Adaptation of Connexin 43-Hemichannel Prostaglandin Release to Mechanical Loading

Abstract

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...

Amyloid β-Induced Death in Neurons Involves Glial and Neuronal Hemichannels

“…1G). Because it has been suggested that Gap27 or Gap26 are not specific in their action on Cx43 hemichannels (Wang et al, 2007), we used the Cx43 E2 antibody directed against the second loop of Cx43 that specifically blocks Cx43 hemichannels, but not gap junction channels (Siller-Jackson et al, 2008). Cx43 E2 (1:500) reduced the maximal A␤ 25-35 -induced Et uptake in snapshot experiments in microglia (from 100 to 50.2 Ϯ 10.3%; n ϭ 5) (Fig.…”

“…Anti-Cx43 monoclonal antibody was obtained from BD Biosciences. Cx43 E2 antibody specifically for blocking hemichannels was generated and affinity purified as previously described (Siller-Jackson et al, 2008).…”

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

“…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 integrinassociated 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.…”

“…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).…”

Connexin43 Potentiates Osteoblast Responsiveness to Fibroblast Growth Factor 2 via a Protein Kinase C-Delta/Runx2–dependent Mechanism

“…It is important to note that although we model that the effects of Cx43 function are mediating gap junction intercellular communication, a compelling and expanding body of research has demonstrated that Cx43 can also function as unopposed hemichannels to regulate responses to fluid flow (Cherian et al, 2005;Genetos et al, 2007;Siller-Jackson et al, 2008) and the antiapoptotic actions of bisphosphonates (Plotkin et al, 2002;Plotkin et al, 2005) in osteoblast or osteocytes-like cells. None of the approaches we have utilized in this study can specifically rule out that the results we observe could be attributable to hemichannel function.…”