MOLECULAR MECHANISMS UNDERLYING OSTEOCYTE APOPTOSIS AND THE ASSOCIATED OSTEOCLASTOGENESIS IN CX43-DEFICIENCY AND AGING Hannah Marie Davis SubmitteD to the Faculty oF the University GraDuate School in partial Fulfillment oF the requirements for the degree Doctor oF Philosophy in the Department of Anatomy anD Cell Biology, InDiana University June 2019 AccepteD by the GraDuate Faculty oF InDiana University, in partial fulfillment of the requirements for the degree of Doctor oF Philosophy. Doctoral Committee ______________________________________ Lilian I. Plotkin, Ph.D., Chair ______________________________________ Joseph P. BiDWell, Ph.D. April 26, 2019 ______________________________________ MattheW R. Allen, Ph.D. ______________________________________ Angela Bruzzaniti, Ph.D. ii © 2019 Hannah Marie Davis DEDICATION To my loveD ones Thank you For your endless support and For alWays reminDing me of what truly matters. iv ACKNOWLEDGEMENTS I WoulD like to thank the National Institutes of Health anD the Cagiantas family for their funDing, Which alloWeD me to conDuct these experiments anD gave me the opportunity to present my Work at numerous scientiFic conferences. I WoulD like to extend my thanks to my committee For their aDvice anD support throughout my graDuate training. Particularly, Dr. BiDwell, thank you For the countless hours of therapy sessions over the years. Dr. Plotkin thank you for Welcoming me into your lab anD proviDing me With enDless guidance and encouragement throughout my graduate training. You have been a wonDerFul mentor anD I Will alWays be thankful For the countless opportunities you have given me over the years. I Would like to thank the numerous inDiviDuals Who helpeD contribute to these studies, as their efForts led to several publications. In particular, I thank all of the members of the Plotkin lab as Well as the numerous other inDividuals in the Department oF Anatomy and Cell Biology for all of their assistance and support with these studies. I woulD like to thank the Department of Anatomy anD Cell Biology, the IBMG program, and Indiana University School of Medicine For providing me With this opportunity. Lastly and most important of all, I WoulD like to thank my family and frienDs for their endless support and patience With me throughout my graduate studies. v Hannah Marie Davis MOLECULAR MECHANISMS UNDERLYING OSTEOCYTE APOPTOSIS AND THE ASSOCIATED OSTEOCLASTOGENESIS IN CX43-DEFICIENCY AND AGING OlD age is associateD With increaseD bone Fragility anD risk oF Fracture as a result of skeletal alterations, including low bone density and cortical thinning. Further, apoptotic osteocytes accumulate in old mice and humans. We have previously shown that mice lacking osteocytic connexin (Cx) 43 (Cx43ΔOt) exhibit a phenotype similar to that of the aging skeleton, With elevated osteocyte apoptosis and an associated increase in osteoclastogenesis. These finDings suggest that osteocyte apoptosis results in the release of Factors that recruit osteoclasts to bone surFaces close to areas that contain apoptotic osteocytes. However, the speciFic chemotactic signals, the events meDiating their release, and the mechanisms of their action remain unknown. Consistent with this notion, We also founD that HMGB1 releaseD by Cx43-deficient (Cx43def) MLO-Y4 osteocytic cells enhances osteoclastogenesis in part by increasing osteocytic RANKL, which promotes osteoclastogenesis, and, at the same time, directly stimulating osteoclastogenesis. Further, expression of the pro-survival microRNA (miR), miR21, is loW in Cx43def cells and bones From old Female mice, and low miR21 levels increase osteocyte apoptosis. However, surprisingly, mice lacking miR21 (miR21ΔOt) have decreased osteoclast number and activity even under conditions of elevated osteocyte apoptosis; suggesting that osteocytic miR21 may meDiate osteoclast precursor recruitment/survival inDuceD by apoptotic osteocytes. However, Whether HMGB1/miR21 are released by osteocytes, anD if the HMGB1 receptors, receptor for aDvanceD glycation enD proDucts (RAGE) and/or toll- like receptor (TLR4) are involved in osteoclast recruitment in Cx43ΔOt and old mice is unknown. The overall objectives of this series of studies were to eluciDate the mechanisms vi underlying osteocyte apoptosis in Cx43-deficiency and aging and identiFy the signaling molecules that link osteocyte apoptosis and osteoclast recruitment. We hypothesize that enhanced extracellular release of the signaling factors HMGB1 anD miR21 recruits osteoclasts to areas containing apoptotic osteocytes and promotes osteoclast activity, leaDing to targeteD bone resorption. Overall, these stuDies Will proviDe insights into the mechanisms governing osteocyte apoptosis anD the associateD targeteD osteoclast resorption, alloWing for the Development of novel therapeutics for preserving the structure and strength of the aging skeleton. Lilian I. Plotkin, Ph.D., Chair vii TABLE OF CONTENTS Chapter 1. IntroDuction ...................................................................................................... 1 Chapter 2. ReDuceD osteocyte apoptosis anD preservation of cortical bone quality with aDvanceD age in mice with osteocytic Cx43 overexpression ................................... 12 Chapter 3. Disruption oF the Cx43/miR21 pathway leaDs to osteocyte apoptosis anD increaseD osteoclastogenesis With aging ........................................................................ 42 Chapter 4. Sex divergent role of osteocytic miR21 in the maintenance of osteocyte viability and regulation of bone turnover .......................................................................... 77 Chapter 5. Short-term pharmacologic RAGE inhibition DifFerentially eFFects bone anD skeletal muscle in aging ................................................................................................. 126 Chapter 6. High mobility group box1 (HMGB1) protein regulates osteoclastogenesis through direct actions on osteocytes anD osteoclasts in vitro ........................................ 167 Chapter 7. Summary oF FinDings anD Future Directions ................................................ 189 Bibliography ................................................................................................................... 199 Curriculum Vitae viii LIST OF TABLES Table 4-1. 9-Plex cell-signaling array results in bones From Female miR21∆ot mice ....... 119 Table 4-2. 9-Plex cell-signaling array results in bones From male miR21∆ot mice .......... 119 Table 4-3. Mechanical strength in cortical bone in Female miR21∆ot mice ..................... 120 Table 4-4. Mechanical strength in cortical bone in male miR21∆ot mice ........................ 121 Table 4-5A. ConDitioneD meDia cytokines in Female miR21∆ot mice .............................. 122 Table 4-5B. ConDitioneD meDia cytokines in male miR21∆ot mice ................................. 123 Table 4-6A. Circulating serum cytokines in female miR21∆ot mice ................................ 124 Table 4-6B. Circulating serum cytokines in male miR21∆ot mice ................................... 125 Table 5-1. Biomechanical properties measureD in the Femoral miD-diaphysis by 3-point bending test ....................................................................................................... 166 LIST OF FIGURES Figure 1-1. Illustration of the cellular and tissue-level skeletal alterations With aging ........ 1 Figure 1-2. Graphic illustration of the bone compartment-speciFic cellular changes with aging ........................................................................................................................... 2 Figure 1-3. Schematic illustration of a Cx43 molecule ....................................................... 4 Figure 1-4. Skeletal efFects of Cx43 deletion in osteoblastic lineage cells at diFFerent diFFerentiation stages .......................................................................................................... 5 Figure 1-5. Illustration of the skeletal phenotype resulting from osteocytic Cx43 removal .............................................................................................................................. 7 Figure 1-6. Schematic summary of the possible mechanisms of HMGB1 signaling in osteocytes anD osteoclasts ............................................................................................ 8 Figure 1-7. Aims of the current studies ............................................................................ 11 Figure 2-1. EFFective osteocyte targeted Cx43 transgene expression in DMP1-8kb- Cx43/GFP (Cx43OT) mice ................................................................................................ 30 Figure 2-2. Expression of Cx43 in osteocytes reduces osteocyte apoptosis induced with aging ......................................................................................................................... 32 Figure 2-3. 14-month-old Cx43OT mice exhibit enhanceD enDocortical bone formation anD decreaseD resorption ................................................................................ 33 Figure 2-4. Cx43OT mice exhibit increaseD cortical bone resilience to Fracture that is maintaineD With aDvanceD age ..................................................................................... 35 Figure 2-5. Cx43OT mice are not protecteD against age-inDuceD loss oF cancellous bone mass ....................................................................................................................... 36 Figure 2-6.