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Stem Cells 2005;23:252-263 www.StemCells.com
© 2005 AlphaMed Press

Roles of Stat3 and ERK in G-CSF Signaling

Kenjirou Kamezakia,b, Kazuya Shimodaa,b, Akihiko Numataa,b, Takashi Haroa,b, Haruko Kakumitsua,b, Masumi Yoshiec, Masahiro Yamamotoc, Kiyoshi Takedad, Tadashi Matsudae, Shizuo Akirad, Katsuhiro Ogawac, Mine Haradaa,b

a The First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan;
b Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan;
c Department of Pathology, Asahikawa Medical College, Asahikawa, Japan;
d Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan;
e Department of Immunology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan

Key Words. Signal transduction • Hematopoiesis • Signal transducer and activator of transcription • Mitogen-activated protein kinase • G-CSF

Correspondence: Kazuya Shimoda, M.D., The First Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan. Telephone: 81-92-642-5230; Fax: 81-92-642-5247; e-mail: kshimoda{at}intmed1.med.kyushu-u.ac.jp


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
G-CSF specifically stimulates the proliferation and differentiation of cells that are committed to the neutrophil-granulocyte lineage. Although Stat3 was thought to be essential for the transduction of G-CSF–induced cell proliferation and differentiation signals, mice deficient for Stat3 in hematopoietic cells show neutrocytosis and infiltration of cells into the digestive tract. The number of progenitor cells in the neutrophil lineage is not changed, and G-CSF–induced proliferation of progenitor cells and prolonged neutrophil survival were observed in Stat3-deficient mice. In hematopoietic cells from Stat3-deficient mice, trace levels of SOCS3, a negative regulator of granulopoiesis, were observed, and SOCS3 expression was not induced by G-CSF stimulation. Stat3-null bone marrow cells displayed a significant activation of extra-cellular regulated kinase 1 (ERK1)/ERK2 under basal conditions, and the activation of ERK was enhanced and sustained by G-CSF stimulation. Furthermore, the augmented proliferation of Stat3-deficient bone marrow cells in response to G-CSF was dramatically decreased by addition of a MEK1 inhibitor. These results indicate that Stat3 functions as a negative regulator of G-CSF signaling by inducing SOCS3 expression and that ERK activation is the major factor responsible for inducing the proliferation of hematopoietic cells in response to G-CSF.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The proliferation and differentiation of hematopoietic precursor cells are regulated by a family of cytokines. In particular, granulocyte colony-stimulating factor (G-CSF) specifically stimulates the proliferation and differentiation of cells that are committed to the neutrophil lineage [1]. The biological functions of G-CSF are mediated through binding to a cell-surface receptor that is predominantly expressed on neutrophilic progenitor cells and mature neutrophilic granulocytes, although the receptor is also expressed on hematopoietic progenitor cells[2].The binding of G-CSF to its receptor induces the tyrosine phosphorylation of Jak1, Jak2, and Tyk2 [3–6], which are members of the Janus family of protein tyrosine kinases (Jaks) [7]. Activated Jaks phosphorylate residues in the cytosolic tails of G-CSF receptors, allowing subsequent recruitment of various signaling proteins to the receptor complex. Members of the signal transducers and activators of transcription (Stat) family are recruited and phosphorylated by Jak kinase, translocate to the nucleus, and bind the promoter regions of target genes [8,9]. G-CSF stimulation results in the specific phosphorylation of Stat3 [4,10] and, more infrequently, Stat5 and Stat1 [6,11]. Generally, this Jak-Stat signaling pathway is thought to be essential for the transduction of cytokine signaling [12], but other cytoplasmic protein tyrosine kinases, including lyn and syk [13,14], are also phosphorylated and activated in response to G-CSF signaling.

The role of Jak kinases in the G-CSF signaling pathway was initially examined by the use of cell lines that were deficient for each of the Jak kinases. G-CSF induces the tyrosine phosphorylation and activation of Jak1, Jak2, and Tyk2, and the absence of one Jak does not preclude G-CSF–induced tyrosine phosphorylation of the remaining Jaks. However, in the absence of Jak1, G-CSF does not induce receptor tyro-sine phosphorylation, and the induced tyrosine phosphorylation of Stat proteins is greatly reduced [6]. Although multiple Jaks are activated by G-CSF, Jak1 is in a unique position to phosphorylate the receptor and thereby affect Stat protein tyrosine phosphorylation because of either location within the receptor complex or substrate specificity. However, Jak1-deficient mice do not show neutropenia, and Jak1 deficiency has no effect on the colony formation of bone marrow cells induced by G-CSF [15]. Furthermore, deletion of either Jak2 or Tyk2 has no effect on G-CSF–induced colony formation of bone marrow cells [16,17]. These results indicate that there is redundancy among the Jak kinases in G-CSF signaling, and the lack of one Jak kinase may be compensated for by the activation of other Jak kinases. Interestingly, the involvement of Jak kinases in G-CSF signaling seems to be different from that in other cytokine-signaling pathways. For example, Jak1 is essential for gp130, interferon (IFN), and interleukin (IL-7)–mediated signaling [15], Jak2 is essential for erythropoietin (Epo) signaling [17], Jak3 is essential for IL-2 receptor common gamma chain–mediated signaling [15], and Tyk2 is essential for IL-12 signaling [16].

In addition, we and others have used Stat-deficient mice to demonstrate that Stats play an essential and nonredundant role in cytokine signaling [18–22]. G-CSF stimulation activates mainly Stat3 and, to a lesser extent, Stat1 and Stat5 in bone marrow cells. Stat1 and Stat5a/b-deficient mice have normal neutrophil numbers, although colony formation by bone marrow cells in response to G-CSF stimulation is decreased by the absence of Stat5a/b [18,21]. However, the expression of dominant-negative Stat3 in 32Dcl3 cells, which differentiate into neutrophils after G-CSF treatment, does not prevent them from proliferating in response to G-CSF [23]. Additionally, transgenic mice with a targeted mutation of the G-CSF receptor that abolishes G-CSF–dependent Stat3 activation show severe neutropenia, with an accumulation of immature myeloid precursors in the bone marrow [24]; constitutively active Stat3 partially rescues this neutropenia. Stat3 is then thought to transduce the proliferation and differentiation signal of G-CSF. Ablation of Stat3 produced early embryonic lethality [25], and selective ablation of Stat3 in neutrophils and monocytes by Cre recombinase-dependent gene deletion directed by the macrophage lysozyme promoter did not affect neutrophil production, suggesting that Stat3 is required at an early stage of neutrophil development. To clarify the role of Stat3 in the G-CSF signaling pathway, we have examined the role of Stat3 in hematopoiesis by selective ablation of the Stat3 gene in hematopoietic progenitor cells. In contrast to expectations, mice that were deficient for Stat3 in hematopoietic cells show neutrocytosis and infiltration of cells into the digestive tract.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Stat3flox/– mice were generated by mating Stat3flox/+ mice, in which the DNA base pairs encoding the tyrosine phosphorylation site in Stat3 are flanked by two loxP sites (Stat3flox/flox) [26], with Stat3+/– mice, in which exons 20 through 22 are replaced by a neomycin resistance gene in the knockout allele [27]. To establish mice with a conditional knockout of Stat3 in hematopoietic cells, Stat3flox/– mice were mated with a transgenic line bearing Cre recombinase driven by the IFN-inducible Mx promoter [28]. Genotyping was performed by polymerase chain reaction analysis of genomic tail DNA. The primer sequences were as follows: Stat3-flox: 5'-cctgaagac-caagttcatctgtgtgac-3' and 5'-cacacaagccatcaaactctggtctcc-3'; Stat3–: 5'-agcagctgacaacgctggctgagaagct-3' and 5'-atcgccttc-tatcgccttcttgacgag-3'; Mx1-Cre: 5'-ggacatgttcagggatcgc-caggcg-3' and 5'-gcataaccagtgaaacagcattgctg-3'. Expression of Cre was induced by injecting mice intraperitoneally with 250 µg of polyinocinic-polycytidylic acid (pIpC) (Sigma, St. Louis) three times at 2-day intervals as previously described [28]. Age-matched Stat3flox/– and Mx1-Cre:Stat3flox/– were injected with pIpC and used for further experiments 2 weeks later, except where noted in the text. Mice were housed and bred in the Kyushu University Animal Center.

Histological and Hematological Analysis
Tissues were fixed in 10% phosphate-buffered formalin, and paraffin-embedded tissue sections were stained with hematoxylin and eosin using standard techniques.

Complete blood counts were analyzed using Celltac {alpha} (Nihon Kohden, Tokyo). Peripheral and bone marrow blood cells were prepared on slide glasses and stained with Giemsa solution. Differential cell counts were scored visually.

Preparation of Neutrophils from the Peritoneal Cavity
Mice were injected intraperitoneally with 2 ml of 4% thioglycollate. After 4 hours, peritoneal neutrophils were obtained by peritoneal lavage with 10 ml of ice-cold phosphate-buffered saline (PBS). After an 8-hour incubation in plastic dishes, nonadherent cells were harvested and used for further experiments. These cells were >90% Gr-1+, as determined by flow cytometry.

Evaluation of Apoptosis
To evaluate apoptosis, cells were incubated with fluorescein isothiocyanate (FITC)–conjugated Gr1 (Becton, Dickinson, San Jose, CA) and propidium iodide for 30 minutes at 4°C, washed twice in PBS, and analyzed on a FACS Calibur (Becton, Dickinson). To exclude the dead cells, the gated nucleated cells were used for further examination.

Chemotaxis Assay
The neutrophil chemotaxis assay was performed as described [29]. Briefly, the lower well contained 800 µl of Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum (FCS) and 1 x 10–7 M formyl-methionyl-leucyl-phenylalanine (fMLP) (Sigma), and the upper chemotaxicell (Kurabo, Osaka) chamber contained 400 µl of a neutrophil suspension. After incubation for 1 hour at 37°C, the cells that had passed through the membrane were counted.

Hematopoietic Progenitor Cell Assays
The frequency of hematopoietic progenitor cells was determined by clonogenic assays in methylcellulose, as described previously [30]. Briefly, bone marrow or peripheral blood mononuclear cells were separated by Ficoll-Paque sedimentation, and 1 x 105 or 2 x 105 cells were cultured in methylcellulose containing IL-3 (20 ng/ml), stem cell factor (SCF) (20 ng/ml), and Epo (4 U/ml) for the colony-forming unit-granulocyte, macrophage (CFU-GM), BFU-E, and CFU-culture (CFU-C) assays or G-CSF (50 ng/ml) only for the CFU-G assay. CFU-GM, CFU-G, and BFU-E were measured after 10 days in culture.

In Vitro Proliferation Assays
Bone marrow mononuclear cells (1 x 106/ml) were incubated for 3 days in IMDM supplemented with 30% FCS in the presence or absence of IL-3 (1 ng/ml) and G-CSF (10 ng/ml). To assess the effects of an MEK inhibitor, dimethyl sulfoxide (DMSO) or U0126 (Cell Signaling, Beverly, MA) dissolved in DMSO was added. After 72 hours, 0.5 µCi 3H-thymidine was added and the cells were incubated for an additional 8 hours. Proliferative activity was determined by 3H-thymidine incorporation.

In Vivo Administration of G-CSF or Thrombopoietin
Mice were injected subcutaneously with either G-CSF (50 µg/kg daily for the indicated period) or with thrombopoietin (TPO) (30 µg/kg for 5 days). Peripheral blood was collected on the indicated day.

Dextran Sulfate Sodium–Induced Colitis (DSS)
Colitis was induced by feeding mice drinking water supplemented with 4% DSS (Wako, Osaka, Japan), as described previously [31]. Control mice were fed drinking water without DSS.

Western Blotting
Bone marrow cells were treated with G-CSF for the indicated time and were then lysed in lysis buffer as previously described [32]. Cell lysates were centrifuged at 12,000g for 15 minutes to remove debris. Total cell lysates were resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were probed using the indicated antibodies and visualized with an ECL detection system (Amersham, Uppsala, Sweden). Anti-phospho-ERK1/2, anti-phospho-p38, anti-ERK2, anti-phospho-Stat3, and anti-phospho-Stat5 antibodies were purchased from Cell Signaling. Anti-Stat3 and anti-Stat5 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-SOCS3 antibody was purchased from IBL (Gunma, Japan).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increased Numbers of Circulating Neutrophils and Myeloid Cells in the Bone Marrow in Stat3-Deficient Mice
To determine whether Stat3 plays a role in hematopoiesis, we used the Cre-loxP recombination system. To decrease the amount of residual Stat3 protein after Cre-mediated deletion, we crossed Stat3flox/– mice with a transgenic line bearing Cre recombinase driven by the IFN-inducible Mx promoter. Only trace amounts of Stat3 were detected after induction of Mx-Cre by treatment with pIpC (Fig. 1AGo). When bone marrow cells from pIpC-treated Mx1:Stat3flox/– mice were treated with G-CSF, the phosphorylation of Stat3 was diminished, whereas Stat5 was phosphorylated at the same extent as wild-type mice by IL-3 stimulation.



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  		Figure 1. (A): Phosphorylation of Stat3 or Stat5 in response to G-CSF or IL-3 stimulation of bone marrow cells from Stat3flox/– and Mx1:Stat3flox/– mice. Bone marrow cells from Stat3flox/– and Mx1:Stat3flox/– mice were incubated for 8 hours in the absence of cytokines and then stimulated with G-CSF (50 ng/ml) or IL-3 (10 ng/ml) for 30 minutes. Total cell lysates were analyzed by Western blot with the indicated antibodies. (B): Bone marrow analysis from Stat3flox/– and Mx1:Stat3flox/– mice. Bone marrow differential counts were performed on preparations from Stat3flox/– and Mx1:Stat3flox/– mice that were euthanized 2 weeks after treatment with polyinocinic-polycytidylic acid. Abbreviation: IL, interleukin.

 
We next examined the role of Stat3 in granulopoiesis in vivo. Blood cell counts from wild-type and Mx1:Stat3flox/– littermates 2 weeks after deletion of Stat3 are shown in Table 1Go. Morphologically identifiable neutrophils were detected in the peripheral blood of mice lacking Stat3 in hematopoietic cells, and the number of circulating neutrophils in Mx1:Stat3flox/– mice was 2.3-fold greater than in control mice. There were no differences in the numbers of lymphocytes, erythrocytes, or platelets due to ablation of Stat3. In bone marrow, the number of mature myeloid cells was greater in Mx1:Stat3flox/– mice than in control littermates, which is consistent with the observations in peripheral blood (Fig. 1BGo).


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  		Table 1. Complete blood counts
 
Effects of Stat3 Deletion on the Frequency of Myeloid and Erythroid Progenitor Cells and on the Response to G-CSF in Bone Marrow Cells
Because the number of neutrophils was higher in the Mx1: Stat3flox/–mice, we next determined the number of hematopoietic progenitor cells in the bone marrow. Bone marrow from pIpC-treated control or Mx1:Stat3flox/– mice was seeded in methylcellulose containing IL-3, SCF, and Epo to determine CFU-GM or in methylcellulose containing G-CSF to determine CFU-G. The CFU-GM and BFU-E cloning efficiencies were comparable in Stat3flox/– and Mx1:Stat3flox/– bone marrow cells in the presence of IL-3, SCF, and Epo (Fig. 2AGo). Similarly, the total colony numbers were not altered due to the absence of Stat3 (data not shown). The number of CFU-G induced by G-CSF was also unaffected by the absence of Stat3 (Fig. 2AGo).



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  		Figure 2. (A): In vitro colony formation by bone marrow cells from Stat3flox/– and Mx1:Stat3flox/– mice. Bone marrow cells (1 x 105/plate) from Stat3flox/– or Mx1:Stat3flox/– mice were plated in methylcellulose containing IL-3 (20 ng/ml), stem cell factor (20 ng/ml), and erythropoietin (4 U/ml) for the CFU-GM and BFU-E assays or G-CSF (50 ng/ml) only for the CFU-G assay. CFU-GM, BFU-E, and CFU-G were measured after 10 days in culture. (B): Proliferative activity of bone marrow cells in response to IL-3 and G-CSF. Bone marrow cells (1 x 106/ml) were incubated for 3 days in the presence of IL-3 (1 ng/ml) or G-CSF (10 ng/ml). Proliferative activity was measured by 3H-thymidine incorporation. (C): Survival of neutrophils due to G-CSF stimulation. Neutrophils from the peritoneal cavity were cultured in IMDM supplemented with 10% FCS and 10 ng/ml G-CSF. Apoptosis of neutrophils was examined by flow cytometry after staining with PI and FITC-conjugated Gr1 on day 2. (D): Prevention of apoptosis of neutrophils in response to G-CSF. Neutrophils from the peritoneal cavity were cultured in IMDM supplemented with 10% FCS and 10 ng/ml G-CSF. Cell viability was determined by the trypan blue exclusion assay on the indicated days. Abbreviations: CFU-GM, colony-forming unit-granulocyte, macrophage; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; IL, interleukin; IMDM, Iscove’s modified Dulbecco’s medium; PI, propidium iodide.

 
Because the number of myeloid progenitor cells was not affected by the absence of Stat3, we next examined the effect of Stat3 deletion in bone marrow cells for the proliferation by cytokine stimulation. Bone marrow cells taken from Mx1:Stat3flox/– mice and Stat3flox/– mice 14 days after pIpC treatment responded to IL-3 to a similar extent. In contrast, the proliferation of bone marrow cells after G-CSF stimulation was enhanced in Mx1:Stat3flox/– mice 14 days after pIpC treatment compared with control littermates (Fig. 2BGo).

To examine the possibility that Stat3 is involved in G-CSF–mediated cell survival, we determined the susceptibility of neutrophils to apoptosis in the presence of 10 ng/ml of G-CSF (Fig. 2CGo). After 48 hours in media supplemented with G-CSF, there was an increased number of apoptotic neutrophils (21%) from wild-type mice. In contrast, almost all of the neutrophils from Stat3-deficient mice were protected from apoptosis (Fig. 2CGo). Consistent with this observation, the survival of Stat3-deficient neutrophils was enhanced in culture with G-CSF (especially after a 2-day incubation) (Fig. 2DGo).

Stat3 Deficiency Enhances the Mobilization of Hematopoietic Progenitor Cells into the Peripheral Blood after In Vivo Administration of G-CSF
To assess the role of Stat3 in the G-CSF signaling pathway, we first treated mice with pIpC and then, after 14 days, treated them with daily injections of G-CSF for 7 days. After G-CSF treatment, the number of neutrophils was measured. Control littermates responded to G-CSF, and the number of peripheral blood neutrophils increased. Mx1: Stat3flox/– mice also responded to G-CSF, and the number of neutrophils after G-CSF treatment was almost the same in both cases (Fig. 3AGo). In addition to increasing the number of mature neutrophils in the peripheral blood, G-CSF also mobilizes hematopoietic progenitor cells from the bone marrow into the peripheral blood. Before treatment with G-CSF, no colony-forming hematopoietic progenitor cells were found in the peripheral blood (data not shown). In vivo G-CSF administration to control mice mobilized hematopoietic progenitor cells into the peripheral blood; this phenomenon could be assessed by colony-forming assay. In Mx1:Stat3flox/– mice, the number of hematopoietic progenitor cells mobilized by G-CSF was increased compared with the number mobilized in control mice (Fig. 3BGo). However, the chemotactic activity of neutrophils toward fMLP was almost identical in wild-type and Stat3-deficient cells (Fig. 3CGo).



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  		Figure 3. (A): In vivo administration of G-CSF. Stat3flox/– and Mx1:Stat3flox/– mice were injected subcutaneously with G-CSF from days 1 through 7 at 50 µg/kg. Peripheral blood was collected on the indicated day, 6 hours after G-CSF injection. (B): Mobilization of progenitor cells after administration of G-CSF in vivo. Stat3flox/– and Mx1:Stat3flox/– mice (n = 5) were injected subcutaneously with G-CSF from days 1 through 5 at 50 µg/kg. At day 5, peripheral blood mononuclear cells (2 x 105 cells/plate) were plated in methylcellulose containing interleukin-3 (20 ng/ml), stem cell factor (20 ng/ml), and erythropoietin (4 U/ml). The number of mobilized progenitor cells (CFU-C) was measured after 10 days in culture. (C): Chemotactic activity of neutrophils. Neutrophils from the peritoneal cavity were placed in the upper chamber and were attracted by fMLP in the lower chamber for 1 hour. The cells that passed through the membrane were counted, and the results are shown as the chemotactic index. (D): In vivo administration of TPO. Stat3flox/– and Mx1: Stat3flox/– mice were injected intraperitoneally with TPO from days 1 through 5 at 30 µg/kg. Peripheral blood was collected at the indicated day, 6 hours after TPO injection. Abbreviations: CFU-C, colony-forming unit-culture; TPO, thrombopoietin.

 
In addition to G-CSF, TPO also activates Stat3 in hematopoietic cells. Administration of TPO for 5 days increased the number of platelets in mice, and the deletion of Stat3 in hematopoietic cells did not alter this effect (Fig. 3DGo).

Stat3-Deficient Mice Develop Enterocolitis and Are Susceptible to DSS-Induced Colitis
Because mice with conditional deletion of Stat3 in macrophages and neutrophils (generated using LysM-Cre) developed colitis [26] and mice with conditional deletion of Stat3 in the bone marrow and endothelial cells (generated using Tie2-Cre) developed Crohn’s-like disease [33], we performed histological analysis on the Mx1cre:Stat3flox/– mice 2 weeks after pIpC treatment. This histological analysis demonstrated a reduction in goblet cell number, inflammatory cells infiltrating the lamina propria, and formation of crypt abscesses (Fig. 4AGo). Neutrophils and monocytes infiltrated the submucosal, muscular, and serosa layers. These observations indicate that Stat3 deficiency in hematopoietic lineages enhanced inflammation in the intestine.



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  		Figure 4. (A): Histological analysis of colitis. Stat3flox/– and Mx1:Stat3flox/– mice were treated with or without 4% DSS for 7 days. At day 7, mice were euthanized, and histological analysis was performed. Histological sections of the colon were stained with hematoxylin and eosin. Magnification x200. (B): Time course of DSS-induced body weight loss. Stat3flox/– and Mx1:Stat3flox/– mice (n = 5) were treated with 4% DSS for 7 days, and body weight was measured at the indicated day. Relative body weight compared with the day-1 baseline was plotted. Abbreviation: DSS, dextran sulfate sodium.

 
Next we investigated the role of Stat3 in an experimental inflammatory bowel disease model, DSS-induced colitis. Seven days after DSS administration, Mx1:Stat3flox/– mice developed severe colitis characterized by loss of weight (Fig. 4BGo), extensive leukocyte infiltration, and necrosis of the lamina propria (Fig. 4AGo). These changes were also observed in control mice, but the degree of weight loss and inflammation were extremely mild compared with the Mx1:Stat3flox/– mice. Two of five Mx1:Stat3flox/– mice after DSS administration died, whereas all of the control mice survived. These data suggest that Stat3 plays a negative regulatory role in intestinal inflammation.

Absence of SOCS3 Induction by G-CSF in Stat3-Deficient Bone Marrow Cells
The cytokine signaling is negatively regulated by SOCS family protein. Among them, SOCS3 is induced by G-CSF, and SOCS3 binds to phosphorylated G-CSR receptors to prevent Jak kinase activation. As shown in Figure 5Go, SOCS3 was induced by G-CSF stimulation in bone marrow cells from wild-type mice. By contrast, the expression level of SOCS3 protein in Stat3-deficient bone marrow cells is a trace, and it is not augmented by G-CSF stimulation.



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  		Figure 5. Induction of the SOCS3 protein in response to G-CSF stimulation of bone marrow cells from Stat3flox/– and Mx1: Stat3flox/– mice. Bone marrow cells from Stat3flox/– and Mx1: Stat3flox/– mice were incubated for 8 hours in the absence of G-CSF and were then stimulated with G-CSF (50 ng/ml) for the indicated period. Total cell lysates were analyzed by Western blot with the indicated antibodies.

 
Enhanced ERK Phosphorylation in Stat3-Deficient Bone Marrow Cells
Neutrophilia and colitis are observed in mice with conditional deletion of Stat3 in hematopoietic cells. G-CSF is the main cytokine regulating the proliferation and differentiation of cells in the granulocyte lineage [1], and the binding of G-CSF to its receptor activates the Ras–mitogen-activated protein kinase (MAPK) signaling cascade and the Jak-Stat signaling pathway [13,14]. Therefore, we examined the intracellular signaling pathways induced by G-CSF. In bone marrow cells from control mice, G-CSF stimulation promptly activated extracellular regulated kinase 1 (ERK1)/ERK2, and the activation diminished 60 minutes after stimulation (Fig. 6AGo). Stat3 activation occurred 30 minutes after G-CSF stimulation and was still apparent 120 minutes after G-CSF stimulation. In bone marrow cells from Mx1: Stat3flox/– mice 14 days after pIpC treatment, G-CSF–induced activation of Stat3 did not occur. However, ERK phosphorylation was observed in the absence of G-CSF stimulation. Furthermore, G-CSF stimulation increased the already high basal levels of ERK phosphorylation, and the activation was prolonged until 120 minutes after stimulation (Fig. 6AGo).



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  		Figure 6. (A): Phosphorylation of ERK1/2 in response to G-CSF stimulation of bone marrow cells from Stat3flox/– and Mx1: Stat3flox/– mice. Bone marrow cells from Stat3flox/– and Mx1: Stat3flox/– mice were incubated for 8 hours in the absence of G-CSF and were then stimulated with G-CSF (50 ng/ml) for the indicated period. Total cell lysates were analyzed by Western blot with the indicated antibodies. (B): Inhibition of G-CSF–mediated proliferative activity by the MEK1/2 inhibitor U0126. Bone marrow cells (1 x 106/ml) were incubated for 3 days in the presence of G-CSF (10 ng/ml) with 1 µM of the MEK1/2 inhibitor U0126 or DMSO. Proliferative activity was measured by 3H-thymidine incorporation. Abbreviations: DMSO, dimethyl sulfoxide; ERK, extracellular regulated kinase.

 
ERK1/2 is autonomously activated in bone marrow cells with specific deletion of Stat3; therefore, it is possible that the G-CSF–mediated hyperproliferation in Stat3-deficient bone marrow cells is attributable to the augmented activation of MAPK. We next examined the effects of MAPK activation on the G-CSF–mediated proliferation of bone marrow cells. G-CSF induced the proliferation of bone marrow cells from control mice, and the MEK kinase inhibitor U0126 almost completely inhibited this proliferative activity (Fig. 6BGo). Stat3-deficient bone marrow cells showed an enhanced proliferative activity after G-CSF treatment compared with wild-type cells. A large part of the augmented proliferative activity induced by G-CSF in Stat3-deficient bone marrow cells was abolished by the addition of U0126. These data indicate that MAPK activation is responsible for most of the enhanced proliferative activity of G-CSF–stimulated Stat3-deficient bone marrow cells. Furthermore, the activation of Stat3 by G-CSF negatively regulates the MAPK activation.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
G-CSF specifically stimulates the proliferation and differentiation of cells that are committed to the neutrophil-granulocyte lineage. Because mice lacking G-CSF or the G-CSF receptor had impaired production of mature granulocytes [34,35], G-CSF is thought to be the major regulator of differentiation and activation in the granulocyte lineage. The binding of G-CSF to the G-CSF receptor induced activation of the Jak-Stat pathway [3–6, 10, 11] and the Ras-Raf-ERK pathway [10,11]. In the Jak-Stat pathway, Jak1, Jak2, Tyk2, Stat1, Stat3, and Stat5 are tyro-sine phosphorylated in response to G-CSF [3–6, 10, 11]. Among the Stats, Stat3 is mainly tyrosine phosphorylated [4,10], and transgenic mice with a targeted mutation of the G-CSF receptor (d715F) that abrogates Stat3 activation have granulopenia [24]. We now report the generation of mice lacking Stat3 in the hematopoietic system to analyze Stat3 functions in vivo.

Although mice lacking G-CSF or the G-CSF receptor showed a decrease in peripheral neutrophils [34,35], mice with the Stat3 deficiency in the hematopoietic system do not have neutropenia. Rather, these mice have granulocytosis, consistent with the result reported by Welte et al. [33] or Lee et al. [36]. Furthermore, the number of neutrophils in the bone marrow was greater in mice deficient for Stat3 in the hematopoietic system (Fig. 1BGo). There was no difference in the number of progenitor cells (CFU-G, CFU-GM) in the bone marrow between wild-type mice and mice deficient for Stat3 in the hematopoietic system, as assessed by the number of colony-forming cells (Fig. 2AGo). Therefore, we next examined the proliferative response of bone marrow cells to G-CSF stimulation. As shown in Figure 2BGo, bone marrow cells from the mice deficient for Stat3 in the hematopoietic system responded more strongly to G-CSF stimulation than cells from wild-type mice. Furthermore, apoptosis in cultures supplemented with G-CSF was suppressed by ablation of Stat3 in neutrophils (Fig. 2CGo). Thus, the enhanced proliferation of neutrophils observed in mice deficient for Stat3 in the hematopoietic system is attributable both to the augmented proliferation of neutrophilic progenitor cells in response to G-CSF and prolonged neutrophil survival in response to G-CSF.

The G-CSF–induced mobilization of hematopoietic progenitor cells from the bone marrow into the peripheral blood is also augmented by the absence of Stat3 in hematopoietic cells (Fig. 3BGo). The mechanism by which hematopoietic progenitor cells are mobilized from the bone marrow into the peripheral blood in response to G-CSF has not been elucidated. G-CSF causes neutrophils to secrete proteases, which allows hematopoietic progenitor cells to release from the bone marrow microenvironment into the peripheral blood. Because the absence of Stat3 in hematopoietic cells had no effect on the number of hematopoietic progenitor cells in the bone marrow (Fig. 2AGo), the augmented mobilization induced by G-CSF in Stat3-deficient mice must be attributable to hyperactivity of the Stat3-deficient neutrophils in response to G-CSF stimulation. On the other hand, the fMLP-induced chemotactic activity of neutrophils was not affected by the absence of Stat3 in neutrophils (Fig. 3CGo).

We assessed the presence of pathological abnormalities caused by the deletion of Stat3 in the hematopoietic system. There was some infiltration of inflammatory cells into the mucosa propria in the stomach and small intestine (data not shown), and moderate inflammation and the disappearance of goblet cells were observed in the colon (Fig. 4AGo). No inflammation was observed in the brain, liver, or lung. Welte et al. [33] also reported that pathological abnormalities similar to those observed in Crohn’s disease occurred in mice with conditional deletion of Stat3 driven by the Tie2 promoter (Stat3CFF) [33]. Stat3CFF mice had widespread inflammatory disease in the digestive tract, and close to 100% of the animals died by 4– 6 weeks after birth. Although our Stat3-deficient mice developed colitis, they survived under specific pathogen-free conditions. Because Tie-2 is expressed in hemangioblasts [37], the deletion of Stat3 in the blood vessels may lead to severe inflammation in the digestive tract.

Conditional knockout of Stat3 in macrophages and neutrophils resulted in chronic enterocolitis with age [26]. Mice deficient for Stat3 in hematopoietic cells developed colitis in their youth. Thus, the deletion of Stat3 not only in mature neutrophils and macrophages but also in immature myeloid cells might augment the development of colitis.

We next investigated the role of Stat3 in the pathology of DSS-induced colitis. Stat3 is mainly activated by G-CSF, IL-6, and IL-10, and elevation of IL-6 levels has been reported in DSS-induced colitis. The degree of emaciation after DSS treatment is more severe in mice lacking Stat3 in hematopoietic cells than in control mice, and two of five Stat3-deficient mice died during DSS administration (Fig. 4BGo). After DSS treatment, the disappearance of the gland duct, necrosis of the proper mucosa, and infiltration of inflammatory cells into the serosa were observed in mice deficient for Stat3 in the hematopoietic system, although moderate infiltration also occurred in wild-type mice (Fig. 4AGo). The deletion of Stat3 in hematopoietic cells augmented the infiltration of inflammatory cells into the digestive tract in DSS-induced colitis. Taken together, these data indicate that the ablation of Stat3 in hematopoietic cells enhanced the response (proliferation and inflammation) to cytokines, including G-CSF, which is likely due to the loss of negative regulatory signals.

The suppressor of cytokine signaling (SOCS) family is a group of negative regulators of cytokine signaling [38–42]. These proteins are induced as part of the cellular response to cytokines. In particular, SOCS3 expression is induced by G-CSF stimulation [43]. SOCS3 binds selectively to phosphorylated tyrosine residues in the G-CSF receptor (Y729 in human and Y728 in murine G-CSF receptor) through its SH2 domain and inhibits the catalytic activity of Jaks [43]. Therefore, we examined whether the expression level of SOCS3 was affected by the deletion of Stat3 in hematopoietic cells. Only trace levels of SOCS3 were expressed in bone marrow cells from mice deficient for Stat3 in the hematopoietic system, and this expression was not induced by G-CSF stimulation. In contrast, G-CSF treatment induced the expression of SOCS3 in bone marrow cells from wild-type mice (Fig. 5Go). Taken together, these data indicate that Stat3 is required for induction of SOCS3 in response to G-CSF signaling. Furthermore, the absence of SOCS3 negative feedback may allow prolonged Jak activation, resulting in enhanced signaling and increased proliferation. Certainly the phenotype of mice deficient for SOCS3 in hematopoietic cells [44,45] is extremely similar to the phenotype of mice deficient for Stat3 in hematopoietic cells. SOCS3-deficient mice developed neutrophilia, and cells from the neutrophil-granulocyte lineage of mice deficient for SOCS3 in hematopoietic cells displayed an enhanced cellular response to in vitro stimulation with G-CSF.

Stat3 is the principal protein activated by G-CSF [6,10]. 32Dcl3 cells normally differentiate into neutrophils after treatment with G-CSF; however, when these cells express dominant-negative Stat3 (32Dcl3/DNStat3), they proliferate in the presence of G-CSF, but they maintain immature morphologic characteristics without evidence of differentiation [23]. Additionally, transgenic mice with a targeted mutation in the G-CSF receptor, which abolishes G-CSF–dependent Stat3 activation, have severe neutropenia with an accumulation of immature myeloid precursors in the bone marrow [24]. These data suggest that Stat3 transduces the differentiation signal of G-CSF. However, our study clearly shows that Stat3 actually transduces an inhibitory signal by the induction of SOCS3 in response to G-CSF signaling. This observation led us to ask what molecule transduces the proliferation and differentiation signals of G-CSF. G-CSF is known to activate Stat1 and Stat5, each of which might be involved in regulating cell proliferation. However, the deletion of Stat1 does not affect granulopoiesis [18]. The Stat5a/Stat5b double-knockout mouse has normal levels of neutrophils and monocytes, although G-CSF–stimulated colony formation of bone marrow cells is slightly affected by the absence of both Stat5a and Stat5b [21]. These phenotypes, together with the phenotype of mice deficient for Stat3 in the hematopoietic system, strongly suggest that ERK is a plausible candidate as a downstream mediator of G-CSF signaling responsible for proliferation and differentiation. This is consistent with a recent report indicating that Tyr-764 of the G-CSF receptor is the most important element for G-CSF–induced proliferation, which was reversed by inhibition of ERK activity [46].

Therefore, we evaluated the role of MAPK in the G-CSF signaling pathway (Figs. 6A, 6BGo). Wild-type bone marrow cells displayed a transient activation of ERK1/2 after G-CSF stimulation. In contrast, Stat3-null bone marrow cells displayed a significant activation of ERK1/2 even under basal conditions. In these cells, ERK activation was enhanced by G-CSF stimulation, and it was more sustained, remaining significantly elevated 60 minutes after G-CSF stimulation (Fig. 6AGo). Because enhanced proliferation of granulocytes in response to G-CSF in Stat3-null cells might result from the enhanced and prolonged activation of ERK, we studied the effects of the MEK1 inhibitor U0126, which blocks activation of ERK1 and ERK2, on G-CSF–induced proliferation of wild-type and Stat3-deficient bone marrow cells. As shown in Figure 6BGo, addition of U0126 to the cultures inhibited G-CSF–induced cell proliferation in wild-type mice. Surprisingly, the proliferative activity of Stat3-deficient bone marrow cells in response to G-CSF was dramatically decreased upon addition of U0126. These data indicate that MAPK activation is responsible for most of the proliferative activity of hematopoietic cells in response to G-CSF.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We would like to thank M. Sato and M. Ito for their excellent technical assistance and Drs. A. Nonami and A. Kimura (Kyushu University) for discussion. This work was supported in part by a grant from the Japanese Leukemia Foundation, a Grant for Clinical Research, and Grants-in-Aid for Scientific Research (13218096 and 15390302) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Demetri GD, Griffin JD. Granulocyte colony-stimulating factor and its receptor. Blood 1991;78:2791–2808.[Free Full Text]

  2. Shimoda K, Okamura S, Harada N et al. High-frequency granuloid colony-forming ability of G-CSF receptor possessing CD34 antigen positive human umbilical cord blood hematopoietic progenitors. Exp Hematol 1995;23:226–228.[Medline]

  3. Nicholson SE, Oates AC, Harpur AG et al. Tyrosine kinase JAK1 is associated with the granulocyte-colony-stimulating factor receptor and both become tyrosine-phosphorylated after receptor activation. Proc Natl Acad Sci U S A 1994;91:2985–2988.[Abstract/Free Full Text]

  4. Nicholson SE, Novak U, Ziegler SF et al. Distinct regions of the granulocyte colony-stimulating factor receptor are required for tyrosine phosphorylation of the signaling molecules JAK2, Stat3, and p42, p44MAPK. Blood 1995;86:3698–3704.[Abstract/Free Full Text]

  5. Shimoda K, Iwasaki H, Okamura S et al. G-CSF induces tyrosine phosphorylation of the JAK2 protein in the human myeloid G-CSF responsive and proliferative cells, but not in mature neutrophils. Biochem Biophys Res Commun 1994;203:922–928.[CrossRef][Medline]

  6. Shimoda K, Feng J, Murakami H et al. Jak1 plays an essential role for receptor phosphorylation and Stat activation in response to granulocyte colony-stimulating factor. Blood 1997;90:597–604.[Abstract/Free Full Text]

  7. Ihle JN. Cytokine receptor signalling. Nature 1995;377:591–594.[CrossRef][Medline]

  8. Ihle JN, Nosaka T, Thierfelder W et al. Jaks and Stats in cytokine signaling. STEM CELLS 1997;15(suppl 1):105–111.

  9. Ihle JN. STATs: signal transducers and activators of transcription. Cell 1996;84:331–334.[CrossRef][Medline]

  10. Tian SS, Lamb P, Seidel HM et al. Rapid activation of the STAT3 transcription factor by granulocyte colony-stimulating factor. Blood 1994;84:1760–1764.[Abstract/Free Full Text]

  11. de Koning JP, Dong F, Smith L et al. The membrane-distal cytoplasmic region of human granulocyte colony-stimulating factor receptor is required for STAT3 but not STAT1 homodimer formation. Blood 1996;87:1335–1342.[Abstract/Free Full Text]

  12. Darnell JE Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994;264:1415–1421.[Abstract/Free Full Text]

  13. Corey SJ, Burkhardt AL, Bolen JB et al. Granulocyte colony-stimulating factor receptor signaling involves the formation of a three-component complex with Lyn and Syk protein-tyrosine kinases. Proc Natl Acad Sci U S A 1994; 91:4683–4687.[Abstract/Free Full Text]

  14. Matsuda T, Hirano T. Association of p72 tyrosine kinase with Stat factors and its activation by interleukin-3, inter-leukin-6, and granulocyte colony-stimulating factor. Blood 1994;83:3457–3461.[Abstract/Free Full Text]

  15. Rodig SJ, Meraz MA, White JM et al. Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic responses. Cell 1998;93:373–383.[CrossRef][Medline]

  16. Shimoda K, Kato K, Aoki K et al. Tyk2 plays a restricted role in IFN alpha signaling, although it is required for IL-12-mediated T cell function. Immunity 2000;13:561–571.[CrossRef][Medline]

  17. Parganas E, Wang D, Stravopodis D et al. Jak2 is essential for signaling through a variety of cytokine receptors. Cell 1998;93:385–395.[CrossRef][Medline]

  18. Meraz MA, White JM, Sheehan KC et al. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 1996;84:431–442.[CrossRef][Medline]

  19. Shimoda K, van Deursen J, Sangster MY et al. Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature 1996;380:630–633.[CrossRef][Medline]

  20. Thierfelder WE, van Deursen JM, Yamamoto K et al. Requirement for Stat4 in interleukin-12-mediatedresponses of natural killer and T cells. Nature 1996;382:171–174.[CrossRef][Medline]

  21. 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]

  22. Takeda K, Tanaka T, Shi W et al. Essential role of Stat6 in IL-4 signalling. Nature 1996;380:627–630.[CrossRef][Medline]

  23. Nakajima H, Ihle JN. Granulocyte colony-stimulating factor regulates myeloid differentiation through CCAAT/enhancer-binding protein epsilon. Blood 2001;98:897–905.[Abstract/Free Full Text]

  24. McLemore ML, Grewal S, Liu F et al. STAT-3 activation is required for normal G-CSF-dependent proliferation and granulocytic differentiation. Immunity 2001;14:193–204.[CrossRef][Medline]

  25. 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.[Abstract/Free Full Text]

  26. Takeda K, Clausen BE, Kaisho T et al. Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils. Immunity 1999;10:39–49.[CrossRef][Medline]

  27. Takeda K, Kaisho T, Yoshida N et al. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient mice. J Immunol 1998;161:4652–4660.[Abstract/Free Full Text]

  28. Kuhn R, Schwenk F, Aguet M et al. Inducible gene targeting in mice. Science 1995;269:1427–1429.[Abstract/Free Full Text]

  29. Ludwig A, Petersen F, Zahn S et al. The CXC-chemokine neutrophil-activating peptide-2 induces two distinct optima of neutrophil chemotaxis by differential interaction with interleukin-8 receptors CXCR-1 and CXCR-2. Blood 1997;90:4588–4597.[Abstract/Free Full Text]

  30. Kato K, Kamezaki K, Shimoda K et al. Intracellular signal transduction of interferon on the suppression of haematopoietic progenitor cell growth. Br J Haematol 2003;123:528–535.[CrossRef][Medline]

  31. Kanauchi O, Nakamura T, Agata K et al. Effects of germinated barley foodstuff on dextran sulfate sodium-induced colitis in rats. J Gastroenterol 1998;33:179–188.[CrossRef][Medline]

  32. Aoki K, Shimoda K, Oritani K et al. Limitin, an interferon-like cytokine, transduces inhibitory signals on B-cell growth through activation of Tyk2, but not Stat1, followed by induction and nuclear translocation of Daxx. Exp Hematol 2003;31:1317–1322.[CrossRef][Medline]

  33. Welte T, Zhang SS, Wang T et al. STAT3 deletion during hematopoiesis causes Crohn’s disease-like pathogenesis and lethality: a critical role of STAT3 in innate immunity. Proc Natl Acad Sci U S A 2003;100:1879–1884.[Abstract/Free Full Text]

  34. Lieschke GJ, Grail D, Hodgson G et al. Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization. Blood 1994;84:1737–1746.[Abstract/Free Full Text]

  35. Liu F, Wu HY, Wesselschmidt R et al. Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice. Immunity 1996;5:491–501.[CrossRef][Medline]

  36. Lee CK, Raz R, Gimeno R et al. STAT3 is a negative regulator of granulopoiesis but is not required for G-CSF-dependent differentiation. Immunity 2002;17:63–72.[CrossRef][Medline]

  37. Maisonpierre PC, Suri C, Jones PF et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 1997;277:55–60.[Abstract/Free Full Text]

  38. Endo TA, Masuhara M, Yokouchi M et al. A new protein containing an SH2 domain that inhibits JAK kinases. Nature 1997;387:921–924.[CrossRef][Medline]

  39. Kamura T, Sato S, Haque D et al. The Elongin BC complex interacts with the conserved SOCS-box motif present in members of the SOCS, ras, WD-40 repeat, and ankyrin repeat families. Genes Dev 1998;12:3872–3881.[Abstract/Free Full Text]

  40. Naka T, Narazaki M, Hirata M et al. Structure and function of a new STAT-induced STAT inhibitor. Nature 1997;387:924–929.[CrossRef][Medline]

  41. Starr R, Willson TA, Viney EM et al. A family of cytokine-inducible inhibitors of signalling. Nature 1997;387:917–921.[CrossRef][Medline]

  42. Sasaki A, Yasukawa H, Suzuki A et al. Cytokine-inducible SH2 protein-3 (CIS3/SOCS3) inhibits Janus tyrosine kinase by binding through the N-terminal kinase inhibitory region as well as SH2 domain. Genes Cells 1999;4:339–351.[Abstract]

  43. Hortner M, Nielsch U, Mayr LM et al. Suppressor of cytokine signaling-3 is recruited to the activated granulocyte-colony stimulating factor receptor and modulates its signal transduction. J Immunol 2002;169:1219–1227.[Abstract/Free Full Text]

  44. Kimura A, Kinjyo I, Matsumura Y et al. SOCS3 is a physiological negative regulator for granulopoiesis and granulocyte colony-stimulating factor receptor signaling. J Biol Chem 2004;279:6905–6910.[Abstract/Free Full Text]

  45. Croker BA, Metcalf D, Robb L et al. SOCS3 is a critical physiological negative regulator of G-CSF signaling and emergency granulopoiesis. Immunity 2004;20:153–165.[CrossRef][Medline]

  46. Hermans MH, van de Geijn GJ, Antonissen C et al. Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells. Blood 2003;101:2584–2590.[Abstract/Free Full Text]

Received August 3, 2004; accepted for publication October 6, 2004.



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