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INTRODUCTION |
Hematopoiesis in vertebrates, which is a complicated multistep
process, is a paradigm for the development of diverse specialized cell
types from multipotent progenitors. How specific signaling pathways
control hematopoiesis is a major focus in the area of hematopoiesis. A
widely used model system for studying early steps in megakaryocytic
differentiation of hematopoietic stem cells is
PMA1-induced differentiation
of K562 cells (1). It has been well documented that K562 cells, human
chronic myelogenous leukemic cells carrying Philadelphia chromosome
(2), can be induced by PMA, a protein kinase C activator, to
differentiate into cells with megakaryocytic characteristics (3-6).
These include changes in cell morphology and adhesive properties, cell
growth arrest, and expression of markers associated with the megakaryocytes.
Despite recent advances in studying the molecular events associated
with PMA-induced megakaryocytic differentiation of K562 cells, the
exact signaling mechanism is not fully understood. We have previously
shown that PMA-induced differentiation of K562 cells is inhibited by
the treatment with pyrrolidine dithiocarbamate, an inhibitor of NF-
B
(7), and NF-
B subunit-transfected cells are more sensitive to
PMA-induced differentiation than their parental cells, and PMA-induced
differentiation was enhanced by the pretreatment with I
B
antisense oligonucleotide (8). In contrast, it has been reported
recently that overexpression of the constitutively active mutants of
MEK induce megakaryocytic differentiation in K562 cells, and blockade
of MEK activation by PD98059 reverses both the growth arrest and the
morphological changes of K562 cells induced by PMA treatment (9-11).
This suggested that a sustained activation of the ERK/MAPK pathway is
necessary to induce a differentiation program along the megakaryocyte
lineage in K562 cells.
In our previous study (8), although there were no differences in the
basal or PMA-stimulated activities of ERK/MAPK between the parental and
NF-
B subunit-transfected K562 cells, PMA-induced differentiation was
almost completely inhibited in both parental and NF-
B
subunit-transfected cells after preventing PMA-stimulated activation of
MAP kinase by the pretreatment of PD98059. Therefore, it is likely that
the MAP kinase pathway is required for the PMA-induced megakaryocytic
differentiation of K562 cells, suggesting that activation of MAP kinase
works through NF-
B activation, or activation of both NF-
B and MAP
kinase pathways are involved.
Two recent reports have shown that the 90-kDa ribosomal S6 kinase
(RSK1) is an essential kinase required for phosphorylation and
subsequent degradation of I
B
in response to mitogens, including PMA (12, 13). Therefore, RSK1 is a good candidate to connect the
ERK/MAPK pathway and the NF-
B pathway in PMA-induced megakaryocytic differentiation of K562 cells. In this study, the role of RSK1 in
PMA-induced megakaryocytic differentiation of K562 cells was investigated.
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MATERIALS AND METHODS |
Cell Culture--
K562 cells were grown in suspension in RPMI
1640 medium (Life Technologies, Inc.) supplemented with 10% (v/v)
heat-inactivated fetal bovine serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 µg/ml streptomycin (Sigma). For the
experiment, exponentially growing K562 cells were collected (1,200 × g, 10 min) and resuspended in fresh culture medium.
Ribosomal S6 Kinase Assay--
The S6 kinase assays were
performed using S6 kinase Assay Kit from Upstate biotechnology,
Inc., according to manufacturer's protocol. Cells (1 × 106/ml) were harvested, washed twice in ice-cold PBS, and
then scraped into 150 µl of lysis buffer (20 mM Tris, pH
7.4, 150 mM NaCl, 5 mM EDTA, 1 mM
EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
-glycerophosphate, 1 mM
Na3Vo4, 1 µg/ml leupeptin, and 1 mM PMSF). After incubation for 30 min at 4 °C, lysates
were centrifuged at 20,000 × g for 20 min at 4 °C.
Cell lysates (200 µg of protein) were mixed with 2 µg of
anti-p90RsK1 antibody (rabbit polyclonal IgG) and then
immunoprecipitated with gentle rocking overnight at 4 °C. The
immunocomplexes were collected by protein A-Sepharose beads for 3 h at 4 °C.
For in vitro kinase assay, the immunoprecipitates/protein
A-Sepharose beads were washed once with ice-cold cell lysis buffer and
three times with an assay dilution buffer (ADB: 20 mM MOPS, pH 7.2, 25 mM
-glycerol phosphate, 5 mM
EGTA, 1 mM sodium orthovanadate, and 1 mM
dithiothreitol). The immunoprecipitates/protein A-Sepharose beads were
then resuspended in ADB containing substrate mixture (50 µM substrate peptide), inhibitor mixture (4 µM protein kinase C inhibitor peptide, 0.4 µM protein kinase A inhibitor peptide, and 4 µM Compound 24571 in ADB), and [
-32P]ATP
mixture. The reaction mixture was incubated for 10 min at 30 °C, and
then 20 µl of the reaction mixture was spotted onto p81
phosphocellulose squares (1.5 × 1.5 cm, Upstate biotechnology, Inc.). The papers were washed five times for 5 min each with 0.75% phosphoric acid and once for 3 min with acetone. Dried papers were
wrapped, and radioactivity was detected with a Molecular Imager
(GS-525, Bio-Rad).
Plasmids and DNA Transfection--
pcDNA3-Rsk1,
pcDNA3-Rsk1- D205N, and pCMV-I
B
-32A36A (kindly donated by
Dr. Zantema, Leiden University, Netherlands) have been described
elsewhere (13). pcDNA3-Rsk1-D205N has a mutated ATP binding
site of the amino-terminal kinase domain of RSK1, and
pCMV-I
B
-S32AS36A contains mutated alanines at the serine phosphorylation sites. pCMV-p50 and -p65 are expression plasmids in
which the p50 and p65 subunits of NF-
B were cloned in
HindIII/XbaI of pRc/CMV (Invitrogen),
respectively (8). Electroporation was carried out using an
Electroporator II (Invitrogen) at 1,500 V/cm, 50 microfarads
condition. After electroporation, stable transformants were selected
with G418 (475 µg/ml) and identified by Western blot analysis using
polyclonal antibody against RSK1 (Upstate biotechnology, Inc.) and
I
B
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assays--
Nuclear extracts were prepared as described
previously (8). 2 × 106 cells were harvested and
washed with cold phosphate-buffered saline and resuspended in 50 µl
of lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol, and 0.5 mM PMSF). The cells were allowed to
be swollen on ice for 10 min, after which the cells were resuspended in
30 µl of lysis buffer containing 0.05% Nonidet P-40. Then the tube
was vigorously mixed on a vortex machine three times for 10 s, and
the homogenate was centrifuged at 250 × g for 10 min
to pellet the nuclei. The nuclear pellet was resuspended in 40 µl of
ice-cold nuclear extraction buffer (5 mM HEPES, pH 7.9, 26% glycerol (v/v), 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM PMSF), incubated on ice for 30 min with intermittent
mixing, and centrifuged at 24,000 × g for 20 min at
4 °C. The nuclear extract was either used immediately or stored at
70 °C for later use.
Electrophoretic mobility shift assay was performed by incubating 10 µg of nuclear extract for 20 min at room temperature with 17.5 fmol
of 32P-end labeled 22-mer double-stranded NF-
B
oligonucleotide (5'-AGT TGA GGG GAT TTT CCC AGG C-3') from the
light chain enhancer (15) according to the manufacturer's manual
(Promega, Co.). For gel mobility supershift assay, 1 µg of anti-p50
antibody was incubated with whole cell extracts for 20 min at 21 °C
prior to NF-
B binding reaction.
Nonspecific Esterase Staining--
The cytocentrifuged
slides were stained with a nonspecific esterase staining kit (Muto Pure
Chemicals, Ltd., Tokyo, Japan) according to manufacturer's
protocol. Nonspecific esterase-stained slides were examined
microscopically for intracellular dark red granules.
Kinase Assays for MAP Kinase/ERK Activity--
MAP kinase
activity was measured with a p44/42 MAP Kinase Assay Kit (New
England Biolabs, Inc.) according to manufacturer's protocol. Cells
were lysed in buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
-glycerophosphate, 1 mM Na3VO4,
1 µg/ml leupeptin, and 1 mM PMSF for 15 min at 4 °C.
200 µl of cell lysate (~200 µg of protein) were mixed with
monoclonal phospho-MAPK antibody (1:50 dilution) and incubated with
gentle rocking for overnight at 4 °C. Immunoprecipitates were
collected by protein A-Sepharose beads (10-20 µl) for 2 h at
4 °C. Beads were washed twice with cold lysis buffer and twice with
500 µl of kinase buffer (20 mM Tris, pH 7.5, 5 mM
-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM MgCl2). Kinase assay was performed by incubating the suspended pellet with kinase buffer containing 100 µM ATP and GST-Elk1 fusion protein for 30 min at
30 °C. The samples were analyzed by 12% SDS-polyacrylamide gel
electrophoresis. Phospho(Ser383)-Elk1 was detected with
specific antibody using Western blot analysis.
Flow Cytometric Analysis--
Cells were harvested after
treatment with PMA for 72 h and washed in PBS with 1% bovine
serum albumin (PBS-B). 2-5 × 105 cells were
incubated at 30 °C in 100 µl of PBS-B containing 10 µl
anti-CD41-PE or anti-CD61-FITC (Becton-Dickinson, Mountain View, CA).
Cells were washed in PBS-B, resuspended in PBS with 1% glycerol, and
analyzed with a fluorescence-activated cell sorter using CellQuest 8.6 (Becton-Dickinson, Mountain View, CA).
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RESULTS |
Activation of RSK1 after Treatment of PMA--
As we mentioned
previously, the ribosomal S6 kinase (RSK1) is a good candidate
connecting the ERK/MAPK pathway and the NF-
B pathway, both of which
are known to be involved in PMA-induced megakaryocytic differentiation
of K562 cells. Therefore, we examined whether RSK1 could be activated
by PMA in K562 cells.
To measure the PMA-stimulated RSK1 activity, an immune complex
kinase assay was done. When K562 cells were treated with various concentrations of PMA for 1 h, RSK1 was activated in a
dose-dependent manner up to 10 nM of PMA (Fig.
1A). After treatment with 10 nM PMA for the indicated time (Fig. 1B), a
sustained activation of RSK1 was shown over at least 12 h with
~10-fold activation at 1 h peak time. This result is consistent
with sustained activation of the ERK/MAPK during PMA-induced
megakaryocytic differentiation of K562 cells (11).

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Fig. 1.
Activation of RSK1 after treatment of PMA in
K562 cells. A, cells (1 × 106/ml)
were treated with the indicated doses of PMA for 1 h.
B, cells were treated with 10 nM PMA for the
indicated time. Cells were harvested, and S6 kinase assay was done as
described under "Materials and Methods."
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Isolation of Wild-type or Dominant Inhibitory Mutant of
RSK1-transfected K562 Cells--
To clarify the discrepancy between
our previous study showing that activation of NF-
B mediates the
PMA-induced differentiation of K562 cells (8) and other studies showing
that a sustained activation of the ERK/MAPK pathway is required for
induction of differentiation along the megakaryocyte lineage in K562
cells (9-11), K562 cells were transfected with wild-type RSK1 or a
dominant inhibitory mutant (D205N) of RSK1 and then stable
transformants were isolated. After stimulation of PMA, K562-RSK1 or
K562-RSK1 (D205N) cells have higher or lower RSK1 activity compared
with parental K562 cells, respectively (Fig.
2A). During the
transfection of wild-type RSK1, we observed some adherent cells with a
morphological resemblance to the PMA-stimulated population (Fig.
2B), suggesting that the RSK1-dependent pathway
might be involved in megakaryocytic differentiation of K562 cells.
Practically, these cells, which may be high expressors of RSK1, could
not be expanded due to a severely retarded growth rate. On the other
hand, all isolated RSK1 transfectants showed no or very weak evidence
of differentiation, such as morphological change, nonspecific esterase
activity, or expression of surface markers (see Figs 5 and
7B). Empty vectors did not affect the PMA-induced
megakaryocytic differentiation of K562 cells (data not shown).

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Fig. 2.
Isolation of wild-type or dominant inhibitory
mutant (D205N) of RSK1-transfected K562 cells. A,
comparison of RSK1 activities among parental and wild-type or dominant
negative inhibitory mutant (D205N) of RSK1-transfected K562 cells. Of
the isolated clones, high expressors of each insert were selected, and
their RSK1 activities were compared with that of parental K562 cells
after treatment of PMA for 1 h. B, adhesion and
spreading of K562 cells following transfection with wild-type RSK1.
Cells in the culture dish were visualized by inverted microscopy.
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Effect of Wild-type or Dominant Inhibitory Mutant of RSK1 on
PMA-induced Activation of NF-
B in K562 Cells--
Since we have
suggested that PMA-induced megakaryocytic differentiation of K562 cells
is dependent on the NF-
B pathway (8), a NF-
B mobility shift assay
was done to examine whether NF-
B activity of K562 cells would be
modulated after transfection with wild-type RSK1 or the dominant
inhibitory mutant (D205N) of RSK1. Overexpression of RSK1 or RSK1
(D205N) enhanced or suppressed PMA-stimulated NF-
B activity,
respectively (Fig. 3, upper
panel). Consistently, degradation of I
B
was enhanced or
suppressed by overexpression of RSK1 or RSK1 (D205N) (Fig. 3,
lower panel). These results are consistent with the RSK1
activities of K562-RSK1 and K562-RSK1 (D205N) cells (Fig.
2A), indicating that activity of NF-
B could be modulated
downstream of RSK1 in K562 cells.

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Fig. 3.
Effect of wild-type or dominant inhibitory
mutant of RSK1 on PMA-induced activation of
NF- B in K562 cells. Upper
panel, activities of NF- B after treatment with PMA in the
parental and wild-type or dominant negative inhibitory mutant (D205N)
of RSK1-transfected K562 cells. Nuclear extracts were prepared after
treatment of PMA for 1 h, and 10 µg of nuclear extracts were
used for mobility shift assay. For supershift assay (last
lane), anti-p50 antibody was incubated with nuclear extracts prior
to the NF- B binding reaction. Lower panel, Western blot
analysis of the cytosolic I B level after treatment of PMA.
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The Effect of RSK1 and NF-
B Activities on the PMA-induced
Megakaryocytic Differentiation of K562 Cells--
If RSK1 mediates
PMA-induced megakaryocytic differentiation of K562 cells via NF-
B
activation, inhibition of NF-
B should lead to blocking of
RSK1-mediated NF-
B activation and differentiation of K562 cells
induced by PMA. To address this hypothesis, we isolated a double
transfectant, which expresses both a dominant inhibitory mutant of
I
B
and a wild-type RSK1 as much as each single transfectant (Fig.
4A). K562 and K562-I
B
(S32A/S36A) cells had comparable activities of RSK1, and K562-RSK1 and
K562-RSK1/I
B
(S32A/S36A) cells had comparable activities of RSK1
(Fig. 4B). As shown in Fig. 4C, overexpression of
I
B
(S32A/S36A) inhibited both PMA-stimulated NF-
B activation
and RSK1-mediated NF-
B activation.

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Fig. 4.
Effect of dominant inhibitory mutant
(S32A/S36A) of I B on
PMA-stimulated and RSK1-mediated activation of
NF- B. A, isolation of I B
(S32A/S36A) or RSK1/I B (S32A/S36A)-transfected K562 cells. A
double transfectant, which expressed both a dominant inhibitory mutant
of I B (upper panel) and RSK1 (lower panel)
as much as each single transfectant, was isolated. B,
comparison of RSK1 activities. The parental K562 cells and
transfectants were treated with PMA for 1 h, and the S6 kinase
assay was done as described under "Materials and Methods."
C, effect of dominant inhibitory mutant (S32A/S36A) of
I B on PMA-stimulated and RSK1-mediated activation of NF- B.
NF- B DNA binding activity was assayed as described in the legend of
Fig. 3.
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Then, we have examined the effect of RSK1 and NF-
B activities on
PMA-induced megakaryocytic differentiation of K562 cells. PMA-induced
differentiation of K562 cells was enhanced or suppressed by
overexpression of RSK1 or RSK1 (D205N), respectively, as shown by
morphology (Fig. 5A) and
nonspecific esterase assay (Fig. 5B), indicating that
PMA-induced differentiation of K562 cells could be affected by RSK1
activity. Overexpression of I
B
(S32A/S36A) prevented
morphological change and activation of nonspecific esterase induced by
PMA and also inhibited RSK1-mediated differentiation of K562 cells
(Fig. 5, A and B). In addition, when K562 cells were transfected transiently with both subunits of NF-
B, some cells
underwent morphological changes and were adherent to the culture dish
(Fig. 5C). These results demonstrate that the RSK/NF-
B pathway mediates PMA-induced megakaryocytic differentiation of K562
cells.

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Fig. 5.
Effect of RSK1 and
NF- B activity on the PMA-induced
megakaryocytic differentiation of K562 cells. The parental,
RSK1-transfected, RSK1 (D205N)-transfected, I B
(S32A/S36A)-transfected, or RSK1/I B (S32A/S36A)-transfected K562
cells were cultured in the absence (left panels) or presence
(right panels) of 10 nM PMA. After 3 days cells
in the culture dish were photographed for morphological assay
(A), and cytospin preparations were stained to examine
nonspecific esterase activity (B). C, adhesion
and spreading of K562 cells following transfection with both subunits
of NF- B. After 72 h of transfection, cells in culture dish were
visualized by inverted microscopy.
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Effect of ERK Activity on RSK1 Activation and RSK1-mediated NF-
B
Activation of K562 Cells--
If the sequential ERK/RSK/NF-
B
pathway plays a role in PMA-induced megakaryocytic differentiation of
K562 cells, pretreatment with PD98059, a specific inhibitor of MEK that
prevents the activation of MEK and subsequently the activation of
ERK/MAPK, would lead to prevention of the activation of the
ERK/RSK/NF-
B pathway and subsequent megakaryocytic differentiation
of K562 cells induced by PMA. When PMA-stimulated activation of ERK was
prevented by the pretreatment with PD98059 in K562 and K562-RSK1 cells
(Fig. 6A), PMA-stimulated
activation of RSK1 and NF-
B was also prevented (Fig. 6, B
and C, respectively), indicating that activation of RSK1 and
NF-
B depends on ERK/MAPK activation. In addition, PMA-induced megakaryocytic differentiation was prevented by the pretreatment with
PD98059 in both K562 and K562-RSK1 cells (Fig. 6D). From these results, it could be concluded that PMA-stimulated activation of
RSK1 and NF-
B is ERK-dependent, and sequential
activation of ERK, RSK1, and NF-
B mediates PMA-induced
megakaryocytic differentiation of K562 cells.

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Fig. 6.
Effect of PD98059 on RSK1 and
NF- B activities and PMA-induced megakaryocytic
differentiation in RSK1-transfected K562cells. After pretreatment
of 40 µM PD98059 for 2 h, the parental and
RSK1-transfected cells were treated with PMA for 1 h, and ERK
(A), RSK1 (B), and NF- B (C)
activities were compared. For morphological and nonspecific esterase
activity assays (D), the parental and RSK1-transfected cells
were treated with 10 nM PMA after pretreatment of 40 µM PD98059 for 2 h. After 3 days cells in the
culture dish were photographed for morphological assay (upper
panels), and cytospin preparations were stained to examine
nonspecific esterase activity (lower panels). For untreated
and PMA-treated controls, refer to the legend of Fig. 5.
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Flow Cytometric Analysis of the Effect of Overexpression of RSK1,
Dominant Inhibitory Mutants of RSK1 and I
B
and PD98059 on CD41
Expression--
To evaluate the role of the ERK/RSK/NF-
B pathway in
PMA-induced megakaryocytic differentiation of K562 more specifically, flow cytometric analysis on expression of CD41, a megakaryocytic surface marker, was performed (Fig. 7).
Consistent with the morphology and nonspecific esterase assay,
overexpression of RSK1 enhanced the expression of CD41 induced by PMA
(Fig. 7B), while overexpression of RSK1 (D205N) suppressed
the expression of CD41 (Fig. 7C). Overexpression of I
B
(S32A/S36A) inhibited the expression of CD41 induced by PMA (Fig.
7D) and enhanced by overexpression of RSK1 (Fig.
7E). In addition, PMA-induced CD41 expression was prevented
by the pretreatment with PD98059 in both parental and RSK1-transfected K562 cells (Fig. 7F). Similar results were observed in CD61
expression (data not shown). These results confirmed that PMA-induced
megakaryocytic differentiation is dependent on the ERK/RSK/NF-
B
pathway.

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Fig. 7.
Flow cytometric analysis of the effect of
overexpression of RSK1, dominant inhibitory mutants of RSK1 or
I B , and PD98059 on
CD41 expression. To analyze the effect of overexpression of RSK1,
dominant inhibitory mutants of RSK1 or I B on CD41 expression, the
parental (A), RSK1-transfected (B), RSK1
(D205N)-transfected (C), I B (S32A/S36A)-transfected
(D), or RSK1/I B (S32A/S36A)-transfected (E)
cells were cultured in the absence (thin solid lines) or
presence (thick solid lines) of 10 nM PMA for 3 days. For analysis of the effect of PD98059 on CD41 expression
(F), the parental (thin solid line) and
RSK1-transfected (thick solid line) cells were treated as
described in the legend of Fig. 6. Dashed lines show isotype
controls.
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DISCUSSION |
The signaling mechanism that mediates the PMA-induced
megakaryocytic differentiation of K562 cells is still controversial. We
have proposed that PMA-induced megakaryocytic differentiation of K562
cells is dependent on the NF-
B pathway (8). In contrast, others
(9-11) have suggested recently that a sustained activation of the
ERK/MAPK pathway is required to induce a differentiation program along
the megakaryocyte lineage in K562 cells. In this study, we have
demonstrated that RSK1 plays a role bridging ERK and NF-
B pathways
during the PMA-induced megakaryocytic differentiation of K562 cells.
Since RSK is activated by ERK/MAPK (16), RSK1 activity was assayed
during PMA-induced differentiation of K562 cells. After treatment with
PMA, a sustained activation of RSK1 was observed over at least 12 h with a 1-h peak time after stimulation. It has been shown that a
sustained activation of the ERK/MAPK pathway is required for
megakaryocytic differentiation of K562 cells (11). Since RSK is a
target molecule of ERK/MAPK (17), a sustained activation of ERK/MAPK
would lead to a sustained activation of RSK1.
Previously we demonstrated that the NF-
B pathway is responsible for
PMA-induced differentiation of K562 cells (8), and it has been shown
that RSK1 is a signal-inducible I
B
kinase required for
phosphorylation and subsequent degradation of I
B
, and
consequently activation of NF-
B in response to mitogens, including
PMA (12, 13). Therefore, there is a possibility that activation of RSK1
may lead to activation of NF-
B and subsequent induction of
megakaryocytic differentiation in K562 cells. In our experiment,
activation of NF-
B stimulated by PMA was enhanced or prevented by
overexpression of RSK1 or the dominant inhibitory mutant of RSK1
(D205N), respectively. These results were followed by an increased or
decreased differentiation of K562 cells as demonstrated with
morphology, nonspecific activity, and expression of megakaryocytic
markers, suggesting that RSK1 plays an important role in PMA-induced
megakaryocytic differentiation.
Since RSK was discovered in Xenopus laevis oocytes as an
intracellular kinase activity that phosphorylated the 40 S ribosomal subunit protein S6 (18), diverse substrates have been identified, including transcription factors like the cAMP response element-binding protein (CREB) (19), the estrogen receptor-
(ER
) (20),
I
B
/NF-
B and c-Fos (12, 13), the transcriptional coactivator
proteins CREB-binding protein (CBP) and p300 (21), several proteins in the ribosomal complex (22), glycogen synthase kinase-3 (GSK3) (23), the
neural cell adhesion molecule, L1 CAM (24), the Ras GTP:GDP exchange
factor, Sos (25), and the p34 cdc2 inhibitory kinase Myt1 (26). The
diversity of these substrates suggests that RSK is involved in
regulation of a wide range of cellular functions (27). Therefore, it is
possible that downstream signals other than the NF-
B pathway might
be involved in megakaryocytic differentiation of K562 cells. In the
present study, overexpression of I
B
(S32A/S36A), which led to
inhibition of RSK1-mediated NF-
B activation, prevented the
RSK1-mediated megakaryocytic differentiation of K562 cells, and
transfection of K562 cells with p65 subunit of NF-
B induced
morphological changes and adherence to the culture dish, demonstrating
that RSK1 mediates the PMA-stimulated signal to NF-
B, activation of
which leads to induction of megakaryocytic differentiation of K562 cells.
Several groups (9-11) have demonstrated recently that a sustained
activation of the ERK/MAPK pathway is necessary and sufficient to
induce a differentiation program along the megakaryocyte lineage in
K562 cells, and PD98059 abrogated the differentiation of K562 cells. In
the present study, PD98059 prevented the PMA-stimulated activation of
RSK1 and NF-
B in both parental and RSK1-transfected K562 cells and
subsequently the megakaryocytic differentiation. Therefore, it could be
suggested that sequential activation of the ERK/RSK/NF-
B pathway
after treatment with PMA resulted in induction of megakaryocytic
differentiation of K562 cells. However, we do not know how NF-
B
regulates megakaryocytic differentiation. Several
megakaryocyte-specific promoters, including platelet factor-4 (28),
glycoprotein (GP) IIb (29-32), GPIb
(33), thrombopoietin receptor
(34), and GPIX (35), have been characterized. These promoters usually
contain cis-acting elements regulated by the Sp1 (36), GATA (36-38),
and Ets (14) families of transcription factors. At least some of
these promoters, such as GPIIb and thrombopoietin receptor, have
several putative NF-
B binding site. Therefore, studies of
transcriptional regulation of megakaryocytic markers via NF-
B would
be required to elucidate an exact role of NF-
B in megakaryocytic differentiation.
Previously, we had failed to show induction of megakaryocytic
differentiation of K562 cells after transfection with
Ha-ras, which activated ERK/MAPK ~2.5-fold (7). Although
we do not know the threshold activity of ERK/MAPK enough to trigger
megakaryocytic differentiation of K562 cells, strong initial activation
followed by long lasting activation of ERK/MAPK is thought to be
required. Our failure to demonstrate the induction of differentiation
of K562 cells after transfection of Ha-ras led us to study
the role of NF-
B during the PMA-induced differentiation of K562
cells, and now we have shown that RSK1 bridges the ERK/MAPK pathway and the NF-
B pathway, each of which has been known to be involved in
megakaryocytic differentiation of K562 cells.