Gap junctions and fluid flow response in MC3T3-E1 cells

M. M. Saunders1, J. You1, J. E. Trosko2, H. Yamasaki3, Z. Li1, H. J. Donahue1, and C. R. Jacobs4

1 Musculoskeletal Research Laboratory, Department of Orthopedics and Rehabilitation, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033; 2 Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, East Lansing, Michigan 48824; and 3 Kwansei Gakuin University, Uegahava, Nishinomiya 662-8501, Japan; 4 Biomechanical Engineering Division, Department of Mechanical Engineering, Stanford University, Stanford, California 94304


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the current study, we examined the role of gap junctions in oscillatory fluid flow-induced changes in intracellular Ca2+ concentration and prostaglandin release in osteoblastic cells. This work was completed in MC3T3-E1 cells with intact gap junctional communication as well as in MC3T3-E1 cells rendered communication deficient through expression of a dominant-negative connexin. Our results demonstrate that MC3T3-E1 cells with intact gap junctions respond to oscillatory fluid flow with significant increases in prostaglandin E2 (PGE2) release, whereas cells with diminished gap junctional communication do not. Furthermore, we found that cytosolic Ca2+ (Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>) response was unaltered by the disruption in gap junctional communication and was not significantly different among the cell lines. Thus our results suggest that gap junctions contribute to the PGE2 but not to the Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> response to oscillatory fluid flow. These findings implicate gap junctional intercellular communication (GJIC) in bone cell ensemble responsiveness to oscillatory fluid flow and suggest that gap junctions and GJIC play a pivotal role in mechanotransduction mechanisms in bone.

prostaglandin E2; calcium; mechanotransduction; gap junctional intercellular communication


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WIDELY ACCEPTED that bone adapts to its physical loading milieu by optimizing mass and mechanical performance in a process known as bone remodeling. In a nonpathological scenario, this process results in normal bone turnover whereby new bone formation is balanced by removal of existing bone. In a pathological scenario, this process results in an imbalance whereby net bone formation (osteopetrosis) or net bone loss (osteopenia) ensues. Although the effects of remodeling have been histologically observed, the exact cellular pathways by which it occurs are incompletely understood. To this end, researchers have recently begun to investigate mechanotransduction mechanisms (15, 17, 32) in an attempt to better uncover the elusive signal transduction pathways by which physical stimuli can affect cellular responses in bone. These studies have found that bone cells can respond to a wide variety of endogenously occurring signals including mechanical stretch (41), streaming potentials, chemotransport, electrical effects (2, 6, 26, 33), and fluid flow (8, 17, 28, 41). In the latter area of research, it has been hypothesized that the fluid flow through the lacunar-canalicular network is pivotal to bone cell responsiveness. Although several hypotheses have been proposed, many believe that the osteocytes in the canalicular spaces sense the fluid flow (1) and in turn signal the osteoblasts to form bone.

Although many accept the theory that osteoblast responsiveness to biophysical effects is linked to the osteocyte (1, 23), few have proposed a mechanism by which this may occur. We propose that bone cell responsiveness to fluid flow is aided by gap junctions that physically connect osteoblasts and osteocytes (39) as well as osteoblasts to other osteoblasts. In this scenario, we hypothesize that gap junctions not only enable osteocytes to transfer signals to osteoblasts but that the responsiveness of osteoblastic networks to the signal is amplified via gap junctions. By coupling the osteoblasts together, gap junctions enable the cells to respond in concert, resulting in a more robust response attained than if an equal number of individual cell responses was achieved. This is the focus of our current work.

Gap junctions are transmembrane protein channels that enable neighboring cells to physically link, thereby facilitating the rapid diffusion of small molecules and ions on the order of 1 kDa in a process known as gap junctional intercellular communication (GJIC). Gap junctions may be homospecific, uniting cells of the same type, or may be heterospecific, uniting cells of unlike type. With the exception of blood cells and muscle fibers, gap junctions have been found in most cells (30), with at least 13 mammalian connexins identified to date and named with respect to molecular weight.

There is growing evidence to support a role for gap junctions in the cellular (and cell ensemble) response to physical stimuli. In bone, gap junctions have been linked to such functions as hormonal responsiveness (34), gene expression (25), and differentiation (7). Intercellularly, gap junctions have been linked to second messenger responses induced by physical stimuli such as Ca2+ release following membrane deformation (21). Interestingly, many fluid flow studies have shown that osteoblastic cells respond to flow in vitro with an increase in such second messengers as Ca2+ (13, 15), cAMP (28), and NO release (20), which have been shown to be physical regulators of gap junction channel opening.

GJIC has been linked to both normal and abnormal cell function. In normal cell function, GJIC has been linked to such processes as proliferation and differentiation, although the findings at times have been inconsistent. For instance, while GJIC is found to maintain cell differentiation status in cultured hepatocytes (40), it is decreased in differentiating keratinocytes compared with proliferating ones (12). Furthermore, we have recently demonstrated that gap junctional function and expression parallel osteoblastic differentiation, contributing to alkaline phosphatase expression (7). Thus gap junction studies are widely dependent on cell line, culture conditions, and experimental environment, and results must be interpreted within these contexts. In abnormal cell function, alterations in GJIC have been linked to disease (30, 36), suggesting that a status quo in gap junctional function is crucial to homeostasis.

In the current study, we set out to examine the role of GJIC in transducing a mechanical stimulus to bone cells. That is, we exposed osteoblastic cells to levels of oscillatory fluid flow that occur in vivo due to habitual loading (35), and we measured prostaglandin E2 (PGE2) release and cytosolic Ca2+ concentration ([Ca2+]i), markers selected for their proposed role in the regulation of bone turnover (4, 10, 16, 19, 27). To correlate these findings with the role of GJIC, we utilized osteoblastic MC3T3-E1 cells, MC3T3-E1 cells expressing a dominant-negative connexin43 (Cx43; DN-8), the predominant gap junction protein in bone, and a control transfectant (DN-VC). Comparisons of changes in PGE2 release and [Ca2+]i in the presence of oscillatory fluid flow in the three cell lines were then used to draw conclusions about the contribution of gap junctions and GJIC to bone remodeling.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Three immortalized osteoblastic cell lines were utilized in this study: MC3T3-E1, DN-VC, and DN-8. The MC3T3-E1 is an immortalized mouse osteoblastic cell line; the DN-8 is a neomycin-sustained transfectant of MC3T3-E1 containing a mutant Cx43; and the DN-VC is a control for the transfection containing an empty plasmid. MC3T3-E1 cells were cultured in Eagles's minimal essential medium (MEM-alpha ; GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1% penicillin/streptomycin (GIBCO BRL). The DN-8 and DN-VC cells were cultured in MC3T3-E1 medium supplemented with neomycin (200 µg/ml). All cell lines were maintained in an incubator at 37°C and 5% CO2, with flow experiments conducted in the appropriate media supplemented with 2% FBS.

The DN-8 line was developed from a dominant-negative strategy as previously described (24). Briefly, a mutant gap junction protein (Cx43Delta ) of Cx43 was developed by the deletion of residues in the internal cytoplasmic loop of the connexin structure. The goal of this strategy was to introduce this mutant gene into both protein channels of each linking cell such that the mutant could oligomerize with only a wild-type species. Unlike previous dominant-negative strategies in which GJIC is obliterated and the resulting connexin oligomers are not transported to the membrane but remain in the cytoplasm, this novel mutation approach affects only permeability, leaving transport intact (24).

Cell preparation. Experiments were conducted on two differently sized microscope slides. For the quantification of oscillatory fluid flow-induced cytosolic Ca2+ (Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>) mobilization, cells were plated on quartz slides (76 mm × 26 mm × 1.6 mm) for imaging. These slides accommodated the relatively few cells needed to conduct the experiments and were made of quartz to allow for ultraviolet visualization. Cells were plated at 1.0 × 105, 0.75 × 105, or 0.5 × 105 cells/slide and cultured for 24, 48, or 96 h, respectively, to achieve 85-90% confluence. For the quantification of oscillatory fluid flow-induced PGE2 production, cells were plated on glass microscope slides (75 mm × 38 mm × 1 mm). These slides were larger so that larger volumes of cells could be evaluated. Cells were plated at 3.5 × 105, 2.75 × 105, or 2.0 × 105 cells/slide and cultured for 24, 48, or 96 h, respectively, to achieve 85-90% confluence. For quantification of GJIC, cells were plated as described for the PGE2 experiments. In addition, cells for double labeling were cultured in round (35-mm-diameter) polystyrene petri dishes in the appropriate media for 24, 48, or 96 h.

GJIC assays. GJIC assays were completed using epifluorescent microscopy and a double-labeling technique, as previously described (39). In this technique, cells are loaded with the fluorescent dyes calcein AM (Molecular Probes, Eugene, OR) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes). The fluorescent dye calcein AM, once in the cell, is cleaved of its AM group and trapped within the cell. However, as a result of its small molecular size (<1 kDa), calcein is gap junction permeable and able to transfer to neighboring cells if functional (open) gap junctions are established. The fluorescent dye DiI is of a larger molecular size, intercalates within cell membranes, and does not transfer to neighboring cells via GJIC. The loaded cells are then dropped onto unloaded cells in a monolayer, and cell transfer is quantified. If functional gap junctions are established, the calcein will transfer to neighboring cells that will then fluoresce green.

Coupling assays were completed to establish the extent of disruption of GJIC in DN-8 cells at 24, 48, and 96 h in culture and were compared with GJIC in the MC3T3-E1 and DN-VC lines at the same time points. After quantification of GJIC, we assessed GJIC in the three cell lines at 96 h in culture simultaneously with PGE2 and [Ca2+]i experiments to minimize passage variables. On the day of the experiments, the preconfluent cells ("donor" cells) plated in the petri dishes were removed from the incubator and washed twice with room temperature phosphate-buffered saline (PBS) followed by aspiration. The donor cells were labeled with a BSA-enriched PBS-fluorescent dye mixture containing 20 µl of calcein AM, 7 µl of DiI, and 20 µl of pluronic acid (Molecular Probes) and incubated for 30 min at 37°C. After being incubated, the dye mixture was aspirated, and the donor cells were washed twice in room temperature PBS. The donor cells were detached from the dishes by trypsinization, centrifuged at 200 g for 8 min, and resuspended in fresh growth medium. The double-labeled (calcein and DiI) donor cells were then dropped onto the glass slides containing confluent monolayers of unlabeled cells at a ratio of ~1:500 cells (labeled to unlabeled) and incubated for 90 min at 37°C. After the incubation period, the slides were removed from the dishes, washed twice with PBS, and covered by round (25-mm-diameter) glass coverslips. The slides were placed on a Nikon fluorescent microscope (Nikon EFD-3; Optical Apparatus, Ardmore, PA) and visualized using fluorescein (lambda excitation = 465-495 nm; lambda emission = 520 nm) and rhodamine (lambda excitation = 541-551 nm; lambda emission = 590 nm) filters to locate the calcein- and DiI-loaded cells, respectively. Coupling was quantified by counting the number of neighboring cells fluorescing green, while the DiI was used to distinguish the labeled cells from those in the monolayer. Thirty cells were randomly selected and counted for each slide. Coupling was considered extensive if individual cells transferred calcein to >15 cells and were not counted past this threshold number.

Parallel plate flow chambers and testing machine. For PGE2 and Ca2+ experiments, bone cells were placed in a parallel plate flow chamber and subjected to oscillatory fluid flow. This system has been previously characterized, and we and others have employed it to expose endothelial cells (8), chondrocytes (37, 38), and bone cells (14, 15, 17, 41) to physiological levels of fluid flow. Briefly, the system imparts a laminar flow to the cells in a monolayer, exposing them to a shear stress governed by the equation (9)
&tgr;=6&mgr;Q/<IT>bh</IT><SUP>2</SUP>
where tau  is the shear stress, µ is the viscosity of the flow medium, Q is the flow rate, and b and h are the width and height of the chamber, respectively. To accommodate the quartz and glass microscope slides, as previously noted, two differently sized chambers were employed. In general, the components for both chambers were the same and are shown in the exploded view of Fig. 1. The chambers consisted of a polycarbonate manifold, a silastic gasket, and a glass slide. This slide containing the cells in a monolayer formed the bottom of the flow chamber when inverted on the manifold. For Ca2+ studies, an 18 ml/min flow rate resulted in a shear stress of 20 dyn/cm2 and a rectangular flow volume of 38 mm × 10 mm × .28 mm; for PGE2 studies, a 43 ml/min flow rate resulted in a shear stress of 20 dyn/cm2 and a rectangular flow volume of 56 mm × 24 mm × 0.28 mm. In all flow experiments, flow rate was monitored with an ultrasonic flow probe (Transonic Systems, Ithaca, NY) connected to the chamber inlet. For Ca2+ imaging, the flow chamber assembly was held together with vacuum pressure; for PGE2 quantification, the flow chamber assembly was placed in a polycarbonate case bolted together to form an air-tight seal. For the latter experiments, the polycarbonate case containing the chamber enabled the system to be placed in an incubator for long-term flow periods (1 h) such that temperature and CO2 levels could be precisely regulated. For both short- (Ca2+) and long-term (PGE2) experiments, the chamber was connected to a pneumatic, closed-loop feedback materials testing machine (EnduraTec, Minnetonka, MN) via tubing and syringes with oscillatory fluid flow delivered in the form of a 1-Hz sine wave (Fig. 1).


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Fig. 1.   Schematic of oscillatory fluid flow delivery system. A: oscillatory fluid flow was delivered via a sinusoidal waveform generated by a materials testing machine connected to the flow chamber using tubing and syringes. B: the flow chamber consisted of a parallel plate design. Cells in a monolayer on the glass slide were inverted on the flow chamber on a silastic gasket. During fluid flow, the assembly was held together with either a vacuum seal or encased in a polycarbonate case (not shown).

PGE2 quantification. PGE2 accumulation in the supernatant was quantified with a commercially available, nonradioactive, competitive binding enzyme immunoassay system (BioTrak; Amersham Pharmaceuticals, Piscataway, NJ). After assay, the optical densities of the samples were read at 450 nm using a microplate reader (Dynex Technologies, Chantilly, VA). Manufacturer-supplied standards were also analyzed and used to construct a standard curve from which the sample concentrations were determined.

Oscillatory fluid flow-induced PGE2 was quantified at the 48- and 96-h time points. On the day of the experiments, preconfluent slides of cells were washed, placed in the parallel plate flow chamber, encased in the polycarbonate case, placed in the incubator, and connected to the fluid flow delivery system. Cells were exposed to flow for 1 h, after which 10 ml of media from the inlet and outlet ports of the chamber and adjacent tubing were collected for PGE2 analysis. These media are referred to throughout as media collected immediately postflow. In addition, the plates of cells were incubated in 10 ml of fresh medium for 1 h postflow, and these media were also collected for PGE2 analysis. These media are referred to throughout as media collected 1 h postflow. Immediately after media collection, aliquots were frozen at -80°C. In addition, for some experiments, the ionophore 4-bromo-calcium (50 µM) was added to a plated slide from each cell line for 15 min at 37°C. The media from these collections were used as positive controls in the PGE2 assays. On the day of assay, samples were thawed at 4°C and vortexed. Assays were completed at room temperature within 1 mo of collection, and degradation assays were completed to ensure that this time period did not adversely affect the results.

The three cell lines were also subjected to oscillatory flow in the presence of thapsigargin, a drug used in our study to empty and prevent refilling of intracellular Ca2+ stores, thus eliminating this source of Ca2+ contributing to changes in [Ca2+]i (42). PGE2 experiments in the presence of thapsigargin were completed at the 96-h time point following the exact protocol previously outlined with one exception: thapsigargin (50 nM) was added to each petri dish of plated cells (30 min before placing it in the flow chambers), the flow medium, and the 10 ml of fresh, 1-h postflow incubation medium.

Because total PGE2 accumulation in the medium is dependent on cell number, prostaglandin accumulation was normalized to total cell protein for each slide. After the 1-h incubation and collection of the additional 10 ml of fresh medium, the cells were removed from each microscope slide by trypsinization, centrifuged at 200 g for 8 min, and resuspended in 0.5 ml of 0.05% Triton X-100 detergent. The suspended cells were placed in 1-ml centrifuge tubes and lysed using three cycles of rapid freezing (-80°C) and thawing. The lysate was frozen at -80°C until analysis with a commercially available assay kit (Bio-Rad, Hercules, CA). After assay, the optical densities of the samples were read at 405 nm using a microplate reader. Manufacturer-supplied standards were analyzed and used to construct the standard curve from which the sample concentrations were determined. Frozen cells and media were assayed at room temperature within 1 mo of collection.

Ca2+ imaging. Ca2+ imaging was completed with fluorescent microscopy and the dual-wavelenth ratiometric dye fura 2-AM (Molecular Probes). This indicator was selected for its ability to exhibit two distinct spectra and two distinct wavelengths based on the presence or absence of Ca2+ binding to the indicator (31). The indicator is loaded in the fura 2-AM form, which allows it to easily enter the cells. After loading, the AM groups are cleaved in an enzymatic process leaving the indicator trapped within the cell.

[Ca2+]i was quantified in the three cell lines at the 96-h time point. On the day of the experiments, preconfluent slides of cells were loaded with 10 µM fura 2-AM in 1 ml of fresh media and incubated at 37°C for 45 min. After being incubated, the cells were washed in the appropriate flow medium (2% FBS), placed on the parallel plate flow chamber, transferred to a fluorescent microscope, and connected to the loading machine. To allow the cells to settle and ensure that the AM hydrolizing process was complete, the cells were allowed to equilibrate on the microscope stage for 30 min immediately before testing. Cells were subjected to 3 min of oscillatory flow preceded by a 3-min no-flow baseline. An image acquisition and analysis software package (Metafluor) was used to capture the images for [Ca2+]i determination.

Data analyses. Ca2+ results were analyzed with a Rainflow counting technique (18). This technique, adapted from the field of mechanical fatigue, enables individual responses to be extracted from data containing multiple responses and has generally been employed to determine the contribution of a particular loading cycle to the overall lifetime of a structure. Rainflow applied to our research enabled individual cell responses to be isolated and separated from background noise with a threshold response defined as a change in [Ca2+]i of >= 20 nM.

PGE2 results were analyzed using a microplate reader and normalized to total protein with total PGE2 accumulation in the medium given in picograms per micrograms. GJIC was quantified by counting cell fluorescence transfers, as previously described. All Ca2+, PGE2, and GJIC data obtained were expressed as means ± SE. To compare results among the cell lines, general linear model ANOVAs with Student-Newman-Keuls post hoc comparisons were completed using a commercially available software program (Instat; GraphPad Software, San Diego, CA) with an a priori significance level of 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Osteoblastic cell line GJIC as a function of time in culture. GJIC was qualitatively evaluated at 24-, 48-, and 96-h time points in the three cell lines with typical dye transfers shown (Fig. 2). In these double-exposed photographs, the green (calcein) fluorescence indicates the coupled cells in the monolayer, whereas the yellow (calcein and DiI) fluorescence indicates double-labeled donor cells. Quantitative results for the 24-, 48-, and 96-h time points (Fig. 3) depict the number of donor cells coupled to individual acceptor cells in the monolayer. We found that the MC3T3-E1 and DN-VC cell line coupling was not dependent on time in culture up to 96 h. At 24 and 48 h, the three cell lines did not exhibit a significant difference in coupling compared with each other. However, at the 96-h time point, DN-8 cells exhibited a significant decrease in coupling compared with the MC3T3-E1 (P < 0.001) and DN-VC (P < 0.001) lines at 96 h, as well as compared with themselves at the 24- (P < 0.001) and 48-h (P < 0.001) time points.


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Fig. 2.   Qualitative results of double-labeling assay at 24 and 96 h in the 3 cell lines examined. MC3T3-E1 (A and B), DN-VC (C and D), or DN-8 (E and F) cells were grown in the monolayer for 24, 48 (not shown), or 96 h and subjected to homospecific gap junctional intercellular communication analysis. Donor cells double labeled with the fluorescent dyes calcein and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were placed in contact with unloaded like cells in the monolayer. Cell transfer was visualized after 90 min. In the dual-exposure photographs, the cells fluorescing green (calcein) are the unlabeled cells in the monolayer demonstrating functional coupling; the cells fluorescing yellow (calcein and DiI) are the dual-labeled donor cells (original magnification, ×400).



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Fig. 3.   Quantitative results of double-labeling assay at 24, 48, and 96 h in the 3 cell lines examined. MC3T3-E1 and DN-VC cell lines were highly coupled at all time points. No significant differences in coupling were found within or between these 2 cell lines at the various time points. The DN-8 cell line was well coupled at 24 and 48 h and not significantly different from the other cell lines at these time points. At 96 h, coupling in the DN-8 line was significantly diminished compared with the DN-8 line at 24 and 48 h (P < 0.001) as well as compared with the MC3T3-E1 and DN-VC lines at this time point (P < 0.001). Each bar is representative of a minimum of 60 cells (maximum 110) and is plotted as means ± SE with individual cell transfers not counted past a maximum of 15 cells. *Significantly different from 24- and 48-h time points within group; +significantly different from 96-h time points in MC3T3-E1 and DN-VC cell lines.

PGE2 accumulation in response to fluid flow. Because GJIC was decreased in DN-8 cells only after 96 h in culture, we first examined PGE2 response to fluid flow at this time point (Fig. 4A). Media from MC3T3-E1 and DN-VC cells collected 1 h postflow accumulated significantly more PGE2 than cells not exposed to flow (P < 0.0005 and P < 0.0001, respectively). However, media from poorly coupled DN-8 cells did not accumulate more PGE2 than control cells. Similar results were obtained when media were collected immediately postflow (not shown).


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Fig. 4.   Results of prostaglandin E2 (PGE2) quantification in the 3 cell lines examined. The numbers are representative of total PGE2 accumulation in the media normalized to total protein and collected after 1-h incubation period. A: at 96 h, the MC3T3-E1 and DN-VC cell lines responded to fluid flow with an increase in PGE2 accumulation, whereas the DN-8 cell line did not respond to fluid flow. Although results are shown for the 1-h collections only, similar trends were exhibited in the 0-h collections (data not shown). Interestingly, baseline levels were elevated in this line at this time point. B: at 48 h, the DN-8 cell line displayed an increase in PGE2 accumulation in response to oscillatory fluid flow with more accumulation obtained from the 1-h postflow samples. At 96 h, the DN-8 cell line responded to flow with no significant increases in PGE2 accumulation from either the 0-h or 1-h collections. All results shown are plotted as means ± SE with each value representative of at least 10 experiments. *Significantly different from no-flow control within group; **significantly different from media collected 1-h postflow at same time point in DN-VC and MC3T3-E1; ***significantly different from media collected 1-h postflow at same time point in MC3T3-E1.

We also examined the effect of fluid flow on PGE2 accumulation in DN-8 cells cultured at 48 h, a period after which DN-8 cells are as well coupled as MC3T3-E1 cells (Fig. 4B). Whereas exposure to fluid flow did not increase PGE2 accumulation in media collected 1 h postflow from DN-8 cells cultured for 96 h, it did increase in media from DN-8 cells cultured for 48 h (P < 0.005 vs. no-flow controls). Similar results were obtained when media were collected immediately postflow (data not shown).

Ca2+ response to oscillatory fluid flow. In cells cultured for 96 h, there was a 7.9-fold increase (P < 0.0007 vs. no flow) in the percentage of MC3T3-E1 cells responding to 3 min of oscillatory fluid flow with an increase in [Ca2+]i, an 8.9-fold increase in DN-VC cells (P < 0.0001), and a 9.3-fold increase in DN-8 cells (P < 0.0003; Fig. 5). The fold increases were not statistically different among the three cell lines. No significant differences in [Ca2+]i amplitude within or among groups were observed (data not shown), a finding also made in our previous work with human fetal osteoblastic cells (41).


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Fig. 5.   Results of cytosolic Ca2+ concentration ([Ca2+]i) imaging at 96 h. In all cell lines examined, oscillatory fluid flow induced a significant increase in the percentage of cells responding with an increase in [Ca2+]i (P < 0.0003, at least). Significant differences were not observed between the groups when comparing the no-flow controls or the flowed samples. All results are shown plotted as means ± SE with each value representative of at least 4 experiments (MC3T3-E1) or 6 experiments (DN-VC and DN-8). *Significantly different from no-flow control within group.

PGE2 accumulation in the presence of thapsigargin. One interpretation of our findings that GJIC contributed to the PGE2 but not the [Ca2+]i response to fluid flow in DN-8 cells is that Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> mobilization may not be critical to fluid flow-induced PGE2 accumulation. To address this issue, we examined the effect of thapsigargin on fluid flow-induced PGE2 accumulation. In the presence of thapsigargin, media from MC3T3-E1 and DN-VC cells collected 1 h postflow had a 92.1% and 278%, respectively, increase in PGE2 accumulation relative to no-flow control. Once again, fluid flow did not increase PGE2 accumulation in DN-8 cells cultured for 96 h and thus coupled poorly. Therefore, thapsigargin did not significantly alter the PGE2 response to fluid flow in any of the cell lines examined (Fig. 6).


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Fig. 6.   Results of PGE2 quantification in the presence of thapsigargin at 96 h in the 3 cell lines examined. At 96 h, the PGE2 response of the cell lines to oscillatory fluid flow was not altered by the presence of thapsigargin. All results are shown plotted as means ± SE with each value representative of a minimum of 2 experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we set out to investigate the role of gap junctions and GJIC in mechanotransduction mechanisms in bone. We applied a novel dominant-negative genetic intervention strategy to MC3T3-E1 osteoblastic parent cells to render them communication deficient. We subjected the resulting cell line to oscillatory fluid flow and measured flow-induced PGE2 release and changes in [Ca2+]i. This is the first study to examine GJIC in bone cell ensemble responsiveness to fluid flow, and, while only the second study to examine the effects of oscillatory fluid flow on [Ca2+]i in osteoblastic cells, it is the first to quantify the PGE2 response. We found that a breakdown in gap junction coupling had no effect on changes in [Ca2+]i but resulted in a significant inhibition of oscillatory fluid flow-induced PGE2 release, suggesting that gap junctions play a pivotal role in the mediation of oscillatory fluid flow-induced PGE2 production in osteoblastic cells.

To verify the effectiveness of the dominant-negative strategy used to render the DN-8 cells communication deficient, we quantified the extent of coupling in the DN-8 cells at 24, 48, and 96 h in culture. These results were compared with coupling experiments conducted at the same time points in the communication-intact, control-transfectant DN-VC cell line. We found an 80.1% decrease in coupling in the DN-8 cells between 48 and 96 h in culture, whereas no significant change was noted in the DN-VC cells over the same time period. These results indicate that GJIC in only the DN-8 cell line was dependent on time in culture. Therefore, because the cells are genetically identical and cultured under the same culture conditions, these cells provide a novel model system in the analysis of GJIC in bone cell responsiveness to fluid flow.

To address the role that gap junctions play in the oscillatory fluid flow-induced PGE2 response, we subjected the cell lines to 1 h of oscillatory fluid flow and measured PGE2 accumulation in the media compared with PGE2 accumulation in media from no-flow controls. At the 48-h time point, when intact GJIC was exhibited in the DN-8 cell line, the application of oscillatory fluid flow resulted in significant increases in PGE2 accumulation. However, at the 96-h time point, when GJIC was inhibited, no increase in oscillatory fluid flow-induced PGE2 accumulation resulted. In contrast, the DN-VC cell line responded at both time points with significant increases in PGE2 accumulation. Thus we found that a breakdown in coupling was accompanied by a significant decrease in PGE2 responsiveness to oscillatory fluid flow. These findings strongly suggest that gap junctions and GJIC are necessary in the signal transduction pathway whereby osteoblastic cells increase production of PGE2 in response to oscillatory fluid flow and that a GJIC-dependent pathway exists.

To address the role that gap junctions play in the mediation of oscillatory fluid flow-induced changes in [Ca2+]i, we subjected the cell lines to 3 min of oscillatory fluid flow and measured [Ca2+]i compared with [Ca2+]i of no-flow controls. At the 96-h time point, DN-8 and DN-VC cells responded to oscillatory fluid flow with significant increases in [Ca2+]i. Moreover, differences in flow-induced [Ca2+]i were not significantly different in the DN-8 and DN-VC lines at this time point. Thus we found that a breakdown in coupling was not accompanied by a significant change in [Ca2+]i and that the Ca2+ responses of the cell lines, regardless of degree of coupling, were equally responsive. These findings strongly suggest that gap junctions and GJIC are not necessary in the signal transduction pathway whereby osteoblastic cells increase [Ca2+]i in response to oscillatory fluid flow and that a GJIC-independent pathway exists.

In this study, we found that although coupling-deficient osteoblastic cells responded to the application of oscillatory fluid flow with significant increases in PGE2 release, changes in [Ca2+]i were not found due to changes in coupling. These findings suggest that the PGE2 and [Ca2+]i responses elicited via oscillatory fluid flow may be unlinked in these osteoblastic cells, a notion contradictory to prevailing opinion. To address this issue, we subjected the cell lines to oscillatory fluid flow in the presence of thapsigargin. We found that the introduction of thapsigargin did not significantly affect PGE2 production, whereas the Ca2+ response was completely annihilated (data not shown). Furthermore, because we have data indicating that the only source of Ca2+ in the MC3T3-E1 cells is from intracellular stores (42) that are emptied by the thapsigargin, our findings provide concrete evidence to suggest a separation of pathways is involved in Ca2+ wave propagation and PGE2 production in osteoblastic cells.

In this study, we set out to investigate the role of gap junctions in mediating oscillatory fluid flow-induced PGE2 response in osteoblastic cells. Inasmuch as this was our goal, we were largely interested in whether the application of oscillatory fluid flow resulted in significant increases in PGE2 production in the cell lines. However, studies have shown that the exact PGE2 time course has yet to be elucidated and that flow-induced PGE2 production is not obliterated with the cessation of the stimulant (22). To address the time-dependent response of oscillatory fluid flow-induced PGE2 release in these cell lines, PGE2 accumulation in the media was measured at two time points after cessation of flow. In the first approach, the flowed media were collected immediately after flow exposure; in the second approach, the flowed cells were placed in an equivalent volume of fresh media immediately postflow and incubated for 1 h. We found that in the coupling-intact DN-VC cells, flow-induced levels of PGE2 accumulation in media from cells incubated for 1 h postflow were significantly higher compared with media from cells collected immediately postflow. Similarly, PGE2 levels in media from no-flow control cells incubated for 1 h postflow were significantly elevated compared with levels from no-flow control cells collected immediately postflow. These findings were similar to those observed in the DN-8 cell line at 48 h when coupling was still intact, suggesting that a comparison of baseline PGE2 accumulation levels from media collected from incubated postflow cells is more appropriate than in media collected immediately postflow and may be more sensitive to extracellular regulation.

Curiously, we found that basal PGE2 levels were elevated in the DN-8 cells at the 96-h time point. Although we are unable to definitively explain this result, it is unlikely that the elevation was a result of the transfection process, since the control transfectant DN-VC cells did not exhibit a similar trend. To further address this issue, we added the ionophore 4-bromo-calcium (50 µM for 15 min) to confluent slides of DN-8 cells and measured PGE2 accumulation levels in excess of those shown in Fig. 4 (data not shown), indicating that increases beyond these basal levels were indeed possible. In any case, we do not feel that these findings detract from the main finding of this paper, namely that GJIC-deficient cells do not respond to oscillatory fluid flow with an increase in PGE2 release.

It is also possible that factors other than GJIC are involved in the responsiveness of a cell ensemble to oscillatory flow by affecting the inherent responsiveness of the individual cells. For instance, it is possible that membrane permeability or morphological changes in the membrane are important and could lead to sensitivity changes in protein receptors, ion channels, and cytoskeletal elements. This may help to explain the increase we observed in basal PGE2 production levels in the DN-8 cells at the 96-h time point. Furthermore, it is also possible that GJIC may affect such changes in cellular sensitivity. For instance, it has previously been shown that GJIC contributes indirectly to morphological changes by contributing to extracellular matrix organization (3).

Although a substantial body of evidence exists linking GJIC and cellular responsiveness to physical stimuli, the work to date has provided only indirect evidence. For instance, several studies have shown that the application of physical stimuli in vitro results in increased Cx43 expression in both osteoblasts (43) and osteocytes (29), a finding that parallels those in smooth muscle (5) and endothelial cells (11). However, these studies do not address changes in coupling or that changes in coupling influences the sensitivity of the cell ensemble. Thus it is important to distinguish between connexin formation and functional coupling, which would provide direct evidence to suggest that gap junctions are important in mechanotransduction.

In this study, we investigated the role of GJIC in oscillatory fluid flow-induced PGE2 production and changes in Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling. We found direct evidence to indicate that the PGE2 response was dependent on gap junctions, demonstrated by the lack of PGE2 released in the gap junction-deficient DN-8 cell line compared with the DN-VC cell line. In addition, by investigating real-time Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> responses in these cell lines, we found that all three cell lines were able to respond to oscillatory fluid flow with an immediate increase in [Ca2+]i. Finally, by blocking the Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> response with thapsigargin, we demonstrated that the PGE2 response in MC3T3-E1 cells to oscillatory fluid flow does not depend on an increase in [Ca2+]i. Together, these findings strongly suggest an important role for gap junctions and GJIC in bone cell mechanotransduction mechanisms.


    ACKNOWLEDGEMENTS

We thank Dr. Zhiyi Zhou for maintenance of the cell lines and technical assistance with the double-labeling assays.


    FOOTNOTES

This work was supported by U.S. Army Grant DAMD17-98-1-8509, National Institutes of Health Grants AR-45989 and AG-15107, and the Whitaker Foundation and National Cancer Institute Grant CA-211042JET.

Address for reprint requests and other correspondence: M. M. Saunders, Dept. of Orthopedics and Rehabilitation, Pennsylvania State Univ. College of Medicine, PO Box 850, 500 Univ. Dr., Hershey, PA 17033-0850 (E-mail: msaunder{at}mrl.hmc.psu.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.

Received 22 November 2000; accepted in final form 21 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 281(6):C1917-C1925
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