extensive washing in
TBS-T, a horseradish peroxidase-conjugated protein G (1:1000 dilution;
Bio-Rad Laboratories, Inc.) was added for 1 h. The
membranes were again subjected to extensive washing in TBS-T, and the
specific protein bands were visualized using enhanced chemiluminescence
detection system (NEN Life Science Products, Boston, MA),
according to the manufacturers instructions.
Transfections
The pRCßactin vector encoding the
I
B
(A32/36) was kindly provided by Michael Karin (University of
California, San Diego, CA). The mutated I
B
(A32/36) protein
contains serine-to-alanine mutations in amino acids 32 and 36,
preventing its phosphorylation and subsequent degradation (53). For
transfection experiments, 107 Ba/F3 GHR cells
were transiently transfected with 30 µg of I
B
(A32/36) encoding
plasmid or 30 µg of the parental pRCßactin
plasmid (53). Cells were electroporated at 330 V, 1500 µF,
R in a
CellJect apparatus (Eurogentec, Seraing, Belgium). Transfected cells
were maintained in normal medium for 24 h before subsequent
treatment.
EMSA
The double-stranded oligonucleotide containing the upstream
NF-
B element from the c-myc gene, termed URE, contains
the following sequence: 5'-AAGTCCGGGTTTTCCCCAACC-3' (with
core NF-
B binding site underlined), as previously
described (54). The DNA was labeled using the Klenow fragment of
Escherichia coli DNA polymerase I (Life Technologies, Inc., Gaithersburg, MD) and
[
-32P]dCTP (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Nuclear extracts used for EMSA
were prepared as described by Dignam et al. (55). Nuclear
extracts (23 µg) were incubated in sample buffer (0.4 µg of
poly(dIdC), 0.1% Triton X-100, 0.5% glycerol, 0.8
mM dithiothreitol, 2 mM
HEPES, pH 7.5) and adjusted to 100 mM KCl in a
final volume of 25 µl. Then, 32P-labeled URE
probe (40,000 cpm,
2 ng) was added to the mixture, which was
incubated for 30 min at room temperature. For competition analysis 10-
and 20-fold molar excess of unlabeled wild-type or mutant (with
conversion of internal two G-to-C residues) URE competitor
oligonucleotides were added to the binding reaction. For supershift
experiments, 1 µl of anti-p65 antibody (27) was added to the mixture
for 1 h at room temperature before the incubation with the labeled
probe. The reaction was resolved on a 4.5% acrylamide gel containing
Tris-Borate-EDTA buffer for 2 h at 150 V. The gel was dried and
subjected to autoradiography at -80 C using screens.
 |
ACKNOWLEDGMENTS
|
---|
We thank M. Karin and N. Rice for generously providing I
B
expression vector and anti-p65 antibody, respectively. B. Bennett and
W. Baumbach are gratefully acknowledged for the gifts of hGH
antagonists and bGH, respectively, and M.T. Bluet-Pajot for assistance
with the GH measurements. We thank INSERM Unité 373 for use of
the fluorescence-activated cell sorter (FACS) and C. Garcia for
technical assistance in the FACS analyses.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Marie-Catherine Postel-Vinay, INSERM U344, Endocrinologie Moléculaire, Faculté Necker-Enfants Malades, 156, rue de Vaugirard, 75015 Paris, France.
1 Current address: Dr. Elena Baixeras, Department of Medicine and Liver
Unit, Medical School, University of Navarra, 31 008 Pamplona,
Spain. 
This work was supported by the Institut National de la Santé et
de la Recherche Médicale (S.J., M.C.P-V., and E.B.), Grant 5363
from Association pour la Recherche sur le Cancer, and by Public Health
Service Grant CA-36355 (G.E.S.) from the National Institute of
Health.
Received for publication November 10, 1999.
Revision received February 1, 2000.
Accepted for publication February 10, 2000.
 |
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