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HEMATOPOIESIS
From the Department of Chemical Engineering,
Northwestern University, Evanston, IL.
External stimuli act in concert with intracellular signals to
regulate a cell's genetic program, activating genes important in
granulocytic lineage commitment, proliferation, and maturation. Signal
transducer and activator of transcription 3 (STAT3), a transcription
factor, has been implicated in mediating granulocytic differentiation.
We have examined the role of STAT3 as a physiologic mediator of
granulocytic kinetics. Distinct isoforms Hematopoietic lineage determination by
transcriptional collaboration with cytokines is well
established.1 In particular, members of the STAT (signal
transducer and activator of transcription) transcription factor family
mediate many cytokine-induced responses in hematopoietic cells,
including proliferation, differentiation, and survival.2
Upon ligand binding, STATs are recruited to activated cell-surface
receptors and become phosphorylated on a specific residue either
directly or through association with a Janus kinase. Subsequently, the
phosphorylated STATs homodimerize or heterodimerize and translocate to
the nucleus, where they regulate transcription by binding to specific
DNA promoter elements.2,3 The STAT family consists of 7 members, most of which are ubiquitously expressed. However, individual
STAT proteins may be differentially activated depending on the cell
type or tissue.2,4-6
During myeloid differentiation, various isoforms of STAT3 and STAT5 are
activated in a cell type- and maturation state-dependent manner.
While STAT5 appears to be important for proliferative responses to
interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor
(GM-CSF), and G-CSF,7,8 STAT3 is activated by G-CSF and
appears to be the major STAT protein driving G-CSF-mediated granulocytic differentiation.9-16 Expression of dominant
negative STAT39 or G-CSF receptor (G-CSFR) mutants lacking
regions in the membrane distal domain, which contains recruitment sites
for STAT3,16-20 blocks granulocytic differentiation,
demonstrating a requirement for STAT3 activation in growth arrest and
morphologic granulocytic differentiation.
Three distinct isoforms of STAT3, all derived from a single gene, have
been identified11,21: STAT3 Analyzing STAT3 signaling in primary cells is key to understanding its
physiologically relevant function. However, as mentioned above, in
primary cells STAT3 has previously been examined only in uncultured
human myeloid cells isolated at 3 different stages of
differentiation.10,27 We designed an ex vivo
granulopoietic system that expands granulocytic cells from
CD34+ cells in sufficient numbers and purity (> 90%) to
examine STAT3 isoform expression and phosphorylation in coordination
with granulocytic proliferation and differentiation. Little is known
about the production of the different STAT3 isoforms Cells and cell culture
Flow cytometric analysis
Western blot analysis Cells numbering 6 × 106 to 12 × 106 (day 0) or 1.5 × 106 to 3.0 × 106 (at later times) were mixed with an equal volume of ice-cold phosphate-buffered saline (PBS) supplemented with 1 mM Na3VO4 and 5 mM NaF and immediately spun down (4°C, 300g, 10 minutes). Cells were rinsed with 1 mL PBS solution and pelleted again at 4°C. Whole-cell extracts were prepared by adding 60 µL of modified radioimmunoprecipitation (RIPA) buffer per 1.0 × 106 cells. Preparation of RIPA buffer and cell lysate was carried out according to the manufacturer (Upstate Biotechnology, Lake Placid, NY)41 with the following modifications to the RIPA solution: 2 µg/mL each of aprotinin, leupeptin, and pepstatin and 20 mM NaF. Protein was quantified in duplicate with the BCA kit (Pierce, Rockford, IL) using bovine serum albumin (BSA) as standard.From each sample, 20 µg total protein was separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Biorad, Hercules, CA) and transferred to a polyvinylidene fluoride membrane (0.45 µm Immobilon-P; Millipore, Bedford, MA). Membranes were incubated with either a mouse monoclonal anti-STAT3 (1:2000; clone 84; Transduction Laboratories, Lexington, KY), rabbit polyclonal anti-STAT3 pTyr 705 (1:1000; Cell Signaling Technology, Beverly, MA), or rabbit polyclonal anti-STAT3 pSer 727 (1:500; Upstate Biotechnology) antibody, followed by horseradish peroxidase-conjugated secondary antibody, either sheep anti-mouse (1:5000; Amersham Pharmacia Biotech, Piscataway, NJ) or donkey anti-rabbit (1:2500; Amersham Pharmacia Biotech), as appropriate. Immunoreactive bands were visualized by using the enhanced chemiluminescence (ECL) Plus detection kit (Amersham Pharmacia Biotech) and directly scanned using the Molecular Dynamics Storm imaging system (Sunnyvale, CA). Signals were quantified using ImageQuant software (Molecular Dynamics). Immunofluorescence microscopy A total of 2 × 104 cells were washed in PBS supplemented with 2% BSA, cytocentrifuged (Cytospin 3, Shandon, Pittsburgh, PA) onto glass microscope slides, fixed in 4% paraformaldehyde for 10 minutes, washed in PBS/0.1 M glycine 5 times for 3 minutes each, and permeabilized in 0.3% Triton X-100 in PBS for 5 minutes. After blocking nonspecific binding sites for 1 hour with 10% normal goat serum (Vector Laboratories, Burlingame, CA) in PBS/2% BSA, cells were incubated with a polyclonal rabbit anti-STAT3 or anti-STAT3 pSer 727 (1:300; Cell Signaling Technology) antibody simultaneously with monoclonal mouse anti-CD15 antibody (1:100; clone 80H5; immunoglobulin M [IgM]; Immunotech, France) in PBS/2% BSA overnight at 4°C. Cells were washed in PBS/0.1 M glycine 5 times for 3 minutes each, incubated with both fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:200; Vector Laboratories) and Texas Red-conjugated goat anti-mouse IgM µ-chain-specific (1:100; Jackson Immunoresearch Laboratories, West Grove, PA) secondary antibodies in PBS/2% BSA containing 2% goat serum for 2 hours in the dark, and washed again. Slides were mounted in Vectashield Mounting Media (Vector Laboratories) containing DAPI. Each fluorochrome was analyzed individually: DAPI fluorescence (white pixels) to distinguish the nuclei, FITC fluorescence (green pixels) to assess STAT3 expression, and Texas Red fluorescence (red pixels) to identify CD15+ cells. No significant background or overlap between fluorescence signals was detected. All images were captured with a Zeiss Axiophot fluorescence microscope using a 40× or 100× oil immersion objective and processed by IPlab software (VayTek, Farfield, IA).Statistical analysis Statistical comparisons of the values representing production of total cells and CD15dim/CD11b ,
CD15bright/CD11b, and
CD15bright/CD11b+ granulocyte subpopulations
and receptor expression levels under different culture conditions were
performed using a 2-tailed paired Student t test. Data are
reported as the mean ± SEM. Immunofluorescence microscopy and
Western analysis were carried out on 3 sets of experiments that
examined the same 4 culture conditions with different donor
CD34+ cells. The results were consistent across
experiments, and thus one representative data set is shown.
Immunofluorescence images are representative of 2 to 3 randomly
selected fields per culture condition for each of the 3 different sets
of experiments.
To control for possible error introduced during protein quantitation
and gel loading, each Western blot was probed for
Manipulation of the experimental conditions under which the CD34+ cells were cultured elicited a variety of distinct cellular responses. Granulocytic differentiation and proliferation were clearly altered in unique ways depending upon exposure to a different pH, pO2, or IL-3 environment. The ability of these 3 factors to independently induce effects on a specific cellular process or stage of maturation allowed potential functional links with STAT3 isoform expression and/or phosphorylation to be explored. As detailed below, this experimental approach entailed a thorough kinetic characterization of each culture as it progressed through different stages of granulocytic development, ie, detecting phenotypic and morphologic changes in conjunction with changes in STAT3 isoform expression and phosphorylation. Characterization of ex vivo granulocytic differentiation of CD34+ cells To assess STAT3 kinetics throughout the granulocytic pathway, we used flow cytometry and fluorescence microscopy to clearly define the differentiation status of our cultures. By monitoring the differential expression of CD15 and CD11b, in combination with nuclear morphology, the neutrophil lineage can be classified into discrete maturational stages. As cells progressed from CD15/CD11b
to CD15dim/CD11b (myeloblasts),
CD15bright/CD11b (promyelocytes/early
myelocytes), and CD15bright/CD11b+ (early
myelocytes/myelocytes/metamyelocytes/bands),35 their morphology and size also changed dramatically from the initial small
size of the CD15/CD11b (CD34+)
cells. The nuclei increased in size to days 5 to 7 (Figure
1) and contained multiple nucleoli that
were readily visible from the lack of STAT3 within their interior
(Figure 2). As the cells matured into
myelocytes, the nucleoli disappeared and the nuclei decreased in size,
becoming more rounded so that the nuclear-cytoplasmic ratio decreased
(Figure 2). With further maturation, cells decreased in size while the
nuclei underwent drastic changes that allowed for the more mature
CD15bright/CD11b+ population to be further
compartmentalized into distinct stages (Figures 1 and 2) going from
the indented, kidney-shaped appearance of the metamyelocyte to the more
elongated, horseshoelike appearance of the band form, which showed the
initial signs of nuclear cleavage that characterizes segmented
neutrophils.
Cultures under all conditions were predominantly granulocytic,
containing at least 90% CD15bright cells by day 15. However, the rate at which the cells matured as well as the expansion
of each granulocytic subpopulation (Figure 3B-D, Table
1) varied under each culture
condition.35,36 Total cell expansion was dramatically
favored in cultures supplemented with IL-3 at low pH (pH 7.25) and low
pO2 (5% O2), with a 2.4- or 2.7-fold
enhancement in cell numbers by day 15 over cultures either at high pH
(pH 7.4) or under high pO2 (20% O2),
respectively (Figure 3A, Table 1). A pH of 7.4 delayed maturation
throughout the entire differentiation pathway compared with control
cultures.35,36 For example, by day 15, cultures at high pH
consisted mostly of metamyelocytes that were just beginning to take on
the appearance of bands and showed very few signs of nuclear cleavage
or lobulations (Figure 2), whereas at low pH, cultures contained more
mature cells, with nuclei segmented into 2 to 5 lobes. In contrast to pH, pO2 did not affect differentiation based on
CD15/CD11b35,36 or morphologic characterization (data not
shown) during the first week of culture and had only minor effects on
differentiation at later time points.
Total cell expansion was significantly reduced by 6.3-fold in cultures
without IL-3 (Figure 3A, Table 1). However, the rate of differentiation
was accelerated compared with that in the presence of
IL-3.36 The kinetic effects of IL-3 on granulocytic
maturation were distinct from the pH effects in that they did not
persist through the entire granulocytic differentiation pathway but,
rather, were isolated to the CD15dim/CD11b G-CSFR up-regulation that occurs with granulocytic maturation was retarded at high pH and accelerated in the absence of IL-3 compared with control cultures (Figure 3E, Table 1). Approximately 90% of the cells acquired a high content of G-CSFR after 9 days at high pH, whereas control cultures approached this level on day 7 and as early as day 5 in cultures without IL-3. An independent set of experiments showed that high pO2 (20% O2) had no effect on G-CSFR up-regulation.35 Localization and expression of STAT3 during granulocytic differentiation Cells were stained with antibodies specific for STAT3 and the granulocytic differentiation marker CD15 to assess protein expression and subcellular distribution. STAT3 was present early in hematopoiesis, staining intensely in day 0 CD34+ cells (Figure 1). On day 3, STAT3 protein appeared in both the nucleus and cytoplasm but not in the nucleoli (Figure 2). Subsequently, STAT3 continued to stain intensely but with an increased preference for the nucleus. Coexpression of STAT3 and CD15 clearly revealed their selective partitioningSTAT3 (green) in the nucleus and CD15 (red) in an
extranuclear location, such as on the cell membranewith minimal
colocalization (yellow). STAT3 expression appeared strongest in
CD15 cells and gradually decreased upon granulocytic
differentiation (Figures 1 and 2). The most remarkable decrease in
STAT3 expression was evidenced in day 15 CD15+ segmented
neutrophils (Figure 2).
Expression of distinct STAT3 isoforms during granulocytic differentiation Western blot analysis was used to discriminate between the different STAT3 proteins present during granulocytic development. A widely used N-terminal anti-STAT3 antibody, generated against amino acids 1 to 178, recognized at least 3 previously characterized STAT3 isoforms with different electrophoretic mobilities: full-length STAT3 (92 kd) and the carboxyl-truncated forms, STAT3 (83 kd) and
STAT3 (72 kd) (Figure 4A). An
additional faster-migrating protein of approximately 64 kd, which we
designated as STAT3 , was detected, likely representing a new
putative truncated isoform. Protein levels of the different STAT3
species were assessed by quantitating the densities of the respective
immunoreactive bands. Unlike the microscopic images, which depict STAT3
expression on a per-cell basis, the Western analysis of Figure 4
reflects changes in STAT3 expression relative to equivalent amounts of
total protein loaded on each day. Total protein per cell decreased as
the cells matured, making it necessary to lyse a greater number of
cells on later days to yield an equivalent amount of protein as on
earlier days of analysis.
The ratio of isoforms shifted markedly during differentiation, creating
a distinct pattern of expression at different stages of maturation
(Figure 4A, culture condition 1). CD34+ cells expressed
more than 65% of STAT3 as STAT3
STAT3 expression under different pH, pO2, and IL-3 conditions Total STAT3 expression per cell (Figures 1 and 2) as well as the kinetics and magnitude of the shift between isoforms (Figure 4A, culture conditions 1-4) varied with culture conditions (pH, pO2, IL-3) and was consistent with differences in culture differentiation. At pH 7.4, STAT3 levels per cell were sustained for a longer period than at pH 7.25. For example, the less differentiated cells at high pH demonstrated a higher intensity of STAT3 in their nuclear portion at days 13 and 15 (Figures 1 and 2). The primary effect on isoform patterns was the maintenance of higher STAT3 levels throughout culture (Figures 4A, culture condition 3, and 5C). At its
lowest level on day 9, STAT3 at pH 7.4 was still 50% higher than at
pH 7.25. This, together with lower levels of STAT3, resulted in a
lower STAT3/STAT3 ratio at pH 7.4 versus 7.25 throughout culture.
In contrast, in the absence of IL-3, total STAT3 levels per cell
decreased more rapidly than in control cultures, along with a faster
rise in the fraction of CD15+ cells and earlier signs of
nuclear cleavage (Figures 1 and 2). The shift from predominantly
STAT3 STAT3 tyrosine phosphorylation A strong correlation is known to exist between tyrosine phosphorylation specifically on residue 705 and rapid nuclear translocation of STAT3 and the formation of STAT3-STAT3-DNA complexes.26,30 We used an antibody against tyrosine-phosphorylated STAT3 (anti-STAT3 pTyr 705) to assess activation of total STAT3 and of the individual isoforms under different culture conditions (Figure 4B, culture conditions 1-4) and thereby determine whether selective phosphorylation of one isoform versus another occurred, leading to its preferential translocation to the nucleus. Although overall levels of STAT3 did not vary much between culture conditions when comparing equal amounts of total protein (Figure 6A), the extent of tyrosine phosphorylation did. Overall levels of tyrosine-phosphorylated STAT3 remained relatively constant under control conditions but were about 2.5 to 3 times greater in the absence of IL-3 (Figure 6B). STAT3 tyrosine phosphorylation at high pH (7.4) also reached levels of 2.5 to 3 times that at the lower pH (7.25) of control conditions but increased more gradually until about day 13 (Figure 6B,C). Under 20% O2, STAT3 tyrosine phosphorylation also appeared to increase steadily, although to a lesser extent (Figure 6B,C).
The most notable difference between trends in expression of the
individual STAT3 isoforms and their respective tyrosine phosphorylation kinetics was the preferential activation of STAT3 The kinetics of STAT3 STAT3 STAT3 serine phosphorylation Immunoreactivity for STAT3 serine phosphorylation was detected only with the STAT3 isoform, reflecting the absence of
serine residue 727 in the carboxyl-truncated isoforms, STAT3 and
STAT3, and in STAT3 (Figure 4C, culture condition 1). Therefore,
unlike all STAT3 isoforms that are potential targets for tyrosine
phosphorylation of residue 705, only STAT3 is subject to serine
phosphorylation on residue 727. Strongest reactivity of anti-STAT3 pSer
727 antibody was observed on day 0, after which there was a dramatic
drop in expression levels (Figure 6D).
The effects of different culture conditions on serine phosphorylation
of STAT3
STAT3 kinetics were maturation stage-dependent and varied
with pH, pO2, and IL-3 in a manner consistent with the
marked differences induced by each condition on granulocytic growth and
differentiation. Overall levels of STAT3 per cell were found to
decrease with granulocytic differentiation (Figures 1 and 2). This
phenomenon, and the disappearance of nucleoli past the myelocyte stage,
may reflect the end of mitosis and the need for new protein synthesis,
while the lack of STAT3 expression in segmented neutrophils could be
representative of their maturation status as the terminal
differentiation stage. These cell-to-cell differences in overall STAT3
protein production were less obvious by Western analysis, especially
because more cells were used on later days to account for decreasing
levels of total protein with differentiation. Western analysis,
however, was essential in revealing that different STAT3
isoforms The most pronounced change in the relative concentrations of the
different STAT3 isoforms occurred with the shift in expression from
predominantly STAT3 Variations in STAT3 A fourth putative STAT3 isoform, STAT3 The balance among the various STAT3 isoforms does indeed appear to be
functionally relevant, given the coordination between differential
expression and/or activation of the different STAT3 isoforms and
distinct granulocytic cell responses induced by pH, pO2,
and IL-3. At high pH, differentiation and proliferation were slower
throughout the granulocytic pathway in parallel with slower G-CSFR
up-regulation. This correlates with a slower shift from STAT3 In contrast to slower overall differentiation at high pH, the
absence of IL-3 accelerated differentiation and G-CSFR up-regulation. These data are suggestive of an antagonistic action of IL-3 on G-CSF-dependent differentiation, which is consistent with earlier cell
line-based data.48-50 It seems likely, therefore, that
the enhanced differentiation rate in Exposure of cells to 20% O2 was also found to increase
STAT3 tyrosine phosphorylation, particularly during the second week of
culture. Given that we found no link between pO2 and G-CSFR expression, a mechanism distinct from the proposed G-CSFR-mediated pathway leading to enhanced STAT3 tyrosine phosphorylation in In contrast to tyrosine phosphorylation, which is obligatory for transcriptional activity of STAT3, the role of serine phosphorylation has been controversial; it can enhance,30-32 inhibit,34,58 or have no effect6,59,60 on tyrosine phosphorylation, DNA binding, and transcriptional activity. Serine phosphorylation in STAT3 occurs primarily on a single residue, serine 727, and in our cultures levels were greatest on early days, when cells occupied relatively immature stages of granulopoiesis and were highly proliferative, but decreased rapidly with differentiation. This suggests that serine phosphorylation may be a more influential determinant of the expansion of primitive cell types (myeloid stem and progenitor cells) than in the differentiation of granulocytic postprogenitors. These data collectively reveal the complexity of STAT regulation and show that STAT3 can integrate diverse signals, ranging from cytokines to pH and pO2, so as to coordinate proliferation and differentiation. The evidence strongly indicates that the multiple STAT3 isoforms, each possessing distinct physiologic functions, potentiate the range of granulocytic responses observed under the different experimental conditions. Selective expression and/or phosphorylation of the different STAT3 isoforms provides a molecular basis for these responses to the culture environment, thereby emphasizing the importance of cellular context in determining granulocytic kinetics. These results are particularly relevant in the physiologic setting where cells are exposed to numerous stimuli simultaneously. Other potential mechanisms that were not investigated here, such as the activation of other STAT family members like STAT5 or functional interactions with other transcriptional coactivators, may also contribute to the balance between proliferation and differentiation and will be important in further defining a molecular model of granulopoiesis.
We thank Amgen for donation of SCF. We are grateful to Dr B. He for numerous valuable discussions on quantitative Western analysis. We also thank Dr M. Swartz, Dr R. Holmgren, and Yu Kuang for technical assistance.
Submitted July 27, 2001; accepted October 26, 2001.
Supported by National Institutes of Health grant R01 HL48276.
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: Eleftherios T. Papoutsakis, Northwestern University, Dept of Chemical Engineering, 2145 Sheridan Rd, Evanston, IL 60208-3120; e-mail: e-paps{at}northwestern.edu.
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  M. S. Redell, A. Tsimelzon, S. G. Hilsenbeck, and D. J. Tweardy Conditional overexpression of Stat3{alpha} in differentiating myeloid cells results in neutrophil expansion and induces a distinct, antiapoptotic and pro-oncogenic gene expression pattern J. Leukoc. Biol., October 1, 2007; 82(4): 975 - 985. [Abstract] [Full Text] [PDF]
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R. T. Sasmono, A. Ehrnsperger, S. L. Cronau, T. Ravasi, R. Kandane, M. J. Hickey, A. D. Cook, S. R. Himes, J. A. Hamilton, and D. A. Hume Mouse neutrophilic granulocytes express mRNA encoding the macrophage colony-stimulating factor receptor (CSF-1R) as well as many other macrophage-specific transcripts and can transdifferentiate into macrophages in vitro in response to CSF-1 J. Leukoc. Biol., July 1, 2007; 82(1): 111 - 123. [Abstract] [Full Text] [PDF]
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 T. Kato, E. Sakamoto, H. Kutsuna, A. Kimura-Eto, F. Hato, and S. Kitagawa Proteolytic Conversion of STAT3{alpha} to STAT3{gamma} in Human Neutrophils: ROLE OF GRANULE-DERIVED SERINE PROTEASES J. Biol. Chem., July 23, 2004; 279(30): 31076 - 31080. [Abstract] [Full Text] [PDF]
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 M. Benekli, M. R. Baer, H. Baumann, and M. Wetzler Signal transducer and activator of transcription proteins in leukemias Blood, April 15, 2003; 101(8): 2940 - 2954. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
IL-3
(absence of IL-3). Granulocytic cell production was greatest at
5% O2, pH 7.25, +IL-3, which was designated as the
"control" condition. Cultures from 3 independent donor samples were
evaluated, except for studies on G-CSFR expression (n = 6). The
CD34+ cell purity and viability were more than 97%, with
viabilities remaining more than 95% throughout culture.
indicates 5%
O2, pH 7.25, +IL-3; 
indicates 20% O2, pH
7.25, +IL-3; 
indicates 5% O2, pH 7.4, +IL-3; 
indicates 5% O2, pH 7.25, 
