From the The STAT5 activation has important roles in cell
differentiation, cell cycle control, and development. However, the
potential implications of STAT5 in the control of apoptosis remain
unexplored. To evaluate any possible link between the erythropoietin
receptor (EpoR) JAK2/STAT5 transduction pathway and apoptosis, we have investigated apoptosis-resistant cells (ApoR) that arose from positive
selection of the erythroid-committed Ba/F3EpoR cells triggered to
apoptosis by ectopic expression of the HOX-B8 homeotic gene. We show
that JAK2 is normally activated by Epo in both Ba/F3EpoR and ApoR
cells. In contrast, both STAT5a and STAT5b isoforms are uniquely
activated in a C-truncated form (86 kDa) only in ApoR cells. Analysis
of ApoR and Ba/F3EpoR subclones confirmed that the switch to the
truncated STAT5 isoform coincides with apoptosis survival and that ApoR
do not derive from preexisting cells with a shortened STAT5. In
addition, ApoR cells die in the absence of Epo. This indicates that
resistance to apoptosis is not because of a general defect in the
apoptotic pathway of ApoR cells. Furthermore, we show that the 86-kDa
STAT5 protein presents a dominant-negative (DN) character. We
hypothesize that the switch to a DN STAT5 may be part of a mechanism
that allows ApoR cells to be selectively advantaged during apoptosis.
In conclusion, we provide evidence for a functional correlation between
a naturally occurring DN STAT5 and a biological response.
Erythropoietin (Epo)1
regulates both the growth and differentiation of immature erythroblasts
through its interaction with specific, high affinity cell surface
receptor (EpoR). EpoR belongs to the cytokine receptor superfamily (1,
2) and associates with JAK2, a member of the Janus protein tyrosine
kinases, through the receptor cytoplasmic, membrane-proximal domain
containing the conserved box1 and box2 motifs (3). The activated JAK2 phosphorylates tyrosine residues on the cytoplasmic tail of the receptors that serve as docking sites for the SH2 domain that occur in
STAT5 (4, 5), a member of the signal transducer and activator of
transcription (STAT) proteins (6, 7). STAT5 dimerizes, translocates to
the nucleus and promotes the expression of genes containing specific
DNA elements homologous to the Apoptosis, or programmed cell death, is a genetically controlled
mechanism for cells to commit suicide in response to specific stimuli such as tumor necrosis factor In the present study, we have investigated the EpoR JAK2/STAT5 signal
transduction pathway in cells that have been positively selected during
the HOX-B8-mediated apoptosis of the erythroid-committed Ba/F3EpoR
cells (28). HOX-B8 is an homeobox-containing gene, and it is among a
variety of transcription factors that have emerged as key components of
the regulatory process for lineage-specific development of early
hematopoietic cells (29). It has been recently described that HOX-B8
inhibits granulocytic differentiation as evident by rapid apoptosis of
the 32DHOX-B8 cells treated with G-CSF (30). Furthermore, HOX-B8 exerts
a contributory role in leukemogenesis: the integration of an
intracisternal A-particle (IAP) genome into the 5' noncoding region
causes the constitutive expression of HOX-B8 and interleukin-3 (IL-3)
in the WEHI-3B myelomonocytic leukemia cell line (31).
We show that cells signaling through dominant-negative STAT5 are
selectively advantaged during the HOX-B8-mediated apoptosis, suggesting
that C-truncated STAT5 isoforms are likely to contribute to a unique
biological response in the cells where they are expressed.
Cell Culture and Electroporations--
Ba/F3 are murine
IL-3-dependent immature progenitor cells (32). Ba/F3EpoR
cells have been previously generated by ectopic expression of the EpoR
and are irrevocably committed to erythroid differentiation (28).
Ba/F3HOX-B8 and Ba/F3EpoRHOX-B8 cells have been generated by
transfecting the MPZenNeo/Hox-2.4 vector (33) (now referred to as
MPZenNeo/HOX-B8, according to the new nomenclature of HOX genes (34),
into Ba/F3EpoR cells, followed by G418 selection in the presence of
Epo. Cell subclones were obtained by limiting dilution in the presence
of Epo and G418. All cells were maintained in RPMI 1640 medium
supplemented with 10% fetal calf serum (HyClone) in 5%
CO2 incubators at 37 °C. Recombinant human Epo was
purchased from Boehringer Mannheim and was used at 0.2 units/ml.
Conditioned medium at 10% from WEHI-3B cells was used as a source of
murine IL-3.
Institutes of Biological Chemistry and
¶ Anatomy,
Institute for Neurological Sciences "C. Besta",
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-interferon activation site (GAS)
(8). STAT5 was originally identified in prolactin-mediated responses
(9-11) but is activated by several other cytokines (12). In mice,
there exist two STAT5 genes that are 96% identical (STAT5a and
STAT5b). These encode proteins of 94 and 92 kDa, respectively (11, 13).
STAT5a is the principal and also an obligate mediator of mammopoietic
and lactogenic signaling (14). Studies on STAT proteins indicate that
the transcriptional activation domains reside in the C terminus
(15-18). Moreover, C-terminal truncated STAT5 a/b isoforms, generated
by protein processing (19, 20), retain high affinity DNA-binding
activity and present dominant-negative (DN) character suppressing
transcription (16-17). However, the potential physiological
significance of DN STAT5 is unknown at present. Another member of the
STAT family, STAT1, exists in two forms of 91 and 84 kDa, and it has
been demonstrated that they differ in their ability to trans-activate
selected genes (21).
(TNF-
) or the
fas ligand (22, 23), or it is activated in response to
various forms of cell injury or stress (24, 25). The potential role of
the JAK/STAT signaling pathway in the induction of apoptosis has been poorly investigated. Recent studies report that activation of STAT1 by
gamma interferon (IFN-
) and epidermal growth factor (EGF) induced
apoptosis in HeLa and A431 cells via caspase-1 activation (26), and
furthermore, that TNF-
failed to induce apoptosis in STAT1-null
cells because of low levels of caspases (27).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
RNA Analysis--
RNase protection assays and -globin
riboprobe synthesis were performed as described previously (28). The
HOX-B8 riboprobe was generated by subcloning the 300-base pair
SacI-BamHI 5' DNA fragment of the HOX-B8 cDNA
into SP72 polylinker (Promega). SP6 polymerase was used to synthesize
the radiolabeled antisense riboprobe.
Apoptosis Assays-- Cells were harvested by centrifugation, resuspended in the appropriate culture medium at a density of 106/ml and allowed to settle on poly-L-lysine (Sigma)-coated glass coverslips for 15 min in a humid chamber. The glass coverslips were washed with phosphate-buffered saline, and the cells were fixed with 4% paraformaldehyde for 5 min at room temperature and stained for 1 min with 1 mg/ml DNA-binding fluorochrome Hoechst 33258 dye. DNA fragmentation was analyzed by pulsed field gel electrophoresis as described by Walker et al. (35).
Cellular Extracts and Electrophoretic Mobility Shift
Assay--
Cells were starved by Epo-depletion for 5 h and
subsequently stimulated with 10 units/ml of Epo for 15 min at 37 °C.
To obtain whole cell extracts (WCE), cells were resuspended in a high
salt buffer (20 mM Hepes, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol supplemented with 1 mM
Na3V04, 50 mM NaF and a standard
protease inhibitors mixture) and lysed by three freeze-thaw cycles in
liquid N2. Electrophoretic mobility shift assays (EMSAs) were performed as described previously (36) using as a probe an
oligonucleotide corresponding to the IFN--responsive region (GRR)
located within the promoter of Fc
RI gene (37).
Antibodies, Immunoprecipitation, and Immunoblotting-- Anti-STAT5a/b mouse monoclonal antibodies raised against amino acids 451-649 were purchased from Transduction Laboratories; two anti-STAT5a/b rabbit polyclonal antibodies, raised against amino acids 5-24 (N-20) and 711-727 (C-17) and anti STAT1 monoclonal antibody raised against amino-terminal region, were purchased from Santa Cruz Biotechnology. Monoclonal antibodies specific for STAT5a C' and STAT5b C' were purchased from R&D Systems. Anti-JAK2 antibodies and anti-phosphotyrosine 4G10 antibodies were purchased from Upstate Biotechnology.
Whole cell extracts from 7 × 107 cells were prepared following the same procedure used to prepare WCE for EMSA. A final NaCl concentration of 150 mM was obtained by diluting the extracts with 50 mM Tris, pH 8, and Triton X-100 was then added to make 1% of the final concentration. Immunoprecipitation and immunoblotting were performed as described previously (38) with some modifications. WCE were immunoprecipitated with a 1:200 dilution of anti-STAT5 (N-20) antibodies overnight at +4 °C. Immunoprecipitates were washed 5 times with washing buffer and once with 1× Tris-buffered saline. In immunoblotting, anti-phosphotyrosine antibodies 4G10 and anti-STAT5 (N-20 and C-17) were used at 1:2000, whereas monoclonal antibodies anti-STAT5 were used at 1:250 dilution.Luciferase Assays--
For luciferase reporter assays, the pGL2
promoter vector (Promega) was used. pGL2 contains the luciferase gene
driven by a simian virus 40 basic promoter without an enhancer. The
GAS-luc. construct (kindly provided by Bernard Mathey-Prevot, Dana
Farber Cancer Institute, Boston, MA) contains four tandem repeat GAS sequences from the murine -casein promoter (core sequence:
ATTTCTAGGAAATCG) upstream of the luciferase element. Transient
transfection experiments were performed as follows. 5 × 106 cells/condition were starved by depletion of Epo for
5 h and then transfected by electroporation with 10 µg of test
plasmid. After electroporation, cells were replaced in complete medium supplemented with Epo at 1.2 units/ml. Luciferase activity, assayed using the Luciferase Assay System (Promega), was determined after 12 h of stimulation. Each construct was tested four times by
independent electroporations with similar results each time.
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RESULTS |
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Overexpression of HOX-B8 in the Progenitor Ba/F3 and in the Erythroid Committed Ba/F3EpoR Cells-- The homeobox gene HOX-B8 is not expressed in Ba/F3 (not shown) and Ba/F3EpoR cells (Fig. 1). To understand whether HOX-B8 interferes with erythroid differentiation, we examined the consequences of ectopic expression of HOX-B8 on erythroid-committed Ba/F3EpoR as well as in the progenitor Ba/F3 cells. Three weeks following electroporation, G418-resistant Ba/F3HOX-B8 and Ba/F3EpoRHOX-B8 cells were available. Unexpectedly, keeping the cells in culture in the presence of Epo, a high rate of cell mortality was observed with the Ba/F3EpoRHOX-B8 cells. The same phenomenon was also observed when Ba/F3EpoRHOX-B8 cells were grown in the presence of IL-3. On the contrary, Ba/F3HOX-B8 cells grew normally. To evaluate whether mortality was because of programmed cell death, staining experiments were performed with the Hoechst 33258 fluorescent dye. The morphological features of nuclear chromatin indicative for apoptosis were observed in Ba/F3EpoRHOX-B8 cells (not shown). DNA fragmentation analysis confirmed that apoptosis was indeed the cause of cell death (not shown). However, a small proportion (approximately 3%) of cells survived apoptosis and generated an apoptosis-resistant cell population, named ApoR.
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A C-terminal Truncated STAT5 Protein Is Phosphorylated upon Epo Stimulation in Cells Surviving Apoptosis-- The fact that apoptosis of the Ba/F3EpoRHOX-B8 cells occurred in the presence of Epo and that the recovered ApoR cells died if Epo was removed from the culture medium, suggested to us that the ApoR cells did not present a general defect in the apoptotic pathway. Therefore, we explored the hypothesis that the EpoR signal transduction pathway, in cells that selectively escaped apoptosis, could have undergone some modification. To study STAT5 activation, we performed EMSA experiments with a radiolabeled GRR probe and WCE from unstimulated or stimulated with Epo, Ba/F3EpoR, and ApoR cells. As shown in Fig. 2, A and B, two bands were detected in both cell types after Epo treatment. To our surprise, however, the two bands from ApoR lysates had higher intensity and migrated slightly faster than the two bands from Ba/F3EpoR lysates. To evaluate whether the retarded bands in ApoR were exclusive to Epo treatment, EMSA experiments were performed with cells stimulated with IL-3 for the same length time. The same pattern bands were observed when ApoR cells were stimulated with IL-3 (not shown).
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The C-truncated STAT5 Protein Present a Dominant-negative
Character--
To further analyze STAT5 activation,
immunoprecipitations with N'-STAT5a/b antibodies followed by
immunoblotting with anti p-Tyr antibodies (4G10) were
performed on WCE from representative Ba/F3EpoR and ApoR subclones.
Interestingly, a unique phosphorylated 86-kDa band was detected in the
ApoR cells (STAT5), whereas two phosphorylated 92-94-kDa bands were
present in Ba/F3EpoR cells (Fig.
4A). To verify whether in ApoR
cells, the phosphorylated 86-kDa protein was truncated at the
C'-terminal, the same filter was stripped and reprobed with C'-STAT5a/b
antibodies. Immunoprecipitated STAT5 proteins were recognized by these
antibodies in the parental cells (Fig. 4B), whereas in the
ApoR cells, STAT5 bands were not detected. Finally, the same filter was
stripped and reprobed with anti-STAT5 antibodies raised against an
internal domain of the protein. These antibodies not only recognized
the same 86-kDa protein detected by 4G10 in ApoR cells, but also the
two 92-94-kDa proteins in Ba/F3EpoR cells (Fig. 4C). To
confirm whether both STAT5a and STAT5b were switched to the truncated
form, Western blot experiments were performed with monoclonal
antibodies for STAT5a and STAT5b. As expected, because both monoclonal
antibodies were recognizing the C terminus of STAT5, no bands were
detected in ApoR cells. On the contrary, different bands corresponding to STAT5a and STAT5b were present in the Ba/F3EpoR cells (not shown).
This result confirmed that both STAT5a and b were processed in ApoR
cells.
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DISCUSSION |
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Our first observation is that HOX-B8 overexpression causes
apoptosis in cells that have irreversibly passed the checkpoint for
erythroid maturation. But it does not do so in the immature progenitor
Ba/F3 cells. The fact that the erythroid phenotype is not permissive
for HOX-B8 expression is supported by the finding that cells which
survived apoptosis lost -globin transcripts. However, before
apoptosis
-globin transcript is easily detectable in the parental
Ba/F3EpoR and in the Ba/F3EpoRHOX-B8 cells. In line with our results,
it has been recently shown that ectopic expression of HOX-B8 in
hematopoietic progenitor cells negatively affects granulocyte
development and positively regulates macrophage development; thus
confirming that inappropriate constitutive HOX-B8 expression can alter
growth, differentiation, and survival of hematopoietic cells (30).
Apoptosis of Ba/F3EpoRHOX-B8 cells was detected only after Neo-selection had occurred. A possible explanation for this is that a period of latency is required before the HOX-B8 interacts with cellular targets and thus exerts its functions. We also notice that HOX-B8 overexpression does not abolish the factor-dependence (IL-3 or Epo) in any of the Ba/F3-derivative cells. This supports the finding that derivative cells are not fully transformed by HOX-B8 alone (34). Accordingly, the enforced expression of HOX-B8 in bone marrow cells yields IL-3-dependent, nontumorigenic cell lines, and the progression to a fully malignant state is favored, as in WEHI-3B cells, by somatic mutations conferring autocrine production of IL-3 (34).
Studying STAT5 activation by Epo, we observe by EMSAs bands with higher intensity and faster migration in ApoR cells than in the Ba/F3EpoR cells. This is consistent with the higher affinity for the DNA target sequence of the C-truncated STAT5 isoform, as previously reported (16, 17). Accordingly, antibodies against the C-terminal STAT5 region do not supershift the ApoR-derived DNA/STAT5 complex. Analysis of the ApoR subclones allowed the isolation of one clone (clone 13) that exhibits an activated full-length STAT5 following Epo stimulation. This shows that within the ApoR cell population, (frozen soon after apoptotic selection) cells exhibiting the full-length STAT5 protein still exist. However, this seems to be rather exceptional because EMSAs from ApoR cellular extracts, obtained at the same time as when the cells were frozen, do not show any slow-migrating band indicative of the full-length STAT5/DNA complex. Thus, activation of the C-truncated STAT5 by Epo is very representative of cells that escaped apoptosis. This is confirmed by the ApoR subclone-13 that undergoes apoptosis. In fact, the derived apoptosis-resistant cells exhibit only the activated C-truncated STAT5 variant, upon Epo stimulation, and retain their Epo-dependence for growth.
Significantly, in the ApoR cells, the phosphorylated 86-kDa STAT5 does not coexist with the phosphorylated full-length 92-94-kDa STAT5a/b proteins, as previously reported for other cell lines (11, 13). Therefore, this is indicative of a complete processing occurring on all the STAT5a/b proteins present in the cells. Furthermore, the 86-kDa size that we report for the phosphorylated STAT5 form in cells that survived apoptosis does not coincide with the 77-80-kDa size previously described for the naturally occurring C-truncated STAT5 isoforms in 32Dcl3 or CTLL cells (40). A possible explanation for the different size of the C-truncated STAT5 is that the 86-kDa form originates from a different STAT5 processing that might be related to the maturation state of ApoR cells. In this context, it has been reported that in immature myeloid cells, IL-3 activates the 77-80 kDa STAT5, whereas in mature myeloid cells IL-3 activates the full-length 94-96 kDa STAT5; thus indicating that IL-3 signals through multiple isoforms of STAT5, depending on the differentiation state of target cells (41). Indeed, we speculate that this might be the case for the ApoR cells that might be reverted to an immature state by HOX-B8. Nevertheless, further studies are required to better characterize the maturation stage of the ApoR.
We also show that the C-truncated STAT5 form presents a
dominant-negative feature since transient expression of the
GAS-luciferase construct in cells that escaped apoptosis have
negligible luciferase activity. As mentioned above, we hypothesize that
the 86-kDa STAT5 isoform accounts for a selective STAT5 processing,
peculiar to ApoR cells, and that the inhibition of transcription by the
dominant-negative STAT5 may be restricted to certain genes. Therefore
it might be that alternative STAT5 processing discriminates between
different functions, for example, as in ApoR cells, allowing
proliferation and inhibiting apoptosis-connected functions. However,
Epo, like a number of cytokines, activates a variety of signaling
pathways such as the tyrosine phosphorylation of SHC and subsequent
activation of the ras pathways (42) or the tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) (43) and
phospholipase C-1 (44). Therefore, we cannot exclude the possibility
that the DN STAT5 isoform in ApoR cells may not be directly involved in
the control of cell proliferation.
In conclusion, our observations that cells presenting a DN STAT5 protein are selectively advantaged during HOX-B8-mediated apoptosis led us to speculate a potential physiological role for C-truncated STAT5 isoforms. Studies are presently under way to determine the antiapoptotic activity exerted by the ApoR-DN STAT5. This could account for the role of Epo in stimulating different responses in ApoR cells, depending on the particular cell-differentiation stage.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Susan Cory for the generous gift of the HOX-B8 expression vector, Bernard Mathey-Prevot for luciferase plasmids and invaluable comments and suggestions, Sergio Ottolenghi and Markus Heimm for critical reviews of the manuscript, Massimo Libonati for moral support, and Chiara Costanzo for technical advice.
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FOOTNOTES |
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* This work was supported in part by the Italian Ministry of University and Scientific Research (60%).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a fellowship from Istituto Superiore di Sanitá. Present address: AIDS Immunopathogenesis Unit, DIBIT, S. Raffaele Scientific Institute, Milano, Italy.
** Recipient of a NATO C.R.G. 970203. To whom reprint requests should be addressed. Tel.: 39-2-4814766; Fax: 39-2-4814755; E-mail: LIBEL{at}borgoroma.univr.it.
The abbreviations used are:
Epo, erythropoietin; EpoR, erythropoietin receptor; JAK, Janus kinase; STAT, signal
transducer and activator of transcription; GAS, gamma interferon
activation site; GRR, gamma interferon-responsive region; IL-3, interleukin-3; TNF-, tumor necrosis factor
; IFN, interferon; DN, dominant-negative; EMSA, electrophoretic mobility shift assay; WCE, whole cell extract.
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REFERENCES |
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