Turnover of cells and matrix occurs in all organs and tissues and is essential to maintain health and mechanical integrity. In bone, cell and matrix turnover occur through bone remodeling. Dysregulation of bone remodeling is the fundamental driver underlying all forms of Osteoporosis. Osteoporosis results from the accelerated birth of new remodeling units and/or alterations in osteoclast or osteoblast kinetics within remodeling units, leading to excessive resorption, too little formation or both. Regulation of the bone remodeling effector cells, osteoclasts and osteoblasts, is well understood. However, our understanding of the critical Activation step or the On-Switch to start bone remodeling is very limited. Recently, we discovered that osteocyte apoptosis activates bone resorption at bone microdamage sites. Furthermore, we found that estrogen loss and disuse cause osteocyte apoptosis that activates bone resorption as well. Finally, we discovered that in bone microdamage-induced remodeling, RANKL production is triggered in viable osteocytes (i.e., the Bystanders) immediately neighboring apoptotic osteocytes. Apoptotic osteocytes themselves do not produce osteoclastogenic cytokines. However, it is the dying osteocytes, not bone microdamage, which triggers this Bystander signaling in viable osteocytes - RANKL production is not turned-on when osteocyte apoptosis is inhibited. How apoptotic osteocytes trigger RANKL production in viable neighbor osteocytes is not known. Our preliminary studies suggest that gap junctions and Pannexin 1 channels play critical roles in this process. Experiments: In the current studies, we will use a combination of immunohistochemical approaches (osteocyte apoptosis vs. RANKL and OPG expressing osteocytes) and bone histomorphometric approaches to assess osteocyte apoptosis and production of osteoclastogenic cytokines by viable osteocyte. 1) In Aim 1, we will use mouse models of estrogen loss and disuse (OVX, HLS) to test whether RANKL production from viable Bystander osteocytes is the generalized mechanism for bone remodeling activation in response to a range of physiological stimuli. 2) In Aim 2, we will determine the role of gap junctions (GJ) vs Cx43 hemichannels in regulating the spread of osteocyte apoptosis and extent of osteoclastogenic signaling at bone microdamage sites. We will use a mouse ulnar fatigue to produce microcracks in bone in vivo. Pharmacological and peptide inhibition of GJ and Cx43 channels will be used to establish their regulation of the extent of osteocyte apoptosis and RANKL expression. 3) Finally, recent studies show apoptotic cells acutely release find me signals that stimulate recruitment of phagocytes that remove dying cells. Primary among these acute signals are nucleotides released through apoptosis-induced opening of Pannexin 1 channels.
In Aim 3, we will determine the role of Pannexin 1 and associated nucleotide signaling in coupling osteocyte apoptosis at bone microdamage sites to activation of Bystander osteocyte signaling using Panx1 knockout mice.
Osteoporosis results from the accelerated birth of new remodeling sites and/or to alterations to the activities of the cells, osteoclasts or osteoblasts, within those remodeling sites, leading to excessive resorption, too little formation or both. Understanding of the critical Activation step or the 'On-Switch' to start bone remodeling is very limited. Our studies have revealed that osteocyte apoptosis, i.e., regulated death of osteocytes, controls activation of bone remodeling in response to skeletal unloading, fatigue (wear & tear) bone microdamage and estrogen loss. In the case of fatigue microdamage in bone, the controlled death of groups of osteocytes triggers their viable neighbor osteocytes to produce RANKL biochemical signals to recruit osteoclasts that resorb bone at the start of remodeling. In the current studies we will test whether this osteocyte apoptosis triggering of signaling from non-dying neighbor cells is actually a universal mechanism for a range of challenges that turn on bone remodeling and can lead to bone loss. In a related series of studies, we will examine how dying osteocytes signal their surviving neighbors to 'call for help.' We will explore parallels wit the cell death and the 'help' signaling responses evoked from surviving cells in stroke and heart attack, with particular focus on the role of gap junctions channels and pannexin channels. Understanding these biological mechanisms may open a new window for novel therapeutic targets that to modulate bone remodeling and prevent osteoporosis and bone fragility.
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