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Item Bone Mass and Strength are Significantly Improved in Mice Overexpressing Human WNT16 in Osteocytes(Springer, 2017-04) Alam, Imranul; Reilly, Austin M.; Alkhouli, Mohammed; Gerard-O’Riley, Rita L.; Kasipathi, Charishma; Oakes, Dana K.; Wright, Weston B.; Acton, Dena; McQueen, Amie K.; Patel, Bhavmik; Lim, Kyung-Eun; Robling, Alexander G.; Econs, Michael J.; Medicine, School of MedicineRecently, we demonstrated that osteoblast-specific overexpression of human WNT16 increased both cortical and trabecular bone mass and structure in mice. To further identify the cell-specific role of Wnt16 in bone homeostasis, we created transgenic (TG) mice overexpressing human WNT16 in osteocytes using Dmp1 promoter (Dmp1-hWNT16 TG) on C57BL/6 (B6) background. We analyzed bone phenotypes and serum bone biomarkers, performed gene expression analysis and measured dynamic bone histomorphometry in Dmp1-hWNT16 TG and wild-type (WT) mice. Compared to WT mice, Dmp1-hWNT16 TG mice exhibited significantly higher whole-body, spine and femoral aBMD, BMC and trabecular (BV/TV, Tb.N, and Tb.Th) and cortical (bone area and thickness) parameters in both male and female at 12 weeks of age. Femur stiffness and ultimate force were also significantly improved in the Dmp1-hWNT16 TG female mice, compared to sex-matched WT littermates. In addition, female Dmp1-hWNT16 TG mice displayed significantly higher MS/BS, MAR and BFR/BS compared to the WT mice. Gene expression analysis demonstrated significantly higher mRNA level of Alp in both male and female Dmp1-hWNT16 TG mice and significantly higher levels of Osteocalcin, Opg and Rankl in the male Dmp1-hWNT16 TG mice in bone tissue compared to sex-matched WT mice. These results indicate that WNT16 plays a critical role for acquisition of both cortical and trabecular bone mass and strength. Strategies designed to use WNT16 as a target for therapeutic interventions will be valuable to treat osteoporosis and other low bone mass conditions.Item C-Mpl Is Expressed on Osteoblasts and Osteoclasts and Is Important in Regulating Skeletal Homeostasis(Wiley, 2016-04) Meijome, Tomas E.; Baughman, Jenna T.; Hooker, R. Adam; Cheng, Ying-Hua; Ciovacco, Wendy A.; Balamohan, Sanjeev M.; Srinivasan, Trishya L.; Chitteti, Brahmananda R.; Eleniste, Pierre P.; Horowitz, Mark C.; Srour, Edward F.; Bruzzaniti, Angela; Fuchs, Robyn K.; Kacena, Melissa A.; Orthopaedic Surgery, School of MedicineC-Mpl is the receptor for thrombopoietin (TPO), the main megakaryocyte (MK) growth factor, and c-Mpl is believed to be expressed on cells of the hematopoietic lineage. As MKs have been shown to enhance bone formation, it may be expected that mice in which c-Mpl was globally knocked out (c-Mpl(-/-) mice) would have decreased bone mass because they have fewer MKs. Instead, c-Mpl(-/-) mice have a higher bone mass than WT controls. Using c-Mpl(-/-) mice we investigated the basis for this discrepancy and discovered that c-Mpl is expressed on both osteoblasts (OBs) and osteoclasts (OCs), an unexpected finding that prompted us to examine further how c-Mpl regulates bone. Static and dynamic bone histomorphometry parameters suggest that c-Mpl deficiency results in a net gain in bone volume with increases in OBs and OCs. In vitro, a higher percentage of c-Mpl(-/-) OBs were in active phases of the cell cycle, leading to an increased number of OBs. No difference in OB differentiation was observed in vitro as examined by real-time PCR and functional assays. In co-culture systems, which allow for the interaction between OBs and OC progenitors, c-Mpl(-/-) OBs enhanced osteoclastogenesis. Two of the major signaling pathways by which OBs regulate osteoclastogenesis, MCSF/OPG/RANKL and EphrinB2-EphB2/B4, were unaffected in c-Mpl(-/-) OBs. These data provide new findings for the role of MKs and c-Mpl expression in bone and may provide insight into the homeostatic regulation of bone mass as well as bone loss diseases such as osteoporosis.Item Foreword: Calcified Tissue International and Musculoskeletal Research Special Issue: Bone Material Properties and Skeletal Fragility.(Springer, 2015-09) Burr, David B.; Allen, Matthew R.; Department of Anatomy & Cell Biology, IU School of MedicineItem Lnk Deficiency Leads to TPO-Mediated Osteoclastogenesis and Increased Bone Mass Phenotype(Wiley, 2017-08) Olivos III, David J.; Alvarez, Marta; Cheng, Ying-Hua; Hooker, R. Adam; Ciovacco, Wendy A.; Bethel, Monique; McGough, Haley; Yim, Christopher; Chitteti, Brahmananda R.; Eleniste, Pierre P.; Horowitz, Mark C.; Srour, Edward F.; Bruzzaniti, Angela; Fuchs, Robyn K.; Kacena, Melissa A.; Orthopaedic Surgery, School of MedicineThe Lnk adapter protein negatively regulates the signaling of thrombopoietin (TPO), the main megakaryocyte (MK) growth factor. Lnk-deficient (-/-) mice have increased TPO signaling and increased MK number. Interestingly, several mouse models exist in which increased MK number leads to a high bone mass phenotype. Here we report the bone phenotype of these mice. MicroCT and static histomorphometric analyses at 20 weeks showed the distal femur of Lnk-/- mice to have significantly higher bone volume fraction and trabecular number compared to wild-type (WT) mice. Notably, despite a significant increase in the number of osteoclasts (OC), and decreased bone formation rate in Lnk-/- mice compared to WT mice, Lnk-/- mice demonstrated a 2.5-fold greater BV/TV suggesting impaired OC function in vivo. Additionally, Lnk-/- mouse femurs exhibited non-significant increases in mid-shaft cross-sectional area, yet increased periosteal BFR compared to WT femurs was observed. Lnk-/- femurs also had non-significant increases in polar moment of inertia and decreased cortical bone area and thickness, resulting in reduced bone stiffness, modulus, and strength compared to WT femurs. Of note, Lnk is expressed by OC lineage cells and when Lnk-/- OC progenitors are cultured in the presence of TPO, significantly more OC are observed than in WT cultures. Lnk is also expressed in osteoblast (OB) cells and in vitro reduced alkaline phosphatase activity was observed in Lnk-/- cultures. These data suggest that both direct effects on OB and OC as well as indirect effects of MK in regulating OB contributes to the observed high bone mass. J. Cell. Biochem. 118: 2231-2240, 2017.Item New Insights Into the Local and Systemic Functions of Sclerostin: Regulation of Quiescent Bone Lining Cells and Beige Adipogenesis in Peripheral Fat Depots(Wiley, 2017-05) Delgado-Calle, Jesus; Bellido, Teresita; Anatomy and Cell Biology, School of MedicineItem Osteoclast-mediated bone loss observed in a COVID-19 mouse model(Elsevier, 2022-01) Awosanya, Olatundun D.; Dalloul, Christopher E.; Blosser, Rachel J.; Dadwal, Ushashi C.; Carozza, Mariel; Boschen, Karen; Klemsz, Michael J.; Johnston, Nancy A.; Bruzzaniti, Angela; Robinson, Christopher M.; Srour, Edward F.; Kacena, Melissa A.; Orthopaedic Surgery, School of MedicineThe consequences of SARS-CoV-2 infection on the musculoskeletal system represent a dangerous knowledge gap. Aging patients are at added risk for SARS-CoV-2 infection; therefore, a greater understanding of the resulting musculoskeletal sequelae of SARS-CoV-2 infection may help guide clinical strategies. This study examined fundamental bone parameters among mice treated with escalating viral loads. Male C57BL/6J (WT, n = 17) and B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (K18-hACE2 transgenic mice, n = 21) expressing human ACE2 (TG) were divided into eight groups (n = 4-6/group) and subjected to intranasal dosing of 0, 1 × 103, 1 × 104, and 1 × 105 PFU (plaque forming units) of human SARS-CoV-2. Animal health was assessed daily by veterinary staff using established and validated scoring criteria (activity, posture, body condition scores and body weight). We report here that mock and WT infected mice were healthy and completed the study, surviving until 12-14 days post infection (dpi). In contrast, the TG mice infected with 1 × 105 PFU all experienced severe health declines that necessitated early euthanasia (6-7 dpi). For TG mice infected with 1 × 104 PFU, 2 mice were also euthanized after 7 dpi, while 3 mice showed signs of moderate disease at day 6 dpi, but recovered fully by day 11 dpi. Four of the 5 TG mice that were infected with 1 × 103 PFU remained healthy throughout the study. This suggests that our study mimics what is seen during human disease, where some patients develop severe disease resulting in death, while others have moderate to severe disease but recover, and others are asymptomatic. At necropsy, femurs were extracted and analyzed by μCT. No difference was found in μCT determined bone parameters among the WT groups. There was, however, a significant 24.4% decrease in trabecular bone volume fraction (p = 0.0009), 19.0% decrease in trabecular number (p = 0.004), 6.2% decrease in trabecular thickness (p = 0.04), and a 9.8% increase in trabecular separation (p = 0.04) among surviving TG mice receiving any viral load compared to non-infected controls. No differences in cortical bone parameters were detected. TRAP staining revealed surviving infected mice had a significant 64% increase in osteoclast number, a 27% increase in osteoclast surface, and a 38% increase in osteoclasts per bone surface. While more studies are needed to investigate the long-term consequences of SARS-CoV-2 infection on skeletal health, this study demonstrates a significant reduction in several bone parameters and corresponding robust increases in osteoclast number observed within 2 weeks post-infection in surviving asymptomatic and moderately affected mice.Item Overexpression of WNT16 Does Not Prevent Cortical Bone Loss Due to Glucocorticoid Treatment in Mice(American Society for Bone and Mineral Research, 2018-10-23) Alam, Imranul; Oakes, Dana K.; Reilly, Austin M.; Billingsley, Caylin; Sbeta, Shahed; Gerard‐O'Riley, Rita L.; Acton, Dena; Sato, Amy; Bellido, Teresita; Econs, Michael J.; Medicine, School of MedicineGlucocorticoids (GC) are commonly used for the treatment of a wide variety of autoimmune, pulmonary, gastrointestinal, and malignancy conditions. One of the devastating side effects of GC use is osteoporotic fractures, particularly in the spine and hip. Bisphosphonates (BP) are the most commonly prescribed pharmacological agents for the prevention and treatment of GC-induced osteoporosis (GIO). However, GIO is marked by reduced bone formation and BP serves mainly to decrease bone resorption. The WNT signaling pathway plays a major role in bone and mineral homeostasis. Previously, we demonstrated that overexpression of WNT16 in mice led to higher bone mineral density and improved bone microarchitecture and strength. We hypothesized that WNT16 overexpression would prevent bone loss due to glucocorticoid treatment in mice. To test our hypothesis, we treated adult wild-type and WNT16-transgenic mice with vehicle and GC (prednisolone; 2.1 mg/kg body weight) via slow-release pellets for 28 days. We measured bone mass and microarchitecture by dual-energy X-ray absorptiometry (DXA) and micro-CT, and performed gene expression and serum biochemical analysis. We found that GC treatment compared with the vehicle significantly decreased femoral areal bone mineral density (aBMD), bone mineral content (BMC), and cortical bone area and thickness in both wild-type and transgenic female mice. In contrast, the trabecular bone parameters at distal femur were not significantly changed by GC treatment in male and female mice for both genotypes. Further, we observed significantly lower level of serum P1NP and a tendency of higher level of serum TRAP in wild-type and transgenic mice due to GC treatment in both sexes. Gene expression analysis showed lower mRNA levels of Wnt16, Opg, and Opg/Rankl ratio in GC-treated female mice for both genotypes compared with the sex-matched vehicle-treated mice. These data suggest that although WNT16 overexpression resulted in higher baseline bone mineral density and bone volume per trabecular volume (BV/TV) in the transgenic mice, this was insufficient to prevent bone loss in mice due to glucocorticoid treatment.Item Sex-specific differences in direct osteoclastic versus indirect osteoblastic effects underlay the low bone mass of Pannexin1 deletion in TRAP-expressing cells in mice(Elsevier, 2022-01-04) Deosthale, Padmini; Hong, Jung Min; Essex, Alyson L.; Rodriguez, Wilyaret; Tariq, Dua; Sidhu, Harmandeep; Marcial, Alejandro; Bruzzaniti, Angela; Plotkin, Lilian I.; Anatomy, Cell Biology and Physiology, School of MedicinePannexin1 (Panx1) is a hemichannel-forming protein that participates in the communication of cells with the extracellular space. To characterize the role of osteoclastic Panx1 on bone, Panx1fl/fl;TRAP-Cre (Panx1ΔOc) mice were generated, and compared to Panx1fl/fl littermates at 6 weeks of age. Total and femoral BMD was ~20% lower in females and males whereas spinal BMD was lower only in female Panx1ΔOc mice. μCT analyses showed that cortical bone of the femoral mid-diaphysis was not altered in Panx1ΔOc mice. In contrast, cancellous bone in the distal femur and lumbar vertebra was significantly decreased in both female and male Panx1ΔOc mice compared to Panx1fl/fl controls and was associated with higher osteoclast activity in female Panx1ΔOc mice, with no changes in the males. On the other hand, vertebral bone formation was decreased for both sexes, resulting from lower mineral apposition rate in the females and lower mineralizing surface in the males. Consistent with an osteoclastic effect in female Panx1ΔOc mice, osteoclast differentiation with RANKL/M-CSF and osteoclast bone resorbing activity in vitro were higher in female, but not male, Panx1ΔOc mice, compared to Panx1fl/fl littermates. Surprisingly, although Panx1 expression was normal in bone marrow stromal-derived osteoblasts from male and female Panx1ΔOc mice, mineral deposition by male (but not female) Panx1ΔOc osteoblasts was lower than controls, and it was reduced in male Panx1fl/fl osteoblasts when conditioned media prepared from male Panx1ΔOc osteoclast cultures was added to the cell culture media. Thus, deletion of Panx1 in TRAP-expressing cells in female mice leads to low bone mass primarily through a cell autonomous effect in osteoclast activity. In contrast, our evidence suggests that changes in the osteoclast secretome drive reduced osteoblast function in male Panx1ΔOc mice, resulting in low bone mass.Item Systemic effects of BMP2 treatment of fractures on non-injured skeletal sites during spaceflight(Frontiers Media, 2022-08-15) Zamarioli, Ariane; Adam, Gremah; Maupin, Kevin A.; Childress, Paul J.; Brinker, Alexander; Ximenez, Joao P. B.; Chakraborty, Nabarun; Gautam, Aarti; Hammamieh, Rasha; Kacena, Melissa A.; Orthopaedic Surgery, School of MedicineUnloading associated with spaceflight results in bone loss and increased fracture risk. Bone morphogenetic protein 2 (BMP2) is known to enhance bone formation, in part, through molecular pathways associated with mechanical loading; however, the effects of BMP2 during spaceflight remain unclear. Here, we investigated the systemic effects of BMP2 on mice sustaining a femoral fracture followed by housing in spaceflight (International Space Station or ISS) or on Earth. We hypothesized that in spaceflight, the systemic effects of BMP2 on weight-bearing bones would be blunted compared to that observed on Earth. Nine-week-old male mice were divided into four groups: 1) Saline+Earth; 2) BMP+Earth; 3) Saline+ISS; and 4) BMP+ISS (n = 10 mice/group, but only n = 5 mice/group were reserved for micro-computed tomography analyses). All mice underwent femoral defect surgery and were followed for approximately 4 weeks. We found a significant reduction in trabecular separation within the lumbar vertebrae after administering BMP2 at the fracture site of mice housed on Earth. In contrast, BMP2 treatment led to a significant increase in trabecular separation concomitant with a reduction in trabecular number within spaceflown tibiae. Although these and other lines of evidence support our hypothesis, the small sample size associated with rodent spaceflight studies limits interpretations. That said, it appears that a locally applied single dose of BMP2 at the femoral fracture site can have a systemic impact on distant bones, affecting bone quantity in several skeletal sites. Moreover, our results suggest that BMP2 treatment works through a pathway involving mechanical loading in which the best outcomes during its treatment on Earth occurred in the weight-bearing bones and in spaceflight occurred in bones subjected to higher muscle contraction.Item The Role of PPARδ-Driven β-Oxidation in Bone Health During Aging(Oxford University Press, 2022-12-20) Prideaux, Matt; O'Connell, Tom; Kitase, Yukiko; Anatomy, Cell Biology and Physiology, School of MedicineMusculoskeletal disorders are a significant complication of aging, leading to increased morbidity and mortality. However, current understanding of the mechanisms by which aging affects skeletal health is limited. Osteocytes are the most numerous and long-lived bone cells and play key roles in maintaining bone mass by responding to anabolic signals such as mechanical loading. Energy metabolism is dysregulated in many cells with aging, however regulation of energy metabolism in osteocytes and how this is affected during aging and by mechanical loading remains undefined. To investigate this, we first used IDG-SW3 osteocyte cells to determine the effects of mechanical loading on osteocytes in vitro by applying fluid flow shear stress (FFSS). FFSS increased Pparδ and Cpt1 expression, key promoters of fatty acid β-oxidation (FAO). Pharmacological antagonism of PPARδ or CPT1 resulted in dysregulated expression of key bone remodeling genes and impaired ATP release in response to FFSS. In vivo, mechanical loading significantly increased FAO in tibia cortical bone. However, FAO was impaired in the bones from aged mice. To further elucidate the role of osteocyte FAO, we deleted PPARδ specifically in osteocytes (PPARδ cKO), which resulted in decreased FAO and bone volume in female PPARδ cKO mice. Lastly, treatment of aging mice with the PPARδ activator GW0742 resulted in significantly increased bone mineral content, density and trabecular bone volume. These findings suggest important functions of osteocyte energy metabolism during aging and with mechanical loading on bone and identify PPARδ-driven FAO as a novel therapeutic target for improving skeletal health with aging.