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 E 2 (PGE 2 ). Cells plated at lower densities release more PGE 2 than cells plated at higher densities. This response was significantly reduced by antisense to Cx43 and by the gap junction and hemichannel inhibitors 18 ␤-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 P2X 7 and the prostaglandin transporter PGT, had no effect on PGE 2 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 PGE 2 in response to mechanical strain.

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...

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...