1 New York Center for Biomedical Engineering, City College of the City University of New York, New York, 10031; and 2 Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461
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ABSTRACT |
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We tested the hypothesis that fluid
shear stress () modifies the expression, function, and distribution
of junctional proteins [connexin (Cx)43, Cx45, and zona occludens
(ZO)-1] in cultured bone cells. Cell lines with osteoblastic (MC3T3-E1
cells) and osteocytic (MLO-Y4 cells) phenotypes were exposed to
-values of 5 or 20 dyn/cm2 for 1-3 h.
Immunostaining indicated that at 5 dyn/cm2, the
distribution of Cx43, Cx45, and ZO-1 was moderately disrupted at cell
membranes; at 20 dyn/cm2, disruption was more severe.
Intercellular coupling was significantly decreased at both shear stress
levels. Western blots showed the downregulation of membrane-bound Cx43
and ZO-1 and the upregulation of cytosolic Cx43 and Cx45 at different
levels of shear stress. Similarly, Northern blots revealed that
expression of Cx43, Cx45, and ZO-1 was selectively up- and
downregulated in response to different shear stress levels. These
results indicate that in cultured bone cells, fluid shear stress
disrupts junctional communication, rearranges junctional proteins, and
determines de novo synthesis of specific connexins to an extent that
depends on the magnitude of the shear stress. Such disconnection from
the bone cell network may provide part of the signal whereby the
disconnected cells or the remaining network initiate focal bone remodeling.
gap junctions; connexin; zona occludens-1; mechanotransduction; bone remodeling
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INTRODUCTION |
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BONE DRAMATICALLY
CHANGES its structure and mass in response to static and dynamic
loads. It has been well established that bending loads in bone cause
small deformations in the bone matrix, which in turn generate fluid
pressure differences that lead to fluid flow from the compression to
the tension side. The resulting fluid shear stress () is proposed to
be one of the factors by which the bone cell network senses mechanical
loading (7, 51, 54, 61). At physiological whole-tissue
strain levels (<0.2%), culture studies have demonstrated that fluid
flow is a more potent stimulator of bone cells than is substrate
deformation itself (38, 59). Previous experimental studies
have shown that bone cells release signaling molecules such as
prostaglandins, nitric oxide (NO), Ca2+, and other second
messengers in response to the fluid shear stress (3, 18, 20, 24,
41).
One mechanism of bone remodeling by which second messenger signals spread throughout the bone cell network involves gap junction channels that connect osteoblasts and bone-lining cells along the surfaces of Haversian and Volkmann canals and osteocytes that are embedded within the bone matrix (15, 22, 62). The connexin proteins that form gap junctions are encoded by a gene family with as many as 21 members in mammals (56). Connexins are expressed with an overlapping pattern of tissue specificity; connexin43 (Cx43) and connexin45 (Cx45) are the gap junction proteins that have been associated with bone cells (5, 33, 34, 48, 57).
Gap junction channels, including those formed by Cx43 and Cx45, are permeable to signaling ions and second messenger molecules (46). Gap junctional communication of such signals between bone cells gives rise to modulation of hormonal responses in the osteoblastic network (53), regulation of gene expression (29), and propagation of intracellular signals (12). Moreover, connexin expression and function are essential for normal osteogenesis and bone mineralization (30, 31, 34).
Connexins may also play roles in addition to the formation of pathways
for intercellular communication. Recent studies have shown that
connexins directly interact with both adherens and tight
junction-associated proteins including zona occludens-1 (ZO-1),
claudins, and -catenin (1, 16, 26, 37, 50). Accordingly, it has been proposed that connexins are part of a multimolecular signaling complex, the "Nexus" (47).
From the standpoint of the studies described here, ZO-1 has been shown to interact with gap junction proteins Cx43 and Cx45 in osteoblastic cells (28) where ZO-1 binding to connexins may play a role
in the organization, trafficking, and/or stabilization of gap junction proteins (28, 49).
Regulation of gap junctional communication in response to load-induced biophysical signals has been examined in both vascular endothelial (6, 10) and bone cells (4, 13, 62, 63). Although such studies have generally reported an increase in Cx43 expression, changes in functional coupling in the bone cell network have not been as consistently demonstrated.
In this study, we tested the hypothesis that fluid shear stress of the magnitude that is expected to occur in bone tissue modifies the expression, distribution, and function of connexins (Cx43 and Cx45) and an associated protein (ZO-1). Well-characterized cell lines, MC3T3-E1 osteoblastic and MLO-Y4 osteocytic cells, were used in our experiments. Our results demonstrate that in both osteoblastic and osteocytic cell lines, fluid shear stress disrupts cell-to-cell junctional communication, rearranges gap junction proteins, and determines de novo synthesis of specific connexins to an extent that depends on the magnitude of the shear stress. Such disconnection from the bone cell network due to fluid shear stress may provide part of the signal whereby the disconnected cells or the remaining network initiates focal bone remodeling.
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MATERIALS AND METHODS |
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Cell culture.
Osteoblastlike MC3T3-E1 cells (obtained from Dr. Kenneth J. McLeod,
SUNY, Stony Brook) were cultured in -MEM (GIBCO-BRL, Grand Island,
NY) that contained 1% penicillin-streptomycin (GIBCO-BRL) and 5%
fetal bovine serum (FBS, Gemini Bio-Products, Woodland, CA), and
osteocyte-like MLO-Y4 cells (obtained from Dr. Lynda F. Bonewald, Univ.
TX Health Science Center) were cultured in
-MEM that contained 1%
penicillin-streptomycin, 10% FBS, and 2.5% calf serum (GIBCO-BRL) at
37°C with 95% O2-5% CO2. For each cell
type, confluent monolayers of cells were grown on glass slides in
static conditions and transferred to the flow apparatus to expose the
monolayers to a fluid
-value of 5 or 20 dyn/cm2 for 1, 2, or 3 h. These durations were chosen based on the turnover rates
of Cx43 and Cx45, which exhibit half-lives of 1.5 and 3 h,
respectively (9).
Flow chamber and experiment.
The fluid-flow setup consisted of a parallel-plate flow chamber
(Cytodyne, La Jolla, CA) and a recirculating flow circuit. This circuit
included a variable-speed peristaltic pump (Taitec, Saitama, Japan),
pulse dampener (Cole-Palmer Instruments, Vernon Hills, IL), and a
reservoir with culture medium (-MEM with 1% FBS) maintained at
37°C with 95% O2-5% CO2. This system
produces laminar flow over a cell monolayer. A flow rate was
chosen to yield a
-value of 5 or 20 dyn/cm2 using
the equation
= 6
µ/bh2, where
is flow rate, µ is medium viscosity, and b and
h are channel width and height, respectively. Control cells
were kept under static conditions with the same culture medium at
37°C.
Alkaline phosphatase staining. To determine whether cells retained the differentiated phenotypes, both cell lines were routinely checked for alkaline phosphatase activity. Cells were fixed with 4% formaldehyde and permeabilized with 70% ethyl alcohol (EtOH). Cells were then rinsed with 0.2 M Tris(hydroxymethyl)-aminomethane (Sigma, St. Louis, MO) and incubated in naphthol AS-BI phosphate (Sigma) with fast red violet LB salt (Sigma) plus Tris · HCl for 30 min (2). After several washes with distilled water, cells were counterstained with Mayer's hematoxylin (Sigma) for 2 min. The cells were then washed with distilled water and mounted for analysis.
Cell viability studies. Cells from both control and shear stress-exposed samples were analyzed for health and viability using the Live/Dead Viability/Cytotoxity kit (Molecular Probes, Eugene, OR). Cells were rinsed a few times with 1× PBS and incubated with 4 µM eithidium homodimer-1 (EthD-1) and 2 µM of calcein-acetoxymethyl ester in 1× PBS for 30 min as recommended by the manufacturer. Live cells, which retained the polyanionic dye calcein, gave rise to green fluorescence; dead cells, which took up EthD-1 through membrane damage, produced red fluorescence. Live and dead cells were counted from 10 cell fields for all of the samples, and percentages of live and dead cells were calculated.
Immunofluorescence studies. Both control and shear stress-exposed cells were fixed with 2% formaldehyde, permeabilized with 0.4% Triton X-100 (Sigma), and blocked with 10% goat serum (GIBCO-BRL) in 1× PBS. The cells were then incubated with primary polyclonal antibodies against Cx43, Cx45 (courtesy of Dr. E. Hertzberg, AECOM and Dr. T. Steinberg, Washington Univ. School of Medicine), ZO-1 (Zymed, South San Francisco, CA), and secondary antibody conjugated to Alexa 488 (Molecular Probes). For F-actin staining, cells were incubated with rhodamine-labeled phalloidin (Sigma) immediately after fixation. The coverslips were mounted on slides, examined on a Nikon Eclipse TE300 microscope, and photographed using a SPOT-RT digital camera (Diagnostic Instruments, Sterling Heights, MI).
Western blot analysis.
Controls and shear stress-exposed (1 and 3 h) samples were lysed
in 80 µl of lysis buffer [10 mM Tris · HCl, pH
7.5, and 2 mM phenylmethylsulfonyl fluoride (PMSF)], sonicated, and
centrifuged (14,000 rpm for 30 min) as described by Guan et al.
(17). Pellets and supernatants from the samples were
collected for crude membrane and cytosolic protein analyses. Samples
were loaded onto 10% SDS-PAGE gels (Bio-Rad Laboratories, Hercules,
CA) for separation and were electrophoretically transferred to
nitrocellulose membranes (Schleicher and Schuell, Keene, NH). The
membranes were probed with primary polyclonal and monoclonal antibodies
to Cx43, Cx45, and ZO-1, polyclonal -actin (Sigma), and monoclonal
GAPDH (Research Diagnostics, Flanders, NJ), followed by secondary
antibody incubation with horseradish peroxidase (HP)-conjugated
anti-rabbit and anti-mouse IgGs (Santa Cruz Biotech, Santa Cruz, CA).
The protein bands were detected using the Amersham ECL detection kit
(Amersham Biosciences, Piscataway, NJ) and were exposed on Fuji X-ray
film. The intensity of the bands was analyzed using Scion NIH Image
software (Scion, Frederick, MD). Measured intensities for all
experiments were first normalized with respect to internal controls
(GAPDH for cytosolic proteins and
-actin for membrane-bound
proteins) and then with respect to controls.
Northern blot analysis. Total RNA from the samples was extracted using TRIzol reagent (GIBCO-BRL) and was quantified as previously described (52). Total RNA (10 µg) from the samples was separated on 1.2% formaldehyde-agarose gel and was transferred onto a Gene Screen hybridization transfer membrane (NEN Life Science Products, Boston, MA). Membranes were hybridized with appropriate denatured random-primed probes. The rat cDNA probes used were full-length Cx43 and Cx45 (obtained from Dr. Eric Beyer, Univ. of Chicago Medical School) and 18S labeled with [32P]dCTP using the Megaprime labeling system (Amersham Biosciences). The membranes were then exposed to the phosphor screen overnight, scanned on a Storm PhosphorImager system, and quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). All acquired data were first normalized with respect to 18S RNA band intensity, and then all experimental data were normalized with respect to control data.
RT-PCR and semiquantitative RT-PCR analyses. RT-PCR was performed as previously described (52). For semiquantitative PCR, a 9:1 ratio of competimers and 18S primers (Ambion, Austin, TX) was added to the PCR mixture. Conditions applied for PCR using a PTC-100 programmable thermal controller (MJ Research, Watertown, MA) were 94°C for 30 s, 55°C for 30 s, 72°C for 30 s for 30 cycles, and 72°C for 8 min. Reaction products were analyzed by electrophoresis on 2% agarose gels and were quantified using Kodak 1D Scientific Imaging Systems. The following primers were used for each cDNA amplification: mCx43, TACCACGCCACCACTGGC (sense), AATCTCCAGGTCATC AGG (antisense); mCx45, AAAGAGGAGAGCCAACCAAA (sense), GTCCCAAACCCTAAGTG AAGC (antisense) (11); mZO-1, CATAGAATAGACTCCCCTGG (sense), GCTTGAGACCTCAT ACCTGT (antisense) (21); and mosteocalcin, gacaaagccttcatgtccaagc (sense), TTTGAG ACCGTCGAGCCGAAA (antisense) (23).
Scrape loading. Quantitative scrape loading as described by Pina-Benabou et al. (40) was used to analyze gap junctional communication after cells were exposed to shear stress. Incisions on the cell monolayers of control and shear stress-exposed samples were made with a fine razor blade in two or three different regions on the slides. The slides were incubated with 0.5% Lucifer yellow (Sigma) at 37°C, rinsed with 1× PBS, and fixed with 2% formaldehyde. The intensity of the dye spread from the damaged cells to the neighboring cells was observed using a Nikon Eclipse TE300 microscope and was photographed with a SPOT-RT digital camera. Extent of dye spread was quantified as linear distance perpendicular to the scrape using Scion NIH Image software.
Statistical analysis. Data were analyzed using one-way ANOVA (SigmaStat, Chicago, IL). Northern blot analyses are presented as means ± SE of six experiments. Western blot, quantitative RT-PCR, and scrape-loading analyses are presented as means ± SE of three experiments. A significant difference compared with controls is indicated as * P < 0.05.
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RESULTS |
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Osteoblast and osteocyte cell lines express gap junction proteins
associated with osteogenesis and tight junction-associated protein
ZO-1.
We used the osteoblastic cell line MC3T3-E1 (57) and the
osteocytic cell line MLO-Y4 (23) to compare expression,
distribution, and function of gap junctions associated with
osteogenesis (34). As shown in Fig.
1A in which the cell
cytoskeleton was stained with phalloidin, these cell lines show quite
different morphologies. Whereas the osteoblast cells exhibit flattened,
epitheliod shapes and a close packing arrangement, the osteocyte cells
exhibit a more stellate shape with rounded somata and multiple
processes extending for variable distances to neighboring cells.
Because phenotypic expression of differentiated cell markers may vary in cell lines maintained under different conditions and after prolonged
passage, we determined expression levels of alkaline phosphatase and
osteocalcin in the MC3T3-E1 and MLO-Y4 cell lines under conditions used
in our studies. Alkaline phosphatase activity was higher in the
osteoblasts than in the osteocytes (Fig. 1B), whereas the
osteocytic cell line but not the osteoblastic cell line expressed
measurable osteocalcin mRNA (Fig. 1C). RT-PCR results showed
that under normal conditions, both types of immortalized cell lines
expressed mRNAs that encode Cx43, Cx45, and ZO-1 (Fig. 1C).
These data indicate that mRNAs corresponding to the osteogenic gap
junction proteins and the tight junction protein ZO-1 are expressed in
both cell lines.
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Fluid shear stress does not affect cell viability but disrupts
cell-to-cell communication and rearranges gap junction proteins Cx43
and Cx45 and associated ZO-1 protein in cultured bone cells.
The overall experimental design critically depends on maintenance of
cell viability during periods of shear stress exposure. To determine
the extent to which cells were injured by the procedure, we performed
the Live/Dead assay on both cell types with no shear stress and with 5 and 20 dyn/cm2 of shear stress for 3 h. Both MC3T3-E1
and MLO-Y4 cells were viable after 3 h of exposure to fluid shear
stress. As shown, applying this assay to osteoblastic (Fig.
2A) and osteocytic (Fig. 2B) cells revealed that the percentages of live cells in
normal and shear stress-exposed cells were all >80%. ANOVA analysis
revealed that the percentage of dead cells was lower than live cells
for controls and for each treatment, but the percentage of dead cells did not significantly differ among the groups.
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Intercellular coupling in cultured bone cells is significantly
decreased by fluid shear stress.
Previous studies (4, 57) have shown that MC3T3-E1 and
MLO-Y4 cells are coupled by functional gap junction channels. In this
study, we used the scrape-loading technique to quantitatively examine
the effects of fluid shear stress on cell-to-cell coupling. Our
scrape-loading data indicated that intercellular coupling significantly
decreased in MC3T3-E1 cells; this change was more pronounced with
higher shear stress and longer duration. As shown in Fig.
5A, the extent of dye spread
in control MC3T3-E1 cells was 270.4 ± 10.7 µm. This
dye-transfer distance was reduced to 237.4 ± 4.7 or 120.8 ± 5.0 µm when cells were exposed to the lower -value of 5 dyn/cm2 for 1 or 3 h, respectively. At the high
-value of 20 dyn/cm2, the degree of dye transfer was
dramatically reduced to 168.4 ± 4.7 µm for the shorter duration
and 106 ± 6.3 µm for the longer duration (Fig. 5, bar graphs).
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Fluid shear stress downregulates ZO-1 and phosphorylated Cx43 in
cultured bone cells.
We performed Western blot analyses for crude membrane and cytosolic
proteins to determine the extent to which fluid shear stress regulates
levels of Cx43, Cx45, and ZO-1 within each cell type. For Cx43,
multiple bands were detected in Western blots corresponding to
different extents and/or types of phosphorylation (35,
36), where NP is the dephosphorylated form and P1 and P2
designate phosphorylated Cx43 species. For both cell lines at all shear
stress levels, there was a similar pattern in which the P2 form of Cx43
from the membrane was significantly decreased in the membrane fraction,
whereas all three forms of Cx43 were dramatically increased in the
cytosolic fraction. For osteoblastic MC3T3-E1 cells, densitometric
analysis of membrane-bound Cx43 bands showed significant downregulation
of the P2 form of Cx43 at 3 h for both 5 and 20 dyn/cm2 of shear stress. By contrast, NP, P1, and P2 forms
of cytosolic Cx43 increased after exposure to both levels of shear
stress (Fig. 6A). Similarly,
densitometric analysis of membrane-bound Cx43 for MLO-Y4 cells revealed
downregulation of the P2 form at both 5 and 20 dyn/cm2 of
shear stress, whereas cytosolic Cx43 noticeably increased in response
to both levels of shear stress, especially to 5 dyn/cm2
(Fig. 6B). In both MC3T3-E1 and MLO-Y4 cells, Cx45 was
detectable only in the cytosolic fraction, and low levels of shear
stress seemed to downregulate Cx45, whereas high levels of shear stress appeared to upregulate Cx45 at both exposure times (Fig.
7A). Western blots using
ZO-1-specific antibodies revealed that for both shear stress levels,
this membrane-bound protein was downregulated at longer exposure times
in both MC3T3-E1 and MLO-Y4 cells (Fig. 7B). These data
indicate that fluid shear stress regulates the level of Cx45 and ZO-1
expression and abundance of all three forms of Cx43. Notably, the P2
form of the membrane-bound Cx43 decreased as the duration of the shear
stress increased. Such a correlation of decreased P2 with decreased dye
coupling is consistent with previous reports on other cell types (see
DISCUSSION).
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Selective regulation of Cx43, Cx45, and ZO-1 expression in response
to different shear stress levels.
Previous studies on endothelial cells have shown that fluid flow
and mechanical stretch alter the expression of Cx43 (6, 10). Therefore, in this study, we analyzed the consequence of fluid shear stress on the expression of Cx43, Cx45, and ZO-1 mRNAs in
osteoblastic and osteocytic cell lines using Northern blot analysis and
semiquantitative RT-PCR. In both cell lines, different levels of shear
stress seemed to regulate expression of Cx43 and Cx45 in a reciprocal
or compensatory manner. As shown in Fig. 8A, Cx43 mRNA levels increased
1.5-2-fold for MC3T3-E1 cells and 1.25- to 1.5-fold for MLO-Y4
cells at 5 dyn/cm2 of shear stress for 1, 2, or 3 h,
yet no apparent changes were found from the controls at 20 dyn/cm2 of shear stress for any of the durations for either
cell types. By contrast, Cx45 mRNA remained the same as the controls at
the lower shear stress level for all durations and increased to 1.25- to 1.75-fold at 20 dyn/cm2 of shear stress for 1, 2, or
3 h (Fig. 8B). With regard to ZO-1 mRNA, there were no
detectable changes with respect to control in MC3T3-E1 cells at the
lower shear stress level, whereas the mRNA level was decreased to half
that of controls at the higher shear stress level (Fig.
9A). However, more dramatic
effects on ZO-1 expression were seen in MLO-Y4 cells, where ZO-1 mRNA
was profoundly decreased at both 5 and 20 dyn/cm2 of shear
stress for 1-, 2-, or 3-h exposure times (Fig. 9B). These
data suggest that for both cell lines, lower shear stress (5 dyn/cm2) upregulates the expression of Cx43, whereas higher
shear stress (20 dyn/cm2) upregulates the expression of
Cx45. By contrast, ZO-1 is downregulated at high shear stress levels in
the osteoblastic cell line and even more strikingly at both shear
stresses in the osteocytic cell line.
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DISCUSSION |
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In this study, we found that steady fluid shear stress modifies expression, function, and distribution of connexins (Cx43 and Cx45) and an associated tight junction protein (ZO-1) in cultured bone cells. Our results strongly suggest that fluid shear stress disrupts cell-to-cell communication and rearranges the gap junction proteins Cx43 and Cx45 and an associated protein ZO-1 in both MC3T3-E1 and MLO-Y4 cells. This disrupted gap junctional communication and rearrangement of Cx43, Cx45, and ZO-1 depended on the magnitude of the shear stress as well as the exposure duration. Our results as well as others (4, 14, 23, 57, 58) indicate that the major gap junction protein that mediates cell-to-cell communication in both cell types is Cx43. Our finding that the selective reduction in the P2 form of membrane-bound Cx43 correlates with the loss of dye coupling is consistent with previous reports, which indicate that Cx43 phosphorylation is important for its function (35, 36). Elevated levels of all three forms (NP, P1, and P2) of cytosolic Cx43 after exposure to fluid shear stress suggest that newly synthesized as well as internalized Cx43 contributed to the increases in cytosolic Cx43 level. We believe that this significant increase in cytosolic Cx43 level was mainly due to the internalization of membrane-bound Cx43, because newly synthesized Cx43 should mostly be in NP form. Furthermore, based on the cytosolic Cx43 analysis, low shear stress seemed to be regulating Cx43 in both cell types, whereas high shear stress appeared to be upregulating cytosolic Cx45. At the level of mRNA, Cx43, Cx45, and ZO-1 all showed interesting changes with fluid-induced shear stress. Expression of Cx43 and Cx45 mRNA was selectively upregulated in response to different shear stress levels, whereas both magnitudes of shear stress inhibited ZO-1 expression. Together, these results show for the first time that in cultured bone cells, fluid shear stress disrupts cell-to-cell junctional communication, rearranges junctional proteins, and determines de novo synthesis of specific connexins in a manner that depends on the magnitude of the shear stress.
There is increasing evidence that fluid shear stress regulates Cx43 in vascular smooth muscle (6), vascular endothelial (10), and cultured bone cells (4). Our observation of the disruption and translocation or internalization of Cx43 from the membrane after exposure to laminar flow in both MC3T3-E1 and MLO-Y4 cells is in agreement with the findings of DePaola and coworkers (10) for endothelial cells in the laminar flow region at 5 h of exposure time. We speculate that fluid shear stress of short duration (1 or 3 h) reduces intercellular communication as a consequence of morphological changes (4, 39), loss and/or internalization of membrane-bound Cx43 (10, 27, 35, 36), and possibly also inhibited trafficking of newly synthesized Cx43 to the membrane. Our finding that plasmalemmal ZO-1 immunostaining decreased in a somewhat similar manner to Cx43 indicates that ZO-1 and Cx43 might interact in MC3T3-E1 and MLO-Y4 cells as has been reported for other cell types (16, 19, 28, 50). Because interaction with ZO-1 has been suggested to stabilize Cx43 at the cell surface (49), the downregulation of ZO-1 by shear stress might lead to redistribution of Cx43 to intracellular compartments.
Functional studies on junctional communication have indicated that during shear stress exposure, dye coupling is reduced as gap junction protein expression decreases on the appositional membranes. Our observation of impaired dye coupling during the early period of laminar shear stress in cultured bone cells was similar to that reported for endothelial cells (10). Hence, these results verify our conclusion that fluid shear stress disrupts cell-to-cell communication. Biochemical evidence for the redistribution of Cx43 due to fluid shear stress is provided by the observed decrease in Cx43 P2 from the membrane, which is believed to be the predominant form of this gap junction protein that forms functional gap junction channels (35, 36) and an increase in all three forms of Cx43 in the cytosol.
Our findings on the distribution of Cx43, intercellular coupling, and the phosphorylation level of Cx43 after the steady shear stress exposure in MLO-Y4 cells differ from a recent report (4), which states that migration of Cx43 from the perinuclear region toward the dendritic processes and intercellular coupling increased after MLO-Y4 cells were subjected to fluid flow. Although differences might have arisen from usage of different antibodies, the analytical methods applied, or the composition of culture media, the previous study noted a lack of correlation between Cx43 distribution and the enhanced intercellular coupling, which suggests that another connexin might upregulate cell-to-cell communication after shear stress exposure. We analyzed both Cx43 and Cx45 in our experiments, and our data suggest that although Cx43 is the major gap junction protein that regulates junctional communication in MC3T3-E1 and MLO-Y4 cells, Cx45 can be upregulated under high shear stress conditions. The previous study also analyzed various osteoblastic cells (2T3, ROS17/2.8, MC3T3-E1), reported no effect of fluid flow on cell-to-cell communication, and concluded that osteoblasts are less responsive than osteocytes to such stimuli (see also Ref. 25). However, numerous previous studies on osteoblastic cells have clearly demonstrated that stress enhances the production of second messengers such as cAMP, NO, Ca2+, and prostaglandin (18, 20, 24, 41, 45) and alters cell morphology including the reorganization of the actin cytoskeleton (39). Our data indicate that osteoblastic MC3T3-E1 cells do respond to fluid shear stress.
We have observed differential mRNA expression of Cx43, Cx45, and ZO-1 in response to different shear stress levels. Low shear stress upregulated Cx43 expression in both cell types, whereas high shear stress upregulated Cx45 expression in both cell types. Previous studies have shown that when Cx43 function is inhibited in avian osteogenic tissue, Cx45 is upregulated to fulfill at least some aspect of the missing functions (33). Such selective upregulation of Cx43 and Cx45 by different levels of shear stress provides evidence that patterns of gene expression are transduced by the mechanical stimulus that would be expected to qualitatively alter the junctional phenotype and may correspond to either differentiation or dedifferentiation of bone cells (13, 31, 33, 34, 42, 44).
Although the mechanochemical transduction cascade leading from shear stress to altered gene expression patterns remains to be fully elucidated, fluid shear stress has been shown to enhance second-messenger production in cultured bone cells. In bone cells, cAMP is regulated by prostaglandin, and both are increased under fluid-flow conditions (41); cAMP has been shown to upregulate both the mRNA and protein of Cx43 and Cx45 in cultured cardiac myocytes (8). Therefore, a possible mechanism by which cultured bone cells might respond to fluid flow would involve disruption of cell-to-cell communication and enhanced production of prostaglandins. This would elevate cAMP, which in turn would upregulate expression and phosphorylation of either Cx43 or Cx45 depending on the magnitude of the shear stress and the duration of the exposure period. Subsequently, cellular differentiation would be initiated, and disconnected cells or the remaining network would begin focal bone remodeling.
The decrease in ZO-1 mRNA and protein expression with increased levels of shear stress that we have observed may also play an important role in bone remodeling. Truncation mutants of the tight junction protein ZO-1 have been shown to disrupt epithelial cell morphology, which suggests that ZO-1 may be involved in the regulation of cellular differentiation (43). Moreover, downregulation of ZO-1 and occludin appear to be related to phenotypic changes associated with epithelial cell transformation (32). Therefore, it seems likely that ZO-1 may also be involved in mediating cell differentiation in cultured bone cells.
The results described here were obtained under in vitro conditions in tissue culture. In vivo, there is increasing evidence that osteocyte cell processes are surrounded by the pericellular matrix with transverse tethering filaments (55), whereas in culture, there is no encircling support structure. In addition, the recent theoretical model by You et al. (60) brings to our attention that in vivo, the fluid shear stresses on the cell body are much smaller than those on the membrane in cell processes, so that the fluid drag force on the transverse filaments in the pericellular matrix is much greater than the fluid shear force on the cell-process membrane. Therefore, it is possible that under in vitro conditions the actin cytoskeleton in the cell body responds to the fluid shear stress rather than the more rigid actin bundle in the cell process. However, You et al. (60) predicted that the fluid drag on the tethering filaments can produce a 20- to 100-fold amplification in the strain on the actin filament bundle in the cell process. This amplification is sufficient to elicit intracellular signaling responses in all cell cultures with deformed substrates.
In summary, we have shown that fluid-induced shear stress is an important biophysical signal in bone mechanotransduction. Our observations suggest that in both osteoblastic and osteocytic cell lines, fluid shear stress of the magnitude expected to occur in bone tissue disrupts junctional communication, rearranges junctional proteins, and determines de novo synthesis of specific connexins to an extent that depends on the magnitude of the shear stress. Such disconnection from the bone cell network due to the fluid shear stress may provide part of the signal whereby the disconnected cells or the remaining network initiates focal bone remodeling.
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ACKNOWLEDGEMENTS |
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We thank Marcia Urban for technical assistance, Dr. Wei Li for advice on separating membrane and cytosolic Cx43, Dr. Karen Cusato for advice on use of the Live/Dead cell assay, Dr. Elliot L. Hertzberg (Albert Einstein College of Medicine) and Dr. Thomas H. Steinberg (Washington University School of Medicine) for generous supply of Cx43 and Cx45 antibodies, and Dr. Kenneth J. McLeod (State University of New York, Stony Brook) and Dr. Lynda F. Bonewald (University of Texas Health Science Center) for graciously providing the MC3T3-E1 and MLO-Y4 cell lines.
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FOOTNOTES |
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This work was supported primarily by National Institutes of Health (NIH) Grant HL-19454 (principal investigator, S. Weinbaum) and a Gillecce Fellowship (City University of New York Graduate School) with additional support provided by NIH Grants DK-41918 and NS-34931 (principal investigator, D. C. Spray).
Address for reprint requests and other correspondence: D. C. Spray, Albert Einstein College of Medicine, Dept. of Neuroscience, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: spray{at}aecom.yu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 3, 2002;10.1152/ajpcell.00052.2002
Received 1 February 2002; accepted in final form 30 September 2002.
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