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Stem Cells, Vol. 17, No. 2, 82-91, March 1999
© 1999 AlphaMed Press

HGF Activates Signal Transduction from EPO Receptor on Human Cord Blood CD34+/CD45+ Cells

Tomoko Iguchia,d, Shinji Sogoe, Hiroko Hishaa, Shigeru Taketanib, Yasushi Adachia, Rika Miyazakic, Hajime Ogatac, Seiji Masudaf, Ryuzo Sasakif, Motoki Itoa,d, Shirou Fukuharad, Susumu Ikeharaa

a First Department of Pathology,
b Department of Hygiene,
c Department of Pediatrics,
d First Department of Internal Medicine, Kansai Medical University, Moriguchi City, Osaka, Japan;
e Cellular Technology Institute, Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan;
f Laboratory of Biosignals and Response, Division of Applied Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

Key Words. HGF • Human cord blood • Signal transduction • Erythropoiesis

Dr. Susumu Ikehara, First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hepatocyte growth factor (HGF) is a multifunctional cytokine with early hematopoiesis-stimulatory activity. Here, we focus on its erythropoiesis-stimulatory effect on highly purified human hematopoietic progenitor cells (CD34+/CD45+ cells) derived from the cord blood.

In immunoblot analyses, c-met protein (a receptor of HGF) was detected in the CD34+/CD45+ cells, although the expression levels were different among samples. The c-met expression was facilitated by incubation of the cells with stem cell factor (SCF) or interleukin 3 (IL-3), even if the expression level had been low. IL-6, G-CSF, or erythropoietin (EPO) did not show such a stimulatory effect on the c-met expression of the cells. When HGF was added to the CD34+/CD45+ cells in the presence of SCF, the numbers of CD36+/CD11b– cells (very early erythroid lineage cells) and BFU-E increased. EPO-dependent tyrosine phosphorylation of Stat 5 also increased, but the EPO receptor (EPO-R) expression remained unchanged in the CD34+/CD45+ cells treated with SCF + HGF. Our present study suggests that stimulation of the HGF/c-met signal is concomitant with induction of c-met protein by SCF. The subsequent enhancement of signal transduction via the activation of Stat 5 from the EPO-R plays a crucial role in the commitment of hematopoietic stem cells into erythroid lineage cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hepatocyte growth factor (HGF)/scatter factor, which was first isolated as a liver-regenerating factor from the plasma of patients with fulminant hepatitis and rat platelets, is now known as a multifunctional factor regulating cell growth, motility, and migration [1-5]. It is also known to regulate tumor invasion [6], morphogenesis [3, 7], and organ regeneration [3, 8].

It has been shown that the serum levels of HGF increase not only in patients with fulminant hepatitis but also in those with leukemia or multiple myeloma [9-11]. The bone marrow (BM) plasma levels of HGF were found to significantly decrease in patients with leukemia after complete remission [10]. These findings indicate that HGF production is related to the progression of these diseases, and it is conceivable that it modifies lymphohematopoietic disorders.

von Schweinitz et al. have reported that both hepatoblastoma (HB) cells and intratumoral cells produce various cytokines, including stem cell factor (SCF), erythropoietin (EPO), G-CSF, GM-CSF, and HGF [12, 13]; hematopoietic foci were detected in all of the 15 HB tumor-bearing patients studied. In the hematopoietic foci, erythroblasts were found in all the HB patients, and megakaryocytes were in 10 of 15, whereas no granulocyte or monocyte precursors were detected. Thus, it is conceivable that HGF, SCF, and EPO function synergistically to induce erythroid- and megakaryocyte-lineage-specific hematopoiesis in the HB tumor. HGF was shown to be produced by various cells, including malignant cells such as lung [14] and breast [15] cancers. It is thus suggested that HGF is involved in hemopoietic disorders of patients with solid tumors.

In mice, HGF was found to increase the colony formation of bone marrow cells in combination with interleukin 3 (IL-3) and GM-CSF [16]. mRNA of c-met (a tyrosine kinase-type receptor of HGF) and its protein are also detected in both NFS-60 (a myeloid progenitor cell line) and unfractionated BM cells [16]. We have previously shown that the mRNA of both HGF and c-met are detected not only in murine BM adherent cells but also in BM stromal cell lines (MS-5 and PA-6) [17]. Recently, it has been reported that human BM stromal cells constitutively produce HGF and express c-met [18]. In Sl/Sld and W/Wv mice, which show disorders of SCF/c-kit signal transduction, the in vivo administration of HGF increases the numbers of WBCs, platelets, and RBCs [19]. This suggests that a compensatory system such as the HGF/c-met system functions in the SCF/c-kit system-deficient mice. Thus, it is speculated that HGF, secreted from the BM stroma, directly affects hematopoietic progenitors in the bone marrow.

In humans, it has not been clarified whether c-met is expressed on the CD34+ multipotent progenitors [18, 20]. In this report, we isolate CD34+ cells from the human cord blood (CB) or BM using a fluorescence-activated cell sorter [21] and show that c-met is expressed on CD34+/CD45+ cells, although the expression levels are different among samples. In addition, we show that HGF preferentially stimulates erythropoiesis of the CD34+/CD45+ cells, in which the expression of c-met is enhanced by SCF. We also discuss the mechanisms underlying the stimulatory effects of HGF.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of CD34+/CD45+ Cells
After fully informed consent, human umbilical CB was collected and suspended in ACD (acid-citrate-dextrose) according to the guidelines of the Cord Blood Bank, Kansai Medical University. Human BM cells were obtained from healthy volunteers and suspended in ACD. Mononuclear cells were separated from the ACD solutions by Lymphoprep (Nycomed Pharma; Oslo, Norway) density gradient centrifugation. The mononuclear cells were then incubated with microbead-conjugated anti-CD34 class II monoclonal antibody (mAb) (Miltenyi Biotec; Bergisch Gladbach, Germany) at 4°C for 20 min and processed through Mini MACS magnetic separation column (Miltenyi Biotec) to obtain a CD34+ cell-enriched fraction. Highly purified hematopoietic CD34+/CD45+ cells were sorted from the CD34+ cell-enriched fraction using a FACStarTM (Becton Dickinson Immunocytometry Systems; San Jose, CA) after staining with fluorescein isothiocyanate (FITC)-conjugated anti-CD34 class III (HPCA-2: Becton Dickinson Immunocytometry Systems) and phycoerythrin-conjugated anti-CD45 mAb (Pharmingen; San Diego, CA) for 30 min at 4°C.

Colony-Forming Assay
The colony-forming ability of sorted CD34+/CD45+ cells was assayed in two methylcellulose assay systems. In some experiments, CD34+/CD45+ cells (250 cells/well) were plated in 12-well plates (ICN Biomedicals, Inc.; Aurora, OH) in a volume of 0.8 ml of Methocult GF H4434 (Stem Cell Technologies Inc.; Vancouver, BC, Canada), consisting of optimal concentrations of cytokines (recombinant human SCF [rHuSCF], EPO, IL-3, GM-CSF, and G-CSF), 30% fetal bovine serum (FBS), 1% bovine serum albumin, 2 mM L-glutamine, 10–4M 2-mercaptoethanol, and 0.9% methylcellulose.

In the other colony-forming assay system, Methocult H4230 (cytokine-free) was supplemented with rHuSCF (30 ng/ ml, Kirin Brewery; Tokyo, Japan) and rHuEPO (2 U/ml, Kirin Brewery). Various concentrations of rHuHGF (2.5-16.0 ng/ml, R&D Systems; Minneapolis, MN) were then added to each methylcellulose culture mixture.

The plates were incubated for 14 days at 37°C under 5% CO2, and colony-forming unit-mixture (CFU-M) and BFU-E colonies were counted under an inverted microscope; the CFU-M colony consisted mostly of monocytes and only very few granulocytes. The average number of colonies and standard error were calculated from quadruplicated wells.

Cell Suspension Culture and Flow Cytometric Analysis
All the suspension cultures of sorted CD34+/CD45+ cells were performed at a concentration of 5 x 105 cells/ml in Iscove's modified Dulbecco's medium (IMDM) (GIBCO; Grand Island, NY) containing 10% heat-inactivated FBS (lot No. HCC 6450, Stem Cell Technologies Inc.). Sorted cells were incubated in combination with the following growth factors: rHuSCF (30 ng/ml), rHuEPO (2 U/ml), rHuIL-3 (10 ng/ml, Boehringer Mannheim Biomedica; Indianapolis, IN), rHuG-CSF (10 ng/ml, Kirin Brewery), and rHuHGF (5 ng/ml).

HGF receptor was detected as follows: the cultured cells were incubated with 200 ng/ml of rHuHGF at 10°C for 3 h, washed twice, and stained with FITC-conjugated rabbit anti-HGF polyclonal Ab (Cellular Technology Institute, Otsuka Pharmaceutical Co., Ltd.; Otsuka, Japan) at 4°C for 30 min. For the detection of CD36 antigen, the cultured cells were triple-stained with biotin-conjugated anti-CD11b mAb (Pharmingen) plus streptoavidin-RED670 conjugate (SA-RED670; GIBCO), FITC-conjugated anti-CD36 mAb (Immunotech SA; Marseille, France), and PE-conjugated anti-CD34 mAb (HPCA-2: Becton Dickinson). Flow cytometric analysis was performed using a FACScanTM (Becton Dickinson Immunocytometry Systems).

Preparation of Cell Lysates and Western Blot Analysis
Sorted CD34+/CD45+ cells (1 x 105) were incubated with various growth factors in IMDM containing 10% FBS. A carcinoma cell line, A431, was used as a positive control for c-met expression. A human leukemia cell line, UT-7 [22], (a positive control for EPO receptor [EPO-R]) was kindly provided by Dr. N. Komatsu (Jichi Medical School; Tochigi, Japan).

The collected cells were lysed in lysis buffer (Buffer A:1M pH 7.9 HEPES-KOH, 1M KCL, 500 mM EDTA, 100 mM EGTA, 100 mM DTT, 574 mM phenylmethylsulfonyl fluoride, 100 mM orthovanadate, 100 mM aprotinin, 1% Nonidet P-40) and incubated for 30 min at 4°C. Unsolubilized material was removed by centrifugation for 10 min at 10,000 x g at 4°C. Supernatants were mixed in sample buffer (0.25M Tris-HCl pH 6.5, 4% SDS, 20% glycerol, 10% mercaptoethanol) and then applied to 6% and 7.5% polyacrylamide gels. After electrophoresis, proteins were then transferred to nitrocellulose filters (Hybond ECL, Amersham; Buckinghamshire, England). Antibodies used were: rabbit anti-c-met Ab (Santa Cruz Biotechnology; Santa Cruz, CA), horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ab (Bio-Rad Laboratories; Hercules, CA), and HRP-conjugated anti-phosphotyrosine mAb (4G10, Upstate Biotechnology; Lake Placid, NY). After washing, the blots were incubated with the enhanced chemiluminescence substrate (ECL, Amersham) and exposed to Hyperfilm ECL (Amersham). Detection of the EPO-R protein was carried out as previously described [23].

Protein concentrations were measured by the Bradford method [24].

Immunoprecipitation of Stat 5
Supernatants of cell lysates prepared as above were precleared with Protein A Sepharose. The proteins were immunoprecipitated from resulting supernatants with anti-Stat 5 (Transduction Laboratories; Lexington, KY), and the immune complex was collected by incubation with Protein A Sepharose. The proteins were analyzed using 7.5% polyacrylamide gel and transferred onto nitrocellulose filters. Proteins were probed with anti-Stat 5 or anti-phosphotyrosine Ab (4G10) and visualized using the ECL system.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) of c-met
Total cellular RNA was prepared using a nucleic acid extractor, TRIZOL reagent (Life Technologies, Inc.; Grand Island, NY) followed by chloroform extraction and isopropanol precipitation. cDNA was synthesized using reverse transcriptase (M-MLV Rtase in RT-PCR high [RT-PCR Kit] Toyobo; Tokyo, Japan) and Oligo (dT) 20.P7 primers (RT-PCR high).

PCR was performed on the cDNA using the following primers for c-met (Maxim Biotech, Inc.; San Francisco, CA.) and G3PDH (RT-PCR high) with thermal cycling amplification using Takara PCR Thermal Cycler MP (Takara; Otsu, Japan). The c-met-specific primers consisted of a sense (4035 to 4058: 5'-AAAGTCAGATGTGTGGTCCT-TTGGC-3') and an antisense (4287 to 4263: 5'-GTCCACCTCATCATCAG-CGTTAT-3').

The samples (sorted 1 x 105 to 1 x 106 CD34+/CD45+ cells) were amplified for 35 cycles at cycling temperatures of 94°C, 1 min; 55°C, 1 min; and 35°C, 3 min for both the G3PDH and the c-met amplification. PCR products were separated on a 1.2% agarose gel (GIBCO) and visualized by ethidium bromide (Nakarai; Kyoto, Japan) staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of HGF on Colony Formation of CD34+/CD45+ Cells
Partially purified CD34+ cells from the human CB were stained with FITC-conjugated anti-CD34 mAb and PE-conjugated anti-CD45 mAb, to avoid the contamination of stromal cells in the CD34+ hematopoietic stem cells. The CD34+/CD45+ cells were sorted as highly purified hematopoietic progenitors (Fig. 1).



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  		Figure 1. A) Typical forward- and side-scatter pattern of purified CD34 class II+ cells. B) CD34 class III- and CD45- expression patterns of cells in the blast window.

 
We examined the stimulatory effect of HGF on colony formation. The sorted cells were cultured in the methylcellulose medium containing optimal doses of SCF, IL-3, GM-CSF, G-CSF, and EPO in the presence of various concentrations of HGF. After a 14-day culture, erythroid (BFU-E) and non-erythroid (CFU-M) colonies emerged. The CFU-M colony consisted mostly of monocytes and only very few granulocytes. The addition of HGF increased the BFU-E colonies in a dose-dependent manner, whereas there was no significant increase in CFU-M colonies (Fig. 2A). The colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM) and CFU-E colonies did not increase (data not shown).



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  		Figure 2. Effects of HGF on colony formation of CD34+/CD45+ cells. Sorted CD34+/CD45+ cells (1 x 103) were assayed using methylcellulose culture medium containing the following growth factors: A) SCF, IL-3, GM-CSF, G-CSF, and EPO; B) SCF (30 ng/ml) and EPO (2 U/ml), in the presence of various concentrations of HGF (2.5-16.0 ng/ml). After a 14-day culture, the number of colonies (BFU-E and CFU-M) were counted. The mean ± SE: *p < 0.05, **p < 0.01

 
The effect of HGF on the modification of differentiation patterns was also examined in the methylcellulose medium containing only two kinds of cytokines (SCF and EPO). A similar increase in BFU-E counts was observed according to the increase in HGF concentrations, but not in CFU-M counts (Fig. 2B). Under both culture conditions, the preferential differentiation into erythroid lineage cells was induced from 2.0 ng/ml of HGF.

When HGF was added to the methylcellulose culture containing G-CSF alone as a cytokine, no increase in the numbers of colonies (BFU-E, CFU-M, and CFU-GEMM) was observed (data not shown).

HGF exhibited a similar stimulatory effect on the BFU-E formation of CD34+/CD45+ human bone marrow cells (data not shown), although not significantly.

Expression of c-met in CD34+/CD45+ Cells
The expression of c-met protein on the surface of sorted CD34+/CD45+ cells was analyzed using flow cytometry. c-met was detected on the CD34+/CD45+ cells of both the CB and BM (Fig. 3A). c-met precursor protein (p170 met) and ß chain (p145 met) were also detected in the lysates of these cells by immunoblotting, although the expression levels were different among samples (Fig. 3B). When blocking peptide of c-met was added to the cell lysates, both bands (p170 and p145 met) disappeared, indicating that these bands represent c-met protein (data not shown). Moreover, RT-PCR analyses of CD34+/CD45+ cells revealed that c-met mRNA was detected in all the CB samples, even if the expression in protein levels was low (Fig. 3C). This finding was confirmed in many CB samples. These results indicate that HGF receptors are expressed on CD34+/CD45+ cells purified from both the CB and BM.



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  		Figure 3. Expression of HGF receptor (c-met) on CD34+/CD45+ cells. The expression of c-met on freshly isolated CB and BM CD34+/CD45+ cells was detected by: A) flow cytometry; B) immunoblotting, and C) RT-PCR. B) Lanes 1-9: sorted CD34+/CD45+ CB cells; lanes 10, 11: sorted CD34+/CD45+ BM cells. C) The expected PCR products of 353 bp (c-met) were detected. Control primers amplified a 450 bp fragment of G3PDH. Lanes 1-7: sorted CD34+/CD45+ CB cells.

 
Enhancement of c-met Expression in SCF-Treated CD34+/CD45+ Cells
Since HGF increases the number of erythroid colonies in the presence of SCF and EPO (Fig. 2B), we next examined whether SCF, EPO, or HGF increases the c-met expression on CD34+/CD45+ cells. As shown in Figure 4, the addition of SCF markedly enhanced the p170 and p145 met expression. EPO and SCF synergized to maximize the induction of c-met, but c-met induction by EPO or HGF alone was only marginally greater than with untreated cells.



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  		Figure 4. Induction of c-met expression by SCF. Sorted CD34+/CD45+ cells (1 x 105) were incubated with HGF or SCF and/or EPO for 24 h. The expression of c-met precursor (170 kDa) and c-met ß-chain (145 kDa) was detected. Lane 1: preincubation; lane 2: medium alone; lane 3: SCF; lane 4: EPO; lane 5: HGF; and lane 6: SCF+EPO, and lane 7: A431 cells.

 
We next examined the time course of the c-met induction by SCF. Equal amounts of cellular protein containing 10 µg protein for each slot of SDS-PAGE were applied. c-met precursor and c-met ß chain increased with time to a maximum level after incubation with SCF for 24-48 h (Fig. 5A). Increasing the concentrations of SCF caused the amounts of c-met proteins to increase, reaching a maximum level at 30 ng/ml (Fig. 5B).



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  		Figure 5. A) Time course of c-met induction by SCF. Sorted CD34+/CD45+ cells (1 x 105) were incubated with SCF (30 ng/ml) for 24-72 h. Lane 1: preincubation; lane 2: 24 h; lane 3: 48 h, and lane 4: 72 h. B) Dose-dependent induction of c-met by SCF. Lane 1: preincubation; lane 2: medium alone; lane 3: SCF (0.3 ng/ml); lane 4: SCF (3 ng/ml), and lane 5: SCF (30 ng/ml). C) Induction of c-met expression by SCF in different samples. Sorted CB CD34+/CD45+ cells (1 x 105) were incubated with SCF (30 ng/ml) for 24 h, and c-met was detected. Lanes 1-2 and 3-4 are pairs of the same sample. Lanes 1 and 3 are preincubation, and lanes 2 and 4 are SCF-treated CD34+ cells. Cellular proteins (10 µg of protein) were analyzed by polyacrylamide gel electrophoresis in the presence of SDS, and immunoblotting was performed.

 
c-met protein is not found on freshly isolated CD34+/CD45+ cells, but the expression increased in these cells after incubation with 30 ng/ml of SCF for 24 h (Fig. 5C: lanes 2, 4).

These results indicate that SCF markedly induces the precursor and ß chain of c-met receptors on CD34+/CD45+ cells at the protein level.

Induction of c-met on CD34+/CD45+ Cells Treated with Various Growth Factors
We next examined the stimulatory effect of various cytokines other than SCF on the expression of c-met in CD34+/CD45+ cells. Flow cytometry analyses revealed that the addition of IL-3 or SCF induces the expression of c-met on CD34+/CD45+ cells, but G-CSF and EPO do not (Fig. 6A). As shown in Figure 6B, both p170 met and p145 met were strongly induced by IL-3 and SCF, respectively, but only weakly by IL-6 and G-CSF. In separate experiments, the other growth factors (Flt-3 ligand, IL-11, and GM-CSF) did not induce the expression of c-met (data not shown). A similar result was obtained when the CD34+/CD45+ cells were cultured in the serum-free medium.



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  		Figure 6. Ability of various cytokines to induce c-met protein on CD34+/CD45+ cells. Sorted CD34+/CD45+ cells (1 x 105) were incubated with the various growth factors for 24 h. A) HGF receptor (c-met) was detected by flow cytometry. B) The expression of c-met precursor (170 kDa) and c-met ß-chain (145 kDa) was detected. Lane 1: medium alone; lane 2: SCF (30 ng/ml); lane 3: IL-3 (10 ng/ml); lane 4: IL-6 (10 ng/ml); lane 5: G-CSF (10 ng/ml), and lane 6: A431 cells.

 
These results suggest that both SCF and IL-3 are more effective inducers than other factors.

HGF Accelerates Induction of CD36+ Cells
It has previously been shown that CD36 is a much earlier erythroid marker than glycophorin A and that the expression of CD36 increases in spite of CD34 disappearance according to erythroid commitment [25, 26].

Sorted CD34+/CD45+ cells were incubated with SCF + EPO in the presence or absence of HGF for seven days. As the CD36 antigen is also expressed on monocytic lineage cells, the CD36 expression was analysed on the gated CD11b– fraction (Fig. 7). The addition of HGF increased the relative number of CD36+ cells. As shown in Table 1, about 66% of CD11b– cells were CD36+ in the cells cultured with HGF, whereas about 50% were CD36+ cells without HGF. No increase in the glycophorin A antigen was observed by the addition of HGF; no expression of glycophorin A was detected after seven-day culture (mean fluorescence intensity; SCF + EPO, 274.59 versus SCF + EPO + HGF, 245.74 ).



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  		Figure 7. Flow cytometric analysis of CD36 antigen expression on CD11b– cells. Sorted CD34+/ CD45+ cells (1 x 105/well) were incubated with SCF (30 ng/ml) + EPO (2 U/ml) or SCF+EPO+HGF (5 ng/ml). After a seven-day culture, the expression of CD36 antigen was analyzed on CD11b– cells by flow cytometry.

 

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  		Table 1. Flow cytometric analysis of CD36 antigen expression on CD11b– cells
 
These results indicate that HGF enhances the number of erythroid progenitors that are more primitive than glycophorin A+ cells.

Augmentation of EPO Signaling in HGF-Treated CD34+/CD45+ Cells
To investigate whether the preferential differentiation of CD34+/CD45+ cells into erythroid lineage cells is caused by an increase in the expression of EPO-R on the cells, immunoblotting analyses were performed (Fig. 8A). The expression level of EPO-R (62-65 kDa) was compared among the treated cells (lanes 1-3). Lane 4 is leukemia cell line UT-7, which is known to express high levels of EPO-R. The addition of HGF did not increase the expression of EPO-R in comparison with the SCF-treated CD34+/CD45+ cells.



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  		Figure 8. A) Analysis of EPO-R expression. Sorted CD34+/CD45+ cells (1 x 105) were incubated with SCF (30 ng/ml) or SCF+HGF (5 ng/ml) for 24 h. Lane 1: preincubation; lane 2: SCF; lane 3: SCF+HGF, and lane 4: UT-7 cells used as EPO-R positive control. B) Augmentation of EPO signaling in HGF-treated CD34+/CD45+ cells. Sorted CD34+/CD45+ cells (1 x 105) were incubated with SCF (30 ng/ml) alone or SCF + HGF (5 ng/ml) for 24 h. The cells were collected and then stimulated with 60 U/ml EPO for 1, 5, and 30 min at 37°C. The time course of tyrosine phosphorylation in response to EPO was detected by immunoblotting. C) The tyrosine phosphorylation of Stat 5 induced by EPO. Lysates of cells treated with SCF and/or HGF were subjected to immunoprecipitation with anti-Stat 5 Ab and subsequently blotted with 4G10 (upper panel). Reprobing with anti-Stat 5 Ab confirmed equal loading (lower panel). Cellular proteins (10 µg of protein) were analyzed by polyacrylamide gel electrophoresis in the presence of SDS, and immunoblotting was performed.

 
We next examined the EPO signaling to clarify the erythroid lineage-specific stimulatory effect by HGF. The effect of HGF-pretreatment on EPO-induced tyrosine phosphorylation was analyzed. Sorted CD34+/CD45+ cells were incubated in the presence of SCF with or without HGF. After 24 h, the cells were stimulated with EPO, and tyrosine phosphorylation in response to EPO was measured. The pretreatment with HGF markedly enhanced EPO-induced tyrosine phosphorylation (Fig. 8B). To identify the phosphotyrosyl protein, immunoprecipitation with anti-Stat 5 was carried out. As shown in Figure 8C, HGF activated the tyrosine phosphorylation of Stat 5 (92kDa) by EPO-mediated signals. These results demonstrate that HGF in combination with SCF augments the tyrosine phosphorylation of Stat 5 by EPO signaling in CD34+/CD45+ cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HGF is a cytokine with many functions targeting various tissues and organs [1-8]. In the hematopoietic system, HGF plays an important role in physiological [16-18, 20] and pathological states [9-15]. An HGF receptor, c-met is expressed in several human epithelial cells and is often overexpressed in carcinomas. In the hematopoietic tissue, c-met is expressed on a small population of BM cells [27]. Recent reports show that c-met is expressed on CD34+ cells from the human CB [20] and BM. In other reports, no c-met is expressed on human CD34+ cells, but it is expressed on the bone marrow stromal cells in an autocrine mechanism [18]. Thus, the expression of c-met on human hematopoietic CD34+ cells is controversial.

In the present study, we used highly purified hematopoietic progenitor cells bearing both CD34 and CD45 antigens for all the experiments. Because CD45 is known as a marker of leukocyte common antigen [21] and is not expressed on stromal cells [28], it seems likely that the CD34+/CD45+ cells represent pure hematopoietic progenitor cells.

There is the possibility that cell numbers of certain populations with different receptors or surface markers have increased as a result of cytokine stimulation. However, as shown in Figure 6A ,c-met was expressed on one population 24 h after incubation with cytokines. The cell numbers remained unchanged after only a 24 h incubation. In addition, even when the sorted CD34+/CD45+ cells were incubated with SCF, EPO, and/or HGF for seven days, the cells with CD36 (an early erythroid marker) showed morphologically uniform characteristics, as seen in basophilic erythroblasts. We therefore think that the observed changes are not the consequence of cell proliferation generating progeny with differing properties but are due to an induced change within the individual cells.

In the present study, we have detected the expression of both the precursor and ß chain of c-met on the sorted CD34+/CD45+ cells in many samples from the CB and BM using flow cytometry and immunoblotting (Figs.3A and 3B). RT-PCR analyses revealed that all the samples have mRNA of c-met (Fig. 3C), indicating that the CD34+/CD45+ cells have the message of c-met even if the c-met protein is not detected in these cells.

We have also found that the addition of SCF increases the expression of c-met on CD34+/CD45+ cells in a dose-dependent manner (Fig. 5). The expression of p145 met protein, as well as p170 met, was enhanced. The p145 met protein is the ß chain of c-met, which is known to undergo autophosphorylation on tyrosine residue when the receptor is activated [29-31]. It has been reported that SCF is required when HGF acts on hematopoietic cells [16, 20, 32]. This is probably because SCF induces c-met expression on hematopoietic cells.

We next examined the capacity of several growth factors other than SCF to upregulate c-met expression on the CD34+/CD45+ cells. IL-3 induced the c-met expression to a greater extent than IL-6 and G-CSF (Fig. 6), whereas GM-CSF and Flt-3 ligand did not induce it (data not shown). IL-3, like SCF, stimulated tyrosine phosphorylation after HGF stimulation (data not shown), indicating that IL-3 and SCF not only induce c-met expression but also enhance signal transduction from HGF receptors. These findings support the observation that IL-3 acts on colony formation synergistically with HGF [16]. In contrast, neither G-CSF nor IL-6 showed any colony-stimulatory activity in the methylcellulose medium containing HGF. G-CSF also did not increase HGF signaling (data not shown).

CD36 antigen is known as an early erythroid marker [25]. CD36 antigen is also expressed at high levels on platelets, monocytes, some types of endothelial cells, and erythroid lineage cells from BFU-E colonies [25, 26]. Here, we have shown that HGF significantly increases the number of CD36+/CD11b– cells (Fig. 7). These cells were morphologically recognizable as basophilic erythroblasts at seven-day culture (data not shown). These findings indicate that HGF functions in the process of proliferation and differentiation of erythroid progenitors.

How does HGF act on the CD34+/CD45+ cells and induce the proliferation and differentiation into erythroid lineage? There are two possibilities: A) HGF enhances the expression of EPO-R on the CD34+ progenitor cells, since it has been reported that EPO-R expression on the cell membrane of progenitor cells is critical during the erythroid differentiation process [33], and B) HGF augments the signal transduction from EPO-R. In the present study, we have shown that the EPO/EPO-R-mediated signals are enhanced markedly, and Stat 5 plays an essential role in the effect of HGF action on early erythropoiesis, whereas the expression level of EPO-R remains unchanged.

SCF is necessary for the development of committed erythroid progenitor cells until the CFU-E stage [34, 35]. The SCF/c-kit system regulates the differentiation and proliferation of erythroid progenitor cells. Since we have used fresh human samples (instead of cell lines), the expression levels of EPO-R seem to vary among individual samples.

The activation of the EPO-R by the SCF/c-kit system and subsequent interactions of these receptors with intracellular proteins are more important than their increased expression [36]. Because the EPO-R is known to lack the kinase-related sequence, JAK/STAT pathway activation of subsequent EPO stimulation is required for biological activity. In addition to activation of EPO-R by the SCF/c-kit system, HGF acts on the erythroid progenitor cells that have already been committed by SCF, and HGF then augments the tyrosine phosphorylation of Stat 5 by EPO-driven signals (Figs. 8B and 8C). Considering that Stat 5 is important in the erythroid differentiation and proliferation [37, 38], it is conceivable that HGF enhances erythropoiesis mediated by signal transduction through Stat 5.

Many studies have been carried out on signal transduction using cell lines [29-31, 34-38]. As these findings have not always been reflected from HGF-related disease, the analysis of signal transduction using fresh human samples is important. As previously mentioned, increases in serum levels of HGF are reported in many patients with multiple myeloma (MM) [11]. In recent reports, the administration of rHuEPO is effective in severely anemic MM patients showing advanced unresponsiveness to chemotherapy [39, 40]. The present study indicates that HGF can enhance the signal from EPO together with SCF. Therefore, EPO administration may be an effective treatment. Taking these findings into consideration, the elevated HGF levels observed in MM patients may be a physiological response for recovering from the low responsiveness to EPO.

Although the enhanced effect of HGF on erythropoiesis was found even in adult bone marrow cultures, the effects varied among individual samples. Therefore, there was no statistically significant difference. We have no idea what differences exist between CB and BM cells; it is possible that the cell population (CD34+/CD45+ cells) differs between the CB and BM.

In conclusion, A) HGF is a direct activator of human hematopoietic progenitors (CD34+/CD45+ cells), in combination with SCF, and B) the activation of Stat 5 by EPO-driven signaling is one of the events underlying the erythropoiesis-stimulatory effects of HGF. Therefore, it can be concluded that HGF regulates early human hematopoiesis, especially erythropoiesis. The possibility that HGF acts on other lineage hematopoiesis, e.g., megakaryopoiesis, cannot be ruled out. We are in the process of examining whether such a stimulatory effect by HGF is also observed in human erythroid or megakaryocytic progenitor cell lines.


   Acknowledgments
 
This work was supported by grants from the Japanese Ministry of Health and Welfare, Grant-in-Aid for Scientific Research on Priority Areas 029250217, and the Science Research Promotion Fund of the Japan Private School Promotion Foundation, Japan.

The authors wish to thank Dr. N. Komatsu (Jichi Medical School; Tochigi, Japan) for donating UT-7. We also thank Mr. F. Ishida (Research Center of Kansai Medical University) for flow cytometric studies, and Ms. K. Ando for manuscript preparation.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication December 21, 1998.



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