Aging of the skeleton is associated with increased prevalence of osteocyte apoptosis in mice and humans. Work leading to this application shows that the skeletal syndrome that ensues with aging is mimicked by osteocyte specific deletion of the gap junction protein connexin (Cx) 43. Mice lacking Cx43 in osteocytes exhibit increased osteocyte apoptosis and accumulation of empty lacunae, and reduced OPG levels, and Cx43- deficient osteocytes express lower levels of the survival-associated microRNA (miR) miR21. In addition, bones from old mice exhibit reduced Cx43, OPG, and miR21 expression. We showed in vitro that apoptotic osteocytes release high mobility group box protein1 (HMGB1), which reduces OPG expression and increases osteoclast recruitment and differentiation through activation of receptors for advanced glycation end products (RAGE). HMGB1 also activated toll-like receptor 4 (TLR4). However, whether activation of HMGB1 receptors RAGE or TLR4 has a role in the skeletal phenotype of Cx43-deficient or old mice is not known. The mechanism by which HMGB1 is released by apoptotic cells is not known. A potential mechanism could involve opening of Pannexin1 (Pax1) channels in cells undergoing apoptosis. Whether this mechanism operates in osteocytes undergoing apoptosis is not known. The long-term goal of our studies is to improve the management of the adverse skeletal effects of aging by targeting osteocyte apoptosis and resorption. The specific objective of this application is to define the signaling pathways involved in the elevated osteocyte apoptosis and bone resorption in aging. We propose that the bone fragility syndrome that ensues with aging is due to increased osteocyte apoptosis as a consequence of reduced expression of osteocytic Cx43, OPG, and miR21. We hypothesize that low OPG and the consequent increase in the RANKL/OPG ratio acts as a permissive event for osteoclast development; whereas low miR21 reduces anti-apoptotic kinase signaling and activates caspases, with the consequent Panx1 channel opening and HMGB1 release, directing osteoclasts to areas with accumulated apoptotic osteocytes. To test this hypothesis, we will pursue a combination of in vivo and in vitro studies that include the use of novel genetically modified mice, and established and primary osteocytic cells.
Aim 1 will examine the role of Cx43/miR21 on osteocyte apoptosis with aging.
Aim 2 will investigate the role of HMGB1 in osteoclast recruitment and differentiation in Cx43 deficiency and aging.
Aim 3 will explore the consequence of osteocytic Panx1 removal on HMGB1 release and osteoclast recruitment. Successful completion of these studies will provide the molecular basis for the in- creased osteocyte apoptosis in the absence of Cx43 and in old animals. In addition, these studies will establish the molecular link between dying osteocytes and targeted bone resorption. Moreover, they will offer the basis for treatments in which the effect of increased osteocyte apoptosis on osteoclast recruitment are counteracted by targeting the HMGB1-RAGE/TLR4 system.

Public Health Relevance

Increased bone fragility with aging has been associated with accumulation of apoptotic osteocytes and empty lacunae in mice and humans. In this application, we propose to advance in our understanding of the molecular mechanisms by which osteocytes from old individuals are less susceptible to survival signals, thereby setting the stage for improved management of bone fragility with aging, a growing public health concern. These studies are relevant to the NIH mission because they will provide evidence for the effectiveness of pharmacological intervention to restrain targeted osteoclast recruitment, thus preserving bone micro- architecture and strength.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
1R01AR067210-01
Application #
8796545
Study Section
Skeletal Biology Structure and Regeneration Study Section (SBSR)
Program Officer
Alekel, D Lee
Project Start
2015-04-01
Project End
2020-02-29
Budget Start
2015-04-01
Budget End
2016-02-29
Support Year
1
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Davis, Hannah M; Pacheco-Costa, Rafael; Atkinson, Emily G et al. (2017) Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging. Aging Cell 16:551-563
Plotkin, Lilian I; Pacheco-Costa, Rafael; Davis, Hannah M (2017) microRNAs and connexins in bone: interaction and mechanisms of delivery. Curr Mol Biol Rep 3:63-70
Plotkin, Lilian I; Davis, Hannah M; Cisterna, Bruno A et al. (2017) Connexins and Pannexins in Bone and Skeletal Muscle. Curr Osteoporos Rep 15:326-334
Pacheco-Costa, R; Davis, H M; Atkinson, E G et al. (2016) Osteocytic connexin 43 is not required for the increase in bone mass induced by intermittent PTH administration in male mice. J Musculoskelet Neuronal Interact 16:45-57
Hammond, Max A; Berman, Alycia G; Pacheco-Costa, Rafael et al. (2016) Removing or truncating connexin 43 in murine osteocytes alters cortical geometry, nanoscale morphology, and tissue mechanics in the tibia. Bone 88:85-91
Plotkin, Lilian I; Laird, Dale W; Amedee, Joelle (2016) Role of connexins and pannexins during ontogeny, regeneration, and pathologies of bone. BMC Cell Biol 17 Suppl 1:19
Plotkin, Lilian I; Stains, Joseph P (2015) Connexins and pannexins in the skeleton: gap junctions, hemichannels and more. Cell Mol Life Sci 72:2853-67
Plotkin, Lilian I; Speacht, Toni L; Donahue, Henry J (2015) Cx43 and mechanotransduction in bone. Curr Osteoporos Rep 13:67-72
Pacheco-Costa, Rafael; Davis, Hannah M; Sorenson, Chad et al. (2015) Defective cancellous bone structure and abnormal response to PTH in cortical bone of mice lacking Cx43 cytoplasmic C-terminus domain. Bone 81:632-43
Plotkin, Lilian I; Bellido, Teresita (2014) Comment on Osteocytes: masters orchestrators of bone. Calcif Tissue Int 95:382-3