|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on August 1, 2002; DOI 10.1182/blood-2002-05-1602.
HEMATOPOIESIS
From the Hematopoiesis Department and the Flow
Cytometry Facility, American Red Cross Holland Laboratory,
Rockville, MD; and the Department of Anatomy and Cell Biology, The
George Washington University, Washington, DC.
Secreted growth factors are integral components of the bone marrow
(BM) niche and can regulate survival, proliferation, and differentiation of committed hematopoietic stem cells (HSCs). However, downstream genes activated in HSCs by early-acting cytokines are not well characterized. To better define intracellular cytokine signaling in HSC function, we have analyzed mice lacking expression of
both signal transducer and activator of transcription 5a (STAT5a) and STAT5b (STAT5ab Hematopoietic stem cells (HSCs) have tremendous
proliferative potential and are required to generate differentiated
progeny cells of all hematopoietic lineages in response to short-term myelosuppression. In addition, HSCs are defined clonally by their ability both to differentiate and to self-renew. Regulation of HSC commitment is not well-defined but is believed to be stochastic and
not regulated by the microenvironment.1,2 However, HSC survival and self-renewal are likely regulated by secreted factors within the bone marrow (BM) niche.3 Following commitment,
early-acting hematopoietic growth factors can drive cell cycle
activation and promote differentiation of HSCs. Growth factor signals
can promote HSC survival and proliferation during in vitro stimulation,
leading to enhanced oncoretroviral-mediated gene
transfer.4 However, little is known about the
intracellular signal transduction pathways that are necessary for
proliferation and differentiation or for self-renewal division of HSCs.
Our studies have focused on activation of Janus kinase (JAK) and the
latent signal transducer and activator of transcription (STAT)
pathway. The JAK/STAT signaling axis is a conserved pathway involved in diverse aspects of development. Recent studies in Drosophila have highlighted the role of the lone STAT
protein in self-renewal of germ cells during
spermatogenesis.5,6 Furthermore, murine STAT3 knockout
leads to early embryonic lethality,7 and its activation is
essential for embryonic stem cell self-renewal in
vitro.8,9 In contrast, STAT5 expression is first evident during differentiation of embryonic stem (ES) cells.10
HSCs from mice lacking expression of both STAT5a and STAT5b
(STAT5ab Difficulties in the characterization of HSC defects in adult
STAT5ab Mice genotyping and drug injections
Bone marrow transplantation and peripheral blood analyses
Antibody staining and flow cytometry BM cells were lineage depleted using a magnetically labeled antibody kit (StemSep; Stem Cell Technologies, BC, Canada), which was followed by staining with a cocktail of phycoerythrin (PE)-conjugated antibodies to lineage markers that included Ly-6G (Gr-1), CD11b (Mac-1), CD45R/B220, CD4 (L3T4), CD8 (Ly-2), Ter119/Ly-76, CD90.2 (Thy1.2), and NK1.1 (NKR-P1B and NKR-P1C). The cells also were stained with antibodies to fluorescein isothiocyanate (FITC)-conjugated Ly-6A/E (Sca-1) and biotin-conjugated CD117 (c-kit). The biotinylated c-kit antibody was detected using a secondary streptavidin-phycoerythrin-Cy5 conjugate (SA-PE-Cy5). All antibodies for these studies were obtained from BD Pharmingen (San Diego, CA). Cells were then analyzed on a fluorescence activated cell sorter (FACS)Vantage SE flow cytometer equipped with an INNOVA 70C laser providing 488 nm of excitation at 70 mW output power (BD Biosciences, San Jose, CA) . For peripheral blood analyses, the percentage of Ly-5.2 donor engraftment in Ly-5.1 recipient mice was quantitated using an FITC-conjugated CD45.2 (anti-Ly-5.2) antibody in combination with either direct PE-conjugated or biotinylated PE-Cy5-conjugated lineage antibodies. Some analyses also were performed on a BD LSR flow cytometer (BD Biosciences). For 4-color analyses of KLS cells and Ly-5.1 on the BD LSR, allophycocyanin (APC)-conjugated antibody to c-kit and PE-Cy5-conjugated antibody to CD45.1 (Ly-5.1) were used.Southern blot analyses Genomic DNA was prepared from BM and spleen tissues from mice that received transplants as previously described.11 DNA was then extracted with an equal volume of phenol/chloroform/isoamyl alcohol 25:24:1 and precipitated with 2.5 volumes of ice-cold ethanol and 1/10 volume sodium acetate. Overnight, 5 µg DNA was digested with NcoI enzyme and separated on a 0.8% agarose gel by electrophoresis. Gels were blotted overnight onto Hybond N+ nylon membrane (Amersham, Arlington Heights, IL), UV cross-linked, and hybridized with a [32P]-labeled mouse Y-chromosome probe. Blots were washed at a final stringency of 0.5X sodium citrate (SSC)/0.5% sodium dodecyl sulfate (SDS) at 65°C, exposed overnight, and autoradiographic images were obtained using a Storm phosphorimager (Amersham Biosciences) and x-ray film (Eastman Kodak, Rochester, NY). [32P]-dCTP was obtained from Amersham.
Long-term repopulating defects are intrinsically dependent on STAT5 activation To eliminate lymphocytes, the C57Bl/6 STAT5ab+/
genotype was crossed onto the C57Bl/6 RAG2/ background
where splenomegaly disease and autoimmune phenotypes were not observed.
BM cells were isolated from both hind limbs of donor C57Bl/6
(hemoglobin single, Hbs) RAG2/
STAT5ab+/+ or RAG2/
STAT5ab/ littermate mice and mixed 1:1 with congenic
wild-type HW80 (hemoglobin diffuse; Hbd) mouse BM cells
using the approach previously described.11 In all
RAG2/ mice, absolute lymphocyte counts were virtually
zero as determined on peripheral blood smear (data not shown). Lethally
irradiated BM transplant (BMT) recipients were analyzed 19 and 18 weeks
later for experiment no. 1 and experiment no. 2, respectively (Figure 1).
At all time points after transplantation, no Hbs was
detectable from the RAG2 Since we previously showed that STAT5-deficient HSCs could engraft
transplant recipients in the absence of a competitor
marrow,11 a second approach was to generate chimeric mice
by transplanting Ly-5.2/Hbs STAT5ab
STAT5-deficient HSCs are not 5-FU sensitive and are noncompetitive during steady-state hematopoiesis Using the BM chimera approach, engraftment was complete at all times from 8 weeks after transplantation, except for the previously described STAT5ab/ T-lymphocyte
chimerism.11 To determine the sensitivity of
STAT5-deficient HSCs to a cell cycle-active drug, between 12 and 16 weeks after transplantation the chimeras were treated with a single
intraperitoneal injection of 5-FU and analyzed serially for the
hematocrit and the engraftment measured by hemoglobin electrophoresis
(Figure 3). Although the myelosuppression
was severe in the STAT5ab/ chimeras, the recovery from
the nadir was not delayed. At 2 weeks after transplantation for
experiment no. 1, the hematocrit of 20% ± 7% for
STAT5ab/ was lower than the 33% ± 2% for wild-type
chimeras. However, in both experiment no. 1 and experiment no. 2, the
hematocrit was able to rebound to levels that unexpectedly were not
different from the wild-type chimeras. In experiment no. 1 at 6 weeks,
the hematocrit was normal (42% ± 3% for STAT5ab/
versus 46% ± 2% for wild-type), and at 15 weeks the
STAT5ab/ chimeras had a hematocrit of 47% ± 3%
(n = 3), and the wild-type chimeras had a hematocrit of 50% ± 6%
(n = 4). In experiment no. 2 at 13 weeks, the
STAT5ab/ chimeras had a hematocrit of 42% ± 2%
(n = 3), and the wild-type chimeras had a hematocrit of 43%
(n = 2). Engraftment was always 100% at all time points as
determined by hemoglobin electrophoresis (data not shown).
Although the HSC pool was not ablated by 5-FU treatment in the context
of the chimeric mouse, we also wanted to determine whether
STAT5-deficient repopulating cells might be more 5-FU sensitive than
wild-type HSCs. For these experiments, we treated both mutant and
wild-type young (3-5 weeks old) mice that had not received transplants
with 150 mg/kg 5-FU, and 2 days later harvested the BM for competitive
repopulation against PBS control-treated BM cells. Two experiments
were performed, one with male treated mice and the other with female
treated mice. In both cases, the male versus female competitive
repopulation also was performed with both PBS-treated mice as a
control. The mice were reconstituted for 16 weeks and then
killed for BM collection. Pooled BM cells were then analyzed by
Southern blot for the percentage of male DNA as determined using a
standard curve of male DNA mixed into mouse Y-negative female DNA
(Figure 4, left panels). Toxicity from
the 5-FU treatment was reflected by an increase in the reconstitution with PBS-treated competitor as shown in the right panels. Modest 5-FU
toxicity was observed on wild-type BM cells, however, relative to the
wild-type samples, the STAT5-deficient 5-FU-treated samples competed
on average 1.7-fold better against its own PBS-treated competitor.
Thus, STAT5 deficiency does not increase 5-FU sensitivity but rather
may decrease sensitivity even in the 3- to 5-week old mutant
microenvironment. The average overall engraftment of the donor cell
pool was used as the measure of repopulating potential to control for
possible variation between individual recipients of
STAT5ab
To test whether STAT5-deficient chimeras would be favorable recipients
for a wild-type BM challenge, chimeric mice were again generated.
STAT5ab
STAT5-deficient KLS cells are defective in long-term repopulating activity in vivo and have reduced cytokine responsiveness in vitro We next wanted to determine whether the effects observed in vivo would be evident in an enriched HSC population isolated from the BM of the STAT5-deficient mice. For some experiments, Sca-1+c-kit+lin (KLS) cells were
transplanted at limiting dilution into lethally irradiated recipient
mice along with 2 × 105 competitor cells (Table
1). In all 3 transplants with wild-type KLS cells, long-term repopulating activity was seen with cell doses as
low as 250 cells. However, in 2 separate KLS transplants using mutant
cells, no detectable engraftment was observed with 10- to 20-fold
higher KLS cell doses. Even following injection of lineage-negative
non-KLS cells from experiment no. 3, no engraftment was detected in 3 mice receiving 14 000 cells, while 1 of 4 mice receiving 43 000
wild-type lineage-negative non-KLS cells did show some engraftment
(6%). This result demonstrates the complete loss of competitive
long-term repopulating activity despite a normal KLS
phenotype.
In other experiments, 200 to 800 KLS cells/well were put into liquid
suspension culture in the presence of cytokine cocktails to stimulate
proliferation in vitro over a 6-day incubation period. Consistent with
the defective repopulating activity in vivo, the KLS cells showed a
reduced expansion potential in vitro (Table 2).
As expected, IL-3 supported the greatest proliferation and was required
for maximal levels of cell expansion. Stimulation with cocktails
containing other early-acting growth factors such as IL36S, IL3S, and
IL3SFT revealed the greatest deficiency for STAT5-deficient KLS cells.
These growth factor combinations are known to synergize with IL-3 to
promote cell proliferation and survival during short-term ex vivo
culture. As described previously, in all experiments the absolute
number of hind limb KLS cells was not different between wild-type and
STAT5-deficient mice. Interestingly, analysis of KLS numbers
from 3 separate experiments 16 weeks following transplantation also
showed equivalent numbers of KLS cells between both hind limbs of the
mice receiving transplants of wild-type BM cells (7945 ± 4738),
STAT5ab
This study sought to determine whether the competitive disadvantage of STAT5-deficient HSCs was due primarily to a cell intrinsic requirement for STAT5 activation or to the other life-threatening phenotypes observed in these mice.13,15 Since previous reports have shown antiapoptosis15 and cell cycle-related12 defects in the adult STAT5-deficient mouse, we have clarified a critical issue regarding the role of STAT5 in long-term repopulating activity. The STAT5-deficient mouse is severely affected by many phenotypes that reduce the life span and that may likely contribute to an autoimmune-mediated effect on HSC survival within the BM cavity. The results of this study indicate that STAT5 activation is required downstream of early-acting cytokines to promote cytokine-mediated repopulating activity of HSCs. The inability to rescue this defect by elimination of lymphocytes or by transplant into a "normal" wild-type microenvironment demonstrates that the primary mechanism for the defect is cell intrinsic. We isolated KLS fractions from young STAT5ab Bromodeoxyuridine-labeling studies have shown that within 30 days, all murine HSCs will have gone through one cell division.25,26 Therefore, rather than a clonal succession model with both proliferating and deeply quiescent HSC populations, HSCs are currently believed to be slowly cycling, but at any one point in time only 4% to 5% of the HSC population will be in S/G2/M. Here we show by competitive repopulation that repopulating STAT5-deficient HSCs are less sensitive to the mild 5-FU effects than wild-type HSCs. This result suggests that less cell cycle activation reduced 5-FU toxicity27 or 5-FU-mediated HSC activation,17 which has been associated with decreased repopulating activity.28 Further studies using 5-FU in combination with SCF will be necessary to test more HSC toxic regimens.27 Possible downstream targets of STAT5 in HSCs could be the cell cycle-associated genes such as the cyclins.29,30 Cyclin D2 is expressed at high levels in long-term repopulating HSCs.26 While we were unable to obtain sufficient numbers of STAT5-deficient mice and KLS cells for cell cycle analysis by FACS, future studies on differential gene expression might identify important downstream genes that are dysregulated. STAT5 is a mediator of p210Bcr-abl signaling in transduced cells,31 and chronic myelogenous leukemia is known to be a clonal stem cell proliferative disease. Constitutively activated mutants of STAT332 and STAT533 are also oncogenic. Therefore, the decreased cytokine responsiveness reported here for STAT5-deficient HSCs provides further support for targeting STAT5 as a treatment for at least some leukemias. However, STAT5 is not essential for myeloproliferative disease in chronic myeloid leukemia models, since STAT5-deficient mice could still develop disease.34 In an acute myeloid leukemia (AML) model for the Tel-JAK2 translocation, STAT5 activation was essential for leukemogenesis,33 and STAT5 activation is associated with AML.35,36 In these studies we have not determined whether antiapoptosis gene expression is dysregulated in the absence of STAT5. This might also comprise a significant component of the deficiency, although increased apoptosis in the HSC compartment would be predicted to reduce the pool size. Survival signaling pathways mediated through c-mpl37 and/or Flt338 might also require STAT5 activation in HSCs. Other signaling components downstream of c-kit could also compensate for the STAT5-mediated defects, possibly resulting in a similar but less severe phenotype than reported for the W/Wv mouse. Our data demonstrate that STAT5 activation is not essential for HSC self-renewal, since the KLS pool was restored to levels equivalent with wild-type mice following transplantation and hematopoietic reconstitution following 5-FU treatment or secondary transplant was not severely reduced. This finding was consistent with the idea that SCF may not be essential for murine HSC self-renewal39-41 but capable of promoting cell cycle entry.42 However, differences might exist between fetal and adult HSCs in this regard.43 Direct demonstration of STAT5 activity in the specific proliferative or antiapoptotic responses to cytokine stimulation will require further study. In summary, this study demonstrates that overall stem cell cytokine responsiveness and repopulating potential are highly STAT5 dependent and that in the STAT5-deficient background, wild-type BM cells have a dominant selective advantage in multiple lineages. Inversely, it remains to be determined whether conditional STAT5 activation might be used to confer a competitive engraftment advantage on wild-type BM cells. Recent demonstration of STAT5-dependent expansion of multipotential hematopoietic cells by signals from JAK2 and c-kit or Flt3 supports this concept.44
The authors would like to thank James N. Ihle for helpful discussions and Robert G. Hawley for critical review of the manuscript. We also thank Albert Forero for technical assistance with the mouse genotyping PCR analyses.
Submitted May 31, 2002; accepted July 3, 2002.
Prepublished online as Blood First Edition Paper, August 1, 2002; DOI 10.1182/blood-2002-05-1602.
Supported by National Institutes of Health grant R01DK059380, The Lauri Strauss Leukemia Foundation (K.D.B.), and the American Red Cross.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kevin D. Bunting, Hematopoiesis Department, American Red Cross Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855; e-mail: buntingk{at}usa.redcross.org.
1. Morrison SJ, Uchida N, Weissman IL. The biology of hematopoietic stem cells. Annu Rev Cell Dev Biol. 1995;11:35-71[CrossRef][Medline] [Order article via Infotrieve]. 2. Morrison SJ, Shah NM, Anderson DJ. Regulatory mechanisms in stem cell biology. Cell. 1997;88:287-298[CrossRef][Medline] [Order article via Infotrieve]. 3. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001;414:105-111[CrossRef][Medline] [Order article via Infotrieve].
4.
Bodine DM, Karlsson S, Nienhuis AW.
Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells.
Proc Natl Acad Sci U S A.
1989;86:8897-8901
5.
Tulina N, Matunis E.
Control of stem cell self-renewal in Drosophila spermatogenesis by JAK-STAT signaling.
Science.
2001;294:2546-2549
6.
Kiger AA, Jones DL, Schulz C, Rogers MB, Fuller MT.
Stem cell self-renewal specified by JAK-STAT activation in response to a support cell cue.
Science.
2001;294:2542-2545
7.
Takeda K, Noguchi K, Shi W, et al.
Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality.
Proc Natl Acad Sci U S A.
1997;94:3801-3804
8.
Niwa H, Burdon T, Chambers I, Smith A.
Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.
Genes Dev.
1998;12:2048-2060 9. Matsuda T, Nakamura T, Nakao K, et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J. 1999;18:4261-4269[CrossRef][Medline] [Order article via Infotrieve]. 10. Nemetz C, Hocke GM. Transcription factor Stat5 is an early marker of differentiation of murine embryonic stem cells. Differentiation. 1998;62:213-220[CrossRef][Medline] [Order article via Infotrieve].
11.
Bunting KD, Bradley HL, Hawley TS, Moriggl R, Sorrentino BP, Ihle JN.
Reduced lymphomyeloid repopulating activity from adult bone marrow and fetal liver of mice lacking expression of STAT5.
Blood.
2002;99:479-487
12.
Snow JW, Abraham N, Ma MC, Abbey NW, Herndier B, Goldsmith MA.
STAT5 promotes multilineage hematolymphoid development in vivo through effects on early hematopoietic progenitor cells.
Blood.
2002;99:95-101 13. Moriggl R, Topham DJ, Teglund S, et al. Stat5 is required for IL-2-induced cell cycle progression of peripheral T cells. Immunity. 1999;10:249-259[CrossRef][Medline] [Order article via Infotrieve].
14.
Socolovsky M, Fallon AE, Wang S, Brugnara C, Lodish HF.
Fetal anemia and apoptosis of red cell progenitors in Stat5a
15.
Suzuki H, Zhou YW, Kato M, Mak TW, Nakashima I.
Normal regulatory alpha/beta T cells effectively eliminate abnormally activated T cells lacking the normal interleukin 2 receptor beta in vivo.
J Exp Med.
1999;190:1561-1572 16. Teglund S, McKay C, Schuetz E, et al. Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell. 1998;93:841-850[CrossRef][Medline] [Order article via Infotrieve].
17.
Randall TD, Weissman IL.
Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment.
Blood.
1997;89:3596-3606
18.
Sato T, Laver JH, Ogawa M.
Reversible expression of CD34 by murine hematopoietic stem cells.
Blood.
1999;94:2548-2554 19. Orlic D, Bodine DM. Pluripotent hematopoietic stem cells of low and high density can repopulate W/Wv mice. Exp Hematol. 1992;20:1291-1295[Medline] [Order article via Infotrieve].
20.
Wright DE, Wagers AJ, Gulati AP, Johnson FL, Weissman IL.
Physiological migration of hematopoietic stem and progenitor cells.
Science.
2001;294:1933-1936 21. Jiao H, Berrada K, Yang W, Tabrizi M, Platanias LC, Yi T. Direct association with and dephosphorylation of Jak2 kinase by the SH2-domain-containing protein tyrosine phosphatase SHP-1. Mol Cell Biol. 1996;16:6985-6992[Abstract].
22.
Lorenz U, Bergemann AD, Steinberg HN, et al.
Genetic analysis reveals cell type-specific regulation of receptor tyrosine kinase c-Kit by the protein tyrosine phosphatase SHP1.
J Exp Med.
1996;184:1111-1126 23. Ryan JJ, Huang H, McReynolds LJ, et al. Stem cell factor activates STAT-5 DNA binding in IL-3-derived bone marrow mast cells. Exp Hematol. 1997;25:357-362[Medline] [Order article via Infotrieve].
24.
Brizzi MF, Dentelli P, Rosso A, Yarden Y, Pegoraro L.
STAT protein recruitment and activation in c-Kit deletion mutants.
J Biol Chem.
1999;274:16965-16972 25. Bradford GB, Williams B, Rossi R, Bertoncello I. Quiescence, cycling, and turnover in the primitive hematopoietic stem cell compartment. Exp Hematol. 1997;25:445-453[Medline] [Order article via Infotrieve]. 26. Cheshier SH, Morrison SJ, Liao X, Weissman IL. In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proc Nat | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
chain knockout mouse, which has an activated T-cell
phenotype that alters repopulating activity.
/
