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INTRODUCTION |
Connexin43
(Cx43)1-mediated gap
junctional communication regulates bone matrix production in
vitro (1) and is required for normal bone formation in
vivo (2). Modulation of gap junctional communication in
vitro alters osteocalcin production by transcriptional regulation.
In vivo, detailed analysis of Cx43-deficient mice has
revealed distinctive skeletal abnormalities. However, the mechanisms by
which Cx43-mediated gap junctional communication alter bone formation
are poorly understood. One such mechanism may be by the coordination of
intercellular calcium signaling among osteoblastic cells. A well
described in vitro model, and one which may mimic the
effects of mechanical forces on bone cells, is that of mechanically
induced intercellular calcium signaling or intercellular calcium waves
(ICW).
Two mechanisms for the propagation of mechanically induced ICW in
osteoblastic cells have been described. In the UMR 106-01 rat
osteoblastic cell line, mechanical stimulation of a single osteoblast
leads to propagation of fast (~15 µm/s) ICW that require activation
of P2Y (purinergic) receptors on neighboring cells, presumably by
released nucleotides, and subsequent generation of inositol
trisphosphate and release of intracellular calcium stores. In the ROS
17/2.5 rat osteoblastic cell line, slow ICW require gap junctional
communication. Unlike gap junction-mediated ICW that have been
identified in many other cell models, gap junction-mediated ICW in ROS
cells do not require release of intracellular calcium stores and do not
require diffusion of IP3 through gap junction pores.
In human osteoblastic cells, both of these mechanisms for the
propagation of ICW have been identified and have different kinetics. Mechanical stimulation of human osteoblast-like cells results in fast
calcium waves that require activation of P2Y receptors. If these waves
are blocked by desensitization of P2Y receptors, slow gap
junction-dependent ICW are unmasked.
In this study we have investigated the mechanism of slow gap
junction-dependent ICW in osteoblastic cells and found that
these ICW involve plasma membrane depolarization, activation of L-type voltage-operated calcium channels, and influx of extracellular calcium.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Chemicals--
The rat osteoblastic cell lines,
ROS 17/2.8 (ROS) and UMR 106-01 (UMR) and UMR cells transfected with
the gene coding for the gap junction protein, connexin43 (UMR/Cx43)
(3), were grown in Minimum Essential Medium supplemented with
10% heat-inactivated calf serum, non-essential amino acids, 1 mM sodium pyruvate, 2 mM
L-glutamine, and penicillin/streptomycin
(Invitrogen). Geneticin (200 µg/ml) was added to the medium of
UMR/Cx43 cells. The cells were maintained in a humidified atmosphere of
5% CO2 at 37 °C with medium change every 2-3 days.
Human osteoblastic cells were isolated from human bone marrow obtained
from healthy volunteers, age 20-36, by puncture of the posterior iliac
spine. All participants had read and signed informed consent, and the
study was approved by the local ethics committee. The marrow material
was collected in phosphate-buffered saline with Ca and Mg (Invitrogen)
containing 100 units/ml Heparin (Sigma). The mononuclear fraction of
the marrow was isolated on a Lymphoprep (Nycomed) gradient and plated.
The next day the supernatant was aspirated to remove the non-adherent
cells. Adherent cells were then grown in Minimal Essential Medium
without phenol red (Invitrogen), supplemented with 10%
heat-inactivated fetal calf serum and penicillin/streptomycin. The
cells were maintained in a humidified atmosphere of 5% CO2
at 37 °C, with medium change every 7 days. After 4-5 weeks of
culture when cells reached ~80% confluence, the medium was
supplemented with 100 nM dexamethasone (Sigma) for 7-10
days to enhance osteoblastic differentiation. For calcium imaging
experiments, cells were plated on 25-mm No. 1 glass coverslips for 2-4
days and used at 70-90% confluence.
The fluorescent calcium indicator, fura-2, was used for calcium imaging
and measurements and purchased from Molecular Probes (Eugene, OR).
Nucleotides were from Roche Molecular Biochemicals. The endoplasmic
reticulum calcium ATPase inhibitor, thapsigargin, was purchased from
Calbiochem and used in a final concentration of 50 nM. All
other chemicals were from Sigma. The gap junction inhibitor, heptanol,
was made fresh on the day of experiment as a 1:4 heptanol:ethanol
solution and used in a final concentration of 3.5 mM.
-glycyrrhetinic acid (AGA), also a gap junction inhibitor, was used
in a final concentration of 2 or 5 µM. Nifedipine, an inhibitor of L-type voltage-operated calcium channels (VOCC) was used
in a concentration of 1, 5, or 10 µM.
Calcium Imaging--
Measurement of intracellular calcium
concentration was performed using the fluorescent calcium indicator,
fura-2. Cells in monolayers adherent to non-coated glass coverslips
were incubated at 37 °C in Minimum Essential Medium
containing 5 µM fura-2/AM for 30 min and then
incubated in fresh medium without dye for an additional 20 min.
Coverslips were affixed to a teflon chamber and mounted in a PDMI-2
open perfusion microincubator (Medical Systems Corp., Greenvale, NY),
maintained at 37 °C with superfused CO2, on a Zeiss
Axiovert 135 microscope. Imaging was performed with a
Metamorph/Metafluor imaging system (Universal Imaging, West Chester,
PA), with excitation wavelengths of 340 and 380 nm for acquiring ratio
images of fura-2. Probenecid (Sigma) at a concentration of 1 mM was added throughout the experiments to prevent dye
leakage. Intercellular calcium waves were initiated by mechanical
stimulation of a single cell using a borosilicate glass micropipette
affixed to an Eppendorf 5171 micromanipulator.
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RESULTS |
ROS Cells, UMR/Cx43 Cells, and Human Osteoblastic Cells
Propagate Intercellular Calcium Waves with Different
Kinetics--
Intercellular calcium waves were initiated by
mechanically stimulating single cells in monolayers. As previously
shown, stimulation of a single ROS cell caused a rise in the
intracellular calcium concentration in the stimulated cell.
Subsequently, an intercellular calcium wave was transmitted to
neighboring cells with a time lag of 15-30 s (Fig.
1). This intercellular calcium wave
propagated to 2.4 cells and with slow kinetics (wave propagation of
2-4 µm/s) as previously defined (1). In UMR/Cx43 cells, mechanical
stimulation of a single cell initiated intercellular calcium waves as
well, but these waves were faster (15 µm/s) and propagated to an
average of 23 cells with only a short time lag between neighboring
cells (Fig. 1). These kinetics are similar to those previously defined in the parental UMR cells and have previously been shown to require activation of P2Y receptors but not gap junctional communication. In
human osteoblastic cells, the mechanically induced intercellular calcium waves propagated with a velocity of 10-12 µm/s and extended to 7.6 cells with an intermediate time lag between cells.

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Fig. 1.
Intercellular calcium waves in ROS 17/2.8,
UMR/Cx43, and human osteoblastic cells (HOB). A
single cell was stimulated mechanically, causing an increase in the
intracellular calcium concentration, which subsequently propagates to
adjacent cells. Numbers indicate seconds after mechanical
stimulation. Scale bar indicates relation between
pseudocolors and intracellular calcium concentrations. Note the
differences in kinetics for the propagation of the waves in the three
cell types.
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ROS Calcium Waves Require Intact Gap Junctional
Communication--
The gap junction inhibitor AGA was used to confirm
that inhibition of gap junctional communication blocked the spread of
intercellular calcium signals in ROS cells. Monolayers of ROS cells
were loaded with fura-2, and ICW were induced before and after the
addition of 2 or 5 µM AGA. Mechanically induced ICW
extended to 2.8 cells before addition of AGA and to 0.3 cells after AGA
was added (Fig. 2). No difference was
seen between 2 or 5 µM AGA. After washing the AGA away
with phosphate-buffered saline containing 0.2% bovine serum albumin,
mechanically induced ICW were partially restored, propagating to 1.4 cells. AGA did not inhibit mechanically induced ICW in UMR cells; waves
propagated to 17 cells before adding AGA and to 24 cells after adding
AGA (2 µM) to the medium.

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Fig. 2.
The maximum calcium wave extension in ROS
17/2.8 cells and in a primary culture of human osteoblastic cells with
and without ATP (100 µM)
desensitization of the P2Y receptors, in columns named HOB
and HOB (after ATP), respectively. The
effectS of different inhibitory conditions are shown. ATP,
desensitization of purinergic P2Y receptors by adenosine-triphosphate.
AGA, the gap junctional inhibitor -glycyrrhetinic acid (2 µM). low
[Ca2+]o, extracellular
calcium is removed. Nifedipine, a blocker of L-type VOCC (1 µM). n.d., not done.
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The gap junction inhibitor heptanol was also used to
confirm the involvement of gap junctions in the propagation of ICW in osteoblastic cells. The initial untreated wave in the ROS cells extended to an average of 2.8 cells. Freshly mixed heptanol was added
to the bathing solution in a final concentration of 3.5 mM.
After 5 min, ICW were almost totally blocked, with a calcium signal
propagated only to 0.3 cells. As with AGA, wave propagation could be
partially restored by removing heptanol (1.3 cells/wave). Thus, the
propagation of intercellular calcium waves in ROS cells, but not in UMR
cells, requires intact gap junctional communication, in agreement with
previous data (4).
ICW in ROS Cells, but Not in UMR Cells, Require Influx of
Extracellular Calcium through L-type Voltage-gated Calcium
Channels--
We have previously shown that intracellular calcium
stores are not required for the propagation of ICW in ROS cells (4). To
confirm that gap junction-dependent ICW required influx of extracellular calcium, ICW were assessed after the bathing medium was
exchanged for calcium-free medium (Fig. 2). ICW propagation was
assessed within 2 min of medium exchange; at this time the cells
retained ionomycin-releasable intracellular calcium stores (not shown).
In contrast, P2-dependent ICW in UMR cells extended farther
in calcium-free medium than in calcium-containing medium (48 cells/wave
versus 30 cells/wave).
The above results implied that a plasma membrane calcium channel was
involved in gap junction-dependent ICW in ROS cells. To
assess the involvement of L-type calcium channels in the wave mechanism, monolayers of ROS cells were treated with nifedipine, an
antagonist of L-type voltage-gated calcium channels. Nifedipine was
used in a final concentration of 1, 5, or 10 µM. At all
concentrations, nifedipine inhibited ICW propagation (Fig. 2).
Replacing the bathing solution with nifedipine-free medium restored the
ability of the cells to propagate ICW. In contrast, UMR waves were not
inhibited by nifedipine treatment, and in fact propagated farther: 28 cells/wave before addition of nifedipine and 50 cells/wave in the
presence of nifedipine.
ROS Calcium Waves Require Membrane Depolarization--
Because
L-type calcium channels are activated by membrane depolarization, prior
membrane depolarization would inhibit ICW by preventing membrane
depolarization at the time of ICW generation. For these experiments,
ROS cells were depolarized using medium containing 70 mM
KCl. Before depolarization, ICW propagated to 2.6 cells/wave. After the
cells were switched to the high potassium medium, there was a calcium
response in all cells because of depolarization-induced activation of
VOCC. After the cells had returned to resting
[Ca2+]i, another mechanical
stimulus was applied, and the wave now propagated only to 0.6 cells.
Changing back to the original medium with physiological potassium
concentration restored ICW propagation (1.8 cells/wave). In UMR cells,
this protocol again had the reverse effect on ICW propagation; the
number of cells in the wave increased from 21 to 33, similar to the
result seen after nifedipine treatment or removal of extracellular calcium.
Nifedipine-sensitive ICW Require Gap Junctional
Communication--
Because ICW in ROS cells require gap junctional
communication, the above data suggest that the L-type calcium channel
activation responsible for ICW is dependent on gap junctional
communication in ROS cells. We tested this hypothesis by examining ICW
in UMR/Cx43 cells, which express both their native P2Y2 receptors and
transfected Cx43 gap junction protein (4). We first confirmed that the fast calcium wave in these cells, like UMR ICW, did not require extracellular calcium as follows. Single cells in fura-2-loaded monolayers of UMR/Cx43 cells were stimulated mechanically. A fast wave
was seen, similar to the wave in untransfected UMR cells. The wave
spread to an average of 24 cells. Next, the medium was exchanged for
calcium-free medium, and ICW were assessed within 2 min. In
calcium-free medium the wave spread to an average of 34 cells/stimulated cell.
Next, we confirmed that the slow ICW unmasked by desensitization of ATP
receptors in UMR/Cx43 required gap junctional communication. The fast
P2 receptor-dependent ICW were blocked by desensitizing the
receptors with 100 µM ATP, which inhibits further
response to ATP without depleting intracellular calcium stores (4). Mechanical stimulation of single UMR/Cx43 cells now revealed a slow
ROS-like wave that extended to 3.0 cells. This slow ICW was absent in
untransfected UMR cells similarly treated. Finally, AGA (2 µM) was added to the bathing solution of UMR/Cx43 cells, and upon mechanical stimulation ICW were almost completely blocked (0.5 cells in wave). Again, the AGA effect was reversed by washing the cells
in solution not containing AGA (2.3 cells in wave).
Finally we asked whether these slow ICW, which were present in UMR/Cx43
but not in UMR, required L-type calcium channels and plasma membrane
depolarization. When ICW were elicited in UMR/Cx43 cells in medium
containing 1 µM nifedipine before desensitization of P2
receptors, ICW propagated to 30 cells compared with 25 cells without
nifedipine. In high potassium experiments performed as described above,
adding potassium increased the wave from 31 cells before to 36 cells
after. In contrast, when these experiments were performed in UMR/Cx43
after desensitization of P2 receptors, both nifedipine- and
potassium-induced membrane depolarization blocked the slow gap
junction-dependent ICW.
Slow ICW in Human Osteoblasts Require Activation of L-type Calcium
Channels--
Human osteoblastic cells propagate both fast P2
receptor-dependent ICW and slow gap
junction-dependent ICW (5). As was seen in UMR/Cx43 cells,
5 µM AGA did not block fast ICW propagation in the human
cells; fast ICW propagated to 7.6 cells before addition of AGA and 6.9 cells after addition of AGA. Addition of ATP to human osteoblastic
cells resulted in a widespread calcium response and desensitization so
that subsequent addition of ATP did not cause calcium transients. After
ATP desensitization, mechanical stimulation elicited a slow ICW that
propagated to an average of 4.7 cells. The slow ICW were inhibited by 5 µM AGA, confirming that the slow ICW unmasked after
desensitization of P2 receptors require gap junctional communication.
We then asked whether gap junction-dependent ICW seen in
ATP-desensitized human osteoblastic cells were inhibited by
removal of extracellular calcium, plasma membrane
depolarization by high external potassium, or VOCC inhibitors as
summarized in Table I. Removal of
extracellular calcium resulted in inhibition of the slow wave
(Fig. 2). Slow ICW were also blocked by the L-type calcium channel
inhibitors nifedipine (Fig. 2), verapamil, or nitrendipine or membrane
depolarization in high extracellular potassium (Fig.
3). To confirm these treatments did not
inhibit the P2Y-dependent wave, experiments were performed
in cells that were not exposed to ATP. No inhibition of fast ICW was
seen after adding nifedipine in a final concentration of 1 µM to the bathing solution, with an average extent of the
wave to 4.9 cells in the wave before nifedipine and 5.1 cells in the
wave after nifedipine (Fig. 2).
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Table I
Maximal extent of calcium transients expressed as number of cells in
wave
Maximal number of cells in a wave after adding different agents. ND,
not done; n, number of experiments; S.E., standard error of
the mean.
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Fig. 3.
The average number of cells in calcium waves
in human osteoblastic cells after ATP desensitization and subsequent
blocking of either L-type VOCC by nifedipine (1 µM) or depolarizing the plasma membrane
by increasing extracellular potassium concentration to 70 mM.
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Fig. 4.
A schematic presentation of the two
mechanisms for mechanically induced calcium wave propagation in human
cells. Top panel, a mechanical stimulus
increases intracellular calcium concentration in the stimulated cell. A
diffusible messenger traverses the gap junctions to the neighboring
cell with subsequent membrane depolarization. Activation of VOCC
mediates extracellular calcium entry into the cytosol, thereby
increasing the intracellular calcium concentration. Bottom
panel, mechanical stimulation of a single cell with subsequent
increase in intracellular calcium concentration. Nucleotides (probably
ATP) are released to the extracellular space, binding to P2 receptors
on the neighboring cell. Inositol trisphosphate (IP3) is
generated in these cells, binding to receptors of the endoplasmic
reticulum, releasing calcium from the intracellular stores.
Cx43 indicates high permeability gap junctions containing
connexin 43.
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Gap Junction Hemichannels Are Not Required for ICW
Propagation--
In low extracellular calcium, some cells form gap
junction hemichannels at the plasma membrane that may mediate release
of ATP and propagation of ICW that appear to be gap
junction-dependent (6). We asked whether osteoblastic cells
express functional gap junction hemichannels by monitoring uptake of
the fluorescent dye Lucifer Yellow added to the extracellular
medium. In ROS cells and UMR/Cx43 cells no uptake of dye was seen in
medium containing either normal calcium or no extracellular calcium
(data not shown). In human osteoblastic cells, no Lucifer Yellow uptake
was seen in medium containing extracellular calcium. However, in
calcium-free medium, Lucifer Yellow uptake was seen in ~10% of cells.
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DISCUSSION |
These studies show that slow gap junction-mediated intercellular
calcium waves in osteoblastic cells require the influx of extracellular
calcium through L-type VOCC, as summarized in Table I. The propagation
of gap junction-dependent ICW was abolished in the absence
of extracellular calcium. Depolarization of the plasma membrane with
high potassium inhibited the waves, presumably by preventing the
activation of L-type calcium channels by rapid changes in the plasma
membrane potential. Finally, L-type calcium channel antagonists blocked
wave propagation. Nifedipine in concentrations as low as 1 µM inhibited ICW; electrophysiological studies have shown
that nifedipine concentrations of 10 µM selectively
blocked the L-type VOCC with no effects on T-type VOCC in osteoblastic cells (7).
Although it is unclear what is diffusing through gap junctions to
mediate these ICW, the requirement for plasma membrane depolarization and activation of VOCC suggests that ionic traffic through gap junctions may be responsible (Fig. 4). This mechanism would be reminiscent of the transmission of calcium transients in excitable tissues such as cardiac myocytes. However, the ICW in osteoblastic cells are much slower than in excitable tissues, suggesting some fundamental difference in mechanism or in receptor density.
This model for the propagation of gap-junction-dependent
ICW differs from the model for ICW propagation originally proposed for
respiratory epithelia and invoked for other cell types, including hepatocytes and glial cells. In this previously described model, ICW
are propagated by diffusion of inositol trisphosphate through gap
junction channels and subsequent release of intracellular calcium
stores (8, 9). Although ROS cells are well coupled by gap junctions
comprised of Cx43, they do not propagate mechanically stimulated ICW
via IP3 diffusion, demonstrated by the observation that
depletion of intracellular calcium stores has no effect on mechanically
induced ICW propagation in these cells (4). It is unclear why these
cells do not propagate ICW by diffusion of IP3 through gap
junctions. One possible explanation is that osteoblastic cells may not
express sufficient levels of Cx43 to permit the amount of
IP3 diffusion that is required for these ICW to occur. Another possibility is that different connexins may be required to
allow ICW mediated by IP3 diffusion.
In primary osteoblastic cells, ICW that require
IP3-mediated release of intracellular calcium stores can
occur. However, these ICW do not require gap junctional communication;
they involve activation of P2 receptors by extracellular ATP. This
mechanism for ICW propagation occurs in many cells because G
protein-coupled P2Y receptors are widely expressed. These P2-mediated
ICW may complicate analysis of ICW propagation and must be excluded
before ICW can be attributed to gap junctional communication. We have previously shown that monolayers of hamster tracheal epithelia (4) and
mouse nasal epithelia (10) propagate
IP3-dependent ICW that do not require gap
junctional communication but do require activation of P2 receptors.
In glioma cells, connexins participate in P2 receptor-mediated ICW by
forming plasma membrane hemichannels that release ATP (6). We
could not detect gap junction hemichannel formation in the rat
osteoblastic cell lines, and in human osteoblastic cells we
demonstrated hemichannel formation in only 10% of cells and only under
low calcium conditions. It is therefore unlikely that hemichannels play
a role in the gap junction-mediated ICW seen here. If gap junction
hemichannels did participate in osteoblast ICW, they would have to do
so by mediating calcium influx rather than by mediating ATP release.
These studies demonstrate that gap junction-mediated ICW in ROS cells
are a good model for the gap junction-dependent
coordination of ICW that occurs in human osteoblastic cells.
Intercellular calcium signaling in human osteoblastic cells is complex
because it involves both gap junction-dependent and
P2Y-receptor-dependent mechanisms, but the two mechanisms
can be studied independently, as demonstrated in this study and our
previous work on ICW in human osteoblastic cells and can be modeled
independently using the ROS and UMR rat osteoblastic cell lines.
In contrast to gap junction-dependent ICW,
P2Y-dependent ICW in UMR cells and primary osteoblastic
cells were actually increased after removal of extracellular calcium,
VOCC inhibition, or addition of potassium to depolarize the cells.
These fast P2Y-mediated waves therefore seem to be inhibited by calcium
influx via VOCC activation for reasons that are not readily apparent.
Gap Junction-mediated intercellular communication is important for
production of bone matrix proteins in vitro (1) and for
normal bone development in vivo (2). The current studies suggest that activation of L-type calcium channels might be an important component of the signaling pathway by which gap junctional communication influences bone development or turnover. The current work
highlights the importance of L-type VOCC in osteoblast calcium signaling and suggests the possibility that regulation of the expression and activity of VOCC may be important in modulating the
coordination of calcium signaling that occurs among osteoblasts. Osteoblasts have been demonstrated to express plasma membrane VOCC that
can mediate calcium entry (11, 12). VOCC have been implicated in
regulation of protein expression in bone cells. Applying strain
to osteoblasts in vitro in the presence of Bay K 8644, an
agonist of L-type VOCC, has been shown to increase the expression of
certain proteins specific for bone formation, such as osteopontin and
osteocalcin (13). In vitro fluid shear stress applied to the
human osteosarcoma cell line SaOS-2 increased TGF-
mRNA
expression (14), a growth factor involved in bone formation. Verapamil,
an antagonist of L-type VOCC, inhibited this increase. Another L-type
VOCC antagonist, nifedipine, prevented loading-related increases in
prostaglandin E2, nitric oxide, and glucose-6-phosphate dehydrogenase
in rat osteoblasts, but not in osteocytes (15). It is possible that the
ICW characterized here are involved in these processes.
In conclusion, we have shown that the gap
junction-dependent propagation of intercellular calcium
signals in ROS rat osteoblastic cells requires influx of extracellular
calcium through L-type VOCC and that the gap
junction-dependent component of intercellular calcium
signaling in human osteoblastic cells occurs by the same mechanism.
These findings suggest that regulation of the expression or activity of
L-type calcium channels may be a means by which calcium signaling is
modulated in bone cells and that modulation of these channels may
influence bone turnover.