Gap junctions formed by connexin 43 (Cx43) play an important role in transmitting signals between bone cells such as osteoblasts and osteoclasts, cells responsible for bone formation and bone remodeling. However, osteocytes express extremely large amounts of Cx43 compared with other bone cell types and this protein is located both on the membrane cell surface as well as in the cytoplasm. Osteocytes, unlike other cell types, only connect and form gap junctions with other cells through the tips of their dendritic processes. This is a very small percentage of the total cell surface area compared to other cells. Our data shows that in addition to gap junctions, primary osteocytes and osteocyte-like MLO-Y4 cells express functional Cx43- forming hemichannels and that these hemichannels mediate the immediate release of prostaglandin by osteocytes in response to fluid flow shear stress. The central hypothesis of the project based on our significant preliminary findings is that hemichannels formed by Cx43 have essential, yet distinct functions from gap junctions in the mediation and regulation of the osteocytic response to mechanical strain. Three specific aims will be pursued: 1). Determine the importance of Cx43 in osteocytes in vivo in response to mechanical strain by the generation of targeted deletion and overexpression of Cx43 in osteocytes in transgenic mouse models;2). Determine the function of hemichannels in osteocytes by the specific blocking of hemichannel function, but not gap junction function;and 3). Determine the role alpha5 Integrin plays in the regulation of the opening of hemichannels induced by fluid flow shear stress. This last specific aim will be accomplished by blocking either expression of aSintegrin or blocking protein interaction with Cx43. One of the innovative aspects of this proposal is the discovery of a novel, unconventional function for hemichannels in osteocytes and regulation of Cx43-forming hemichannels in osteocytes in response to mechanical strain. Moreover, this study combines comprehensive biochemical, molecular, transgenic and functional approaches with unique mechanical engineering applications. It is our expectation that our experimental findings will have a major impact on our understanding of the novel roles hemichannels play in regulating osteocyte response to mechanical strain. Our experimental outcomes will be significant because the new knowledge obtained will contribute to our understanding of how mechanical signals are transduced and modulated between osteocytes and cells on the bone surface. Furthermore, completion of these three specific aims should provide novel contributions to the development of strategies for the treatment of bone diseases such as osteoporosis by providing clues for potential targets for drug action and development.