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Department of Biochemistry (B.C., L.F.B., J.X.J.), Medicine (S.Z., L.F.B.) and Radiology (J.L., E.S.), University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900; and Asahi Chemical Company (Y.K.), Ohito, Japan
Address all correspondence and requests for reprints to: Jean X. Jiang, Ph.D., Department of Biochemistry, MSC 7760, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900. E-mail: jiangj{at}uthscsa.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The application of force to bone causes several potential stimuli for osteocytes, including hydrostatic pressure and fluid flow-induced shear stress. Over the past several years, various theoretical and experimental studies suggest that flow of interstitial fluid is likely the mechanical stress-related stimuli affecting bone cells (2, 6, 7). Fluid flow is more effective than hydrostatic pressure in eliciting a response (4). Mechanical forces applied to bone cause fluid to flow through the canaliculi surrounding the osteocyte, resulting in the deformation of the cell membrane (5, 7, 8). Thus, this type of deformation is likely to stimulate biochemical responses, and the signal molecules generated are likely to be transmitted through gap junction channels connecting all osteocytes through their extensive network of dendritic processes (9, 10).
Gap junctions are transmembrane channels, which connect the cytoplasm of two adjacent cells. These channels permit molecules with molecular mass less than 1 kDa, such as small metabolites, ions, and intracellular signaling molecules (i.e. calcium, cAMP, inositol triphosphate), to pass through (11). These channels have been demonstrated to be important in modulating cell signaling and tissue function in many organs and cells (12, 13, 14, 15, 16, 17, 18). Gap junction channels are formed by members of a family of homologous and structurally related proteins known as connexins (19, 20).
Although morphological observations suggest the existence of gap junctions in osteocytes, there is no published evidence supporting functional intercellular communication in vivo. Because osteocytes are deeply embedded in the mineralized bone matrix, they are not readily accessible for many experimental approaches. Thus, osteocyte cell cultures are required to perform such experiments. An osteocyte-like cell line named MLO-Y4 has been established and characterized, and it seems to have characteristics of primary osteocytes (21). MLO-Y4 cells have extensive dendritic processes, a morphological feature of osteocytes in bone, and express the osteocyte phenotype, as demonstrated by their increased osteocalcin production, low levels of collagen type I, little or no alkaline phosphatase production, and low proliferation rate. More importantly, MLO-Y4 cells express an osteocyte-specific marker protein, E11 (22). Connexin 43 (Cx43) was identified as a major gap junction protein expressed in MLO-Y4 cells (21, 23, 24). Dye-transfer experiments show that MLO-Y4 cells are functionally coupled, and that this coupling is mediated by gap junction channels (24). In addition, Yellowley and co-workers (23) have shown that the osteocyte-like MLO-Y4 cells can couple, through gap junctions, to osteoblast-like MC3T3 cells. Therefore, this cell line is a valuable cell model to study the function of gap junctions in cell-to-cell communication relationships required for the regulation and function of osteocytes.
Primary osteocytes and primary calvarial bone cells have been shown to release PGs in response to fluid flow treatment (4) (25). The response of bone cells to mechanical strain is blocked by inhibitors of PG biosynthesis, suggesting that PG biosynthesis is essential for the bone remodeling process (6, 26). PGs are generally thought to be skeletal anabolic agents, because administration of these agents can increase bone mass in humans, rabbits, and rats (27, 28, 29); stimulate bone formation in vitro in organ culture (30); and increase nodule formation in rat calvarial osteoblasts (31, 32). PGs also have catabolic effects on bone and have been shown to stimulate osteoclastic bone resorption, osteoclast formation, and activation (33, 34, 35, 36, 37). In addition, PG activates intracortical bone remodeling in both intact and ovariectomized female rats in vivo (38).
Fluid flow stimulates PGs’ release in primary osteocyte cells (4, 5). PGE2 has also been shown to stimulate gap junction function and Cx43 expression in osteoblast-like UMR106–01 cells (39). The role of PGE2 in regulation of gap junctions in mechanical-stimulated osteocytes is yet unknown. In our previous studies, we have shown that fluid flow-induced shear stress stimulates gap junction-mediated intercellular communication in osteocyte-like MLO-Y4 cells and increases Cx43 expression. This stimulatory effect is further enhanced during the postfluid flow period (24). In this report, we show that fluid flow increases the release of PGE2 in osteocyte-like MLO-Y4 cells and that PG is involved in the stimulatory effects of fluid flow-induced shear stress on intercellular communication.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EIA kits and
PGE2 Affinity Sorbent were from Cayman Chemical
(Ann Arbor, MI). Super Script II reverse transcriptase and
Taq DNA polymerase were from Life Technologies, Inc. (Baltimore, MD). BCA microprotein assay kit was from
Pierce Chemical Co. (Rockford, IL). Chemiluminescence kit,
ECL, was from Amersham Pharmacia Biotech Pharmacia
(Piscataway, NJ). All other reagents were purchased from
Sigma (St. Louis, MO) or Fisher Scientific
(Pittsburgh, PA).
Cell cultures
MLO-Y4 cells were cultured on collagen-coated (rat tail collagen
type I, 0.15 mg/ml) surfaces, including plastic plates, polyester
sheets, and glass slides. There is no obvious difference of cell growth
on these three collagen-coated surfaces. Cells were grown in -MEM,
supplemented with 2.5% FBS and 2.5% calf serum, and incubated in a
5% CO2 incubator at 37 C as described previously
(21).
Cell shear stress induced by fluid flow
Parallel plate flow chambers were designed to provide either a
5- or a 90-cm2 cell surface area for shear stress
exposure as described previously (40). Each chamber
consisted of two horizontal parallel glass plates, separated by a
spacer/gasket of defined thickness, creating a long rectangular flow
channel whose height was much less than either its length or width.
Fluid flow was established across the width of the channel via a
reservoir at either end of the channel formed by narrow slots in the
top plate cut perpendicular to the direction of flow and placed
immediately beneath the flow connectors. MLO-Y4 cells were cultured
either on collagen-coated large polyester sheets (Regal Plastics, San
Antonio, TX) for protein assay or collagen-coated microscopic slides
for dye transfer and morphological assays. The wall shear stress
experienced by cells in these chambers was directly related to the flow
rate of the circulating medium through the channel and inversely
related to the square of the channel height. Flow was gravity-driven,
and the rate was governed by the height of separation between the upper
and lower medium reservoirs, using a peristaltic pump (Cole Parmer
Instrument, Chicago, IL) to return medium to the upper reservoir. Flow
rate was continually monitored by an in-line flowmeter (Cole Parmer
Instrument). Using this flow system, wall shear stress levels, caused
by steady laminar flow of 16 dynes/cm2, were
generated by adjusting the channel height (using spacers) and medium
flow rate (1–1.8 ml/sec). Under such flow conditions, a low Reynold’s
number was attained, ensuring that the flow was laminar and the
velocity profile was that of a fully parabolic form over more than 98%
of the channel length. The construction design of these chambers also
permitted continuous microscopic visualization and recording of cells
residing within the flow channel during shear stress regimens to
monitor loss or change in cell orientation. All experiments were
repeated at least three times. The circulating medium was identical to
the culture medium. The entire flow system was housed within a large
walk-in CO2 incubator to maintain the circulating
medium and the cell environment at 37 C and pH 7.4. Upon completion of
the flow regimen, the cell-covered sheets or slides were removed for
the analyses described below.
PG measurement and depletion of PGE2 from conditioned
medium of fluid flow
After the fluid flow treatment, for 30 min to 2 h at 16
dynes/cm2, the conditioned medium was collected,
and the amount of PGE2 and 6-keto
PGF1 released into the medium was measured
using PGE2 and 6-keto
PGF1 EIA kit (Cayman Chemical) according to
the manufacturer’s instructions.
A PGE2 affinity column (Cayman Chemical) was used for the removal of PGE2 from the conditioned medium. Ten milliliters of conditioned medium were collected from either the 2-h fluid flow-treated MLO-Y4 cells at 16 dynes/cm2 or from nontreated cells. The medium was then passed through a PGE2 affinity column containing 1 ml Sepharose 4B conjugated with monoclonal anti-PGE2 antibody. The column was preeluted with 10 ml regular growth medium before the application of the conditioned medium, and the flow-through medium was collected. The amount of PGE2 in the flow-through medium was measured as described above.
Scrape-loading dye transfer assay and fluorescence
microscopy
MLO-Y4 cells were grown to approximately 75–85% confluence in
100 mm round tissue culture plates. The Scrape-loading dye transfer
assay was performed based on the modified procedure described by McNeil
and co-workers (41). In this method, cells were scratched,
in the presence of two types of fluorescent dyes [LY
(Mr, 457 Da), which can penetrate through gap
junction channels; and RD (Mr, 10 kDa), which is
too large to pass through the channels, thus serving as a tracer dye
for the cells originally receiving the dye]. Cells were washed three
times with HBSS plus 1% BSA. Then 1% LY and 1% RD, dissolved in PBS,
were applied to the cells, which subsequently were scraped lightly with
a 26-gauge needle. After incubation for 10 min, cells were washed with
HBSS three times, then twice with PBS, and finally fixed in fresh 2%
paraformaldehyde (from 16% stock) for 20 min. The dye transfer results
were examined using a fluorescence microscope (Axioscope, Carl Zeiss, Jena, Germany), in which LY could be detected, using the
filter set for fluorescein; and RD, using the filter set for rhodamine.
To reach statistical significance, more than 500 cells receiving RD
were counted for each assay.
SDS-PAGE and Western blotting
The protein concentration of crude membrane samples of MLO-Y4
cells was determined using the MicroBCA assay (Pierce Chemical Co.) according to the manufacturer’s instructions. Equal
amounts of protein (10 µg) were loaded in each lane of a 10%
SDS-PAGE and transferred to nitrocellulose membranes according to the
method of White et al. (42). Membranes were
incubated with a 1:250 dilution of affinity-purified anti-Cx43 antibody
as described previously (43) or a 1:5000 dilution of
monoclonal anti-ß actin antibody (Sigma). The primary
antibody was detected using peroxidase-conjugated secondary antirabbit
or mouse antiserum and followed by use of a chemiluminescence reagent
kit (ECL, Amersham Pharmacia Biotech) according to the
manufacturer’s instructions. The membranes were exposed to X-OMAT AR
films (Eastman Kodak Co., Rochester, NY) and detected by
fluorography. The intensity of Cx43 bands was quantified by
densitometry (NIH image).
RT-PCR
Total RNA was isolated from the cultures of MLO-Y4 cells
(Biotex Laboratories, Inc., Houston, TX) according to the
manufacturer’s instructions. cDNA was synthesized from 5 µg of the
total RNA in a 20-µl reaction mixture containing 1x first-strand
buffer (20 mM Tris-HCl, pH 8.4; 50 mM KCl;
1.5 mM MgCl2; 200 mM
MgCl2), 500 µM deoxy-NTPs, 10
mM dithiothreitol, 50 ng oligo (dT)12–18 primers, and 280
U Super Script II reverse transcriptase. Also, 0.2% cDNA was amplified
using PCR in a 50-µl reaction mixture containing 1x PCR
buffer, 10 nM 5' and 3' primer, 200 µM
deoxy-NTPs, 1 mM MgCl2, and
2.5 U Taq DNA polymerase. Amplifications were formed in a
DNA Thermal Cycler (Techne Inc., Princeton, NJ) for 20, 25, 30, 35, and
40 cycles following the reaction profile: 94 C for 30 sec, 58 C for 45
sec, and 72 C for 45 sec. The primers for cyclooxygenase (COX) genes
and control ß-actin were used as follows: COX-1 (mouse, accession no.
M34141, 516 bp): sense: 5'-TGGGAGTCCTTCTCCAATGTGAG-3';
antisense: 5'-GTTATGTTCACGAAGCCAGATCG-3'; COX-2 (mouse, accession
no. M64291, 591 bp): sense: 5'-TCCCCTTCCTGCGAAGTTTAAC-3';
antisense: 5'-CATACATCATCAGACCAGGCACC-3'. ß-actin (mouse, accession
no. X03765, 500 bp): sense: 5'-CGGGACCTGACGGACTACCTC-3';
antisense: 5'-CACATCTGCTGGAAGGTGGACA-3'.
Statistical analysis
Data were analyzed using one-way ANOVA and
Student’s-Newman-Keuls multiple-comparison test, with the Instat
biostatistic program (GraphPad Software, Inc., San Diego,
CA). Data are presented as the mean ± SD of three
determinations. Asterisks indicate the degree of significant
differences compared with the controls (*, P < 0.05,
**, P < 0.01, ***, P < 0.001).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, panel a) or with conditioned medium
generated either in the absence (Fig. 1A, panel b) or presence (Fig. 1A, panel c) of fluid flow, and changes in cell morphology were
examined. Fluid flow-conditioned medium enhanced the extended dendritic
processes made by MLO-Y4 cells and enhanced connectivity with their
neighboring cells. These morphological changes are similar to those
observed with MLO-Y4 cells treated directly with fluid flow shear
stress. The increased connections in the fluid flow-conditioned
medium-treated MLO-Y4 cells were confirmed by the increased
intercellular coupling mediated by gap junctions (Fig. 1B). Cx43
expression is also increased, as shown by densitometric measurements
(Fig. 1C). These results show that conditioned medium, generated from
the fluid flow treatment of MLO-Y4 cells, stimulates a change of
osteocyte-like cell morphology and enhances gap junction function,
which is similar to the MLO-Y4 cells treated directly by fluid
flow, as shown previously (24).
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released into the medium was measured (Fig. 2A). The enhancement of
PGE2 release was shown in MLO-Y4 cells under
fluid flow treatment. The amount of PGE2 released
into the medium was directly proportional to the treatment time,
reaching a maximum at 2 h. The 6-keto
PGF1 responses had a pattern similar to that
of the PGE2 responses, although at a much lower
level of production. The results are consistent with previous studies
of primary osteocytes, in which the accumulation of PGs is detected as
early as 15 min after fluid flow treatment (5). A
significant increase was observed in less than 30 min after the
application of fluid flow, suggesting that transcription of
cyclooxygenase mRNA may not be responsible for this early increase
in PG release.
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). Whereas COX-1 mRNA level remained constant, COX-2
mRNA expression was increased at 30 min and at 2 h after fluid
flow. The increase was still evident 24 h after fluid flow.
Regulation of gap junctions and Cx43 expression by
PGE2
We have shown previously that fluid flow increases gap junctions
and Cx43 expression (24). PGE2 has
been shown to have stimulatory effects on gap junctions in
osteoblast-like cells (39). To determine whether
PGE2 could be influencing gap junctions, various
concentrations of PGE2 were used to treat MLO-Y4
cells. Intercellular coupling between MLO-Y4 cells was analyzed using
the Scrape-loading dye transfer assay (Fig. 3A). Functional gap junctions between
MLO-Y4 cells increased significantly with increased concentrations of
PGE2. Immunoblot analysis, combined with
densitometric measurement of PGE2-treated MLO-Y4
cell samples, showed that PGE2 increased Cx43
expression (Fig. 3B), which is consistent with the intercellular
coupling data. Together, the results from both functional and
biochemical analyses show that PGE2 increases
functional gap junctions and Cx43 protein expression in MLO-Y4
cells in a dose-dependent fashion.
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).
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). In addition, the
increase in Cx43 expression of MLO-Y4 cells was significantly depressed
when treated with conditioned medium lacking PGE2
(Fig. 4C). The results suggest that PGE2 is a
factor that increases gap junctions in fluid flow-treated MLO-Y4
cells.
Indomethacin inhibits the stimulatory effects of fluid flow on gap
junctions
To analyze the involvement of PG biosynthesis in regulation
of gap junctions, the PG synthase inhibitor, indomethacin, was used.
The amount of PGE2 released in the presence or
absence of indomethacin was measured. Indomethacin treatment
dramatically decreased the release of PGE2
induced by fluid flow, compared with the nonindomethacin-treated
controls (Fig. 5A). The gap
junction channel permeability was examined using Scrape-loading dye
transfer analyses (Fig. 5B). In the presence of indomethacin, fluid
flow stimulation of intercellular coupling mediated by gap junctions
was significantly attenuated. Similarly, indomethacin treatment also
blocked the stimulatory effect by fluid flow on Cx43 expression, as
shown by densitometric quantification of the Western blot (Fig. 5C).
Together, indomethacin partially inhibited the stimulatory effect of
fluid flow on both gap junction-mediated intercellular coupling and
Cx43 protein levels, further confirming that PGE2
is a regulatory factor for gap junction function under fluid flow shear
stress.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A model is proposed for the role of PGE2 in
regulation of gap junctions in mechanically stimulated osteocytes, as
illustrated in Fig. 6. Application
of mechanical strain in osteocytes results in the redistribution of
Cx43, assembly of gap junction channels, and formation of additional
functional gap junctions (24). Soon after application of
mechanical strain, in less than 30 min, PGE2 is
converted from arachidonic acid and released from the cell. The
released PGE2 functions in an autocrine manner.
We have recently shown that, immediately after a 2-h fluid flow
treatment, gap junction-mediated intercellular communication is
increased, but there is no increase of Cx43 expression
(24). The significant increase of Cx43 protein occurs
2 h after fluid flow treatment. Based on these observations,
formation of functional gap junction induced by
PGE2 with the existing Cx43 seems to happen in
less than 2 h, while the induction of Cx43 biosynthesis by
PGE2 takes place around 4 h. Increased
biosynthesis of Cx43, in turn, generates additional functional gap
junctions, which can accommodate the passage of greater numbers of
signaling molecules between osteocytes. In addition, COX-2
transcription is also increased during the poststress period, possibly
through the autocrine effects of newly formed
PGE2. Fluid flow studies by Reich and Frangos
(46) support this model. They observed that an early phase
of PG production depends on substrate availability and that a later
phase requires de novo protein synthesis. It has been
demonstrated that PGs can induce COX-2 expression via autostimulation,
likely through protein kinase C as well as cAMP pathways (47, 48).
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) that could also play a stimulatory role
in gap junctions. Previous studies have reported that fluid flow shear
stress stimulates the release of PGF2 and
6-keto PGF1 in cultured chicken osteocyte
cells and mouse calvarial bone cells (4, 5, 25).
Consistent with observations using primary osteocytes, we have also
shown the increased release of PGE2 and 6-keto
PGF1 in the fluid flow-treated MLO-Y4 cells,
although the production of 6-keto PGF1 is much
lower than that of PGE2. Indomethacin
inhibits the enzymatic activities of COX, the first enzyme in PG
biosynthesis, and therefore eliminates the generation of all downstream
PG derivatives. In the presence of indomethacin, the release of
PGE2 was completely blocked, but gap junction
activity was still partially (but significantly) inhibited, compared
with the nontreated controls. This result excludes the sole involvement
of PG derivatives in stimulating gap junctions induced by fluid flow.
Either PGE2 or conditioned media from fluid
flow-treated cells significantly increased gap junction function and
Cx43 expression in MLO-Y4 cells. However, the concentration of
PGE2 in conditioned medium generated by fluid
flow is much lower than that needed to stimulate gap junctions.
Together, the results suggest that another factor(s), in addition to
PGs, is likely to play an additive or a synergistic role in regulation
of gap junctions in response to mechanical strain. Published studies (49) have shown that nitric oxide (NO) production is increased dramatically after fluid flow treatment of primary osteocytes. In addition, inhibition of NO biosynthesis prevents the effect of fluid flow on NO as well as PGE2 release, suggesting that NO is a mediator of mechanical stress that leads to enhanced PGE2 release. Conversely, studies by Chow et al. (50) show that, although both PGs and NO are required in mechanically induced osteogenesis, they seem to be generated largely independently of each other. The exploration of the involvement of other autocrine factors, such as NO, will require further investigation.
We have observed that COX-2 expression is increased during the postfluid flow period, suggesting that COX-2 is most likely the enzyme responsible for the biosynthesis of PGE2. These in vitro observations support the in vivo study by Forwood (51). In this study, a specific COX-2 inhibitor completely blocked bone formation induced by mechanical loading in rats. Several in vivo studies using COX inhibitors have found that PGs are necessary for the induction of bone formation by mechanical stimuli (51, 52, 53). Addition of PGE2 allows bone formation to occur in response to lower thresholds of mechanical loading (54). Therefore, increased PG production, through COX-2 induction, that results in increased gap junction function may be one of the means whereby PGs mediate the effects of mechanical loading.
It is not clear how PGE2, released in response to mechanical loading, regulates gap junctions. Many signal transduction mechanisms activated by PGs have been described in bone cells (55). In osteoblastic and osteoclastic cells, PG stimulates cAMP, increases calcium entry, and releases inositol phosphates and diacylglycerol by phospholipase C, as well as release of diacylglycerol from phosphatidylcholine by phospholipase D (37, 56, 57, 58, 59). cAMP is a molecule that is known to be frequently associated with increased gap junction activity and increased total numbers of gap junctions (60). Therefore, it is likely that cAMP is one of the down-stream signaling molecules of PG that up-regulates gap junctions. More work is needed to explore the signaling transduction pathways in regulation of gap junctions induced by mechanical loading and the regulatory mechanism of PGs in osteocytes.
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Acknowledgments |
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Footnotes |
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Abbreviations: COX, Cyclooxygenase; Cx43, connexin 43; RD, rhodamine dextran; LY, Lucifer yellow; NO, nitric oxide.
Received February 21, 2001.
Accepted for publication April 24, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 connexin gene leads to proteolysis and
cataractogenesis in mice. Cell 91:833–843[CrossRef][Medline]
on osteoclast formation in mouse bone
marrow cultures. J Bone Miner Res 6:157–164[Medline]
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