Browsing by Author "Loots, Gabriela G."
Now showing 1 - 10 of 10
Results Per Page
Sort Options
Item Conditional Deletion of Sost in MSC‐derived lineages Identifies Specific Cell Type Contributions to Bone Mass and B Cell Development(Wiley, 2018) Yee, Cristal S.; Manilay, Jennifer O.; Chang, Jiun C.; Hum, Nicholas R.; Murugesh, Deepa K.; Bajwa, Jamila; Mendez, Melanie E.; Economides, Aris E.; Horan, Daniel J.; Robling, Alexander G.; Loots, Gabriela G.; Anatomy and Cell Biology, School of MedicineSclerostin (Sost) is a negative regulator of bone formation and blocking its function via antibodies has shown great therapeutic promise by increasing both bone mass in humans and animal models. Sclerostin deletion in Sost knockout mice (Sost‐/‐) causes high bone mass (HBM) similar to Sclerosteosis patients. Sost‐/‐ mice have been shown to display an up to 300% increase in bone volume/total volume (BV/TV), relative to aged matched controls, and it has been postulated that the main source of skeletal Sclerostin is the osteocyte. To understand the cell‐type specific contributions to the HBM phenotype described in Sost‐/‐ mice, as well as to address the endocrine and paracrine mode of action of sclerostin, we examined the skeletal phenotypes of conditional Sost loss‐of‐function (SostiCOIN/iCOIN) mice with specific deletions in (1) the limb mesenchyme (Prx1‐Cre; targets osteoprogenitors and their progeny); (2) mid‐stage osteoblasts and their progenitors (Col1‐Cre); (3) mature osteocytes (Dmp1‐Cre) and (4) hypertrophic chondrocytes and their progenitors (ColX‐Cre). All conditional alleles resulted in significant increases in bone mass in trabecular bone in both the femur and lumbar vertebrae, but only Prx1‐Cre deletion fully recapitulated the amplitude of the HBM phenotype in the appendicular skeleton and the B cell defect described in the global knockout. Despite wildtype expression of Sost in the axial skeleton of Prx1‐Cre deleted mice, these mice also had a significant increase in bone mass in the vertebrae, but the Sclerostin released in circulation by the axial skeleton did not affect bone parameters in the appendicular skeleton. Also, both Col1 and Dmp1 deletion resulted in a similar 80% significant increase in trabecular bone mass, but only Col1 and Prx1 deletion resulted in a significant increase in cortical thickness. We conclude that several cell types within the Prx1‐osteoprogenitor derived lineages contribute significant amounts of Sclerostin protein to the paracrine pool of Sost, in bone.Item Expression of a Degradation‐Resistant β‐Catenin Mutant in Osteocytes Protects the Skeleton From Mechanodeprivation‐Induced Bone Wasting(Wiley, 2019) Bullock, Whitney A.; Hoggatt, April; Horan, Daniel J.; Lewis, Karl; Yokota, Hiroki; Hann, Steven; Warman, Matthew L.; Sebastian, Aimy; Loots, Gabriela G.; Pavalko, Fredrick M.; Robling, Alexander G.; Anatomy and Cell Biology, IU School of MedicineMechanical stimulation is a key regulator of bone mass, maintenance, and turnover. Wnt signaling is a key regulator of mechanotransduction in bone, but the role of β‐catenin—an intracellular signaling node in the canonical Wnt pathway—in disuse mechanotransduction is not defined. Using the β‐catenin exon 3 flox (constitutively active [CA]) mouse model, in conjunction with a tamoxifen‐inducible, osteocyte‐selective Cre driver, we evaluated the effects of degradation‐resistant β‐catenin on bone properties during disuse. We hypothesized that if β‐catenin plays an important role in Wnt‐mediated osteoprotection, then artificial stabilization of β‐catenin in osteocytes would protect the limbs from disuse‐induced bone wasting. Two disuse models were tested: tail suspension, which models fluid shift, and botulinum‐toxin (botox)‐induced muscle paralysis, which models loss of muscle force. Tail suspension was associated with a significant loss of tibial bone mass and density, reduced architectural properties, and decreased bone formation indices in uninduced (control) mice, as assessed by dual‐energy X‐ray absorptiometry (DXA), micro‐computed tomography (µCT), and histomorphometry. Activation of the βcatCA allele in tail‐suspended mice resulted in little to no change in those properties; ie, these mice were protected from bone loss. Similar protective effects were observed among botox‐treated mice when the βcatCA was activated. RNAseq analysis of altered gene regulation in tail‐suspended mice yielded 35 genes, including Wnt11, Gli1, Nell1, Gdf5, and Pgf, which were significantly differentially regulated between tail‐suspended β‐catenin stabilized mice and tail‐suspended nonstabilized mice. Our findings indicate that selectively targeting/blocking of β‐catenin degradation in bone cells could have therapeutic implications in mechanically induced bone disease.Item Improving Bone Health by Optimizing the Anabolic Action of Wnt Inhibitor Multitargeting(Wiley, 2021-05-06) Choi, Roy B.; Bullock, Whitney A.; Hoggatt, April M.; Loots, Gabriela G.; Genetos, Damian C.; Robling, Alexander G.; Anatomy and Cell Biology, School of MedicineSclerostin antibody (romosozumab) was recently approved for clinical use in the United States to treat osteoporosis. We and others have explored Wnt‐based combination therapy to disproportionately improve the anabolic effects of sclerostin inhibition, including cotreatment with sclerostin antibody (Scl‐mAb) and Dkk1 antibody (Dkk1‐mAb). To determine the optimal ratio of Scl‐mAb and Dkk1‐mAb for producing maximal anabolic action, the proportion of Scl‐mAb and Dkk1‐mAb were systematically varied while holding the total antibody dose constant. A 3:1 mixture of Scl‐mAb to Dkk1‐mAb produced two to three times as much cancellous bone mass as an equivalent dose of Scl‐mAb alone. Further, a 75% reduction in the dose of the 3:1 mixture was equally efficacious to a full dose of Scl‐mAb in the distal femur metaphysis. The Scl‐mAb/Dkk1‐mAb combination approach was highly efficacious in the cancellous bone mass, but the cortical compartment was much more subtly affected. The osteoanabolic effects of Wnt pathway targeting can be made more efficient if multiple antagonists are simultaneously targeted. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.Item Lrp4 Mediates Bone Homeostasis and Mechanotransduction through Interaction with Sclerostin In Vivo(Elsevier, 2019-10-25) Bullock, Whitney A.; Hoggatt, April M.; Horan, Daniel J.; Elmendorf, Andrew J.; Sato, Amy Y.; Bellido, Teresita; Loots, Gabriela G.; Pavalko, Fredrick M.; Robling, Alexander G.; Anatomy and Cell Biology, School of MedicineWnt signaling plays a key role in regulating bone remodeling. In vitro studies suggest that sclerostin's inhibitory action on Lrp5 is facilitated by the membrane-associated receptor Lrp4. We generated an Lrp4 R1170W knockin mouse model (Lrp4KI), based on a published mutation in patients with high bone mass (HBM). Lrp4KI mice have an HBM phenotype (assessed radiographically), including increased bone strength and formation. Overexpression of a Sost transgene had osteopenic effects in Lrp4-WT but not Lrp4KI mice. Conversely, sclerostin inhibition had blunted osteoanabolic effects in Lrp4KI mice. In a disuse-induced bone wasting model, Lrp4KI mice exhibit significantly less bone loss than wild-type (WT) mice. In summary, mice harboring the Lrp4-R1170W missense mutation recapitulate the human HBM phenotype, are less sensitive to altered sclerostin levels, and are protected from disuse-induced bone loss. Lrp4 is an attractive target for pharmacological targeting aimed at increasing bone mass and preventing bone loss due to disuse.Item Sclerostin Depletion Induces Inflammation in the Bone Marrow of Mice(MDPI, 2021-08-24) Donham, Cristine; Chicana, Betsabel; Robling, Alexander G.; Mohamed, Asmaa; Elizaldi, Sonny; Chi, Michael; Freeman, Brian; Millan, Alberto; Murugesh, Deepa K.; Hum, Nicholas R.; Sebastian, Aimy; Loots, Gabriela G.; Manilay, Jennifer O.; Anatomy and Cell Biology, School of MedicineRomosozumab, a humanized monoclonal antibody specific for sclerostin (SOST), has been approved for treatment of postmenopausal women with osteoporosis at a high risk for fracture. Previous work in sclerostin global knockout (Sost−/−) mice indicated alterations in immune cell development in the bone marrow (BM), which could be a possible side effect in romosozumab-treated patients. Here, we examined the effects of short-term sclerostin depletion in the BM on hematopoiesis in young mice receiving sclerostin antibody (Scl-Ab) treatment for 6 weeks, and the effects of long-term Sost deficiency on wild-type (WT) long-term hematopoietic stem cells transplanted into older cohorts of Sost−/− mice. Our analyses revealed an increased frequency of granulocytes in the BM of Scl-Ab-treated mice and WT→Sost−/− chimeras, indicating myeloid-biased differentiation in Sost-deficient BM microenvironments. This myeloid bias extended to extramedullary hematopoiesis in the spleen and was correlated with an increase in inflammatory cytokines TNFα, IL-1α, and MCP-1 in Sost−/− BM serum. Additionally, we observed alterations in erythrocyte differentiation in the BM and spleen of Sost−/− mice. Taken together, our current study indicates novel roles for Sost in the regulation of myelopoiesis and control of inflammation in the BM.Item Sclerostin neutralization unleashes the osteoanabolic effects of Dkk1 inhibition(American Society for Clinical Investigation, 2018-06-07) Witcher, Phillip C.; Miner, Sara E.; Horan, Daniel J.; Bullock, Whitney A.; Lim, Kyung-Eun; Kang, Kyung Shin; Adaniya, Alison L.; Ross, Ryan D.; Loots, Gabriela G.; Robling, Alexander G.; Anatomy and Cell Biology, IU School of MedicineThe WNT pathway has become an attractive target for skeletal therapies. High-bone-mass phenotypes in patients with loss-of-function mutations in the LRP5/6 inhibitor Sost (sclerosteosis), or in its downstream enhancer region (van Buchem disease), highlight the utility of targeting Sost/sclerostin to improve bone properties. Sclerostin-neutralizing antibody is highly osteoanabolic in animal models and in human clinical trials, but antibody-based inhibition of another potent LRP5/6 antagonist, Dkk1, is largely inefficacious for building bone in the unperturbed adult skeleton. Here, we show that conditional deletion of Dkk1 from bone also has negligible effects on bone mass. Dkk1 inhibition increases Sost expression, suggesting a potential compensatory mechanism that might explain why Dkk1 suppression lacks anabolic action. To test this concept, we deleted Sost from osteocytes in, or administered sclerostin neutralizing antibody to, mice with a Dkk1-deficient skeleton. A robust anabolic response to Dkk1 deletion was manifest only when Sost/sclerostin was impaired. Whole-body DXA scans, μCT measurements of the femur and spine, histomorphometric measures of femoral bone formation rates, and biomechanical properties of whole bones confirmed the anabolic potential of Dkk1 inhibition in the absence of sclerostin. Further, combined administration of sclerostin and Dkk1 antibody in WT mice produced a synergistic effect on bone gain that greatly exceeded individual or additive effects of the therapies, confirming the therapeutic potential of inhibiting multiple WNT antagonists for skeletal health. In conclusion, the osteoanabolic effects of Dkk1 inhibition can be realized if sclerostin upregulation is prevented. Anabolic therapies for patients with low bone mass might benefit from a strategy that accounts for the compensatory milieu of WNT inhibitors in bone tissue.Item Sost, independent of the non-coding enhancer ECR5, is required for bone mechanoadaptation(Elsevier, 2016-11) Robling, Alexander G.; Kang, Kyung Shin; Bullock, Whitney A.; Foster, William H.; Murugesh, Deepa; Loots, Gabriela G.; Genetos, Damian C.; Anatomy and Cell Biology, School of MedicineSclerostin (Sost) is a negative regulator of bone formation that acts upon the Wnt signaling pathway. Sost is mechanically regulated at both mRNA and protein level such that loading represses and unloading enhances Sost expression, in osteocytes and in circulation. The non-coding evolutionarily conserved enhancer ECR5 has been previously reported as a transcriptional regulatory element required for modulating Sost expression in osteocytes. Here we explored the mechanisms by which ECR5, or several other putative transcriptional enhancers regulate Sost expression, in response to mechanical stimulation. We found that in vivo ulna loading is equally osteoanabolic in wildtype and Sost-/- mice, although Sost is required for proper distribution of load-induced bone formation to regions of high strain. Using Luciferase reporters carrying the ECR5 non-coding enhancer and heterologous or homologous hSOST promoters, we found that ECR5 is mechanosensitive in vitro and that ECR5-driven Luciferase activity decreases in osteoblasts exposed to oscillatory fluid flow. Yet, ECR5-/- mice showed similar magnitude of load-induced bone formation and similar periosteal distribution of bone formation to high-strain regions compared to wildtype mice. Further, we found that in contrast to Sost-/- mice, which are resistant to disuse-induced bone loss, ECR5-/- mice lose bone upon unloading to a degree similar to wildtype control mice. ECR5 deletion did not abrogate positive effects of unloading on Sost, suggesting that additional transcriptional regulators and regulatory elements contribute to load-induced regulation of Sost.Item Sostdc1 deficiency accelerates fracture healing by promoting the expansion of periosteal mesenchymal stem cells(Elsevier, 2016-07) Collette, Nicole M.; Yee, Cristal S.; Hum, Nicholas R.; Murugesh, Deepa K.; Christiansen, Blaine A.; Xie, LiQin; Economides, Aris N.; Manilay, Jennifer O.; Robling, Alexander G.; Loots, Gabriela G.; Department of Anatomy & Cell Biology, IU School of MedicineLoss of Sostdc1, a growth factor paralogous to Sost, causes the formation of ectopic incisors, fused molars, abnormal hair follicles, and resistance to kidney disease. Sostdc1 is expressed in the periosteum, a source of osteoblasts, fibroblasts and mesenchymal progenitor cells, which are critically important for fracture repair. Here, we investigated the role of Sostdc1 in bone metabolism and fracture repair. Mice lacking Sostdc1 (Sostdc1−/−) had a low bone mass phenotype associated with loss of trabecular bone in both lumbar vertebrae and in the appendicular skeleton. In contrast, Sostdc1−/− cortical bone measurements revealed larger bones with higher BMD, suggesting that Sostdc1 exerts differential effects on cortical and trabecular bone. Mid-diaphyseal femoral fractures induced in Sostdc1−/− mice showed that the periosteal population normally positive for Sostdc1 rapidly expands during periosteal thickening and these cells migrate into the fracture callus at 3 days post fracture. Quantitative analysis of mesenchymal stem cell (MSC) and osteoblast populations determined that MSCs express Sostdc1, and that Sostdc1−/− 5 day calluses harbor > 2-fold more MSCs than fractured wildtype controls. Histologically a fraction of Sostdc1-positive cells also expressed nestin and α-smooth muscle actin, suggesting that Sostdc1 marks a population of osteochondral progenitor cells that actively participate in callus formation and bone repair. Elevated numbers of MSCs in D5 calluses resulted in a larger, more vascularized cartilage callus at day 7, and a more rapid turnover of cartilage with significantly more remodeled bone and a thicker cortical shell at 21 days post fracture. These data support accelerated or enhanced bone formation/remodeling of the callus in Sostdc1−/− mice, suggesting that Sostdc1 may promote and maintain mesenchymal stem cell quiescence in the periosteum.Item Sostdc1 Suppression in the Absence of Sclerostin Potentiates Anabolic Action of Cortical Bone in Mice(Oxford University Press, 2023) Choi, Roy B.; Hoggatt, April M.; Horan, Daniel J.; Rogers, Emily Z.; Loots, Gabriela G.; Robling, Alexander G.; Anatomy, Cell Biology and Physiology, School of MedicineThe development of Wnt-based osteoanabolic agents have progressed rapidly in recent years, given the potent effects of Wnt modulation on bone homeostasis. Simultaneous pharmacologic inhibition of the Wnt antagonists sclerostin and Dkk1 can be optimized to create potentiated effects in the cancellous bone compartment. We looked for other candidates that might be co-inhibited along with sclerostin to potentiate the effects in the cortical compartment. Sostdc1 (Wise), like sclerostin and Dkk1, also binds and inhibits Lrp5/6 co-receptors to impair canonical Wnt signaling, but Sostdc1 has greater effects in the cortical bone. To test this concept, we deleted Sostdc1 and Sost from mice and measured the skeletal effects in cortical and cancellous compartments individually. Sost deletion alone produced high bone mass in all compartments, whereas Sostdc1 deletion alone had no measurable effects on either envelope. Mice with co-deletion of Sostdc1 and Sost had high bone mass and increased cortical properties (bone mass, formation rates, mechanical properties), but only among males. Combined administration of sclerostin antibody and Sostdc1 antibody in WT female mice produced potentiation of cortical bone gain despite no effect of Sostdc1 antibody alone. Sostdc1 inhibition/deletion can work in concert with sclerostin deficiency to improve cortical bone properties.Item Vhl deficiency in osteocytes produces high bone mass and hematopoietic defects(Elsevier, 2018) Loots, Gabriela G.; Robling, Alexander G.; Chang, Jiun C.; Murugesh, Deepa K.; Bajwa, Jamila; Carlisle, Cameron; Manilay, Jennifer O.; Wong, Alice; Yellowley, Clare E.; Genetos, Damian C.; Anatomy and Cell Biology, School of MedicineTissue oxygen (O2) levels vary during development and disease; adaptations to decreased O2 (hypoxia) are mediated by hypoxia-inducible factor (HIF) transcription factors. HIFs are active in the skeleton, and stabilizing HIF-α isoforms cause high bone mass (HBM) phenotypes. A fundamental limitation of previous studies examining the obligate role for HIF-α isoforms in the skeleton involves the persistence of gene deletion as osteolineage cells differentiate into osteocytes. Because osteocytes orchestrate skeletal development and homeostasis, we evaluated the influence of Vhl or Hif1a disruption in osteocytes. Osteocytic Vhl deletion caused HBM phenotype, but Hif1a was dispensable in osteocytes. Vhl cKO mice revealed enhanced canonical Wnt signaling. B cell development was reduced while myelopoiesis increased in osteocytic Vhl cKO, revealing a novel influence of Vhl/HIF-α function in osteocytes on maintenance of bone microarchitecture via canonical Wnt signaling and effects on hematopoiesis.