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1.
Proc Natl Acad Sci U S A ; 109(9): 3359-64, 2012 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-22331870

RESUMO

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 α5ß1 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 α5ß1 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 α5ß1 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.


Assuntos
Conexina 43/metabolismo , Integrina alfa5beta1/fisiologia , Osteócitos/metabolismo , Estresse Mecânico , Androstadienos/farmacologia , Animais , Linhagem Celular , Cromonas/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Separação Imunomagnética , Integrina alfa5beta1/antagonistas & inibidores , Camundongos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Mapeamento de Interação de Proteínas , RNA Interferente Pequeno/farmacologia , Wortmanina
2.
J Biol Chem ; 283(39): 26374-82, 2008 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-18676366

RESUMO

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.


Assuntos
Remodelação Óssea/fisiologia , Conexina 43/metabolismo , Dinoprostona/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular/fisiologia , Osteócitos/metabolismo , Animais , Linhagem Celular , Galinhas , Camundongos , Osteócitos/citologia , Estresse Mecânico , Fatores de Tempo
3.
Mol Biol Cell ; 16(7): 3100-6, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15843434

RESUMO

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.


Assuntos
Conexina 43/química , Osteócitos/metabolismo , Prostaglandinas/metabolismo , Animais , Biotinilação , Western Blotting , Osso e Ossos/metabolismo , Carbenoxolona/química , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Galinhas , Dendritos/metabolismo , Junções Comunicantes , Ácido Glicirretínico/química , Camundongos , Microscopia de Fluorescência , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Ratos , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2X7 , Estresse Mecânico
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