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Proc Natl Acad Sci U S A. Aug 3, 2010; 107(31): 13648–13653.
Published online Jul 19, 2010. doi:  10.1073/pnas.1009382107
PMCID: PMC2922284
Applied Biological Sciences

Dendritic processes of osteocytes are mechanotransducers that induce the opening of hemichannels

Abstract

Osteocytes with long dendritic processes are known to sense mechanical loading, which is essential for bone remodeling. There has been a long-standing debate with regard to which part(s) of osteocyte, the cell body versus the dendritic process, acts as a mechanical sensor. To address this question experimentally, we used a transwell filter system that differentiates the cell body from the dendritic processes. Mechanical loading was applied to either the cell body or the dendrites, and the osteocyte’s response was observed through connexin 43 hemichannel opening. The hemichannels located on the cell body were induced to open when mechanical loading was applied to either the dendritic processes or the cell body. However, no significant hemichannel activity in the dendrites was detected when either part of the cell was mechanically stimulated. Disruption of the glycocalyx by hyaluronidase on the dendrite side alone is sufficient to diminish a dendrite’s ability to induce the opening of hemichannels on the cell body, while hyaluronidase has no such effect when applied to the cell body. Importantly, hyaluronidase treatment to the dendrite side resulted in formation of poor integrin attachments with the reduced ability of the dendrites to form integrin attachments on the underside of the transwell filter. Together, our study suggests that the glycocalyx of the osteocyte dendritic process is required for forming strong integrin attachments. These integrin attachments probably serve as the mechanotransducers that transmit the mechanical signals to the cell body leading to the opening of hemichannels, which permits rapid exchange of factors important for bone remodeling.

It is widely accepted that osteocytes are the principal mechanosensory cells of the bone tissue and are implicated to be involved in bone homeostasis (1). Osteocytes are embedded inside the bone tissue and are surrounded by fluid filled spaces known as lacunae (2). Long dendritic processes of osteocytes form a network connecting the neighboring osteocytes and the cells on the bone surface such as osteoblasts and osteoclasts (3). Osteocytic dendrites are surrounded by canalicular wall, thus forming a lacuno-canalicular network (4). Mechanical signals sensed by osteocytes are converted to chemical signals, and the lacuno-canalicular network plays a critical role in conveying these signals to osteoblasts, osteoclasts, and bone-lining cells (5). Gap junctions are present at the tip of osteocytic dendritic processes and play an important role in communicating cellular signals (6). Gap junctions are formed by proteins known as connexins, and hexameric forms of connexins are called connexons (7). Two such connexons present on adjacent cells dock onto each other to form a gap junction channel. When connexons are present on an unapposed cell membrane they are known as hemichannels (8). Hemichannels are involved in communicating with the extracellular environment and are implicated in the exchange of molecules that are involved in bone remodeling, such as prostaglandin E2 (PGE2) (9). Our previous studies have shown that hemichannels formed by connexin 43 (Cx43) are abundantly expressed in osteocytes and are mechanosensitive (10). We also have shown that their opening is adaptively regulated by different magnitudes of mechanical stimulation (11).

Mechanical loading of bone causes fluid flow in the lacuno-canalicular network that is proposed to result in drag forces that act directly on the tethering filaments that attach the osteocyte cell process membrane to the canalicular wall (12, 13). The flow-induced drag forces on the tethering filaments are then transmitted via linker molecules to the central actin filament bundle of the process. This is proposed to lead to a large amplification of whole tissue strains at the cellular level and result in cytoskeletal reorganization and cellular signaling (12, 14). These strains are concentrated around integrin attachments along the dendritic process, attachments that are absent from the pericellular space surrounding the cell body in its lacuna (15, 16). Mechanical stimulation of osteocytes through fluid flow leads to increased dendritic processes, and osteocytes are shown to arrange in the direction of the flow (17).

Extracellular matrix proteins are implicated in bringing about changes in the cytoskeletal organization (18). The glycocalyx present in the endothelial extracellular matrix has been shown to act as a transducer of mechanical signals in endothelial cells and is involved in changes in the actin cytoskeletal organization (19, 20). In osteocytes, it is shown that the glycocalyx degradation results in reduced PGE2 release after fluid flow shear stress (21). These studies imply that the glycocalyx could be involved in causing changes in the cell membrane leading to actin cytoskeletal reorganization and thereby regulating the release of molecules, such as PGE2. In addition, it has been shown that osteocyte morphology and alignment also affect mechanosensory nature of these cells (22). As these studies implicate the unique role of cell morphology in regulating the mechanosensing by osteocytes, it is imperative to experimentally differentiate which part(s) of these cells, dendrites or the cell body, is involved in mechanotransduction. By taking advantage of a transwell filter system, we show here that osteocyte dendritic processes sense mechanical loading, which is likely to be transmitted through the glycocalyx, leading to the opening of hemichannels on the cell body. This study provides direct evidence suggesting the specific role of dendrites and the glycocalyx in transducing mechanical signals and in the regulation of the opening of hemichannels.

Results

Mechanical Stimulation on Dendrites Led to the Opening of Hemichannels on the Cell Body of Osteocytes.

MLO-Y4 cells were cultured on transwell filters with a pore size of 1 μm in order to separate cell bodies and dendrites (Fig. 1A). In order to confirm the separation of dendrite from cell body through the transwell filters, we performed immunostaining using antibodies against α5 integrin, a membrane marker, and β-actin, a cytoskeletal protein. When antibodies against α5 integrin and β-actin were used for immunostaining, dendrites (green punctae) of MLO-Y4 cells could be visualized passing through the blue autofluorescent pores of the transwell filters (Fig. 2 A and B Bottom). Nuclei of these cells were stained using DAPI. Confocal z-stacking images showed that the cell bodies were retained above the filter membrane and dendrites invade through the filter (Fig. 2A Left). HeLa-43A cells that overexpress Cx43 were used as a control because these cells lack dendrites. We could not obtain any significant fluorescence signal from the bottom side of the filter membrane (presumably the dendritic side) when immunostained with β-actin antibody (Fig. 2C Bottom); however, β-actin could be observed in the cell body (Fig. 2C). These results suggest that the pores on the transwell membrane allow dendrites, but not DAPI-stained cell bodies, to pass through, confirming the effectiveness of this approach in separating the cell body from the dendrites of the osteocytes.

Fig. 1.
Schematic diagram of osteocyte culture on transwell inserts and application of mechanical loading on cell body or dendrites. (A) Transwell inserts with a pore size of 1.0 μm permit the dendritic processes of osteocytes to pass through. ...
Fig. 2.
Transwell separates cell bodies from dendrites. (A) Either side of fiber insert membrane cultured with MLO-Y4 cells were labeled with anti-α5-integrin antibody (green) and DAPI (blue). The α5-integrin staining (green) fills the cells and ...

The total force acting over the approximate area (20 μm × 20 μm) of the cell attached to the substrate resulting from the drop landing on the substrate reaches a peak of 1.2E-5 N at approximately 0.05 s after the impact, with an average force of approximately 1.0E-5 N sustained over a period of approximately 0.10 s. This force results in a computed peak pressure on the substrate of approximately 250 Pa (250 pN/μm2) at approximately 0.10 s. This is the computed peak pressure acting over the approximate area of the cell directly below the center of the drop. The shear stress imparted by the spreading droplet determined via the computational fluid dynamics simulation was computed to range from a peak of 140 dynes/cm2 at a radial distance of 0.3 cm from the center on the drop at impact to 37 dynes/cm2 at a radial distance of 0.9 cm to 1 dyne/cm2 at a radial distance of 1 cm as the droplet spread across the surface of the culture.

We tested the responsiveness of the dendrites and the cell bodies to mechanical stress through the determination of Cx43 hemichannel activity using a dye uptake assay. Hemichannels on the cell bodies of MLO-Y4 cells were open as indicated by Lucifer yellow (LY) uptake when the cell body was mechanically stimulated by dropping the media (Fig. 3A). This resulted in an over 8-fold increase in dye uptake when compared to that of the controls. However, there is no significant dye uptake in dendrites when the cell bodies were stimulated. Similarly, dendrites were stimulated by inverting the filter and dropping the media onto the dendritic side (Fig. 1B). Interestingly, there was about an 8-fold increase in dye uptake in cell bodies when the dendrite side was mechanically stimulated. Hemichannel activity on the dendrites was not observed with mechanical stimulation of the dendritic side. The experiments performed in MLO-Y4 cells were validated in primary osteocytes and the results obtained were analogous (Fig. 3B). The results generated from MLO-Y4 cells and primary osteocytes suggest that hemichannel opening on the cell body can be induced by the application of mechanical stimulation either locally on the cell body or distally from dendritic processes. The data indicate that the dendritic processes are the likely mechanical transducers that transmit the signals to the cell body to activate hemichannels. Also, a higher dye uptake through the cell body could be due to the abundance of hemichannels on the cell body in comparison to the dendritic process.

Fig. 3.
Application of mechanical loading on either cell body or dendritic processes induced the opening of hemichannels. Dye uptake assay after application of normal shear stress on (A) MLO-Y4 cells, (B) primary osteocytes, and (C) HeLa43A. (A) MLO-Y4 (B) primary ...

Similar experiments were performed using HeLa-43A cells that form functional Cx43 hemichannels but lack dendritic processes. There was over a 9-fold increase in dye uptake when the cells were mechanically stimulated on the cell body (Fig. 3C). When the bottom of the filter (assumed to be the dendritic side) was stimulated, unlike in MLO-Y4 and primary osteocytes, no significant dye uptake was observed. Because HeLa-43A cells do not have dendritic processes, these cells served as appropriate negative controls to exclude the possibility of potential stimulation in the absence of dendritic process.

Glycocalyx Around the Dendritic Processes Plays an Important Role in Mechanotransduction.

The extracellular matrix surrounding the osteocytes is considered to be important for sensing the mechanical signals (12). Furthermore, it has been shown that an intact glycocalyx is required for fluid flow-induced PGE2 release in MLO-Y4 cells (21). Weak glycocalyx tethering is required for formation of strong integrin attachments, which are involved in amplification of mechanical strain (14). To understand if the glycocalyx tethering is required for osteocyte mechanosensing and if it has any differential effects on osteocyte cell body compared to osteocyte dendrites, we used hyaluronidase to degrade glycocalyx. Hemichannel function on cell body of MLO-Y4 cells was not affected by hyaluronidase treatment when the cell body side was mechanically stimulated (Fig. 4A). However, when dendrites were treated with hyaluronidase and mechanical stimulation was applied to dendrites, opening of hemichannels was abolished. Similarly when both the cell body and dendrites were treated with hyaluronidase and dendrites were mechanically stimulated, no hemichannel opening was observed on the cell body side. These results suggest that the glycocalyx around dendritic processes is an important extracellular component that transmits the signals to cell bodies to induce the opening of hemichannels.

Fig. 4.
Disruption of glycocalyx on dendrites prevented the opening of hemichannels on cell body. Cell bodies of MLO-Y4 cells treated with hyaluronidase were mechanically stimulated and were incubated with LY and RD mixture. Hyaluronidase was applied to the dendrites, ...

To understand the effect of hyaluronidase treatment on dendrites and cell bodies, the cells cultured on transwell filters were immunostained using α5-integrin antibody. In untreated control cells, the immunostaining pattern is similar to that observed in Fig. 2A. Confocal z-stacking images show that the majority of the dendrites penetrate through the pores present on the transwell filters (Fig. 4B Bottom, arrows). Some punctae that do not line up with the pores are likely the dendritic processes that turn and attach to the surface below the filter (as shown in Fig. 1A). A similar observation was made when the transwell filter side harboring the cell bodies was treated with hyaluronidase (Fig. 4C Bottom). Interestingly, when the dendrite side of the filter was treated with hyaluronidase, almost all punctate α5-integrin signals at the dendritic side disappeared (Fig. 4D). These results suggest that the intact glycocalyx is required for forming integrin attachments and maintenance of the dendritic structure, in order to convey the mechanical signals to the cell body.

Discussion

The location of osteocytes within bone makes them the leading candidate for the mechanosensory cell of the bone tissue (23). Previous studies have shown that the osteocytes are responsive to mechanical loading and release molecules, such as PGE2 and nitric oxide (9, 24, 25). Since osteocytes are surrounded by fluid-filled spaces, they continuously experience fluid flow shear stress (26). Also, osteocytic dendrites may experience higher levels of mechanical strain resulting from fluid flow within the canaliculi compared to the cell bodies because the drag forces acting on the dendritic processes are hypothesized to be significantly greater than those acting on the cell bodies (14, 15). Therefore, osteocyte cell bodies and dendrites could be differentially regulated by mechanical stimulation and could demonstrate differences in the manner of the response.

In this paper, using a transwell system, we differentially stimulated osteocyte cell bodies and dendrites and observed the response through Cx43 hemichannel activity in MLO-Y4 cells and primary osteocytes. The mechanical stimulation of osteocyte cell bodies resulted in opening of Cx43 hemichannel located on cell body, indicating a local effect. In contrast, dendrites exhibit minimal hemichannel opening when stimulated either on the dendritic or cell body side. Alternatively, osteocyte dendritic stimulation leads to the opening of hemichannels on the cell body. The lack of hemichannel activity in dendrites could be due to multiple factors, including scarce Cx43 hemichannels in the dendrites as the surface area of cell bodies is much higher than that of dendrites. It also could be due to the lack of responsiveness to mechanical loading or sensitivity of dye uptake assay. The magnitude of mechanical stimulation applied to the dendrites and cell body in our assay was comparable, but differences in responsiveness of dendrites and cell bodies toward mechanical stimulation were clearly demonstrated. The possible impact of mechanical loading independent of dendritic processes was excluded because hemichannels on the cell body of HeLa-43A cells lacking dendrites failed to induce open when the presumed dendritic side was stimulated. Therefore, these results indicate that both dendrites and cell bodies of osteocytes are mechanosensitive, but functional hemichannels are primarily present on cell body. More importantly, dendritic processes serve as a mechanotransducer that transmits the signals to cell body leading to opening of the hemichannels. Also, dendritic processes that do not transgress the filter could also be involved in mechanosensing when mechanical stimulation was applied to the top side (presumed cell body side) of the filter. However, stimulation of dye uptake of the cell body from dendritic sides suggests that dendrites are mechanical transducer/sensors that lead to the opening of hemichannels. Consistently, a recent report shows that the osteocyte cell bodies and dendritic processes are differentially stimulated and the stimulated osteocyte dendritic processes have higher intracellular calcium response when compared to cell bodies (27). Because the measure for opening of hemichannels used in our experiments is through LY dye uptake, we cannot rule out the possibility that the dye uptake observed could be due to other integrin-associated channels. However, we have previously shown that the dye uptake observed after mechanical stimulation could be blocked by an antibody generated using Cx43 E2 loop as the antigen (11), suggesting that the Cx43 hemichannels are the major mechanosensory channels expressed in osteocytes.

Osteocytic dendritic processes are proposed to be tethered to the canalicular wall through tethering filaments via glycocalyx (14). The glycocalyx is referred to as a pericellular, carbohydrate-enriched network of glycoproteins and proteoglycans (28). Enzymatic degradation (via hyaluronic acid) of the glycocalyx network resulted in the reduction of PGE2 release by osteocytes subjected to fluid flow shear stress (21). We observed that Cx43 hemichannel opening on cell bodies was not affected with hyaluronidase treatment. However, when dendrites were treated with hyaluronidase and were stimulated, hemichannel opening was totally abolished. Confocal imaging showed that after glycocalyx degradation, the punctate integrin signals on the dendritic side disappeared. This could be partially explained by the fact that the cell processes are unable to attach to the surface below the transwell filter after the glycocalyx is degraded. The glycocalyx surrounding the processes consists of hyaluronan, which is bound to the cell membrane of the processes likely by CD44, which are weak, flexible tethering attachments (13, 28). In addition, there are strong, more rigid and sparsely spaced integrin attachments that generate large strains and signaling (16, 18). It is probable that if the weak hyaluronan attachments are compromised, the strong integrin attachments are unable to form resulting in the disruption of signaling from the processes to the cell body. Our observation of the disappearance of the punctate integrin signals from the dendrite side of the filter after disruption of glycocalyx supports this possibility.

One important question that remains unsolved is why hyaluronic acid degradation does not have an effect on the mechanosensing by osteocyte cell bodies. A possible explanation is that it is not known if there is a difference in the amount of hyaluronic acid that is deposited over the cell bodies and dendrites. Because forces generated by fluid flow shear stress are too small to be sensed by osteocyte cell bodies present in lacunae, the cell bodies could be directly sensing the mechanical stimulation. To substantiate this explanation, studies have shown that the direct mechanical stimulation of osteocytes through a microneedle resulted in release of NO (24). In parallel, another study (22) shows that the mechanical stimulation of round osteocytes in solution using optical tweezers results in the release of NO. These studies imply a difference in the mechanosensory nature of osteocytic dendrites and cell bodies, which is substantiated by our findings.

A limitation of our study is that the mechanical stimulation of the cell was applied via a fluid drop impacting the cells cultured on a substrate. The forces and pressures generated on the cell body and cell processes are likely to be larger than those expected to occur in vivo based on the analytical predictions (15). Furthermore, the loading on the cell processes in vivo is predicted to occur due to the drag force generated on the tethering elements from the skeletal loading-induced fluid flow within the canaliculi. Our loading generates compressive forces and pressure on the cultured cell process and cell bodies, and the subsequent flow around these structures may produce a tensile drag force on the tethering elements, particularly when a glycocalyx is present.

Overall, our study suggests that glycocalyx on the surface of dendritic processes plays an essential role in mechanotransduction and a different, local mechanosening mechanism could be involved in lacunae where the cell bodies of the osteocytes are harbored. Therefore, it is likely that hemichannels in osteocytes are regulated distally by dendritic process through glycocalyx in response to mechanical stress. The bone modulators released/uptaken by hemichannels are essential for bone modeling/remodeling process.

Materials and Methods

Isolation of Primary Osteocytes and Cell Culture.

Preparation of primary osteocytes from chick calvaria was based on previously published procedures (29) with some modifications. Briefly, calvarial bone was dissected from 16-day embryonic chicks and minced into very small pieces. The soft tissues and osteoid were removed by several rounds of collagenase treatment followed by decalcification using EDTA. The final particles were treated with collagenase and vigorously agitated to release osteocytes. This preparation contained most of the osteocytes with very few osteoblast/fibroblast cells, which was separated from osteocytes by incubating the cells with collagen-coated culture plates sequentially for 5 min, 1, 2, and 4 h, of which osteoblast/fibroblast was attached to the plates.

Primary osteocytes and MLO-Y4 cells were cultured in α-MEM (minimal essential medium) media supplemented with 2.5% of FBS, 2.5% of bovine calf serum. HeLa-43A cells stably transfected with Cx43 cDNA were cultured in DMEM media supplemented with 10% FBS and G418 (1 mg/10 mL). The cells were plated on rehydrated (250-μL media for 30 min) transwell filters inserts (BIOCOAT® Cell Culture Inserts, 1.0 μm pores) (354585, BD Biosciences) (Fig. 1A). The transwell occupancy rate is approximately 80 to 85% and cells are at about 50% confluency. To study the role of glycocalyx, the cells were treated with hyaluronidase (160 U/mL) (21). Briefly, either side of the filters was incubated with hyaluronidase dissolved in PBS for 1 h at 37 °C. The untreated side of the filter was incubated with PBS alone. The cells were then either subjected to mechanical stimulation or processed for immunostaining.

Mechanical Loading Assay on Transwell Filters.

The mechanical stimulation of the cells (MLO-Y4 or HeLa-43A) in the transwell inserts was conducted by dropping 50 μL of S-minimum essential medium (SMEM) from different heights (59.6 cm, 27.3 cm, and 5.7 cm) through a pipette in the center and in three edges of the membrane (four drops total per well) from either side of the filter (Fig. 1B). After quantifying the hemichannel activity from these three different heights, stimulation of Cx43 hemichannels in MLO-Y4 cells was found to be optimal at 5.7 cm. Hence, we maintained this height throughout our experiments. Either sides of the filter were then placed in a clean well with 500 μL of either SMEM or the dye solution [mixture of 1% LY and 1% rhodamine-dextran (RD)]. After washing with PBS the cells were fixed with 2% paraformaldehyde (PFA) and incubated with 2 ng/μL solution of DAPI to stain nuclei. The transwell membrane was then peeled from the inserts and was mounted onto glass slides using Vectashield Mounting Medium (H-1000, Vector Laboratories). Images were taken with a Zeiss Epifluorescence microscope using the appropriate filters.

Estimated Force, Pressure, and Shear Stress Acting Cell Body and Dendrite due to Drop Loading.

The force transmitted to the osteocyte cell body and dendrite was estimated using an explicit dynamics finite element analysis approach (LS-DYNA, v.9.71, LSTC, Co.). The estimated mechanics loads on the transwell filter are independent of the number of cells or dendrites present on the transwell filter as this estimation does not take into account the microenvironment of the cells. The drop was modeled as an incompressible liquid with the physical properties of water. The 50-μL liquid drop was placed at 5.7 cm above a substrate within a 1-g gravitational field and allowed to fall under the force of gravity simulating the experiment (Fig. 1C). At impact on the substrate, the force generated by the drop on the substrate directly below the center of the drop as a function of time was computed over an area approximating the size of a MLO-Y4 cell (20 μm × 20 μm). In addition, the pressure exerted on this area was computed as a function of time. To determine the shear stress acting on the plated cells, the dynamic impact of a liquid (water) droplet onto the filter was simulated using computational fluid dynamics methods (FLOW-3D). FLOW-3D is a volume-of-fluid-based algorithm that solves the Navier-Stokes equations for flow of two-phase or two-component fluid systems and directly computes the sharp interface between liquid and gas phases accounting for surface tension effects. In this specific problem, a liquid droplet of water is simulated falling through an air-filled environment and impacting onto the filter surface. To expedite the simulation process, the droplet is allowed to fall as a rigid body to a standoff distance of 0.004 cm from the surface. The droplet has then achieved a velocity of 105.8 cm/s. This then defines the initial conditions for the axisymmetric simulations. A uniform grid resolution of 0.002 cm was used to resolve the liquid droplet (~114 cells across the diameter of the droplet), and allowed for an appropriate resolution of the resulting boundary layer flow. For these simulated conditions the mean shear stress is predicted as a function of the radial distance from the center of the drop impact. Details of the computational fluid dynamics simulations can be found in SI Text.

Immunostaining and Image Analysis.

Cells fixed with 2% PFA were permeabilized with 0.5% Triton-X-100 and were blocked using 3% BSA. After blocking, both MLO-Y4 and HeLa-43A cells were incubated with anti-α5 integrin (1[ratio]50) (AF1754, R and D Systems) or β-actin (1[ratio]500) (A5441, Sigma-Aldrich) antibody separately for 1 h at room temperature and were probed with appropriate secondary antibodies. Images for this experiment were taken on an Olympus IX81 Confocal microscope with FluoView software in the UTHSCSA. Z-series scans were obtained to view the staining at different depths.

ImageJ software was used to analyze the fluorescent images from the standardization and transwell experiments. The “Cell Counter” plug-in and “Measure” tools were used to select and measure the intensity of the LY dye uptake. The Cell Counter was used to count the number of cells that took up LY and the total number of cells indicated by DAPI staining. LY-stained cells that also took up RD were excluded from intensity measurements and counting. ImageJ was also used to analyze the stacked images obtained from the immunostaining experiment.

Statistical Analysis.

Data were analyzed using one-way ANOVA and Student Newman—Keul’s comparison test with the Instat biostatistic program (GraphPad software). Data are presented as the mean ± SEM of three determinations. Asterisks indicate the degree of significant differences compared with the controls (*, P < 0.05;**, P < 0.01).

Supplementary Material

Supporting Information:

Acknowledgments.

We thank the Communicating Editor, Dr. Sheldon Weinbaum, for his invaluable comments, suggestions, and extensive communication in improving our study. We thank Dr. Bruce Nicholson at University of Texas Health Center at San Antonio (UTHSCSA) for generously providing HeLa-43A cell and members of Dr. Jiang’s laboratory for critical reading of the manuscript. Confocal images were generated in the Core Optical Imaging Facility, UTHSCSA. This work was supported by National Institutes of Health (NIH) Grant P01 AR46798 (to J.X.J and D.P.N), Welch Foundation Grant AQ-1507 (to J.X.J) and NIH BSURE Undergraduate Research Support (to K.P).

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1009382107/-/DCSupplemental.

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