Extract
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Introduction
Notoginseng is a Chinese medicinal herb which belongs to the panax pseudo-ginseng plant of the
Araliaceae family. It has been used in clinics for thousands of years as a treatment for "antithrombosis and vascular diseases". The effective
components of notoginseng are mainly panax notoginosides
(PNS), which contains the notoginsenoside monomer of
R1, Rg1, Rb1,
Rg2, Rh1, and Rd, etc. Recently, investigators have
found some pharmacological actions of PNS on the central
nervous, cardiovascular, and immune
systems[1,2], but little is known about its effects on human
CD34+ hematopoietic stem/progenitor cells. The mature blood cells are derived
from undifferentiated stem cells, progenitor cells, and
precursor cells through a complex series of proliferation,
differentiation, maturation, and apoptosis. The stem cell
compartment is made up of rare primitive cells, which are
multipotential and maintain the capacity to give rise to large
numbers of progenitor cells; they also have a high self-renewal
capacity. The progenitor cell compartment comprises mainly
of cells with the capacity to differentiate along one lineage.
The glucocorticoid receptor (GR) is a member of the
nuclear hormone receptor superfamily of ligand-activated
transcription factors. The receptor molecules consist of three
functional domains: hormone-binding domain, the
DNA-binding domain, and the transactivation region on the
amino-terminal side[3]. GR is expressed in a wide variety types of
cell and tissues, but its expression level is different.
Inactivated GR is bound to a large protein complex and localized in
the cytoplasm. When glucocorticoid (GC) binds to GR, the
protein complex dissociates and the GC/GR complex
translocates to the nucleus where it binds to GC response elements
resulting in the transcriptional upregulation of gene
transcription[4, 5].The influence on the nuclear transcription
factor of the glucocorticoid receptor (GR-NTF) initiated by PNS
has not been reported yet. In this study, the proliferation
effects of PNS, extracted from the notoginseng herb on
CD34+ hematopoietic stem cells, granulocytic, erythrocytic, and
megakaryocytic progenitor cells of human bone marrow, were
observed by colony forming assay of all lineage mixtures,
granulocytic, erythrocytic, and megakaryocytic progenitor
cells (CFU-GEMM, CFU-GM, CFU-E, and CFU-MK) in
semisolid culture. The GR-NTF was detected to recognize the GC
signaling pathway initiated by PNS and to elucidate its
action related with proliferation of the human hematopoietic
cells lines of granulocytic HL-60, erythrocytic K562,
megakaryocytic CHRF-288, and Meg-01 cells.
Materials and methods
Preparation of bone marrow nuclear cells Bone marrow
samples were obtained from human resected ribs during
thoracotomy without hematology disorders, with the
permission of the patients, and approval by The Human Ethics
Committee at The First Affiliated Hospital of Zhejiang
Chinese Medical University (Hangzhou, Zhejiang). The marrow
smear showed a normal hematological cellular profile. The
mononuclear cells were isolated by Ficoll-Paque gradient
centrifugation (specific gravity 1.077 g/mL) from bone marrow,
and were suspended in Iscove's Modified Dulbecco's
medium (IMDM) after being washed 3 times in the medium.
The number of mononuclear cells was counted before plating.
Purification of CD34+ stem/progenitor cells
The method was described previously in our
study[6] and modified in this paper. The
CD34+ cells were isolated from mononuclear cells
of bone marrow using immune beads of Dynal CD34 cell
selection system (Dynal, Oslo, AS, Norway). The
immuno-magnetic beads was washed 3 times and resuspended in 1
mL isolation buffer. The mononuclear cells were then added
to the isolation buffer containing
immunomagnetic beads and shaken gently for 30 min at 4 °C. The
CD34+ cells, conjugated with the
immunomagnetic beads, were obtained by putting them on a magnetic particle concentrator (Dynal,
Norway) after co-culture. For the detachment of the
immunomagnetic beads from the purified
CD34+ cells, 0.1 mL detachment buffer was added to the mixture and incubated
for 15 min at 37 °C in a water bath, and then 2 mL isolation
buffer was added and thoroughly shaken with a vortex.
Finally, the tube was placed on a magnetic particle
concentrator to separate the beads from the
CD34+ cells. The CD34+ cells in the isolation buffer were harvested by centrifugation
and suspended in the medium; some of the cells were stained
with trypan blue for their survival. The purity of the
CD34+ cells was detected as 86%_93% by flow cytometry with the
monoclonal antibody against CD34.
CD34+ cell culture for the CFU-GEMM colony and
cytological identification The semi-solid culture system of
CFU-GEMM was similar to our previous
report[6] and modified in this study, which was composed of 20% fetal bovine serum
(Gibco, Grand Island, NY, USA), 1% bovine serum albumin
(BSA, Sigma, St Louis, MO, USA), 1×10-5
mol/L 2-mercapto-ethanl, 0.3% agar, 10 ng/mL stem cell factor (SCF, Pepro Tech,
Rehovot, Israel), 20 ng/mL interleukin-3 (IL-3, Strathmann,
Hanover, Germany), and 50 ng/mL granulocyte-colony
stimulating factor (G-CSF, Strathmann, Hanover, Germany). The
CD34+ cells, from the bone marrow of 9 people, were
expos-ed to PNS at a final concentration of 0, 10, 25, 50, and 100
mg/L, respectively. The culture system described above
was mixed and plated into 24-well plate in triplicate for each
sample with 5×103 CD34+ cells/ 0.5 mL per well, then
incubated at 37 oC in a humidified atmosphere supplemented with
5% CO2. The number of CFU-GEMM colonies
(³100 cells) was counted under an inverted microscope on d 14 after
initial plating. The CFU-GEMM colonies were made up of 3
lineage blood cells, including granulocytes, erythrocytes,
and megakaryocytes. For the identification of these cells,
granulocytes within the colonies were revealed as black with
peroxidase staining, erythrocytes displayed as red wine
colour with dimethoxybenzidine staining, and human
megakaryocytes were identified with monoclonal antibodies.
Bone marrow culture for CFU-E and CFU-GM colonies
The mononuclear cells from the bone marrow of 10 people
were plated to semi-solid culture. The culture system of
CFU-E was similar to the authors' previous
study[7] which contained 20% fetal bovine serum,
1×10-5 mol/L 2-mercapto-ethanal, 5% phytohemagglutinin-leukocyte conditioned
medium, 2 U/mL recombinant human erythropoietin (EPO,
Amgen, Thousand Oaks, CA, USA), and 0.3% agar of IMDM
medium. The culture system described above was mixed
and plated in triplicate wells for each sample with
105 mononuclear cells/0.5 mL per well, and subsequently incubated at
37 oC for 7 d within a humidified atmosphere supplemented
with 5% CO2. The nuclear erythrocytes within the CFU-E
colony were revealed as red with dimethoxybenzidine
staining in situ[8], which was helpful for determining the
erythro-cytes. The CFU-E (³8 cells) colonies, which consisted of
positive cells, were counted using an inverted microscope.
The culture system of CFU-GM contained 20% fetal bovine
serum, 10 ng/mL recombinant human
granulocyte/macrophage-colony stimulating factor (GM-CSF, Sandoz, São Paulo,
Brazil), and 0.3% agar, which was plated in triplicate with
1×105 bone marrow mononuclear cells/0.5 mL per well. The
culture system was then incubated at 37
oC for 7 d. The colony numbers of CFU-GM
(³40 cells) were scored on an inverted microscope.
Bone marrow culture for the CFU-MK colony and
identification of megakaryocytes The mononuclear cells from
the bone marrow of 10 people were plated to semisolid
culture. The culture system of CFU-MK, set up as described
in another study with modifications[8], was composed of 20%
fetal bovine serum, 1% BSA, 1×10-5
mol/L 2-mercaptoethanl, recombinant human thrombopoietin (rHu TPO, Pepro Tech,
Rehovot, Israel) with final concentration of 30 ng/mL, and
0.8% methylcellulose as viscous support. The semisolid
culture of CFU-MK was performed in triplicate wells for each
sample with 2×105 nuclear cells/0.5 mL per well, and the colony
numbers of CFU-MK (³4 cells) were evaluated on d 14 after
initial planting. The megakaryocytes of the CFU-MK colony
could be identified directly according to their large size, and
well demarcated translucent as well as hyaline cytoplasm
under an inverted microscope of high optical quality. For
the identification of the megakaryocytes, all cells of the
colony were harvested for preparation slides by washing
out methylcellulose on d 14 after plating. The
megakaryocytes on the slides were identified with monoclonal
antibodies against CD41 and
CD42 by streptavidin-alkaline phosphatase (SAP) enzyme conjugation reaction. The positive
cells with red were regarded as megakaryocytes.
Culture of human hematopoietic cell lines
Three lineage cell lines of human granulocytic HL-60, erythrocytic
K562, megakaryocytic CHRF-288, and Meg-01 cells were
incubated in IMDM supplemented with 10% new-born
bovine serum without any growth factors. CHRF-288 and
Meg-01 cells were generously gifted from Prof
Beng Hock CHONG (St George Hospital, Sydney, Australia) who
purchased the cells from ATCC (Manassas, VA, USA).
PNS stimulating test for hematopoietic progenitor cells
PNS was extracted and purified from the notoginseng herb
by Sanxi Zhengkang Pharmaceutical Company (Xi'an, China),
which dissolves drastically in water. It is a clinical treatment
drug, administered by muscle or vein injection, and contains
50 mg/mL notoginosides per vial (certification
No ZZ-5599-1995-000806). The suitable dilution of PNS was added to the
semisolid culture system of CFU-GEMM, CFU-E, CFU-GM,
and CFU-MK, with a final concentration of 0, 10, 25, 50, and
100 mg/L, respectively. Each group of PNS concentration
was performed in triplicate wells for every sample. For the
human cell lines, HL-60, K562, CHRF-288, and Meg-01 cells
were incubated respectively in the presence of PNS with
final concentration of 20 mg/ L for 14 d. Before harvest, all
cells were starved in IMDM lacking new-born bovine serum
for 18 h at 37 oC, then
1×106 cells/ mL was incubated in
medium with 50 mg/ L PNS or 1×10-7
mol/L of dexamethasone (Dex) for 2 h as the PNS-treated group or Dex-positive control,
respectively, and no-PNS treated cells duration of culture
were used as a negative control.
Preparation of nuclear extracts The nuclear extracts
were similarly prepared to those of our previous
study[9,10]. After washing with ice-cold phosphate-buffered saline, the
cells were resuspended in ice-cold hypotonic buffer A
[10 mmol/L Hepes (pH 7.9), 10 mmol/ L KCl, 1 mmol/L
EDTA, 1 mmol/L dithiothreitol (DTT), 0.5 mmol/L
phenymethyl-suphonyl fluoride (PMSF), 10 mg/ mL aprotinin, leupeptin,
antipain, and pepstatin (Sigma, St Louis, MO, USA)] for 15
min. Subsequently, 0.6% Nonidet P-40 was added, and the
sample was vortexed for 10 s. Nuclei were separated from
the cytosol by centrifugation at 13 000 r/min for 15 min and
resuspended in hypotonic buffer C (20 mmol/ L Hepes, 25%
glycerol, 0.4 mmol/ L NaCl, 1 mmol/ L EDTA, 1 mmol/ L DTT,
0.5 mmol/ L PMSF, and 10mg/ mLleupeptin, antipain, aprotein,
and pepstatin) and briefly sonicated on ice. Nuclear extracts
were obtained by centrifugation at 13 000 r/min for 20 min at
4 oC. The supernatant fluid was the nuclear extracts which
were measured by the Bradford method using the protein
dye reagent (Bio Rad, Hercules, CA, USA) for the protein
concentration of each sample.
Western blot analysis Western blotting was performed
as our previous study[10,11]. 10 μg nuclear protein was
loaded,with an equal volume of 2×electrophoresis sample buffer,
and separated by SDS-PAGE with 7.5% acrylamide. The
proteins were transferred to a nitrocellulose membrane
(Amer-sham, Buckinghamshire, UK) using an electroblotting
apparatus (Bio Rad, Hercules, CA, USA). The membranes
were submerged in a blocking buffer containing 1.0%
bovine serum albumin in TBS solution (150 mmol/ L NaCl, 50
mmol/L Tris, pH 7.5) for 1 h at room temperature
Sub-sequently, the membranes were incubated in primary
antibody (Santa Cruz, CA, USA) at a dilution of 1:1000 against
the amino terminus (E-20) or carboxyl terminus (P-20) of
GR-NTF for an additional 1 h at room temperature. After washed
3 times, the membranes were incubated with anti-rabbit
horseradish peroxidase-conjugated secondary antibody (Santa
Cruz, CA, USA) at a dilution of 1:2000 for 45 min at room
temperature. After washed again, the membranes were
visualized. The special bands from the conjugation reaction
of the protein antigen and antibody were visualized by the
ECL kit (Santa Cruz, CA, USA), and the density of the bands
was analyzed after scanning image on X film (Kodak,
Shanghai, China). The experiment was repeated 3 times.
Electrophoretic mobility shift assay
(EMSA) EMSA was performed as our previous
study[9,11]. For the binding reaction, 10
mg nuclear extracts from each sample were incubated in a 25 µL total reaction volume containing 20 mmol/ L
Hepes (pH 7.9), 50 mmol/ L NaCl, 0.1 mmol/ L EDTA, 1
mmol/ L DTT, 5% glycerol, 200 mg/mL BSA, and 2.5 mg poly
(dI/dC) (Boehringer Ingelheim, Germany) for 15 min at 4
oC. The probe used in this study was the double-stranded
nuclear transcription factor of the GR (Santa Cruz, CA, USA)
consensus oligonucleotide containing the binding site for
GR-NTF: 5'-AGA GGA TCT GTA CAG GAT GTT CTA GAT-3' GR-NTF consensus ds-oligonucleotide was radiolabeled with
the polynucleotide kinase (Boehringer Ingelheim, Ingelheim,
Germany) and [α-32P]ATP (Amersham, Buckinghamshire,
England). 50 000 cpm GR-NTF oligonucleotide was added to
the reaction mixture and incubated for 30 min at room
temperature. The reaction products were analyzed by
electrophoresis in 6% polyacrylamide gel with 0.25-fold TBE
running buffer (22.3 mmol/ L Tris, 22.2 mmol/ L borate, and 0.5
mmol/ L EDTA). The gel was dried and the complexes of
GR-NTF with DNA were visualized by autoradiography. The
experiment was repeated 3 times.
Statistical analysis Triplicate experimental results were
pooled and expressed as mean±SD. Student's
t-test was used to determine statistical difference between the
experimental groups and control groups. P<0.01 or
P<0.05 were considered statistically significant.
Results
Effects of PNS on the proliferation of
CD34+ cells The CFU-GEMM colonies, derived from the culture of
CD34+ cells, were made up of 3 lineage blood cells, including granulocytes,
erythrocytes, and megakaryocytes. The colony growth
pattern was seen (³100 cells) in vitro as shown in Figure 1A.
Table 1 shows a control of non-PNS treatment; CFU-GEMM
colony numbers were 47.8±11.1/5×103
CD34+ cells on d 14 after initial planting, while the
CD34+ cells from the 9 samples were exposed to PNS at the concentrations of 10, 25, and 50
mg/L, the frequencies of CFU-GEMM colony were
significantly more than those of the non-PNS control
(P<0.01 and 0.05). The highest colony formation was noticed at 25 mg/ L
PNS with an increasing rate of 34.7±16.0%
(P<0.01). The results indicated that PNS could enhance the proliferation of
CD34+ hematopoietic stem cells to increase the formation of
CFU-GEMM colony in vitro.
Effects of PNS on the proliferation of CFU-GM and
CFU-E The results of CFU-GM and CFU-E colony assay in the
semisolid culture system from 10 samples exposed to PNS at
concentrations of 0, 10, 25, 50, and 100 mg/L on d 7 after
initial planting. The colony formation of CFU-GM was
49.4±4.2/1×105 mononuclear cells in the non-PNS control,
while the frequencies of CFU-GM colony in 3 groups of PNS
10, 25, and 50 mg/ L were significantly more than those of the
no-PNS control, respectively (P<0.01), with an increasing
rate of 22.5%, 37.4%, and 39.3% over the non-PNS control,
respectively. Meanwhile, CFU-E colony (shown as Figure
1B with Wright staining) numbers from bone marrow culture
were 35.7±3.0/1×105 mononuclear cells in the non-PNS
control, while in the presence of PNS (20 and 50 mg/ L), the
colony numbers of CFU-E were significantly more than those
of the non-PNS control (P<0.01), respectively. These
results indicated that PNS could enhance the proliferation of
human granulocytic and erythrocytic progenitor cells in a
dose-dependent manner to increase the formation of
CFU-GM and CFU-E colonies in vitro.
Effects of PNS on the proliferation of CFU-MK
The results of CFU-MK colony assay in the semi-solid culture
system from the 10 samples exposed to PNS at
concentrations of 0, 10, 25, 50, and 100 mg/ L on d 14 after initial
planting. Megakaryocytic colonies could be divided into 2
kinds according to their growth pattern under the inverted
microscope: loose aggregates of 3_10 cells without
additional hematopoietic cell lineages present, and
megakaryocytes within mixed colonies which contained the other
lineages of erythrocytic and granulocytic cells. The cells in red
were regarded as megakaryocytes labeled with monoclonal
antibody against CD41 or
CD42 by SAP enzyme conjugation reaction (shown as Figure 1C with
CD41 labeling). Table 2 shows that
the CFU-MK colony numbers from 10 samples were
42.8±5.2/1×105 mononuclear cells in the non-PNS
control, while in response to PNS (25 and 50 mg/ L), the
colony numbers of CFU-MK were significantly more than
those of the non-PNS control (P<0.01), respectively. The
results suggest that PNS could enhance the proliferation of
megakaryocytic progenitor cells to increase the formation of
CFU-MK colony in vitro.
Upregulated expression of the GR-NTF protein induced
by PNS The expression level of the GR-NTF protein was
determined by Western blotting in 4 cell lines of HL-60, K562,
CHRF-288, and Meg-01 after treated with PNS, respectively
(Figure 2). By using the antibodies against the amino
terminus and carboxyl terminus, respectively, the specific bands
of 95 kDa that appeared were derived from the conjugation
reaction of the GR-NTF protein with the antibodies againstthe
amino terminus and carboxyl terminus, respectively. The
band density of the GR-NTF protein (amino terminus)
initiated by PNS was 2.6-, 2.8-, and 2.4-fold (Figure 2, lanes 4, 6,
and 8), respectively higher than those of the non-PNS
control (Figure 2, lanes 3, 5, and 7) in 3 cell lines of K562,
CHRF-288, and Meg-01. The band density of the GR-NTF protein
(carboxyl terminus) initiated by PNS was 1.3-, 3.9-, and
2.7-fold (Figure 2, lanes 4, 6, and 8), respectively, higher than
those of the non-PNS control in 3 cell lines of K562,
CHRF-288, and Meg-01. However, there was no obvious difference
of the expression level of the GR-NTF protein (both the amino
terminus and carboxyl terminus) between the PNS-treated
and untreated HL-60 cells. Meanwhile, the positive control
of Dex also raised the expression of the GR-NTF protein
(both the amino terminus and carboxyl terminus) compared
with the untreated K562 and CHRF-288 cells. The results in
Figure 2 indicate that PNS can increase the expression level
of the GR-NFT protein in K562, CHRF-288, and Meg-01cells,
but not in HL-60 cell. Since Dex was used for the positive
control only, and the effect of PNS in K562 and CHRF-288
cells was more obvious than those in HL-60 and Meg-01
cells, we selected 2 target cells to study the expression level
of the GR-NTF protein induced by Dex. The results
suggested that Dex also could increase the expression level of
the GR-NFT protein in K562 and CHRF-288 cells.
Upregulation of GR-NTF binding activity induced by PNS
Evidence from the colony assay of CFU-GEMM, CFU-GM,
CFU-E, CFU-MK, and GR-NTF protein expression by
Western blotting supported the hypothesis that GR-NTF may be
involved in the effects of PNS on the proliferation in
hematopoietic cells. To confirm this possibility, EMSA was
performed with a 32P-labelled GR-NTF consensus
oligonucleotide(Figure 3). The binding complex of the GR-NTF protein with
DNA revealed a single major band in the nuclei of cells. This
complex band was competitively abolished by 500-fold
excess of unradiolabeled GR-NTF consensus oligonucleotide
(data not shown). The GR-NTF binding activity initiated by
PNS was apparently elevated to form higher density bands
(the complex of GR-NTF and DNA) in the nuclei of K562 and
CHRF-288 cells (Figure 3, lanes 4 and 6). There was little
binding activity of GR-NTF, which appeared as a shallow
band in the nuclei of HL-60 cells treated with PNS, although
there was no detectable band in the untreated cells. The
binding activity of GR-NTF was no obvious difference
between the PNS-treated and untreated Meg-01 cells (Figure
3). The results in Figure 3 indicate that PNS could induce the
upregulation of GR-NFT binding activity in K562 and
CHRF-288 cells, to a lesser extent in HL-60, but not in Meg-01 cells.
Meanwhile, the positive control of Dex also could induce
the upregulation of GR-NFT binding activity in K562 and
CHRF-288 cells.
Discussion
GC have been shown to enhance the formation of mouse
erythroid colonies in vitro[12]. In the presence of GC, lower
concentrations of erythropoietin were required to induce
maximal proliferation of erythroid progenitor cells
in vivo, GC can restore normal erythropoiesis in pediatric aplastic
anemia[13]. Lindern et al reported that the addition of the GR
ligand Dex to EPO and SCF allowed the establishment of
mass cultures of normal erythroid progenitors from
mononuclear cells of human umbilical cord blood, bone marrow,
and peripheral blood[14]. Erythroid progenitor cells could be
induced to terminal erythroid differentiation upon the
removal of SCF and Dex. Furthermore, GR-NTF is a key
regulator of the decision between self-renewed and
differentiation in erythroid progenitor
cells[15].
The hematopoietic stem cells are characterized by the
expression of the CD34 antigen, which distinguishes them
from other immature cell types. In the early stage of
hematopoietic cells, the expression of the CD34 antigen is at its
highest level, and the CD34 antigen gradually decreases and
finally disappears along with cell differentiation and
maturation[16]. Therefore,
CD34+ hematopoietic stem cells are the best target cells for the investigation of hematopoiesis
regulation, and they are applied for stem cell transplantation
and gene therapy. Traditionally, transplantation of
hematopoietic stem cell is mainly used for the treatment of
malignant blood disorders. Recent advances have made the
transplantation of CD34+ hematopoietic stem cells a hot research
field, which will provide new therapeutic approaches to the
treatment of solid tumors, immunological disorders, and
heredity diseases. In this study, we obtained
CD34+ cells with high purity from human bone marrow by immunomagnetic
beads. The amount of CD34+ cells was enough for the
semisolid culture to observe the proliferation effects of PNS on
human CD34+ cells.
Wang et al reported that PNS promoted the proliferation
of granulocytic progenitors in both normal and aplastic
anemia mice[17]. The supernatant prepared from the culture of
mice spleen cells and fibroblastic cells after PNS treatment
revealed stimulating activity on hematopoietic progenitor
cells. It was suggested that PNS might induce fibroblastic
cells and lymphocytes to secrete various kinds of cytokines
for hematopoiesis[18]. The report also indicated that PNS
induced differentiation of the leukemia cell line K562 into
granulocytic lineage cells. Other investigators found that
PNS was effective in preventing 60Co-irradiated mice from
hematopoiesis suppression through an increasing quantity
of hemoglobin and number of granulocytes and enhancing
the proliferation of progenitors in radiation
mice[19]. We have reported the effects of PNS on hematopoiesis through a
series of studies, including the mouse model with aplastic
anemia, the proliferation test of hematopoietic cells, and
intracellular transcription regulation. It was indicated that
PNS could increase the number of peripheral white blood
cells, improve hematopoiesis function of bone marrow, and
promote the proliferation of hematopoietic progenitors of
CFU-GM and CFU-E in mice with immune-mediated aplastic
anemia when bone marrow was
suppressed[20]. Also, PNS could induce
CD34+ hematopoietic stem cell differentiation
committed towards granulocytes by exposure to PNS at
optimal concentrations in the suspension culture. The
percentage of the granulocyte-specific marker
CD33+ and CD15+ cells were much higher than those of the no-PNS control by
analysis of flow cytometry[21]. Also, we observed the
differentiation effects of PNS on the blastic cell lines above with
flow cytometry analysis; the results showed no obvious
differentiation phenomena when treated with PNS, which may
be explained by their abnormal heterogeneity, incapable of
differentiation, which is different from normal hematopoietic
progenitor cells. In thisstudy, we observed that PNS could
not only promote the proliferation of
CD34+ cells and significantly raise the colony numbers of
CFU-GEMM in vitro, but also enhance the proliferation of granulocytic, erythrocytic,
and megakaryocytic progenitors of bone marrow to increase
CFU-GM, CFU-E, and CFU-MK colony formation in
vitro. Our results suggest that PNS might act as a growth factor or
synergistic efficacy with growth factors, such as SCF, EPO,
GM-CSF, and IL-3 in the proliferation of hematopoietic
stem/progenitor cells. It may provide useful evidence for the
possible application of PNS in treating blood diseases in the
future.
In order to explore the intracellular signal pathway
correlated with proliferation and differentiation induced by PNS
in hematopoietic cells, we observed the regulation of GATA
family transcription factors, GATA-1 and GATA-2, AP-1
family transcription factors NF-E2, c-Jun, c-Fos, and
NF-kB family transcription factors, c-Rel and NF-kB proteins, by
Western blotting. The majority of these transcription factors were
upregulated at distinct degrees in response to PNS, although
displaying different susceptibility to PNS among 4 cell lines
of HL-60, K562, CHRF-288, and Meg-01
cells[22,23]. EMSA results indicated that the DNA binding activity of GATA and
AP-1 initiated by PNS was apparently elevated. The immune
precipitation showed that both GATA-1 and GATA-2
proteins were at a phosphorylated
status[20,21]. It suggested that PNS might play a role in the upregulation of gene
expression related to proliferation and differentiation in
hematopoietic cells, through increasing synthesis, DNA
binding activity of multiple transcription factors, and their
phosphorylated status.
In this study, the nuclear transcription factor of GR was
detected to elucidate whether PNS was involved in the
signaling pathway similar to GC in relation to the proliferation
of hematopoietic cells. Western blotting showed that
GR-NTF protein levels of either the amino or carboxyl terminus
in 3 cell lines of K562, CHRF-288, and Meg-01 treated with
PNS increased by 2.4_2.8-fold and 1.3_3.9-fold over the
untreated cells, respectively. Meanwhile, the positive
control of Dex also elevated the expression of the GR-NTF
protein. GR-NTF binding activity initiated by PNS or Dex
apparently elevated to form higher density bands (the
complex of GR-NTF and DNA) in K562 and CHRF-288 cells. The
little binding activity of GR-NTF appeared as a shallow band
in HL-60 cells treated with PNS, although there was no
detectable band in the untreated cells. These data suggest
that the intracellular signal pathway of PNS is involved in
GR-NTF, which might play a role in the upregulation of gene
expression, correlated with the proliferation in
hematopoietic cells by increasing its synthesis and DNA binding activity.
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