The connexin 43 (Cx43) hemichannel (HC) in the mechanosensory osteocytes is a major portal for the release of factors responsible for the anabolic effects of mechanical loading on bone formation and remodeling. However, little is known about how the Cx43 molecule responds to mechanical stimulation leading to the opening of the HC. Here, we demonstrate that integrin α5β1 interacts directly with Cx43 and that this interaction is required for mechanical stimulation-induced opening of the Cx43 HC. Direct mechanical perturbation via magnetic beads or conformational activation of integrin α5β1 leads to the opening of the Cx43 HC, and this role of the integrin is independent of its association with an extracellular fibronectin substrate. PI3K signaling is responsible for the shear stress-induced conformational activation of integrin α5β1 leading to the opening of the HC. These results identify an unconventional function of integrin that acts as a mechanical tether to induce opening of the HC and provide a mechanism connecting the effect of mechanical forces directly to anabolic function of the bone.

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.The skeleton regulates its architecture and mass to meet structural and metabolic needs. To fulfill its structural functions, this complex tissue must adapt to loading and unloading while simultaneously regulating the metabolic demands of the skeleton. Numerous in vivo animal studies show the essential role of mechanical loading for bone formation and remodeling; however, the underlying molecular mechanisms, in particular how bone cells adapt to mechanical stimulation, remain largely uncharacterized.When mechanical forces are applied to bone, several potential stimuli occur including changes in hydrostatic pressure, direct cell strain, fluid flow, and electric potentials. These changes lead to fluid movement through the bone (1-3). Shear stress induced by mechanical loading facilitates the exchange of nutrients and bone modulators, and elicits biochemical responses. Osteocytes are well positioned in the bone to sense the magnitude of mechanical strain and are essential for the skeleton adaptive response to load. Experimental studies have shown that osteocytes are sensitive to stress applied to both intact bone tissue and in cell culture (4 -6). Encased within mineralized tissue, their dendritic morphology allows them to connect through small tunnels called canaliculi to form a threedimensional network not only with adjacent osteocytes but also to connect to cells on the bone surface and bone marrow.Connexins (Cx), 2 gap junction-forming proteins, belong to a multigene family expressing four transmembrane domains. The regions corresponding to transmembrane and extracellular domains are highly conserved. Cx43 has been identified in most types of bone cells (7-13) and is the major connexin expressed in osteocyte-like MLO-Y4 cells and primary osteocy...

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.

Gap junctions formed by connexins (Cx) play an important role in transmitting signals between bone cells such as osteoblasts and osteoclasts, cells responsible for bone formation and bone remodeling, respectively. Gap junction intercellular communication (GJIC) has been demonstrated to mediate the process of osteoblast differentiation and bone formation. Furthermore, GJIC propagates Ca 2+ signaling, conveys anabolic effects of hormones and growth factors, and regulates gene transcription of osteoblast differentiation markers. GJIC is also implicated to regulate osteoclast formation, survival and apoptosis. Compared with other bone cells, the most abundant type are osteocytes, which express large amounts of connexins. Mechanosensing osteocytes connect and form gap junctions with themselves and other cells only through the tips of their dendritic processes, a relatively small percent of the total cell surface area compared to other cells. Recent studies show that in addition to gap junctions, osteoblasts and osteocytes express functional hemichannels, the un-opposed halves of gap junction channels. Hemichannels are localized at the cell surface and function independently of gap junctions. Hemichannels in osteocytes mediate the immediate release of prostaglandins in response to mechanical stress. The major challenges remaining in the field are how the functions of these two types of channels are coordinated in bone cells and what the asserted, distinct effects of these channels are on bone formation and remodeling processes, and on conveying signals elicited by mechanical loading. KeywordsBone; Gap Junctions; Hemichannels; Cx43; Connexin; Osteoblast; Osteocyte; Mechanical Stress; Review INTRODUCTION Gap Junctions in Bone CellsGap junctions are transmembrane channels, which connect the cytoplasm of adjacent cells. These channels permit molecules with molecular weights approximately less than 1 kD a such as small metabolites, ions, and intracellular signaling molecules (i.e. calcium, cAMP, inositol triphosphate) to pass through. Gap junction channels have been demonstrated to be important in modulating cell signaling and tissue function in many organs, such as heart, liver, peripheral nerve, ovary, ear and lens of the eye (1-8). Gap junctions are formed by members of a family of sequentially and structurally related proteins known as connexins. Approximately twenty connexins have been identified and cloned from various tissues and cells (9-11). Six monomers of connexins are joined head-to-head across the extracellular "gap" between two adjacent cells (21,26,27). However, we found that Cx45 protein is expressed in bone marrow, but not in osteoblasts, osteocytes and osteoclasts in the alveolar bone tissues of the tooth (28).Functional gap junctions in osteoblasts were first demonstrated with electrical conductance and dye injection (29). Voltage-sensitive gap junction currents were detected in osteoblastic cells derived from calvarias of new-born rats with a single gap junction channel conductance of approxim...

Connexin (Cx) 43 serves important roles in bone function and development. Targeted deletion of Cx43 in osteoblasts or osteocytes leads to increased osteocyte apoptosis, osteoclast recruitment, and reduced biomechanical properties. Cx43 forms both gap junction channels and hemichannels, which mediate the communication between adjacent cells or between cell and extracellular environments, respectively. Two transgenic mouse models driven by a DMP1 promoter with the overexpression of dominant negative Cx43 mutants were generated to dissect the functional contribution of Cx43 gap junction channels and hemichannels in osteocytes. The R76W mutant blocks gap junction channel, but not hemichannel function, and the Δ130-136 mutant inhibits activity of both types of channels. Δ130-136 mice showed a significant increase in bone mineral density compared to WT and R76W mice. MicroCT analyses revealed a significant increase in total tissue and bone area in midshaft cortical bone of Δ130-136 mice. The bone marrow cavity was expanded, whereas the cortical thickness was increased and associated with increased bone formation along the periosteal area. However, there is no significant alteration in the structure of trabecular bone. Histologic sections of the midshaft showed increased apoptotic osteocytes in Δ130-136, but not in WT and R76W, mice which correlated with altered biomechanical and estimated bone material properties. Osteoclasts were increased along the endocortical surface in both transgenic mice with a greater effect in Δ130-136 mice which likely contributed to the increased marrow cavity. Interestingly, the overall expression of serum bone formation and resorption markers were higher in R76W mice. These findings suggest that osteocytic Cx43 channels play distinctive roles in the bone; hemichannels play a dominant role in regulating osteocyte survival, endocortical bone resorption and periosteal apposition, and gap junction communication is involved in the process of bone remodeling.

Background: Opening of Cx43 hemichannels by mechanical stress releases factors important for bone remodeling; however, the regulatory mechanism is unknown. Result: Upon mechanical stimulation, AKT phosphorylates integrin ␣5 and Cx43, increases interaction, and opens hemichannels. Conclusion: Phosphorylation of Cx43 and ␣5 by AKT is critical for hemichannel opening. Significance: This is the first report demonstrating the functional importance of AKT in regulation of Cx43 hemichannels.

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Intracellular signaling in osteocytes activated by mechanical loading is important for bone formation and remodeling. These signaling events are mediated by small modulators released from Cx43 hemichannels (HC). We have recently shown that integrin a5 senses the mechanical stimulation and induces the opening of Cx43 HC; however, the underlying mechanism is unknown. Here, we show that both Cx43 and integrin a5 interact with 14-3-3h, and this interaction is required for the opening of Cx43 HC upon mechanical stress. The absence of 14-3-3h prevented the interaction between Cx43 and integrin a5, and blocked HC opening. Furthermore, it decreased the transport of Cx43 and integrin a5 from the Golgi apparatus to the plasma membrane. Mechanical loading promoted the movement of Cx43 to the surface which was associated not only with an increase in 14-3-3h levels but also its interaction with Cx43 and integrin a5. This stimulatory effect on forward transport by mechanical loading was attenuated in the absence of 14-3-3h and the majority of the Cx43 accumulated in the Golgi. Disruption of the Golgi by brefeldin A reduced the association of Cx43 and integrin a5 with 14-3-3h, further suggesting that the interaction is likely to occur in the Golgi. Together, these results define a previously unidentified, scaffolding role of 14-3-3h in assisting the delivery of Cx43 and integrin a5 to the plasma membrane for the formation of mechanosensitive HC in osteocytes.