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. Author manuscript; available in PMC: 2007 Feb 13.
Published in final edited form as: Cell Commun Adhes. 2006;13(1-2):115–125. doi: 10.1080/15419060600634619

Mechanical Loading Stimulates Expression of Connexin 43 in Alveolar Bone Cells in the Tooth Movement Model

JELICA GLUHAK-HEINRICH 1, SUMIN GU 2, DUBRAVKO PAVLIN 1, JEAN X JIANG 2
PMCID: PMC1797153  NIHMSID: NIHMS16616  PMID: 16613785

Abstract

Bone osteoblasts and osteocytes express large amounts of connexin (Cx) 43, the component of gap junctions and hemichannels. Previous studies have shown that these channels play important roles in regulating biological functions in response to mechanical loading. Here, we characterized the distribution of mRNA and protein of Cx43 in mechanical loading model of tooth movement. The locations of bone formation and resorption have been well defined in this model, which provides unique experimental systems for better understanding of potential roles of Cx43 in bone formation and remodeling under mechanical stimulation. We found that mechanical loading increased Cx43 mRNA expression in osteoblasts and bone lining cells, but not in osteocytes, at both formation and resorption sites. Cx43 protein, however, increased in both osteoblasts and osteocytes in response to loading. Interestingly, the upregulation of Cx43 protein by loading was even more pronounced in osteocytes compared to other bone cells, with an appearance of punctate staining on the cell body and dendritic process. Cx45 was reported to be expressed in several bone cell lines, but here we did not detect the Cx45 protein in the alveolar bone cells. These results further suggest the potential involvement of Cx43-forming gap junctions and hemichannels in the process of mechanically induced bone formation and resorption.

Keywords: Cx43, gap junctions, mechanical loading of bone, tooth movement model

INTRODUCTION

Gap junctions and hemichannels formed by con-nexin (Cx) 43 in osteoblasts and osteocytes are reported to modulate biological responses induced by mechanical loading (Saunders et al. 2001; Saunders et al. 2003; Cherian et al. 2005). Signals generated by mechanical stimulation are hypothesized to be transmitted between bone cells through gap junction channels, and between cells and extracellular matrix through hemichannels. Cx43 mutations have been reported to cause the pleiotropic phenotypes of oculodentodigital dysplasia (Paznekas et al. 2003). Cx43-null mice have delayed ossification, craniofacial abnormalities, and osteoblast dysfunction (Lecanda et al. 2000).

Gap junction channels that connect the cytoplasm of two adjacent cells have been shown to be important in regulating cell and tissue functions in many organs (Goodenough et al. 1996). The existence of gap junction structures between osteoblasts, osteoblastosteocytes, and between osteocytes has been revealed by morphological studies in bone tissue (Doty 1981). Gap junction channels are formed by members of a family of protein known as connexins. Cx43 is found as predominant gap junction protein expressed by bone cells and bone marrow stromal cells, although Cx45 and Cx46 were also identified in these cells (Palumbo et al. 1990; Edelson 1990; Su et al. 1997; Vander Molen et al. 1996; Steinberg et al. 1994; Schirrmacher et al. 1992; Mason et al. 1996; Ilvesaro et al. 2002; Durig et al. 2000). We and others have shown that connexins, in addition to being the major component of gap junction channels, exist and function in the form of unapposed halves of gap junction channels called hemichannels (Goodenough et al. 2003). These hemichannels modulate physiological functions of bone cells, such as effects of bisphosphate in prevention of apoptosis and release of prostaglandin in response to mechanical loading (Cherian et al. 2005; Plotkin et al. 2002).

Similar to other skeletal tissues, homeostasis of alveolar bone is maintained by dynamic process between bone formation and resorption (Sodek et al. 2000). Exposure of teeth to mechanical loading activates bone remodeling (King et al. 1997; Pavlin et al. 2000b; Pavlin et al. 2000a; Alhashimi et al. 2000; Guajardo et al. 2000). Our recent in vivo studies showed mechanical loading induced by tooth movement model stimulated a specific temporal pattern of gene expression in osteoblasts and matrix of alveolar bone (Pavlin et al. 2001; Gluhak-Heinrich et al. 2003). Previous report showed the increased expression of Cx43 in rat mandibular bone and periodontal ligament cells during experimental tooth movement (Su et al. 1997). Osteocytes are the primary mechanosensory cells in bone (Cowin et al. 1991; Aarden et al. 1994; Burger and Klein-Nuland 1999). In our previous studies, we have reported that mechanical loading on osteocytes induces the release of prostaglandin (PG) E2 and this release is mediated through hemichannels formed by Cx43 (Cherian et al. 2005). Released PGE2 acts in an autocrine fashion to stimulate gap junction function and Cx43 expression (Cheng et al. 2001b).

Gap junctions and hemichannels appear to be crucial for bone growth and function. However, it is not clear how the expression of connexin is coordinated during bone formation and remodeling process in vivo in response to mechanical stimulation. Mechanical loading induced by tooth movement permits us to assess the levels of Cx43 expression at both formation and resorption sites within the same sample. The differential expression of Cx43 mRNA and protein in response to loading at these two sites suggests the crucial role of Cx43 in bone formation and remodeling process.

MATERIALS AND METHODS

Mechanical Loading of Alveolar Bone

Manipulation and treatment of animals were performed according to the protocol approved by the Institutional Animal Care and Usage Committee and was described previously (Pavlin et al. 2000a, 2000b). Briefly, the mice were anesthetized before insertion of appliance. Orthodontic tooth movement, calibration of appliance, and biomechanical characterization of the model were conducted as described (Pavlin et al. 2000a, 2000b). The orthodontic appliance consisted of a coil spring bonded directly to the incisors and maxillary first molar. A continuous force (10–12 g) was applied for various periods of time from 6 h to 7 days. Mechanically loaded and control alveolar bone sites adjacent to the palatal and distobuccal roots of the molar were obtained for analysis.

Preparation of Histological Sections

Mouse maxillae were dissected and fixed in 4% paraformaldehyde overnight. After demineralization in 15% EDTA for 6 weeks at pH 7.5 and 4°C, samples were dehydrated in increasing concentrations of methanol (on ice), embedded in paraffin, and sectioned at 6–8 μm thickness.

In Situ Hybridization Methods and Quantification

Preparation of Probe

RNA probe for Cx43 was used for hybridization. Mouse Cx43 antisense and sense RNA probes of 1.1 kb were prepared from mouse Cx43 bluescript plasmid DNA, linearized with BamHI and EcoRI, and transcribed with T3 or T7 polymerase, respectively (Gluhak-Heinrich et al. 2003). RNA probes were transcribed in vitro in the presence of 32P-rUTP and hydrolyzed in 40 mM NaHCO3/60 mM Na2CO3, pH 10.2, for the desired time at 60°C. Sizes of the RNA probes were confirmed by electrophoresis on 5% PAGE gels containing 15 M urea.

In situ hybridization

The in situ hybridization was performed using a modification of the procedure described previously (Gluhak-Heinrich et al. 2003). Briefly, after deparaffinization, sections were treated with proteinase K. Hybridization was performed at 55°C overnight with 32P rUTP labeled Cx43 RNA probe. After hybridization, sections were incubated with RNase. Consecutive 5 min washes at 57°C were conducted with 2X SSC, 0.5X SSC, and 0.1X SSC. For autoradiography, slides were dipped in photographic emulsion (Kodak NTB 3) and exposed for 3 weeks. The slides were counter-stained with hematoxylin, dehydrated through ethanol, cleared in xylene and mounted with Permount (Fisher Scientific, Pittsburgh, PA).

Quantification of Hybridization Signal and Image Analysis

Total number of osteocytes and osteocytes expressing the Cx43 mRNA was determined by counting osteocytes embedded in bone or osteoid within 200 μm of alveolar bone adjacent to the coronal 2/3 of the molar root. The percent of osteocytes expressing Cx43 mRNA was determined by counting cells in three independent fields for both mesial (resorption) and distal (formation) sites, using the number of osteocyte expressing target mRNA and total number of osteocytes. An osteocytes expressing Cx43 as counted positive if the grain count was at least three times the background of the adjacent area. A two-tailed unpaired Student’s t-test was used to determine the significance of differences in hybridization signal in osteocytes from loaded and control unloaded alveolar bone.

The intensity of the hybridization signal of Cx43 in osteoblasts with no more than 2–3 layers of cells on bone surface was quantified by video image microscopy as described previously (Gluhak-Heinrich et al. 2003). These sections were generated from only distal and mesial root of the mouse first molar. With tooth movement model we analyzed palatal and distal roots as two different samples, since direction of force was perpendicular only to these roots, not to mesial root. Direction of the applied force in areas around root apex and adjacent to mesial root were under different angles, therefore could not be defined as resorption or formation sites (Pavlin et al. 2000b). Variability of the overall hybridization signal from slide to slide was corrected using internal control. Briefly, two to three images of identical nonoverlapping fields along the length of the root, overlaying osteoblasts on the bone surface and fibroblasts in the center of periodontium, were captured on in situ autoradiographs. The level of Cx43 mRNA in fibroblasts was used as an internal standard to determine the mRNA changes in osteoblasts. The osteoblast-specific hybridization signal in treated periodontal sites was compared to that in control untreated sites using a two-tailed unpaired Student’s t-test to determine the significance.

Immunocytochemistry of Cx43 and Cx45 Proteins

Immunohistochemistry with antibodies against Cx43 and Cx45 was performed on sequential sections used for in situ hybridization. The rabbit antimouse Cx43 polyclonal antibody was generated in our laboratory previously (He et al. 1999). After deparaffinization and rehydration, endogenous peroxidase in the tissue was inactivated by treatment with the 3% H2O2 for 30 min. Retrieval of signals was performed with Vector demasking solution according manufactures instruction. Avidinbiotin-peroxidase kit for immunohistochemistry (Vector laboratories, Burlingame, CA) was used to detect Cx43 expression. After that, sections were blocked in PBS containing 1% goat serum at room temperature for 1 h. The rabbit antimouse-Cx43 polyclonal antibody was added on sections at dilution of 1:100 in PBS containing 1% BSA and 1% rabbit serum overnight at 4°C. After washing the sections, biotinylated antigoat antibody (Vector) was added at dilution of 1:200 and the sections were incubated at room temperature for 1 h. The sections were washed again and incubated with the ABC reagent (Vector) at room temperature for 30 min. Alkaline phosphatase substrate solution was used to visualize immunoreaction sites. The sections were mounted with 70% glycerol in PBS. Negative control was obtained by substituting the primary antibody with rabbit IgG.

For Cx45 immunohistochemical staining we used monoclonal mouse Cx45 antibody MAB3100 obtained from Chemicon International (Temecula, Ca) and diluted 1 to 50. Detection was performed with 3. 3′ diaminobenzidine after treatment with biotinylated antimouse secondary antibody (Dako Animal Research Kit Peroxidase for mouse antibodies; Dako, Fort Collins, CO).

RESULTS

Expression of Tartrate-Resistant Acidic Phosphatase (TRAP) Positive Cells at Resorption, but not Formation, Site of Mechanical Loading of Tooth Movement

To investigate the effect of mechanical loading on bone cells and their native environment, we utilized the mouse tooth movement model (Pavlin et al. 2000a, 2000b, 2001). Our previous studies have shown that using the mouse tooth movement model bone formation (in tension site) and resorption (in compression site) can be monitored simultaneously in vivo (Pavlin et al. 2000a). Our results showed positive TRAP staining in cells close to bone surface on compression (resorption) sites after 4 days of treatment (Figure 1, a). Staining was absent in cells on bone surface in tension (formation sites) sites (Figure 1, b). The pattern of TRAP staining suggests that, in this loading model compression sites represent bone resorption and osteoclast formation, whereas tension sites represent bone formation, which is consistent with our previous observations (Pavlin et al. 2000a, 2001).

Figure 1.

Figure 1

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Localization of TRAP positive cells in mouse maxillary first molar after 4 days of mechanical loading. Osteoclasts as TRAP positive cells (big filled arrowheads) are only shown in resorption site (a) compared to formation site (b). F, direction of the orthodontic force and tooth movement; T, tooth; P, periodontium; B, bone; BM, bone marrow space; white arrows, osteocytes; small filled arrows, osteoblasts.

Cx43 mRNA Expression in Periodontium under Mechanical Loading

Using in situ hybridization, we first analyzed the expression level of Cx43 mRNA in osteocytes of alveolar bone adjacent to growing incisors in 10-week-old mice. We then determined the changes in Cx43 mRNA expression in osteocytes and osteoblasts in response to mechanical loading in the tooth movement model and compared them to those in the untreated animals where no loading was applied. The control (left maxillae) and treated (right maxillae) sections were taken from the same animal at the respective time points. There was no difference in Cx43 mRNA expression between untreated and control (left maxillae) of treated animals. A uniform hybridization signal was present across the periodontium in the control sites (Figure 2, a–c) and after mechanical loading of 6 h, on both bone formation (d and e) and bone resorption (d and f) sites, and after 2 days of loading on resorption site (i). Increased hybridization signal was present in osteoblasts and cells closer to bone surface (small filled arrows) compared to the rest of the periodontium after 2 (h) and 4 days (k) of loading in formation periodontal sites. On the bone resorption sites, after 4 days of loading there was higher hybridization signal present in multinucleated osteoclasts close to the bone surface (l, big arrowheads). There was also an increase in Cx43 signal in preosteocytes in osteoid after 4 days of treatment (k, big filled arrows). However, mRNA signal in osteocytes (empty arrows) showed little transient increase after 2 days of treatment on both formation (h) and resorption sites (i). Cx43 mRNA signals in osteoblasts and bone lining cells were quantified in loaded and control samples using relative quantitative video image analysis as described previously (Pavlin et al. 2000a) (Figure 3). Cx43 mRNA was significantly up-regulated in alveolar osteoblasts after 2 days of treatment (73%) with 156%, 96% and 104% increase during 3, 4 and 7 days of loading, respectively (Figure 3, a). In resorption site, there was a significant increase of Cx43 mRNA with 27% after 4 and 32% after 7 days of loading, although the increase appeared to be less dramatic compared to that in formation sites (Figure 3, b). The increase of Cx43 mRNA expression at formation sites became even more apparent by comparing the ratio of signals at loaded vs. control sites (Figure 3, c). These results show a stimulatory effect of mechanical loading on Cx43 mRNA in osteoblasts and bone lining cells in bone formation site, and a slightly less in resorption site. Loading, however, failed to stimulate Cx43 mRNA expression in osteocytes.

Figure 2.

Figure 2

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In situ hybridization using Cx43 antisense probe in mouse maxillary first molar after mechanical loading using tooth movement. Ten-week-old mice were subjected to mechanical loading of tooth movement for 6 h, 2 days, and 4 days. The tissue sections were in situ hybridized with Cx43 antisense probe. Dark-field autoradiographs represent control (a), 6 h (d), 2 days (g) and 4 days (j) of loading. Light-field photomicrographs of untreated control (b, c), and loaded 6 h (e, f), 2 days (h, i), and 4 days (k, l) periodontal sides represent higher magnification of boxed areas in figures a, d, g, and j, respectively. F, direction of the force used for the tooth movement; T, tooth; P, periodontium; B, bone, O, osteoid, periodontal osteoblasts and fibroblasts, small filled arrows; preosteocytes, big filled arrows (panel k); osteocytes, empty arrows; multinucleated osteoclasts, big arrowheads (panel l).

Figure 3.

Figure 3

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The effect of mechanical stimulation of tooth movement on Cx43 mRNA in osteoblasts versus fibroblasts at formation and resorption sites. Ten-week-old mice were subjected to mechanical loading of tooth movement for 6 and 12 h, and 1, 2, 3, 4, and 7 days. The tissue sections were in situ hybridized with Cx43 antisense probe. The expression of Cx43 mRNA was quantified using video imaging microscopy. Each point is the mean ± standard error of the ratio of hybridization intensity in osteoblasts and fibroblasts from 3–6 samples. During 7 days of orthodontic treatment, the relative expression of Cx43 mRNA in osteoblasts and bone lining cells in formation and resorption sites is presented in (a) and (b), respectively. (c) shows a ratio of hybridization intensity in loaded and control sites.

Increased Accumulation of Cx43 Protein in Response to Mechanical Loading of Tooth Movement

Immunocytochemistry of Cx43 (dark stain) in the formation site at 6 h and 4 days after loading is shown in Figure 4. The same slide sets that were used in the in situ hybridization experiments were also used for immunocytochemistry experiments. Cx43 protein was detected using immunolabeling after 6 h and 4 day of loading using a polyclonal antibody specific for carboxyl-terminal of Cx43 that we have developed previously (He et al. 1999) (Figure 4). No signals were detected with preimmune antiserum (Figure 4, panel a). Cx43 protein was detected only in bone marrow spaces of control untreated sections (b). After 6 h of loading there were no significant changes in overall levels of Cx43 protein in either bone formation or bone resorption sites (c and d, respectively). Cx43 protein was significantly higher in alveolar osteoblasts after 4 days of treatment on bone formation sides. Increased distribution, although lower than in osteoblasts, was noticeable in periodontal fibroblasts on both bone formation and bone resorption sides (e and f). Significant increase of Cx43 protein was also observed in cells on bone surface on bone resorption sites (f) in TRAP positive cells (Figure 1, a) after 4 days of treatment, suggesting involvement of Cx43 in osteoclast formation. Interestingly, in contrast to Cx43 mRNA, distribution of Cx43 protein in osteocytes was markedly increased in response to loading. Cx43 protein appeared as punctate labeling, the typical pattern of gap junctions, between the osteocytes indicating staining along the dendritic processes within the bone canaliculi (Figure 4, f). These immunocytochemistry experiments have been repeated with at least two independent animals. These results suggest that longer periods of loading significantly stimulated the accumulation of Cx43 protein, but not Cx43 mRNA, in osteocytes and support the hypothesis that Cx43 protein is undergoing dynamic changes in level of distribution after mechanical loading.

Figure 4.

Figure 4

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The effect of mechanical stimulation by tooth movement on the level of Cx43 protein at formation and resorption sites. Immunocytochemistry of Cx43 expression of antigenic reactivity in negative control using preimmune (a), control unloaded alveolar bone (b), and after mechanical loading of 6 h (c, d), and 4 days (e, f). The bone formation sides after mechanical loading of alveolar are shown in (c) and (e) while the bone resorption sides are shown in (d) and (f). The hollow arrows indicate Cx43 immunoreactivity in the lacunae of the osteocyte. Star indicates Cx43 immunoreactivity appearing as punctate labeling along the dendritic processes within the canaliculi of the bone (f). F with arrows, direction of the orthodontic force and tooth movement; T, tooth; P, periodontium; B, bone; BM, bone marrow space.

Effect of Loading on Cx43 Protein in Osteoid and Osteocytes at Formation Site

The increased amount of Cx43 protein by 4 days of mechanical loading was further analyzed in osteoid and osteocytes at formation site (Figure 5). Immunostaining with Cx43 antibody shows elevation of Cx43 protein in periodontal osteoblasts and fibroblasts (small filled arrows), and preosteocytes (big filled arrows) (Figure 5, b). There are strong positive signals of Cx43 in osteoid (O) and embedded osteocytes (empty arrows) in bone. Cx43 staining appear as a punctate distribution along osteocytic cell-cell contacts and in osteocyte processes (labeled as stars), a typical expression pattern of gap junctions. This result confirms the upregulation of Cx43 protein distribution in osteoid and osteocytes in response to mechanical loading.

Figure 5.

Figure 5

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Expression of Cx43 in osteoid at bone formation site after mechanical loading by tooth movement. After 4 h of treatment, tissue sections were immunostained with anti-Cx43 antibody. Photomicrograph of hematoxylin-eosin staining (a) represents a consecutive section to that shown in (b). Immunostaining image (b) shows the expression of Cx43 protein in periodontal osteoblasts and fibroblasts (small filled arrows), preosteocytes in osteoid (O) (big filled arrows) and osteocytes (empty arrows). The reversal line between osteoid and mineralized bone is visible in (a) and labeled R (in callout). Punctate stating (*) between osteocytes suggests Cx43 expression in intercellular canaliculi.

Lack of Expression of Cx45 in the Alveolar Bone

Cx45 is reported to be expressed in osteoblast and osteocyte cell lines (Steinberg et al. 1994). We examined Cx45 protein in the alveolar bone sections of maxilla. There was no detectable specific staining for Cx45 protein in alveolar osteocytes, osteoblasts, and fibroblasts of mouse maxillae before or after loading by tooth movement (Figure 6, b). High distribution of Cx45 protein was detected only in blood vessels and bone marrow spaces of maxillae sections (a) and 12 day embryos with predominant expression in neural tube tissue (a). The expression pattern of Cx45 protein in mouse embryos was consistent with the previous published study (Delorme et al. 1997). The results suggest that Cx45 is unlikely to be the major connexin isoform in alveolar bone tissues.

Figure 6.

Figure 6

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Lack of Expression of Cx45 protein in alveolar bone cells. Cx45 immunostaining was detected in neural tube of 12-day-old embryo (a), and bone marrow spaces (BM) and blood vessels (b, small filled arrow) after 4 days of orthodontic treatment in periodontium. No Cx45 signals were detected in osteoblasts, and osteocytes on formation site (b). F, direction of the orthodontic force and tooth movement; T, tooth; P, periodontium; B, bone; BM, bone marrow space; osteocytes, big filled arrow; blood vessel, small filled arrow.

DISCUSSION

This study showed the differential expression of Cx43 in mouse alveolar bone in response to mechanical loading induced by tooth movement model. The stimulation of Cx43 mRNA expression by mechanical loading was observed in osteoblasts and lining cells on formation sites, and osteoclasts on resorption sites. However, there was no discernible increase of Cx43 mRNA expression in osteocytes. In contrast to mRNA, level of Cx43 protein in alveolar osteocytes was highly upregulated in response to mechanical loading. Appearance of the punctate staining was shown around cell body and, remarkably between osteocytes, suggesting the formation of gap junctions between osteocytes and possibly hemichannels in the absence of cell contacts. The differential regulation of Cx43 distribution in major cells of alveolar bone suggests the involvement of Cx43 in mechanically-induced process of bone formation and resorption.

Cx43 is expressed in all major bone cells including osteoblasts, osteocytes, osteoclasts and bone marrow stromal cells (Goodenough et al. 1996; Schirrmacher et al. 1992; Mason et al. 1996; Ilvesaro et al. 2002; Durig et al. 2000). Cx43 is also identified in mandibular bone and periodontal ligament cells of rat teeth (Su et al. 1997). Cx45 is expressed in UMR-102 and hFOB 1.19 osteoblastic, and MLO-Y4 osteocytic cell lines, but its role in these cells is unknown (Steinberg et al. 1994; Thi et al. 2003; Donahue et al. 2000b). In our study of Cx45 protein, although prominent in neural tubes and some cells in bone marrow, Cx45 was not detected in osteoblasts, osteocytes, and osteoclasts and did not show any response to mechanical treatment.

There are multiple molecular pathways that mediate mechanical signaling in bone (for recent review, see Rubin et al. 2005). It has been proposed that gap junctions through the propagation of intracellular signals contribute to mechanotransduction in bone (Donahue 2000a). Cyclical stretching enhances the phosphorylation of Cx43 and gap junction communication in primary osteoblastic cells (Ziambaras et al. 1998). A dominant negative mutant of Cx43 diminishes fluid flow-induced release of PGE2, but not Ca2+ responses (Saunders et al. 2003). In addition, the fluid flow-induced PGE2 response of osteoblastic ROS17/2.8 cells is gap junction-mediated and independent of intracellular Ca2+ (Saunders et al. 2003). Limited studies were reported to examine the expression and regulation of Cx43 in response to mechanical stimulation using in vivo animal models. By using a rat model system of experimental tooth movement, Su et al. (1997) reported that Cx43 is increased in osteoclasts and periodontal ligament cells in compression zones, and in ostebolasts and osteocytes in tension zones of the periodontal ligament. This observation partially agrees with our observation. However, in contrast to our observation, they observed high expression of Cx43 mRNA in osteocytes, although the specific punctate staining is not visible in their study. There are several major discrepancies between their studies and our work presented here. First, the rat model system of experimental tooth movement in their studies was conducted by extracting maxillary first molars to initiate supra-eruption of opposing mandibular molars. Our mouse model of tooth movement is well controlled by which the orthodontic appliance consisted of a coil spring boned directly to the incisors and maxillary first molar and a continuous force was applied. The advantage of the our tooth movement model is the possibility of monitoring bone resorption and bone formation simultaneously during first 7 days of the treatment with defined force (10–12 g) and reproducibility (Pavlin et al. 2000a; 2000b). Second, the duration of the loading in their studies is relatively shorter with maximum time of 48 h, while our loading regimes lasted up to 7 days. Lastly, there are variations with regard to animal models, probes and antibody used.

Osteocytes are thought to be mechanosensors in bone (Cowin et al. 1991; Aarden et al. 1994; Burger et al. 1999). The application of force to bone results in several potential stimuli including hydrostatic pressure and fluid flow shear stress. Over the years, prevailing views favor that flow of interstitial fluid driven by extravascular pressure is likely the stress-related factor that informs osteocytes about mechanical loading (Weinbaum et al. 1993; Wang et al. 2003; Knothe Tate 2003). Cx43 is abundantly expressed in primary osteocytes and MLO-Y4 osteocyte cell line (Mason et al. 1996; Cheng et al. 2001b). Gap junction activities and accumulation of Cx43 protein in osteocytes are regulated by mechanical loading (Cheng et al. 2001b; Alford et al. 2003). We recently found that in addition to gap junctions, Cx43 forming hemichannels mediate the biological responses elicited by fluid flow, such as the release of PGE2 (Cherian et al. 2005). A recent report shows that PGE2 release induced by fluid flow is decreased by the degradation of the glycocalyx on the cell surface, a hypothesized mechanosensor in osteoyctes (Reilly et al. 2003). Here we found that in response to loading Cx43 protein levels show dynamic upregulation that do not correlate to mRNA expression. There was weak Cx43 mRNA expression detected by in situ hybridization, while the immunocytochemistry of matched sections showed a dramatic increase in Cx43 protein signals. These results imply that stimulation of Cx43 protein by loading appeared not to be regulated at the level of mRNA since there was no alteration of Cx43 mRNA expression after loading. It is not likely that difference between Cx43 mRNA and protein is caused by differences in sensitivity of the two different techniques. First, nondetected signals of Cx43 mRNA in osteocytes with 32P labeling are unlikely to be false negative because this method is more sensitive than immunocytochemistry based on alkaline phosphatase reaction. Second, other bone cells show the strong signals of 32P labeling while they have similar or lower immunostaining signals as compared to osteocytes. For both techniques, we used closest possible adjacent tissue sections derived from the same animal. The upregulation of Cx43 could be resulted from upregulation of protein biosynthesis or/and delayed protein turnover. The alternate, but less likely, explanation is that after loading Cx43 protein undergoes conformational changes and promotes its interactions with other proteins that block interactions with this antibody. However, no previous studies report the blockade of antigenic reaction due to potential interactions of Cx43 with other proteins. Nevertheless, these intriguing results show that Cx43 protein, but not mRNA, level is significantly increased in osteocytes of alveolar bone as a result of mechanical loading. A possible mediator for increases in Cx43 in response to mechanical load could be PGE2. PGE2 increases the skeletal response to mechanical loading and increases Cx43 expression in osteocytic MLO-Y4 cells (Tang et al. 1997; Cheng et al. 2001a). Whether Cx43 expression is induced by PGE2 in vivo remains to be determined.

This study shows that alveolar bone cells in their native bone environment respond differentially to mechanical loading with respect to distribution of Cx43 and further suggests that Cx43-forming gap junctions and hemichannels play important roles in regulation of mechanotransduction in bone.

Acknowledgments

This study was supported by the National Institutes of Health grant AR46798 (J. G-H, D.P. and J.X.J), DE11005 (D.P and J.G-H), and the Welch Foundation grant AQ-1507 (J.X.J).

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