Cell Density and Growth-dependent
Down-regulation of Both Intracellular Calcium Responses to Agonist
Stimuli and Expression of Smooth-surfaced Endoplasmic Reticulum in
MC3T3-E1 Osteoblast-like Cells*
Toshiyuki
Koizumi
,
Hisako
Hikiji
§¶,
Wee Soo
Shin§**,
Tsuyoshi
Takato
,
Satoru
Fukuda
,
Takahiro
Abe
,
Noboru
Koshikiya
,
Kuniaki
Iwasawa§**, and
Teruhiko
Toyo-oka§**
From the Departments of
Oral and Maxillofacial
Surgery,
Pathology, and ** Organ Pathology and
Internal Medicine, Faculty of Medicine, and § Health Service
Centre, University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan
Received for publication, October 7, 2002, and in revised form, December 4, 2002
 |
ABSTRACT |
A two-dimensional intracellular
Ca2+ ([Ca2+]i)
imaging system was used to examine the relationship between
[Ca2+]i handling and the
proliferation of MC3T3-E1 osteoblast-like cells. The resting
[Ca2+]i level in densely cultured
cells was 1.5 times higher than the
[Ca2+]i level in sparsely
cultured cells or in other cell types (mouse fibroblasts, rat vascular
smooth muscle cells, and bovine endothelial cells). A high resting
[Ca2+]i level may be specific for
MC3T3-E1 cells. MC3T3-E1 cells were stimulated with ATP (10 µM), caffeine (10 mM), thapsigargin (1 µM), or ionomycin (10 µM), and the effect
on the [Ca2+]i level of MC3T3-E1
cells was studied. The percentage of responding cells and the degree of
[Ca2+]i elevation were high in
the sparsely cultured cells and low in densely cultured cells. The rank
order for the percentage of responding cells and magnitude of the
Ca2+ response to the stimuli was ionomycin > thapsigargin = ATP > caffeine and suggests the existence of
differences among the various [Ca2+]i channels. All
Ca2+ responses in the sparsely cultured MC3T3-E1 cells,
unlike in other cell types, disappeared after the cells reached
confluence. Heptanol treatment of densely cultured cells restored the
Ca2+ response, suggesting that cell-cell contact is
involved with the confluence-dependent disappearance of the
Ca2+ response. Immunohistological analysis of type 1 inositol trisphosphate receptors and electron microscopy showed
distinct expression of inositol trisphosphate receptor proteins and
smooth-surfaced endoplasmic reticulum in sparsely cultured cells but
reduced levels in densely cultured cells. These results indicate that
the underlying basis of confluence-dependent
[Ca2+]i regulation is
down-regulation of smooth-surfaced endoplasmic reticulum by cell-cell contacts.
 |
INTRODUCTION |
The calcium ion (Ca2+) acts as an intracellular
messenger and regulates a diverse array of functions in many types of
cells (1). Various substances that influence bone remodeling modify the
intracellular Ca2+
([Ca2+]i) concentration in
osteoblasts (2). There are two sources of
[Ca2+]i in osteoblasts: 1) inflow
from the extracellular space and 2) release from intracellular stores,
such as the endoplasmic reticulum. The release of Ca2+ from
the endoplasmic reticulum is a ubiquitous signal in many cells
including osteoblasts (3, 4). ATP and caffeine cause inositol
trisphosphate
(IP3)1-induced
Ca2+ release (IICR) and Ca2+-induced
Ca2+ release (CICR), respectively, from intracellular
Ca2+ stores.
We previously reported that the response of the Ca2+ level
in vascular smooth muscle cells (5) to various stimuli is heterogeneous and that it is dependent on the stage of cell growth (6). A heterogeneous Ca2+ response has also been reported in an
osteosarcoma cell line stimulated by parathyroid hormone (7) and among
sub-cell lines of MC3T3-E1 cells treated with bradykinin (8). In the
present study, we examined the effects of cell density and growth on
[Ca2+]i-handling mechanisms in
MC3T3-E1 cells using a two-dimensional fura-2 imaging system,
immunocytochemistry, and electron microscopy, and we identified unique
characteristics of these cells. We studied the effect of growth on
[Ca2+]i-handling mechanisms in
MC3T3-E1 cells by studying the Ca2+ dynamics in cells
seeded in the sparse or dense condition.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
All reagents used were of analytical grade.
Thapsigargin (TG; Sigma Chemical) and ionomycin (IM; Sigma Chemical)
were dissolved in dimethyl sulfoxide (Me2SO) and then
diluted with phosphate-buffered saline (PBS). The final concentration
of Me2SO was less than 0.1% (v/v) and had no action on the
handling of [Ca2+]i in the
MC3T3-E1 cells (data not shown). Caffeine and ATP were purchased from
Wako Pure Chemicals (Osaka, Japan). Each reagent was administered by
replacing half the volume of extracellular medium with medium
containing 2-fold concentrated reagent (5, 6). In a pilot study, we
attempted to identify suitable reagents and their appropriate
concentrations to analyze the transient [Ca2+]i currents. We used 10 mM caffeine, 10 µM ATP, 1 µM TG, and 10 µM ryanodine or IM in the present study.
Cell Culture--
MC3T3-E1 mouse clonal osteogenic cells (a
generous gift from Prof. S. Yamamoto, Ohu University, Koriyama,
Japan) were seeded onto dishes (radius, 1 cm) made of
fluorescence-free glass at a density of 1.0 × 104
cells/dish (sparse condition) or 2.0 × 105 cells/dish
(dense condition). The cells were cultured in
-minimal essential
medium (Invitrogen) containing 10% fetal bovine serum (Bioserum, Victoria, Australia), 100 units/ml penicillin, 100 µg/ml
streptomycin, and 0.25 µg/ml amphotericin B (Sigma Chemical). The
medium was changed every 48 h after seeding. Caffeine, ATP, TG, or
IM was added between 0 and 120 h after seeding, and the peak
[Ca2+]i level upon stimulation was
studied. The resting [Ca2+]i level
in MC3T3-E1 cells seemed to be higher than that in other cells. We
compared the [Ca2+]i level in
MC3T3-E1 cells with that in endothelial cells (ECs) from the bovine
aorta, cloned rat vascular smooth muscle cells (A7r5), and
cloned mouse fibroblasts (NIH3T3), which have been used in studies on
[Ca2+]i dynamics (5, 6). In
single-cell line cultures, each cell line was seeded at a density of
1.0 × 104 cells/dish and cultured for 48 h. In
cocultures, EC, A7r5, or NIH3T3 cells were seeded together
with MC3T3-E1 cells at a density of each cell line of 1.0 × 104 cells/dish and cultured for 48 h.
To examine the effect of cell-cell contact, MC3T3-E1 cells were seeded
in dishes at a density of 2.0 × 105 cells/dish (dense
condition). After 24 h, 3 µM heptanol, a
gap-junctional inhibitor (9, 10), was added to the dishes and the cells were cultured for an additional 48 h. And then, ATP, TG, or IM was
added, and the peak [Ca2+]i level
upon stimulation was studied.
Two-dimensional (2D) Image Analysis of
[Ca2+]i--
The
[Ca2+]i level within individual
cells was analyzed as described previously (11-13). Briefly, the cells
were washed with PBS and incubated in PBS(+) containing 137 mM NaCl, 2.7 mM KCl, 8.1 mM
Na2HPO4, 1.5 mM KH2
PO4, 0.5 mM MgCl2, and 1 mM Ca2+, pH 7.4. The Ca2+-free
solution consisted of PBS in which CaCl2 was replaced by 1.0 mM EGTA. Cells were loaded with 4 µM
fura-2/acetoxymethyl ester in the same buffer at 37 °C for 30 min,
rinsed twice with PBS(+), and then preincubated at 37 °C for an
additional 10 min. The recorded fluorescence images were digitized
using an on-line image processor (Olympus-Merlin, Tokyo, Japan; Fig.
1A). Fig. 1B shows
the change in the relative value of the 340- to 380-nm fluorescence
ratio (F340/F380) in
MC3T3-E1 cells upon treatment with ATP. Because of the difficulty in
measuring the absolute [Ca2+]i
concentration caused by problems intrinsic to fura-2 fluoroscopy, the
maximum amplitude of the [Ca2+]i
elevation in response to each stimulant was expressed as the relative
value of F340/F380 in
relation to the value of F340/F380 in the resting
state (5, 6).

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Fig. 1.
Two-dimensional images of the
Ca2+ response (A) and
Ca2+ dynamics (B) in MC3T3-E1
cells induced by ATP (10 µM).
ATP was applied at 60 s. A, 1-6.
[Ca2+]i response at 0, 70, 85, 95, 105, and 115 s, respectively, after ATP stimulation. B,
the relative level of [Ca2+]i is
shown (expressed as the relative value of
F340/F380 compared with
the F340/F380 in the
resting state) at various time points after ATP was
applied.
|
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Caffeine transiently increased the
[Ca2+]i level, in both the
presence and the absence of extracellular Ca2+
([Ca2+]o). The degree of
[Ca2+]i elevation evoked by
caffeine in the Ca2+-free solution became progressively
smaller as the incubation time increased (data not shown). To avoid
fluctuations in the response, we applied caffeine to cells in the
presence of 1 mM [Ca2+]o. Ryanodine (10 µM; Wako Pure Chemicals) was also added to keep the CICR
channels open (14) and to prevent the activation of additional CICR.
All measurements were made 24 h after seeding to avoid the effects
of trypsin, which was used in seeding, on the Ca2+ channels.
Immunohistochemistry--
MC3T3-E1 cells incubated in plastic
dishes (60 mm in diameter) were fixed with a mixture of equal volumes
of ethanol and acetone. The nonspecific binding of antibody was blocked
by goat serum, and then the cells were treated with
anti-IP3 type-1 receptor (IP3-R1)
polyclonal antibody or non-immunized rabbit IgG at room temperature for
1 h, followed by an LSAB2 kit/horseradish peroxidase (DAKO
Corporation, Carpinteria, CA). The immunoproduct was visualized by
treatment with amino-9-ethylcarbazol (DAKO) and micrographs were taken
at 100× (AX-80; Olympus, Tokyo, Japan).
Electron Microscopy--
For transmission electron microscopy,
the MC3T3-E1 cells were detached from the culture dishes by treatment
with trypsin and washed three times with PBS, pH 7.4. After fixing
overnight in 2% paraformaldehyde plus 2.5% glutaraldehyde in
phosphate buffer, the cells were sedimented at 2000 rpm at room
temperature for 5 min, postfixed with 1% osmium tetroxide, dehydrated
in graded alcohol, and then embedded in Epon (Epon 812; TAAB Lab,
Berkshire, England) overnight at 60 °C. Ultrathin sections (60 nm)
were stained with uranyl acetate and lead citrate and observed under an
electron microscope (H-7000; Hitachi, Tokyo, Japan).
Statistical Analysis--
Data were expressed as the mean ± S.E. and were analyzed by one-way analysis of variance. Treatment
pairs were compared by Scheffé's F test. All differences
were considered to be statistically significant at p < 0.05.
 |
RESULTS |
[Ca2+]i in the Resting State--
The
[Ca2+]i level in MC3T3-E1 cells in
the resting state that had been seeded in the sparse or dense condition
was measured at 0, 24, 48, 72, and 120 h after the first medium
change (Fig. 2). The
[Ca2+]i level of the densely
seeded cells was 1.5 times higher than that of the sparsely seeded
cells at 0 h (1.19 ± 0.01 versus 0.80 ± 0.02, p < 0.01). The
[Ca2+]i level of the densely
seeded cells increased slightly, although significantly, up through
120 h from 1.19 ± 0.01 to 1.33 ± 0.01 (p < 0.01). The
[Ca2+]i level of the sparsely
seeded cells increased significantly from 0.80 ± 0.02 to
1.30 ± 0.01 at 120 h (p < 0.01) and reached the same level as that of the densely seeded cells at 120 h
(1.33 ± 0.01, densely seeded, versus 1.30 ± 0.01, sparsely seeded), with no significant change thereafter. These
results indicate that the [Ca2+]i
level of MC3T3-E1 cells increased as proliferation progressed.

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Fig. 2.
Relationship between the resting
[Ca2+]i level in
MC3T3-E1 cells and length of the culture period. The
[Ca2+]i level of densely seeded
cells ( ) was 1.5 times higher than that of sparsely seeded cells
( ) at 0 h. Each point represents the mean ± S.E. of 60 responder cells in three cultures. *, p < 0.05, significant difference between sparsely and densely seeded
MC3T3-E1 cells. #, significant difference compared with 0 h.
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To confirm that densely seeded MC3T3-E1 cells in the resting state have
a high [Ca2+]i level, we measured
the [Ca2+]i level in both
isocultures and cocultures with other cell lines, including mouse
fibroblasts (NIH3T3), rat vascular smooth muscle cells
(A7r5), and ECs from the bovine aorta. The resting state
[Ca2+]i level of MC3T3-E1 cells
was higher than that of the other cell lines both in the isocultures,
in which one cell line each had been harvested (Fig.
3A), and in the cocultures at
48 h, in which two cell lines had been cultured in the same dish (Fig. 3B). These results suggest that the high
[Ca2+]i level in resting state
MC3T3-E1 cells may exert a specific function in calcifying tissue.

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Fig. 3.
Comparison of the resting state
[Ca2+]i level
among MC3T3-E1, EC, A7r5, and NIH3T3 cells.
The resting state Ca2+ level of MC3T3-E1 cells was higher
than that of the other cells both in isoculture (A) and
coculture (B). *, p < 0.05, significant
difference compared with that in MC3T3-E1 cells. Each bar
represents the mean ± S.E. of three cultures.
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Effects of ATP and Caffeine on [Ca2+]i
Release from Intracellular Ca2+ Stores at Different Cell
Densities during Cell Proliferation--
In the following experiments,
MC3T3-E1 cells were incubated in PBS in which CaCl2 had
been replaced with EGTA (1.0 mM) to prevent
Ca2+ influx into cells and to selectively evaluate
Ca2+ release from intracellular calcium stores. ATP (10 µM), which stimulates IICR (15, 16), elevated the
[Ca2+]i level in 84% of the
sparsely seeded cells when it was applied at 24 h after seeding,
in 100% of the cells when applied at 48 h, and in 98% of the
cells when applied at 72 h (Fig.
4A). In contrast, ATP elevated
the [Ca2+]i level in only 18-23%
of the densely seeded cells when it was applied between 24 and 120 h. The relative value of
F340/F380 upon treatment
with ATP at 24 and 48 h in each responder cell in the culture
under the sparsely seeded condition was 1.675 ± 0.066 and
1.770 ± 0.068, respectively. Then, it sharply decreased to
1.279 ± 0.023 and 1.038 ± 0.003 when ATP was applied at 72 and 120 h, respectively, when the cell density, because of
proliferation, reached that of the densely seeded cells (Fig.
4B).

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Fig. 4.
Effect of ATP on the
[Ca2+]i level in
MC3T3-E1 cells. A, percentage of ATP-responding cells
under sparsely ( ) and densely ( ) seeded conditions during cell
proliferation. B, relative value of
F340/F380 upon ATP
stimulation in responder cells under sparse ( ) and dense ( )
conditions. Each point represents the mean ± S.E. of
60 responder cells in three cultures. *, p < 0.05, significant difference between MC3T3-E1 cells seeded in sparse and
dense conditions. #, significant difference compared with that at
24 h.
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Caffeine, which stimulates CICR (14), increased the
[Ca2+]i level in 61 and 79% of
the sparsely seeded cells when it was applied at 24 and 48 h after
seeding, respectively. When caffeine was applied at 72 or 120 h,
only 0-3% of the sparsely seeded cells had an increased
[Ca2+]i level (Fig.
5A). In the densely seeded
cells, caffeine had no effect on the
[Ca2+]i level when it was applied
between 24 and 120 h after seeding. In sparsely seeded cells, the
relative value of
F340/F380 was 1.115 ± 0.009 when caffeine was applied at 24 h, and it increased to
1.199 ± 0.028 when applied at 48 h (Fig. 5B).
These results indicate that caffeine affected the
[Ca2+]i level in MC3T3-E1 cells
more heterogeneously than ATP and that the degree of heterogeneity upon
treatment with caffeine or ATP was affected by the cell density.

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Fig. 5.
Effect of caffeine on the
[Ca2+]i level in
MC3T3-E1 cells. A, percentage of caffeine-responding
cells under sparsely ( ) and densely ( ) seeded conditions during
cell proliferation. B, relative value of
F340/F380 upon treatment
with caffeine in the sparsely ( ) and densely ( ) seeded
conditions. Each point represents the mean ± S.E. of
60 responder cells in three cultures. *, p < 0.05, significant difference between MC3T3-E1 cells seeded in sparse and
dense conditions. #, significant difference compared with that at
24 h.
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In the sparsely seeded condition, the relative value of
F340/F380 upon treatment
with ATP in caffeine responding cell was larger than that upon
treatment with ATP in caffeine non-responding cell at 24 and 48 h
(Fig. 6). These results indicate that
cells that responded to caffeine also responded to ATP and suggest that the source of [Ca2+]i release was
the same smooth-surfaced endoplasmic reticulum (sER) via CICR or IICR,
a finding that differs from that in vascular smooth muscle cells under
the sparsely seeded condition (5, 6).

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Fig. 6.
Comparison of the relative value of
F340/F380
upon treatment with ATP between caffeine-responded (black
columns) and non-responded (white columns)
MC3T3-E1 cells at 24 or 48 h in the sparse condition. Each
bar represents the mean ± S.E. of three cultures. *,
p < 0.05, significant difference between caffeine
responded cells and caffeine non-responded cells.
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Effects of Thapsigargin (TG) and Ionomycin (IM) on the
[Ca2+]i Level at Different Cell
Densities--
TG blocks the uptake of Ca2+ from the
cytoplasm to the sER (17). When TG was applied at 24, 48, or 72 h,
the [Ca2+]i level increased in
97.5-100% of the sparsely seeded cells; however, upon application of
TG at 120 h, none of the cells had an increased
[Ca2+]i level (Fig.
7A). The percentage of densely
seeded cells with elevated [Ca2+]i
was only 3-8% when TG was applied between 24 and 120 h after
seeding. In sparsely seeded cells, the relative value of
F340/F380 in the
responders was 1.445 ± 0.026, 1.375 ± 0.025, and 1.376 ± 0.023 when TG was applied at 24, 48, and 72 h, respectively (Fig. 7B). However, the relative value of
F340/F380 in sparsely seeded cells upon treatment with TG at 120 h and the relative values of F340/F380 in
densely seeded cells that had been treated with TG at 24, 48, 72, or
120 h did not significantly differ.

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Fig. 7.
Effect of thapsigargin on the
[Ca2+]i level in
MC3T3-E1 cells. A, percentage of
thapsigargin-responding cells under the sparsely ( ) and densely
( ) seeded conditions during cell proliferation. B,
relative value of
F340/F380 in responder
cells upon treatment with thapsigargin among sparsely seeded cells
( ) at 24, 48, or 72 h is high. The
F340/F380 did not change
in the densely seeded cells upon treatment with thapsigargin between 24 and 120 h ( ). Each point represents the mean ± S.E. of 60 responder cells in three cultures. *, p < 0.05, significant difference between MC3T3-E1 cells seeded in the
sparse and dense conditions. #, significant difference compared with
that at 24 h.
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All Ca2+ ions in internal stores can be non-specifically
released by the Ca2+ ionophore, IM (2). IM elevated the
[Ca2+]i level in 100% of the
sparsely and densely seeded cells. In the sparsely seeded cells, the
relative value of
F340/F380 upon treatment
with IM at 24, 48, 72, or 120 h after seeding was 2.110 ± 0.067, 2.058 ± 0.078, 1.910 ± 0.042, and 1.421 ± 0.021, respectively (Fig. 8). When
densely seeded cells were treated with IM, the
[Ca2+]i level increased by a
smaller degree. All data on the Ca2+ dynamics in the TG and
IM experiments supported the dependence of the
[Ca2+]i response on cell density.
Furthermore, the Ca2+ response was smaller upon treatment
with TG than upon treatment with IM at each time point. This may
indicate that Ca2+ was released from other internal stores
in addition to the sER.

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Fig. 8.
Relative value of
F340/F380
upon treatment with ionomycin in responder cells under sparse
( ) and dense ( ) conditions. Each point represents
the mean ± S.E. of 60 responder cells in three cultures. *,
p < 0.05, significant difference between MC3T3-E1
cells seeded in sparse and dense conditions. #, significant difference
compared with that at 24 h.
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Effect of Heptanol on the [Ca2+]i
Response--
The relative value of
F340/F380 upon treatment
with ATP, caffeine, TG, or IM in the responders was much lower
throughout the experiment in the densely seeded cells than in the
sparsely seeded cells (Figs. 4, 5, 7, 8). Heptanol, a gap-junctional
inhibitor (9, 10), was used to evaluate the effect of cell-cell contact in the densely seeded cells. When densely seeded cells were treated with 3 µM heptanol, the
[Ca2+]i elevation and the relative
value of F340/F380 upon treatment with ATP, TG, or IM recovered (Fig.
9). These results suggest that cell-cell
contact may inhibit the Ca2+ response of densely seeded
cells to ATP, caffeine, TG, and IM.

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Fig. 9.
Effect of heptanol on the increase in
F340/F380
induced by ATP (A), thapsigargin
(B), and ionomycin (C). Each
bar represents the mean ± S.E. of 40 responder cells
in two cultures. *, p < 0.05, significant difference
between densely seeded control cells (black bars)
and 3 µM heptanol-treated cells (white
bars).
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Immunodetection of
IP3-R1--
Ca2+ was released from
CICR channels in response to caffeine only in sparsely
seeded cells up to between 48 and 72 h. The Ca2+
efflux from IICR channels was also closely related to the
Ca2+ released from the sER. These results suggest that IICR
may play a more significant role than CICR in calcium handling in
osteoblasts. To date, three isoforms of the IP3 receptor
have been cloned, and the type-1 receptor is believed to be ubiquitous.
Using a specific antibody to IP3-R1, we
investigated the expression of this receptor in sparsely and densely
seeded cells to determine whether it is involved in the
Ca2+ response. A high level of
IP3-R1 protein was detected in sparsely seeded
cells upon culture for 24 h (Fig.
10B), and a low level was
detected upon culture for 120 h (Fig. 10C). The level
of immunostaining was low in densely seeded cells at both 24 and
120 h (Fig. 10, D and E). The level of
IP3-R1 protein expression was associated with
the amount of Ca2+ released from IICR channels.

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Fig. 10.
Immunodetection of
IP3-R1.
A, negative control in sparsely seeded cells at 24 h.
B, a high level of IP3-R1 was
detected in sparsely seeded cells at 24 h. C, the level
of IP3-R1 in sparsely seeded cells decreased at
120 h. The level of staining was weak in densely seeded cells both
at 24 (D) and 120 h (E). Bar, 60 µm.
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Electron Microscopy of sER--
Transmission electron microscopy
was used to ascertain the relationship between the
[Ca2+]i level and the amount of
sER in osteoblasts. Many organelles, including sER, were observed in
the sparsely seeded cells at 24 h (Fig.
11, A and B).
However, at 120 h of culture, the sER had been reduced, and many
rough-surfaced endoplasmic reticula and mitochondria were detected
after cell proliferation (Fig. 11, C and D). Many
rough-surfaced endoplasmic reticula and mitochondria were observed and
reduced number of sER were seen in densely seeded cells that had been
cultured for 24 (Fig. 11, E and F) or 120 h (Fig. 11, G and H). Thus, the reduced
Ca2+ response of densely seeded cells to ATP, caffeine, TG,
and IM can be explained by the reduced number of sER. These results
suggest that the expression of sER is regulated by cell
proliferation.

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Fig. 11.
Electron microscopic observation of
sER. Many organelles, including sER, are observed in sparsely
seeded cells at 24 h (A and B). However, the
sER disappeared and many rough-surfaced endoplasmic reticula and
mitochondria are observed at 120 h (E and
F), when the cell density became similar to that of densely
seeded cells after cell proliferation. In densely seeded cells, many
rough-surfaced endoplasmic reticula and mitochondria are observed and
no sER are seen at 24 (C and D) and 120 h
(G and H). Bar in A,
C, E, and G, 0.5 µm; in
B, D, F, and H, 0.2 µm.
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 |
DISCUSSION |
Heterogeneity of the [Ca2+]i
level among MC3T3-E1 cells was observed even before stimulation and was
dependent on cell density (sparsely seeded or densely seeded). The
[Ca2+]i level in cells in the
resting state ranges from 50 to 400 nM and varies according
to cell type and function (14). Because estimation of the absolute
[Ca2+]i level by fura-2
fluorescence is inaccurate because of several problems intrinsic to
fura-2 fluorescence (11-13), the [Ca2+]i level in the resting state
obtained in previous reports cannot be simply compared with that
obtained in the present study. The relative value of
F340/F380 in reference to
F340/F380 in the resting
state (11) was used to evaluate the Ca2+ response. To
compare the [Ca2+]i levels of
various cells at the resting state, we used F340/F380 in iso- and
cocultures of MC3T3-E1 cells and other established cell lines (EC,
A7r5, and NIH3T3).
It is unclear why the [Ca2+]i
level was higher in the densely seeded MC3T3-E1 cells than in the
sparsely seeded cells or other cell lines. We consider that this
characteristic is specific for bone-forming MC3T3-E1 osteoblast-like
cells. In the densely seeded MC3T3-E1 cells, the number of sER was
reduced, indicating a reduced amount of stored Ca2+.
Consequently, it is suggested that in the resting state, the [Ca2+]i level increases. However,
the [Ca2+]i level in densely
seeded cells was about 1.5 times higher than that in the sparse
condition or other cell lines, and the degree of the increase in
[Ca2+]i level was not necessarily
so high that it would be toxic for cells. The presence of an
increased [Ca2+]i level in the
resting state of MC3T3-E1 cells, especially in densely seeded cells,
seems to be informative for estimating the physiological significance
of bone-forming cells. We believe that the increased
[Ca2+]i level may be advantageous
for the release of Ca2+ during calcification from the point
of view of ionic strength.
We focused on the cell density- and growth-dependent
changes in the [Ca2+]i response of
osteoblast-like cells to various stimuli. There is a study that focused
on the downstream signaling mechanism via Ca2+, not only
from internal stores but also from extracellular Ca2+ in
osteoblasts (18). Whether the
[Ca2+]i response of
osteoblast-like cells to various stimuli creates cross-talk with
extracellular Ca2+-sensing pathways is of much interest and
requires further investigation (19). Among the agonists employed in the
present study, ATP, which stimulates IICR, caused
[Ca2+]i elevation when applied
between 24 and 120 h to both sparsely and densely seeded cells. In
contrast, caffeine, which opens CICR channels, induced
[Ca2+]i elevation in only the
sparsely seeded condition. CICR may be inhibited more strongly than
IICR in densely seeded cells. On the other hand, cells showing CICR
channel function reacted to ATP, and the other cells that did not
reveal an IICR channel response did not respond to caffeine either.
Therefore, IICR channels mainly handle the calcium response in MC3T3-E1
cells. We previously showed that the expression of CICR and IICR
channels, in addition to voltage-dependent Ca2+
channels, is influenced by cell growth and enhanced by the
differentiation of vascular smooth muscle cells and that they are
independently regulated from each other (5, 6). The percentage of
responding cells and the magnitude of the Ca2+ response
were much lower in sparsely seeded vascular smooth muscle cells than in
densely seeded cells. Thus,
[Ca2+]i handling in MC3T3-E1 cells
differs from that in vascular smooth muscle cells.
TG depletes Ca2+ from the sER and induces
[Ca2+]i elevation via capacitative
Ca2+ entry. A sufficient dose of IM, an ionophore that
forms artificial [Ca2+]i channels
in membranes and releases all Ca2+ from the sER, also
increased the [Ca2+]i level even
when Ca2+ entry did not occur in PBS(
). The rank order of
the degree of [Ca2+]i increase was
IM > TG = ATP > caffeine. These results indicate that
there is a large amount of Ca2+ in the sER of sparsely
seeded MC3T3-E1 cells and that IICR channels of the sER mainly control
[Ca2+]i elevation in MC3T3-E1
cells. These findings are fundamentally compatible with the results of
the study of Zach et al. (19). In sparsely seeded MC3T3-E1
cells, the presence of a high level of IP3-R1
and a large number of sER was morphologically confirmed by
immunohistology and electron microscopy, respectively.
The [Ca2+]i level in osteoblasts
has been reported to be correlated with the seeded cell density, the
cell line, morphology, and receptor distribution (7, 8, 12). Various
factors influence the [Ca2+]i
level in individual cells at rest and after agonist administration. A
previous study suggested that gap junctions and gap-junctional
intercellular communication play pivotal mechanotransduction mechanisms
in bone (20). In this study, the results of heptanol treatment revealed
that gap-junctional cell-cell contact also contributes to the cell
density-dependent, heterogeneous
[Ca2+]i response to agonists. In
the proliferating phase of osteoblasts, cells without cell-cell contact
have both CICR and IICR channels or abundant sER. We
previously showed that IP3-R1 may
regulate the proliferation of vascular smooth muscle cells (21). During
bone formation in physiological growth or pathological repair after a
bone fracture, enhanced IP3-R1 expression may
be required for the proliferation of osteoblasts. To elucidate the precise significance of IICR modulation, more specific and detailed studies should be conducted in the future.
 |
FOOTNOTES |
*
This study was financially supported by grants-in-aid from
the Ministry of Education, Culture, Sports, Science, and Technology and
the Ministry of Health, Labor, and Welfare.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.
¶
To whom correspondence should be addressed: University of
Tokyo, 7-3-1, Hongo, Bunkyo-ku 113-8655, Tokyo, Japan. Tel.:
81-3-5800-8669; Fax: 81-3-5800-6832; E-mail:
hikiji-ora@h.u-tokyo.ac.jp.
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M210243200
 |
ABBREVIATIONS |
The abbreviations used are:
IP3, inositol trisphosphate;
IICR, inositol trisphosphate-induced
Ca2+ release;
CICR, Ca2+-induced
Ca2+ release;
F340/F380, 340- to 380-nm
fluorescence ratio;
TG, thapsigargin;
IM, ionomycin;
PBS, phosphate-buffered saline;
EC, endothelial cell(s);
IP3-R1, inositol trisphosphate type-1 receptor;
sER, smooth-surfaced endoplasmic reticulum.
 |
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