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Renal Division (B.D.B., U.A., K.A.H.), Departments of Medicine and Cell Biology, Barnes-Jewish Hospital, Washington University School of Medicine, St. Louis, Missouri 63110
Address all correspondence and requests for reprints to: Brian Bennett, Renal Division, Barnes-Jewish Hospital, Washington University School of Medicine, 216 South Kingshighway, Mailstop #90–32-648, St. Louis, Missouri 63110. E-mail: bbennett{at}im.wustl.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phospholipase C may mediate [Ca2+]e-induced effects in OCs, because [Ca2+]e increases production of inositol 1,4,5-triphosphate (IP3) in GCT23 osteoclast-like cells (5) and chicken OCs (Miyauchi, A., and K. A. Hruska, unpublished data). IP3 activates receptor linked PLC mediated Ca2+ signaling by binding IP3 receptors, which release Ca2+ from internal stores. Evidence exists for [Ca2+]e-induced release of Ca2+ from intracellular stores in OCs, beginning with studies showing marked reduction of [Ca2+]e-induced [Ca2+]i rises by the release inhibitor TMB-8 (6). Release via IP3 receptors is often accompanied by stimulation of other intracellular channels such as the ryanodine receptor, which may explain why the ryanodine receptor (RYR) inhibitor dantrolene reduces [Ca2+]e-induced [Ca2+]i rises in chicken OCs (3). PLC mediated release of intracellular stores initiates refilling of stores by store-operated influx through transient receptor potential (trp) channels (7, 8, 9). Receptor driven release through either the IP3 receptor or the RYR initiates store-operated influx through trp channels putatively via direct coupling of these intracellular release channels to the plasma membrane influx channel (9, 10). However, trp channels also mediate receptor operated influx that is not dependent on thapsigargin sensitive stores (11, 12, 13). Trp3 can operate in a store-sensitive or store-insensitive mode (14) and trp6 is regulated by DAG or DAG metabolites after emptying internal stores (12). Also, receptor operated trp4 and trp5 channels are activated by agonist in a store insensitive manner (13). Preliminary results in OCs derived from the RAW 264.7 rodent macrophage cell line showed expression of several trp homologs including trp3, trp4 and trp6 (15), leading us to ask if osteoclast calcium sensing requires filled stores. The data presented here demonstrate that Ca2+ entry into chicken osteoclasts stimulated by high [Ca2+]e did not require release of store contents, occurred while stores remained empty, and maintained PLC dependency, all hallmarks of store-independent influx supported by trp channels. Thus, PLC activated intracellular Ca2+ store independent channels are a mechanism of extracellular Ca2+ sensing by osteoclasts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of avian osteoclasts
Avian osteoclasts were prepared as described previously in
accordance with protocols approved by the Animal Studies Committee of
the Washington University School of Medicine (16).
Briefly, osteoclast precursors were isolated from bone marrow of
egg-laying hens maintained on Ca2+-deficient
diets. Partially purified preparations of mononuclear cells were
recovered from the interface of Ficoll/Hypaque gradients. Nonadherent
cells were separated from the adherent population after 18–24 h in
culture. The nonadherent cells were sedimented, resuspended in fresh
medium, and plated on 25-mm glass coverslips in six-well plates at
5 x 105/well. After culture in the presence
of cytosine arabinoside (5 µg/ml), multinucleated osteoclast giant
cells (>100 µm diameter) formed between 3 and 6 days in culture.
Ninety-five percent of the cells in the preparation were
tartrate-resistant acid phosphatase-positive cells, of which 90% were
multinucleated giant cell osteoclasts.
[Ca2+]i imaging and analysis
Cells plated on circular coverslips were rinsed and incubated
with 2 mM Fura2/AM and 0.01% Pluronic-127 (Molecular Probes, Inc., Eugene, OR) in KRB-HEPES, 130
mM NaCl, 5.0 mM KCl, 1 mM
MgCl2, 1 mM
CaCl2, 10 mM glucose, 17
mM HEPES at 25 C for 1 h. After one change in
KRB-HEPES, the coverslip was transferred to a PDMI-2 thermostated
perifusion chamber (Medical Systems Corp., Greenvale, NY) mounted on a
Carl Zeiss Axiovert 35 microscope (Carl Zeiss Inc., Thornwood, NY). Imaging was performed using an IM-4000
system (Georgia Instruments, Roswell, GA). Fura-2 ratio images were
acquired using excitation wavelengths of 340 and 380 nm.
[Ca2+]i in individual
cells was monitored during 37 C perifusion at 1 ml/min in either a 0.5
ml (for TPEN experiments) or 1 ml chamber. FURA-2 was calibrated by the
standard two-point method (17). Individual
Ca2+ dynamics from osteoclasts were taken across
three separate osteoclast preparations used on the same day after
initiation of differentiation. When monocytes were compared with
osteoclasts they were paired within each of three separate
preparations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In the giant
cell OC shown, THG induced the expected transient rise in
[Ca2+]i due to passive
Ca2+ extrusion from the ER. Next
[Ca2+]e was taken from 1
mM (Ca1) to 20 mM (Ca20), a change that induces
IP3 production in OCs (5) and activates
heterologously expressed calcium sensing receptors (19).
Ca20 evoked a significant increase in
[Ca2+]i in this
osteoclast even after Ca2+ uptake into the ER was
inhibited. Similar Ca20 evoked responses were seen in 30 osteoclasts
across multiple preparations. However, the THG-induced
Ca2+ transient occurred in only 5 of these 30
osteoclasts, suggesting that THG would not normally cause passive
depletion of Ca2+ stores in our OCs. Ionomycin
was not able to induce Ca2+ release from stores
when THG was added first (Fig. 1B). However, without THG pretreatment
ionomycin caused a significant
[Ca2+]i rise (Fig. 1C).
Thus, ionomycin reveals the THG block on SERCA in our OCs. The effect
of THG on ionomycin associated stores was not seen unless the
calcium-free incubation period was short, suggesting again that the OC
stores were prone to depletion. The data suggest that
[Ca2+]i rises linked to
[Ca2+]e contain a
component that does not require filled thapsigargin-sensitive
Ca2+ stores and concur with previous
observations, which also suggest that influx is an important component
of osteoclast calcium sensing (3, 5, 20). Further, since
Ca2+ store-operated influx (SOI) is intimately
linked to intracellular stores and release from them, it appears that
the osteoclast calcium sensing seen above bypasses SOI
regulation.
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, left). Also, repeated Ca20 volleys gave the same result seen
for OCs in Fig. 2. The results demonstrate that mononuclear cells do
not sense calcium, and thus, PLC catalyzed IP3 production
in response to [Ca2+]e is
likely limited. To activate SOI in these monocytes in a PLC independent
manner we depleted stores with TPEN. TPEN activates SOI in RBL cells by
selectively chelating high Ca2+ stored in
internal calcium stores (22). In Fig. 2, cells were
challenged with Ca20 again after treatment with 1
mM TPEN in calcium free buffer (Fig. 2, right). After TPEN treatment mononuclear cells now responded
to Ca20. In contrast, TPEN treatment did not affect the Ca20-induced
[Ca2+]i rise in giant
cells. For giant cells, this again demonstrates the store-independence
of osteoclast calcium sensing as shown by the THG result. However,
monocytes did respond to TPEN with an increase in influx when Ca20 was
readded, providing an internal control for TPEN-induced SOI. That TPEN
evoked similar SOI in both monocytes and giant cells was demonstrated
by readding Ca1 rather than Ca20. After TPEN treatment, Ca1 readdition
led to the same increase in
[Ca2+]i in OCs and
monocytes (Fig. 3A). Also, the increased
influx was TPEN specific since Ca0 alone did not elicit the large
increase in [Ca2+]i upon
Ca1 readdition (Fig. 3B).
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demonstrates that, after TPEN treatment, changing the
[Ca2+]e from Ca0 to Ca1
raised [Ca2+]i by 130
nM in mononuclear cells, whereas a 160 nM
increase was seen after Ca20. Ca1 raised
[Ca2+]i by the same
amount in osteoclasts as in mononuclear cells. However, Ca20 increased
[Ca2+]i by 400
nM in osteoclasts after TPEN treatment. Quantitative
analysis (Fig. 4B) shows that the
[Ca2+]i rise due to SOI
alone, as represented by monocytes, was rather shallow. However,
osteoclasts gave a much steeper response. The data demonstrate that a
calcium sensing effector must be activated to increase influx; calcium
activation of SOI alone is not enough.
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), osteoclasts exposed to THG (Fig. 5B), and osteoclasts treated with U73122 in addition to THG (Fig. 5C).
Thus the increase in
[Ca2+]i caused by basal
extracellular calcium (Ca1) was unaffected by store depletion or PLC
inhibition. Also, the
[Ca2+]i increase due to
Ca20 was unaffected by THG (Fig. 5B), thus confirming store-insensitive
calcium sensing. However, the store-insensitive influx due to Ca20 was
blocked by 10 µM U73122 (Fig. 5C). In Fig. 6, quantitative analysis shows that THG
alone did not significantly reduce calcium sensing due to Ca20, but
addition of U73122 at 1 µM or 10 µM
produced significant inhibition. Because store-insensitive PLC
dependent influx is a functional hallmark of trp channel-mediated
receptor operated influx, we tested the effects of SKF-96365, a known
blocker of receptor operated trp3 channel activity (24).
Figure 7 demonstrates that store depleted
osteoclasts do not respond to Ca20 in the presence of 50
µM SKF-96365.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Store depletion by TPEN is known to initiate SOI in RBL-1 cells (22). Thus, TPEN "preactivates" store-operated channels, which leads to enhanced influx of Ca2+. Increasing [Ca2+]e to high levels associated with calcium sensing should lead to increased flux through "preactivated" SOI channels, while flux through channels activated simultaneously by store independent means would add to the influx through preactivated SOI. The change in [Ca2+]i due to influx of 1 mM [Ca2+]e after TPEN addition was similar for giant cell OCs and mononuclear cells. Therefore, TPEN appeared to activate similar levels of SOI in each cell type. However, the effects of 20 mM [Ca2+]e were quite different for giant cells compared with mononuclear monocytes. With TPEN-activated SOI present, 20 mM [Ca2+]e produced about the same [Ca2+]i rise as 1 mM [Ca2+]e in monocytes. In contrast, 20 mM [Ca2+]e caused a much greater response than 1 mM [Ca2+]e in giant cell osteoclasts. The monocyte data suggests that influx follows increased [Ca2+]e monotonically when SOI is preactivated, thus changing [Ca2+]i by increased mass action. The much higher level of giant cell response to 20 mM [Ca2+]e under SOI activating conditions are not easily explained by mass action. Though SOI contributes to some influx of 20 mM [Ca2+]e under SOI preactivation conditions, it cannot account for the large [Ca2+]i changes seen in giant cells. Most likely, 20 mM [Ca2+]e produces some form of signaling. We suggest PLC linkage to cation channels enhances the influx over and above that possible by SOI alone because high [Ca2+]e activates PLC in OCs (5) and PLC is linked to such influx activation (25). That monocytes give a muted response to Ca20 during SOI activation conditions compared with giant cells may reflect the lack of intracellular signal generation in monocytes, which is consistent with the monocyte insensitivity to Ca20 seen without TPEN pretreatment.
We also related SERCA store pumps to OC calcium sensing by blocking SERCA with thapsigargin. In Ca1, SERCA inhibition with THG increased [Ca2+]i in only 20% of the giant cell OC population, indicating that SERCA stores are prone to depletion in giant cells. However, ionomycin released Ca2+ from intracellular stores when OCs were bathed in zero calcium buffer, and this ionomycin-sensitive pool was not released when THG was added first. Thus, SERCA associated Ca2+ stores in ER and Golgi lumen were affected by THG. The difficulty detecting passive Ca2+ efflux from THG sensitive stores may arise due to slow efflux or close proximity of SERCA in the ER with PM CaATPase efflux pumps or rapid basal release that may leave SERCA stores relatively empty. An alternative, in which OCs express THG resistant SERCA pumps seems unlikely, because all known SERCA isoforms are THG sensitive, and ionomycin-linked pools were emptied by THG. The latter concept originally led us to a rationale where basal store depletion would engender chicken giant cell OCs with "constitutively" active store-operated influx through which elevated [Ca2+]e could flow and promote calcium sensing. This idea was influenced by studies showing that cells cloned during prolonged thapsigargin treatment have constitutively open Ca2+ entry channels coupled to empty or partially emptied thapsigargin-sensitive Ca2+ stores (26). However, we showed here that store-operated influx "constitutively" activated by TPEN cannot account for the bulk of OC calcium sensing. Rather, Ca20 produces similarly large increases in [Ca2+]i with or without interference from THG or TPEN. Combined, the data from THG/TPEN store perturbation methods reveal a calcium sensing component that does not require the normal operation of filling and maintenance of high calcium in intracellular stores.
Because PLC is activated by
[Ca2+]e in OCs, a variety
of PLC linked influx pathways may be involved in calcium sensing. For
example, Falsolato et al. (27) have shown that
receptors operating through
G
q and PLC concurrently
mediate both store and store-independent influx pathways. These
original functional data have received considerable molecular
description by the activities attributed to the trp family of ion
channels. For example, several trp homologous support store and
store-insensitive receptor-mediated influx. Often the same trp homolog
is store sensitive in one heterologous expression system, but
store-insensitive in another (28). The rules of trp
regulation are currently unfolding. Because OCs derived from mouse RAW
264.7 macrophages express several trp homologs that support
store-insensitive influx, we took clues from receptor-operated trp3.
trp3 supports store-independent, PLC-dependent, and
SKF-96363-sensitive influx (24). We suggest that PLC
activation by our OCs sensing high calcium increases
[Ca2+]i by both store and
store-independent influx. The modes of store perturbation used here
preactivate SOI so that concurrent store-independent influx adds to
SOI much like that seen for store-independent trp3 mediated influx
(24).
The PLC dependency we see in chicken OC calcium sensing suggest the possible involvement of a plasma membrane delimited calcium sensing receptor, such as the parathyroid (pCaR). However, OCs do not respond to the CaR agonist ion, Gd3+, suggesting that the pCaR is not functional in OCs. This is consistent with the lack of pCaR expression in osteoclast like GCT23cells (5). But, other reports show expression of the pCaR in osteoclasts and osteoclast precursors (29, 30). Added complexity arises from data showing that osteoblasts from CaR knock-out mice respond to high [Ca2+]e and Gd3+ (31). If involved in calcium sensing, such a receptor would likely elicit signaling through Gaq and PLCß (32). Here, we provide more evidence that PLC is involved in OC calcium sensing and look to modes of [Ca2+]i regulation known to be mediated by the GPCR-PLC couple.
Calcium signals mediated by receptor activation are often modified by RYR channels. [Ca2+]e-induced RYR channel activation in OCs was originally indicated by sensitivity to dantrolene (14). Kiselyov et al. 2000 (10) have demonstrated functional and physical coupling of trp3 to ryanodine receptors, suggesting that the PM channel-ER-RYR structure of muscle cells is also present in nonexcitatble cells. Also, synthetic catalysis of cyclic ADP-ribose, the RYR agonist, by CD38 appears to be activated downstream of GPCRs because the first phase of the [Ca2+]i rise due to acetylcholine-induced muscarinic receptor activation is muted by CD38 knockout (33). Therefore, a complex relationship between trp, RYR, and G protein-coupled receptors can be envisioned. However, in OCs special consideration of the RYR is required, because the RYR-type2 isoform (RYR2) is expressed in OCs (34). Though the bulk of RYR2 localizes throughout the OC in other nonexcitable cells, the peripheral orientation seen in OCs may impart a topology that puts a low affinity Ca2+ binding site in the extracellular space where it could sense high [Ca2+]e. Such a PM topography has was also suggested for the IP3-receptor intracellular release channel in an early proposal for IP3R-mediated SOI. Ion conductances for neither IP3R or RYR for ions flowing from the extracellular space to the cytosol have been demonstrated, indicating that functional pools are relegated to intracellular membrane delimited stores. Further support for an RYR-mediated calcium sensing component comes from the increase in [Ca2+]i caused by extracellular application of the potent RYR agonist cADP-ribose (35) and by OC expression of CD38 (36), the ubiquitious enzyme that catalyzes synthesis of cADP-ribose (37). cADP-ribose is cell impermeant and usually requires cell permeabilization to effectively activate the cytoplasmic binding domain of RYRs. However, cADP-ribose has also been shown to be transported to the cytosol where it can diffuse to its binding domain on the RYRs (38). Possibly uptake of cADP-ribose by OCs could explain its effects on OC calcium signaling. Overall, OC calcium sensing appears to be mediated by effectors normally associated with receptor activation, but specific molecular approaches are lacking to date.
In summary, the published literature before this report indicated that OC Ca sensing is related to a Ca sensor/receptor induced release of intracellular stores and Ca2+ entry through undefined pathways (3, 5, 15). The data reported here are the first to characterize a Ca2+ sensor activated influx as Ca2+-store independent, activities supported by various trp receptor-activated channels. We propose that the calcium sensing receptor of the OC activates PLCß and releases Ca2+ stores through the action of IP3 and concurrently stimulates Ca2+ entry even when stores are empty, through the action of Ca2+ store independent trp channels.
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Acknowledgments |
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Footnotes |
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Received May 25, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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