OF JOURNAL BONE AND MINERAL RESEARCH
Volume 20, Number 1, 2005
Published online on October 18, 2004; doi: 10.1359/JBMR.041009
2005 American Society for Bone and Mineral Research
Fluid Shear-Induced ATP Secretion Mediates Prostaglandin Release in
MC3T3-E1 Osteoblasts
Damian C Genetos,1 Derik J Geist,2 Dawei Liu,2 Henry J Donahue,1,3 and Randall L Duncan2
ABSTRACT: ATP is rapidly released from osteoblasts in response to mechanical load. We examined the
mechanisms involved in this release and established that shear-induced ATP release was mediated through
vesicular fusion and was dependent on Ca2+ entry into the cell through L-type voltage-sensitive Ca2+ channels.
Degradation of secreted ATP by apyrase prevented shear-induced PGE2 release.
Introduction: Fluid shear induces a rapid rise in intracellular calcium ([Ca2+]i) in osteoblasts that mediates
many of the cellular responses associated with mechanotransduction in bone. A potential mechanism for this
increase in [Ca2+]i is the activation of purinergic (P2) receptors resulting from shear-induced extracellular
release of ATP. This study was designed to determine the effects of fluid shear on ATP release and the
possible mechanisms associated with this release.
Materials and Methods: MC3T3-E1 preosteoblasts were plated on type I collagen, allowed to proliferate to
90% confluency, and subjected to 12 dynes/cm2 laminar fluid flow using a parallel plate flow chamber. ATP
release into the flow media was measured using a luciferin/luciferase assay. Inhibitors of channels, gap
junctional intercellular communication (GJIC), and vesicular formation were added before shear and maintained in the flow medium for the duration of the experiment.
Results and Conclusions: Fluid shear produced a transient increase in ATP release compared with static
MC3T3-E1 cells (59.8 15.7 versus 6.2 1.8 nM, respectively), peaking within 1 minute of onset. Inhibition
of calcium entry through the L-type voltage-sensitive Ca2+ channel (L-VSCC) with nifedipine or verapamil
significantly attenuated shear-induced ATP release. Channel inhibition had no effect on basal ATP release in
static cells. Ca2+-dependent ATP release in response to shear seemed to result from vesicular release and not
through gap hemichannels. Vesicle disruption with N-ethylmaleimide, brefeldin A, or monensin prevented
increases in flow-induced ATP release, whereas inhibition of gap hemichannels with either 18 -glycyrrhetinic
acid or 18 -glycyrrhetinic acid did not. Degradation of extracellular ATP with apyrase prevented shearinduced increases in prostaglandin E2 (PGE2) release. These data suggest a time line of mechanotransduction
wherein fluid shear activates L-VSCCs to promote Ca2+ entry that, in turn, stimulates vesicular ATP release.
Furthermore, these data suggest that P2 receptor activation by secreted ATP mediates flow-induced prostaglandin release.
J Bone Miner Res 2005;20:4149. Published online on October 18, 2004; doi: 10.1359/JBMR.041009
Key words: ATP release, mechanotransduction, Ca2+ signaling, osteoblasts, fluid shear
tonic swelling, substrate strain, and fluid shear stress (FSS),
have been developed to study the cellular responses and
mechanisms involved in the perception of mechanical
stimuli by bone cells. Whereas none of these models completely replicate the stresses endured by bone, most produce osteoblastic responses that are considered anabolic in
vivo. These responses include transients in intracellular calcium levels ([Ca2+]i),(3) changes in gene expression,(46) and
prostaglandin release.(7,8) We have focused this study on
the effects of fluid shear because we have shown that fluid
shear, and not physiologic levels of mechanical strain, increases the expression of osteopontin, c-fos, cyclooxygenase
2, and TGF (4,9)
Osteoblasts respond to FSS with a rapid increase in in-
INTRODUCTION
B
ONE IS A dynamic organ, with its architecture constantly
changing in accordance with the mechanical usage required of it. As external forces placed on bone decrease,
which occurs in prolonged bed rest, immobilization, or microgravity, the skeleton undergoes net resorption, resulting
in significant bone loss.(1) Conversely, increased external
forces on the skeleton can produce net bone accumulation.(2) Various in vitro loading techniques, including hypo-
Dr Donahue served as a consultant for Merck, Osteoporosis
Thought Leader, and Orthologic. All other authors have no conflict of interest.
1
Departments of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA;
Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA; 3Orthopaedics and Rehabilitation, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.
2
41
J0403135R1
41 49
42
GENETOS ET AL.
tracellular Ca2+ that is dependent on both extracellular
Ca2+ entry and intracellular Ca2+ release.(3) Whereas we
have shown that FSS-induced increases in gene expression
are mediated by intracellular Ca2+ release in osteoblasts,(10)
Ca2+ entry in response to shear has been shown to be required for release of prostaglandins,(11) NO,(12) and
TGF .(13) Rapid Ca2+ entry must occur through ion channels, and two potential candidates for mediation of FSSinduced Ca2+ entry in osteoblasts are the mechanosensitive,
cation-selective channel (MSCC) and the L-type, voltagesensitive Ca2+ channel (L-VSCC).(14) The MSCC has been
shown to be important in the release of prostaglandins(11)
and TGF ,(13) whereas inhibition of the L-type VSCC has
been shown to inhibit NO release in bone organ cultures(15)
and reduce loading-induced bone formation in vivo.(16)
There is a significant body of evidence showing that ATP
in the extracellular milieu induces a host of physiologic responses on activation of ATP-binding purinergic (P2) receptors. These receptors are found in a wide variety of cell
types and tissues and have been shown to alter Ca2+ signaling in numerous cell types. P2 receptors can be divided into
two families of receptors: metabotropic P2Y receptors that
induce intracellular Ca2+ release through activation of Gproteins and ionotropic P2X receptors that are ligand-gated
channels. Osteoblasts express a variety of P2Y and P2X
receptors,(17) and activation of these receptors have been
shown to increase [Ca2+]i, propagate calcium waves,(18) induce c-fos,(17) and increase proliferation.(19,20) Release of
ATP from the cytosol to the pericellular environment is a
regulated process, and its extracellular availability for P2
receptor binding is limited by the presence of membranebound nucleotidases.(21) The mechanism(s) of ATP release
are unclear, yet chloride-conducting channels,(22,23) gap
junctional hemichannels, (24,25) and vesicular mechanisms(26,27) have been implicated in the controlled release
of ATP.
In this study, we examined the effects of fluid shear stress
on ATP release in MC3T3-E1 osteoblasts. We showed that
shear transiently increases ATP and that this release is
Ca2+-dependent. We further show that the shear-induced
release of ATP is blocked by inhibition of the L-VSCC
mediated by vesicular fusion. Most significant is the observation that ATP activation of P2 receptors is important for
shear-induced prostaglandin E2 (PGE2) release.
MATERIALS AND METHODS
Cell culture
MC3T3-E1 cells, a murine osteoblast-like cell line (a gift
from Dr Mary C Farach-Carson, University of Delaware),
were grown in -MEM containing 10% FBS (Gibco, New
York, NY, USA), 100 U/ml penicillin G, and 100 g/ml
streptomycin. Cells were maintained in a humidified incubator at 37C with 5% CO2/95% air and subcultured every
72 h. For shear studies, 80,000 cells were seeded onto rattail type I collagencoated (100 g/ml; BD, Franklin Lakes,
NJ, USA) glass slides. Fluid shear experiments were performed 2 days later, when the cells were 8085% confluent.
Flow media consisted of -MEM containing 0.5% FBS, 100
U/ml penicillin G, 100 g/ml streptomycin, and 20 mM
HEPES (pH 7.4).
Fluid flow experiments
Fluid flow was applied to cells in a parallel plate flow
chamber using a closed flow loop, as described previously(28) (Cytodyne, San Diego, CA, USA). This system
uses a constant hydrostatic pressure head to drive media
through the channel of the flow chamber to subject the cell
monolayer to steady laminar flow resulting in a welldefined fluid shear stress of 12 dynes/cm2. The apparatus
was maintained at 37C throughout the duration of experimentation. The correlation between shear and flow rate
was calculated using the equation
=
6Q
bh2
(Eq. 1)
where Q is the flow rate (cm3/s); is the viscosity of the
flow media (0.01 dynes/cm2); h is the height of the channel
(0.022cm); b is the slit width (3.2cm); and is the wall shear
stress (dyne/cm2). For time course studies of ATP release,
a programmable Harvard Syringe Pump (PHD programmable; Harvard Apparatus, Hollison, MA, USA) was used
to perfuse the flow chamber with fresh media at the aforementioned shear rate of 12 dynes/cm2.
Luciferin/luciferase-dependent detection of ATP
An ATP bioluminescence assay containing luciferin/
luciferase reagent was used to detect ATP (ATP Bioluminescence Assay kit HS II; Roche, Indianapolis, IN, USA).
This assay uses the conversion of D-luciferin by luciferase
into oxyluciferin and light that requires ATP as a cofactor.
The resultant luminescence, measured using a Monolight
3010 (BD Biosciences Pharmingen, San Diego, CA, USA),
reflects ATP concentration. Conditioned media samples
were acquired in two separate protocols. To determine the
time course of ATP release, media samples were taken at
each time-point and immediately frozen at 80C for further analysis. For studies using the closed flow loop, a
known volume of media was added to the flow loop before
fluid shear exposure for 5 minutes. After 5 minutes of fluid
shear, 1 ml of media was removed and stored as above.
Controls were performed with each drug solution to ensure
that the added drugs had no effect on luciferase activity.
Results were normalized to cellular protein concentration,
as determined by the amido black method.
Pharmacologic agents
All drugs tested were purchased from Sigma Chemical
(St Louis, MO, USA) and dissolved in distilled water unless
otherwise specified. Drugs were added 45 minutes before
the onset of flow and remained present for the duration of
the experiment. The following concentrations were used: 5
M nifedipine (from 5 mM stock in ethanol), 10 M verapamil, 5 M 18 -glycyrrhetinic acid (AGA; from 15 mM
stock in DMSO), 5 M 18 -glycyrrhetinic acid (BGA; from
15 mM stock in DMSO), 100 M monensin (from 100 mM
SHEAR-INDUCED ATP RELEASE IN OSTEOBLASTS
stock in MeOH), 10 M brefeldin A (BFA; 35 mM stock in
EtOH), and 100 M N-ethylmaleimide (NEM; from 100
mM stock).
Assessment of plasma membrane integrity
Cell damage was assessed quantitatively by measuring
the samples of recovered media for lactate dehydrogenase
(LDH). Analysis of LDH levels was performed using the
CytoTox96 Nonradioactive Cytotoxicity Assay (Promega,
Madison, WI, USA). This assay uses NADH, generated by
oxidation of lactate into pyruvate, with the conversion of
iodonitrotetrazolium into a red formazan product in the
presence of diaphorase. The absorbance at 490 nm is proportional to the amount of LDH in the media sample. For
a positive control, cells were disrupted by lysing in 0.1%
Triton X-100. Serial dilutions of this positive control were
compared with LDH levels from media samples.
Gap junctional intercellular communication assays
Gap junctional intercellular communication (GJIC)
assays were performed using double labeling and immunofluorescence as described previously.(29,30) In these experiments, cells were loaded with the dyes calcein-AM
(Molecular Probes, Eugene, OR, USA) and 1,19dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes). Because of its small molecular weight (<1 kDa), calcein is gap junction-permeable
and able to transfer to neighboring cells in the presence of
functional gap junctions, whereas DiI, a larger, lipophilic
dye, incorporates into the membrane and is unable to pass
through functional gap junctions. These double-labeled
(donor) cells were dropped onto nonlabeled (acceptor)
cells in monolayer. If functional GJIC existed, calcein
would be transferred to the acceptor cells, whereas DiI
would stay in the donor cell. Donor cells can be distinguished from acceptor cells through double exposure with
fluorescein and rhodamine filters: donor cells fluoresce yellow because of the presence of both calcein (green) and DiI
(red), whereas acceptor cells only fluoresce green.
Two days before experimentation, MC3T3-E1 cells were
seeded onto 35-mm glass coverslips in 6-well plates at a
density of 60,000 cells/well (acceptor cells). On the day of
the experiments, the preconfluent donor cells were removed from the incubator and washed with PBS followed
by aspiration. The donor cells were labeled in a solution
composed of 2 ml HBSS, 2% BSA, 7 l DiI, 20 l calceinAM, and 20 l pluronic acid (Molecular Probes) and incubated at 37C for 30 minutes. After 30 minutes, the dye was
aspirated, and the donor cells were detached by trypsinization and resuspended in fresh growth medium. A cell pellet
was obtained by 5 minutes of centrifugation at 200g. Five
hundred double-labeled donor cells were dropped onto the
acceptor cells and incubated for 2 h at 37C. After incubation, the coverslips were removed from the dishes, washed
twice in HBSS, and mounted onto a glass slide using Fluoromount-G (Fischer Scientific). The slides were placed on a
Nikon fluorescent microscope (Nikon EFD-3; Optical Apparatus, Ardmore, PA, USA) and visualized using fluorescein and rhodamine to locate the calcein- and DiI-loaded
43
cells, respectively. For studies using AGA or BGA to inhibit GJIC, 5 M AGA or BGA was added to acceptor
cells 45 minutes before the addition of donor cells and
maintained in the incubation media until the coverslips
were mounted.
Quinacrine staining of intracellular ATP
Osteoblasts were seeded onto type I collagencoated
glass slides as described above. Two days later, when the
slides were 8085% confluent, the cells were incubated in
25 M quinacrine for 30 minutes, rinsed twice in HBSS, and
mounted with Fluoromount G. The slides were immediately examined using the same Nikon fluorescent microscope as for GJIC assays.
Prostaglandin measurement
For measurement of PGE2 in sheared cells, experiments
were conducted as described above but exposed to fluid
shear for 60 minutes instead of 5 minutes. After the 60minute shear treatment, slides of cells were overlaid with 1
ml of fresh flow media (with or without drug, as appropriate) and incubated for 30 additional minutes at 37C with
5% CO2. The media were collected, and PGE2 was measured using commercially available ELISA kits (Amersham
Biosciences, Piscataway, NJ, USA) according to the manufacturers instructions. Results were normalized to cellular
protein levels. The effect of exogenous ATP addition to
static cells was also addressed. Experiments were performed as above for PGE2 release, but overlaid with 1 ml of
ATP (at concentrations of 100 nM1 mM) in flow media for
30 minutes, after which time the media were collected and
analyzed by ELISA.
Statistical analysis
A minimum of two slides per treatment was examined
each day on at least 3 different days. Two-way ANOVA
analyses were used to compare ATP release from MC3T3E1 cells treated with pharmacological agents. When a significant difference was found between samples, a Fishers
PLSD was performed to localize the significant difference.
Statistical significance was considered at p < 0.05, and
samples are presented as mean SE.
RESULTS
Fluid shear stress induces ATP release
MC3T3-E1 cells exhibited a basal release of ATP (6.2
1.8 nM) that was significantly increased 10-fold (59.8 15.7
nM; p < 0.001) when cells were subjected to 12 dynes/cm2
FSS (Fig. 1A). Because cytosolic ATP concentrations are in
the millimolar range,(31) it was necessary to determine
whether the changes in extracellular ATP levels resulted
from an active release rather than shear-induced cell lysis.
To ensure that membrane damage did not contribute to
shear-mediated ATP release, we analyzed the conditioned
media from sheared cells for the presence of the cytosolic
enzyme, lactate dehydrogenase (LDH): if ATP release resulted from cellular damage, LDH would be found in the
conditioned media. We consistently found that fluid shear-
44
GENETOS ET AL.
FIG. 1. (A) Effects of fluid shear stress at 12 dynes/cm2 on ATP
release in MC3T3-E1 osteoblasts. After 5 minutes of flow, the
flow media was removed for analysis of ATP content using an
ATP-dependent luciferinluciferase reaction. Fluid shear increased ATP release 5-fold compared with static controls (ap <
0.01 vs. static cells). Each bar represents the mean SE of 10
experiments. (B) Time course of ATP release in response to
shear. ATP was released rapidly from MC3T3-E1 cells, peaking
within 1 minute of shear application. ATP release returned to
near, but elevated, baseline levels with sustained shear (ap < 0.01
vs. static cells at same time-point).
FIG. 2. ATP release is dependent on extracellular Ca2+ entry.
(A) MC3T3-E1 cells were treated with ionomycin (1 M), a calcium ionophore, for 10 minutes. The media were collected for
ATP analysis. The addition of ionomycin significantly increased
ATP release compared with control cells (ap < 0.02 vs. vehicle
control). (B) Effects of channel blockers on ATP release. MSCC
inhibition with GdCl3 (10 M) had no effect on either basal or
FSS-induced ATP release, whereas L-type VSCC inhibition with
nifedipine (5 M) or verapamil (10 M) attenuated FSS-induced
ATP release but not basal release (ap < 0.05 vs. static cells in same
conditions; bp > 0.05 vs. static cells in same conditions).
induced ATP release occurred in the absence of significant
plasmalemmal damage compared with static controls (data
not shown).
ATP release in response to a variety of stimuli has been
shown to be transient, occurring rapidly after the stimulus
and decreasing to basal levels over a period of 3060 minutes.(32) To determine if a similar pattern occurs in osteoblastic MC3T3-E1 cells in response to FSS, we examined
ATP release at time-points before and after 5 minutes using
a programmable Harvard Syringe Pump to produce 12
dynes/cm2 shear. We found that FSS induced a rapid release of ATP within one minute of the onset of FSS that
returned to preflow levels with prolonged fluid shear
(Fig. 1B).
channels have been implicated in the intracellular Ca2+ response to mechanical load.(3,4,15,33) To test whether these
channels are involved in shear-induced ATP release, we
blocked the MSCC with GdCl3 (10 M) and the L-VSCC
with nifedipine (5 M) and verapamil (10 M). Nifedipine
significantly attenuated the shear-induced ATP release
(Fig. 2B) in a manner similar to verapamil inhibition (data
not shown). Neither inhibitor altered basal ATP release.
Block of the MSCC with GdCl3 did not alter either basal or
shear-induced ATP release.
Shear-induced ATP release requires calcium entry
To determine whether ATP release was Ca2+-dependent,
we exposed static MC3T3-E1 cells to the Ca2+ ionophore,
ionomycin (1 M), for 10 minutes, and removed the bathing medium for ATP analysis. Addition of ionomycin produced a 3-fold (p < 0.05) increase in ATP release compared
with untreated controls (Fig. 2A). L-VSCC and MSCC
Shear-induced ATP release does not require GJIC
Gap junctions and GJIC have been implicated in the
mechanism through which other cell types release ATP in
response to a mechanical signals.(24,25) Because serum proteins can bind to AGA and titrate the effective AGA concentration,(34) it was first necessary to show that GJIC was
inhibited under the flow conditions used. GJIC was evaluated using the double-labeling technique as described previously. In Fig. 3A, the green (calcein) fluorescence indicates the coupled cells in the monolayer, whereas the
yellow (calcein and DiI) fluorescence indicates the donor
SHEAR-INDUCED ATP RELEASE IN OSTEOBLASTS
45
tion with quinacrine, a high level of punctuated fluorescence was seen, localized primarily within the cytosol of the
cells (Fig. 4A). To assess the role of vesicular exocytosis in
ATP release, we used three pharmacologic agents: BFA,
which causes disruption of the Golgi apparatus(26); monensin, which prevents vesicle formation from the Golgi apparatus(26,27); and NEM, which prevents vesicle fusion to the
plasma membrane by interfering with vesicle-associated
NSF proteins.(26,27) Fluid shear in the presence of each of
these antagonists significantly attenuated ATP release compared with untreated controls (Figs. 4B4D). These data,
combined with the highly granular and punctate intracellular localization of ATP as visualized by quinacrine staining,
suggest that ATP is released from murine osteoblasts in a
vesicular manner.
Extracellular ATP is required for flow-induced
increases in PGE2
FIG. 3. The role of GJIC in ATP release. (A) GJIC was assessed
in MC3T3-E1 cells by dual-label dye transfer. Donor cells double
labeled with the fluorescent dyes calcein and 1,1 -dioctadecyl3,3,3 ,3 -tetramethylindocarbocyanine perchlorate (DiI) were
placed in contact with unloaded cells in the monolayer. Dye transfer was evaluated after 2 h. In the dual-exposure photographs,
calcein has transferred to acceptor cells, which are green, showing
functional GJIC; cells fluorescing yellow contain both calcein and
DiI and are the dual-labeled donor cells. Preincubation with 5 M
18 -glycyrrhetinic acid inhibited GJIC. (B) MC3T3-E1 cells pretreated with vehicle control (DMSO) or GJ inhibitors, 18 glycyrrhetinic acid or 18 -glycyrrhetinic acid, showed that inhibition of GJIC had no significant effect on FSS-induced ATP
release (ap < 0.01 vs. static cells in same conditions).
cells. We found that 5 M AGA pretreatment in flow media (containing 0.5% FBS) effectively inhibited GJIC in
MC3T3-E1 osteoblasts. Under these conditions, we found
that osteoblasts released ATP in the presence of 5 M
AGA when exposed to fluid shear stress (Fig. 3B). Similar
results were found when cells were sheared in the presence
of another GJ inhibitor, BGA (5 M). These results suggest
that shear-induced ATP release in murine osteoblasts does
not require GJIC or hemichannels.
Localization of intracellular ATP stores
We examined the localization of intracellular ATP stores
in MC3T3-E1 osteoblasts using quinacrine, a cell-permeant
fluorophore that binds to ATP. After a 30-minute incuba-
Fig 3A live 4/C
Prostaglandins are rapidly in released response to shear
in osteoblasts,(8,11) and their formation is required for loadinduced bone formation.(35) To determine whether shearinduced PGE2 release was mediated by extracellular ATP,
we added apyrase (5U/ml), a nucleotidase that degrades
nucleotide triphosphates into nucleotide monophosphates,
to the flow medium. Apyrase attenuated flow-induced increases in PGE2 release (Fig. 5A). Similar results were obtained from experiments performed with the nonspecific P2
antagonist PPADS, and additional experiments with heatinactivated apyrase confirmed that the enzymatic activity of
apyrase, and not some secondary, nonspecific effect, was
responsible for attenuating PGE2 release (data not shown);
Furthermore, when exogenous ATP was added to static
cells, we observed significant increases in PGE2 release
(Fig. 5B) without induction of Cox-2 (data not shown).
These data suggest that shear-induced ATP secretion mediates the release of prostaglandins, paracrine factors that
have been implicated in the anabolic response to exogenous
mechanical load.
DISCUSSION
Several mechanisms have been proposed for stimuliinduced ATP release from various cell types. These proposed mechanisms include release through a chloride
conducting pathway,(36) through gap junctional hemichannels,(24,37) and through Ca2+-dependent vesicular exocytosis.(26) Our data indicate that shear-induced ATP release
results from Ca2+ dependent vesicular release. However,
we did observe that the general chloride channel blocker,
4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS), inhibited both basal and shear-induced ATP release (data not
shown). While this is a focus of another study, previous
reports suggest that DIDS does not block ATP release
through inhibition of a chloride conductance, but rather, by
binding to a sulfonylurea receptor on the vesicle to prevent
fusion of the vesicle to the membrane.(38) Another potential pathway, ATP conductance through gap junctional
hemichannels seems to be involved in other cell types,(24,39)
but not in the MC3T3-E1 cell model. We showed that gap
junction inhibition with either AGA or BGA prevented
46
GENETOS ET AL.
FIG. 4. (A) Quinacrine staining of MC3T3-E1 cells shows grainy, punctate localization of ATP, suggesting that ATP is contained in
vesicles. (B) Brefeldin A, an agent that disrupts the Golgi and thereby prevents vesicle formation, attenuated FSS-induced increases
in ATP release but had no effect on basal ATP release. (C) Monensin, which maintains the Golgi structure but prevents vesicle budding
from the Golgi, similarly attenuated FSS-induced ATP release without affecting static ATP release. (D) N-ethylmaleimide prevents
vesicular exocytosis by inhibiting NSF proteins. Addition of NEM also inhibited FSS-induced ATP release. (ap < 0.01 vs. static cells in
same conditions).
dye movement through the junctional complex, but had no
effect on FSS-induced ATP release. These data support a
previous report that found no difference in mechanically
stimulated ATP release in human osteoblast-like cells overexpressing the Cx43 connexin compared with wildtype controls.(25) Quinacrine, a ATP-binding fluorophore, showed
punctate, granular staining, suggesting that ATP can be localized in vesicles in MC3T3-E1 osteoblasts; further experimentation, such as density gradient fractionation, however,
is required to unambiguously show vesicular ATP localization.
Similar to a previous report in epithelial cells,(26) we
show that FSS-induced ATP release from osteoblastic cells
is the result of Ca2+-dependent vesicular binding to the
membrane. Three pharmacologic agents, which cause disruption of the Golgi apparatus,(26) prevent vesicle formation from the Golgi apparatus(26,40) or prevent vesicle
fusion to the plasma membrane by interfering with vesicleassociated NSF proteins(26,40) all significantly decreased
FSS-induced ATP release compared with untreated controls. Interestingly, none of these agents completely
blocked either basal or FSS-induced ATP release. This observation would suggest that either each of these agents
does not totally block vesicular fusion to the membrane or
that a secondary pathway for ATP release exists.
Whereas numerous studies have shown that ATP binding
to P2 receptors results in an increase in [Ca2+]i that is dependent on both extracellular Ca2+ entry and intracellular
Ca2+release, few have examined the role of [Ca2+]i in ATP
release. Osteoblasts and osteocytes respond to fluid shear
with a rapid increase in intracellular Ca2+(3) that is essential
for shear-induced changes in actin cytoskeletal organization
and gene expression.(10) Whereas this [Ca2+]i response has
been shown to be dependent on both extracellular Ca2+
entry and intracellular Ca2+ release, we have shown that
only IP3-mediated Ca2+ release is required for the subsequent changes in cell architecture and protein production.(10) However, others have reported that Ca2+ entry
through ion channels is important to shear-induced secretion of prostaglandins,(11) TGF ,(13) and NO(15) from bone
cells, leading us to postulate that Ca2+ entry mediates signal
amplification, whereas intracellular Ca2+ release results in
changes in gene expression in osteoblasts. Katsuragi et
al.(41) have shown that inhibition of phospholipase C/IP3mediated intracellular Ca2+ release blocks angiotensin II
stimulated ATP release in smooth muscle cells. However,
SHEAR-INDUCED ATP RELEASE IN OSTEOBLASTS
FIG. 5. (A) Addition of apyrase (5 U/ml), which hydrolyzes
extracellular ATP, significantly decreased PGE2 release from
sheared osteoblasts, suggesting that FSS-induced ATP secretion
mediates PGE2 release. (B) Addition of exogenous ATP to static,
nonsheared cells dose-dependently increased PGE2 release (ap <
0.01 to static control; bp < 0.01 to shear in the absence of apyrase).
in this study, we found no consistent effect of inhibition of
phospholipase C with U73122 on either basal or shearinduced ATP release. This lack of consistency may be
caused by the subsequent inhibition by U73122 of protein
kinase C, which mediates a number of cellular responses,
including phosphorylation of Ca2+ channels.
Calcium entry through membrane ion channels could
also mediate ATP release in response to shear. This postulate is strengthened by the observation that, when extracellular Ca2+ is removed, ATP release in response to mechanical load is attenuated.(26) Osteoblasts express a number of
ion channels capable of conducting Ca2+,(14) but to date,
only two have been shown to play a role in the [Ca2+]i
response to shear: the mechanosensitive, cation-selective
channel (MSCC)(3,33) and the dihydropyridine- and voltage-sensitive L-type Ca2+ channel (VSCC).(33) Activation
of the MSCC in response to loading has been associated
with prostaglandin, TGF , and NO release.(11,13,15) Activation of the L-type VSCC has been linked to cellular responses of osteoblasts or osteocytes to shear(15) and hormonal stimulation. (33) We have recently shown that
inhibition of this channel with either nifedipine or verapamil significantly reduces bone formation in mechanically
loaded rat tibias and ulna, in vivo,(16) indicating the importance of this channel in mechanotransduction in bone.
How L-type VSCCs are activated by mechanical perturbation is unclear. We have postulated that shear-induced
47
activation of the MSCC results in a membrane depolarization that, in turn, activates the VSCC current. However, the
data reported here fail to support this premise. Inhibition of
the MSCC with 10 M GdCl3 did not significantly block
ATP release, although a reduced ATP release level in response to shear was observed. Thus, either FSS depolarizes
the membrane through a separate mechanism to activate
the L-VSCC, or this stimulus can directly activate the LVSCC.
One mechanism through which the L-VSCC could be
activated directly is by the autocrine/paracrine action of
ATP. ATP released from the cell can bind to P2 receptors
that modulate a number of second messenger pathways,
including [Ca2+]i. There are two subtypes of P2 receptors:
ionotropic (P2X) receptors that allow entry of ions through
receptor-mediated channels, and metabotropic (P2Y) receptors that induce G-proteinmediated intracellular Ca2+
release.(42) Whereas the P2Y receptor has been linked with
IP3-mediated intracellular Ca2+ release, P2X receptors have
been shown to activate both K+ channel and Ca2+ channels,
including the L-type VSCC.(43,44) A number of isoforms for
each of these subtypes have been described, based on the
nucleotide binding selectivity of the receptor and inhibition.
Osteoblasts exhibit many of these isoforms for both P2X
and P2Y,(17) and ATP binding to these receptors has also
been associated with fast, gap junctionindependent Ca2+
waves(45) and potentiation of the [Ca2+]i response to PTH
in osteoblasts.(46) Here, we showed that MC3T3-E1 osteoblasts respond to a defined fluid shear with release of ATP
within 1 minute of shear onset. Because the [Ca2+]i response to shear exhibits a time lag from onset to intracellular response of 3060 s,(3,33) it is possible that ATP initiates this event to enhance its own release.
Prostaglandin synthesis and release have been shown to
occur rapidly in osteoblasts in response to shear(7,8,11) and
are essential for the anabolic response of bone to mechanical loading.(35) PGE2 release from osteoblasts and osteocytes has been shown to be released in two stages. On application of shear, a brief burst of PGE2 is observed,
peaking at 510 minutes and returning to levels near baseline.(7,11) This is followed at 4560 minutes by a large, continuous increase in release that corresponds to an increase
in Cox-2 production.(7,8) The importance of Cox-2 function
in bone formation was shown when the Cox-2 specific inhibitor, NS398, was given to rats before mechanical loading:
the presence of NS398 completely abolished tibial bone
formation in response to four-point bending compared with
loaded controls.(35) Whereas these results implicate Cox-2
in bone formation in response to exogenous load, it should
be noted that prostaglandin synthesis and release does not
directly correlate with bone formation, because prostaglandins can similarly promote bone resorption.(47) The addition
of exogenous ATP to static MC3T3-E1 cells induced the release of PGE2, and the hydrolysis of ATP released in response
to shear blocked flow-induced increase in PGE2 release, suggesting that FSS-induced ATP secretion induces
PGE2 release through activation of a P2 receptor. Because
Reich and Frangos(48,49) showed that flow-induced PGE2 release was mediated through a Gq-protein, we hypothesize that
a metabotropic P2Y receptor is involved in this response.
48
GENETOS ET AL.
FIG. 6. Proposed model for data presented herein. Fluid shear
stress activates the L-type VSCC calcium channel through some
unknown mechanism. Calcium influx through the L-type VSCC
mobilizes vesicular stores of ATP to the plasmalemma, where
vesicular fusion releases ATP into the extracellular environment.
ATP diffuses to P2 receptors to function in an autocrine or paracrine manner, activating either ionotropic P2X or metabotropic
P2Y receptors to increase PGE2 release.
Whereas the measured amount of secreted ATP in response to shear was on the order of 100 nM (i.e., 1000-fold
less than the amount required to induce PGE2 release in
static conditions), it should be noted that the data presented
in Figs. 14 represents ATP found in solution. This does not
accurately show the local ATP concentration at the cell
surface immediately on its release from the cytosol or the
local concentration at P2 receptors. Furthermore, whereas
their expression has not yet been shown in osteoblasts,
other ATP-releasing cells, such as chondrocytes and endothelial cells, express membrane-bound NTP-degrading enzymes (ecto-5 -nucleotidases and exonucleotidases) that
regulate the extracellular availability of ATP.
Figure 6 shows our working model for the action of ATP
release in osteoblasts. We show that osteoblasts exhibit a
basal release of ATP in static cells and respond to a welldefined fluid shear regimen with a significant increase in
ATP release. This release is mediated by Ca2+-dependent
vesicular fusion to the membrane, although only the shearinduced release is sensitive to Ca2+. We further showed that
Ca2+ entry through the L-type VSCC, but not Ca2+ release
from intracellular stores, is important to this response.
PGE2 release in response to shear seems to be mediated by
secreted ATP, suggesting that purinergic signaling may be
an important component of the mechanotransduction response
of bone.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health
NIAMS Grants P01 AR45218 and R01 AR43222 (RLD),
NIA Grant 13087-09 (HJD), and the National Aeronautics
and Space Administration Predoctoral Fellowship Grant
NGT5-50366 (DCG). We thank Dr Suzanne M Norvell for
assistance with prostaglandin assays and Dr Robert M
Bigsby for assistance with the luciferin/luciferase assay.
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Address reprint requests to:
Randall L Duncan, PhD
Department of Orthopaedic Surgery
Indiana University School of Medicine
541 Clinical Drive, Suite 600
Indianapolis, IN 46202, USA
E-mail: [e-mail]
Received in original form March 4, 2004; revised form July 20,
2004; accepted August 11, 2004.