(Received for publication, November 18, 1996, and in revised form, February 18, 1997)
From the Division of Signal Transduction, Beth Israel
Deaconness Medical Center, Department of Medicine, Harvard Institutes
of Medicine, Boston, Massachusetts 02115 and § Department of
Orthodontics, Harvard School of Dental Medicine,
Boston, Massachusetts 02115
The discovery of a calcium receptor has stimulated interest in the signaling events underlying extracellular calcium ([Ca2+]o)-induced cell-specific responses. In osteoblasts, elevated levels of extracellular calcium mediate both mitogenesis and chemotaxis. Here we provide evidence that [Ca2+]o-stimulated chemotaxis of MC3T3-E1 osteoblast-like cells involves a G-protein-linked calcium-sensing receptor. [Ca2+]o promotes chemotaxis in a concentration-dependent manner. Pertussis toxin blocked almost all of [Ca2+]o-stimulated chemotaxis but had only a small effect on platelet-derived growth factor (PDGF)-stimulated chemotaxis. Consistent with the signaling model for PDGF-mediated chemotaxis, activation of phospholipase C played a critical role in [Ca2+]o-initiated chemotaxis: U-73122, an inhibitor of the activation of phospholipase C, blocked approximately 50% of PDGF-stimulated chemotaxis but blocked nearly all of the [Ca2+]o-stimulated chemotaxis. Down-regulation of protein kinase C also blocked about 50% of PDGF-stimulated chemotaxis but did not block [Ca2+]o-stimulated chemotaxis. Thus, unlike PDGF-mediated chemotaxis, chemotaxis stimulated by [Ca2+]o does not appear to require protein kinase C activation. This finding suggests events downstream of inositol 1,4,5-trisphosphate production rather than diacylglycerol production are critical to [Ca2+]o-promoted chemotaxis of MC3T3-E1 cells. The signal transduction mechanism underlying PDGF-induced chemotaxis involves the activation of phosphoinositide 3-kinase, as judged by the in vivo production of phosphatidylinositol 3,4-diphosphate and 3,4,5-trisphosphate and the partial sensitivity of chemotaxis to wortmannin, an inhibitor of phosphoinositide 3-kinase. In contrast, [Ca2+]o-stimulated chemotaxis was not blocked by wortmannin and elevations in [Ca2+]o did not increase the production of lipid products of phosphoinositide 3-kinase. Overall, [Ca2+]o-promoted chemotaxis of osteoblasts appears to utilize a unique signaling mechanism via a calcium-sensing receptor.
Multiple cell types sense extracellular calcium (1). Cellular responses to alterations in external calcium are diverse and cell-specific. Elevations in extracellular calcium signal parathyroid cells to reduce parathyroid hormone secretion (2). Renin production by kidney cells is inhibited by high [Ca2+]o (3). During bone remodeling, osteoclasts (bone-resorbing cells) and osteoblasts (bone-forming cells) function in local environments characterized by dramatic fluctuations in [Ca2+]o concentrations (4). Elevated external calcium in the resorption lucunae acts as a negative feedback on osteoclasts, inhibiting their resorptive capacity (5). In contrast, high [Ca2+]o concentrations enhances DNA synthesis and promotes chemotaxis of osteoblasts (6, 7). The signal transduction mechanisms underlying [Ca2+]o-stimulated mitogenesis and chemotaxis have yet to be defined; however, [Ca2+]o is capable of producing a transient elevation of intracellular calcium in osteoblasts at concentrations that initiate these two physiological processes (8). Since the transient rise in intracellular calcium was not a result of a [Ca2+]o influx, the existence of a cation-sensing receptor on the surface of osteoblasts was suggested, similar to that reported earlier in parathyroid cells (9).
Recently, calcium receptor cDNA has been cloned and transcripts of this gene were recovered from bovine and human parathyroid, kidney, thyroid and brain cells (10). This 120-kDa polyvalent receptor, BoPCaR1, consists of a seven-transmembrane region and belongs to the G-protein-linked receptor superfamily (10). Recent reports indicate that MC3T3-E1 osteoblast-like cells may have a [Ca2+]o-sensing receptor that is homologous, yet different, to the BoPCaR1 (11).
The aim of this report is to define the initial post-receptor signal transduction events in [Ca2+]o-stimulated chemotaxis of the murine osteoblast clonal cell line MC3T3-E1. Platelet-derived growth factor (PDGF)1 also promotes chemotaxis in these cells, and we examined the effects of perturbations of various signaling pathways on both PDGF- and [Ca2+]o-promoted chemotaxis. In previous studies of other cells, the signaling mechanisms underlying PDGF-induced chemotaxis were examined by mutating specific PDGF receptor tyrosine residues, and it was observed that PDGF-stimulated chemotaxis relies on both phospholipase C and the phosphoinositide 3-kinase (12). Hepatocyte growth factor also activates both PI 3-kinase and PLC, and both signaling events are necessary for hepatocyte growth factor-promoted chemotaxis (13, 14). However, not all chemoattractants use this dual pathway paradigm. For example, basic fibroblast growth factor mediates chemotaxis without activating PLC (15). Therefore, the signal transduction mechanism utilized among chemoattractants is not universal.
We report that PDGF-mediated chemotaxis of MC3T3-E1 osteoblast-like cells is dependent on both PLC and PI 3-kinase. In contrast, extracellular calcium-stimulated chemotaxis requires PLC activation but not PI 3-kinase activation. Our results suggest that a G-protein-linked [Ca2+]o-sensing receptor is present on osteoblasts. The presence of such a receptor would allow extracellular calcium to act as a coupling agent, linking the bone resorptive phase of bone remodeling to bone formation.
All chemicals were reagent grade or better.
-modified Eagle's medium (
-MEM), fetal calf serum,
penicillin/streptomycin, and wortmannin were purchased from Sigma.
Dulbecco's modified Eagle's medium, amphotericin B (Fungizone), and
phorbol 12-myristate 13-acetate (PMA) were obtained from Life
Technologies, Inc. Human PDGF-BB was acquired from Upstate Biochemicals
Inc. (Lake Placid, NY). U-73122 and U-73433 were purchased from Biomol
(Plymouth Meeting, PA). List Biologicals (Campbell, CA) supplied the
pertussis toxin (product no. 180). [32P]ATP (specific
activity, 3,000 Ci/mmol) and
ortho-[32P]phosphate (specific activity,
8500-9120 Ci/mmol) was obtained from DuPont NEN. The
anti-phosphotyrosine antibody (anti-Tyr(P)), a murine monoclonal
antibody, was generously contributed by Dr. Thomas Roberts (Dana-Farber
Cancer Institute, Boston, MA).
MC3T3-E1 cells (16), a clonal line of normal
murine osteoblasts, were kindly provided by Peter Hauscka (The
Children's Hospital, Boston, MA). The cells were grown in a 95% air,
5% CO2 atmosphere in 100-mm diameter dishes containing
-MEM plus 10% fetal calf serum, 100 µg/ml streptomycin, 100 IU/ml
penicillin, and 0.025 µg/ml amphotericin B at 37 °C. All
experiments were conducted with cells at or near confluency. Where
noted, serum-starvation of the MC3T3-E1 cells was accomplished through
exposure to low serum medium (
-MEM plus 0.1% fetal calf serum)
12 h prior to stimulation. Protein kinase C down-regulated cells
were generated by 12-h incubation of cells in low serum
-MEM
containing 1 µM PMA. G-protein inhibition was
accomplished by 12-h exposure of cells to low serum
-MEM containing
100 ng/ml pertussis toxin. Strict cell culture parameters were
maintained, and cells were used early in their passage to avoid
phenotypic maturation that might accompany long term cell culture. For
example, we observed that U-73122 had an enhanced inhibitory effect on
chemotaxis of late passage osteoblasts (data not shown).
-MEM has a basal calcium concentration of 1.8 mM. In all experiments, alterations in the extracellular
calcium concentration from basal (1.8 mM) levels were
produced by the addition of CaCl2 (Sigma) to the final
concentration specified.
A 48-well modified Boyden chemotaxis chamber
(NeuroProbe Inc., Baltimore, MD) and polyvinylpyrrolidone-free
polycarbonate filters (8-µm pore size) (Poretics Corp., Livermore,
CA) were utilized as described previously (17) with the following
alterations. The filters were coated with 50 µg/ml rat type I
collagen (Collaborative Biomedical Products, Bedford, MA). Chemotactic
agent ± inhibitor were diluted in low serum -MEM, which was
then added to the lower wells of the chamber. When dimethyl sulfoxide
was used as a vehicle for an inhibitor, the final concentration of
dimethyl sulfoxide was less than 0.01%. A coated filter was fixed atop
the bottom wells. Cells were diluted in low serum
-MEM to a final
concentration of 4 × 105 cells/ml. Inhibitors were
added to the cell suspension where noted, and 50 µl of this
suspension were loaded into each of the upper wells. In each
experiment, at least 6 of the chamber's 48 wells were used for each
condition examined. The chamber was incubated for 4 h at 37 °C
in a 95% air, 5% CO2 atmosphere.
Following incubation, the chamber was disassembled, and the cells on the upper surface of the filter were removed. The cells on the lower surface were fixed and stained with Diff-Quik (Baxter Healthcare Corp., Miami, FL). Chemotaxis was quantified by counting the number of cells on the lower surface of the filter in each well using 100 nonoverlapping fields (× 200 magnification). For the purpose of comparison between multiple assays, the data were normalized by expressing it as the fold increase in cell chemotaxis relative to that of the control.
Assay of in Vitro Phosphoinositide 3-Kinase ActivityGrowth
factors and inhibitors were exposed to serum-starved cells for the
indicated time at 37 °C. The cells were washed twice with 4 ml of
cold phosphate buffered saline (137 mM NaCl, 15.7 mM NaH2PO4, 1.47 mM
KH2PO4, 2.68 mM KCl (pH 7.4)) and
were lysed in 1 ml lysis buffer (137 mM NaCl, 20 mM Tris, 1 mM MgCl2, 0.2 mM vanadate, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 4.5 mM sodium
pyrophosphate, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM ZnCl2, 48 mM NaF, 9 mM -glycerophosphate). The lysates were vortexed and
centrifuged at 16,000 × g (Eppendorf 5414 microcentrifuge). The cleared supernatants were transferred to new
Eppendorf tubes and incubated with anti-phosphotyrosine (6.6 µg/ml)
for 1 h at 4 °C, followed by the addition of protein A-Sepharose (4 mg/ml lysate) for an additional 2 h at 4 °C.
The immunoprecipitates were pelleted, washed three times in
phosphate-buffered saline, 1% Nonidet P-40, twice in 0.1 M
Tris (pH 7.5), 0.5 M LiCl, and twice in TNE (10 mM Tris, 100 mM NaCl, 1 mM EDTA (pH
7.5)). All three wash solutions contained 200 µM vanadate. To assay the phosphoinositide 3-kinase activity of the immunoprecipitates, sonicated phosphatidylinositol (PI) (0.2 mg/ml final concentration, in 10 mM HEPES, 1 mM EGTA
(pH 7.5)) and [-32P]ATP (10-20 µCi/sample) were
added to the immunoprecipitates for 10 min at room temperature. The
reaction was stopped by the addition of 80 µl of HCl and 160 µl of
methanol:chloroform (1:1 (v/v) mixture). The lipid-containing organic
phase was separated on oxalate-coated thin-layer chromatography
(Silica Gel 60; MCB Reagents, Merck) and developed in
chloroform:methanol:water:ammonium hydroxide (60:47:11.3:2, v/v).
Radiolabeled spots corresponding to phosphatidylinositol 3-phosphate
were quantified using a phosphoimager (Bio-Rad).
MC3T3-E1 cells were serum-starved for 24 h in
-MEM containing 0.1% (v/v) fetal calf serum. The cells were
incubated in 5 ml of phosphate-free Dulbecco's modified Eagle's
medium for 45 min, followed by exposure to 2 ml of the same medium plus
carrier-free ortho-[32P]phosphate (0.5-2
mCi/ml). The cells were allowed to incorporate the radioisotope at
37 °C for 3 h in a humidified atmosphere containing 5%
CO2. PDGF (10 ng/ml) or calcium (5 mM final
concentration) was subsequently added to the dishes for 1-5 min. Cells
were washed twice with 20 mM Tris (pH 7.5), followed by
addition of 750 µl of 1 M HCl:methanol (1:1, v/v). The
cells were scraped with a cell scraper, and the mixture was vortexed in
a 2-ml microcentrifuge tube. Chloroform (380 µl) was added to the
lysates and vortexed vigorously. The samples were centrifuged briefly
at 16,000 × g. The lower chloroform phase was
extracted twice with 400 µl of methanol:EDTA (0.1 M, pH
7.5) in a 1:0.9 (v/v) mixture. The organic (lower) was transferred to
glass scintillation vials and dried under N2. Deacylation
of the lipids was performed using methylamine reagent as described
previously (18).
The glycerophosphoinositides were resuspended in 120 µl of distilled water and filtered using a 0.2-µm filter. The glycerophosphoinositides were then separated by HPLC using a 12.5-cm Partisphere 5-µm strong anion exchange column (SAX, Whatman) and a salt gradient from 0 to 1.0 M (NH4)2HPO4 (pH 3.8), as described previously (18). The HPLC system was connected to an on-line continuous flow liquid scintillation detector (Flow-One/Beta CT; Beckman Instruments).
DataData given as the means ± the S.E. of the means, with the number of determinations (n) representing separate experiments.
There was a dose-dependent relationship between
the extracellular calcium concentration and the rate of chemotaxis of
the MC3T3-E1 osteoblast-like cells. The maximum chemotactic effect was
attained at a calcium concentration of 5 mM (Fig.
1). A substantial enhancement of chemotaxis was also
seen at 3 mM calcium, a concentration less than twice the
basal concentration (1.8 mM). At 10 mM calcium, the rate of chemotaxis was greater than the basal rate, but was less
than the peak rate. Chemotaxis of the MC3T3-E1 cells was not increased
when sodium chloride was used to produce osmolar increases equivalent
to the CaCl2 additions, suggesting that slight increases in
medium osmolality were not responsible for the initiation of chemotaxis
by increases in [Ca2+]o (data not shown). Using
the Zigmond-Hirsch (19) checkerboard analysis, the
[Ca2+]o-induced cell movement was found to be
directed chemotaxis rather than random chemokinesis (data not
shown).
PDGF-BB also promoted an increase in chemotaxis of the MC3T3-E1 osteoblasts. In 12 experiments in which the effects of PDGF and [Ca2+]o were compared directly, PDGF (10 ng/ml) increased chemotaxis to 20.3 ± 1.2 times basal (no stimulus) levels, and [Ca2+]o (5 mM) increased chemotaxis to 7.9 ± 0.4 times basal levels. Previous studies in this cell line have described a dose-dependent relationship between PDGF concentration and chemotaxis, and PDGF-promoted chemotaxis has been observed in other cell lines (20-22). In contrast, the chemotactic effect of extracellular calcium appears to be cell type specific. [Ca2+]o-induced increases in chemotaxis were not seen using NIH-3T3 fibroblasts exposed to 5 or 10 mM calcium, although PDGF (10 ng/ml) stimulated chemotaxis of these cells to 9 times the basal level (data not shown).
Pertussis Toxin Inhibits Extracellular Calcium-stimulated Chemotaxis of MC3T3-E1 CellsPrevious studies have reported that
several cell types have a G-protein-coupled calcium receptor (1). Since
the stimulatory effect of extracellular calcium on chemotaxis suggests
that MC3T3-E1 cells have calcium receptors, we investigated whether the
chemotaxis response in MC3T3-E1 cells was sensitive to pertussis toxin.
Pertussis toxin prevents receptor activation of some G-proteins by
catalyzing ADP-ribosylation of the G-protein -subunits (23).
Following 12-h exposure to 100 ng/ml pertussis toxin,
[Ca2+]o-directed chemotaxis of the MC3T3-E1 cells
was almost completely inhibited (Table I). In contrast,
pertussis toxin had hardly any effect on PDGF-induced chemotaxis of
these cells (Table I). These data suggest that a G-protein-linked
calcium receptor plays a critical role in
[Ca2+]o-stimulated chemotaxis, whereas a
pertussis toxin-sensitive G-protein activation is not involved in
PDGF-directed chemotaxis.
|
U-73122 inhibits the activation of phosphatidylinositol-specific phospholipase C (PI-PLC) (24). Although the precise mechanism of inhibition has yet to be defined, U-73122 probably disrupts the link between G-proteins and PLC (25). [Ca2+]o-directed chemotaxis was almost completely inhibited by this compound, whereas PDGF-stimulated chemotaxis was reduced by only about 50% (Table I). U-73433, an inactive analog of U-73122, had no effect on either [Ca2+]o or PDGF-stimulated chemotaxis (data not shown). Previous studies have shown that PLC activation is involved in PDGF-directed chemotaxis (12). Our results indicate that PLC activation may be responsible for about half of the PDGF-directed chemotaxis response in MC3T3-E1 cells. However, the role of PI-PLC is even more critical in [Ca2+]o-directed chemotaxis of MC3T3-E1 cells, and accounts for nearly the entire response. Consistent with the data obtained using pertussis toxin, these findings suggest the chemotactic effect of [Ca2+]o is mediated by a calcium receptor linked to PLC.
Extracellular Calcium-induced Chemotaxis of MC3T3-E1 Cells Does Not Rely on Protein Kinase C ActivationActivation of PLC leads to hydrolysis of phosphatidylinositol-4,5-P2, generating inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (26). IP3 enhances calcium release from intracellular stores, and DAG recruits and activates protein kinase C (PKC), a serine/threonine kinase (26). After PKC was down-regulated by a 12-h exposure of the cells to 1 µM PMA, about half of the PDGF-stimulated chemotaxis was blocked (Table I). In contrast, [Ca2+]o-directed chemotaxis of the MC3T3-E1 cells was not reduced by PKC down-regulation (Table I). Thus, while the U-73122 studies indicate that both [Ca2+]o- and PDGF-stimulated chemotaxis utilize PLC activation, only PDGF-directed chemotaxis involved PKC.
When added to low serum media in the bottom wells of the modified Boyden chamber, PMA (100 nM) did not stimulate chemotaxis of MC3T3-E1 relative to basal (0.8 times relative to basal, n = 2). Therefore, phorbol ester-mediated PKC activation alone is not sufficient to induce chemotaxis in this cell line, even though PKC plays an important role in PDGF-stimulated chemotaxis. In other cell lines, phorbol ester does function as a chemoattractant capable of producing a robust chemotactic effect (27, 28). The absence of PMA-directed chemotaxis in MC3T3-E1 cells may be indicative of the unique nature of the signal transduction mechanism that drives chemotaxis of these osteoblast-like cells.
PI 3-Kinase Is Involved in PDGF-directed Chemotaxis, but Not Extracellular Calcium-stimulated Chemotaxis of MC3T3-E1 CellsWortmannin, a fungal metabolite, acts as a specific irreversible inhibitor of the catalytic p110 subunit of PI 3-kinase (29). Previous studies have shown that wortmannin will inhibit both PDGF- and hepatocyte growth factor-stimulated chemotaxis, linking PI 3-kinase activation to chemotaxis (13, 14, 30). Consistent with these findings, PDGF-stimulated chemotaxis of MC3T3-E1 cells was inhibited by at least 50% when 100 nM wortmannin was present in both the upper and lower chambers of the Boyden apparatus (Table I). In contrast, wortmannin did not significantly alter the chemotactic effect of extracellular calcium in MC3T3-E1 cells (Table I). These results suggest that the promotion of chemotaxis by extracellular calcium differs from that promoted by PDGF, since PI 3-kinase does not appear to be involved in the signal transduction pathway for [Ca2+]o-stimulated chemotaxis of MC3T3-E1 cells.
PDGF, but Not Extracellular Calcium, Activates PI 3-Kinase in MC3T3-E1 CellsIn anti-phosphotyrosine immunoprecipitates from
MC3T3-E1 cells exposed to 10 ng/ml PDGF, there was an elevation of PI
3-kinase activity to 12.1 ± 2.2 (n = 8) times
that found under basal conditions (Fig. 2). In contrast,
there was no increase (1.1 ± 0.1, n = 3) in PI
3-kinase activity above basal levels in anti-phosphotyrosine immunoprecipitates from calcium (5 mM)-treated cells. This
suggests that elevation in external calcium does not affect PI 3-kinase activity. To examine the in vivo production of
phosphoinositides phosphorylated on the D-3 position of the inositol
ring, the lipid products of PI 3-kinase, the osteoblasts were loaded
with inorganic ortho-[32P]phosphate and then
exposed to PDGF (10 ng/ml) or increases in extracellular calcium (5 mM final concentration). PDGF produced substantial
increases in D-3 lipids. There was a 3-fold elevation in
PI-3,4-P2 and a 13-fold increase in PI-3,4,5-P3
levels after a 1-min exposure to PDGF (Fig. 3). A 5-min
exposure to PDGF produced a 5- and 13.5-fold elevation in
PI-3,4-P2 and PI-3,4,5,-P3, respectively. In
contrast, extracellular calcium (1 or 5 min) did not enhance production
of either PI-3,4-P2 or PI-3,4,5,-P3 in MC3T3-E1
cells (Fig. 3). These results are consistent with the in
vitro immunoprecipitation data in that extracellular calcium did
not activate PI 3-kinase in MC3T3-E1 cells, whereas PDGF-BB
significantly increased PI 3-kinase activity.
Pretreatment of 32P-labeled cells with 100 nM wortmannin (20 min) produced a complete inhibition of 5-min PDGF-stimulated PI-3,4-P2 production and a 60% reduction in PDGF-induced PI-3,4,5-P3 production (Fig. 3). Wortmannin had a negligible effect on the D-3 lipids produced in cells exposed to 5 mM extracellular calcium, consistent with the lack of effect of calcium on PI 3-kinase activity in vivo (Fig. 3). These results support the wortmannin-sensitivity of the chemotaxis assay which indicated a requirement for PI 3-kinase activation in PDGF-stimulated, but not [Ca2+]o-directed, chemotaxis of MC3T3-E1 cells (Table I).
The results of the present study suggest that different chemoattractants can stimulate chemotaxis of the same cell type through chemoattractant-specific signal transduction mechanisms. PDGF produced large increases in PI 3-kinase activity, and the activation of PI 3-kinase and PLC appeared to contribute equally to PDGF-promoted chemotaxis (Table I). In contrast, while elevation in extracellular calcium promoted the chemotaxis of MC3T3-E1 cells, this response appeared to be dependent on the activation of PLC. Extracellular calcium did not activate PI 3-kinase in MC3T3-E1 cells, and [Ca2+]o-stimulated chemotaxis was not sensitive to wortmannin, an inhibitor of PI 3-kinase activity. Our results strongly suggest that MC3T3-E1 cells have a pertussis toxin-sensitive G-protein-linked calcium receptor, and that activation of this receptor stimulates chemotaxis utilizing a PLC-dependent mechanism.
Brown et al. (10) recently cloned a calcium-sensing receptor from bovine parathyroid cells. In addition to parathyroid cells, BoPCaR1 mRNA transcripts were found in kidney, brain, and thyroid cells (10). The BoPCaR1 belongs to the seven-transmembrane receptor family and is linked via a G-protein to PLC (10). Activation of this receptor by extracellular calcium leads to elevation in IP3 with subsequent increases in the intracellular calcium concentration (1). A recent report suggests that MC3T3-E1 osteoblast-like cells do not express the BoPCaR1, at least not in great abundance, but may produce a calcium-sensing receptor that is similar, yet distinct, to BoPCaR1 (11).
Evidence for receptor-mediated signaling by extracellular calcium in MC3T3-E1 cells was indicated by the results obtained using two inhibitors, pertussis toxin and U-73122. Pertussis toxin, an inhibitor of G-protein activation, blocked almost all of the [Ca2+]o-stimulated chemotaxis (Table I). This result suggests that a G-protein-linked receptor must be involved in [Ca2+]o-mediated chemotaxis. In some cells, including osteoblasts (31), pertussis toxin has been shown to inhibit agonist-stimulated transient elevations in intracellular calcium. Pertussis toxin also effects the activation of adenylate cyclase through inhibition of Gi. The role that adenylate cyclase and cAMP play in [Ca2+]o-stimulated chemotaxis of MC3T3-E1 cells has yet to be examined. Several other chemoattractants activate G-protein-linked receptors. For example, neutrophil chemotaxis in response to leukotriene B4 and formyl-methionyl-lucyl-phenylalanine is inhibited by pertussis toxin (32, 33). The limited effect of pertussis toxin on PDGF-mediated chemotaxis (Table I) was expected, since PDGF acts through a tyrosine kinase receptor rather than a G-protein-linked receptor (34).
Signaling events downstream of both G-protein-linked receptors and receptor tyrosine kinases activation include the stimulation of PLC activity. In the presence of U-73122, which blocks the activation of PI-PLC, [Ca2+]o-directed chemotaxis was almost completed inhibited (Table I). This result indicates that [Ca2+]o-mediated chemotaxis of MC3T3-E1 cells requires PLC. U-73122 had a substantial, but less complete, inhibitory effect on PDGF-directed chemotaxis of these cells, confirming previous evidence suggesting that PLC is one of two signal transduction pathways that are critical for PDGF-directed chemotaxis (12). The fact that PDGF promotes a substantial chemotaxis response in U-73122-treated cells suggests that the near complete inhibition of [Ca2+]o-directed chemotaxis by U-73122 was not due to a nonspecific toxic effect of this inhibitor on MC3T3-E1 cells.
Activation of PLC leads to hydrolysis of PI-4,5-P2 to form
DAG and IP3 (26). DAG activates PKC, a serine/threonine
kinase that is involved in multiple signaling events (26). In studies in which cells were exposed to phorbol ester to down-regulate PKC, we
found that PKC is critical to PDGF-stimulated chemotaxis of MC3T3-E1
cells (Table I). In contrast, [Ca2+]o-stimulated
chemotaxis did not appear to require PKC activation, even though PLC
activation was necessary. Alternatively, it is possible that a PKC
isoform(s) which is relatively slowly down-regulated or is insensitive
to PMA (e.g. PKC ) contributes to
[Ca2+]o-promoted chemotaxis. When PMA (100 nM) was added to the bottom wells of the Boyden chamber,
direct migration of MC3T3-E1 cells was not observed, suggesting that
PMA alone is not a chemoattractant for these cells. Hemolytically
inactive complement component complex C5b67 induces chemotaxis of
neutrophils through a G-protein-linked receptor which activates PLC,
but not PKC (34). Our data suggests that the IP3 pathway
rather than the DAG pathway is important in
[Ca2+]o-initiated chemotaxis of MC3T3-E1 cells.
Since extracellular calcium and PDGF both have been reported to elevate
the intracellular calcium concentration, it is possible that a rise in
intracellular calcium contributes to chemotaxis in MC3T3-E1 cells.
Consistent with this, both PDGF- and extracellular calcium-stimulated
chemotaxis were reduced in cells loaded with the calcium-chelator BAPTA
(data not shown). However, this may have been due to an effect on
regulatory components in cell migration/chemotaxis that are far
downstream from receptor activation.
PDGF increased PI 3-kinase activity and the production of PI-3,4-P2 and PI-3,4,5-P3 in MC3T3-E1 cells (Figs. 2 and 3). Wortmannin, a PI 3-kinase inhibitor, significantly reduced both the in vivo and in vitro PDGF-mediated elevations of the D-3 phosphorylated lipids. Furthermore, PDGF-directed chemotaxis of the MC3T3-E1 cells was significantly inhibited by wortmannin (Table I). Thus, PDGF-stimulated chemotaxis requires PI 3-kinase activity in addition to PLC activation, as others have reported in other cells (12). In contrast, cells exposed to increases in [Ca2+]o did not display an increase in PI 3-kinase activity in the in vitro assays of anti-phosphotyrosine immunoprecipitates (Fig. 2). To rule out activation of PI 3-kinase via a G-protein-linked receptor (35), which may not recruit PI 3-kinase to a tyrosine phosphorylated protein, we measured the D-3 lipid production in vivo. PI-3,4-P2 and PI-3,4,5-P3 levels were not elevated by an increase in extracellular calcium (Fig. 3). In addition, wortmannin had an insignificant effect on [Ca2+]o-stimulated chemotaxis of MC3T3-E1 cells (Table I). These data suggest that PI 3-kinase is not involved in extracellular calcium-stimulated chemotaxis. In neutrophils, similar findings demonstrated that PI 3-kinase was essential for PDGF-mediated chemotaxis but was not necessary for chemotaxis directed by chemoattractants that act through G-protein-linked receptors (30).
Previous studies have described a dose-dependent relationship between extracellular calcium concentration and chemotaxis in MC3T3-E1 osteoblast-like cells (6). Our results are consistent with these studies in that concentration-dependent increases in chemotaxis were observed between 1.8 and 5 mM calcium. In contrast to our results shown in Fig. 1, Sugimoto et al. (6) reported that the chemotactic effect of [Ca2+]o was further enhanced at a 10 mM concentration. We found that the stimulation of chemotaxis of MC3T3-E1 cells at this calcium concentration was less than that found at 5 mM. Other investigators have described a reduction of chemotaxis in the presence of high concentrations of chemoattractant and have attributed this effect to a more rapid dissolution of the chemoattractant gradient responsible for directed cell movement (36).
Increases in extracellular calcium may act as a physiological stimulus for bone cells. Bone remodeling consists of two components, a resorptive phase followed by a reparative phase (37). Osteoclasts resorb bone by creating a local environment capable of demineralizing bone and proteolyzing bone matrix proteins (38). The initiation of the reparative phase, known as reversal, requires the recruitment and activation of osteoblasts (37). Several cytokines and growth factors, including PDGF, have been implicated as coupling agents, capable of inducing reversal (39, 40). Osteoblasts incorporate the growth factors into the bone matrix during the reparative phase of bone remodeling (39). These coupling agents are then released from the bone matrix throughout the resorptive phase (39-41). In addition to liberating growth factors, osteoclasts dramatically alter extracellular calcium concentration at local sites of resorption. The extracellular calcium concentration can reach 40 mM within the resorption lucunae (4). By inhibiting osteoclastic activity while stimulating chemotaxis and mitogenesis of osteoblasts, extracellular calcium may serve as a key coupling agent between bone formation and resorption (5, 6, 42). Understanding the signaling events responsible for the induction of extracellular calcium events in this system will lead to further developments in the treatment of bone metabolism disorders.
We are grateful to Melanie Derman and Lloyd Cantley for discussion about the chemotaxis assay. We thank Petros Damoulis and Peter Hauschka for supplying the MC3T3-E1 cells. We also thank Lucia Rameh and Brian Duckworth for advice regarding the in vivo phosphoinositide 3-kinase assay.