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Browsing by Author "Robling, Alexander"
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ItemCombining Sclerostin and DKK1 Inhibitors to Improve Bone Properties in the Aged Skeleton(Oxford University Press, 2022-12-20) Choi, Roy; Robling, Alexander; Anatomy, Cell Biology and Physiology, School of MedicineTargeting the secreted Wnt inhibitor sclerostin has been an attractive strategy to improve skeletal health. Sclerostin antibody (romosozumab-aqqg; Evenity) was recently approved by the FDA to treat patients at increased risk of fracture. However, an increased risk of cardiovascular events was reported, resulting in issue a ‘black box warning’ requirement for romosozumab. One potential solution to lower the risk of adverse events is to reduce the medication dose. Previously, we found that dual inhibition of sclerostin and Dkk1 produced extremely potent synergistic bone anabolic effects, in both genetic and pharmacological models. While Dkk1 inhibition alone has no consistent bone-building effects, combining antibodies that target sclerostin (Scl-mAb) and Dkk1 (Dkk1-mAb) at 3:1 ratio resulted in 2-3X more bone gain as Scl-mAb alone. Further, much lower total doses of dual antibody treatment, given at optimized proportions, generated equivalent bone anabolic effects as Scl-mAb alone (at much higher doses), suggesting that a combinational strategy has obvious translational benefits. Finally, we tested whether low-dose combination therapy can maintain the same osteogenic effect as Scl-mAb in adult (6 month) and aged (20 month) mice. Outcome measures derived from radiographic, biomechanical, and histomorphometric assays revealed that a 3:1 ratio of Scl-mAb:Dkk1-mAb at 12.5mg/kg was as efficacious as 25mg/kg of Scl-mAb alone, in both age groups. Moreover, cortical porosity—a significant factor contributing to skeletal fragility in the aged skeleton—was significantly reduced by both Scl-mAb and low-dose combination treatment. In conclusion, our findings suggest that optimized low-dose combinational therapy is viable strategy for improving skeletal fragility. ItemControl of Bone Anabolism in Response to Mechanical Loading and PTH by Distinct Mechanisms Downstream of the PTH Receptor(Wiley, 2017-03) Delgado-Calle, Jesus; Tu, Xiaolin; Pacheco-Costa, Rafael; McAndrews, Kevin; Edwards, Rachel; Pellegrini, Gretel G.; Kuhlenschmidt, Kali; Olivos, Naomie; Robling, Alexander; Peacock, Munro; Plotkin, Lilian I.; Bellido, Teresita; Anatomy, Cell Biology and Physiology, School of MedicineOsteocytes integrate the responses of bone to mechanical and hormonal stimuli by poorly understood mechanisms. We report here that mice with conditional deletion of the parathyroid hormone (PTH) receptor 1 (Pth1r) in dentin matrix protein 1 (DMP1)-8kb-expressing cells (cKO) exhibit a modest decrease in bone resorption leading to a mild increase in cancellous bone without changes in cortical bone. However, bone resorption in response to endogenous chronic elevation of PTH in growing or adult cKO mice induced by a low calcium diet remained intact, because the increased bone remodeling and bone loss was indistinguishable from that exhibited by control littermates. In contrast, the bone gain and increased bone formation in cancellous and cortical bone induced by daily injections of PTH and the periosteal bone apposition induced by axial ulna loading were markedly reduced in cKO mice compared to controls. Remarkably, however, wild-type (WT) control littermates and transgenic mice overexpressing SOST injected daily with PTH exhibit similar activation of Wnt/β-catenin signaling, increased bone formation, and cancellous and cortical bone gain. Taken together, these findings demonstrate that Pth1r in DMP1-8kb-expressing cells is required to maintain basal levels of bone resorption but is dispensable for the catabolic action of chronic PTH elevation; and it is essential for the anabolic actions of daily PTH injections and mechanical loading. However, downregulation of Sost/sclerostin, previously shown to be required for bone anabolism induced by mechanical loading, is not required for PTH-induced bone gain, showing that other mechanisms downstream of the Pth1r in DMP1-8kb-expressing cells are responsible for the hormonal effect. ItemThe Effect of Overexpression of Lrp5 on the Temporomandibular Joint(Sage, 2021) Utreja, Achint; Motevasel, Hengameh; Bain, Carol; Holland, Robert; Robling, Alexander; Orthodontics and Oral Facial Genetics, School of DentistryObjective: The temporomandibular joint (TMJ) is a unique fibrocartilaginous joint that adapts to mechanical loading through cell signaling pathways such as the Wnt pathway. Increased expression of low-density lipoprotein receptor-related protein 5 (Lrp5), a co-receptor of the Wnt pathway, is associated with a high bone mass (HBM) phenotype. The objective of this study was to analyze the effect of overexpression of Lrp5 on the subchondral bone and cartilage of the TMJ in mice exhibiting the HBM phenotype. Design: Sixteen-week-old Lrp5 knock-in transgenic mice carrying either the A214V (EXP-A) or G171V (EXP-G) missense mutations, and wildtype controls (CTRL) were included in this study. Fluorescent bone labels, calcein, alizarin complexone, and demeclocycline were injected at 3.5, 7.5, and 11.5 weeks of age, respectively. The left mandibular condyle was used to compare the subchondral bone micro-computed tomography parameters and the right TMJ was used for histological analyses. Cartilage thickness, matrix proteoglycan accumulation, and immunohistochemical localization of Lrp5 and sclerostin were compared between the groups. Results: Subchondral bone volume (BV) and percent bone volume (BV/TV) were significantly increased in both EXP-A and EXP-G compared with CTRL (P < 0.05) whereas trabecular spacing (Tb.Sp) was decreased. Cartilage thickness, extracellular matrix production, and expression of Lrp5 and Sost were all increased in the experimental groups. The separation between the fluorescent bone labels indicated increased endochondral maturation between 3.5 and 7.5 weeks. Conclusions: These data demonstrate that Lrp5 overexpression leads to adaptation changes in the mandibular condylar cartilage of the TMJ to prevent cartilage degradation. ItemIdentification of Extracellular Wnt Inhibitors for Novel Synergistic Anabolic Action on the Skeleton(2023-07) Choi, Roy Byung-Jun; Robling, Alexander; Bidwell, Joseph; Thompson, William; Pavalko, Fredrick; White, KennethThe Wnt pathway has been an obvious target for designing skeletal therapies, mainly based on the high-bone-mass phenotypes in patients with activating mutations in the Wnt co-receptors Lrp5 and Lrp6, or with inactivating mutations in the Lrp5/6 inhibitor Sost. An attractive property of the Wnt pathway is that it stimulates anabolic action in bone cells. The powerful anabolic potential of manipulating Wnt signaling in bone has been demonstrated by the recent FDA approval of sclerostin antibody (Scl-mAb) EvenityTM (Romosozumab) for the treatment of osteoporosis. However, an increased risk of cardiovascular events was reported, triggering the FDA to issue a ‘black box warning’ requirement for romosozumab. One potential solution to lower the risk of adverse events is to reduce the medication dose. Reducing the dose of Scl-mAb, while maintaining the anabolic potential of the drug, will likely provide a safer and more cost-effective strategy to harness Wnt for fracture prevention. Here, we found that dual inhibition of sclerostin and Dkk1 produced extremely potent synergistic bone anabolic effects in the cancellous compartment, using both genetic and pharmacological models. Further, much lower total doses of dual antibody treatment, given at optimized proportions, generated equivalent trabecular bone anabolic effects as Scl-mAb alone. On the contrary, we looked for other candidates that might potentiate the cortical effects of sclerostin inhibition. We found that either Sostdc1 or Notum deletion results in high bone mass, specifically in the cortical compartment, with little to no effect in the cancellous compartment. Inhibition/deletion of Sostdc1 or Notum alone had no detectable effects (Sostdc1) or mild (Notum) cortical effects, but suppression of either target while co-supressing/deleting Sost improved bone mass disproportionately. In summary, these findings highlight the potential therapeutic role that combinational inhibition of different Wnt inhibitors generates, resulting in synergistic bone anabolic action in different/select skeletal compartments. ItemMechanical Loading-Driven Tumor Suppression Is Mediated by Lrp5-Dependent and Independent Mechanisms(MDPI, 2021-01-13) Feng, Yan; Liu, Shengzhi; Zha, Rongrong; Sun, Xun; Li, Kexin; Robling, Alexander; Li, Baiyan; Yokota, Hiroki; Biomedical Engineering, School of Engineering and TechnologyBone is mechanosensitive and lipoprotein receptor-related protein 5 (Lrp5)-mediated Wnt signaling promotes loading-driven bone formation. While mechanical loading can suppress tumor growth, the question is whether Lrp5 mediates loading-driven tumor suppression. Herein, we examined the effect of Lrp5 using osteocyte-specific Lrp5 conditional knockout mice. All mice presented noticeable loading-driven tumor suppression in the loaded tibia and non-loaded mammary pad. The degree of suppression was more significant in wild-type than knockout mice. In all male and female mice, knee loading reduced cholesterol and elevated dopamine. It reduced tumor-promoting nexin, which was elevated by cholesterol and reduced by dopamine. By contrast, it elevated p53, TNF-related apoptosis-inducing ligand (TRAIL), and chemerin, and they were regulated reversely by dopamine and cholesterol. Notably, Lrp5 overexpression in osteocytes enhanced tumor suppression, and osteoclast development was inhibited by chemerin. Collectively, this study identified Lrp5-dependent and independent mechanisms for tumor suppression. Lrp5 in osteocytes contributed to the loaded bone, while the Lrp5-independent regulation of dopamine- and cholesterol-induced systemic suppression. ItemMechanical stimulations can inhibit local and remote tumor progression by downregulating WISP1(Wiley, 2020-09) Liu, Shengzhi; Wu, Di; Sun, Xun; Fan, Yao; Zha, Rongrong; Jalali, Aydin; Teli, Meghana; Sano, Tomonori; Siegel, Amanda; Sudo, Akihiro; Agarwal, Mangilal; Robling, Alexander; Li, Bai-Yan; Yokota, Hiroki; Biomedical Engineering, School of Engineering and TechnologyMechanical stimulations can prevent bone loss, but their effects on the tumor-invaded bone or solid tumors are elusive. Here, we evaluated the effect of knee loading, dynamic loads applied to the knee, on metastasized bone and mammary tumors. In a mouse model, tumor cells were inoculated to the mammary fat pad or the proximal tibia. Daily knee loading was then applied and metabolic changes were monitored mainly through urine. Urine samples were also collected from human subjects before and after step aerobics. The result showed that knee loading inhibited tumor progression in the loaded tibia. Notably, it also reduced remotely the growth of mammary tumors. In the urine, an altered level of cholesterol was observed with an increase in calcitriol, which is synthesized from a cholesterol derivative. In urinary proteins, knee loading in mice and step aerobics in humans markedly reduced WNT1-inducible signaling pathway protein 1, WISP1, which leads to poor survival among patients with breast cancer. In the ex vivo breast cancer tissue assay, WISP1 promoted the growth of cancer fragments and upregulated tumor-promoting genes, such as Runx2, MMP9, and Snail. Collectively, the present preclinical and human study demonstrated that mechanical stimulations, such as knee loading and step aerobics, altered urinary metabolism and downregulated WISP1. The study supports the benefit of mechanical stimulations for locally and remotely suppressing tumor progression. It also indicated the role of WISP1 downregulation as a potential mechanism of loading-driven tumor suppression. ItemNmp4 Suppresses Osteoanabolic Potency(2023-07) Heim, Crystal Noelle; Bidwell, Joseph; Wek, Ronald; White, Kenneth; Robling, Alexander; Plotkin, LilianTreating severe osteoporosis is limited to two strategies: 1. Stimulation of the parathyroid hormone receptor with analogs for parathyroid hormone (PTH) or parathyroid hormone related peptide, and 2. Stimulation of Wnt signaling via neutralization of sclerostin, a natural inhibitor of this pathway, with a monoclonal antibody (romosozumab-aqqg, Scl-mAb). Despite mobilizing distinct molecular and cellular pathways to stimulate bone gain, all their efficacies rapidly diminish. Identifying the barrier to enhancing potency is a clinical priority. We recently reported that mice harboring the conditional loss of the transcription factor Nmp4 (Nuclear Matrix Protein 4) in mesenchymal stem/progenitor cells (MSPCs) exhibited no measurable baseline effect on the skeleton but showed a significantly enhanced increase in bone formation during PTH therapy. Remarkably, (and unexpectedly) skeletal response to PTH therapy was not improved when Nmp4 was conditionally disabled at the osteoblast or osteocyte stages. For the present study, we hypothesized that the potency of any osteoanabolic drug is pre-programmed (and can be re-programmed) in osteoprogenitors. To test this hypothesis, we treated our global Nmp4-/- mice, various conditional knockout mice, and their controls with Scl-mAb. We observed a similar pattern of improved bone response in our mouse models, which we previously observed with the PTH therapy. That is, removal of Nmp4 early in osteoblast differentiation (MSPC) was required for an exaggerated bone-formation response to Scl-mAb therapy. Disabling Nmp4 later in osteogenic differentiation did not increase the potency of Scl-mAb. These data suggest that Nmp4 is part of a common barrier to improving the efficacy of any osteoanabolic. Potential pathways and actors that comprise the re-programming of Nmp4-/- MSPCs to support the exaggerated osteoanabolic effect on the skeleton are discussed. ItemOverexpression of Lrp5 enhanced the anti-breast cancer effects of osteocytes in bone(Springer Nature, 2021-07-06) Liu, Shengzhi; Wu, Di; Sun, Xun; Fan, Yao; Zha, Rongrong; Jalali, Aydin; Feng, Yan; Li, Kexin; Sano, Tomohiko; Vike, Nicole; Li, Fangjia; Rispoli, Joseph; Sudo, Akihiro; Liu, Jing; Robling, Alexander; Nakshatri, Harikrishna; Li, Bai-Yan; Yokota, Hiroki; Biomedical Engineering, School of Engineering and TechnologyOsteocytes are the most abundant cells in bone, which is a frequent site of breast cancer metastasis. Here, we focused on Wnt signaling and evaluated tumor-osteocyte interactions. In animal experiments, mammary tumor cells were inoculated into the mammary fat pad and tibia. The role of Lrp5-mediated Wnt signaling was examined by overexpressing and silencing Lrp5 in osteocytes and establishing a conditional knockout mouse model. The results revealed that administration of osteocytes or their conditioned medium (CM) inhibited tumor progression and osteolysis. Osteocytes overexpressing Lrp5 or β-catenin displayed strikingly elevated tumor-suppressive activity, accompanied by downregulation of tumor-promoting chemokines and upregulation of apoptosis-inducing and tumor-suppressing proteins such as p53. The antitumor effect was also observed with osteocyte-derived CM that was pretreated with a Wnt-activating compound. Notably, silencing Lrp5 in tumors inhibited tumor progression, while silencing Lrp5 in osteocytes in conditional knockout mice promoted tumor progression. Osteocytes exhibited elevated Lrp5 expression in response to tumor cells, implying that osteocytes protect bone through canonical Wnt signaling. Thus, our results suggest that the Lrp5/β-catenin axis activates tumor-promoting signaling in tumor cells but tumor-suppressive signaling in osteocytes. We envision that osteocytes with Wnt activation potentially offer a novel cell-based therapy for breast cancer and osteolytic bone metastasis. ItemThe Role of Wnt Signaling in Bone Mechanotransduction(2019-11) Bullock, Whitney Ann; Robling, Alexander; Bidwell, Joseph; Plotkin, Lilian; Sankar, Uma; White, KennethThe aging US population is experiencing a growing incidence of osteoporosis, characterized by increased fracture risk and low bone mass. In skeletal tissue, canonical Wnt signaling is a critical regulator of bone mass, and dysregulation of the Wnt pathway has been implicated in numerous skeletal displasias. Some components of the Wnt signaling pathway have a clear role in bone homeostasis, particularly in the response of bone to altered mechanical environment. Other pathway components are more poorly defined. One important intracellular signal transduction node in the Wnt cascade is β- catenin, which modulates gene expression and cell-cell junctions, among other functions. During periods of disuse, β-catenin is degraded, leading to inhibition of Wnt targets. Here, I characterize the role of β-catenin in bone during a disuse challenge, using a genetic mouse model expressing an inducible constitively-active mutant form of β- catenin in the osteocyte population. I hypothesize that prevention of β-catenin degradation during disuse will prevent the bone wasting effects of mechanodeprivation. As a second goal, I focus on upstream (membrane-bound) modulation of Wnt. Here, I investigate the low-density lipoprotein receptor-related receptor 4 (Lrp4), in the regulation of bone mass and mechanotransduction. I generated an Lrp4 knockin mouse model harboring a missense mutation found among human patients with abnormally high bone mass. I hypothesize that the mutation compromises sclerostin action on bone cells. Understanding how each of these components of the Wnt signaling pathway interact, may lead to novel therapeutic targets for treatment of bone diseases. ItemSonic Hedgehog Signaling in Inner Ear Organoid Development(2019-08) Longworth-Mills, Emma; Hashino, Eri; Jones, Kathryn; Robling, Alexander; Zimmers, Teresa; Chen, JinhuiLoss of the finite cochlear hair cells of the inner ear results in sensorineural deafness. Human cochlear hair cells do not regenerate, and there is no cure for deafness. Our laboratory has established a three-dimensional culture system for deriving functional sensory hair cells from human pluripotent stem cells. A major limitation of this approach is that derived hair cells exhibit a morphological and gene expression phenotype reflective of native vestibular hair cells. Previous studies have shown that establishment of localized domains of gene expression along the dorso-ventral axis of the developing otic vesicle is necessary for proper morphogenesis of both auditory and vestibular inner ear structures. Sonic hedgehog (SHH) signaling has been shown to play a key role in specification of the ventral otic vesicle and subsequent cochlear development. Here, SHH treatment was pursued as a potential strategy for inducing a patterning phenotype permissive to cochlear induction in vitro. Single-cell RNAsequencing analysis revealed that while treatment with the SHH pathway agonist Purmorphamine reduced expression of markers for the vestibular-yielding dorsal otic vesicle, upregulation of ventral otic marker genes was modest. More strikingly, the number of otic progenitors exhibiting a neuroprogenitor phenotype increased in response to Purmorphamine treatment. These results suggest that SHH pathway modulation in early-stage inner ear organoids may bias their differentiation toward a neural lineage at the expense of an epithelial lineage. The present study is the first to evaluate the patterning phenotype of human stem cell derived otic progenitors, and sheds light on the transcriptomic profile at this critical point of inner ear development. This study may also cultivate future efforts to derive cochlear cell types as well as inner ear neural cell types from human pluripotent stem cells, and contribute to the establishment of a more complete in vitro model of inner ear development.