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

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.     

Mechanical loading stimulates expression of connexin 43 in alveolar bone cells in the tooth movement model

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.     

Osteocytic connexin 43 channels affect fracture healing

The cross-talk between cells is very critical for moving forward fracture healing in an orderly manner. Connexin (Cx) 43-formed gap junctions and hemichannels mediate the communication between adjacent cells and cells and extracellular environment. Loss of Cx43 in osteoblasts/osteocytes results in delayed fracture healing. For investigating the role of two channels in osteocytes in bone repair, two transgenic mouse models with Cx43 dominant negative mutants driven by a 10 kb-DMP1 promoter were generated: R76W (gap junctions are blocked, whereas hemichannels are promoted) and Δ130–136 (both gap junctions and hemichannels are blocked). R76W mice (promotion of hemichannels) showed a significant increase of new bone formation, whereas delayed osteoclastogenesis and healing was observed in Δ130–136 (impairment of gap junctions), but not in R76W mice (hemichannel promotion may recover the delay). These results suggest that gap junctions and hemichannels play some similar and cooperative roles in bone repair.     

Gap junctions and hemichannels in signal transmission, function and development of bone

Gap junctional intercellular communication (GJIC) mediated by connexins, in particular connexin 43 (Cx43), plays important roles in regulating signal transmission among different bone cells and thereby regulates development, differentiation, modeling and remodeling of the bone. GJIC regulates osteoblast formation, differentiation, survival and apoptosis. Osteoclast formation and resorptive ability are also reported to be modulated by GJIC. Furthermore, osteocytes utilize GJIC to coordinate bone remodeling in response to anabolic factors and mechanical loading. Apart from gap junctions, connexins also form hemichannels, which are localized on the cell surface and function independently of the gap junction channels. Both these channels mediate the transfer of molecules smaller than 1.2kDa including small ions, metabolites, ATP, prostaglandin and IP(3). The biological importance of the communication mediated by connexin-forming channels in bone development is revealed by the low bone mass and osteoblast dysfunction in the Cx43-null mice and the skeletal malformations observed in occulodentodigital dysplasia (ODDD) caused by mutations in the Cx43 gene. The current review summarizes the role of gap junctions and hemichannels in regulating signaling, function and development of bone cells. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Gap junctional regulation of signal transduction in bone cells

The role of gap junctions, particularly that of connexin43 (Cx43), has become an area of increasing interest in bone physiology. An abundance of studies have shown that Cx43 influences the function of osteoblasts and osteocytes, which ultimately impacts bone mass acquisition and skeletal homeostasis. However, the molecular details underlying how Cx43 regulates bone are only coming into focus and have proven to be more complex than originally thought. In this review, we focus on the diverse molecular mechanisms by which Cx43 gap junctions and hemichannels regulate cell signaling pathways, gene expression, mechanotransduction and cell survival in bone cells. This review will highlight key signaling factors that have been identified as downstream effectors of Cx43 and the impact of these pathways on distinct osteoblast and osteocyte functions.     

Adaptation of connexin 43-hemichannel prostaglandin release to mechanical loading

Bone tissues respond to mechanical loading/unloading regimens to accommodate (re)modeling requirements; however, the underlying molecular mechanism responsible for these responses is largely unknown. Previously, we reported that connexin (Cx) 43 hemichannels in mechanosensing osteocytes mediate the release of prostaglandin, PGE(2), a crucial factor for bone formation in response to anabolic loading. We show here that the opening of hemichannels and release of PGE(2) by shear stress were significantly inhibited by a potent antibody we developed that specifically blocks Cx43-hemichannels, but not gap junctions or other channels. The opening of hemichannels and release of PGE(2) are magnitude-dependent on the level of shear stress. Insertion of a rest period between stress enhances this response. Hemichannels gradually close after 24 h of continuous shear stress corresponding with reduced Cx43 expression on the cell surface, thereby reducing any potential negative effects of channels staying open for extended periods. These data suggest that Cx43-hemichannel activity associated with PGE(2) release is adaptively regulated by mechanical loading to provide an effective means of regulating levels of extracellular signaling molecules responsible for initiation of bone (re)modeling.     

Gap junction channels formed by coexpressed connexin40 and connexin43

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V(j)) compared with homotypic gap junctions and/or an asymmetrical V(j) dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.     

Expression of connexin 43 mRNA in microisolated murine osteoclasts and regulation of bone resorption in vitro by gap junction inhibitors

Several studies have demonstrated that connexin 43 (Cx43) mediates signals important for osteoblast function and osteogenesis. The role of gap junctional communication in bone resorption is less clear. We have investigated the expression of Cx43 mRNA in osteoclasts and bone resorption cultures and furthermore, the functional importance of gap junctional communication in bone resorption. RT-PCR analysis demonstrated Cx43 mRNA expression in mouse bone marrow cultures and in osteoclasts microisolated from the marrow cultures. Cx43 mRNA was also expressed in bone resorption cultures with osteoclasts and osteoblasts/stromal cells incubated for 48h on devitalized bone slices. An up-regulation of Cx43 mRNA was detected in parathyroid (PTH)-stimulated (0.1 nM) bone resorption. Two inhibitors of gap junction communication, 18alpha-glycyrrhetinic acid (30 microM) and oleamide (100 microM), significantly inhibited PTH- and 1,25-(OH)(2)D(3)-stimulated osteoclastic pit formation. In conclusion, our data indicate a functional role for gap junction communication in bone resorption.     

Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin

Mechanosensing bone osteocytes express large amounts of connexin (Cx)43, the component of gap junctions; yet, gap junctions are only active at the small tips of their dendritic processes, suggesting another function for Cx43. Both primary osteocytes and the osteocyte-like MLO-Y4 cells respond to fluid flow shear stress by releasing intracellular prostaglandin E2 (PGE2). Cells plated at lower densities release more PGE2 than cells plated at higher densities. This response was significantly reduced by antisense to Cx43 and by the gap junction and hemichannel inhibitors 18 beta-glycyrrhetinic acid and carbenoxolone, even in cells without physical contact, suggesting the involvement of Cx43-hemichannels. Inhibitors of other channels, such as the purinergic receptor P2X7 and the prostaglandin transporter PGT, had no effect on PGE2 release. Cell surface biotinylation analysis showed that surface expression of Cx43 was increased by shear stress. Together, these results suggest fluid flow shear stress induces the translocation of Cx43 to the membrane surface and that unapposed hemichannels formed by Cx43 serve as a novel portal for the release of PGE2 in response to mechanical strain.     

Parathyroid hormone regulates osteoblast differentiation in a Wnt/β-catenin-dependent manner

Intermittent parathyroid hormone (PTH) administration shows an anabolic effect on bone. However, the mechanisms are not fully studied. Recent studies suggest that Wnt signaling is involved in PTH-induced bone formation. The current study was to examine if Wnt/β-catenin pathway is required during PTH-induced osteoblast differentiation. Osteoblastic MC3T3-E1 cells were treated with human PTH (1-34) (hPTH [1-34]) and expression levels of osteoblast differentiation markers were detected by real-time PCR. RNA levels of β-catenin, Runx2, Osteocalcin, Alkaline phosphatase, and Bone sialoprotein were significantly up-regulated after treatment with 10(-8) M of hPTH (1-34) for 6 h. Alkaline phosphatase activity and protein expression of β-catenin were also increased after 6 days of intermittent treatment with hPTH (1-34) in MC3T3-E1 cells. hPTH (1-34) significantly enhanced Topflash Luciferase activity after 6 h of treatment. More important, PTH-induced Alkaline phosphatase activity was significantly inhibited by knocking down β-catenin expression in cells using siRNA. Real-time RT-PCR results further showed down regulation of Runx2, Osteocalcin, Alkaline phosphatase, Bone sialoprotein gene expression in β-catenin siRNA transfected cells with/without PTH treatment. These results clearly indicate that PTH stimulates Wnt/β-catenin pathway in MC3T3-E1 cells and osteoblast differentiation markers expression was up-regulated by activation of Wnt/β-catenin signaling. Our study demonstrated that PTH-induced osteoblast differentiation mainly through activation of Wnt/β-catenin pathway in osteoblastic MC3T3-E1 cells.     

Enhancement of connexin 43 expression increases proliferation and differentiation of an osteoblast-like cell line

Bone cells form a functional syncytium as they are coupled by gap junctions composed mainly of connexin 43 (Cx43). To further understand the role of Cx43 in bone cell growth and differentiation, we stably transfected Cx45-expressing UMR 106-01 cells with Cx43 using an expression vector containing rat Cx43 cDNA. Three stably transfected clones were analyzed, all of which showed altered expression of Cx43 and/or Cx45 as was obvious from immunocytochemistry and Northern blotting. Double whole-cell patch clamping revealed single-channel conductances of 20 (Cx45) and 60 pS (Cx43). The overexpression of Cx43 led to an increase in dye coupling concomitant with elevated gap-junctional conductance. The phenotype of the transfected clones was characterized by an increased proliferation (4- to 7-fold) compared to controls. Moreover, a transfectant clone with 10- to 12-fold enhanced Cx43 expression showed a significantly increased calcium content of the extracellular matrix and enlarged mineralization nodules, while alkaline phosphatase was moderately increased. We conclude that enhanced gap-junctional coupling via Cx43 significantly promotes proliferation and differentiation of UMR cells.     

Hemichannels formed by connexin 43 play an important role in the release of prostaglandin E(2) by osteocytes in response to mechanical strain

Osteocytes embedded in the matrix of bone are mechanosensory cells that translate strain into signals and regulate bone remodeling. Our previous studies using osteocyte-like MLO-Y4 cells have shown that fluid flow shear stress (FFSS) increases connexin (Cx) 43 protein expression, prostaglandin E(2) (PGE(2)) release, and intercellular coupling, and PGE(2) is an essential mediator between FFSS and gap junctions. However, the role of Cx43 in the release of PGE(2) in response to FFSS is unknown. Here, the FFSS-loaded MLO-Y4 cells with no or few intercellular channels released significantly more PGE(2) per cell than those cells at higher densities. Antisense Cx43 oligonucleotides and 18 beta-glycyrrhetinic acid, a specific gap junction and hemichannel blocker, significantly reduced PGE(2) release by FFSS at all cell densities tested, especially cells at the lowest density without gap junctions. FFSS, fluid flow-conditioned medium, and PGE(2) increased the activity of dye uptake. Moreover, FFSS induced Cx43 to migrate to the surface of the cell; this surface expressed Cx43 developed resistance to Triton-X-100 solublization. Our results suggest that hemichannels formed by Cx43, instead of intercellular channels, are likely to play a predominant role in the release of intracellular PGE(2) in response to FFSS.     

Hemichannels Formed by Connexin 43 Play an Important Role in the Release of Prostaglandin E2 by Osteocytes in Response to Mechanical Strain

Osteocytes embedded in the matrix of bone are mechanosensory cells that translate strain into signals and regulate bone remodeling. Our previous studies using osteocyte-like MLO-Y4 cells have shown that fluid flow shear stress (FFSS) increases connexin (Cx) 43 protein expression, prostaglandin E₂ (PGE₂) release, and intercellular coupling, and PGE₂ is an essential mediator between FFSS and gap junctions. However, the role of Cx43 in the release of PGE₂ in response to FFSS is unknown. Here, the FFSS-loaded MLO-Y4 cells with no or few intercellular channels released significantly more PGE₂ per cell than those cells at higher densities. Antisense Cx43 oligonucleotides and 18 β-glycyrrhetinic acid, a specific gap junction and hemichannel blocker, significantly reduced PGE₂ release by FFSS at all cell densities tested, especially cells at the lowest density without gap junctions. FFSS, fluid flow-conditioned medium, and PGE₂ increased the activity of dye uptake. Moreover, FFSS induced Cx43 to migrate to the surface of the cell; this surface expressed Cx43 developed resistance to Triton-X-100 solublization. Our results suggest that hemichannels formed by Cx43, instead of intercellular channels, are likely to play a predominant role in the release of intracellular PGE₂ in response to FFSS.     

Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium

Gap junction number and size vary widely in cardiac tissues with disparate conduction properties. Little is known about how tissue-specific patterns of intercellular junctions are established and regulated. To elucidate the relationship between gap junction channel protein expression and the structure of gap junctions, we analyzed Cx43 +/- mice, which have a genetic deficiency in expression of the major ventricular gap junction protein, connexin43 (Cx43). Quantitative confocal immunofluorescence microscopy revealed that diminished Cx43 signal in Cx43 +/- mice was due almost entirely to a reduction in the number of individual gap junctions (226 +/- 52 vs. 150 +/- 32 individual gap junctions/field in Cx43 +/+ and +/- ventricles, respectively; P < 0.05). The mean size of an individual gap junction was the same in both groups. Immunofluorescence results were confirmed with electron microscopic morphometry. Thus when connexin expression is diminished, ventricular myocytes become interconnected by a reduced number of large, normally sized gap junctions, rather than a normal number of smaller junctions. Maintenance of large gap junctions may be an adaptive response supporting safe ventricular conduction.     

Hemichannels Formed by Connexin 43 Play an Important Role in the Release of Prostaglandin E2by Osteocytes in Response to Mechanical Strain

Osteocytes embedded in the matrix of bone are mechanosensory cells that translate strain into signals and regulate bone remodeling. Our previous studies using osteocyte-like MLO-Y4 cells have shown that fluid flow shear stress (FFSS) increases connexin (Cx) 43 protein expression, prostaglandin E₂(PGE₂) release, and intercellular coupling, and PGE₂is an essential mediator between FFSS and gap junctions. However, the role of Cx43 in the release of PGE₂in response to FFSS is unknown. Here, the FFSS-loaded MLO-Y4 cells with no or few intercellular channels released significantly more PGE₂per cell than those cells at higher densities. Antisense Cx43 oligonucleotides and 18 β-glycyrrhetinic acid, a specific gap junction and hemichannel blocker, significantly reduced PGE₂release by FFSS at all cell densities tested, especially cells at the lowest density without gap junctions. FFSS, fluid flow-conditioned medium, and PGE₂increased the activity of dye uptake. Moreover, FFSS induced Cx43 to migrate to the surface of the cell; this surface expressed Cx43 developed resistance to Triton-X-100 solublization. Our results suggest that hemichannels formed by Cx43, instead of intercellular channels, are likely to play a predominant role in the release of intracellular PGE₂in response to FFSS.     

Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells

During the assembly of gap junctions, a hemichannel in the plasma membrane of one cell is thought to align and dock with another in an apposed membrane to form a cell-to-cell channel. We report here on the existence and properties of nonjunctional, plasma membrane connexin43 (Cx43) hemichannels. The opening of the hemichannels was demonstrated by the cellular uptake of 5(6)-carboxyfluorescein from the culture medium when extracellular calcium levels were reduced. Dye uptake exhibited properties similar to those of gap junction channels. For example, using different dyes, the levels of uptake were correlated with molecular size: 5(6)-carboxyfluorescein (approximately 32%), 7- hydroxycoumarin-3-carboxylic acid (approximately 24%), fura-2 (approximately 11%), and fluorescein-dextran (approximately 0.4%). Octanol and heptanol also reduced dye uptake by approximately 50%. Detailed analysis of one clone of Novikoff cells transfected with a Cx43 antisense expression vector revealed a reduction in dye uptake levels according to uptake assays and a corresponding decrease in intercellular dye transfer rates in microinjection experiments. In addition, a more limited decrease in membrane resistance upon reduction of extracellular calcium was detected in electrophysiological studies of antisense transfectants, in contrast to control cells. Studies of dye uptake in HeLa cells also demonstrated a large increase following transfection with Cx43. Together these observations indicate that Cx43 is responsible for the hemichannel function in these cultured cells. Similar dye uptake results were obtained with normal rat kidney (NRK) cells, which express Cx43. Dye uptake can be dramatically inhibited by 12-O-tetradeconylphorbol-13-acetate-activated protein kinase C in these cell systems and by a temperature-sensitive tyrosine protein kinase, pp60v-src in LA25-NRK cells. We conclude that Cx43 hemichannels are found in the plasma membrane, where they are regulated by multiple signaling pathways, and likely represent an important stage in gap junction assembly.