JOURNAL OF BONE AND MINERAL RESEARCH
Volume 21, Number 11, 2006
Published online on July 31, 2006; doi: 10.1359/JBMR.060722
© 2006 American Society for Bone and Mineral Research
PTH-Induced Actin Depolymerization Increases Mechanosensitive
Channel Activity to Enhance Mechanically Stimulated Ca2+ Signaling
in Osteoblasts*
Jinsong Zhang,1,2,3 Kimberly D Ryder,2,4 Jody A Bethel,1 Raymund Ramirez,1 and Randall L Duncan1,3
ABSTRACT: Disruption of the actin cytoskeleton with cytochalasin D enhanced the mechanically induced
increase in intracellular Ca2+ ([Ca2+]i) in osteoblasts in a manner similar to that of PTH. Stabilization of actin
with phalloidin prevented the PTH enhanced [Ca2+]i response to shear. Patch-clamp analyses show that the
MSCC is directly influenced by alterations in actin integrity.
Introduction: PTH significantly enhances the fluid shear-induced increase in [Ca2+]i in osteoblasts, in part,
through increased activation of both the mechanosensitive, cation-selective channel (MSCC) and L-type
voltage-sensitive Ca2+ channel (L-VSCC). Both stimuli have been shown to produce dynamic changes in the
organization of the actin cytoskeleton. In this study, we examined the effects of alterations in actin polymerization on [Ca2+]i and MSCC activity in MC3T3-E1 and UMR-106.01 osteoblasts in response to shear ± PTH
pretreatment.
Materials and Methods: MC3T3-E1 or UMR-106.01 cells were plated onto type I collagen–coated quartz
slides, allowed to proliferate to 60% confluency, and mounted on a modified parallel plate chamber and
subjected to 12 dynes/cm2. For patch-clamp studies, cells were plated on collagen-coated glass coverslips,
mounted on the patch chamber, and subjected to pipette suction. Modulators of actin cytoskeleton polymerization were added 30 minutes before the experiments, whereas channel inhibitors were added 10 minutes
before mechanical stimulation. All drugs were maintained in the flow medium for the duration of the experiment.
Results and Conclusions: Depolymerization of actin with 1–5 ␮M cytochalasin D (cyto D) augmented the peak
[Ca2+]i response and increased the number of cells responding to shear, similar to the increased responses
induced by pretreatment with 50 nM PTH. Stabilization of actin with phalloidin prevented the PTH enhanced
[Ca2+]i response to shear. Inhibition of the MSCC with Gd3+ significantly blocked both the peak Ca2+ response
and the number of cells responding to shear in cells pretreated with either PTH or cyto D. Inhibition of the
L-VSCC reduced the peak [Ca2+]i response to shear in cells pretreated with PTH, but not with cyto D.
Patch-clamp analyses found that addition of PTH or cyto D significantly increased the MSCC open probability
in response to mechanical stimulation, whereas phalloidin significantly attenuated the PTH-enhanced MSCC
activation. These data indicate that actin reorganization increases MSCC activity in a manner similar to PTH
and may be one mechanism through which PTH may reduce the mechanical threshold of osteoblasts.
J Bone Miner Res 2006;21:1729–1737. Published online on July 31, 2006; doi: 10.1359/JBMR.060722
Key words: mechanotransduction, PTH, intracellular Ca2+, actin cytoskeleton, fluid shear, osteoblasts
INTRODUCTION
S
KELETAL INTEGRITY AND bone homeostasis are dependent on the physical forces exerted on bone during
movement. Frost(1) has proposed that discrete thresholds of
strain magnitude exist where bone mass is lost, maintained,
Parts of these data were presented at the 50th Annual Meeting
of The Orthopaedic Research Society, San Francisco, CA, March
7–10, 2004.
The authors state that they have no conflicts of interest.
or increased. Martin and Burr(2) have shown that these
thresholds are well defined and are species and size independent, but that the strain threshold where formation is
greater than resorption is at or beyond the limit of physiologic strain. Frost also proposed that these strain thresholds may be lowered by biochemical factors so that lesser,
more physiologic, strains could promote bone formation.
Several potential candidates for the modulation of mechanical thresholds have been proposed. Of these, PTH
may be the strongest contender, producing a synergistic
effect on loading-induced bone formation.(3,4) Because
1
Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA; 2These authors contributed
equally to this study; 3Department of Biological Sciences, University of Delaware, Newark, Delaware, USA; 4Cellular and Integrative
Physiology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
1729
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ZHANG ET AL.
both PTH and mechanical stimulation activate similar second messenger pathways in osteoblasts, we hypothesize
that PTH may sensitize one of these pathways to lower the
threshold for mechanical stimulation.
Both PTH and mechanical stimulation produce a rapid
increase in intracellular Ca2+ ([Ca2+]i) that is dependent on
extracellular Ca2+ entry and intracellular Ca2+ release.(5,6)
Ca2+ entry requires the activation of multiple ion channels
in osteoblasts. One of these, the mechanosensitive cationselective channel (MSCC), is activated by membrane deformation,(7,8) and when inhibited by the nonspecific
blocker, gadolinium (Gd3+), the shear-induced increase in
[Ca2+]i is significantly reduced.(5,9) We have shown that addition of PTH to UMR 106.01 cells increases the MSCC
stretch sensitivity and channel open probability,(10) suggesting that by increasing these kinetic parameters of the
MSCC, PTH may increase Ca2+ signaling in osteoblasts in
response to mechanical stimulation. L-type voltagesensitive channels (L-VSCCs) have also been implicated in
the [Ca2+]i response to mechanical stimulation.(5) L-VSCCs
are voltage-gated and dihydropyridine-sensitive, but do not
seem to be responsive to membrane perturbation. However, we have shown that the PTH-enhanced [Ca2+]i response to fluid shear requires the activation of both MSCCs
and VSCCs in osteoblasts.(9) Furthermore, PTH stimulation alone has been shown to modulate L-VSCC activity in
osteoblasts(6,11) and that inhibition of this channel significantly reduces Ca2+ signaling induced by PTH in osteoblasts.(11)
Osteoblasts also respond to both PTH and mechanical
stimulation with an alteration in the actin cytoskeletal organization. The actin cytoskeleton is a dynamic structural
network that is essential for the regulation of a number of
cellular events, including mechanotransduction.(12,13) Mechanical stimulation rapidly reorganizes the actin cytoskeleton into stress fibers in the osteoblast,(14,15) and disruption
of actin stress fibers can lead to changes in the response of
osteoblasts to mechanical stimulation.(15) PTH also alters
the organization of actin in osteoblasts resulting in changes
in cell morphology within minutes of stimulation,(16,17)
leading to the postulate that actin cytoskeletal reorganization may contribute to the functional response of osteoblasts to PTH.(18)
Several ion channels have been shown to be directly
linked to, or modulated by, the actin cytoskeleton, including the epithelial Na+ channel,(19) mechanosensitive channels,(20) and the L-VSCC.(21,22) In this study, we postulate
that PTH sensitizes osteoblasts to mechanical stimulation
by modulating channel kinetics of the MSCC through repolymerization of the actin cytoskeleton. To test this hypothesis, we disrupted or stabilized the actin network with
cytochalasin D (cyto D) or phalloidin before PTH treatment or application of fluid shear and determined the
[Ca2+]i response and the activation of the MSCC.
MATERIALS AND METHODS
Cell culture
The mouse osteoblast-like cell line, MC3T3-E1 (passages
6–19), and the rat osteosarcoma cell-line, UMR106.01 (pas-
sages 27–45), were kind gifts from Dr Mary C FarachCarson (University of Delaware) and Dr Nicola Partridge
(St Louis University), respectively. These cells were grown
in ␣-MEM (MC3T3-E1) and DMEM (UMR-106.01) containing 10% FCS (Gibco, New York, NY, USA), 100 U/ml
penicillin G, and 100␮g/ml streptomycin. Cells were maintained in a humidified incubator at 37°C with 5% CO2/95%
air and subcultured every 72 h. All cell culture media and
antibiotics were purchased from Sigma Chemical, St Louis,
MO, USA.
Materials
The 1-34 fragment of bovine PTH [bPTH(1-34)]
(Bachem, Torrance, CA, USA) was dissolved in distilled
water and used at a final concentration of 50 nM. Gadolinium chloride (GdCl3), an inhibitor of MSCCs, was dissolved
in water at a stock concentration of 1 mM and used at a
final concentration of 10 ␮M. Nifedipine, an inhibitor of
L-VSCCs, was dissolved in 100% ethanol at a stock concentration of 3 mM and used at a final concentration of 5
␮M. Cytochalasin D (cyto D), an actin cytoskeleton depolymerizing agent, phalloidin (phall), an actin cytoskeleton
stabilizer, and nocodazole (noca), a microtubule disruptor,
were dissolved in dimethyl sulfoxide at stock concentrations
so that the final concentration of the solvent was ⱕ0.1%.
All drugs were obtained from Sigma, unless otherwise indicated.
Ca2+ imaging fluid flow experiments
MC3T3-E1 cells were grown for 4 days on type I collagen–coated (10 ␮g/cm2; Collaborative Biomedical, Bedford, MA, USA) quartz slides. For flow experiments, cells
were rinsed two times with Hanks’ balanced saline solution
(HBSS). Cells were loaded with 3 ␮M fura-2/AM (Molecular Probes, Eugene, OR, USA), a fluorescent Ca2+ probe,
in HBSS for 45 minutes at 37°C. Loaded cells were incubated for an additional 15 minutes with HBSS alone to
ensure complete de-esterification of the fluorescent molecule, yet minimize intracellular compartmentalization.
A parallel-plate flow chamber with a uniform flow channel height of 250 ␮m was used to subject the cells to fluid
shear, as previously described.(9) Flow was introduced to
the chamber through a syringe mounted on a Harvard Syringe Pump (PHD Programmable; Harvard Apparatus,
Holliston, MA, USA) that controlled the flow rate. To establish a fluid-flow [Ca2+]i baseline, cells were exposed to
fluid shear of 1 dyne/cm2 for 3 minutes. Fluid shear magnitude remained at 1 dyne/cm2 or was increased to 12 or 25
dynes/cm2 for 3 minutes. Corresponding flow rates for each
of the fluid shear levels were 1, 15, and 30 ml/min, respectively.
A ratiometric video-image analysis apparatus (Intracellular Imaging, Cincinnati, OH, USA) was used to record
changes in [Ca2+]i. Fura-2 fluorescence was visualized with
a Nikon inverted microscope using a Nikon 30× fluor objective. The cells were illuminated with a Xenon lamp
equipped with quartz collector lenses. A shutter and filter
changer containing the two different interference filters
(340 and 380 nm) was computer controlled. In this system,
CYTOSKELETAL CONTROL OF CA2+ SIGNALING IN OSTEOBLASTS
emitted light is passed through a 430-nm dichroic mirror,
filtered at 510 nm, and imaged with an integrating CCD
video camera. The ratio of emitted light at 340- and 380-nm
excitation was determined (F340/F380) from consecutive
frames, and the [Ca2+]i for each cell was calculated from
this ratio by comparison with fura-2 free acid standards.
Computer-generated individual Ca2+ traces are population
means derived from simultaneous recording of Ca2+ in the
4–12 single cells in the field of view.
Immunocytochemistry and fluorescence microscopy
MC3T3 cells were seeded on type I collagen–coated (10
␮g/cm2; Collaborative Biomedical) coverslips for 4 days.
Cells were washed with PBS (Sigma) and treated for 0.5 h
with bPTH(1-34) or cytoskeletal modifiers in ␣-MEM. After treatment, cells were washed in PBS and fixed in 4%
paraformaldehyde in PBS for 15 minutes. Cells were permeabilized with 0.2% Triton-X100 (Sigma) in PBS for 5
minutes. After permeabilization, cells were rinsed for 5
minutes with PBS. Cells were incubated with 10 ␮g/ml
FITC-phalloidin (Molecular Probes) in PBS for 30 minutes
and washed three times for 5 minutes with PBS. Images
were recorded using a Nikon Optiphot II microscope
through a ×100 objective.
Patch-clamp studies
UMR106.01 cells were plated on glass coverslips and incubated for 48 h. Coverslips were transferred to the patch
chamber and bathed in an isotonic Na+ Ringers consisting
of (in mM) 137 NaCl, 5.5 KCl, 1 CaCl2, 1 MgCl2, 3 glucose,
and 20 HEPES, titrated to a pH of 7.3 with NaOH. Triethylammonium chloride (TEA; 1 mM) was added to the bath
to block K channel activity. Fire polished, 5- to 10-M⍀
borosilicate glass pipettes were backfilled with Na+ Ringer,
and a conventional, cell-attached seal (>15 G⍀) was obtained. The membrane voltage was clamped at −40 mV, and
basal MSCC single channel activity and kinetics were determined by application of suction (15 mmHg, 1 Hz) to the
backside of the pipette. After determination of basal MSCC
activity, either 50 nM bPTH(1-34), 1 ␮M cytochalasin D, or
1 ␮M phalloidin was directly added to the chamber, and
changes in MSCC kinetics were monitored for 30 minutes.
The osmolality of all solutions was checked with a freezingpoint depression osmometer (Precision Systems) and adjusted to 300 ± 5 mosmol/kgH2O. Single channel currents
were recorded with a List EPC-7 amplifier (Medical Systems, Great Neck, NY, USA), filtered at 1 kHz, and digitized at a sampling frequency of 2–5 kHz using pCLAMP
8.0 software (Axon Instruments).
Statistical analysis
Mean peak [Ca2+]i response was expressed as a percentage increase in [Ca2+]i over baseline [Ca2+]i levels. The percentage of cells responding was determined by dividing the
number of cells that responded with a 100% or greater
increase in [Ca2+]i by the total number of cells. Data are
presented as mean ± SE and were obtained from at least
five separate passages of cells. The data were pooled because there was no significant difference for a treatment
1731
between different passages of cells. Significance of all experiments was determined using one-way ANOVA, and
the Bonferroni posthoc test was used to determine significance when multiple comparisons in the study were made.
Differences were considered significant when p < 0.05.
RESULTS
Effects of PTH and actin organization on the
shear-induced Ca2+ increase
MC3T3-E1 osteoblasts respond to fluid shear with a
rapid increase in [Ca2+]i as shown in the representative
trace shown in Fig. 1A. Baseline [Ca2+]i levels were determined during application of 1-dyn/cm2 shear. Fluid shear
was stepped to 12 dynes/cm2 for 3 minutes. We defined a
responding cell as one that had an increase in [Ca2+]i of at
least 100% over its baseline [Ca2+]i level. Defined by this
criterion, the percent increase in the mean peak [Ca2+]i
response in sheared control MC3T3-E1 cells was 185 ± 28%
(Fig. 2A), with 62 ± 3% (66 of 105 cells) of the cells responding with this increase (Fig. 2B). Pretreatment of
MC3T3-E1 cells for 10 minutes before shear with 50 nM
bPTH(1-34) produced a mean peak [Ca2+]i response of 295
± 44% (p < 0.05) above baseline. PTH pretreatment also
significantly increased the number of cells responding to 82
± 4% (55/67 cells; p < 0.05).
To determine the role of the actin cytoskeleton in the
response of mean peak [Ca2+]i and the number of cells
responding, we pretreated MC3T3-E1 cells with cyto D,
which disrupts the actin cytoskeleton and promotes the formation of short chained actin filaments. Pretreatment of
MC3T3-E1 cells with cyto D increased the mean peak
[Ca2+]i response to shear to 271 ± 32% (p < 0.01 compared
with sheared control), with 80 ± 5% (45/56 cells; p < 0.05)
of the cells responding to shear (Fig. 2). Whereas the absolute value of the peak [Ca2+]i response to shear + cyto D
was much higher than that with PTH pretreatment (Fig.
1C), cyto D also significantly increased baseline [Ca2+]i
from control levels of 87 ± 7 to 147 ± 15 nM (p < 0.01), thus
making the percent increase in mean peak [Ca2+]i response
approximately the same as PTH pretreatment. When the
actin cytoskeleton was disrupted with cyto D, followed by
addition of PTH before shear, we observed no significant
increase in the mean peak [Ca2+]i response or the number
of cells responding compared with the PTH + shear group.
We next stabilized the actin cytoskeleton during shear with
phall, an agent that binds to actin and prevents reorganization (Fig. 1D). Pretreatment of MC3T3-E1 cells with phall
for 30 minutes before shear reduced the mean peak [Ca2+]i
response to shear (100 ± 15%; p < 0.05) and PTH + shear
(151 ± 22%; p < 0.01). Phall also decreased the number of
cells responding to shear to 37 ± 3% (18/48; p < 0.05) and
PTH + shear to 39 ± 4% (15/38; p < 0.01). These data
suggest that the actin cytoskeleton plays an important role
in the [Ca2+]i response to both shear alone and PTH +
shear.
To determine the effects of disruption of microtubules on
the [Ca2+]i response to shear and shear + PTH, we pretreated MC3T3-E1 cells for 30 minutes with nocodazole (5
1732
ZHANG ET AL.
FIG. 1. Representative traces showing the
intracellular Ca2+ response to 12 dynes/cm2
fluid shear in MC3T3-E1 osteoblasts exposed
to the following: (A) untreated cells, (B) 50
nM PTH pretreatment for 10 minutes, (C)
cytochalasin D (1 ␮M) pretreatment for 30
minutes, and (D) phalloidin (1 ␮M) pretreatment for 30 minutes followed by 50 nM PTH
for 10 minutes. All drugs were maintained in
the medium during shear. After mounting on
the flow chamber, MC3T3-E1 cells were subjected to a preflow of 1 dyne/cm2 for 3 minutes to establish a baseline before the increase to 12 dynes/cm2 shear. PTH produced
a significant increase in peak [Ca2+]i compared with untreated sheared controls. Addition of cytochalasin D also significantly increased [Ca2+]i; however, this increase was
not different from PTH pretreatment because the baseline Ca2+ levels were elevated.
Addition of phalloidin before PTH pretreatment completely blocked the enhanced
[Ca2+]i response to shear.
␮M) before application of shear. Figure 2 shows that nocodazole did not significantly alter either the mean peak
[Ca2+]i response to shear alone (166 ± 15%) or PTH + shear
(228 ± 25%). Nocodazole also did not change the percentage of the number of cells responding to shear alone or
PTH + shear. These data suggest that disruption of the
microtubule structure has little effect on the Ca2+ signaling
response to shear or shear + PTH.
Role of ion channels in the [Ca2+]i response to
shear, PTH, and cytoskeletal reorganization
We have previously shown that inhibition of the MSCC
with Gd3+ significantly reduced the [Ca2+]i response and
the number of cells responding to shear and shear + PTH,
but that inhibition of the L-VSCC with nifedipine was only
able to significantly block the [Ca2+]i response in sheared
cells pretreated with PTH.(9) To determine which of these
channels are important to the increase in the [Ca2+]i response to shear during actin cytoskeletal disruption, we
added gadolinium or nifedipine separately to cells pretreated with cyto D. Gadolinium (Gd3+; 10 ␮M) significantly reduced the mean peak [Ca2+]i response to shear and
shear + PTH (Fig. 3A). Gd3+ also significantly reduced the
number of cells responding to shear in cells pretreated with
cyto D (Fig. 3B). However, inhibition of the L-VSCC with
nifedipine only partially blocked the mean peak [Ca2+]i response in the shear + PTH group and failed to significantly
reduce the peak [Ca2+]i response to shear alone or shear +
cyto D. These data suggest that the MSCC is important to
the [Ca2+]i response induced by shear and that PTH is able
to control the activity of this channel by altering the actin
cytoskeleton. These data further show that part of the PTHenhanced response is mediated through the L-VSCC and
that this channel does not seem to be directly controlled by
the actin cytoskeleton.
Effect of PTH and cytoskeletal modifiers on the
actin cytoskeleton
We examined the time-course of the effect of PTH on
cell morphology and actin cytoskeletal organization in
MC3T3-E1 cells (Fig. 4). MC3T3-E1 cells grown on type I
collagen are characterized by a flattened morphology with
a prevalence of actin stress fibers traversing the cell. Within
10 minutes of addition of 50 nM PTH, MC3T3-E1 cells
exhibited a more stellated morphology, and the stress fibers
were dramatically reduced compared with control cells. Cytoskeletal changes were maximal at 30 minutes with heavy
staining for f-actin appearing in the perinuclear region of
the cell. Cyto D (1 ␮M) produced similar changes in
MC3T3-E1 cell morphology and actin organization as PTH
treatment. However, after 30 minutes of cyto D treatment,
f-actin staining was punctuated throughout the cell rather
than focused in the perinuclear region as with PTH treatment. When PTH was added to cells after 30-minute treatment with cyto D, PTH did not further alter the cellular
morphology or actin organization or localization (data not
shown). Addition of phalloidin (1 ␮M), which stabilizes
actin and prevents reorganization, did not change the cell
morphology or actin stress fiber organization. Furthermore,
addition of phalloidin before stimulation of MC3T3-E1
CYTOSKELETAL CONTROL OF CA2+ SIGNALING IN OSTEOBLASTS
FIG. 2. Effects of cytoskeletal disruption and stabilization on the
(A) mean peak [Ca2+]i response and (B) the number of responding cells in MC3T3-E1 osteoblasts exposed to 12 dynes/cm2 fluid
shear alone or with 50 nM PTH pretreatment. PTH pretreatment
significantly increased both the mean peak [Ca2+]i response and
the number of cells responding to shear. Cytochalasin D (cyto D;
1 ␮M) also significantly increased peak [Ca2+]i and the number of
cells responding to shear compared with shear alone. However,
PTH did not enhance the effects of actin disruption by cyto D
above the effects of cyto D and shear. Stabilization of actin with
phalloidin (phall; 1 ␮M) before shear or shear + PTH significantly
reduced both the mean peak [Ca2+]i and number of cells responding, suggesting that disruption of actin is required not only for
PTH enhancement of these parameters but also for the responses
to shear alone. Addition of the microtubule disruption agent, nocodazole (noco; 5 ␮M), did not significantly alter either the mean
peak [Ca2+]i response or the number of cells responding to either
shear alone or shear + PTH. (ap < 0.05 compared with shear
controls; bp < 0.01 compared with shear controls; cp < 0.05 compared with shear + PTH controls).
cells with PTH prevented the PTH-induced morphological
changes in cell shape and reduction of stress fiber within the
cell.
Effects of PTH and the cytoskeleton on
MSCC kinetics
To determine the effects of cytoskeletal reorganization
on MSCC kinetics, we used patch-clamp analyses to deter-
1733
FIG. 3. Effects of MSCC and L-VSCC inhibition on the (A)
mean peak [Ca2+]i response and (B) the number of responding
MC3T3-E1 osteoblasts to shear, shear + PTH, or shear + cyto D.
Addition of the MSCC inhibitor, gadolinium chloride (10 ␮M),
significantly reduced both the mean peak [Ca2+]i response and the
number of cells responding compared with the shear control in
each of the groups. Addition of the L-VSCC specific inhibitor,
nifedipine (5 ␮M), significantly reduced the mean peak [Ca2+]i
response and the number of responding cells in the shear + PTH
group, but failed to inhibit the mean peak [Ca2+]i response in
either the shear alone or the shear + cyto D groups. Nifedipine did
significantly inhibit the number responsive cells in the shear +
cyto D group compared with the shear + cyto D shear control. (ap
< 0.05 compared with shear + cyto D control; bp < 0.05 compared
with shear controls in each group; cp < 0.01 compared with shear
controls in each group).
mine changes in channel activity in UMR-106.01 cells in
response to PTH stimulation and actin cytoskeletal organization. Using the cell-attached patch configuration to record single channel activities, we activated MSCC channels
by application of 15 mmHg suction to the back of the pipette. After determination of basal activity in untreated
cells, UMR-106.01 cells were treated with either PTH (50
nM) for 10 minutes, cyto D (1 ␮M) for 30 minutes, phal-
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ZHANG ET AL.
FIG. 4. Effects of PTH and cytoskeletal
agents on actin filaments in MC3T3-E1 cells
using rhodamine phalloidin to stain f-actin
filaments. Control static MC3T3-E1 cells exhibit a high degree of f-actin organized into
stress fibers. Addition of 50 nM PTH over 30
minutes decreased stress fibers in MC3T3-E1
cells in a time-dependent manner, so that by
30 minutes, few stress fibers remained and
most of the staining was localized to the perinuclear region of the cell. Cytochalasin D
(cyto D) also disrupted the f-actin network;
however, the staining pattern differed from
that of PTH in that actin appeared to collapse to the points of attachment of the cell.
If actin was stabilized with phalloidin for 30
minutes before 30-minute treatment with
PTH, f-actin organization did not appear different from static control cells.
loidin (1 ␮M) for 30 minutes, or phalloidin for 30 minutes
followed by PTH addition for 10 minutes. Representative
traces from each group are shown in Fig. 5A. To determine
channel open probability (NPo), we measured the time the
channel was open over the time that suction was applied.
Thus, if one channel was open the duration of suction application, the NPo value would be 1.0. We observed little, if
any, spontaneous MSCC activity; however, suction increased NPo in control cells to 0.41 ± 0.03. As we have
previously shown,(10) PTH pretreatment increased spontaneous MSCC activity and increased NPo during suction to
1.44 ± 0.08 (p < 0.0001). Disruption of actin cytoskeletal
organization with cyto D also increased NPo to 1.15 ± 0.13
(p < 0.0001; Fig. 5B). When PTH was added to cyto
D–treated cells, NPo was increased above that of either
cyto-D or PTH alone to 1.80 ± 0.22. Stabilization of the
actin cytoskeleton with phalloidin reduced the MSCC NPo
to 0.30 ± 0.07 (p < 0.05 compared with untreated controls)
and completely blocked the increased NPo elicited by PTH
(0.37 ± 0.08; p < 0.05 compared with PTH-treated group).
These data indicate that disruption of the actin cytoskeleton
significantly increases MSCC activity similar to the increase
in activity with PTH treatment. However, the NPo values
from both the PTH alone and the cyto D + PTH groups
were significantly greater than the NPo from the cyto D
alone group (p < 0.01 and p < 0.001, respectively).
DISCUSSION
The mechanical forces placed on the skeleton through
locomotion define the architecture and mass of bone. When
a novel mechanical load is encountered, bone cells have the
ability to perceive these vectorial changes in force and initiate a cascade of cellular events that result in alterations in
bone architecture to adapt to these new loads by resorbing
bone in areas of low strain and forming new bone in regions
of increased strain. Whereas bending of a bone during locomotion undoubtedly produces strains on bone cells, the
movement of fluid within the canaliculi and Haversian canals of bone has also been proposed to be a significant
mechanical signal.(23,24) Estimates of the magnitudes of
fluid shear forces generated in bone as the result of physiologic loads range from 8 to 30 dyn/cm2.(24) Whereas in vivo
and in vitro studies have yet to determine what mechanical
signal plays the dominant role in mechanotransduction,
most mechanical forces activate many of the same second
messenger pathways and signaling molecules that may be
important to the osteogenic response to mechanical loading.(25) However, we have shown that fluid shear, but not
physiologic levels of mechanical stretch, increases the expression of osteopontin, c-fos, cyclooxygenase 2, and TGF␤.(26) Therefore, in this study, the principal mechanical
stimulus used was laminar fluid flow.
Well-defined thresholds for the magnitudes of force re-
CYTOSKELETAL CONTROL OF CA2+ SIGNALING IN OSTEOBLASTS
FIG. 5. (A) Representative single channel traces of MSCC activity in control MC3T3-E1 cells or cells pretreated with 50 nM
PTH, 1 ␮M cytochalasin D, 1 ␮M phalloidin, or 1 ␮M phalloidin
for 30 minutes followed by 10-minute treatment with PTH. MSCC
channels were activated by application of suction to the back of
the pipette. (B) Average open probability (NPo) of MSCC from at
least 12 patched cells from each group. NPo was only determined
during application of suction to the pipette. PTH significantly
increased NPo 3-fold during application of mechanical stimulus
and induced spontaneous activity of the channel. Introduction of
cyto D increased NPo, as well, and produced spontaneous activity
of the MSCC. However, addition of PTH to cyto D–treated cells
did not significantly increase NPo above that of cyto D alone.
Phalloidin did not prevent activation of the MSCC on suction to
the pipette, but did prevent PTH enhancement of channel activity.
(ap < 0.05 vs. control untreated; bp < 0.05 vs. PTH control; cp <
0.01 vs. PTH control).
quired for net bone resorption and formation have been
determined, however the threshold for net bone formation
exceeds the levels of strain that occur under physiologic
conditions.(1,2) We, and others,(1,3,4,9) have postulated that
parathyroid hormone (PTH) can interact with the signaling
mechanisms of mechanotransduction to lower the mechanical threshold and prime the osteogenic cells of bone to
respond to lesser magnitudes of mechanical stimulation to
promote bone formation. The effects of PTH on bone are
paradoxical in that PTH is released in response to low serum Ca2+ and stimulates bone resorption to increase the
serum Ca2+ levels. However, when given in low, intermit-
1735
tent doses, PTH increases bone formation in intact
rats,(27,28) ovariectomized rats,(29) and humans.(30) These
anabolic effects are quite similar to the effects of mechanical stimulation on osteoblasts and bone. PTH has been
shown to induce a number of genomic responses, but perhaps most relevant to the mechanical effects on bone is the
stimulation of prostaglandin synthesis by increasing COX-2
production.(31) Inhibition of COX-2 with NS398, a specific
COX-2 blocker, has been shown to completely abrogate
mechanically induced bone formation in vivo.(32) PTH has
also been shown to reverse the effects of mechanical unloading in hindlimb suspended rats and even produce trabecular bone formation greater than controls.(3) Chow et
al.(4) have also shown that PTH is essential for the mechanical responsiveness of bone to mechanical stimuli. Application of mechanical loading to rat tail vertebrae had no effect
in thyroparathyroidectomized rats, yet a single dose of PTH
re-established mechanical responsiveness. These observations indicate that a synergistic interaction between PTH
and mechanical loading of bone exists to stimulate bone
formation, and we hypothesize that this interaction may be
the mechanism behind the anabolic response of bone to low
intermittent doses of PTH.
PTH and mechanical loading also activate many of the
same second messenger pathways and the two stimuli together produce even greater activation of these pathways.
Addition of PTH to rat dentoalveolar cells before mechanical stimulation significantly elevated inositol trisphosphate,
cyclic AMP, and protein kinase C levels compared with
mechanical stimulation alone.(33) One of the initial responses of osteogenic cells to either mechanical stimulation
or PTH treatment is a rapid rise in [Ca2+]i.(5,6,34) Like other
second messengers, this increase in [Ca2+]i in response to
shear is significantly enhanced when osteoblasts are pretreated with PTH.(9) Because the [Ca2+]i response to either
stimuli has been linked to both changes in gene expression
and release of factors associated with signal amplification,
the mechanisms involved in the increase in intracellular
Ca2+ are likely candidates for determining the mechanical
thresholds for mechanotransduction and that PTH can alter
these thresholds by sensitizing these mechanisms. This increase in [Ca2+]i to either stimulus is dependent on both
extracellular Ca2+ entry and intracellular Ca2+ release.(5,6)
Two ion channels have been shown to be involved in extracellular Ca2+ entry in response to fluid shear: the MSCC
and the L-VSCC.(5,9) Activation of these channels by fluid
shear has been linked to release of a number of autocrine/
paracrine factors,(35–38) suggesting that these channels are
important in amplification of the mechanical stimulus to
augment the number of cells responding to the stimulus.
Both MSCC and L-VSCC exhibit increased activation in
response fluid shear in MC3T3-E1 osteoblasts pretreated
with PTH compared with cells subjected to fluid shear
alone.(9) The mechanisms behind this increased activation
are still unclear. However, ion channels can be phosphorylated or dephosphorylated by a number of second messenger pathways that alter channel activity and kinetics.(39)
PTH activates several protein kinases that are known to
phosphorylate L-VSCC channels, including protein kinase
A (PKA) and protein kinase C (PKC).(40) We showed that
1736
the enhanced increase in [Ca2+]i in response to fluid shear
in PTH pretreated MC3T3-E1 osteoblasts is predominantly
the result of PKA phosphorylation of the L-VSCC.(9) However, this enhanced response required the activation of the
MSCC. This synchronization between the MSCC and LVSCC in response to mechanical loading is similar to the
synergistic interaction of 1,25(OH) 2 -vitamin D 3 and
PTH.(41) We also showed that the kinetics of the MSCC are
also regulated by PKA. Addition of PTH before mechanical stimulation increased both the open probability and the
conductance of the MSCC in UMR106.01 cells.(10) Furthermore, pretreatment of UMR cells with 8br cAMP increased
the conductance of the channel but did not alter the open
probability, suggesting that the increase in open probability
was controlled through another mechanism.
Although phosphorylation from other kinases, such as
tyrosine kinase, could be involved in this increase in open
probability of the MSCC, the nature of the gating mechanism of this channel suggests a structural control by the cell.
Whereas deformation of the lipid bilayer has been implicated in the activation of mechanically gated channels, such
as the MscL,(42) the cytoskeleton of the cell has been linked
to regulation of several types of ion channels in different
tissues, including the epithelial Na+ channel (ENaC),(43)
L-VSCC,(44) voltage and ATP gated K+ channels,(45,46) and
stretch-activated cation channels.(47) Prat et al.(43) found
that disruption of actin with cyto D increased ENaC channel activity. Furthermore, if monomeric g-actin was added
to excised patches at a concentration that promoted shortchained actin filaments, channel activity was also increased.
Cyto D collapses the actin cytoskeleton without significantly altering the g-actin/f-actin ratio,(48) suggesting that,
like the ENaC channel, the MSCC becomes more responsive when actin is disrupted into shorter filaments.
PTH has been shown to significantly alter osteoblast
morphology(16) and disrupt the actin cytoskeleton.(18) As
we show here, the disruption of actin filaments by PTH is
rapid and correlates with the increase in MSCC activity.
How PTH alters actin integrity is still unclear; however,
both PKA and PKC are activated by PTH and have been
implicated in the control of actin organization.(49,50)
Whereas PKA has been associated with loss of actin association with integrins,(49) we find that activation of PKA
only increases single channel conduction of the MSCCs and
does not increase open probability. PKC has been shown to
target several proteins associated with the cytoskeleton, including integrin- and actin-associated proteins that can cap
or sever actin filaments.(50) Thus, PTH activation of PKC
may be responsible for the increase in MSCC activation and
is the subject of ongoing studies.
In summary, we found that loss of actin filament integrity
mimics the effects of PTH pretreatment on the intracellular
Ca2+ response to fluid shear in MC3T3-E1 osteoblasts.
Whereas we have previously shown that much of this enhanced response can be blocked when the L-VSCC is inhibited, we believe that activation of the L-VSCC is dependent on the depolarization event initiated by the MSCC.(10)
Thus, increased MSCC activity will increase L-VSCC activation that, in turn, leads to an increased intracellular Ca2+
response. We have previously shown that PTH pretreat-
ZHANG ET AL.
ment will increase both MSCC single channel conductance
and open probability and that the increase in single channel
conductance resulted from PTH-induced activation of
PKA. In this study, we showed that the increase in MSCC
open probability correlates with actin filament disruption
and that stabilization of the actin cytoskeleton before PTH
stimulation prevents this increase. Whereas we can not conclude a direct linkage of the cytoskeleton to the MSCC,
these data indicate that PTH can prime osteoblasts to respond to mechanical stimulation through alteration in cellular structural integrity.
ACKNOWLEDGMENTS
This work was supported by NIH Grants NIDDK
DK058246 and NIAMS AR043222 (RLD) and NASA Predoctoral Fellowship Grant NGT5-5023 (KDR).
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Address reprint requests to:
Randall L Duncan, PhD
Departments of Biological Sciences and Mechanical
Engineering
University of Delaware
319 Wolf Hall
Newark, DE 19716, USA
E-mail: rlduncan@udel.edu
Received in original form May 28, 2006; revised form July 10, 2006;
accepted July 26, 2006.