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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 846-854
HEMATOPOIESIS
Opposing effects of engagement of integrins and stimulation of
cytokine receptors on cell cycle progression of normal human
hematopoietic progenitors
Yuehua Jiang,
Felipe Prosper, and
Catherine M. Verfaillie
From the Stem Cell Laboratory and Division of Hematology, Oncology
and Transplantation, Department of Medicine and Cancer Center,
University of Minnesota, Minneapolis, MN, and the Department of
Hematology and Medical Oncology, Hospital Clinico Universitario,
University of Valencia, Valencia, Spain.
 |
Abstract |
We evaluated the effect of  1-integrin receptor engagement on the
expression and activity of cell cycle regulatory proteins in
CD34+ cells under conditions that mimic the steady-state
marrow microenvironment and in the presence of supraphysiological
concentrations of interleukin-3 (IL3) and stem cell factor (SCF).
Adhesion of CD34+ progenitors to fibronectin (FN) was
similar whether IL3 or SCF was present or absent. Engagement of
1-integrins blocked S-phase entry of CD34+ cells in
the absence of IL3 or SCF, whereas addition of 10 ng/mL IL3 or SCF
prevented such a block in S-phase entry. In the absence of IL3 or SCF,
cyclin-E levels were significantly lower and p27KIP1 levels
significantly higher in FN-adherent than in FN-nonadherent cells, or
than in poly-L-lysine (PLL)-adherent or (PLL)-nonadherent cells.
Cyclin-dependent-kinase (cdk)-2 activity was decreased and levels of
cyclin-E-cdk2 complexes were lower in FN-adherent than in PLL-adherent
cells. In contrast, cyclin-E and p27KIP1 protein levels and
cdk2 activity in cells adherent to FN in the presence of IL3 or SCF
were similar to those in PLL-adherent and FN-nonadherent or
PLL-nonadherent cells. In conclusion, under physiological cytokine
conditions, integrin engagement prevents S-phase entrance of
CD34+ cells, which is associated with elevated levels of
the contact-dependent cyclin kinase inhibitor p27KIP1.
Supraphysiological concentrations of IL3 or SCF prevent
p27KIP1 elevation and override the integrin-mediated
inhibition of entry into S phase.
(Blood. 2000;95:846-854)
© 2000 by The American Society of Hematology.
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Introduction |
1-integrins on human CD34+ cells are
responsible for their adhesion to fibronectin (FN) and to vascular cell
adhesion molecules.1-3 In addition, a number of studies
from our laboratory have shown that adhesion of normal human
CD34+ colony-forming cells (CFC) to FN or direct engagement
of 1-integrins on CD34+ cells with adhesion-blocking
antibodies prevents CFC from entering the S phase of the cell cycle and
inhibits expansion of CFC and more primitive long-term culture
initiating cells (LTC-IC) in long-term bone marrow (BM)
cultures.4-7 The mechanism through which engagement of
1-integrins inhibits CD34+ progenitor proliferation is
not known.
Integrin-mediated signaling has been extensively studied in cells of
mesenchymal origin and in platelets.8-15 Integrins are divalent cation-dependent cell surface glycoproteins consisting of an
 and a chain. They are responsible for cell-extracellular matrix (ECM) and cell-cell adhesion events. Integrins
have a large, heterodimeric extracellular domain responsible for ligand
recognition and binding, a short transmembrane domain, and a short
cytoplasmic tail. The cytoplasmic tails of integrins have no intrinsic
kinase activity. However, engagement of integrins results in the
assembly of focal contacts in which a number of kinases become
activated.8-15 Integrins activate the focal adhesion kinase
(Fak) or the related kinase, Pyk-2.16,17 Phosphorylated Fak
or Pyk-2 serve to bind and activate a number of Rous sarcoma
(Src)-homology domain SH2- and SH3-containing adaptor
proteins, including Src, paxillin, CrkL,
Grb-2, p130Cas, p120Cbl, and the p85 subunit of
phospho-inositol-3 kinase.18-24 Although the exact
mechanism or mechanisms through which engagement of integrins affects
cell proliferation, differentiation, or survival are not known,
integrins can activate the phospho-inositol-3-kinase pathway23 and the Ras/mitogen-activated protein kinase
pathway,24 both of which mediate signals regulating growth.
Further, integrin engagement induces immediate-early inflammatory
response genes.24
Growth of adherent cells such as fibroblast requires signals not only
from growth factor receptors but also from
integrins.13-15,25 Recent studies have shown that
integrin-mediated adhesion of fibroblasts is associated with increased
levels of cyclin-D1, leading to hyperphosphorylation of the
retinoblastoma protein (Rb) and transition of the cell through the
restriction-point (R) in the G1/S phase of the cell cycle.26,27 Other studies have shown that adhesion of
fibroblasts to ECM also results in up-regulation of cyclin-E/cdk2
activity owing to decreases in the cdki's p21CIP1 and
p27KIP1, and up-regulation of cyclin-A
levels.26,27 It is not known whether or how these
observations made in mesenchymal cells relate to signals emanating from
integrins following their engagement on human hematopoietic
progenitors, which are nonadherent cells.
Signals initiated by integrins can be modified or enhanced by
costimulation of cells with cytokines or growth factors.15 In the hematopoietic system, a number of investigators have examined the effect of cytokine stimulation on integrin-mediated
adhesion.28-34 Cytokines, including interleukin 3 (IL3),
granulocyte-macrophage (GM)-colony stimulating factor (CSF), and stem
cell factor (SCF), may increase at least short-term integrin-mediated
adhesion of cytokine-starved CD34+ to FN. Other studies
show that IL3 or SCF do not affect adhesion of progenitor cells.
Another group of reports has demonstrated a synergistic effect of
cytokines and FN on the expansion of progenitors when cultured in the
absence of stromal feeders, thus linking signals initiated following
integrin and cytokine receptor stimulation.
In this study, we show that engagement of 1-integrins on normal
human CD34+ progenitors results in the inhibition of
S-phase entry/progression and increased levels of the cyclin-kinase
inhibitor p27KIP1, which inhibits cyclin-E-cdk2 complex
formation and cdk2 activity. However, costimulation of cells with
cytokines, such as IL3 or SCF, prevents accumulation of
p27KIP1, leading to continued S-phase entry/progression in
progenitors even though they are adherent to FN.
| |
Materials and methods |
We obtained antibodies and reagents from various
sources. Human plasma FN was purified as a by-product of Factor VIII.
Poly-L-lysine (PLL) and bovine serum albumin (BSA; 98%) were purchased
from Sigma Chemical Co (St. Louis, MO).
Blocking antibodies against the integrins 4 (P4C2), 5 (P1D6),
1 (P4C10), and 2 (P1E6) were purchased from
Gibco-BRL (Grand Island, NY). The activating anti-1-antibody, 8A2,
was a kind gift from Dr Kovach, University of Washington (Seattle,
WA).35 Antibodies against CD34 and CD62L were purchased
from Becton Dickinson (Mountain View, CA), and mouse immunoglobulin G
(IgG) was purchased from Sigma. FITC-conjugated goat
antimouse antibody was obtained from Biosource International,
Camarillo, CA.
For the flow-cytometric assessment of the cell cycle, unconjugated
antibodies against p21CIP1, p27KIP1,
p16INK4A, and cyclin-E and fluorescein (FITC)-conjugated
antibodies against cyclin-A and cyclin-D1 + 2 + 3 were
obtained from Pharmingen Inc (San Diego, CA). FITC-coupled anti-PCNA antibodies were obtained from DAKO Inc.
Secondary goat antimouse FITC antibodies as well as
isotype-control antibodies were also purchased from Pharmingen Inc.
For use in immunoprecipitation and Western blot, antibodies against
p21CIP1, p27KIP1, Cyclin-E, Cdk2, and -actin
were obtained from Pharmingen Inc. Secondary goat antimouse horseradish
peroxydase (HRP)-conjugated antibodies were obtained
from Pharmingen Inc.
The following cytokines were purchased from R&D Systems (Minneapolis,
MN): IL3, IL6, leukemia inhibitory factor (LIF), and macrophage-inflammatory protein (MIP)-1. SCF was a kind gift from
Amgen Inc (Thousand Oaks, CA); fetal liver tyrosine kinase-3 ligand
(Flt3-L) was a kind gift from Immunex Inc (Seattle, WA). GM-CSF was
purchased from Immunex, and erythropoietin and G-CSF were purchased
from Amgen.
We obtained 50 mL of heparinized BM from normal donors under
steady-state conditions. To obtain mobilized peripheral blood (PB)
progenitors, we selected normal donors using the standard criteria of
the American Association of Blood Banks for blood donors. The donors
received a daily dose of 10 µg/kg/d G-CSF subcutaneous for 5 days. On
day 6 donors underwent an apheresis procedure, as previously
described.36 All donors signed an informed consent according to the guidelines from the Committee for the Protection of
Human Subjects at the University of Minnesota.
Steady-state BM- and G-CSF-mobilized PB mononuclear cells were
separated by Ficoll Hypaque centrifugation (specific gravity, 1077)
(Sigma). CD34+ cells were selected either by 2 passages
over the MACS CD34 Isolation Kit (Miltenyi Biotec, Sunnyvale, CA) or by
sequential selection with the Ceprate SC device for clinical scale stem
cell concentration (CellPro, Bhotell, WA) followed by the MACS CD34
Isolation Kit.36 CD34+ populations were more
than 95% pure.
As a low-dose cytokine, serum-free medium, we used Iscove's Modified
Dulbecco's Medium (IMDM, Gibco) containing 20 mg/mL BSA, 10 µg/mL
insulin (Sigma), 200 µg/mL transferrin (Sigma), 10-4
mol/L 2-mercapto-ethanol (Bio-Rad, Hercules, CA), 100 U/mL penicillin and 100 U/mL streptomycin (Gibco), and the following
cytokines37: 200 pg/mL GM-CSF, 1000 pg/mL G-CSF, 200 pg/mL
SCF, 50 pg/mL LIF, 200 pg/mL MIP-1, and 1000 pg/mL IL-6.
Integrins were engaged in two ways. One way was to cause them to adhere
to FN. In this method, CD34+ cells suspended in serum-free
IMDM with or without cytokines were plated onto wells coated with 100 µg/mL FN or 10 µg/mL PLL in a humidified atmosphere at
37°C.3,5,7 Adherent and nonadherent cells were
collected after 2 hours to assess adhesion and at 12 to 16 hours to
assess cell cycle status as described.3,5,7
Integrins were also engaged in solution. In this method,
CD34+ cells were incubated in IMDM, with or without
cytokines and with adhesion-blocking antibodies against the 1-,
2-, 4- and 5-integrins, CD62L (all at 10 µg/mL), or mouse
IgG control for 30 minutes at 37°C.7 Cells were washed
and incubated with goat antimouse antibody (1:500 dilution) for 8 to 12 hours at 37°C in a humidified atmosphere.
We assessed cell adhesion in two ways. In the 51Cr labeled
adhesion assay, CD34+ cells were labeled with 0.1 mCi
51Cr (specific activity 734.5 mCi/mg; NEN, Boston, MA) for
1 hour at 37°C and washed twice. 51Cr-labeled
CD34+ cells suspended in IMDM with or without cytokines
were plated in ligand-coated dishes for 2 hours. Nonadherent cells were
collected. Adherent cells were lysed with triton-X-100 (Sigma), wells
were harvested, and 51Cr emission was counted with the use
of a Gamma 4000 Counting System (Beckman Instruments, Irvine,
CA).37 The percentage of adhesion was calculated as
follows: % = (cpm emission in adherent cells  cpm
background) / (cpm emission in all cells cpm
background) × 100.
We also assessed cells for adherence to CFC. In this method,
CD34+ cells incubated in IMDM with or without cytokines
were plated in ligand-coated dishes for 2 hours. Nonadherent cells were
collected in 3 washes, as described.3,5 Adherent cells were
collected after trypsinization for 7 minutes. The percentage of
adhesion of CFC was determined by replating adherent and nonadherent
CD34+ cells in methylcellulose assay and enumerating CFC in
the adherent and nonadherent portion.3,5
The percentage of adhesion was determined as follows: % adhesion = (CFC in adherent cells) / (CFC in
adherent + nonadherent portion) × 100.
We performed various procedures to assess cell cycle and cell cycle
regulatory elements. A flow-cytometric evaluation of cell cycle38 was performed in the following way:
CD34+ cells incubated in IMDM with or without cytokines
were plated in ligand-coated dishes for 12 to 16 hours. Nonadherent
cells were collected in 3 washes, as described.3,5 Adherent
cells were collected after trypsinization for 7 minutes. We have
previously shown that trypsin does not affect assessment of cell
cycle.5 Freshly collected CD34+ cells or
adherent or nonadherent CD34+ cells recovered after 12 hours of adhesion to BSA, PLL, or FN were fixed in 75% ethanol and
stained with propidium iodide (50 µg/mL propidium iodide  , and 10 µg/mL RNase in phosphate buffer saline (PBS)
[Gibco]).39 Cells were
analyzed with a FACS-Calibur flow cytometer (Becton Dickinson). Cell
cycle phase distribution was calculated with the use of ModFit LT
software (Verity Software House Inc). In some experiments,
CD34+ cells selected from G-CSF-mobilized PB were
cocultured with BSA, FN, or PLL in the presence of low-dose cytokines
with or without IL3 or SCF. After 60 hours, cell cycle status was
evaluated by fluorescence-activated cell sorter (FACS).
Flow cytometric evaluation of cell cycle proteins was performed as
follows: CD34+ cells recovered in the adherent and
nonadherent portion of adhesion assays were fixed in
75% ethanol, then incubated overnight at 20°C.40 For assessment of cyclin-D, cells were
fixed in 1% formaldehyde (Sigma) for 15 minutes before fixation in
ethanol. Cells were washed and permeabilized with 0.15% Triton X-100
(Sigma) for 5 minutes, washed, and then incubated with antibodies
directed at PCNA, cyclin-D1 + 2 + 3, cyclin-E,
cyclin-A, p16INK4A, p21CIP1, and
p27KIP1 or with isotype control antibody at room
temperature for 30 minutes. When unconjugated antibodies were used,
cells were washed and incubated with FITC-conjugated goat antimouse Ig
for 30 minutes in the dark. Cells were washed and resuspended in 50 µg/mL propidium iodide. Cells were analyzed with a FACS-Calibur flow
cytometer with the use of Cell Quest and ModFit LT software. In some
experiments, CD34+ cells selected from G-CSF-mobilized PB, which had
not been cultured with low doses of cytokines to induce cell
proliferation, were cocultured with FN or PLL in the presence of
low-dose cytokines with or without IL3 or SCF. After 60 hours, levels
of p27KIP1 were assessed by FACS and Western blot.
A thymidine suicide assay was performed as follows: CFC proliferation
was assessed in 3H-thymidine suicide assays as
described.5,7 (1) Adherent and nonadherent
CD34+ cells recovered after 12-hour adhesion to BSA, PLL,
or FN or (2) CD34+ cells incubated with blocking or
nonblocking anti-integrin antibodies or control antibodies for 12 hours
were incubated at 37°C in serum-free IMDM for 30 minutes with or
without 5 mCi 3H thymidine (specific activity, 6.7 Ci/mmol,
NEN), washed with excess cold thymidine (500 mg/mL
in IMDM plus 20% FCS), and plated in methylcellulose assays. The
percentage of CFC in S phase was calculated as follows: % CFC in
S-phase = [(number of CFC without
3H-thymidine Number of CFC with
3H-thymidine) × 100] / [number of CFC
without 3H-thymidine].
In Western blotting and immunoprecipitation,39 cells
recovered in the adherent and nonadherent portion of
adhesion assay were lysed in NP-40 lysis buffer (50 mmol/L Tris HCl, pH 7.4, 250 mmol/L NaCl, 2 mmol/L
ethylenediaminetetraacetic acid, 1% NP-40, 1 mmol/L
phenylmethyl-sulfonyl fluoride (PMSF), 10 µg/mL aprotinin, 10 µg/mL leupeptin, 50 mmol/L NaFl, 0.1 mmol/L
Na3VO4, all from Sigma), and lysates were
recovered by centrifugation. Total protein from each sample to be used
in Western blots or immunoprecipitations was normalized with the use of
the Bradford assay.
In Western blotting, protein lysates from more than
2 × 106 adherent, nonadherent, or unmanipulated
cells were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred onto nitrocellulose with the
use of a Semidry Transfer Apparatus (Bio-Rad). Immunoblots were blocked
in freshly prepared TBS (Pierce, Rockford, IL)
containing 5% nonfat dry milk. Blots were incubated with 1 ng/mL
primary antibody in blocking buffer for 2 hours at room temperature.
After 4 washes in TBST (TBS supplemented with 0.05% Tween 20), blots
were incubated for 1 hour with a goat antimouse HRP-conjugated antibody
(1:10 000 dilution). Bands were visualized with the use of an ECL
detection system (E.I. du Pont de Nemours & Co, Boston, MA).
Quantitative differences in protein levels in different conditions were
evaluated by scanning images with a GS-700 Imaging Densitometer
(Bio-Rad); the images were then quantitated with the use of Molecular
Analyst software (Bio-Rad).
In immunoprecipitation, protein lysates were precleared with 50 µL protein-G-agarose (Boehringer Mannheim,
Indianapolis, IN) for at least 3 hours on a rocking platform, and
nonbound material was recovered by centrifugation. We added 1 µg/mL
anti-cyclin-E or anti-cdk2 antibody, and the mixture was gently rocked
for at least 2 hours at 4°C. Soluble immune complexes were
incubated with 100 µL protein-G-agarose beads for 3 hours and
bead/protein complexes recovered by centrifugation. Beads were washed 3 times for 20 minutes with lysis buffer, and bound material was eluted by boiling in 1% SDS. The immune complexes were resolved by SDS-PAGE and blots probed as described above. Differences were evaluated with the use of a GS-700 Imaging Densitometer (Bio-Rad), and the images
were then quantitated with the use of Molecular Analyst software.
A histone H1 kinase assay was performed as follows: Cdk2-associated
kinase activity was assayed in cdk2- or cyclin-E-immune complexes.
Cell lysates were prepared as described above, and cyclin-E or cdk2 was
immunoprecipitated from similar quantities of protein. Bead/protein
complexes were washed 3 times with lysis buffer and twice with kinase
buffer (50 mmol/L Tris-HCl [pH 7.5], 10 mmol/L MgCl2, and
1 mmol/L dithiothreitol). Then, 5 µg histone H1 (Boehringer
Mannheim), 1 µM ATP, and 10 µCi [r-32P] were added to
the kinase buffer for 30 minutes at 30°C. The reaction was stopped
by adding Laemmli sample buffer and boiling for 3 minutes. Reaction
products were resolved by SDS-PAGE. The gel was dried and exposed to
X-ray film. Differences were evaluated by scanning images with the use
of a GS-700 Imaging Densitometer (Bio-Rad); the images were then
quantitated with the use of Molecular Analyst software.
Results of experimental points obtained from multiple experiments were
reported as the mean ± SEM. Significance levels were determined by
a 2-sided Student t test.
| |
Results |
Cell cycle status of mobilized PB progenitors
We have shown that 25% to 30% of CFC present in normal,
steady-state BM are in S phase,5,7 and that entry in S
phase is inhibited following coculture with FN5 or when
1-integrins are engaged directly by adhesion-blocking
antibodies.7 We now show that 95% to 99% of mobilized PB
CFC and CD34+ are in G0/G1
(n = 5); this is consistent with other published reports.41,42 Culture for 48 hours with cytokines at
concentrations found in the BM microenvironment (200 pg/mL GM-CSF, 1000 pg/mL G-CSF, 200 pg/mL SCF, 50 pg/mL LIF, 200 pg/mL MIP-1, and 1000 pg/mL IL-6)37 caused 23 ± 3% of mobilized PB
CD34+ cells (n = 4) and 29 ± 4% of mobilized PB
CFC (n = 14) to enter S phase. We then evaluated whether
G-CSF-mobilized PB CD34+ cells undergo similar
adhesion-mediated proliferation inhibition as CD34+ cells
in steady-state BM.
In a first set of experiments, CD34+ cells were induced to
proliferate by culturing in low doses of cytokines for 48 to 72 hours.
Cultured PB CD34+ cells were then plated for 12 hours in
FN-, BSA-, or PLL-coated wells. We have previously shown that although
mobilized PB CD34+ cells express fewer 41 integrins,
and therefore interact less well with fibronectin, expression of
41 increases to normal levels after 24-to-48-hour culture with
cytokines ex vivo. Coculture with FN, but not with BSA or PLL, resulted
in a significant decrease in the portion of CFC
(8.5 ± 3%) (Figure 1A) and
CD34+ cells (11.5% ± 2%) (Figure 1B) adherent to FN
in S phase. Several observations indicate that this is not due to
selective adhesion of G0/G1 cells: (1) The
percentage of CFC in S phase in the adherent and the
nonadherent populations combined was significantly lower for cells
cocultured with FN than for cells cultured with either BSA or PLL; (2)
Incubation of CD34+ cells with the
1-integrin-activating antibody 8A2 increased CD34+ cell
adhesion (without 8A2, 10 ± 1%; with 8A2, 40 ± 1.4%). The cell cycle status of CD34+ cells present in the FN-adherent
and FN-nonadherent portions of assays performed in the
presence or absence of 8A2 was, however, equivalent (FN-adherent
CD34+ cells: without 8A2 = 11 ± 3% S-phase,
n = 3; with 8A2 = 11.5 ± 1.6% S phase, n = 9;
FN-nonadherent CD34+ cells: without 8A2 = 21 ± 4%
S phase, n = 3; with 8A2 = 22.5 ± 2% S phase, n = 9); (3)
Engagement of 1-integrins (4 ± 2.4% S phase), 5-integrins
(10 ± 5.8% S phase), and 4-integrins (10.5 ± 6.8% S
phase), but not the 2-integrin or CD62L, with adhesion-blocking antibodies and cross-linking with a secondary goat antimouse antibody decreased the portion of CFC in S phase (Figure
2). Although the portion
of CFC exposed to anti-4-b anti-5-b or anti-1-integrin antibodies and FN-adherent CFC that were in S phase was similar, the
percentage of CFC cultured in FN-coated wells in S phase (ie, in
FN-adherent plus FN-nonadherent portions) was higher
than in antibody-exposed CFC. This is consistent with the fact that
most CD34+ cells express 1-integrins, and
antibody-mediated engagement of 1 therefore suppresses S-phase entry
in the majority of cells, whereas only 10% to 15% adhere to FN in the
absence of 8A2. Adhesion via 1-integrins therefore inhibits cell
cycle progression in the adherent portion only. Thus,
these studies are consistent with the concept that block in cell cycle
progression in FN-adherent cells is caused by engagement of integrins
by FN and not by selective adhesion of
G0/G1 cells.
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| Fig 1.
Adhesion to FN.
Adhesion to FN decreases portion of blood and BM
CD34+ cells and CFC that are in S phase. CD34+
cells from steady-state BM or mobilized PB were cultured for 48 hours
in serum-free medium with low-dose cytokines. Cells were then plated in
wells coated with PLL or FN for 12 to 16 hours. We collected adherent
(Adh) and nonadherent (NA) cells separately. We analyzed the S phase of
CFC by thymidine suicide assay and analyzed the S phase of all
CD34+ cells by labeling cells with propidium iodide and
analysis by FACS. (A) Thymidine suicide assay (n = 4 for BM
and n = 4 for PB). Data are shown as mean ± SEM. Comparison
between the FN-Adh and the FN-NA portion for PB or BM:
* = P < .01. (B) FACS analysis of propidium iodide
labeled cells (n = 12). A representative experiment is shown.
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| Fig 2.
Antibody-mediated engagement of 1-integrins.
Antibody-mediated engagement of 1-integrins inhibits S-phase entry
of cultured blood CFC. Mobilized PB CD34+ cells cultured
for 48 hours in serum-free medium with low-dose cytokines were
incubated with adhesion-blocking antibodies against the 2 (n = 3),
4 (n = 6), 5 (n = 5), or 1 (n = 6)
integrins or CD62L (n = 3) or mouse IgG (n = 6). After incubation
for 30 minutes at 4°C, cells were washed and incubated with goat
antimouse antibody. Cells were incubated for 8 to 12 hours at 37°C,
and the percentage of CFC in S phase was assessed by thymidine suicide
assay. Data are shown as the mean ± SEM. Comparison between
anti-1, anti-4b and anti-5 group and IgG control group:
* = P < .01.
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To confirm this further, we tested the hypothesis that culture of
freshly isolated PB CD34+ cells, which are in
G0/G1, on FN rather than on BSA- or
PLL-containing wells would delay entry of CD34+ cells into
S phase (Figure 3). Freshly sorted
CD34+ cells (n = 2) suspended in low-dose
cytokine-containing serum-free medium were cultured in wells coated
with FN or PLL. After 24, 48, and 60 hours, adherent and nonadherent
cells were collected, and cell cycle status was assessed by FACS. On
day 0, 0.9% and 1% of CD34+ cells were in S phase. For
PLL-adherent cells, this increased to 5% and 3% after 24 hours, to
17% and 12% after 48 hours, and to 17% and 16% after 60 hours. For
cells in the FN-nonadherent CD34+ cell
portion, 6% and 4% were in S phase after 24 hours,
17% and 13% after 48 hours, and 18% and 16% after 60 hours. In
contrast, for CD34+ cells present in the FN-adherent
portion, only 3% and 2% were in S phase after 24 hours, 7% and 4% after 48 hours, and 10% and 4% after 60 hours. This confirms that contact with FN prevents S-phase entry.
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| Fig 3.
Coculture with FN.
Coculture with FN prevents entry into S phase and is associated with
elevated p27KIP1 levels. G-CSF-mobilized PB
CD34+ cells were analyzed fresh or after coculture with FN
or PLL for 60 hours in the presence of serum-free medium supplemented
with low concentrations of cytokines. Adherent and nonadherent cells
were collected separately and labeled with propidium iodide (FACS
analysis cell cycle status, n = 3) or subjected to Western blot
(representative example of 2 experiments; levels of
p27KIP1) as described in "Materials and methods."
Quantitative differences in protein levels were evaluated by scanning
images with the use of a GS-700 Imaging Densitometer, and the images
were quantitated using Molecular Analyst software.
Values under Western blot represent relative density of each band
compared with day 0 levels of p27KIP1.
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|
G1/S blockade is associated with increased
p27KIP1 levels but decreased cyclin-E protein levels
and decreased cdk2 kinase activity
We next examined the effect of adhesion to FN on cell cycle protein
expression and activity. CD34+ cells were plated for 48 to
72 hours in low-dose cytokine-containing medium to induce entry of
cells into S phase. Cells were then plated in dishes coated with FN or
PLL for 12 to 16 hours, and adherent and nonadherent cells were
collected separately, permeabilized, and stained with antibodies
directed at cell cycle-associated proteins. Since no significant
differences were seen in the cell cycle status of cells assayed in the
presence (FN-adherent: 11.4 ± 3.1% S phase; FN-nonadherent:
26.7 ± 4.9% S phase, n = 3) or absence of low-dose cytokines
(FN-adherent: 11.5 ± 2% S phase; FN-nonadherent:
22.5 ± 2.3% S phase, n = 9), or in the presence or absence of
the activating antibody 8A2 (see above), results from assays with or
without 8A2 or with or without low-dose cytokines during the adhesion
assay were pooled.
Cyclin-E protein levels were lower in FN-adherent compared with
FN-nonadherent cells, and FN-adherent cells had elevated levels of
p27KIP1 compared with FN-nonadherent cells (Figure
4, representative example of 5 individual
experiments). FN-adherent cells contained slightly less cyclin-A than
FN-nonadherent cells, and levels of PCNA,
cyclin-D1 + 2 + 3, p16INK4A, and
p21CIP1 were similar in FN-adherent and FN-nonadherent
cells (not shown). These results were confirmed by immunoprecipitation
and Western blot. All blots were analyzed by densitometry to obtain
quantitative results. Levels of p27KIP1 were
1.99 ± 0.1-fold higher (P < .01) in FN-adherent
compared with FN-nonadherent cells (Figure
5, representative experiment of 3 individual experiments). Levels of cyclin-E were 4.4 ± 0.9-fold lower in FN-adherent compared with FN-nonadherent cells (Figure 5). We
also immunoprecipitated cdk2 from FN-adherent and FN-nonadherent cells.
Immunoprecipitates were then evaluated for the amount of cdk2 present
and the activity of the kinase. In FN-adherent cells, cdk2 activity was
3.46 ± 0.16 lower than in FN-nonadherent cells (Figure
5). However, the total cdk2-protein level was not significantly different between FN-adherent and FN-nonadherent cells (Figure 5).
Further, the amount of cdk2 that coimmunoprecipitated with cyclin-E was
3.8 ± 0.25-fold lower in FN-adherent than FN-nonadherent cells
(not shown). No differences between cells that were adherent or
nonadherent to PLL were seen in PCNA,
cyclin-D1 + 2 + 3, cyclin-E, cyclin-A, cdk2,
p16INK4A, p21CIP1, and
p27KIP1 protein levels and in cdk2-activity (Figures 4, 5,
and not shown).
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| Fig 4.
Adhesion to FN.
Adhesion to FN leads to increased levels of p27KIP1 and
decreased levels of cyclin-E. Mobilized blood CD34+ cells,
cultured for 48 hours in serum-free medium with low-dose cytokines,
were incubated with the activating anti-1-integrin antibody 8A2 for
30 minutes at 37°C and plated in PLL- or FN-coated dishes for 12 to
16 hours. Adherent and nonadherent cells were collected separately.
Cells were fixed, permeabilized, and incubated with antibodies directed
at p27KIP1 and cyclin-E (open histogram) or isotype control
(closed histogram) antibody at room temperature for 30 minutes followed
by FITC-conjugated goat antimouse immunoglobulin for
30 minutes in the dark. Cells were washed and resuspended in 50µg/mL
propidium iodide. Cells were analyzed on a FACS-Calibur flow
cytometer with the use of CellQuest software. A representative
example of 5 individual experiments is shown.
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| Fig 5.
Adhesion to FN.
Adhesion to FN is associated with increased levels of
p27KIP1 and decreased cdk2-kinase activity. Mobilized blood
CD34+ cells cultured for 48 hours in serum-free medium with
low-dose cytokines were incubated with 8A2, washed, and plated in PLL-
or FN-coated dishes for 12 to 16 hours. Adherent (Adh) and nonadherent
(NA) cells were collected separately, and cells were lysed. A
representative example of 3 individual experiments is shown. For
p27KIP1, protein extracts were separated by SDS-PAGE,
transferred onto nitrocellulose, and incubated with antibodies against
p27KIP1 and goat antimouse HRP-conjugated antibody.
Cyclin-E was immunoprecipitated from protein-G-agarose
beads. Immune complexes were separated by SDS-PAGE and
blots probed with anti-cyclin-E antibodies and goat antimouse
HRP-conjugated antibody. Protein bands were visualized with the use of
the ECL detection system, and cdk2 was immunoprecipitated with the use
of protein-G-agarose beads. The immune complexes were separated by
SDS-PAGE, and blots were probed with anti-cdk2 antibodies and goat
antimouse HRP-conjugated antibody. Cdk2 activity was assayed by adding
5 µg histone and 10 µCi [r-32P]. Reaction products
were resolved by SDS-PAGE, and the gel was exposed to X-ray film.
Differences in protein levels were evaluated by scanning images with a
GS-700 Imaging Densitometer and quantitated with the use of Molecular
Analyst software. Relative protein levels/kinase activity values are
shown below all lanes (PLL-nonadherent is arbitrarily 1).
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When freshly isolated CD34+ cells were cultured for 60 hours on PLL- or FN-coated dishes in the presence of low doses of
cytokines, similar results were seen: p27KIP1 levels were
1.5-fold higher in the FN-adherent portion compared with FN-nonadherent portion, and 1.8-fold and 2.3-fold
compared with PLL-adherent and PLL-nonadherent
portions, respectively. Interestingly, the level of
p27KIP1 was 2.13-fold higher in CD34+ cells
cultured for 60 hours in FN-coated wells in the presence of low doses
of cytokines compared with freshly isolated and uncultured CD34+ cells, even though both populations contained only a
small portion of cells in S phase. Thus,
contact with FN, rather than G0/G1 state
of the cell, is associated with increased levels of p27KIP1
(Figure 3).
IL3 or SCF does not alter adhesion of CD34+ cells
and CFC to FN but overrides p27KIP1-mediated
G1/S blockade following 1-integrin engagement
We next examined if supraphysiological concentrations of cytokines
known to stimulate progenitor growth would affect integrin-mediated functions. Cells were cultured for 48 to 72 hours in low-dose cytokine-containing medium to induce S-phase entry. Cells were then
resuspended in serum-free medium either without cytokines or with 10 ng/mL IL3, GM-CSF, SCF, or Flt-3L. The portion of
CD34+ cells or CFC that adhered to FN was similar when
adhesion assays were done in the absence or presence of any of the
following: IL3, GM-CSF, SCF, or Flt-3L (Table
1). In contrast to CFC
cultured in contact with FN in the absence of cytokines, contact with
FN in the presence of 10 ng/mL IL3, GM-CSF, SCF, or Flt-3L prevented inhibition of G1/S-phase progression of CFC, and contact
with FN in the presence of IL3 or SCF also prevented the
G0/G1 blockade in CD34+
cells (Table 2).
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Table 1.
Adhesion of CD34+ cells and CFC to FN in
the presence or absence of supraphysiological concentrations of
cytokines
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Table 2.
Percentage of CD34+ cells and CFC in S
phase following coculture with FN in the presence or absence of
supraphysiological concentrations of cytokines
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We next analyzed the expression level and activity of cell cycle
proteins in FN-adherent or FN-nonadherent cells in cultures supplemented with IL3 or SCF (n = 3). To increase the
portion of FN-adherent cells, we added the activating
anti-1-integrin antibody 8A2. Except for an increase in the
portion of adherent cells, no differences were seen in
the proliferative status of FN-adherent and FN-nonadherent cells or in
the levels of cell cycle-associated proteins in the presence or
absence of 8A2 (not shown). Levels of cyclin-E were higher in
CD34+ cells adherent to FN in the presence of either IL3
(Figure 6) or SCF (data not shown) compared
with CD34+ cells adherent to FN in the absence of
cytokines. In contrast to cytokine-free assays, p27KIP1
levels were not elevated in CD34+ cells adherent to FN in
the presence of IL3 (Figure 6) or SCF (data not shown). Levels of the
other cell cycle proteins, including PCNA,
cyclin-D1 + 2 + 3, p21CIP1, and
p16INK4A, were similar in the presence or absence of IL3 or
SCF (not shown). Immunoprecipitation and Western blot and kinase assays
(n = 3) confirmed that IL3 prevents the accumulation of
p27KIP1 and allows activation of cdk2, even when
CD34+ cells were adherent to FN (Figure
7).
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| Fig 6.
Presence of IL3 during the adhesion assay.
Presence of IL3 during the adhesion assay prevents up-regulation of
p27KIP1 and allows up-regulation of cyclin-E. Mobilized
blood CD34+ cells cultured for 48 hours in serum-free
medium with low-dose cytokines were incubated with 8A2 and plated, with
or without 10 ng/mL IL3, in PLL- or FN-coated wells for 12 to 16 hours.
Adherent and nonadherent cells were collected separately, fixed,
permeabilized, washed, and incubated with antibodies directed at
p27KIP1 and cyclin-E (open histogram) or isotype control
(closed histogram) at room temperature for 30 minutes, followed by
FITC-conjugated goat antimouse immunoglobulin. A
representative example of 3 individual experiments is shown. Adhesion
to PLL in the presence or absence of IL3 did not affect the expression
level of cyclin-E or p27KIP1 (not shown). However,
up-regulation of p27KIP1 and down-regulation of cyclin-E
levels are prevented when IL3 is present during the adhesion assay.
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| Fig 7.
Presence of IL3 or SCF during the adhesion assay.
Presence of IL3 or SCF during the adhesion assay prevents up-regulation
of p27KIP1 and inhibition of cdk2-kinase. Mobilized blood
CD34+ cells cultured for 48 hours in serum-free medium with
low-dose cytokines were incubated with 8A2 and plated, with or without
10 ng/mL IL3, in PLL- or FN-coated dishes for 12 to 16 hours. Adherent and nonadherent cells were collected
separately, and cells were lysed. Methods used to demonstrate presence
and activity of p27KIP1, cyclin-E, and cdk2-kinase activity
are as described in legend to Figure 5. Differences in protein levels
were evaluated by scanning images with a GS-700 Imaging Densitometer
and quantitated with the use of Molecular Analyst software. A
representative example of 2 individual experiments is shown. Relative
protein levels/kinase activity values are shown below all lanes
(PLL-nonadherent is arbitrarily 1).
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Discussion |
We still do not know which signals are responsible for the
regulation of hematopoietic progenitor proliferation, quiescence, or
differentiation. More than 30 hematopoietic cytokines and growth factors that increase or decrease progenitor proliferation and differentiation have been cloned and characterized. Although the molecular effect of these factors on hematopoietic cells has been extensively studied, it remains unclear how the combination of these
factors regulates the hematopoietic process. In vivo hematopoiesis normally occurs in close proximity with BM stromal elements. Coculture of progenitors with BM stromal feeders in vitro inhibits progenitor proliferation through mechanisms that are as yet not
understood.4-6,43,44 Progenitors, as well as cytokines, can
bind specifically with ligands on cells and extracellular matrix
present in the BM,1-3,45,46 resulting in the colocalization
of progenitors at a specific stage of differentiation with a specific
array of cytokines.47 This is thought to provide one level
of regulation of growth and differentiation. There is also mounting
evidence that contact interactions between progenitors and BM stromal
ligands may be equally, or even more, important for the regulation of
the hematopoietic process. Recent studies have shown that engagement
of, for instance, selectins48 and mucins49,50
may profoundly affect progenitor survival and growth. Likewise,
engagement of integrins may enhance progenitor survival in
culture.51 We describe here how integrin engagement alters
progenitor growth.
We used 2 experimental assays to investigate these questions. Almost
all CD34+ cells collected from the PB of individuals
treated with G-CSF are in G0/G1. In 1 set of studies, we showed that FN coculture of PB CD34+
cells that are in G0/G1 prevents entry
into S phase compared with cells cocultured with PLL or BSA, suggesting
that engagement of 1-integrins prevents entry into cell
cycle. This is consistent with what we have previously
shown for steady-state marrow-derived CD34+
cells4-7 and what we show here for CD34+ cells
present in cultured (and hence proliferating) mobilized PB
CD34+ cell populations: when cocultured with FN, but not
PLL, the portion of CD34+ cells in S phase
declines. Using blocking monoclonal antibodies, we showed that
induction of G1/S blockade by adhesion-receptor engagement
in human hematopoietic progenitors is 1-integrin specific: in
contrast to 1-integrins, engagement of other transmembrane adhesion
receptors, such as CD44,7 CD34,7 and CD62L
(data shown here), did not affect the proportion of progenitors that is
in S phase.
1-integrin-mediated block in G1/S
transition in hematopoietic cells occurs in late G1 and is
associated with elevated levels of p27KIP1 and inactivation
of cyclin-E/cdk2 complexes. Of interest is the fact that levels of
p27KIP1 were significantly less elevated in uncultured
CD34+ cells selected fresh from mobilized PB than in
CD34+ cells cultured for 60 hours with low concentrations
of cytokines in contact with FN. This suggests strongly that elevation
of p27KIP1 is not merely a reflection of presence in
G0/G1 status but is correlated with
engagement of integrins.
This is in contrast to what has been described in the majority of other
biological systems: engagement of integrins usually activates
cyclin-D/cdk4 and cyclin-E/cdk2 or cyclin-A/cdk2 complexes leading to cell proliferation but not growth
arrest.13-15,25-27 In contrast to the CD34+
cells studies here, those reports describe the effect on cell cycle proteins in cells that require adhesion for cell growth, such as
fibroblasts, endothelial cells, and myocytes. A few examples have
been described of adhesion-mediated G1-phase arrest
mediated through mechanisms similar to what we show here for
hematopoietic cells: growth arrest occurred late in
G1 and was due to elevated levels of the "contact"
cdki p27 KIP1, which inhibits cyclin-E/cdk2 kinase
activity.52-55 When 51-mediated adhesion of the
FET colon carcinoma cell line to FN was prevented, FET
cells were induced to proliferate.51 This was associated with activation of extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase-2 and significantly elevated levels of cdk4, phosphorylation of Rb, and increased cyclin-A/cdk2 and
cyclin-E/cdk2-associated kinase activity. Thus, as we
show here for hematopoietic cells, 51 engagement in FET colon
carcinoma cells suppresses cyclin-dependent kinase activity, leading to growth arrest. G1-phase growth arrest was also seen when
arterial smooth muscle cells were cultured on polymerized type I
collagen.53 This was associated with elevated levels of
p27KIP1 and p21CIP1 and decreased
cyclin-E-associated cdk2 kinase activity. In contrast, when
cultured on monomer collagen, arterial smooth muscle cell proliferation was induced rather than growth arrest. Like FN-adherent human CD34+ cells, which are round even in the adherent
state, arterial smooth muscle cells adherent to polymeric collagen
adhere in a rounded state and do not spread. These results suggest
that, like lateral association and ligand-binding-site
occupation,10 cell shape may be an additional parameter
that dictates the type of molecules recruited to focal adhesions and
therefore the type of signal pathways that are activated or blocked.
This is consistent with recent studies by Chen et al54
demonstrating that cell death is influenced not only by integrin
engagement by a substrate but also by cell shape.
That cell-cell contact causes normal cells to stop proliferating has
long been known in normal organ development. More recently it has
become clear that such contact-mediated growth arrest is mediated by
up-regulation of the cdki, p27KIP1,56,57 which
inactivates cyclin-E/cdk2 and cyclin-A/cdk2 complexes. This is
illustrated in mutant p27 KIP1 / mice that
display generalized increased body size and a significantly expanded
hematopoietic progenitor pool.56,57 This appears to be
consistent with our observation that cell-ECM interaction elevates levels of p27 KIP1 and inhibits G1/S
progression of CD34+ progenitors. Regulation of
p27KIP1 levels is complex.58,59 Elevated levels
can be due to increases in transcription, messenger RNA (mRNA)
stabilization, or decreased protein degradation. Preliminary results
from ribonucleotide protection assays (results not shown) suggest that
the regulation of p27KIP1 by integrin engagement on
CD34+ cells may not be at the transcriptional level.
Finally, we found that integrin-mediated block in G1/S
transition can be modulated when external conditions change: addition of supraphysiological concentrations of IL3 and GM-CSF, which bind to a
common receptor in the hematopoietic receptor binding family,60 or SCF and Flt-3L, which signal via tyrosine
kinase receptors,61 counteracts the adhesion-mediated block
in G1/S progression. Recent studies from other groups have
shown that IL3 and SCF activate protein kinase C (PKC) and tyrosine
kinases that lead to enhanced adhesive capacity of 1-integrins
present on serum- and cytokine-starved CD34+ cells and
CFC.28-30 In contrast, we show that
supraphysiological concentrations of IL3, GM-CSF, SCF, or Flt-3L did
not affect adhesion of human CFC or CD34+ cells when they
were maintained under physiological conditions. Our results are
consistent with studies from Strobel et al32 and Schofield
et al31 demonstrating that
supraphysiological concentrations of cytokines do not alter the
adhesive function of integrins under serum/cytokine-replete
conditions. Even though adhesion of CD34+ cells to FN was
not affected by the presence of IL3 or SCF, presence of these
supraphysiological concentrations of cytokines prevented adhesion-mediated increased p27KIP1 levels and prevented
adhesion-mediated inhibition of cdk2 activity. Signal pathways
activated by cytokine stimulation have been extensively studied.
Stimulation of either hematopoietic or tyrosine kinase receptors leads
to recruitment and activation of signal and adaptor proteins and to
activation of signal pathways that are also recruited/activated by
integrin stimulation. Like integrin engagement, stimulation of c-kit
leads to phosphorylation of paxillin and CrkL, activation of
PI3-kinase, Ras, and MAPK.60,61 Likewise, IL-3
phosphorylates CrkL and paxillin and activates PI3-kinase and MAPK,
molecules known to be recruited to and activated in focal
adhesions.62-68 Thus, significant overlap exists between
signal pathways activated following cell adhesion or stimulation with
cytokines. Since the mechanism(s) that underlie adhesion-mediated
signaling in hematopoietic cells are unknown, we can only speculate how
the combined activation of integrin and cytokine receptors affects
hematopoietic cell growth. However, the characterization of conditions
that allow adhesion without cell cycle arrest and conditions that
induce adhesion with cell cycle arrest should prove useful in
deciphering the signal pathways that are activated by engagement of
integrins leading to growth arrest of human hematopoietic progenitors.
In conclusion, we demonstrate that cell cycle arrest and reduced
proliferation of CD34+ cells, which are adherent to FN
under physiological cytokine conditions, are mediated by the cdki,
p27KIP1. However, costimulation of
FN-adherent progenitors with certain growth-promoting cytokines
overrides this integrin-dependent elevation of p27KIP1 and
allows S-phase entry of CD34+ cells. Future studies will
identify the molecular mechanisms underlying the reversible inhibition
of entry in S phase mediated by changes in p27KIP1.
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Footnotes |
Submitted July 6, 1999; accepted September 26, 1999.
Supported in part through grants from the National Institutes of Health
(RO1 HL-49930 and RO1-DK-53673) and the Bone Marrow Transplant Research
Fund to C. M. V. and a grant from Fondo De Investigaciones
Sanitarias(FIS 98/0863) to F.P. C. M. V. is a scholar of the Leukemia
Society of America.
Y. J. and F. P. contributed equally to this paper.
Reprints: Catherine M. Verfaillie, Box 806 UMHC, 420 Delaware St SE, Minneapolis, MN 55455; e-mail: verfa001{at}tc.umn.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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References |
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Abstract
Introduction