INSERM Unit 403, Hôpital E. Herriot, Pavillon F, 69437 Lyon Cedex 03, France
* Author for correspondence (chenu{at}lyon151.inserm.fr)
Accepted 24 July 2002
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Summary |
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Key words: Osteoclasts, Cell movement, Potassium channels, Calcium-activated
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Introduction |
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Osteoclasts are multinucleated cells that are primarily responsible for
bone resorption. They alternate between motile and resorptive phases of
activity (Kanehisa and Heersche,
1988), both of which are necessary for effective bone resorption.
During its motile phase, the osteoclast extends large lamellipodia (thin
organelle-free processes involved in plasma membrane extension) along the
movement axis while the membrane retracts at the opposite side of the cell.
Osteoclasts cultured on glass coverslips or bone spontaneously spread and can
exhibit two morphologies; `spread' osteoclasts are flattened and have
lamellipodia, whereas `rounded' resorbing osteoclasts have a dome shape and
lack lamellipodia. During spreading, cells form lamellipodia along one or more
radial axes without retraction. The relationship between osteoclast
electrophysiological activity and its morphological changes has been poorly
documented. It was demonstrated that internal application of GTP analogues
(GTP
S) induces osteoclast spreading and suppresses the inward
rectifying potassium current expressed by these cells
(Arkett et al., 1994
). Calcium
transients were observed spontaneously in osteoclasts
(Radding et al., 1999
) and
were also induced by osteoclast attachment to bone
(Teti et al., 1989
) or by
external stimuli such as ATP and PAF (platelet-activating factor)
(Zheng et al., 1993
;
Weidema et al., 1997
).
However, no study has yet investigated how ionic channel activity is linked to
calcium handling and membrane movements.
In this study, we have quantified spontaneous osteoclast spreading kinetics simultaneously with whole cell membrane current. In particular, we have examined the intracellular calcium dependence of ionic currents. We show for the first time that spontaneous membrane spreading coincides with the activation of a calcium-dependent potassium current. Moreover, the quantitative study of cell membrane movement kinetics demonstrates a strong temporal correlation between instantaneous membrane spreading rate and spontaneous activation of the current. Our results demonstrate that activation of this current is involved in osteoclast spreading and efficient bone resorption.
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Materials and Methods |
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Experiments were carried out according to the guidelines defined by animal welfare committee of the `Institut National de la Santé et de la Recherche Médicale' (INSERM).
Electrophysiology
Perforated whole-cell patch-clamp, cell-attached and inside-out
configurations were used. Currents were recorded with a RK-400 amplifier
(Bio-Logic, Grenoble, France), and data were registered and analysed using
pClamp6 software (Axon Instruments). Macroscopic current signals were filtered
at 3 kHz and digitised at 10 kHz, and microscopic currents were filtered at
300 Hz and digitised at 1 kHz using a DigiData 1200 (Axon instruments)
analogue/digital converter. Borosilicate glass pipettes had a resistance of
2-3 M, and seal resistance was always superior to 1G
.
Current-voltage (I-V) relationships were obtained using a voltage ramp
protocol. Cells were held at -20 mV (Hp), then pulsed to -130 mV for 45
milliseconds (to deactivate all currents activated at -20 mV and to check for
inward rectifier activation), followed by a ramp from -130 mV to +60 mV over
450 ms (0.422 V/second). This protocol was repeated every 2 seconds. The time
course of the background current was obtained off-line by measuring the
current at different spots of the ramp protocol (Hp, -100 mV, 0 mV, +40 mV).
Experiments were performed at room temperature (21-25°C). Cells were
continuously superfused at 3 ml/min using a bath solution flowing by gravity
from a set of five capillaries set at 10-20 µm away from the cell (time
change of solutions 2-3 seconds controlled by [K+]o
variations). The control solution contained NaCl 145 mM, KCl 5 mM,
CaCl2 2 mM, glucose 5 mM, HEPES 10 mM, pH 7.4 adjusted with NaOH.
The pipette filling solution contained in mM: KCl 145 mM, NaCl 5 mM, HEPES 10
mM, pH 7.2 adjusted with KOH (all chemicals were obtained from Sigma).
Pharmacology
The toxin sensitivity of the calcium-dependent potassium current in
osteoclasts was tested on the whole-cell current, which was activated by 10
µM of the calcium ionophore A-23187 (Molecular Probes). Charybdotoxin (CTX)
and apamin (APA) (Alomone, Jerusalem, Israel, and Latoxan, Valence France)
were applied externally at concentrations of 10 nM and 0.5 nM.
Osteoclast area measurement
Osteoclasts were cultured on glass coverslips for 24 hours in the presence
or absence of toxins (APA and CTX at 50 nM). After 24 hours, the
multinucleated osteoclast area was measured for each treatment using Lucia
software (Nikon Imaging analysis system).
Bone resorption in vitro
The bone resorption activity of osteoclasts was tested in vitro, using a
previously validated model (Itzstein et
al., 2000). Briefly, osteoclasts were seeded onto cortical bone
slices in 96-well plates and incubated for 24 hours in the presence or absence
of APA and CTX. At the end of the culture, cells were removed and bone slices
were stained with toluidine blue to score the number of resorption pits.
Resorption was evaluated by the number of resorption pits compared to the
control.
Time lapse
Time-lapse recordings were made with a video CCD camera connected to an
inverted microscope (Olympus IX50). The camera was controlled using `Premiere'
software (Adobe, San Jose, CA.). Images were acquired every two seconds and
stored as TIFF stack files.
Image analysis
Coverslips containing the osteoclasts were flushed with ionic control
solution to wash out culture medium and remove non-adherent cells. For all
experiments, a common cell appearance with a central nuclei zone surrounded by
a narrow cytoplasm band was chosen to record current and membrane kinetics at
the beginning of cell spreading. Recorded cells (n=20) showed
membrane spreading without cellular movement. They were chosen at the
beginning of the spreading process. Time-lapse analysis was performed with
Image Tool (UTHSC, San Antonio, CA) and the Lab Talk programming language
(Microcal Software, Northampton, MA). We have developed a method to
automatically detect the position of the membrane leading edge for every
image. Briefly, our method is as follows:
|
Cell movement can be summarised by a movement map as shown in Fig. 1e, which illustrates the membrane movement rate measured every 30° (vertical axis) against time (horizontal axis). Colors indicate the time-space regions where lamellipodia were extended during cell spreading.
Signal analysis
The spreading rate time course along 12 radial axes was normalised to the
maximum rate observed. The current measured at one potential during the ramp
protocol was normalised to its maximum peak. Mean and slow baseline variations
of each signal were subtracted. The current and the spreading rate signal were
compared using a cross-correlation function
(Bendat and Piersol, 1971):
![]() | (1) |
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Signal frequency analysis was performed using a coherence function
(Bendat and Piersol, 1971):
![]() | (2) |
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Statistical analysis
The effects of toxins on osteoclast spreading and bone resorption were
tested by a Mann-Whitney test (InStat, GraphPad Software Inc).
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Results |
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At the same time, the whole cell membrane current of the same osteoclast
was recorded using the amphotericinperforated patch configuration, which
prevents complete dialysis of the cytosol and therefore allows physiological
changes in the intracellular calcium concentration
([Ca2+]i) (Korn et
al., 1991). The membrane current recorded in osteoclasts remained
stable for 30 to 90 minutes. Spreading osteoclasts, defined as cells that
showed visible forward movements in one region of the cell at least, exhibited
spontaneous peaks of an outward current (recorded at -20 mV holding
potential). This is the first demonstration of a spontaneous electrical
activity in osteoclasts. Spontaneous intracellular calcium variations were
nevertheless described in these cells, consisting of either irregular
[Ca2+]i fluctuations
(Malgaroli et al., 1989
) or
spikes (Radding et al., 1999
)
with non-periodic frequency. We evaluated the degree of periodicity of the
spontaneous current peaks observed in our study using the auto-correlation
function. For a quasi-periodic signal, the auto-correlation is a periodic
function composed of peaks corresponding to the periodic component variance of
the signal. About 40% of osteoclasts exhibited a periodic current
(Fig. 2a,c), whereas others did
not (Fig. 2b,d). This periodic
pattern of membrane current reveals an intracellular cyclic mechanism that
controls the ionic membrane conductance (i.e. cyclic
[Ca2+]i variations). Such a mechanism has been well
documented in other cells during adherence and migration
(Birnbaumer et al., 2000
;
Scherberich et al., 2000
). 95%
of spreading osteoclasts exhibited the spontaneous peaks of background
current, whereas 80% of non-spreading osteoclasts showed no variation in the
current, indicating a strong link between the activation of the oscillatory
current and the cell-spreading process
(Fig. 2e). Similarly, calcium
transients were observed in human neutrophils during spreading, whereas
rounded cells had no [Ca2+]i fluctuations
(Jaconi et al., 1991
).
|
The outward current linked to membrane movement is a KCa
current
The ionic nature of the outward current was investigated using a voltage
clamp protocol (Fig. 3a,
insert). Fig. 3a illustrates
the current-voltage relationship (I-V curve) obtained during the ramp, with
the baseline (resting current) and oscillation peak displayed. A strong inward
rectification was observed owing to the presence of the inward rectifier
potassium current previously described in these cells
(Dixon et al., 1993). Only the
outward current was drastically changed during the spontaneous current
oscillations (Fig. 3a). The
difference current reversed at -75 mV, exhibited an outward rectification and
a negative inflexion at positive potentials
(Fig. 3b). This reversal
potential was close to the potassium equilibrium potential (EK=-80
mV) and suggested a potassium current. To test this hypothesis, the difference
current measured at the peak of spontaneous oscillations was recorded on the
same osteoclast bathed in different external potassium concentrations: 5 mM,
41 mM and 117 mM K+ (Fig.
3c). Reversal potentials (Erev) shifted to positive
potentials (Fig. 3c) and plots
of Erev versus logarithm of external potassium concentrations
showed a linear relationship with a slope of 57.3 mV±1.7 mV per decade,
confirming a potassium current (Fig.
3d).
|
The slight decline of the I-V curve observed at positive potentials
suggested a dependence of the current on calcium influx, which decreased at
positive potentials. Activation of a calcium-dependent potassium current has
been observed in osteoclasts in response to [Ca2+]i
increase induced by extracellular ATP or by A-23187 calcium ionophore
application (Weidema et al.,
1997). To test the internal calcium dependence of the current,
non-spreading osteoclasts were bathed in control solution and voltage clamped
using the protocol described in Fig.
3a. Then, A-23187 solution was applied until the outward current
reached a peak and then washed out. Fig.
4a illustrates the current pattern measured at -20 mV. The
internal calcium increase caused by A-23187 induced an outward current. I-V
curves of total current (Fig.
4b) and difference current
(Fig. 4c) displayed during the
ramp-clamp protocol were similar to those observed spontaneously during
membrane spreading (reversion at EK, outward rectification, slight
inward rectification at positive potentials)
(Fig. 3b). Isolated channel
recordings, performed in the inside-out patch configuration (isotonic 145 mM
K+), demonstrated that calcium application at the patch membrane
inner face activated a channel of about 20-25 pS, which showed rapid
open-closed switching during calcium activation (called `burst openings')
(Fig. 4d).
|
The activity of a single channel was recorded in the central region of a
spreading osteoclast using a cell-attached mode
([K+]pipette and [K+]bath=145 mM;
Fig. 5a). We observed
spontaneous burst openings of a channel of 22 pS
(Fig. 5b), which is similar to
the channel activated by calcium in the inside-out configuration
(Fig. 4d). The open probability
of the unitary current plotted against time showed an oscillatory pattern with
short periods of intense activity followed by rest intervals
(Fig. 5c). This pattern was
comparable to the spontaneous oscillations observed for whole cell current
(Fig. 2a,b). These results link
the calcium-dependent channels observed in the inside-out configuration with
those spontaneously activated during membrane movement and confirm the
calcium-dependent activation of the potassium current. We did not observe any
large conductance BK-type channels (>50 pS) in any of the single channel
recordings (inside-out and cell-attached).
|
Pharmacological sensitivity of the IKCa current expressed
by osteoclasts
Calcium-dependent potassium (KCa) channels are characterised by
their conductance () and pharmacological sensitivity to venom toxins,
such as charybdotoxin (CTX) and apamin (APA). CTX is a specific blocker of big
(BK;
>150 pS) and intermediate (IK/SK4;
=50 to 150 pS)
conductance channels (Joiner et al.,
1997
), whereas APA is a specific blocker of small conductance
channels (SK1,2,3;
<50 pS). The 25 pS unitary conductance measured
in isolated patch recordings indicated small or intermediate KCa
channels. The effects of CTX and APA were therefore tested on the current
activated by calcium ionophore A-23187 in osteoclasts
(Fig. 6a). The IKCa
current was totally blocked by 10 nM CTX and partially inhibited by 0.5 nM
APA. TEA (tetraethyl ammonium), which is known to block BK channels at 1 mM
(Iwatsuki and Petersen, 1985
),
did not inhibit this current at concentrations up to 5 mM (L.E., L.P., C.O. et
al., unpublished). The KCa channels observed in our study have
pharmacological characteristics that are different from classic BK, IK and SK
channels described in other cells. They are also different from the
KCa previously described in osteoclasts
(Weidema et al., 1997
), which
has a 54 pS unitary conductance and is insensitive to venom toxins (CTX, APA
and kaliotoxin).
|
Venom toxins inhibit osteoclast spreading and bone resorption in
vitro
To investigate the role of KCa channels in osteoclast
physiology, the effects of CTX and APA on osteoclast spreading and in vitro
bone resorption were examined. As it was impossible to discriminate between
spontaneous modifications of membrane spreading during patch-clamp and
time-lapse recordings (Fig. 2b)
and modifications induced by toxins, we investigated the effects of toxins on
osteoclast spreading by measuring cell area after their chronic application.
Osteoclast cell area was measured after 24 hours of culture with or without
APA or CTX at 50 nM. Our results showed that osteoclast area cultures with APA
and CTX were 46% and 36% smaller, respectively, than control cells
(Fig. 6b). Similar results were
obtained after 6 hours of exposure to toxins. To evaluate bone resorption, we
used a model of disaggregated osteoclasts cultured on cortical bone slices, as
previously described (Arnett and Dempster,
1987). Both toxins significantly inhibited the number of
resorption lacunae on bone slices compared with the control
(Fig. 6c).
IKCa current is correlated with membrane-spreading
kinetics
Osteoclasts move and spread on glass and bone substrate by lamellipodia
formation (Chambers and Magnus,
1982). No study has previously investigated the time correlation
function between ionic membrane conductances and membrane-spreading kinetics.
However, Arkett et al. have shown that lamellipodia extension and inward
rectifier K+ current in osteoclasts are regulated by different
types of G proteins (Arkett et al.,
1994
), suggesting two different signalling pathways that are
independently regulated. Our aim was to investigate the temporal correlation
between membrane spreading and spontaneous activation of calcium-dependent
potassium current in osteoclasts. When osteoclasts were cultured on glass
coverslips, we observed spreading either along all cell radial axes
(Fig. 7, cell 1) or in only one
or two directions (Fig. 7,
cells 2,3). To correlate membrane movement with the whole cell
electrophysiological signal, we had to take account of the membrane position
changes in all cell regions during the time of the experiment. The movement
map described in the Materials and Methods and
Fig. 1e showed that cell
spreading is discontinuous. Movement of the margin edge is indicated by
isolated `peaks' of the movement rate followed by periods of rest or
retraction. This process started a few seconds before the beginning of the
current oscillations that lasted until the end of cell spreading.
|
To test whether the discontinuous spreading kinetics of the membrane movement rate were time-correlated with current spontaneous oscillations, we performed a multiple regression study between the movement rate along each cell axis and the background current. Fig. 8 shows the relationship analysis between spreading and membrane current for the three cells illustrated on Fig. 7. Time-lapse analysis allowed us to sample every 2 seconds the intersection co-ordinates of the membrane leading edge with each one of the 12 axes. For each time, the intersection points were linked by segments that allowed us to build up a 12-segment polygon representing the cell perimeter (Fig. 8a). Fig. 8b illustrates the correlation coefficient that quantifies the degree of similarity between current and movement rate along each axis. The polar diagram (Fig. 8b) shows that the degree of correlation was larger for regions where cell spreading was the most intense. The multiple regression analysis produces a dependent variable calculated as the best linear combination of the 12 spreading rate signals (see Materials and Methods). This variable showed a strong significant correlation with the IKCa time course (Fig. 8c, note the coincidence between current and spreading rate peaks). Coherence function (see Materials and Methods), which evaluates the frequencies at which two signals are in phase, was calculated for the oscillatory current and the movement rate for each axis. It showed peaks for axes located in cell regions with higher spreading (Fig. 8d). These peaks revealed shared frequencies that may indicate a common periodic activation of the spreading mechanism and IKCa.
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Discussion |
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We show for the first time a spontaneous and transient calcium-dependent
electrical activity in osteoclasts that correlated with the spreading rate of
lamellipodia and is involved in the control of bone resorption in vitro.
Although STOC (spontaneous transient outward currents) were already shown in
many cells (Hoyer et al.,
1998; Schwab,
2001
), this is the first demonstration of a strong temporal
correlation between STOC and movement signals. IKca current
oscillations were concomitant with osteoclast spreading, and a multiple
regression study showed a correlation between the movement rate along all cell
axes and the current. Since frequency analysis of current and movement
identified common frequencies for both signals, these results indicate a link
between these events or the existence of a single common cause.
Spontaneous calcium transients have already been described in osteoclasts
without any periodicity (Radding et al.,
1999). Our results demonstrate the existence of an electric
periodic rhythm in
40% of these cells, revealing a frequency-regulated
mechanism of [Ca]i that involves KCa channel activation.
Multiple pathways can lead to [Ca]i changes, but recent results
indicate that oscillatory opening of inositol triphosphate
[InsPtd(1,4,5)P3] receptors largely contributes to
cytosolic Ca2+ spikes (Taylor
and Thorn, 2001
). An InsPtd(1,4,5)P3-mediated
release of Ca2+ from intracellular stores was demonstrated in cells
that exhibit similar calcium transients and potassium current oscillations to
those observed in our study (Chen et al.,
1997
), indicating that this mechanism may be involved in calcium
transients in osteoclasts required for activation of IKca.
The time course of spontaneous activation of osteoclast calcium-dependent
potassium current was very similar to the STOC described in smooth muscle
cells (Schwab, 2001) that was
generated by `big conductance' (BK) KCa channels. In osteoclasts,
this current is generated by KCa channels with particular
characteristics. The fact that they are blocked by CTX should exclude SK
channels (Castle et al., 1989
);
however, their low unitary conductance and insensitivity to TEA at 1 mM also
exclude BK type channels and their sensitivity to apamin rules out SK4
intermediate channels (Joiner et al.,
1997
). An IKCa current was described in HL-60
granulocytes that was also activated by spontaneous [Ca]i increase
during cell attachment (Varnai et al.,
1993
). Interestingly, this current was generated by an
intermediate KCa channel of
35 pS that is sensitive to
charybdotoxin and insensitive to apamin. Our preliminary studies using RT-PCR
analysis revealed the presence in osteoclasts of mRNA for the alpha subunit of
the BK (Slo) channel and SK3 channels (Espinosa et al., 2001). These
differences between conventional KCa channels and those expressed
by osteoclasts could be attributed to several hypothetical mechanisms: a
strong regulation of BK channels by beta subunits, which would modify their
pharmacological sensitivity (Behrens et
al., 2000
), a cell-specific channel isoform or chimera channels
composed of SK and BK subunits. The hypothesis that BK, IK and SK channels
contribute together to generate the whole cell current could be rejected owing
to the fact that we have always observed only one type of channel in single
channels recordings and that its conductance was too small to be attributed to
BK channels.
The link between cell motility and bone resorption has been suggested by a
study showing that osteoclasts isolated from c-src-deficient mice have a
decreased motility and a strong reduction of their bone resorption activity
(Soriano et al., 1991;
Lowell and Soriano, 1996
). Our
correlation study indicates that osteoclast lamellipodia extension actually
involves IKCa activation. Since we also demonstrate that long-term
inhibition of osteoclast IKCa current induces a strong block of
both osteoclast spreading and bone resorption, we consider that
IKCa current participates in the control of bone resorption through
its role in the osteoclast membrane-spreading mechanism. In transformed kidney
cells, inhibition of IKCa has been shown to induce a diminution of
cell motility (Schwab and Oberleithner,
1996
). It has been hypothesised that activation of a potassium
conductance plays a role in the cell retraction zone by contributing to the
decrease in osmotic pressure and by inducing a local cell shrinkage
(Cantiello et al., 1993
;
Schwab et al., 1994
;
Schwab, 2001
). This hypothesis
was supported by the inhibition of cell movement after local application of
CTX at the rear of migrating cells (Schwab
et al., 1995
). However, we have demonstrated in our study that
activation of potassium channels is correlated with cell protrusion,
suggesting that KCa channels are directly involved in lamellipodia
extension. Moreover, cell-attached recordings have shown that single channel
activity is correlated with membrane spreading in the nuclei region of the
cell, far away from the membrane edge. These observations suggest a role for
KCa channels that is independent from osmotic pressure control.
Recent findings have indicated that ionic channels may be considered as
enzymes (Cahalan, 2001) and are
involved in cellular signalling through ion-independent mechanisms.
Interactions between tyrosine kinases, such as Src and PYK2, and potassium
channels have been demonstrated in transfected cell lines
(Felsch et al., 1998
;
Ling et al., 2000
;
Nitabach et al., 2001
).
Although potassium channels may be regulated by such tyrosine kinases
(Jonas and Kaczmarek, 1996
),
these channels may also be regarded as transducing proteins. In their open
conformation (i.e. linked to Ca2+), they can interact with some
protein kinases and subsequently recruit and activate other kinases involved
in cell function (Rezzonico et al.,
2002
). Independent from their ion channel activity, KCa
channels might therefore play a role in lamellipodia extension in osteoclasts
through a pathway involving tyrosine phosphorylation of PYK2 and Src. These
tyrosine kinases are highly expressed by osteoclasts and are essential for the
formation of structures called podosomes required for cell spreading and
movement (Ochoa et al., 2000
;
Pfaff and Jurdic, 2001
;
Sanjay et al., 2001
).
Furthermore, tyrosine phosphorylation of PYK2, leading to modulation of ion
channel function, is rapidly induced by signals that elevate intracellular
Ca2+ (Lev et al.,
1995
). In spreading osteoclasts, cytosolic Ca2+
increase might result from Ca2+ release from intracellular stores
through an activation of
vß3, the major
osteoclast integrin (Zimolo et al.,
1994
; Palecek et al.,
1997
), and/or from Ca2+ influx via stretch-activated
channels (Wiltink et al.,
1995
; Lee et al.,
1999
).
Analysis of membrane movement kinetics during osteoclast spreading has
demonstrated a temporal correlation with the calcium-dependent potassium
current involved in the control of membrane spreading and bone resorption.
Oscillations of the electrophysiological activity and internal calcium
concentrations are general mechanisms linked to membrane movement
(Schwab et al., 1994;
Gomez and Spitzer, 1999
;
Lee et al., 1999
;
Scherberich et al., 2000
;
Schwab, 2001
). Quantitative
analysis of cell morphology performed simultaneously with variations of the
membrane current, as in this study, may provide a better understanding of the
role of ionic channels in the regulation of membrane movement and particularly
during osteoclast spreading.
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Acknowledgments |
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