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Prepublished online as a Blood First Edition Paper on September 12, 2002; DOI 10.1182/blood-2002-05-1549.
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Blood, 15 January 2003, Vol. 101, No. 2, pp. 498-507
HEMATOPOIESIS
Levels of phospho-Smad2/3 are sensors of the interplay between
effects of TGF- and retinoic acid on monocytic and granulocytic
differentiation of HL-60 cells
Zhouhong Cao,
Kathleen C. Flanders,
Daniel Bertolette,
Lyudmila A. Lyakh,
Jens U. Wurthner,
W. Tony Parks,
John J. Letterio,
Francis W. Ruscetti, and
Anita B. Roberts
From the Laboratory of Cell Regulation and
Carcinogenesis, National Cancer Institute, Bethesda, MD, and the Basic
Research Laboratory, National Cancer Institute (NCI)-Frederick,
Frederick, MD.
 |
Abstract |
We have investigated the role of Smad family proteins, known to be
important cytoplasmic mediators of signals from the transforming growth
factor- (TGF-) receptor serine/threonine kinases, in TGF--dependent differentiation of hematopoietic cells, using as a
model the human promyelocytic leukemia cell line, HL-60. TGF--dependent differentiation of these cells to monocytes, but not
retinoic acid-dependent differentiation to granulocytes, was accompanied by rapid phosphorylation and nuclear translocation of Smad2
and Smad3. Vitamin D3 also induced phosphorylation of Smad2/3 and monocytic differentiation; however the effects were indirect, dependent on its ability to induce expression of TGF-1. Simultaneous treatment of these cells with TGF-1 and
all-trans-retinoic acid (ATRA), which leads to
almost equal numbers of granulocytes and monocytes, significantly
reduced the level of phospho-Smad2/3 and its nuclear accumulation,
compared with that in cells treated with TGF-1 alone. TGF-1 and
ATRA activate P42/44 mitogen-activated protein (MAP)
kinase with nearly identical kinetics, ruling out its involvement in
these effects on Smad phosphorylation. Addition of the
inhibitor-of-protein serine/threonine phosphatases, okadaic acid,
blocks the ATRA-mediated reduction in TGF--induced phospho-Smad2 and shifts the differentiation toward monocytic end points. In HL-60R
mutant cells, which harbor a defective retinoic acid receptor- (RAR-), ATRA is unable to reduce levels of
TGF--induced phospho-Smad2/3, coincident with its inability to
differentiate these cells along granulocytic pathways. Together, these
data suggest a new level of cross-talk between ATRA and TGF-,
whereby a putative RAR--dependent phosphatase activity limits the
levels of phospho-Smad2/3 induced by TGF-, ultimately reducing the
levels of nuclear Smad complexes mediating the TGF--dependent
differentiation of the cells to monocytic end points.
(Blood. 2003;101:498-507)
© 2003 by The American Society of Hematology.
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Introduction |
The growth and differentiation of
hematopoietic cells are regulated by a number of cytokines, in vitro
and in vivo. HL-60, a human promyelocytic cell line, has
been extensively used as an in vitro model for studying the effects of
factors that regulate growth and differentiation of hematopoietic cells
in general, and of myeloid leukemia cells in particular.1
These cells proliferate as promyelocytes, yet retain the capacity to
undergo terminal myeloid or monocytic differentiation in response to
various inducing agents. In the presence of
all-trans-retinoic acid (ATRA), HL-60 cells undergo
differentiation to granulocytes, whereas
1,25(OH)2-vitamin D3 (Vit D3)
and phorbol ester induce differentiation into
monocytes/macrophages.2 Transforming growth factor-
(TGF-), known to be a negative regulator of growth at all stages of
hematopoiesis,3,4 induces differentiation of HL-60 cells
to promonocytes, and has been shown to act synergistically with Vit
D3, tumor necrosis factor (TNF), or the combination of ATRA
plus TNF to induce monocytic differentiation of several other myeloid
leukemic cell lines.6,7 Other studies showing that induction of terminal differentiation by retinoids and Vit
D3 requires TGF-1 as an autocrine mediator suggest that
endogenous TGF-1 plays a critical role in the differentiation of
leukemia cells.8-10
TGF- binds to a heteromeric cell-surface complex consisting of 2 type II and 2 type I transmembrane-receptor serine/threonine kinases in
which ligand binding induces phosphorylation and activation of the type
I receptors by the type II receptor kinases.11,12 Signaling is mediated in part by Smad proteins, which are activated directly by the TGF- type I receptor kinase by phosphorylation on
their C-terminus and then translocate to the nucleus in complex with
the common mediator Smad4 to regulate transcription of target genes.
The principal receptor-activated Smads involved in signaling from
TGF- receptors are Smad2 and Smad3. In the nucleus, Smad proteins
can bind directly to their cognate DNA-binding sites and/or interact
with an increasing number of transcription factors, transcriptional
coactivators, or transcriptional repressors.13,14 The
TGF-1 response can also be regulated by Smad6 and Smad7, which
inhibit the TGF--induced activation of Smad2 and
Smad315,16 and which target the receptor complex for
proteasomal degradation.17 The complexity of the TGF-
responses and their dependence on the physiological context and cell
type may relate to the relative levels of different Smad proteins and
their cooperativity with specific transcription factors, which
themselves may be regulated by different signaling
pathways.18 Ras, extracellular signal-regulated kinase
1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), or p38 mitogen-activated protein kinase (MAPK) may also be regulated by TGF-
in different cell lines.19 Yet other studies show the
involvement of S6 kinase in inhibition of growth20,21 and
phosphatidylinositol 3 (PI-3) kinase in
epithelial-to-mesenchymal transdifferentiation mediated by
TGF-.22
In HL-60 cells, Vit D3 and ATRA have each been shown to
stimulate MAP/ERK kinase (MEK)-dependent activation of
ERK2,23,24 causing subsequent hypophosphorylation of p53
and retinoblastoma protein (pRb), cell differentiation, and
G0 arrest. The MAPK inhibitor PD98059 has been shown to
block ATRA-induced granulocytic differentiation.24 In this
study, we have investigated the role of Smad proteins in
TGF-1-induced monocytic differentiation in HL-60 cells and in the
interplay between MAPK pathways and Smad-signaling pathways. We have
found that whereas ATRA, Vit D3, or TGF-1 each activates ERK1/2 in HL-60 cells, only TGF-1 or Vit D3, each of
which results in monocytic differentiation, results in phosphorylation
of Smad2 and Smad3. Moreover, in cells treated simultaneously with
TGF-1 and ATRA, leading to differentiation of cells along both
granulocytic and monocytic pathways, ATRA decreases the levels of
phosphorylated Smad2/3, possibly by retinoic acid receptor-
(RAR-)-dependent activation of a putative phosphatase
activity, as suggested by experiments using the inhibitor-of-protein
serine/threonine phosphatases, okadaic acid. Together, our data suggest
that levels of phosphorylated Smad2/3 are sensors of the interplay
between ATRA and TGF- or Vit D3 on HL-60 cells and
contribute to the balance achieved between granulocytic and monocytic
pathways of differentiation.
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Materials and methods |
Cell culture and induction of differentiation
The HL-60 cells (American Type Culture Collection, Rockville,
MD) and HL-60R cells resistant to retinoic acid-induced
differentiation (a gift of Steve Collins, Fred Hutchinson Cancer
Center, Seattle, WA) were maintained in RPMI 1640 medium supplemented
with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL
streptomycin, 4.0 mM glutamine, nonessential amino acids, 10 mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (Life Technologies, Gaithersburg, MD).
Experiments were carried out in the same medium supplemented with 5%
fetal bovine serum. Differentiation was induced by exposing 3 to
5 × 105 cells per milliliter to 10 nM ATRA (Sigma, St
Louis, MO), 10 ng/mL TGF-1 (a kind gift of R&D Systems, Minneapolis,
MN), or 10 nM ATRA plus 10 ng/mL TGF-1 or 100 nM Vit D3
(Roche, Basel, Switzerland) for the indicated time periods. For
determination of cell morphology, cytospins were made following 4 days
of culture with the various treatments, and slides were stained with
Wright Giemsa and photographed at 400 × magnification. PD98059 (Cell Signaling Technology, Beverly, MA) was diluted from a 2-mM stock in
dimethyl sulfoxide (DMSO) and added to cells 2 hours prior to
addition of TGF- or ATRA. TGF- control (12H5) or neutralizing (1D11) antibodies (Genzyme Diagnostics, Cambridge, MA) were used at 30 µg/mL. In the experiments in Table 2, a murine immunoglobulin G1 (IgG1) (BD Pharmingen, San
Diego, CA) served as the isotype control. For effects of okadaic acid
(LC Laboratories, Woburn, MA) on cellular differentiation,
cells were cultured in 2.5 nM okadaic acid for 48 hours (cells showed
greater than 60% viability cultured in 2.5 nM okadaic acid for 72 hours).
Flow cytometry analyses
Aliquots of 1 × 106 cells were harvested, washed
twice with phophate-buffered saline (PBS), and incubated for
45 minutes at room temperature with 0.5 µL fluorescein isothiocyanate
(FITC)-antihuman CD66b (Becton Dickinson, Mountain View, CA),
0.5 µL FITC-antihuman CD15 (Becton Dickinson), or 0.5 µL
phycoerythrin (PE)-antihuman CD14 (Becton Dickinson) to
analyze the expression of these surface markers. Cells were then washed
3 times with ice-cold PBS, and resuspended in 1 mL PBS. Two-parameter
analysis was performed by means of a FACS Calibur Flow Cytometer
(Becton Dickinson, Franklin Lakes, NJ). Isotypic mouse IgG1 or IgM was
used to set threshold parameters. Functional activity was assessed by
cytochemical determination of monocytic nonspecific serine esterase
(NSE)5 or nitroblue tetrazolium (NBT)
reduction,25 as previously described.
Immunohistochemical staining
For intracellular localization of Smad proteins, cytospins
collected at the different treatment time points were fixed in 10%
neutral-buffered formalin for 5 to 10 minutes. Staining was performed
by means of the Optimax Plus 2.0 Automated Cell Staining System with research software from BioGenex (San Ramon,
CA). Following blocking of endogenous peroxidase, nonspecific
protein binding was blocked with a solution containing 1% bovine serum
albumin (BSA) and 5% goat serum for 30 minutes. Sections were
incubated for 2 hours with rabbit anti-Smad2 IgG (Santa Cruz
Biotechnology, CA), rabbit anti-Smad3 IgG (Zymed Laboratories, South
San Francisco, CA), or normal rabbit IgG at 4 µg/mL in tris-buffered
saline (TBS)/1% BSA. Antigen-antibody complexes were detected
by means of the Vectastain Elite ABC peroxidase kit from Vector
Laboratories (Burlingame, CA) according to the manufacturer's
instructions. After a 30-minute incubation with biotinylated secondary
antibody and a 30-minute incubation with ABC reagent, a 5-minute
reaction with 3,3'diamino-benzidine (DAB)/H2O2 (BioGenex) was
used to detect the bound peroxidase. Carazzi hematoxylin was used
as the counterstain. Cells were assessed for the presence of
nuclear staining of the Smad proteins by means of a × 200 microscope.
Immunofluorescence staining and confocal microscopy
HL-60 cells were treated for the indicated times, and the cells
were attached to slides by cytospin, fixed in cold 3.5%
paraformaldehyde for 5 minutes, washed with PBS, and permeabilized in
methanol at  20°C for 2 minutes as described
previously.26 After blocking with 10% normal rabbit
serum, the cells were incubated overnight with rabbit anti-Smad2 (1:50)
(Santa Cruz Biotechnology) or Smad3 antibodies (1:50) (Zymed
Laboratories) in PBS containing 5% normal rabbit serum. Cells were
washed with PBS; incubated with antirabbit FITC secondary antibody
(1:500) (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature; washed; mounted with medium containing DAPI
(4,6-diamidino-2-phenylindole) (Vectashield, Vector
Laboratories); and examined by means of a confocal
immunofluorescence microscope.
Reverse transcriptase (RT)-PCR assays
Total RNA was extracted from HL-60 cells cultured with various
inducing agents for different periods of time with the use of TRIzol
Reagent (Life Technologies); cDNA was synthesized with the use
of 1 µg total RNA primed with oligo d(T)
(deoxy-thymidine) in 50-µL reactions. To test for
contamination by genomic DNA, additional reactions were done without
adding reverse transcriptase. The resulting total cDNA was then used in
the polymerase chain reaction (PCR) to measure the mRNA levels
of Smads with the use of primers as described below; the mRNA level of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as
internal control. Linear amplification cycles were determined
separately for each Smad as published elsewhere.27 Smad2,
Smad3, Smad4, and Smad7 primers were ordered from BioServe Biotechnologies (Laurel, MD) with the following sequences:
Smad2 forward, 5'-TCAAGCTTGAGTGTAAACCCTTACCACTATC-3'; Smad2
reverse, 5'-TAGCGGCCGCGAAAGCTATGATTAACAGGGG-3'; Smad3 forward,
5'-TCAAGCTTGAACACCAGTTCTACCTCCTG-3'; Smad3 reverse,
5'-TAGCGGCCGCGAAATGTCTCCCCGACGCGCTG-3'; Smad4 forward, 5'-TCAAGCTTGATGATCTCTCAGGATTAACAC-3'; Smad4 reverse,
5'-TAGCGGCCGCGAACACCAATACTCAGGAGCAG-3'; Smad7, forward,
5'-GGCTGTGTTGCTGTGAATCTTACG-3'; Smad7 reverse, 5'-CAGTGTGGCGGACTTGATGAAG-3'; GAPDH forward,
5'-CGTTCCCAAAGTCCTCCTGTTTC-3'; and GAPDH,
reverse-5'-TTTTTTTCCGCAGCCGCCTG-3'.
As negative controls, tubes without cDNA were included. The PCR
products were checked on a 1.5% agrose gel with DNA molecular weight
markers (Life Technologies).
P44/42 MAPK kinase assays
Cell lysates (200 µL containing 200 µg total protein) were
added to 15 µL (15 µg) resuspended immobilized phospho-p44/42 MAP kinase (Thr202/Tyr204) monoclonal antibody (Cell Signaling Technology), and incubated with gentle rocking overnight at 4°C. Samples were then
microcentrifuged for 30 seconds at 4°C, and the pellet was washed
twice with 500 µL 1 × lysis buffer and twice with 500 µL 1 ×
kinase buffer before being resuspended in 50 µL 1 × kinase buffer
supplemented with 200 µM adenosine triphosphate (ATP) and 2 µg Ets-like transcription factor-1 (Elk-1) fusion protein
and incubated for 30 minutes at 30°C. The reaction was terminated with 25 µL 3 × sodium dodecyl sulfate (SDS) sample buffer,
boiled for 5 minutes, vortexed, and micocentrifuged for 2 minutes. The sample (30 µL) was loaded on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting with the use of
the phospho-Elk-1 antibody (1:100 dilution) (Cell Signaling Technology).
Western blot analysis
Western blot analysis was performed as described
previously.26 Briefly, cells were lysed in 0.5 mL Triton
X-100 lysis buffer (25 mM HEPES at pH 7.5, 150 mM NaCl, 10%
glycerol, 5 mM EDTA (ethylenediaminetetraacetic acid), 1%
Triton X-100) in the presence of phosphatase and protease inhibitors.
Whole cell lysates (40 µL) were separated by SDS-PAGE and transferred
onto Immobilon-P membranes (Millipore, Bedford, MA). The
membrane was incubated for 1 hour in blocking buffer PBS containing 0.05% polysorbate 20 and 5% nonfat
dry milk) followed by a 2-hour incubation with anti-phospho-Smad2
antibody (Upstate Biotech, Lake Placid, NY) or with anti-Smad2 antibody
(Santa Cruz Biotechnology) in blocking buffer. After extensive washing,
the blot was incubated with secondary antibody for 1 hour and processed with the use of Chemiluminescence Reagent according to the
manufacturer's directions (Pierce, Rockford, IL). For experiments with
okadaic acid and MG-132 (Calbiochem, San Diego, CA), the cells were
shifted to medium containing the inhibitors and 0.2% serum for 3 hours, then washed 3 times with 5% serum-containing medium, and
incubated in the presence of TGF-, ATRA, or the combination for
another 24 hours in the absence of inhibitors.
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Results |
Induction of differentiation of HL-60 cells by TGF-1 and
ATRA
It has previously been shown that TGF-1 and ATRA inhibit the
growth of HL-60 cells in a dose-dependent fashion.5
Morphological studies revealed that whereas treatment with TGF-1
alone induced differentiation of HL-60 cells to promonocytes (68%),
treatment with both TGF-1 and ATRA induced both monocytic (54%) and
granulocytic (46%) differentiation.5 We obtained similar
results from assessment of the morphology of the cells treated with
these 2 agents (data not shown) and confirmed the nature and extent of
differentiation induced by ATRA or TGF-1 by examining the expression
of lineage-specific markers on HL-60 cells by fluorescence-activated
cell sorter (FACS) analysis (Table
1). Addition of ATRA to the culture
medium induced the expression of the granulocyte marker CD66b
in about 90% of the cells, whereas addition of TGF- induced
expression of the monocyte-specific marker CD14 in about 23% of the
cells (Table 1). When treated with both TGF-1 and ATRA, a mixed cell
population appeared in which approximately half of the cells expressed
CD14 and half of them CD66b; only 5% of the cells exhibited both
monocytic and granulocytic characteristics (not shown). Similarly to
previously reported results,5 TGF- treatment induced
expression of the cytoplasmic enzyme, NSE5 in
about 22% of the cells, whereas about 26% of the cells expressed NSE
following treatment with both ATRA and TGF-1 (Table 1). The
increased expression of CD14 and NSE following induction of
differentiation in these cells by TGF-1 confirms previous
observations that TGF-1 stimulates monocytic differentiation of
HL-60 cells. It is noteworthy that while TGF-1 enhances the
commitment of HL-60 cells to the monocytic lineage, it can induce
differentiation only to promonocytes, which variably express either no
CD14 or low CD14.5 ATRA, in combination with TGF-, acts
both to drive the differentiation of these committed cells all the way
to mature, CD14+ monocytes, and to enhance the
differentiation of cells committed to the granulocytic lineage to
CD66b+ granulocytes.
Expression of Smad mRNAs in HL-60 cells is not modulated by
TGF- or ATRA
To ascertain whether treatment with TGF- or ATRA might alter
the expression of Smads in the cells, we examined the expression of
Smad2, 3, 4, and 7 mRNAs in HL-60 cells by semiquantitative RT-PCR.
Each Smad was expressed in the cells, and the levels of expression were
invariant whether cells were treated with 10 nM ATRA, 10 ng/mL
TGF-1, or 10 nM ATRA plus 10 ng/mL TGF-1 and whether
they were treated for 2, 4, or 7 days (Figure
1).
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| Figure 1.
Effect of ATRA versus TGF-1 on expression of Smad
mRNAs.
Expression of Smad mRNAs is not changed following induction of
differentiation of HL-60 cells to monocytes or granulocytes by 10 nM
ATRA or 10 ng/mL TGF-1. Human Smad-specific primer pairs were
selected from the corresponding cDNA sequence information obtained from
the National Institutes of Health (NIH) database as indicated
in "Materials and methods." Primer pairs were used to amplify
Smad-specific fragments from reverse-transcribed total RNA isolated
from the indicated samples as template. In all cases, 1 µg
total RNA, quantified by spectrophotometry and agarose gel analysis,
was used for reverse transcription. Smad2, Smad3, Smad4, Smad7, and
GAPDH (as an internal control) were amplified by PCR and analyzed on an
agarose gel.
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TGF-1 induces the phosphorylation and nuclear accumulation of
endogenous Smad2/3
The phosphorylation of Smad2 on Ser465/467 and subsequent
dimerization with Smad4 and translocation to the nucleus are known to
be required for signal transduction by the TGF- receptor, leading to
the transcriptional activation of target genes.13 To
investigate if Smad2 is phosphorylated following treatment of HL-60
cells with ATRA, TGF-1, or the combination of ATRA plus TGF-1,
the levels of C-terminally phosphorylated Smad2 in cell lysates were
measured by Western blot. Immunoblotting with an antibody that
specifically recognizes Ser465/467-phosphorylated Smad2 showed that
whereas ATRA alone had no detectable effect on the phosphorylation of
Smad2 at 2 hours or 24 hours, treatment with TGF-1 strongly induced
Smad2 phosphorylation (Figure 2A). Combined treatment with ATRA plus TGF-1 significantly reduced the
level of phospho-Smad2 in the total cell population compared with that
seen in cells treated with TGF- alone (Figure 2B). The expression
level of total Smad2 remained unchanged, as shown by reblotting the
membranes with an anti-Smad2 antibody (Figure 2A-B). Although an
antibody for phospho-Smad3 is not available, assessment of the binding
of Smad3 to a biotinylated CAGA Smad-binding oligonucleotide28,29 showed that it was activated in a
pattern similar to that of Smad2 (data not shown).
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| Figure 2.
Western blot analysis of activation of endogenous Smad2
in HL-60 cells.
Protein prepared from HL-60 cells untreated or treated with 10 ng/mL
TGF-1 or 10 nM ATRA (A) or treated with both (B) for the indicated
times, as described in "Materials and methods," were directly
subjected to immunoblotting with antibodies against Smad2 (Total
Smad2) or C-terminally phosphorylated Smad2 (P-Smad2).
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To ascertain whether these observed changes in the phosphorylation of
Smad2 and Smad3 were accompanied by changes in the intracellular distribution of these Smad proteins in HL-60 cells, we used specific antibodies30 to assess the intracellular localization of
endogenous Smad2 and Smad3 after treatment of the cells with 10 ng/mL
TGF-1, 10 nM ATRA, or a combination of 10 nM ATRA plus 10 ng/mL
TGF-1 for 18 hours. In the control group, endogenous Smad2 and Smad3 are found predominantly in the cytoplasm (Figure
3A). TGF-1 stimulation resulted in the
translocation of Smad2/Smad3 to the nucleus. Quantitation of the
percentage of cells with nuclear staining showed that at 18 hours about
75% and 41% of the cells showed staining for Smad3 or Smad2,
respectively, in the nucleus (Figure 3B-C). Significantly, treatment
with ATRA alone reduced the basal number of cells showing Smad2/Smad3
nuclear staining, and treatment with both ATRA and TGF-1 strongly
reduced the number of cells exhibiting nuclear Smad2/3, compared with
that seen following treatment with TGF-1 alone. These changes in the
subcellular localization of Smad2/3 in particular subpopulations of
cells were confirmed by confocal microscopy, which clearly demonstrated
the nuclear translocation of Smad2 induced by treatment with TGF-1
alone, and the presence of a mixed population of cells with either
nuclear or cytoplasmic localization following treatment with both ATRA
and TGF-1 (Figure 4). Together, these
results demonstrate that TGF-1 induces phosphorylation and nuclear
translocation of Smad2/Smad3, which are likely to play a critical role
in activation of the transcriptome responsible for
TGF-1-induced monocytic differentiation of HL-60 cells. Moreover, the data show that treatment with ATRA decreases the number of cells in
which there is detectable nuclear Smad2/3 staining compared with cells
treated with TGF-1 alone, consistent with the hypothesis that the
subset of cells lacking nuclear Smad2/3 may then be committed to
differentiate to granulocytes.
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| Figure 3.
Immunohistochemical staining of Smad3/Smad2 in HL-60 cells.
(A) Expression of Smad3 as detected by anti-Smad3 antibody in
HL-60 cells untreated (white) or treated with 10 nM ATRA (gray
striped), with 10 ng/mL TGF-1 (black), or with both (black
and white striped) for 18 hours as described. Original
magnification × 200. (B-C) Following immunohistochemical staining,
the percentage of cells with Smad2 (B) or Smad3 (C) staining
predominantly or exclusively in the nucleus was determined among 1000 cells in 5 different fields. Values are expressed as the means ± SEMs of 3 experiments.
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| Figure 4.
Localization of endogenous Smad2 in HL-60 cells by confocal microscopy.
After 18-hour incubation with 10 ng/mL TGF-1, 10 nM ATRA, or both,
the cells were fixed, subjected to immunochemistry with primary
antibodies against Smad2 and secondary antibodies linked to fluorescein
isothiocyanate, and mounted with medium containing 4,6- DAPI.
The red arrow indicates that Smad2 is localized both in the
nucleus and in the cytoplasm. Original magnification for all panels,
× 630. The result is representative of 3 similar
experiments.
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Vitamin D3 induces phosphorylation of Smad proteins
indirectly through a mechanism dependent on TGF-
It has been reported that the induction of terminal
differentiation by Vit D3 requires TGF-1 as an autocrine
mediator in U937 cells.6 We have shown that treatment of
HL-60 cells with 100 nM Vit D3 induces monocytic
differentiation to CD14+ cells (not shown) characterized by
NSE activity (Table 2). To investigate if
Smad2 and Smad3 are also activated in Vit D3-induced monocytic differentiation, we examined the level of phosphorylated Smad2. Strong phosphorylation of Smad2 can be seen after 24 hours' incubation with 100 nM Vit D3 (Figure
5A) coincident with nuclear translocation
of Smad2/Smad3 (Figure 5B). After incubation with 100 nM Vit
D3 for 18 hours, about 74% and 39% of the cells showed nuclear staining for Smad3 or Smad2, respectively, similar to the
pattern seen in cells treated with TGF- (compare with Figure 3B-C).
To test whether these effects of Vit D3 on Smad
phosphorylation were dependent on induction of TGF- activity, we
treated cells with either control antibodies or antibodies to TGF-.
As shown in Figure 5C, the TGF-1-neutralizing antibody 1D11 blocked
both Vit D3- and TGF--induced phosphorylation of
Smad2, whereas the control antibody (IgG1) had no
effect. To ascertain that the effects of the blocking antibody on Smad2
phosphorylation paralleled effects on differentiation, we assessed the
functional activity of HL-60 cells 6 days after treatment with Vit
D3 or TGF-, using the NSE assay for monocytic
differentiation and the reduction of NBT as a measure of granulocytic
differentiation. As shown in Table 2, addition of the 1D11 TGF-
blocking antibody to cells treated with Vit D3 reduced the
proportion of NSE+ cells from 66% to 23%, whereas an
isotype control antibody had no effect. For comparison, addition of the
antibody to cells treated with TGF- reduced the number of
NSE+ cells from 16% to 1%. Together, these results
strongly suggest that Vit D3 induces monocytic
differentiation of HL-60 cells, at least in part, through expression of
TGF- and subsequent activation of the Smad-signaling
pathway.
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| Figure 5.
Vitamin D3
induction of activation of Smad2/Smad3 in HL-60 cells.
Vitamin D3 induces activation of Smad2/Smad3 in HL-60 cells
in a TGF--dependent manner. (A) Western blot analysis of
phosphorylation of endogenous Smad2 in HL-60 cells untreated or treated
with 100 nM Vit D3 for 24 hours. (B) Immunohistochemical
staining of Smad3 in HL-60 cells untreated (Control) or treated with
100 nM Vit D3 at 18 hours. Original magnification × 400. (C) TGF-1-neutralizing antibodies block the
phosphorylation of Smad2 induced by treatment of Hl-60 cells with
either 10 ng/mL TGF- or 100 nM Vit D3. Lysates from
cells incubated in either the presence or absence of 12.5 µg/mL
TGF-1 control (12H5) or neutralizing (1D11) antibody at 2 hours or
24 hours were directly subjected to immunoblotting with antibodies
against Smad2 (not shown) and phosphorylated Smad2.
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Induction of both monocytic and granulocytic differentiation of
HL-60 cells is accompanied by activation of ERK1/2
Activation of the ERK1/2 MAPK pathway is essential for
granulocytic differentiation of HL-60 cells by retinoic
acid.24 Since this same pathway has been shown to inhibit
nuclear translocation of Smad2/Smad3 induced by TGF-,31
we investigated whether cross-talk between the ERK1/2 MAPK pathway and
the Smad pathway could alter the pattern of phosphorylation or nuclear
accumulation of Smads in HL-60 cells treated with either TGF-1 alone
or the combination of ATRA and TGF-1. A specific inhibitor of
ERK1/2, PD98059 (1.0 and 10 µM), inhibited phosphorylation of ERK1/2
in a dose-dependent manner (data not shown), but had no effect on
either TGF--induced phosphorylation of Smad2 (Figure
6A) or nuclear translocation of Smad3 in
HL-60 cells treated with either TGF-1 alone or the combination of
TGF-1 and ATRA (Figure 6A). Moreover, when we examined the effects
of ATRA, TGF-1, ATRA plus TGF-1, or Vit D3 on the
activation of the ERK1/2 MAPK pathway in HL-60 cells, each of these
treatments increased activation of this pathway with a similar time
course, as measured by phosphorylation of the substrate Elk-1 in an in
vitro kinase assay (Figure 6C). The somewhat delayed induction of ERK
activation by Vit D3 may suggest that it, like Vit
D3-induced Smad phosphorylation, is mediated indirectly by
TGF-1. Together, these data show that TGF- activates both the
Smad and ERK1/2 pathways in HL-60 cells, but that these pathways appear
to act independently, since activation of ERK1/2 occurs at an early
stage of differentiation independently of the effects of the various
differentiating agents on activation of Smad2/Smad3 or on commitment to
specific lineages.
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| Figure 6.
The effect of inhibition of MAPK on phosphorylation and
nuclear translocation of Smad2 induced by TGF-1.
(A) Phosphorylation of Smad2 was not affected by treatment with 10 µM
PD98059 for 2 hours followed by addition of 10 ng/mL TGF-1 for an
additional 24 hours. (B) There was no significant difference observed
in the percentage of nuclei positive for Smad2 immunostaining (of 1000 cells counted in 5 different fields) 18 hours after treatment with 10 ng/mL TGF-1 and 10 nM ATRA with or without the addition of 10 µM
PD98059 (10 µM PD) at 2 hours prior to the other treatments
(means ± SEMs, n = 3). (C) The p44/42 MAP kinase activity is
activated independently of the particular pathway of differentiation.
Proteins were immunoprecipitated with anti-phospho-p44/42 MAP kinase
antibody as described in "Materials and methods," and the
immunoprecipitates were subjected to an in vitro kinase assay with the
use of the Elk-1 fusion protein. Reaction mixtures were separated by
SDS-PAGE and immunoblotted with anti-phospho-Elk-1 antibody. The data
shown are representative of 3 experiments with similar
results.
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ATRA increases dephosphorylation of Smad2
Since ATRA decreases levels of phospho-Smad2/Smad3 and reduces the
number of cells with nuclear staining for Smad2/Smad3 in cells treated
with TGF-1, we investigated whether pre-exposure of HL-60 cells to
ATRA or TGF-1 would affect the subsequent ability of these agents to
activate Smad2. HL-60 cells were incubated for 2 days in the presence
of either TGF-1, ATRA, or the combination; washed to remove these
effectors; and then incubated with a different factor for another 24 hours prior to analysis of the levels of phospho-Smad2 (Figure
7A). These experiments showed that the
level of phospho-Smad2 in cells that had been treated with TGF-1 for 2 days was lower when treatment was followed by addition of ATRA for 24 hours than when cells were treated for the final 24 hours with only
control medium. As before, there was no change in the expression level of total Smad2. We interpreted these data to suggest
that treatment with ATRA actively reduced the level of existing
phospho-Smad2 in cells previously treated with TGF-1.
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| Figure 7.
Effect of ATRA on dephosphorylation of Smad2.
ATRA increases dephosphorylation of Smad2. (A) ATRA reduced the levels
of phospho-Smad2 induced by TGF-1 in HL-60 cells. HL-60 cells
treated sequentially showed a lower level of phospho-Smad2 when cells
were incubated with 10 ng/mL TGF-1 for 48 hours, washed,
and then treated with 10 nM ATRA for 24 hours, as compared with cells
similarly pretreated with 10 ng/mL TGF-1, washed, and treated with
control medium. (B-D) Cells were changed to 0.2% serum, and okadaic
acid or MG-132 was added. After 3 hours, cells were washed 3 times with
medium containing 5% serum and incubated for an additional 24 hours
with addition of TGF-, Vit D3, ATRA, or TGF- plus
ATRA, in the absence of the inhibitors. Lysates were analyzed by
immunoblotting with an antibody specific to C-terminally
phosphorylated Smad2. (B) The proteasome inhibitor MG-132 (50 µM) did not have specific effects on cells treated with
ATRA. (C) Treatment with okadaic acid (100 nM) blocks the ability of
ATRA (10 nM) to decrease levels of phospho-Smad2 induced by TGF-1
(10 ng/mL). (D) Treatment with okadaic acid (100 nM) alone enhances
detection of phospho-Smad2, in addition to its ability to augment
levels induced by Vit D3.
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To address possible mechanisms whereby ATRA might decrease levels of
phospho-Smad2/3 in cells treated with TGF-1, we investigated whether
either proteasome-mediated degradation of phospho-Smad32 or phosphatase-mediated dephosphorylation might be involved. Addition of the proteasome inhibitors MG-132 or lactacystin (not shown) had only
a small effect, slightly increasing the levels of phospho-Smad2 seen in
cells treated with either TGF-1 alone or the combination of TGF-1
and ATRA (Figure 7B). In contrast, preincubation of HL-60 cells with
okadaic acid, an inhibitor of protein serine/threonine phosphatases,33 blocked the ability of ATRA to
reduce the level of phospho-Smad2 in cells treated with both TGF-
and ATRA (Figure 7C). Surprisingly, treatment of the cells with okadaic
acid alone resulted in detectable phospho-Smad2, although at levels
lower than that observed by treatment of the cells with TGF- (Figure 7D). Together, these data suggest that endogenous protein
serine-threonine phosphatases probably control the basal level of
phospho-Smad2 and that ATRA reduces levels of phospho-Smad2 through
induction of such phosphatases.
We then assessed the effect of okadaic acid on the differentiation of
HL-60 cells treated with ATRA, TGF-, or both to test the hypothesis
that induction of the putative serine/threonine phosphatase would favor
granulocytic differentiation and that inhibition of serine/threonine
phosphatase activity would shift the balance toward monocytes. As shown
in Table 3, treatment of HL-60 cells with
ATRA for 2 days followed by incubation for an additional 2 days with
2.5 nM okadaic acid increased expression of both CD14 and NSE activity
and decreased expression of CD15, compared with cells treated only with
ATRA. Okadaic acid also strongly enhanced the ability of TGF- to
induce differentiation to functional monocytes and, when added for the
second 2 days to cells treated with the combination of ATRA and
TGF-, reduced the expression of CD15 and enhanced expression of both
CD14 and NSE activity. These data show that okadaic acid enhances the
proportion of HL-60 cells expressing monocytic features, independently
of the presence of other differentiating agents, and consistent with its effects on phospho-Smad2 (Figure 7C-D). When cells are treated with
both ATRA and TGF-, which results in reduced nuclear phospho-Smad2/3 compared with cells treated with TGF- alone (Figures 2-3),
the addition of okadaic acid inhibits the ATRA-dependent suppression of
Smad2/3 phosphorylation (Figure 7C) simultaneously with its skewing the
differentiation toward monocytes (Table 3).
The ability of ATRA to decrease levels of phospho-Smad2/3 is
dependent on RAR-
To further address the mechanism by which ATRA modulates the
levels of phospho-Smad2 and to link these effects to the ability of
ATRA to induce granulocytic differentiation of HL-60 cells, we used
retinoic acid-resistant cells (HL-60R), which have a point mutation in
the RAR- ligand-binding domain that confers dominant-negative activity.34 ATRA is unable to induce granulocytic
differentiation of HL-60R cells (Robertson et al,34 and
data not shown), whereas the ability of TGF-1 to induce monocytic
differentiation, as assessed by expression of CD14, is unimpaired
(Figure 8A). The ability of ATRA, when
added with TGF-, to stimulate maturation of promonocytic
HL-60R cells stands in strong contrast to its inability to stimulate
granulocytic differentiation, suggesting different roles of RAR- in
these 2 processes. Immunoblotting with the anti-phospo-Smad2 antibody
demonstrated that ATRA is unable to reduce levels of phospho-Smad2 in
HL-60R cells treated with ATRA and TGF-1, with the result
that the level of phosphorylated Smad2 is similar in cells
treated with TGF-1 alone or with the combination of ATRA and
TGF-1 (Figure 8B). Consistent with this, immunohistochemical
staining showed that the percentage of cells with nuclear staining for
Smad2/Smad3 was also similar in cells treated with either TGF-1
alone or the combination of ATRA and TGF-1 (Figure 8C,E).
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| Figure 8.
The effect of RAR- on the ability of ATRA to decrease
levels of phospho-Smad2/Smad3.
The ability of ATRA to decrease levels of phospho-Smad2/Smad3 is
dependent on RAR-. (A) Treatment for 4 days with ATRA together with
TGF-1 enhances the expression of CD14 by HL-60R compared
with cells treated with TGF-1 alone (means ± SEMs). (B) In
contrast to wild-type HL-60, ATRA is unable to reduce levels of
phospho-Smad2 induced by TGF-1 in RAR--mutant HL-60 cells
(HL-60R) treated simultaneously with ATRA and TGF-1 (lane 3 compared
with lane 4). Treatments were for 24 hours. (C-E) Immunohistochemical
staining showed that ATRA is unable to reduce the TGF-1-dependent
Smad3 nuclear localization in RAR--mutant HL-60 cells treated for
18 hours with ATRA and TGF-1 (C) as seen in wild-type HL-60 cells
(D). Original magnification × 400. The arrow on the left
shows a cell with both nuclear and cytoplasmic staining, and the arrow
on the right shows a cell with only cytoplasmic staining. (E)
Quantitation of the data in panel C was obtained by assessment
of the staining patterns in 1000 cells in 5 different fields of treated
HL-60R cells. For panels A, B, and E, treatments were (1)
vehicle (gray); (2) ATRA, 10 nM (black and gray striped); (3) TGF-1,
10 ng/mL (black); or (4) the combination of ATRA and TGF-1 (black
and white striped).
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Discussion |
This study provides new insights into mechanisms by which
hematopoietic cells interpret and integrate the multiplicity of extracellular signals that ultimately specify distinct lineage decisions. Using the model system of HL-60 cells, a human myeloblastic leukemia with promyelocytic features, we have shown that the interplay of signals from ATRA, which specifies differentiation to granulocytes, or TGF-1/Vit D3, which specify commitment to monocytic
differentiation, is mediated, in part, through a balance between
protein serine/threonine phosphatase activity and levels of
phosphorylated Smad2 and Smad3. Thus, we have shown that TGF-1
induces phosphorylation of Smad2/3 and that addition of ATRA together
with TGF-1 reduces the level of phospho-Smad2/3 and increases the
extent of commitment to the granulocytic lineage at the expense of the
monocytic lineage. Conversely, okadaic acid, which inhibits protein
serine/threonine phosphatases and which enhances the level of
phospho-Smad2/3 in cells, pushes the balance toward monocytic
differentiation. Together, these data suggest that monocytic
differentiation is favored by lower protein phosphatase activity and/or
increased levels of nuclear Smad2/3 (if the inducing agents are TGF-
or Vit D3) and that granulocytic differentiation is favored
by higher protein phosphatase activity and/or reduced nuclear Smad2/3.
In the specific case in which ATRA and either TGF- or Vit
D3 are acting on the cell simultaneously, the data suggest
that the induction of protein serine/threonine phosphatase activity by
ATRA can modulate the levels of phospho-Smad2/3 induced by TGF- and
thereby alter the partitioning between the granulocytic and
monocytic pathways.
Signal transduction pathways are regulated by dynamic interplay between
protein kinases and phosphatases. Numerous reports have examined the
ability of ATRA to alter phosphatase activity in HL-60 cells during the
process of differentiation to granulocytes.35-38 Thus, Src
homology 2 (SH2)-containing protein tyrosine phosphatase-1 (SHP-1), a protein tyrosine phosphatase, was shown to be
induced by ATRA in HL-60 cells,35 but another report
showing it to be also elevated by phorbol ester-induced monocytic
differentiation of these same cells39 raises questions
regarding the lineage specificity of this effect. Most germane to our
findings is the report that ATRA elicits a transient and reversible
interconversion of the protein phosphatase 2A (PP2A) holoenzyme at the
G1/S boundary during ATRA-induced granulocytic
differentiation of HL-60 cells.36 PP2A accounts for the
majority of the serine/threonine phosphatase activity in most
cells and is inhibited by okadaic acid.40 Although several
other studies show down-regulation of the catalytic subunit of PP2A
beginning about 48 hours after treatment with ATRA and continuing for 3 to 5 days,37,38 it is probably the transient changes in
the regulatory subunit at 18 at 24 hours,36 which are
predicted to result in a change in substrate specificity, that are most
likely to affect levels of phospho-Smads at these times, as observed
(Figures 2B a |
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Abstract
Introduction