Browsing by Author "Childress, Paul"
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Item Aging negatively impacts the ability of megakaryocytes to stimulate osteoblast proliferation and bone mass(Elsevier, 2019) Maupin, Kevin A.; Himes, Evan R.; Plett, Artur P.; Chua, Hui Lin; Singh, Pratibha; Ghosh, Joydeep; Mohamad, Safa F.; Abeysekera, Irushi; Fisher, Alexa; Sampson, Carol; Hong, Jung-Min; Childress, Paul; Alvarez, Marta; Srour, Edward F.; Bruzzaniti, Angela; Pelus, Louis M.; Orschell, Christie M.; Kacena, Melissa A.; Orthopaedic Surgery, School of MedicineOsteoblast number and activity decreases with aging, contributing to the age-associated decline of bone mass, but the mechanisms underlying changes in osteoblast activity are not well understood. Here, we show that the age-associated bone loss critically depends on impairment of the ability of megakaryocytes (MKs) to support osteoblast proliferation. Co-culture of osteoblast precursors with young MKs is known to increase osteoblast proliferation and bone formation. However, co-culture of osteoblast precursors with aged MKs resulted in significantly fewer osteoblasts compared to co-culture with young MKs, and this was associated with the downregulation of transforming growth factor beta. In addition, the ability of MKs to increase bone mass was attenuated during aging as transplantation of GATA1low/low hematopoietic donor cells (which have elevated MKs/MK precursors) from young mice resulted in an increase in bone mass of recipient mice compared to transplantation of young wild-type donor cells, whereas transplantation of GATA1low/low donor cells from old mice failed to enhance bone mass in recipient mice compared to transplantation of old wild-type donor cells. These findings suggest that the preservation or restoration of the MK-mediated induction of osteoblast proliferation during aging may hold the potential to prevent age-associated bone loss and resulting fractures.Item Development of a step-down method for altering male C57BL/6 mouse housing density and hierarchical structure: Preparations for spaceflight studies(Elsevier, 2018-05) Scofield, David C.; Rytlewski, Jeffrey D.; Childress, Paul; Shah, Kishan; Tucker, Aamir; Khan, Faisal; Peveler, Jessica; Li, Ding; McKinley, Todd O.; Chu, Tien-Min G.; Hickman, Debra L.; Kacena, Melissa A.; Orthopaedic Surgery, School of MedicineThis study was initiated as a component of a larger undertaking designed to study bone healing in microgravity aboard the International Space Station (ISS). Spaceflight experimentation introduces multiple challenges not seen in ground studies, especially with regard to physical space, limited resources, and inability to easily reproduce results. Together, these can lead to diminished statistical power and increased risk of failure. It is because of the limited space, and need for improved statistical power by increasing sample size over historical numbers, NASA studies involving mice require housing mice at densities higher than recommended in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). All previous NASA missions in which mice were co-housed, involved female mice; however, in our spaceflight studies examining bone healing, male mice are required for optimal experimentation. Additionally, the logistics associated with spaceflight hardware and our study design necessitated variation of density and cohort make up during the experiment. This required the development of a new method to successfully co-house male mice while varying mouse density and hierarchical structure. For this experiment, male mice in an experimental housing schematic of variable density (Spaceflight Correlate) analogous to previously established NASA spaceflight studies was compared to a standard ground based housing schematic (Normal Density Controls) throughout the experimental timeline. We hypothesized that mice in the Spaceflight Correlate group would show no significant difference in activity, aggression, or stress when compared to Normal Density Controls. Activity and aggression were assessed using a novel activity scoring system (based on prior literature, validated in-house) and stress was assessed via body weights, organ weights, and veterinary assessment. No significant differences were detected between the Spaceflight Correlate group and the Normal Density Controls in activity, aggression, body weight, or organ weight, which was confirmed by veterinary assessments. Completion of this study allowed for clearance by NASA of our bone healing experiments aboard the ISS, and our experiment was successfully launched February 19, 2017 on SpaceX CRS-10.Item Forces associated with launch into space do not impact bone fracture healing(Elsevier, 2018-02) Childress, Paul; Brinker, Alexander; Gong, Cynthia-May S.; Harris, Jonathan; Olivos, David J.; Rytlewski, Jeffrey D.; Scofield, David C.; Choi, Sungshin Y.; Shirazi-Fard, Yasaman; McKinley, Todd O.; Chu, Tien-Min G.; Conley, Carolynn L.; Chakraborty, Nabarun; Hammamieh, Rasha; Kacena, Melissa A.; Orthopaedic Surgery, School of MedicineSegmental bone defects (SBDs) secondary to trauma invariably result in a prolonged recovery with an extended period of limited weight bearing on the affected limb. Soldiers sustaining blast injuries and civilians sustaining high energy trauma typify such a clinical scenario. These patients frequently sustain composite injuries with SBDs in concert with extensive soft tissue damage. For soft tissue injury resolution and skeletal reconstruction a patient may experience limited weight bearing for upwards of 6 months. Many small animal investigations have evaluated interventions for SBDs. While providing foundational information regarding the treatment of bone defects, these models do not simulate limited weight bearing conditions after injury. For example, mice ambulate immediately following anesthetic recovery, and in most cases are normally ambulating within 1-3 days post-surgery. Thus, investigations that combine disuse with bone healing may better test novel bone healing strategies. To remove weight bearing, we have designed a SBD rodent healing study in microgravity (µG) on the International Space Station (ISS) for the Rodent Research-4 (RR-4) Mission, which launched February 19, 2017 on SpaceX CRS-10 (Commercial Resupply Services). In preparation for this mission, we conducted an end-to-end mission simulation consisting of surgical infliction of SBD followed by launch simulation and hindlimb unloading (HLU) studies. In brief, a 2 mm defect was created in the femur of 10 week-old C57BL6/J male mice (n = 9-10/group). Three days after surgery, 6 groups of mice were treated as follows: 1) Vivarium Control (maintained continuously in standard cages); 2) Launch Negative Control (placed in the same spaceflight-like hardware as the Launch Positive Control group but were not subjected to launch simulation conditions); 3) Launch Positive Control (placed in spaceflight-like hardware and also subjected to vibration followed by centrifugation); 4) Launch Positive Experimental (identical to Launch Positive Control group, but placed in qualified spaceflight hardware); 5) Hindlimb Unloaded (HLU, were subjected to HLU immediately after launch simulation tests to simulate unloading in spaceflight); and 6) HLU Control (single housed in identical HLU cages but not suspended). Mice were euthanized 28 days after launch simulation and bone healing was examined via micro-Computed Tomography (µCT). These studies demonstrated that the mice post-surgery can tolerate launch conditions. Additionally, forces and vibrations associated with launch did not impact bone healing (p = .3). However, HLU resulted in a 52.5% reduction in total callus volume compared to HLU Controls (p = .0003). Taken together, these findings suggest that mice having a femoral SBD surgery tolerated the vibration and hypergravity associated with launch, and that launch simulation itself did not impact bone healing, but that the prolonged lack of weight bearing associated with HLU did impair bone healing. Based on these findings, we proceeded with testing the efficacy of FDA approved and novel SBD therapies using the unique spaceflight environment as a novel unloading model on SpaceX CRS-10.Item Genome-Wide Mapping and Interrogation of the Nmp4 Antianabolic Bone Axis(Oxford University Press, 2015-09) Childress, Paul; Stayrook, Keith R.; Alvarez, Marta B.; Wang, Zhiping; Shao, Yu; Hernandez-Buquer, Selene; Mack, Justin K.; Grese, Zachary R.; He, Yongzheng; Horan, Daniel; Pavalko, Fredrick M.; Warden, Stuart J.; Robling, Alexander G.; Yang, Feng-Chun; Allen, Matthew R.; Krishnan, Venkatesh; Liu, Yunlong; Bidwell, Joseph P.; Department of Anatomy & Cell Biology, IU School of MedicinePTH is an osteoanabolic for treating osteoporosis but its potency wanes. Disabling the transcription factor nuclear matrix protein 4 (Nmp4) in healthy, ovary-intact mice enhances bone response to PTH and bone morphogenetic protein 2 and protects from unloading-induced osteopenia. These Nmp4(-/-) mice exhibit expanded bone marrow populations of osteoprogenitors and supporting CD8(+) T cells. To determine whether the Nmp4(-/-) phenotype persists in an osteoporosis model we compared PTH response in ovariectomized (ovx) wild-type (WT) and Nmp4(-/-) mice. To identify potential Nmp4 target genes, we performed bioinformatic/pathway profiling on Nmp4 chromatin immunoprecipitation sequencing (ChIP-seq) data. Mice (12 w) were ovx or sham operated 4 weeks before the initiation of PTH therapy. Skeletal phenotype analysis included microcomputed tomography, histomorphometry, serum profiles, fluorescence-activated cell sorting and the growth/mineralization of cultured WT and Nmp4(-/-) bone marrow mesenchymal stem progenitor cells (MSPCs). ChIP-seq data were derived using MC3T3-E1 preosteoblasts, murine embryonic stem cells, and 2 blood cell lines. Ovx Nmp4(-/-) mice exhibited an improved response to PTH coupled with elevated numbers of osteoprogenitors and CD8(+) T cells, but were not protected from ovx-induced bone loss. Cultured Nmp4(-/-) MSPCs displayed enhanced proliferation and accelerated mineralization. ChIP-seq/gene ontology analyses identified target genes likely under Nmp4 control as enriched for negative regulators of biosynthetic processes. Interrogation of mRNA transcripts in nondifferentiating and osteogenic differentiating WT and Nmp4(-/-) MSPCs was performed on 90 Nmp4 target genes and differentiation markers. These data suggest that Nmp4 suppresses bone anabolism, in part, by regulating IGF-binding protein expression. Changes in Nmp4 status may lead to improvements in osteoprogenitor response to therapeutic cues.Item Improving Combination Osteoporosis Therapy in a Preclinical Model of Heightened Osteoanabolism(Oxford University Press, 2017-09-01) Shao, Yu; Hernandez-Buquer, Selene; Childress, Paul; Stayrook, Keith R.; Alvarez, Marta B.; Davis, Hannah; Plotkin, Lilian I.; He, Yongzheng; Condon, Keith W.; Burr, David B.; Warden, Stuart J.; Robling, Alexander G.; Yang, Feng-Chun; Wek, Ronald C.; Allen, Matthew R.; Bidwell, Joseph P.; Medical and Molecular Genetics, School of MedicineCombining anticatabolic agents with parathyroid hormone (PTH) to enhance bone mass has yielded mixed results in osteoporosis patients. Toward the goal of enhancing the efficacy of these regimens, we tested their utility in combination with loss of the transcription factor Nmp4 because disabling this gene amplifies PTH-induced increases in trabecular bone in mice by boosting osteoblast secretory activity. We addressed whether combining a sustained anabolic response with an anticatabolic results in superior bone acquisition compared with PTH monotherapy. Additionally, we inquired whether Nmp4 interferes with anticatabolic efficacy. Wild-type and Nmp4-/- mice were ovariectomized at 12 weeks of age, followed by therapy regimens, administered from 16 to 24 weeks, and included individually or combined PTH, alendronate (ALN), zoledronate (ZOL), and raloxifene (RAL). Anabolic therapeutic efficacy generally corresponded with PTH + RAL = PTH + ZOL > PTH + ALN = PTH > vehicle control. Loss of Nmp4 enhanced femoral trabecular bone increases under PTH + RAL and PTH + ZOL. RAL and ZOL promoted bone restoration, but unexpectedly, loss of Nmp4 boosted RAL-induced increases in femoral trabecular bone. The combination of PTH, RAL, and loss of Nmp4 significantly increased bone marrow osteoprogenitor number, but did not affect adipogenesis or osteoclastogenesis. RAL, but not ZOL, increased osteoprogenitors in both genotypes. Nmp4 status did not influence bone serum marker responses to treatments, but Nmp4-/- mice as a group showed elevated levels of the bone formation marker osteocalcin. We conclude that the heightened osteoanabolism of the Nmp4-/- skeleton enhances the effectiveness of diverse osteoporosis treatments, in part by increasing hyperanabolic osteoprogenitors. Nmp4 provides a promising target pathway for identifying barriers to pharmacologically induced bone formation.Item LymphTF Database- A Database of Transcription Factor Activity in Lymphocyte Development(2006-07-26T15:52:21Z) Childress, PaulStudy of the transcriptional regulation of lymphocyte development has advanced greatly in the past 15 years. Owing to improved techniques and intense interest in the topic, a great many interactions between transcription factors and their target genes have been described. For these B and T cells, a more clear picture is beginning to emerge of how they start with a common progenitor cell, and progressively restrict potential to give many different types of terminally differentiated cells. As B and T cells develop they both follow a roughly similar path that involves early stepwise progression to later stages where multiple developmental options are available. To progress in the developmental regime they share requirements for proper anatomical location and successful rearrangements of the germ line DNA to give the plethora of antibodies and T cell receptors seen in the immune system. Because the amount of information is quickly becoming more than can be assimilated by researchers, a knowledge gap has opened between what is known about the transcription factor activities during this process and what any one individual can recall. To help fill this gap, we have created the LymphTF Database. This database holds interactions between individual transcription factors and their specific targets at a given developmental time. It is our hope that storing the interactions in developmental time will allow for elucidation of regulatory networks which guide the process. Work for this project also included construction of a custom data entry web page that automates many tasks associated with populating the database tables. These tables have also been related in multiple ways to allow for storage of incomplete information on transcription factor activity. This is done without having to replace existing records as details become available. The LymphTF DB is a relational MySQL database which can be accessed freely on the web at http://www.iupui.edu/~tfinterx/.Item Megakaryocytes: Regulators of Bone Mass and Hematopoiesis(Office of the Vice Chancellor for Research, 2016-04-08) Alvarez, Marta B.; Xu, LinLin; Himes, Evan R.; Chitteti, Brahmananda R.; Cheng, Yinghua; Engle, Andrew; Olivos, David; Childress, Paul; Srour, Edward F.; Kacena, Melissa A.Emerging evidence demonstrates that megakaryocytes (MK) play a key role in regulating skeletal homeostasis and hematopoiesis. Recent reports show that MK reside in close proximity to hematopoietic stem cells (HSC). Genetic depletion of MK resulted in mitotic activation of HSC suggesting that MK maintain HSC quiescence. Other studies demonstrated that following irradiation, surviving MK migrate to endosteal surfaces where osteoblast (OB) lineage cells dramatically increase and promote engraftment of transplanted HSC. Here we investigated if MK directly impact hematopoiesis or whether they indirectly support HSC function through their interaction with OB-lineage cells. Our data suggests that LSK (Lin-Sca+CD117+, an enriched HSC population) co-cultured with MK and OB generate significantly higher numbers of colony forming cells (HSC function) compared to LSK cocultured with either MK or OB alone. The functionality of this in vitro data was confirmed in vivo with transplantation studies which showed increased engraftment in mice transplanted with LSK cells co-cultured with OB and MK compared to LSK cells co-cultured with OB alone. To test if loss of MK negatively impacts osteoblastogenesis, we generated conditional knockout mice where cMpl, the receptor for the main MK growth factor, thrombopoietin (TPO), was deleted in MK (cMplfl/fl x PF4Cre). Unexpectedly, these mice exhibited a 10-fold increase in platelet numbers, megakaryocytosis, a dramatic expansion of phenotypically defined hematopoietic precursors, and a remarkable 20-fold increase in the bone volume fraction. Collectively, these data indicate that while MK modulate HSC function, this activity is in part mediated through interactions with OB and suggest a complex role for TPO and MK in HSC regulation. While work is needed to further elucidate mechanisms, understanding the coordinated interaction between MK, OB, HSC, and TPO/Mpl should inform the development of novel treatments to enhance HSC recovery following myelosuppressive injuries, as well as bone loss diseases, such as osteoporosis.Item Nmp4/CIZ suppresses the response of bone to anabolic parathyroid hormone by regulating both osteoblasts and osteoclasts(2011-07) Childress, Paul; Philip, Binu K.; Robling, Alexander G.; Bruzzaniti, Angela; Kacena, Melissa A.; Bivi, Nicoletta; Plotkin, Lilian I.; Heller, Aaron; Bidwell, Joseph P.How parathyroid hormone (PTH) increases bone mass is unclear, but understanding this phenomenon is significant to the improvement of osteoporosis therapy. Nmp4/CIZ is a nucleocytoplasmic shuttling transcriptional repressor that suppresses PTH-induced osteoblast gene expression and hormone-stimulated gains in murine femoral trabecular bone. To further characterize Nmp4/CIZ suppression of hormone-mediated bone growth, we treated 10-week-old Nmp4-knockout (KO) and wild-type (WT) mice with intermittent human PTH(1–34) at 30 μg/kg daily or vehicle, 7 days/week, for 2, 3, or 7 weeks. Null mice treated with hormone (7 weeks) gained more vertebral and tibial cancellous bone than WT animals, paralleling the exaggerated response in the femur. Interestingly, Nmp4/CIZ suppression of this hormone-stimulated bone formation was not apparent during the first 2 weeks of treatment. Consistent with the null mice enhanced PTH-stimulated addition of trabecular bone, these animals exhibited an augmented hormone-induced increase in serum osteocalcin 3 weeks into treatment. Unexpectedly, the Nmp4-KO mice displayed an osteoclast phenotype. Serum C-terminal telopeptide, a marker for bone resorption, was elevated in the null mice, irrespective of treatment. Nmp4-KO bone marrow cultures produced more osteoclasts, which exhibited elevated resorbing activity, compared to WT cultures. The expression of several genes critical to the development of both osteoblasts and osteoclasts was elevated in Nmp4-KO mice at 2 weeks, but not 3 weeks, of hormone exposure. We propose that Nmp4/CIZ dampens PTH-induced improvement of trabecular bone throughout the skeleton by transiently suppressing hormone-stimulated increases in the expression of proteins key to the required enhanced activity and number of both osteoblasts and osteoclasts.Item Skeletal adaptations in young male mice after 4 weeks aboard the International Space Station(Nature Research, 2019-09-24) Maupin, Kevin A.; Childress, Paul; Brinker, Alexander; Khan, Faisal; Abeysekera, Irushi; Aguilar, Izath Nizeet; Olivos, David J., III; Adam, Gremah; Savaglio, Michael K.; Ganesh, Venkateswaran; Gorden, Riley; Mannfeld, Rachel; Beckner, Elliott; Horan, Daniel J.; Robling, Alexander G.; Chakraborty, Nabarun; Gautam, Aarti; Hammamieh, Rasha; Kacena, Melissa A.; Orthopaedic Surgery, School of MedicineGravity has an important role in both the development and maintenance of bone mass. This is most evident in the rapid and intense bone loss observed in both humans and animals exposed to extended periods of microgravity in spaceflight. Here, cohabitating 9-week-old male C57BL/6 mice resided in spaceflight for ~4 weeks. A skeletal survey of these mice was compared to both habitat matched ground controls to determine the effects of microgravity and baseline samples in order to determine the effects of skeletal maturation on the resulting phenotype. We hypothesized that weight-bearing bones would experience an accelerated loss of bone mass compared to non-weight-bearing bones, and that spaceflight would also inhibit skeletal maturation in male mice. As expected, spaceflight had major negative effects on trabecular bone mass of the following weight-bearing bones: femur, tibia, and vertebrae. Interestingly, as opposed to the bone loss traditionally characterized for most weight-bearing skeletal compartments, the effects of spaceflight on the ribs and sternum resembled a failure to accumulate bone mass. Our study further adds to the insight that gravity has site-specific influences on the skeleton.