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INTRODUCTION |
Cyclosporin A (CsA)1 is
a cyclic endecapeptide immunosuppressant that is widely used to prevent
organ rejection after transplantation. Despite the effectiveness of CsA
in preventing rejection, its clinical application is limited by various
side effects (1), including cardiotoxicity, nephrotoxicity, and
gingival overgrowth (2). Previous studies have suggested that
perturbation of intracellular Ca2+ signaling (3) is a
common mechanism for CsA-induced cardiotoxicity (4) and many other
side effects. In cardiac muscle cells, CsA inhibits contractility by
altering the function of Ca2+ release channels and
deregulating mitochondrial ion homeostasis (5). CsA induces
mitochondrial dysfunction by binding to cyclophilin D (6), a
mitochondrial protein that regulates conductance of the mitochondrial
membrane permeability transition pore (PTP), a nonspecific, high
conductance ion-permeable channel in mitochondrial membranes. CsA
induces cyclophilin to detach from the PTP (7) and thus facilitates
closure. However, the mechanism(s) by which CsA causes gingival
overgrowth is unknown (2).
Under physiological conditions, gingival connective tissues exhibit a
remarkably rapid rate of collagen turnover (8). The abundant synthesis
of collagen in these tissues is balanced by an equally rapid rate of
intracellular degradation (9), a process that is mediated by the
phagocytic pathway in fibroblasts (10). In CsA-induced gingival
overgrowth, there is a net increase in collagen (11), but CsA does not
increase collagen expression (12) and does not substantially alter
collagenase levels (13). Instead, the net increase in collagen is
apparently due to reduced phagocytosis by fibroblasts (11). Indeed,
although CsA-induced inhibition of collagen phagocytosis has been
demonstrated in several in vivo studies (14, 15), the
mechanisms and the intracellular locus of this inhibition are not defined.
In "professional" phagocytes such as neutrophils, efficient
phagocytosis is dependent on calcium release from intracellular stores
(16), which, in turn, is reliant on inositol 1,4,5-trisphosphate receptor function to regulate calcium levels within endoplasmic reticulum (ER) stores (17). These receptors are translocated to
periphagosomal sites during particle internalization (18), suggesting
that phagocytosis may be affected by calcium levels in the
intracellular stores. Notably,
2
1
integrin-mediated collagen phagocytosis in gingival fibroblasts is
regulated by intracellular calcium (19), in which a
calcium-dependent feedback loop may alter integrin affinity
of matrix molecules and thereby enhance integrin-mediated cell adhesion
(20). Since CsA binding to cyclophilin perturbs the function of the PTP
(6) and can independently inhibit calcium release from ER stores (5,
21), we considered that CsA may inhibit collagen phagocytosis by
deregulating calcium homeostasis in mitochondrial and/or ER stores.
To obtain an improved understanding of the role of calcium signaling in
phagocytosis and CsA-induced gingival overgrowth, we have used a well
characterized in vitro model of
2
1 integrin-mediated collagen
phagocytosis in gingival and Rat2 fibroblasts (19, 22-24). Cells were
stimulated with collagen beads, and the effect of CsA on intracellular
calcium signaling in cytosolic, mitochondrial, and ER stores was
examined. The data show that CsA inhibits the binding step of collagen
phagocytosis through a calcium-regulated pathway involving ER and
mitochondrial stores.
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EXPERIMENTAL PROCEDURES |
Reagents--
Carbonyl cyanide
m-chlorophenylhydrazone (CCCP) and cyclosporin A were
obtained from Sigma. Mouse anti-human cytochrome oxidase subunit I
monoclonal antibodies, BAPTA/AM, fura-2/AM, JC-1
(5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimidazolecarbocyanine iodide), mag-fura-2/AM, MitoTracker Green®,
Pluronic® F-127, and rhod-2/AM were obtained from
Molecular Probes, Inc. (Eugene, OR). Ionomycin and thapsigargin were
obtained from Calbiochem. Fluorescent and nonfluorescent latex beads
were obtained from Polysciences (Warrington, PA).
Cell Culture--
Human gingival fibroblasts (HGFs; passages
8-15) were derived from primary explant cultures as described earlier
(25). Cells from passages 8-15 were grown in
-minimal essential
medium, 15% (v/v) fetal bovine serum (Flow Laboratories, McLean, VA),
and antibiotics. The cells were maintained at 37 °C in a humidified incubator containing 5% CO2 and were passaged with 0.01%
trypsin (Life Technologies, Inc., Burlington, Ontario, Canada). Rat2
cells (ATCC CRL 1764, American Type Culture Collection, Manassas, VA) were incubated in Dulbecco's modified Eagle's medium (high glucose) containing 10% fetal bovine serum and antibiotics.
Mitochondrial Depletion--
Rat2 cells were grown in
Dulbecco's modified Eagle's medium (high glucose) containing 10%
fetal bovine serum and antibiotics as described above. Cells were grown
in the presence of 100 ng/ml ethidium bromide for 30 passages before
the dose was increased gradually to 1 µg/ml by passage 40. The
ethidium bromide-treated Rat2 cells (Rat2EtBr) were always cultured at
1 µg/ml ethidium bromide for at least 4 weeks before using for
experiments to maintain mitochondrial depletion as described (26).
[Ca2+]i Measurement--
For
measurement of [Ca2+]i, cells on glass coverslips
were incubated at 37 °C with 3 µM fura-2/AM in
bicarbonate-free medium (Life Technologies, Inc.) for 25 min. The
attached cells were washed twice with bicarbonate-free calcium buffer
(145 mM NaCl, 5 mM KCl, 5 mM
MgCl2, 10 mM D-glucose, 10 mM HEPES, and 1 mM CaCl2, pH 7.4;
osmolarity = 291 mosM) and transferred to a microscope
mounting stage. CaCl2 was omitted from the buffer solution
where indicated. Measurements of [Ca2+]i were
made on single cells using a Nikon Diaphot II inverted microscope
optically interfaced to an epifluorescence spectrofluorometer and
analysis system (Photon Technology International Inc., London, Ontario)
operating on a 386SX personal computer. The dual-excitation
fluorochrome fura-2 was excited at alternating (~100 Hz) wavelengths
of 346 and 380 nm from dual monochromators with slit widths set at 2 nm. Emitted fluorescence was collected by a 40× quartz 1.30-NA
oil-immersion Nikon Fluor objective, passed through a 520/30-nm
barrier filter (Omega Optical Inc., Brattleboro, VT), and detected by a
photomultiplier tube. A variable-aperture intrabeam mask was used to
restrict measurements to single cells. Estimates of
[Ca2+]i independent of the precise intracellular
concentration of fura-2 were calculated from dual-excitation emitted
fluorescence according to the equation of Grynkiewicz et
al. (27) (i.e. [Ca2+]i = Kd × Sf2/Sb2 × (R
Rmin)/(Rmax
R).
For analysis of calcium in ER compartments, cells were incubated with
4.5 µM mag-fura-2/AM for 140 min at 37 °C as described (28). Mag-fura-2 measurements were made with the Photon Technology International instrument and single cell photometry. For
analysis of mitochondrial calcium, Rat2 fibroblasts were loaded with
4.5 µM rhod-2/AM in 0.005% (v/v) Pluronic F-127 gel for
30-140 min at 37 °C. The cells were washed twice and incubated in
calcium buffer for imaging. The magnitude of fluorescence of
rhod-2-stained samples was observed in a Nikon inverted microscope
equipped with a CCD camera (Pentamax, Princeton Research
Instruments Inc., Princeton, NJ) and analyzed using the Winview
software program (Princeton Research Instruments Inc.). As ratio
imaging cannot be used to overcome problems of dye leakage and
photobleaching in time course experiments with rhod-2, single
excitation/single emission imaging analyses were conducted with a
number of corrective procedures as described earlier (29). First, the
fluorescence intensity of rhod-2 was measured in small sampling grids
(~4 µm2) in the lamellipodia of well spread cells to
avoid the inclusion of nucleoli, which stain brightly with rhod-2.
Second, background fluorescence and adjustments for
time-dependent photobleaching (29) were made for each
measurement by calculating the bleach-induced rate constant
(i.e. time-dependent loss of fluorescence) in
untreated cell samples that were not incubated with collagen-coated
beads. Third, for verification of the appropriate spatial localization of the rhod-2 staining and to ensure that rhod-2 was not sequestered to
lysosomes (30), in some experiments, Rat2 fibroblasts were co-loaded
with 100 nM MitoTracker Green and 4.5 µM
rhod-2/AM in 0.005% (v/v) Pluronic F-127 for 30 min. The cells were
washed twice and left in calcium buffer before imaging.
Flow Cytometric Analysis of Phagocytosis--
Green fluorescent
microbeads (2 µm in diameter) were incubated with 1 ml of a 2.9 mg/ml
acidic bovine collagen solution (Vitrogen, Celtrix Laboratory, Palo
Alto, CA) neutralized with 1 N NaOH to produce
collagen-coated beads (22). In some experiments, beads coated with
either bovine serum albumin or poly-L-lysine were used as
controls (19). Binding of beads was assessed by flow cytometry as
described (22, 23). Cell samples were analyzed with a FACStar Plus flow
cytometer (Becton Dickinson FACS Systems, Mountain View, CA).
Photomultiplier tube voltage settings were determined for each
experiment on the basis of thresholds established from negative and
positive control samples for each sample that was analyzed. To reduce
the likelihood of measuring cells with loosely attached,
nonspecifically bound beads, the cells were washed with calcium- and
magnesium-free phosphate-buffered saline and trypsinized prior to
analysis by flow cytometry.
Analysis of Mitochondrial Membrane Potential
(
m)--
Changes in
m were estimated using
JC-1. This cyanine dye accumulates in the mitochondrial matrix under
the influence of
m and forms J-shaped aggregates that
have characteristic absorption and emission spectra (31). Untreated
cells and cells treated with CsA were incubated in 3 ml of
phosphate-buffered saline supplemented with 10% serum containing 0.5 µM JC-1 for 1 h. As a control for cells in which
m was dissipated, a group of cells were treated with the
uncoupling agent CCCP before labeling with JC-1 (32). Cell suspensions
were prepared for flow cytometry (33), and the 488-nm line of an argon
ion laser was used for excitation. Orange and green emitted
fluorescence was collected through 585/42-nm (FL2) and 530/30-nm (FL1)
band-pass filters. Flow cytometry was performed on a FACStar Plus flow
cytometer and analyzed using FACStation software (Becton
Dickinson FACS Systems). After gating out noncellular
debris, 10,000 cells were analyzed for each sample. Bivariate plots of
FL2 versus FL1 and the computation of FL2:FL1 ratios were
used to estimate
m as described (32, 34).
Western Blot Analysis--
Whole cell extracts were prepared by
rinsing trypsinized cells with calcium- and magnesium-free
phosphate-buffered saline containing protease inhibitors (0.5 µg/ml
leupeptin, 0.5 µg/ml pepstatin, and 1 mM
phenylmethylsulfonyl fluoride). Cells (5 × 106
cells/ml) were pelleted, solubilized for 30 min in calcium- and magnesium-free phosphate-buffered saline containing the above protease
inhibitors plus 1.5% lauryl maltoside at 4 °C, and centrifuged for
20 min at 16,600 × g, and the supernatants were saved
for biochemical analysis. Equivalent amounts of protein (by Bio-Rad assay) were separated on polyacrylamide gels and transferred
electrophoretically to 0.2-µm nitrocellulose membranes. The blots
were developed with chemiluminescent reagents and exposed to x-ray
films, which were developed and analyzed.
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RESULTS |
Effect of CsA on Collagen Bead Phagocytosis--
For study of CsA
regulation of collagen phagocytosis, we utilized an in vitro
model in which collagen bead binding to gingival fibroblasts
facilitates analysis of regulatory processes involved in collagen
internalization (19, 22-24). To overcome the problems of phenotypic
variability associated with primary isolates of gingival fibroblasts
(35), we first rationalized the use of Rat2 fibroblasts, a stable and
readily propagated cell line previously used for studies of collagen
phagocytosis and that also exhibits many of the characteristic features
of periodontal fibroblasts (24). We compared collagen bead-induced
phagocytosis in HGFs and Rat2 fibroblasts by flow cytometry, a protocol
that enables quantitative and unbiased evaluations of the bead binding
step of phagocytosis (36). This integrin-dependent step of
collagen phagocytosis is rate-limiting for subsequent steps involving
intracellular collagen degradation (19, 22, 23). In HGFs, incubation
with collagen-coated beads for 1 h produced a bead
number-dependent relationship with phagocytosis (eight
beads/cell, 70.2 ± 0.7% phagocytic cells; four beads/cell,
45.4 ± 1.2% phagocytic cells; and two beads/cell, 21.4 ± 0.5% phagocytic cells). At eight beads/cell, binding of BSA-coated
beads was >4-fold less compared with collagen-coated beads (17.2 ± 1.4%; p < 0.001). In Rat2 cells, incubation with collagen-coated beads at two beads/cell for 1 h produced a result similar to that obtained with HGFs (19.0 ± 1.5%). In CsA-treated HGFs, addition of very low doses of CsA (10 nM) induced a
large reduction (>2-fold; p < 0.05) in the mean
percent of phagocytic cells and the number of beads/cell
(p < 0.05) (Fig.
1A), which was also seen in
Rat2 fibroblasts (19.0% in controls compared with 6.6 ± 0.7%
after CsA; p < 0.001). When Rat2 cells were treated with increasing doses of CsA for 30 min prior to 1-h incubations with
collagen-coated beads, we found that CsA doses resembling tissue levels
obtained therapeutically in humans (37) produced a
dose-dependent reduction of phagocytosis (Fig.
1B). These reductions of collagen bead binding were not due
to direct interference of CsA in the binding of collagen to integrins
because preincubation of collagen beads with equivalent concentrations
of CsA followed by washing and subsequent incubations with cells and no
CsA caused no significant inhibition of short-term (i.e. 5 min) bead binding.

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Fig. 1.
Effect of CsA on collagen bead binding.
A, flow cytometric analysis of the binding step of collagen
phagocytosis in human gingival fibroblasts after incubation with 10 nM CsA for 30 min. Note the marked reduction of collagen
bead fluorescence (FL1-height) in the CsA-treated cells.
n = 10,000 cells per analysis. B,
quantification of means ± S.E. of percent collagen bead binding
in Rat2 cells after 30-min preincubations with CsA at the indicated
doses. CsA caused dose-dependent reduction of bead binding
(p < 0.01; n = five separate cell
samples/group).
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Phagocytic Response and Calcium Signaling--
Integrin activation
by extracellular matrix ligands stimulates calcium rises (38, 39) that
may be important in regulating integrin affinity and the bead binding
step of phagocytosis. Accordingly, we assessed the role of
intracellular calcium signaling in the regulation of collagen
bead-induced phagocytosis. The treatment of cells with thapsigargin (1 µM, 10 min) to inhibit store uptake and to prevent
subsequent store release dramatically reduced the phagocytic capacity
of Rat2 fibroblasts when incubated with collagen beads for 1 h
(10.2-fold reduction, from 19.0 to 1.86 ± 0.45% of phagocytic
cells; p < 0.01; a reduction that was comparable in
size to the 8-fold decrease seen when Rat2 fibroblasts were pretreated
with 10 µM CsA (p < 0.02); n = five samples/group). A similarly sized reduction of phagocytosis was
obtained after pretreatment with BAPTA/AM, which chelates intracellular
calcium (from 19.0 to 2.2 ± 0.3%; p < 0.01;
n = five samples/group). These data indicate that
intracellular calcium signaling may have an important regulatory
function in collagen bead-induced phagocytosis.
We compared calcium signaling in HGFs and Rat2 cells after collagen
bead stimulation to rationalize the use of Rat2 cells as surrogates for
HGFs in studies of calcium signaling in phagocytosis. For all collagen,
BSA, or poly-L-lysine bead incubations, a ratio of eight
beads to one cell was used. In fura-2/AM-loaded cells, the response of
[Ca2+]i to collagen bead-induced phagocytosis was
similar (the mean [Ca2+]i rise above the
base-line level in HGFs was 124 ± 31 nM, and that in
Rat2 fibroblasts was 130 ± 9 nM; p > 0.2; n = 10 cells/group). The rise to maximum
[Ca2+]i after collagen bead stimulation also
proceeded over a similar time course (time to peak: HGFs, 105 s;
and Rat2 cells, 110 s). In both types of cells, incubation with
BSA- or poly-L-lysine-coated beads produced no significant
rise in [Ca2+]i above the base-line levels (<5
nM; n = five cells). These data indicate
that analogous to the phagocytosis data, Rat2 cells are a sensitive and
ligand-specific model for study of collagen bead-induced calcium
signaling in gingival fibroblasts.
Prior to measuring the impact of CsA on collagen bead-induced calcium
signaling, we examined the effect of CsA alone on Ca2+
mobilization. In Rat2 fibroblasts, CsA induced rapid increases in
[Ca2+]i, which returned to base-line levels
within 100 s (Fig. 2a).
The rapid removal of Ca2+ from the extracellular buffer (5 mM EGTA immediately before addition of CsA) completely
abolished the CsA-induced cytosolic calcium transients, indicating the
importance of extracellular calcium influx for the CsA mechanism of
action (Fig. 2b). To determine whether these cytosolic
calcium changes involved release of calcium from thapsigargin-sensitive
stores, we pretreated cells with thapsigargin (1 µM) to
deplete internal Ca2+ stores (Fig. 2c) and then
added CsA (Fig. 2d). The increases in
[Ca2+]i were reduced in comparison with control
cells, in which thapsigargin stores were not depleted; there was a very slow subsequent reduction of Ca2+ after peak levels were
attained (Fig. 2d), an expected result due to the reduced
uptake of calcium into the ER. Mag-fura-2-loaded cells treated with CsA
also showed no discharge of Ca2+ from the ER (Fig.
2e). These results are consistent with earlier data on
CsA-induced calcium fluxes in LLC-PK1 cells (40) and indicate that influx of extracellular calcium is likely important for
CsA-mediated effects on collagen phagocytosis.

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Fig. 2.
CsA regulation of calcium signaling in Rat2
cells. a, in fura-2-loaded cells, CsA (10 nM) induced a steep and rapid increase in intracellular
calcium, which returned to base-line levels within 50 s in normal
calcium-containing medium. b, cells switched to a medium
containing 5 mM EGTA showed no calcium increase after CsA
treatment. c, cells induced with thapsigargin
(Tg; 1 µM) in normal calcium-containing medium
showed rapid calcium flux, indicating emptying of ER stores.
d, cells in normal medium preincubated with thapsigargin (1 µM) and then treated with CsA showed a large increase in
intracellular calcium, but the calcium did not return to base-line
levels. e, in cells loaded with mag-fura-2 to measure
calcium in ER stores, CsA caused no change in calcium concentration
when cells were in normal medium. These data are representative of five
separate experiments for each treatment. Tx, pretreated.
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Following incubation with collagen beads, there was a steep rise in
[Ca2+]i, which dissipated within 100 s (Fig.
3, A and B,
panel a). In experiments using CsA pretreatment, there was a
large decrease in [Ca2+]i responses to collagen
bead-induced phagocytosis (3.4-fold; p < 0.01; cells
pretreated with 10 nM CsA for 1 h) (Fig.
3A). This effect may be due to CsA-induced inhibition of
intracellular calcium store release. Accordingly, the
Ca2+i levels were investigated after intracellular
stores of calcium were depleted with 1 µM thapsigargin
pretreatment or cytoplasmic calcium was chelated with 3 µM BAPTA/AM. For thapsigargin, the response to collagen
bead-induced phagocytosis was reduced by >3-fold (Fig. 3A).
Following calcium chelation with BAPTA/AM, the amplitude of the
[Ca2+]i transients induced by collagen beads was
reduced by >5-fold (Fig. 3). In comparison with vehicle controls,
pretreatment with CsA reduced the amplitude of the collagen
bead-induced calcium responses an additional 20 nM when
cells were treated with thapsigargin, but there was no additional
reduction conferred by CsA when cells were pretreated with BAPTA/AM.
These data indicate that CsA inhibits the calcium responses induced by
collagen bead binding to integrins.

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Fig. 3.
Collagen bead-induced calcium signaling in
Rat2 cells. A, shown are histograms (means ± S.E.) of peak calcium amplitudes induced by collagen beads in cells
treated with CsA (10 nM) or vehicle (untreated).
In controls, CsA reduced the collagen bead-induced calcium peak by
>3-fold (p < 0.01). In cells pretreated with
thapsigargin (Tg), collagen bead-induced calcium signals
were also reduced by >3-fold (p < 0.01), and CsA
reduced the signal by an additional 20 nM. With BAPTA/AM
pretreatment, the collagen bead-induced calcium signal was reduced by
>6-fold (p < 0.01), and no additional reduction was
produced by CsA. Data are means ± S.E. (n = five
cells/group). B, Rat2 cells were loaded with fura-2/AM and
subsequently incubated with collagen-coated beads (ccb).
Panel a, in medium containing 1 mM
Ca2+; panel b, bead-induced calcium signal in
medium with reduced calcium (0.1 mM) suggesting a
requirement of calcium influx for collagen bead-induced calcium
changes. These individual traces are representative of five
independent experiments.
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We next studied the role of collagen-induced influx of extracellular
calcium using culture medium in which nominal amounts of calcium (100 µM) were present. This approach was employed since the
collagen receptor most abundantly expressed in these cells (
2
1 integrin) (22) requires cations for
ligand binding (e.g. Ca2+ and Mg2+)
to maintain their active conformation (41). In low calcium medium,
there was a small rise in [Ca2+]i after
incubation with collagen-coated beads (Fig. 3B, panel
b). These data indicate that both intracellular and extracellular
calcium sources are important in the [Ca2+]i
responses during the bead binding step of collagen phagocytosis and
that CsA may affect both pathways.
To obtain an improved understanding of cellular Ca2+
homeostasis and the role of the ER calcium stores in response to
collagen bead stimulation, we loaded fibroblasts with mag-fura-2/AM for 140 min according to published methods (42-44). Briefly, cells were
incubated with mag-fura-2/AM for 140 min at 37 °C. In contrast with
cells loaded for 30 min, which showed diffuse fluorescence (Fig.
4A, panel a), cells
loaded for 140 min exhibited preferential accumulation in discrete
organelles (panel b), and virtually no dye was released by
subsequent digitonin permeabilization of the plasmalemma (panel
c) (45). We treated mag-fura-2-loaded cells with ATP to determine
whether the presumptive ER store-loaded mag-fura-2 was reporting
changes in an inositol 1,4,5-trisphosphate-sensitive internal store
(Fig. 4A, panel c). Mag-fura-2-loaded cells
showed rapid reductions of ER calcium following ATP treatment, which were followed by a rapid store refilling back to base-line calcium levels. When cells were incubated with collagen-coated beads, there
were rapid reductions of ER calcium, which preceded increases in
cytosolic calcium measured by conventional fura-2 (Fig. 3B, panel a). Following incubation with BSA-coated beads, there
was no significant change over 1000 s (Fig. 4B,
panel b), indicating that the collagen bead response was
indeed specific. Pretreatment with CsA reduced the base-line levels of
ER Ca2+ and also inhibited the bead-induced reduction of ER
[Ca2+] by >5-fold (Fig. 4B, panel
c).

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Fig. 4.
Response of calcium in ER stores to collagen
beads. A, in cells loaded with mag-fura-2 for 30 min, fluorescence was diffusely distributed throughout the cytoplasm
(panel a). In contrast, when cells were loaded for 140 min,
discrete fluorescence labeling identified the putative ER stores
(panel b), which was confirmed by digitonin permeabilization
(Dig) (panel c). Treatment with ATP caused rapid
loss of calcium and subsequent refilling. B, in cells loaded
with mag-fura-2 as described for panel b in A,
addition of collagen-coated beads (CCB) caused a rapid
calcium efflux from ER stores, followed by a slow refilling as measured
by ratio fluorometry (panel a). Addition of BSA beads caused
no significant change in the mag-fura-2 ratio (panel b).
Pretreatment of cells with CsA (10 nM) reduced the
base-line mag-fura-2 ratio and blunted the collagen bead-induced efflux
of calcium (panel c). Treatment with CCCP (10 µm) alone
caused no change in the mag-fura-2 ratio (panel d), and
pretreatment with CCCP blocked the collagen bead-induced calcium efflux
(panel e). The traces are representative of five
independent experiments. Tx, pretreated.
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Mitochondrial Function--
A role for mitochondria in the
regulation of collagen bead-induced calcium signaling was suggested by
experiments in which the protonophore CCCP (10 µM)
completely blocked collagen bead-induced efflux of calcium from ER
stores (Fig. 4, B, panels d and e). As
CsA has also been shown to affect mitochondrial function (32, 34),
which, in turn, could regulate phagocytic processes, we assessed if
dissipation of the mitochondrial membrane potential with CCCP (10 µM) or inhibition of ATP formation with sodium azide (0.1%) would affect the collagen binding step in phagocytosis. After
short-term (10 min) treatments with either CCCP or sodium azide
followed by 1-h collagen bead incubations, both agents significantly reduced collagen phagocytosis to a level similar to that seen with 100 nM CsA (CCCP, >3-fold reduction, 5.5 ± 0.7%; sodium
azide, >4-fold reduction, 3.5 ± 0.8%; and controls, 19.0%;
p < 0.01). These data suggest that CsA may exert its
effects on phagocytosis by regulating mitochondrial ion homeostasis.
Accordingly, we investigated if the mitochondrial PTP was a candidate
locus of action for CsA in Rat2 cells. To determine whether CsA was
indeed affecting mitochondrial function, we assessed the ability of CsA
to block the dissipation of
m, an effect that is stimulated
by suspension culture of fibroblasts (34).
m was estimated
in JC-1-loaded cells by ratio flow cytometry (32). Treatment of Rat2
fibroblasts in suspension with increasing concentrations of CsA was
accompanied by an elevated ratio of JC-1 aggregates to monomers
(untreated suspended cells, 1.3 ± 0.12; CCCP-treated controls,
0.013 ± 0.001; 10 nM CsA, 1.5 ± 0.08; 100 nM CsA, 2.9 ± 1.07; 1 µM CsA, 3.0 ± 0.08; and 10 µM CsA, 3.5 ± 0.06), indicating
that CsA preserved
m as expected (32) and that CsA was
affecting the function of the PTP.
Mitochondrial calcium changes were monitored with compartmentalized
rhod-2. Double labeling with the vital mitochondrial dye rhod-2 (29)
(Fig. 5A, panel a)
and MitoTracker Green (panel b) demonstrated that rhod-2
fluorescence and MitoTracker Green were completely coincident in Rat2
cells (panel c), indicating that rhod-2 loading reported
calcium changes associated with mitochondria and not with lysosomes
(30). Comparison of rhod-2-loaded cells before (Fig. 5, A,
panel d) and after (panel e) treatment with CCCP
showed that dissipation of
m by CCCP caused loss of
mitochondrion-specific fluorescence and the spread of diffuse fluorescence throughout the cytoplasm. When rhod-2-loaded cells were
stimulated with collagen-coated beads alone, there were progressive and
linear reductions of mitochondrial calcium that occurred over a 20-min
interval, thereby exhibiting much slower kinetics than the discharge of
calcium from the ER (Fig. 5B). The collagen bead-induced reduction of fluorescence was not due to photobleaching or dye loss, as
these factors were already compensated for during the data acquisition
step using previously described methods (29). Incubation with
poly-L-lysine- or BSA-coated beads caused no substantial reduction of fluorescence over time (data not shown). Notably, pretreatment with CsA (10 or 100 nM) caused only small
reductions (p > 0.2, not statistically significant) in
the overall slope (i.e. kinetics) of calcium discharge from
the mitochondria (Fig. 5B), although there was a
statistically significant reduction (p < 0.05) at 10 min. This result may be due to a short-term effect of CsA on calcium
discharge rates. Collectively, these data show that binding of collagen
beads can induce the specific discharge of mitochondrial
Ca2+, but that CsA, at doses that strongly inhibit
phagocytosis, does not substantially alter the overall calcium
discharge rates from mitochondria.

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Fig. 5.
Mitochondrial calcium.
A, shown is the colocalization of the calcium
indicator rhod-2 (panel a) and MitoTracker Green
(panel b) in Rat2 fibroblasts. Panel c shows
colocalization. Rat2 cells loaded with the mitochondrion-specific dye
JC-1 showed discrete staining in mitochondria (panel d).
After dissipation of m with CCCP, JC-1 fluorescence was
diffusely distributed throughout the cytoplasm (panel e).
B, mitochondrial calcium (means ± S.E.) was estimated
by fluorescence after adjustment for photobleaching and dye leakage as
described under "Experimental Procedures." Note that following
incubation with collagen-coated beads at time 0, there was a slow and
progressive reduction of mitochondrial calcium. There was no
significant difference in the rate of reduced fluorescence in
CsA-treated cells and vehicle-treated controls (p > 0.2; n = 10 cells/group; slope for controls, 1.9 × 104 fluorescence units/min; slope for CsA-treated cells,
1.7 × 104 fluorescence units/min). Note that there
was a statistically significant individual difference at 10 min between
controls and CsA-treated cells (p < 0.05).
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Previous reports have emphasized the spatially localized nature of the
interaction between ER and mitochondrial calcium stores (29, 46). As
mitochondria can regulate the release kinetics of calcium from ER
stores (46), we considered that CsA may indirectly affect calcium
signaling by virtue of local interactions with the ER stores (29).
Accordingly, we reduced the numbers of potential ER/mitochondrial
interactions in Rat2 cells using chronic treatment with ethidium
bromide according to well established methods (26). Long-term
monitoring of cells showed that this protocol did not significantly
affect cell viability or the ability of the cells to be passaged in
culture. After staining with JC-1 (0.5 µM) and rhod-2 (4 µM) to localize mitochondria (Fig.
6A, panels a and c), the depleted Rat2EtBr cells showed fewer JC-1-stained
mitochondria and fewer organelles stained with rhod-2 (panels
b and d). Immunoblotting also showed >3-fold less of
the mitochondrial-specific protein cytochrome oxidase I in comparison
with normal cells (panels e and f). These data
indicate that the numbers of mitochondria/cell were reduced and that,
correspondingly, potential ER/mitochondrial interactions were likely
decreased. Similar to Rat2 cells without EtBr treatment (Fig.
5B), after collagen bead incubation, there was a linear
reduction of mitochondrial calcium over 25 min in EtBr-treated cells,
indicating that the reduction of mitochondrial numbers per
se did not substantially affect the regulation of mitochondrial
calcium by collagen bead phagocytosis. However, in contrast to normal
Rat2 cells, CsA pretreatment of these cells induced a 2-fold reduction
of mitochondrial [Ca2+] at base-line levels, and the
discharge rate of calcium from the mitochondrial stores following
collagen bead incubation was ~55% slower than from the untreated
Rat2EtBr cells. These data indicate that when ER/mitochondrial
interactions are curtailed by simply reducing the numbers of
mitochondria, regulation of mitochondrial calcium is impaired. Notably,
collagen phagocytosis in Rat2EtBr cells was reduced by 2-fold (from
19.0% in controls to 9.8% in Rat2EtBr cells), an effect that could be
due in part to CsA causing deregulation of mitochondrial and ER
calcium signaling. Collectively, these data indicate that mitochondria
play an important but perhaps not absolutely central role in the
collagen-induced calcium signaling that is required for collagen
phagocytosis.

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Fig. 6.
Mitochondrial depletion and
calcium signaling. A, Rat2 cells were chronically
treated over 30 passages with ethidium bromide to reduce numbers of
mitochondria and ER/mitochondrial interactions. In control cells
stained with JC-1, mitochondria were brightly stained and numerous
(panel a), whereas ethidium bromide-treated cells exhibited
more sparse staining (panel b). rhod-2 fluorescence
identified calcium in brightly staining organelles in normal Rat2 cells
(panel c); but following ethidium bromide treatment, there
was a substantial reduction in the number (but not the brightness) of
individual rhod-2-stained mitochondria (panel d).
Immunoblotting for the mitochondrial protein cytochrome oxidase I in
Rat2 cells (panel e) and in mitochondrion-depleted cells
(panel f) showed >3-fold reduced blot density.
B, following collagen bead incubation, rhod-2 fluorescence
decreased linearly over 30 min of sampling ( 1.5 × 104 fluorescence units/min), but CsA treatment diminished
this by 60% ( 0.8 × 104 fluorescence units/min;
p < 0.05). Data are means ± S.E.
(n = 10 cells/group).
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DISCUSSION |
Although some of the modes of action of CsA as an
immunosuppressant are understood (37), the mechanism(s) by which CsA
induces gingival overgrowth is unknown (2). The main findings in this study are that the CsA-induced inhibition of collagen phagocytosis observed in vivo (11, 14, 15) can be replicated in the
collagen bead model and that CsA mediates phagocytic inhibition by
deregulating intracellular calcium signaling. In this study, we have
considered previous observations that CsA binding to cyclophilin
inhibits the conductance of the mitochondrial PTP (6) and can
independently act upon ER stores to inhibit calcium release (5, 21).
Furthermore, several functional interactions between the regulation of
calcium in mitochondrial and ER calcium stores have been characterized (46) that underline the importance of communication between these
organelles in calcium homeostasis (48). Accordingly, we have shown here
that calcium signaling is an important regulatory mechanism of collagen
bead phagocytosis and that CsA markedly perturbs calcium responses to
collagen bead incubations. Collagen phagocytosis was strongly inhibited
by clamping [Ca2+]i to low levels with BAPTA, by
depleting intracellular stores with thapsigargin, or by treatment with
pharmacologically relevant doses of CsA. These data implicate
intracellular calcium homeostasis and CsA perturbation of collagen
bead-induced calcium signaling as important target processes in CsA
inhibition of collagen phagocytosis.
Phagocytosis and [Ca2+]i
Signals--
When [Ca2+]i was monitored during
collagen bead-induced phagocytosis, Rat2 fibroblasts exhibited a
calcium peak within 150 s, a similar but more rapid response than
that seen in the spreading of endothelial cells on fibronectin
substrata (38) or after incubation of kidney epithelial cells with
RGD-coated beads (20). This calcium response was evidently important
for the collagen bead binding step of phagocytosis because chelation of
intracellular calcium with BAPTA/AM or inhibition of ER stores with
thapsigargin strongly inhibited phagocytosis. These results are
consistent with the previous demonstration of a requirement for calcium
fluxes in cells forming productive adherent interactions with beads
(20). However, the important requirement of calcium release from ER
stores that we have shown here is in marked contrast to previous data
showing that matrix protein-coated beads induce calcium rises that are
largely dependent on extracellular sources (20). In this context, the
importance of ER calcium stores in collagen bead-induced phagocytosis
was shown by direct measurement of ER calcium stores with mag-fura-2
and by thapsigargin pretreatment, in which collagen beads induced only
a small calcium rise; this effect was seen in both Rat2 fibroblasts and
Rat2 fibroblasts pretreated with CsA. The chelation of
Ca2+i with BAPTA also caused a significant decrease
in the [Ca2+]i response compared with untreated
cells, and the magnitude of this response was similar to that following
thapsigargin treatment. Notably and consistent with previous findings
(20, 38), we found that [Ca2+]i responses to
collagen phagocytic stimuli also involve entry of extracellular calcium
and thus are not due solely to release from intracellular stores (47).
However, in this study, we have concentrated on the role of CsA in
regulating intracellular calcium responses to collagen beads since
previous data showed that CsA can down-regulate calcium release from
both ER (5, 21) and mitochondrial (48, 49) stores and thus is likely to
suggest a mechanism for how CsA inhibits collagen phagocytosis (11, 14,
15).
ER and Mitochondrial [Ca2+] Responses to Collagen
Phagocytic Stimuli--
Preincubation of Rat2 fibroblasts with CsA
caused a rapid and significant inhibition of the calcium efflux from
the ER in response to collagen beads. Previous data (38) suggest that a
large part of the increase in [Ca2+]i following
cell spreading on matrix proteins may be due to calcium release from
intracellular calcium stores, and our direct measurements of ER
[Ca2+] with mag-fura-2 show that ER stores are indeed a
locus for this inhibition. Notably, CsA inhibits inositol
1,4,5-trisphosphate binding to its cognate receptors and inhibits
calcium release from ER stores in macrophages (21); CsA can also alter
the characteristics of the calcium release channel in the ER of cardiac
cells (5). In view of these data, we suggest that an important
mechanism for CsA inhibition of collagen phagocytosis is blocking
calcium efflux from ER stores; this would also account for the greatly reduced [Ca2+]i following CsA pretreatment and
collagen bead stimulation. The CsA-induced attenuation of this calcium
signal may be sufficient to inhibit collagen binding to collagen
receptors, perhaps by regulating the affinity of the
2
1 integrin (20, 41).
Collagen bead-induced phagocytosis provoked a
time-dependent and equivalent decrease in mitochondrial
[Ca2+] in untreated and CsA-pretreated Rat2 cells. Since
CsA greatly attenuated [Ca2+]i responses to
collagen beads, it seems unlikely that the efflux of calcium from
mitochondrial stores contributed significantly to the increased
[Ca2+]i after bead incubation. This result was
not because CsA had no effect on mitochondrial function: indeed, CsA
pretreatment preserved
m following suspension challenge
(34), indicating that the PTP was inhibited, as reported earlier (32).
A more likely explanation for the lack of effect of CsA on
mitochondrial calcium is that calcium ions can exit via other channels
or pumps that are not affected by CsA (48). In view of these data, does mitochondrial regulation of calcium impact on collagen bead binding? Conceivably, and as suggested by recent data on ER/mitochondrial store
interactions (29, 46), mitochondria may sense high local concentrations
of calcium within the cell and act to buffer [Ca2+] (44).
We investigated this possibility using mitochondrial depletion methods
previously described for studies of ER/mitochondrial calcium store
interactions (26). In mitochondrion-depleted cells, collagen
bead-induced calcium efflux from mitochondria was inhibited by CsA,
indicating that ER/mitochondrial regulation of calcium signaling is
dependent on functional interactions between the two sets of
organelles. Conceivably, the effect of CsA on mitochondrial regulation
of calcium may serve to inhibit the explosive release of calcium from
ER stores (46); when these processes are dampened, the
phagocytosis-induced calcium signal in the cytosol is inhibited.
We are aware of the potent inhibition of the serine/threonine
phosphatase calcineurin by CsA (50) and the possibility that CsA may
modulate integrin affinity for collagen through a calcineurin pathway.
However, in the context of this study, we note that FK506, which also
potently inhibits calcineurin, has no effect on collagen phagocytosis
and does not cause gingival overgrowth (51). Consequently, we conclude
that the effect of CsA perturbation on the whole cell is independent of
a calcineurin-mediated decrease in collagen binding and phagocytic
efficiency. In the CsA-treated gingival fibroblast, this inhibition
leads to decreased collagen phagocytosis, a net increase in matrix
proteins, and gingival overgrowth. Although the exact role of the
mitochondrial calcium stores is not shown by these data, the importance
of signaling interactions between discrete calcium stores is evident.