From the Department of Experimental
Pathology and Oncology, University of Firenze, Viale G. B. Morgagni, 50, 50134 Firenze, Italy, the § U. O. Hematology, Policlinico di Careggi, 50134 Firenze, Italy, the
** Department of Biotechnology and Biosciences, University of Milano
Bicocca, Via Emanueli, 12, 20126 Milano, Italy, and the
Department of Genetics, Biology and Biochemistry, University of
Torino, Via Santena 5bis, 10126 Torino, Italy
Received for publication, June 28, 2000, and in revised form, October 27, 2000
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ABSTRACT |
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Integrin receptors have been
demonstrated to mediate either "inside-to-out" and
"outside-to-in" signals, and by this way are capable of regulating
many cellular functions, such as cell growth and differentiation, cell
migration, and activation. Among the various integrin-centered
signaling pathways discovered so far, we demonstrated that the
modulation of the electrical potential of the plasma membrane
(VREST) is an early integrin-mediated signal, which
is related to neurite emission in neuroblastoma cells. This modulation
is sustained by the activation of HERG K+ channels, encoded
by the ether-à-go-go-related gene (herg). The involvement of integrin-mediated signaling is being discovered in
the hemopoietic system: in particular, osteoclasts are generated as
well as induced to differentiate by interaction of osteoclast progenitors with the stromal cells, through the involvement of integrin
receptors. We studied the effects of cell interaction with the
extracellular matrix protein fibronectin (FN) in a human leukemic preosteoclastic cell line (FLG 29.1 cells), which has been
demonstrated to express HERG currents. We report here that FLG 29.1 cells indeed adhere to purified FN through integrin receptors, and that
this adhesion induces an osteoclast phenotype in these cells, as
evidenced by the appearance of tartrate-resistant acid phosphatase, as
well as by the increased expression of
CD51/ The integrin family of adhesion receptors functions not only as
ligands for the extracellular matrix
(ECM),1 but can influence
many aspects of cell behavior, including morphology, adhesion,
migration, as well as cellular proliferation and differentiation. In
fulfilling these functions, integrins are not simply adhesion receptors, but can affect many signaling pathways, and therefore impinge upon complex cellular activities. Therefore integrin function itself is highly regulated, largely through the formation of specific associations with both structural and regulatory components within the
cell. Recently, much research has focused on elucidating the molecular
mechanisms, which control integrin function and transduce integrin-mediated signaling events.
In particular, in the hemopoietic system, it is well known that
adhesive interactions between hemopoietic precursors and the bone
marrow microenvironment play a critical role in regulating hemopoiesis
(1, 2), and that such interactions are often mediated by integrin
receptors (3, 4). Among hemopoietic precursors, the progenitors of
osteoclasts are formed through a contact-dependent
interaction between bipotential osteoclast-macrophage precursors and
stromal cells, which express osteoclast forming activity (5, 6).
Furthermore, osteoclast differentiation and activation leading to bone
remodeling is finely regulated by interaction of osteoclasts with
stromal cells (reviewed in Ref. 7) through the involvement of
integrin receptors, which are numerous on the osteoclast plasma
membrane (8). Therefore, osteoclast precursors can be envisaged as a
good model wherein studying the relationships between the ECM,
integrin-mediated signals, and the induction of cell
differentiation/activation.
Among the various integrin-centered signaling pathways discovered so
far (9, 10), we demonstrated that the modulation of VREST
is an early integrin-mediated signal, which is related to neurite
emission in neuroblastoma cells (11-13). This modulation is sustained
by the activation of a peculiar type of K+ channels, the
HERG channels (14, 15), encoded by the
ether-à-go-go-related gene (herg). These
are channels with limited hyperpolarizing potency, clamping
VREST to substantially depolarized values (around We report here that FLG 29.1 cells indeed adhere to purified FN through
integrin receptors, and that this adhesion is accompanied by the
appearance of osteoclast differentiation markers, namely tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CtR),
as well as CD51/ Cell Culture--
FLG 29.1 were obtained in Dr. P. A. Bernabei's laboratory (Hematological Unit, Florence, Italy) as
previously reported (22). Cells were routinely cultured in RPMI medium
(Hyclone) supplemented with 10% fetal calf serum (Hyclone)
(complete medium) and incubated at 37 °C in 10% CO2.
For experiments, cells were harvested from freshly seeded preparatory
cultures and resuspended (2.5 × 105 cells/ml) in RPMI
containing 250 µg/ml heat-inactivated bovine serum albumin (BSA)
(RPMI + BSA) (for cell adhesion experiments) or in complete medium (for
differentiation experiments). Cells were then plated onto either 35-mm
Petri dishes (Costar) (for patch clamp experiments) or dishes in which
glass slides had been accommodated (for immunocytochemistry
experiments) or 10-cm Petri dishes (for RNA or protein extraction).
Preparation of Substrates and Coating of Culture
Dishes--
Heat-inactivated BSA was prepared as described previously
(11). Coating of culture dishes with FN or vitronectin (VN) was performed by adding the two proteins, prepared as reported in Ref. 11,
at 100 µg/ml in RPMI at 37 °C for 1 h. Thereafter, FN or VN
solution was poured off and dishes were further incubated for 30 min
with RPMI + BSA to saturate the free binding sites of the culture dish
surface. Dishes were then immediately used for experiments or stored
with PBS at 4 °C and used afterward (1 or 2 days).
Experiments with substrate-bound as well as soluble anti-integrin
antibodies were performed by using the following antibodies: (a) anti- Adhesion Assay--
Adhesion assay was performed essentially
according to Arcangeli et al. (11). Briefly, stock cultures
were radiolabeled during 36 h of exponential growth in RPMI medium
containing 1 µCi/ml [methyl-3H]thymidine
(specific activity 24 Ci/mmol). After this time, cells were harvested,
pelleted, and resuspended in RPMI + BSA. Aliquots of cells (14 × 103) were inoculated into each well of 96-well clusters
(Corning-Costar), previously coated with the adhesive proteins (see
above). In the appropriate samples, blocking antibodies to FN-receptor
( Induction of Cell Differentiation--
For induction of cell
differentiation, TPA (Sigma) was dissolved in dimethyl sulfoxide at
10 Pertussis Toxin Treatment--
Pertussis toxin (Calbiochem)
dissolved in water was added to cell cultures at a final concentration
of 100 ng/ml, and cells were incubated in its presence for 14-20 h.
After this time, cells were harvested and resuspended in RPMI + BSA in
the absence of the toxin, and seeded on BSA-, FN-, or
anti- Patch Clamp Recordings--
Cells plated on dishes were
incubated at 37 °C for various times. Patch clamp experiments were
performed at room temperature with an amplifier Axopatch 1-D (Axon
Instruments, Foster City, CA), replacing the Petri dishes every 30 min.
The whole cell configuration of the patch clamp technique (27) was
employed using pipettes (borosilicate glass; Hilgenberg, Germany) whose
resistance was in the range 3-5 M RNA Extraction and Northern Blot--
Total RNA was extracted
from FLG 29.1 cells by the guanidinium/isothyocyanate method (28). Ten
or 20 µg of total RNA were loaded on a formaldehyde-formamide
reducing agarose gel run at 80 mA for 3-4 h. Six micrograms of an RNA
ladder (Life Technologies, Inc.) were also loaded. After staining with
ethidium bromide, the gel was photographed and the position of the
standards marked. RNA was then transferred by Northern blot onto a
nylon membrane (Hybond N+; Amersham Pharmacia Biotech) and
hybridized in Church Buffer (Na phosphate monobasic, 0.5 M,
pH 7.2, 7% SDS, 1 mM EDTA) containing 108
cpm/mg DNA of the appropriate probe (see below), at 65 °C overnight. Filter were then washed twice in Na phosphate 50 mM, SDS
1% for 5 min at room temperature, and once at 65 °C for 30 min, and
exposed to x-ray film (Hyperfilm; Amersham Pharmacia Biotech) overnight at Probes and Plasmids--
A BamHI-HindIII
full-length (3.5 kilobase) fragment of the HERG gene cloned in SP64
vector (29) kindly gifted by Dr. M. Keating (University of Utah, Salt
Lake City, UT) was random priming labeled using [32P]dCTP
(Amersham Pharmacia Biotech), and the probe purified on Sephadex
columns as described (28). An 18S probe (Ambion) was labeled as above.
Detection of Calcitonin Receptor by
Reverse-Transcriptase-Polymerase Chain Reaction (RT-PCR)--
Total
RNA was extracted as reported above; cDNA was then synthesized from
1 µg of RNA using 200 units of reverse transcriptase SuperScript II
(Life Technologies), plus 200 µM of each dNTP and 2.5 µM random hexamers, in a 20-µl final reaction
volume, for 50 min at 42 °C. 5 µl of such reaction were used to
perform the specific amplification of the CtR, using 2.5 units of
Platinum Taq polymerase (Life Technologies), 200 µM of each dNTP, 1.5 mM MgCl2,
and 0.5 µM of the specific primers (see below).
Amplification was performed in a Robocycler (Stratagene), after an
activation step at 94 °C for 2 min, for 30 cycles with 30 s at
94 °C, 30 s at 55 °C, 60 s at 72 °C. The primers
used were: 5' sense oligonucleotide, GTATTGTCCTATCAGTTCTGCC; and 3'
antisense oligonucleotide, AGAGATAATACCACCGCAAGCG. These primers
encompass a nucleotide region from 265 to 842 of the human CtR
cDNA, and give rise to two bands, one 577 bp long, corresponding to
the human CtR1 and one 529 bp long, corresponding to the human CtR2
(30). Preliminary experiments (not reported) demonstrated that under
the conditions used in these experiments the amplification reaction was
in the exponential phase, as reported by Beaudreuil et al.
(30), thus allowing a semiquantitative analysis of the PCR products
(see below). Moreover, the two bands obtained from such experiments
were purified and sent off for sequencing, resulting in being 100%
identical to the CtR sequences present in GeneBankTM. The
human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as
internal control, from 2 µl of the same cDNA, under the above
conditions, and using the following primers, which comprise a sequence
between nucleotide 457 and 595 of the gapdh gene: 5' primer
sense, AACAGCCTCAAGATCATCAGCAA; 3' primer antisense,
CAGTCTGGGTGGCAGTGAT. Samples of the PCR products (35 µl) were
separated by gel electrophoresis on 3% agarose, and the bands,
acquired by an HP 4C scanner, were analyzed by Scion Image software.
Signals of the CtR cDNA were normalized using the values of the
corresponding products from the gapdh amplifications.
Immunoprecipitation of Integrins from Cell Surface Biotinylated
Cells--
Cells prepared as above were placed on ice and washed twice
with Hank's balanced salts (1.3 mM CaCl2, 0.4 mM MgSO4, 5 mM KCl, 138 mM NaCl, 5.6 mM D-glucose, 25 mM HEPES, pH 7.4) and biotinylated by adding 0.5 mg/ml
Sulfo-NHS-LC-Biotin (Pierce) in Hank's buffer for 15 min. Cells were
washed twice with Hank's solution, labeling was repeated and the
reaction was stopped by washing three times with serum-free Dulbecco's
modified Eagle's medium + 0.6% BSA. Labeled cells were lysed in ice
for 20 min with lysis buffer (Tris-HCl 20 mM, pH 7.4, NaCl
150 mM, glycerol 10%, Triton X-100 1%,
phenylmethanesulfonyl fluoride 1 mM, aprotinin 0.15 units/ml, leupeptin 10 µg/ml, NaF 100 mM, Na vanadate 2 mM). The supernatant was then cleared by centrifugation at
16,000 × g.
For immunoprecipitation of integrin subunits, 0.5-1 mg of total cell
lysate was incubated overnight at 4 °C with polyclonal antibodies
raised against the cytoplasmic tails of Immunoblot--
Immunoblot with anti-PKC
Immunoblot with anti-HERG antibodies was performed using a crude
membrane fraction obtained from cells seeded on 10-cm Petri dishes,
covered with BSA or FN, respectively. For membrane extraction, all
steps were performed at 4 °C. Cells were washed with PBS, scraped
off, and homogenized into 2.5 ml of TE buffer (Tris 10 mM,
pH 7.5, EDTA 1 mM) plus a protease inhibitor mixture
(Pefabloc 0.5 mM, pepstatin 1 µM, benzamidine
1 mM, aprotinin 4 µg/ml, iodacetamide 1 mM,
phenanthroline 1 mM, leupeptin 1 µg/ml). Debris and
nuclei were removed by centrifugation at 3,600 × g for
10 min, and membrane fraction was pelleted by centrifugation at
110,000 × g for 40 min. The membrane-enriched pellet
was then solubilized in 50 mM Tris-HCl, 15 mM
In any case, membrane as well as total cell proteins were then heated
in reducing Laemmli buffer at 95 °C for 3 min, separated by 7.5%
(for experiments with anti-PKC Immunocytochemistry--
Cells were seeded on glass slides
covered with BSA or FN, and incubated for 24 h as above. After
this time, cells were air-dried, fixed, and then treated with a 1:50
dilution of the primary antibody (anti-vitronectin receptors, Celbio)
in 0.05 M Tris-HCl, pH 7.6, solution in a humidified
chamber for 30 min. Then the slides were treated with an alkaline
phosphatase anti-alkaline phosphatase kit (TOP-Line Histology)
according to manufacturer's procedure. Slides were then counterstained
with Giemsa, and photographed through a Nikon microscope, then acquired
through a Minolta scanner.
Tartrate-resistant Acid Phosphatase Staining--
Cells were
cultured in slide-containing Petri dishes (see above), in complete
medium. In the appropriate samples TPA (10 To study the relationships between ECM and integrin-mediated
signals in FLG 29.1 leukemic cells, these cells were seeded on a
FN-enriched substratum and the mechanisms sustaining cell adhesion were
evaluated. In fact it had been previously shown that these cells could
adhere to endothelial cells through the FN molecules covering
endothelial cell surfaces (23) (see Introduction). Since FLG 29.1 cells
express various classes of integrin receptors, mainly belonging to the
v
3 integrin and calcitonin receptor.
An early activation of HERG current (IHERG), without any
increase in herg RNA or modifications of HERG protein was
also observed in FN-adhering cells. This activation is apparently
sustained by the
1 integrin subunit activation, through
the involvement of a pertussis-toxin sensitive Gi protein, and appears to be a determinant signal for the up-regulation of
v
3 integrin, as well as for the increased
expression of calcitonin receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
30 mV),
and are expressed in the heart (16-18), embryonic neuroblasts (19), as
well as in tumor cells of various histogenesis (12). We found that a
human leukemia cell line, FLG 29.1 cell, expressed herg, and
an HERG current (IHERG) with a very fast deactivation kinetics, which apparently justifies the low, depolarized value of
their VREST (12, 20). These cells derive from a patient with an M5a-type leukemia (21) and represent immature preosteoclastic precursors, as revealed by their treatment with phorbol esters (22).
They are capable of adhering to bone endothelium, possibly through
fibronectin (FN) molecules produced by the endothelial cells (23), and
this interaction could influence the maturation of these cells through
the osteoclastic pathway. We therefore used the
preosteoclastic FLG 29.1 cells to study HERG K+
channel-centered signals, elicited by integrin-mediated adhesion to the
ECM molecule FN, as a model for hemopoietic precursors, and their
relationships with the process of osteoclastic differentiation.
v
3 integrin. FN also
induces the activation of IHERG, without any increase in
herg RNA or modifications of HERG protein. This activation
is apparently sustained by the
1 integrin subunit
activation, through the involvement of a pertussis toxin-sensitive
Gi protein, and appears to be a determinant signal for the
up-regulation of
v
3 integrin, as well as
for the increased expression of CtR.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 monoclonal antibodies: mAb BV7
(24) and mAb TS2/16; (b) anti-
3 monoclonal
antibody (mAb B212) (25); (c) anti-
3 monoclonal antibody (F2) (26), and following the procedure essentially according to Arcangeli et al. (13). Briefly, the antibodies were diluted at 20 µg/ml in RPMI, and added to Petri dishes at 37 °C for 1 h. After coating the plastic surface, dishes were further incubated for 30 min with RPMI + BSA, as above, and thereafter immediately used for patch clamp experiments (see below). For experiments with soluble anti-
1 antibody (mAb TS2/16),
the antibody was diluted at 20 µg/ml in RPMI medium + BSA, and cells
were incubated in this solution at 2.5 × 105/ml for
10 min at room temperature, then transferred to BSA-coated Petri
dishes, and incubated for further 15 min at 37 °C.
FN-R; 1:50 dilution) (11),
3 subunit (mAb B212, 9 µg/ml) (25), or
v subunit (mAb L230) (10 µg/ml) were
added at time 0. When the mAb B212 was used, cells were preincubated
with the antibody for 20 min at 4 °C, according to Defilippi
et al. (25). When needed, GRGDSP (Telios) or GRGESP (Telios)
were added just before cell seeding at 0.5 µg/ml (final
concentration). After 60 min of incubation, the medium was aspirated
off and adherent cells gently rinsed twice with PBS containing divalent
cations; the cells were then solubilized with 50 µl of 1% SDS in 0.1 N NaOH for 1 h. Radioactive solubilized cells were
quantified by scintillation counting and compared on a percent basis
with the radioactivities of the FN-seeded cells.
3 M and added to complete medium at a final
concentration of 10
6 M.
1 antibody (mAb TS2/16)-coated dishes for 1 h.
. Extracellular solutions were
delivered through a 9-hole (0.6 mm), remote-controlled linear
positioner placed near the cell under study. The standard extracellular
solution contained (mM): NaCl 130, KCl 5, CaCl2
2, MgCl2 2, Hepes-NaOH 10, glucose 5, pH 7.4. The standard
pipette solution at [Ca2+] = 10
7
M contained (mM): K+ aspartate 130, NaCl 10, MgCl2 2, CaCl2 4, EGTA-KOH 10, Hepes KOH 10, pH 7.4. Gigaseal resistance were in the range 1-10 G
. Whole
cell currents were filtered at 2 KHz. For precise measurement of the
gating parameters of the inward rectifier channels, we carefully
compensated pipette and cell capacitance and the series resistance
before each voltage-clamp protocol run. The density of inward
IHERG was calculated at [K+]o = 40 mM as the peak current elicited at a voltage of
120 mV
after 20 s preconditioning at 0 mV, normalized by the cell
capacitance. The values of the peak current refer to the current
obtained after subtraction of the traces obtained in the presence of 5 mM Cs+ or 1 µM WAY 123,398, according to Ref. 19. The IHERG activation curves were
measured according to Refs. 14 and 15. Resting potential
(VREST) was measured at 5 mM
[K+]o in current-clamp mode (I = 0). The
leakage conductance gL was calculated at 5 mM
[K+]o by a ramp protocol ranging from
100 to
+60 mV and lasting 1280 ms, from a holding potential of 0 mV, as the
slope of the current trace obtained in the range
80 to
40 mV. The relatively slow rate of voltage change produced a negligible capacitive current. The cell capacitance was obtained directly by reading the
position of the amplifier knob of the cell capacitance compensation. Input resistance of the cells was in the range 2-6 G
. For data acquisition and analysis, pClamp software (Axon Instruments) and Origin
(Microcal Software, Northampton, MA) were routinely used.
70 °C for HERG probe, 5 min at
70 °C for 18S probe.
v or
1 integrins (a gift from Prof. G. Tarone, University of
Torino, Italy). Immunocomplexes were then bound to protein A-Sepharose
beads and recovered by centrifugation. Bound material was eluted by
boiling beads in nonreducing Laemmli buffer (Tris-HCl 62.5 mM, pH 6.8, glycerol 10%, SDS 0.2%, bromphenol blue
0.00125%), analyzed by 6% SDS-PAGE under nonreducing conditions, and
transferred to a nitrocellulose sheet. The membrane was incubated 30 min at 42 °C in T-TBS (150 mM NaCl, 20 mM
Tris-HCl, pH 7.4, 0.3% Tween), containing 5% BSA, washed with T-TBS,
and then biotinylated integrins were detected by incubating blots in
TBS, 1% BSA, containing 0.2 µg/ml horseradish peroxidase-conjugated
streptavidin (Pierce) for 30 min, washed again with T-TBS, and
developed with a chemiluminescent substrate (ECL, Amersham Pharmacia
Biotech). Images were then acquired by means of an HP4C scanner, and
densitometric analysis performed using the QuantiScan software.
antibodies was performed using a total cell protein extract. Cells,
plated on 60-mm Petri dishes covered with BSA or FN, respectively, were
collected by centrifugation, washed three times with ice-cold PBS and
lysed on ice with lysis buffer (Tris-HCl 20 mM, pH 7.4, NaCl 150 mM, glycerol 10%, Triton X-100 1%,
phenylmethanesulfonyl fluoride 1 mM, aprotinin 0.15 units/ml, leupeptin 10 µg/ml, NaF 100 nM, Na vanadate 2 mM). The supernatant was then cleared by centrifugation at
16,000 × g, for 10 min at 4 °C.
-mercaptoethanol, and 1% SDS.
antibodies) or 6% (for
experiments with anti-HERG antibodies) SDS-PAGE and transferred to a
nitrocellulose sheet. After transfer, membranes were blocked for 4 h at room temperature with PBS + Tween-20 0.1% (T-PBS), containing 5%
BSA (T-PBS-BSA) and then incubated overnight at 4 °C with
anti-PKC
antibodies (Santa Cruz Biotechnology), diluted 1:3000 in
T-PBS-BSA or with purified rabbit polyclonal anti-HERG antibody (a kind gift from Dr. Nerbonne, University of Washington, St. Louis, MO) diluted 1:500 in T-PBS-BSA. Membranes were washed 3 times with T-PBS and incubated with anti-rabbit peroxidase-conjugated secondary antibodies (Sigma) diluted 1:10,000 in T-PBS-BSA for 1 h
at room temperature. After 3 washes with T-PBS, the immunoreactivity was determined by a chemiluminescent reaction (ECL) (Amersham Pharmacia Biotech).
6
M, final concentration) was added at time 0. At different
times of incubation, cells in suspension were harvested and processed for cytospin slide preparations; cytospin preparations as well as cells
remaining adherent on the glass slides were fixed in 60% acetone in
citrate buffer, pH 5.4, for 30 s, washed twice in distilled water,
air-dried, and then stained for TRAP using a commercially available kit
(Sigma), and following the manufacturer's instructions. Stained
cultures were examined under light microscopy at a magnification
of × 250, photographed, and acquired as above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
3 classes, and associated with different
subunits (
2,
3,
4,
5 and
v)
(23)2 the role of these two
different integrin classes on cell adhesion was evaluated. As shown in
Fig. 1, about 50% of the FLG 29.1 cells adhere to FN, while this adhesion is significantly lower (about 35%)
on VN, another integrin-recognized substrate. Cell adhesion to FN is
impaired by blocking antibodies to FN receptor
(
5
1), while unaffected by blocking
antibodies to
3 subunit (25), ruling out the involvement
of
3 subunit in leukemia cell adhesion to FN. As to cell
adhesion to vitronectin, it is 40% inhibited by blocking antibodies to
FN receptor, while only slightly affected by blocking antibodies to the
3 subunit (see Fig. 1). However, FLG 29.1 adhesion to VN
is impaired by RGD containing peptides, as well as
anti-
v antibodies, suggesting the involvement of an integrin receptor containing the
v subunit in this
adhesion. Since the polyclonal anti-FN-R antibodies contain a great
amount of antibodies against epitopes belonging to the
1
subunit, the most plausible explanation for this finding is that FLG
29.1 cells recognize VN through their
V
1
integrins, a heterodimer that is indeed expressed in these cells (see
also Fig. 3). On the whole, data presented in Fig. 1 confirm that FLG
29.1 cells firmly adhere to FN, mainly through their
5
1 integrin receptors.
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Fig. 1.
Effects of various treatments on FLG 29.1 cell adhesion. Cells were seeded on FN- or VN-coated substrates
for 60 min and the adhesion test performed as described under
"Experimental Procedures." Anti-FN-R antibodies (dilution 1:50),
anti- v antibodies (mAb L230, 10 µg/ml), GRGDSP
(Telios) and GRGESP (Telios) peptides (0.5 mg/ml) were added at time 0 just before cell seeding. Anti-
3 antibodies (mAb B212, 9 µg/ml) were added to the cell suspension and preincubated at 4 °C
for 20 min before cell seeding. Values are expressed as the percentage
of adherent cells in the various samples on the total number of cells
inoculated. Values are mean ± S.E. of data carried out in
quadruplicate samples and obtained in three separate experiments.
The effect of FLG 29.1 cell adhesion to FN on cell differentiation was
then evaluated: since FLG 29.1 cells show a preosteoclastic phenotype,
as revealed by the appearance of osteoclastic markers upon TPA
treatment (22), three of these markers, namely the CD
51/V
3, CtR (30), as well as TRAP (31)
were studied. When FLG were left to adhere to FN for 24 h (in
complete medium) the appearance of immunoreactivity to CD 51 was first
detectable: in fact, this marker cannot be easily detected with this
method in control cells (Fig.
2A), while it appears in
FN-seeded cells (Fig. 2B). CD51 is also strongly reactive in
cells treated with TPA (Fig. 2C), i.e. the
classic inducer of osteoclastic differentiation of these cells (22),
indicating that the increase in immunoreactivity to CD51 correlates
with cell differentiation. The expression of TRAP could be also
detected after longer times of incubation: as shown in the figure,
while control cells barely (less than 10% of cells) express TRAP
(panel D), about 68% of cells cultured on FN-coated dishes
for at least 6 days display the marker (panel E), which is
detectable either in resuspended (E) and spread
(E') cells. Here again the expression of TRAP in TPA-treated
cells is reported as an internal control (panel F). In
panel G is reported the expression pattern of CtR as
revealed by RT-PCR: two thin PCR bands of 577 and 529 bp, corresponding
to hCtR1 and hCtR2, respectively (as assessed by sequencing the PCR
products, see "Experimental Procedures") can be detected in control
cells (lane 1). The intensity of either bands is
significantly increased (see also inset to panel
G) in cells cultured for 1 week on FN-coated dishes (lane
2), and, at a roughly identical extent, in TPA-treated cells
(lane 3).
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Since CD51 represents the vitronectin receptor
v
3, the modulation of this integrin was
studied in more detail. As shown in Fig. 3A, FLG 29.1 control cells express the
v integrin subunit, which
associates with the
1 and, although at very low levels, with the
3 subunit (see legend to the figure). These
results were confirmed by cytofluorimetric experiments performed with antibodies against external epitopes of the three integrin subunits (not shown). Moreover, expression of
V
3
integrin was 50% increased in FN-seeded cells (see inset to
Fig. 3A and the legend to the figure), as compared with control BSA-seeded cells. This up-regulation is induced, although at a much higher extent, also by treatment with
TPA (see lane c of Fig. 3A, and inset
to the figure), indicating here again that the increase in
V
3 integrin correlates with cell
differentiation. As shown in panel B, another integrin
expressed on FLG 29.1 plasma membrane, namely the
1
subunit and its associated
(s), is not affected by adhesion of
leukemic cells to FN. Moreover, a cytoplasmic protein, namely PKC
,
was not affected at all by cell adhesion to FN (panel C),
ruling out any aspecific potentiating effect on protein synthesis
operated by this adhesion.
|
On the whole, FLG 29.1 cells appear to be a suitable model for studying
the electric signals induced by integrin activation, since these cells
undergo a strong 5
1-mediated adhesion to
FN and this adhesion is capable of inducing the differentiation of these cells through the osteoclastic pathway, and, in this process, of
sustaining an up-regulation of another integrin expressed in these
cells, namely the
v
3. The effect of
integrin activation on various biophysical parameters was then studied
in FLG 29.1, using the whole cell configuration of the patch clamp
technique, after seeding the cells for different times on a FN- or a
BSA-coated substratum, the latter taken as a control.
In Fig. 4 the time course of
VREST is reported: this potential starts to be
hyperpolarized in FN-seeded FLG 29.1 cells as soon as 15 min after
seeding, reaching a value significantly different from that of the
BSA-seeded control cells (28.6 mV ± 2.6 versus
12.9 mV ± 2.3; p < 0.03) after 1 h of
incubation. Thereafter, VREST of FN-seeded FLG 29.1 cells
returns to starting values, identical to that of control cells.
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Since we previously demonstrated that only two currents contribute to regulate VREST in FLG 29.1 cells (12, 20), namely IHERG and the leakage, values relative to these parameters were evaluated and are reported in Table I: IHERG density is significantly increased after 1 h of cell adhesion to FN, as compared with the controls. This activating effect of FN on IHERG declines thereafter, so that the IHERG density in FN-seeded cells displays values even lower than those of control cells, after 2 h of incubation. This increase in IHERG density is not accompanied by variations in biophysical characteristics of the current, such as the activation or the deactivation properties (not shown). On the other hand, the leakage conductance is not significantly increased by cell adhesion to FN: in fact, this parameter is even higher in BSA-seeded cells after 1 h of incubation, while is not significantly different in the two experimental conditions after 2 h. This effect on the leakage conductance exerted by cell adhesion is different from that elicited by laminin in human neuroblastoma cells (13), where a concomitant activation of IHERG and leakage conductance is observed.
|
The integrin involvement in the mechanism underlying the FN-induced
HERG channel activation was further evaluated at 1 h of incubation
and results are reported in Table II: as
shown in the table, the effect of FN is not unique, since also the
adhesion to VN induces the activation of IHERG in FLG 29.1 cells, although at a slightly lower extent as compared with FN. This
result suggests, even in this case as in neuroblastoma cells (13), the
involvement of the 1 subunit of integrin receptors in
the increment of IHERG: this is clearly proven by the fact
that FLG 29.1 cell adhesion to dishes coated with mAb BV7 or TS2/16 to
the
1 subunit (24, 13) (bound anti-
1-ab
in Table II) induces a significant increase in IHERG
density, whose intensity is even greater than that induced by adhesion
on the whole FN molecule. Moreover, IHERG density was also
increased at the same extent by antibody-induced clustering of
1 integrin subunits on FLG 29.1 cells kept in suspension
(soluble anti-
1-ab in Table II). These data indicate
that antibody-induced
1 clustering is sufficient to
trigger HERG channel activation. The specificity of the involvement of
the
1 subunit in HERG channel activation is confirmed by
data, also reported in Table II, showing that dishes coated with
antibodies directed against either an
subunit
(anti-
3) or the
3 subunit do not
substantially increase IHERG density. On the whole, data
reported in Fig. 4 and Tables I and II demonstrate that FLG 29.1 adhesion on FN induces a cycle of VREST hyperpolarization,
which peaks after 1 h of incubation and is conceivably sustained
by a concomitant increase in IHERG, the latter being
induced by the engagement of
1 integrin subunit with its
ligand.
|
We then analyzed whether the above reported increment in
IHERG density could be reconducted to variations in
herg RNA level and/or modification of the HERG protein on
the plasma membrane. In Fig. 5 the
results of this analysis are reported. Fig. 5A shows a
Northern blot performed with RNA extracted from cells seeded on BSA- or
FN-coated dishes for 1 h, and probed with herg or a control gene (ribosomal 18S). It is evident that FLG 29.1 cell adhesion to FN is not accompanied by any variations in
herg RNA level as compared with the controls. The effect of
cell adhesion on HERG protein expression on the plasma membrane is
shown in Fig. 5B, where an immunoblot performed on membrane
proteins extracted from FLG 29.1 cells seeded on BSA or FN for 1 h, and revealed with an anti-HERG antibody is reported. Two protein
bands are evident in FLG 29.1 membrane extracts, one upper band of 155 kDa, and a lower band of 135 kDa. According to data reported so far (32),3 the 155-kDa isoform
represents the completely mature, fully glycosylated form of the
protein, expressed on the plasma membrane, while the 135-kDa band is
the immature, core-glycosylated protein. It is evident that FN adhesion
does not alter this HERG protein profile, neither quantitatively, nor
qualitatively.
|
Therefore, the FN-induced increment in IHERG could be
realized by the action of a short range modulator of channel activity: a possible candidate could be envisaged in a pertussis toxin-sensitive Gi protein interposed between 1 integrins
and channel protein, as in neuroblastoma cells (11). And in fact, as
shown in Fig. 5C, when FLG 29.1 cells are seeded on FN-
(lane d) or activating anti-
1 antibody-coated
dishes (lane e), in the presence of PTX, no increase in
IHERG density can be observed as compared with BSA-seeded
control cells. It is worth noting that PTX-treated cells do adhere to
FN, but do not spread on the substrate (panels c and
c'), an effect already reported for PTX on melanoma cells (33). It is important to note that patch clamp experiments were performed either on adherent round or spread cells, and no difference in IHERG density as well as biophysical characteristics of
the current were detected in the two conditions (not shown). This rules
out that the lack of effect of FN on IHERG in PTX-treated cells can be due to the incapacity of the cells to undergo cell spreading.
The question now arises as to whether the FN-induced,
1-mediated electric signal is a necessary step in the
pathway leading to osteoclastic differentiation of leukemic cells.
Since we showed that this pathway can be evidenced by at least three
markers (CD 51/
v
3 integrin, TRAP, and CtR
receptor), experiments were performed evaluating the expression of such
markers in cells incubated on a BSA- or FN-enriched substratum for
different times, with or without a specific inhibitor of HERG channels,
namely WAY 123,398. In the latter conditions, the IHERG was
completely and irreversibly blocked (not shown) (15). As shown in Fig.
6, panel A, incubation of FLG
29.1 cells with 40 µM WAY 123,398 for 24 h
(lane b) impairs the increase in
v
3 expression induced by cell adhesion to
FN (which is reported for comparison in lane c). Moreover,
WAY 123,398 addition can also impair the increased expression of CtR
(panel B, lanes 2 and 3), which can be observed
after a 1-week incubation of FLG 29.1 cells on FN-coated dishes.
|
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DISCUSSION |
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---|
Integrin receptors are known to mediate either "inside-to-out"
and "outside-to-in" signals (9), and, in this way, can regulate many cellular functions, such as cell growth and differentiation, cell
migration and/or activation. We present evidence that
1 integrin receptors can elicit the activation of a
hyperpolarizing, HERG-sustained, K+ current in leukemic
hemopoietic precursors (FLG 29.1 cells), and that this signal
apparently modulates the increased expression of
v
3 integrins on the plasma membrane, a
phenotypic marker of osteoclastic differentiation in these cells.
An integrin-mediated electric signal had been previously reported to occur in murine erythroleukemic (34), as well as murine and human neuroblastoma cells (11, 13). While in murine leukemic cells, adhesion to FN leads to the activation of Ca2+-dependent K+ channels, the electric signal elicited by FN in FLG 29.1 leukemic cells is represented by a hyperpolarization of VREST, mediated by the activation of HERG K+ channels, as occurs in neuroblastoma cells (11, 13). Therefore, HERG channels appear to be frequently involved in the signaling pathway induced by integrins in cells of different lineages.
An association between integrins and K+ channels has been
reported to occur between 1 subunit and GIRK channels in
transfected cells (35), although integrins do not appear to be
essential for a functional GIRK expression (36), despite the physical association of the two proteins. A functional association between integrins and a K+ channel different from HERG involves the
Kv 1.3 channels in human lymphocytes: in these cells, however, the
modulation of the K+ current is the trigger for
1 integrin activation, which leads to an enhanced
functioning of the adhesive receptor (37).
In our case, the integrin-induced HERG activation does not imply any
modulation in herg RNA or HERG protein level, but is mediated by a short-range mechanism of activation. In particular, in
human leukemic, as in neuroblastoma cells (11), a Gi
protein is involved in the functional association between integrins and HERG channels. Integrins are known to modulate the activity of the Rho
family of small GTPases (reviewed in Ref. 38), but also a physical and
functional association between integrins, the integrin-associated protein and trimeric Gi proteins has been reported (39,
40). These proteins constitute a signaling complex, involved in many cellular functions, such as chemotaxis (41) and platelet aggregation (39), possibly through the activation of 3 integrins. In
such models, the stimulation of Gi induces an inhibition of
adenyl cyclase, which ultimately leads to a decrease in intracellular cAMP. We have no evidence whether such a mechanism can be also invoked
in our model, or whether a direct effect of Gi occurs on
HERG channels as reported for other types of K+ channels
(42).
Another point to be stressed here is that HERG channels, and their
mediated electric variation of VREST, appear to be more frequently involved in processes of cell differentiation (11, 13, 43,
44). In fact, we show here that leukemic cells apparently start
undergoing a process of osteoclastic differentiation, after adhesion to
FN, as evidenced by the appearance of osteoclast markers, such as TRAP,
CtR, as well as CD51/v
3 integrin. This is
an important demonstration of a differentiating effect of the ECM on
cells of the hemopoietic lineage, which couples with recent evidence showing a cooperative effect of FN with cytokines and growth factors on
the maintenance and growth of CD34+ hemopoietic stem cells (45).
Moreover, data reported in this paper demonstrate that this
differentiation process includes the appearance of immunoreactivity to
CD51, i.e. by the increased expression of
v
3 integrin on the plasma membrane, the
integrin involved in osteoclast adhesion to the bone matrix, a
necessary step for bone readsorption (8). The demonstration of an
up-regulation of an integrin receptor, induced by the engagement of a
different integrin subunit with the ECM deserves more attention.
Moreover, we show here that drugs, which specifically block HERG
channels, such as anti-arrhythmic drugs, impair either this
up-regulation of
v
3 integrin or the increased expression of CtR. Therefore, the activation of HERG channels
is capable of mediating the up-regulation of membrane receptors,
including integrins, an interesting finding unexplored so far. In fact,
in T lymphocytes, the activation of Kv 1.3 channels induced an
increased function of
1 integrins, possibly through a
conformational change of the integrin molecule; however, an increased
expression of this integrin subunit on the plasma membrane, as in our
case, is not reported. While the effect of HERG channels on CtR appears
to happen at the RNA level, the mechanism leading, in our model, to the
up-regulation of
3 integrins remains to be explored, and
a transcriptional activation could be invoked. However, a direct
electrostatic effect on the protein could also occur, thus candidating
some classes of integrins as voltage-dependent proteins
(46), capable of being influenced themselves by the biophysical
modifications of the plasma membrane. On the whole, a mechanism of
cross-talk between integrin subunits could be hypothesized, centered on
the activity of K+ HERG channels, which can be influenced
by integrins (
1 subunits, mainly), and can activate
diverse proteins, either membrane-endowed, such as integrins themselves
(
3 subunits mainly), and hormone receptors, or
cytoplasmic, such as pp125FAK (47).
On the whole, these data support the hypothesis that HERG channels
represent an important molecular device, involved both in the
integrin-mediated outside-to-in and inside-to-out signaling and by this
way in some signaling pathways controlling cell differentiation in the
hemopoietic system.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge Prof. G. Tarone (University of
Torino) and Dr. A. Becchetti (University of Milano Bicocca), for the
kind revision of the manuscript, Prof. P. Bruni (University of Firenze)
for the gift of anti-PKC antibodies, Prof. G. Mugnai (University of
Firenze) for supplying vitronectin, Prof. G. Delfino (University of
Firenze) for the acquisition of cytochemistry images, and Dr. R. Caporale (Hematology Unit, Firenze) for flow cytometry control measurements.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Associazione Italiana contro le Leucemie (AIL) Firenze, Ministero dell'Università e della Ricerca Scientifica e Tecnologica COFIN `99 (to A. A.) and COFIN '97 (to E. W.), Telethon project m.1046 (to E. W.), and the Associazione Italiana per la Ricerca sul Cancro (AIRC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a fellowship from the Fondazione Italiana per la Ricerca sul Cancro.
To whom correspondence should be addressed: Dept. of
Experimental Pathology and Oncology, Viale Morgagni 50, 50134 Firenze, Italy. Tel.: 39-055-411131/414814; Fax: 39-055-4282333; E-mail: annarosa.arcangeli@unifi.it.
Published, JBC Papers in Press, November 15, 2000, DOI 10.1074/jbc.M005682200
2 G. Hofmann, P. Defilippi, and A. Arcangeli, unpublished results.
3 L. Guasti, D. Crociani, and A. Arcangeli, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ECM, extracellular matrix; herg, human eag-related gene; HERG, herg-encoded protein; IHERG, HERG current; FN, fibronectin; VN, vitronectin; VREST, membrane resting potential; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TPA, 12-O-tetradecanoylphorbol-13-acetate; TRAP, tartrate-resistant acid phosphatase; RT-PCR, reverse transcriptase polymerase chain reaction; CtR, calcitonin receptor; mAb, monoclonal antibody; bp, base pair(s); PKC, protein kinase C; PTX, pertussis toxin; gapdh, glyceraldehyde-3-phosphate dehydrogenase.
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