RESUMEN
Wnt signaling pathways are transmitted via 10 homologous frizzled receptors (FZD1-10) in humans. Reagents broadly inhibiting Wnt signaling pathways reduce growth and metastasis of many tumors, but their therapeutic development has been hampered by the side effect. Inhibitors targeting specific Wnt-FZD pair(s) enriched in cancer cells may reduce side effect, but the therapeutic effect of narrow-spectrum Wnt-FZD inhibitors remains to be established in vivo. Here, we developed a fragment of C. difficile toxin B (TcdBFBD), which recognizes and inhibits a subclass of FZDs, FZD1/2/7, and examined whether targeting this FZD subgroup may offer therapeutic benefits for treating breast cancer models in mice. Utilizing 2 basal-like and 1 luminal-like breast cancer models, we found that TcdBFBD reduces tumor-initiating cells and attenuates growth of basal-like mammary tumor organoids and xenografted tumors, without damaging Wnt-sensitive tissues such as bones in vivo. Furthermore, FZD1/2/7-positive cells are enriched in chemotherapy-resistant cells in both basal-like and luminal mammary tumors treated with cisplatin, and TcdBFBD synergizes strongly with cisplatin in inhibiting both tumor types. These data demonstrate the therapeutic value of narrow-spectrum Wnt signaling inhibitor in treating breast cancers.
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Toxinas Bacterianas , Neoplasias de la Mama , Clostridioides difficile , Neoplasias Mamarias Animales , Humanos , Animales , Ratones , Femenino , Vía de Señalización Wnt , Neoplasias de la Mama/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , CisplatinoRESUMEN
Insulin resistance impairs postprandial glucose uptake through glucose transporter type 4 (GLUT4) and is the primary defect preceding type 2 diabetes. We previously generated an insulin-resistant mouse model with human GLUT4 promoter-driven insulin receptor knockout (GIRKO) in the muscle, adipose, and neuronal subpopulations. However, the rate of diabetes in GIRKO mice remained low prior to 6 months of age on normal chow diet (NCD), suggesting that additional factors/mechanisms are responsible for adverse metabolic effects driving the ultimate progression of overt diabetes. In this study, we characterized the metabolic phenotypes of the adult GIRKO mice acutely switched to high-fat diet (HFD) feeding in order to identify additional metabolic challenges required for disease progression. Distinct from other diet-induced obesity (DIO) and genetic models (e.g., db/db mice), GIRKO mice remained leaner on HFD feeding, but developed other cardinal features of insulin resistance syndrome. GIRKO mice rapidly developed hyperglycemia despite compensatory increases in ß-cell mass and hyperinsulinemia. Furthermore, GIRKO mice also had impaired oral glucose tolerance and a limited glucose-lowering benefit from exendin-4, suggesting that the blunted incretin effect contributed to hyperglycemia. Secondly, GIRKO mice manifested severe dyslipidemia while on HFD due to elevated hepatic lipid secretion, serum triglyceride concentration, and lipid droplet accumulation in hepatocytes. Thirdly, GIRKO mice on HFD had increased inflammatory cues in the gut, which were associated with the HFD-induced microbiome alterations and increased serum lipopolysaccharide (LPS). In conclusion, our studies identified important gene/diet interactions contributing to diabetes progression, which might be leveraged to develop more efficacious therapies.
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Diabetes Mellitus Tipo 2 , Dieta Alta en Grasa , Intolerancia a la Glucosa , Transportador de Glucosa de Tipo 4 , Hiperglucemia , Resistencia a la Insulina , Animales , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Dieta Alta en Grasa/efectos adversos , Transportador de Glucosa de Tipo 4/biosíntesis , Transportador de Glucosa de Tipo 4/metabolismo , Hiperglucemia/sangre , Hiperglucemia/etiología , Hiperglucemia/metabolismo , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Musculoskeletal diseases such as muscular dystrophy, cachexia, osteoarthritis, and rheumatoid arthritis impair overall physical health and reduce survival. Patients suffer from pain, dysfunction, and dysmobility due to inflammation and fibrosis in bones, muscles, and joints, both locally and systemically. The Interleukin-6 (IL-6) family of cytokines, most notably IL-6, is implicated in musculoskeletal disorders and cachexia. Here we show elevated circulating levels of OSM in murine pancreatic cancer cachexia and evaluate the effects of the IL-6 family member, Oncostatin M (OSM), on muscle and bone using adeno-associated virus (AAV) mediated over-expression of murine OSM in wildtype and IL-6 deficient mice. Initial studies with high titer AAV-OSM injection yielded high circulating OSM and IL-6, thrombocytosis, inflammation, and 60% mortality without muscle loss within 4 days. Subsequently, to mimic OSM levels in cachexia, a lower titer of AAV-OSM was used in wildtype and Il6 null mice, observing effects out to 4 weeks and 12 weeks. AAV-OSM caused muscle atrophy and fibrosis in the gastrocnemius, tibialis anterior, and quadriceps of the injected limb, but these effects were not observed on the non-injected side. In contrast, OSM induced both local and distant trabecular bone loss as shown by reduced bone volume, trabecular number, and thickness, and increased trabecular separation. OSM caused cardiac dysfunction including reduced ejection fraction and reduced fractional shortening. RNA-sequencing of cardiac muscle revealed upregulation of genes related to inflammation and fibrosis. None of these effects were different in IL-6 knockout mice. Thus, OSM induces local muscle atrophy, systemic bone loss, tissue fibrosis, and cardiac dysfunction independently of IL-6, suggesting a role for OSM in musculoskeletal conditions with these characteristics, including cancer cachexia.
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Cardiopatías , Interleucina-6 , Animales , Caquexia , Fibrosis , Inflamación , Interleucina-6/farmacología , Ratones , Ratones Noqueados , Atrofia Muscular , Oncostatina M/farmacología , ARNRESUMEN
Skeletal homeostasis is sensitive to perturbations in Wnt signaling. Beyond its role in the bone, Wnt is a major target for pharmaceutical inhibition in a wide range of diseases, most notably cancers. Numerous clinical trials for Wnt-based candidates are currently underway, and Wnt inhibitors will likely soon be approved for clinical use. Given the bone-suppressive effects accompanying Wnt inhibition, there is a need to expose alternate pathways/molecules that can be targeted to counter the deleterious effects of Wnt inhibition on bone properties. Activation of the Pi3k/Akt pathway via Pten deletion is one possible osteoanabolic pathway to exploit. We investigated whether the osteopenic effects of ß-catenin deletion from bone cells could be rescued by Pten deletion in the same cells. Mice carrying floxed alleles for Pten and ß-catenin were bred to Dmp1-Cre mice to delete Pten alone, ß-catenin alone, or both genes from the late-stage osteoblast/osteocyte population. The mice were assessed for bone mass, density, strength, and formation parameters to evaluate the potential rescue effect of Pten deletion in Wnt-impaired mice. Pten deletion resulted in high bone mass and ß-catenin deletion resulted in low bone mass. Compound mutants had bone properties similar to ß-catenin mutant mice, or surprisingly in some assays, were further compromised beyond ß-catenin mutants. Pten inhibition, or one of its downstream nodes, is unlikely to protect against the bone-wasting effects of Wnt/ßcat inhibition. Other avenues for preserving bone mass in the presence of Wnt inhibition should be explored to alleviate the skeletal side effects of Wnt inhibitor-based therapies.
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Proteínas de la Matriz Extracelular/genética , Neoplasias/tratamiento farmacológico , Fosfohidrolasa PTEN/genética , beta Catenina/genética , Animales , Enfermedades Óseas Metabólicas/tratamiento farmacológico , Enfermedades Óseas Metabólicas/genética , Proliferación Celular/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Neoplasias/genética , Osteoblastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
The functions of the paralogous transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in bone are controversial. Each has been observed to promote or inhibit osteogenesis in vitro, with reports of both equivalent and divergent functions. Their combinatorial roles in bone physiology are unknown. We report that combinatorial YAP/TAZ deletion from skeletal lineage cells, using Osterix-Cre, caused an osteogenesis imperfecta-like phenotype with severity dependent on allele dose and greater phenotypic expressivity with homozygous TAZ vs. YAP ablation. YAP/TAZ deletion decreased bone accrual and reduced intrinsic bone material properties through impaired collagen content and organization. These structural and material defects produced spontaneous fractures, particularly in mice with homozygous TAZ deletion and caused neonatal lethality in dual homozygous knockouts. At the cellular level in vivo, YAP/TAZ ablation reduced osteoblast activity and increased osteoclast activity, in an allele dose-dependent manner, impairing bone accrual and remodeling. Transcriptionally, YAP/TAZ deletion and small-molecule inhibition of YAP/TAZ interaction with the transcriptional coeffector TEAD reduced osteogenic and collagen-related gene expression, both in vivo and in vitro. These data demonstrate that YAP and TAZ combinatorially promote bone development through regulation of osteoblast activity, matrix quality, and osteoclastic remodeling.-Kegelman, C. D., Mason, D. E., Dawahare, J. H., Horan, D. J., Vigil, G. D., Howard, S. S., Robling, A. G., Bellido, T. M., Boerckel, J. D. Skeletal cell YAP and TAZ combinatorially promote bone development.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Desarrollo Óseo , Matriz Ósea/metabolismo , Remodelación Ósea , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular , Eliminación de Gen , Ratones , Ratones Noqueados , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/patología , Fosfoproteínas/genética , Transactivadores , Proteínas Señalizadoras YAPRESUMEN
Indoleamine 2,3 dioxygenase (IDO) plays an important role in immunoregulation as it is involved in downregulating immune responses to infections. We sought to characterize IDO activity in histoplasmosis and to do so, C57Bl6 mice were infected intranasally with Histoplasma capsulatum. After infection, lung and spleen IDO activity was assessed by HPLC and IDO expression by qRT-PCR. The distribution of IDO was determined by immunohistochemical staining. Cytokine levels were measured in lung and spleen homogenates using cytokine bead array. Fungal burden was quantified by culture. Subcutaneous pellets containing methyltryptophane (1-MT) were employed to inhibit IDO in vivo. Histoplasma infection strongly induced functional lung IDO, with activity at its highest at weeks 1 and 2 and then decreased thereafter as the mice cleared the infection. Lung IDO activity positively correlated with the fungal burden (Rho = 0.845), interferon-γ (Rho = 0.839) and tumor necrosis factor-α (Rho = 0.791) levels, P < 0.001. In contrast, spleen IDO activity was not induced despite high infection burden and cytokine levels. IDO expressing cells were predominately located at the ring edge of Histoplasma-induced granulomas. IDO inhibition prior to infection reduced fungal burdens and inflammation in lungs and spleen. Histoplasma preferentially induces lung IDO, as early as one week after infection. IDO appears to modulate the immune response to Histoplasma infection.
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Histoplasma/inmunología , Histoplasma/patogenicidad , Histoplasmosis/inmunología , Histoplasmosis/patología , Indolamina-Pirrol 2,3,-Dioxigenasa/biosíntesis , Animales , Cromatografía Líquida de Alta Presión , Recuento de Colonia Microbiana , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Inmunohistoquímica , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Bazo/microbiología , Bazo/patologíaRESUMEN
The development of Wnt-based osteoanabolic agents has 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 coreceptors 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 codeletion 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 wild-type female mice produced potentiation of cortical bone gain despite no effect of Sostdc1 antibody alone. In conclusion, Sostdc1 inhibition/deletion can work in concert with sclerostin deficiency to improve cortical bone properties. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
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Glicoproteínas , Péptidos y Proteínas de Señalización Intercelular , Masculino , Femenino , Animales , Ratones , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Glicoproteínas/metabolismo , Huesos/metabolismo , Hueso Cortical/metabolismo , Hueso Esponjoso/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
Activation of bone anabolic pathways is a fruitful approach for treating severe osteoporosis, yet FDA-approved osteoanabolics, eg, parathyroid hormone (PTH), have limited efficacy. Improving their potency is a promising strategy for maximizing bone anabolic output. Nmp4 (Nuclear Matrix Protein 4) global knockout mice exhibit enhanced PTH-induced increases in trabecular bone but display no overt baseline skeletal phenotype. Nmp4 is expressed in all tissues; therefore, to determine which cell type is responsible for driving the beneficial effects of Nmp4 inhibition, we conditionally removed this gene from cells at distinct stages of osteogenic differentiation. Nmp4-floxed (Nmp4fl/fl ) mice were crossed with mice bearing one of three Cre drivers including (i) Prx1Cre+ to remove Nmp4 from mesenchymal stem/progenitor cells (MSPCs) in long bones; (ii) BglapCre+ targeting mature osteoblasts, and (iii) Dmp1Cre+ to disable Nmp4 in osteocytes. Virgin female Cre+ and Cre- mice (10 weeks of age) were sorted into cohorts by weight and genotype. Mice were administered daily injections of either human PTH 1-34 at 30 µg/kg or vehicle for 4 weeks or 7 weeks. Skeletal response was assessed using dual-energy X-ray absorptiometry, micro-computed tomography, bone histomorphometry, and serum analysis for remodeling markers. Nmp4fl/fl ;Prx1Cre+ mice virtually phenocopied the global Nmp4-/- skeleton in the femur, ie, a mild baseline phenotype but significantly enhanced PTH-induced increase in femur trabecular bone volume/total volume (BV/TV) compared with their Nmp4fl/fl ;Prx1Cre- controls. This was not observed in the spine, where Prrx1 is not expressed. Heightened response to PTH was coincident with enhanced bone formation. Conditional loss of Nmp4 from the mature osteoblasts (Nmp4fl/fl ;BglapCre+ ) failed to increase BV/TV or enhance PTH response. However, conditional disabling of Nmp4 in osteocytes (Nmp4fl/fl ;Dmp1Cre+ ) increased BV/TV without boosting response to hormone under our experimental regimen. We conclude that Nmp4-/- Prx1-expressing MSPCs drive the improved response to PTH therapy and that this gene has stage-specific effects on osteoanabolism. © 2022 American Society for Bone and Mineral Research (ASBMR).
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Células Madre Mesenquimatosas , Osteogénesis , Animales , Femenino , Humanos , Ratones , Huesos , Densidad Ósea , Proteínas de Homeodominio/genética , Ratones Noqueados , Hormona Paratiroidea/farmacología , Microtomografía por Rayos XRESUMEN
Severe osteoporosis is often treated with one of three Food and Drug Administration (FDA)-approved osteoanabolics. These drugs act by (1) parathyroid hormone (PTH) receptor stimulation using analogues to PTH (teriparatide) or PTH-related peptide (abaloparatide) or by (2) monoclonal antibody neutralization of sclerostin, an innate Wnt inhibitor (Scl-mAb, romosozumab-aqqg). The efficacies of both strategies wane over time. The transcription factor Nmp4 (Nuclear Matrix Protein 4) is expressed in all tissues yet mice lacking this gene are healthy and exhibit enhanced PTH-induced bone formation. Conditional deletion of Nmp4 in mesenchymal stem progenitor cells (MSPCs) phenocopies the elevated response to PTH in global Nmp4-/- mice. However, targeted deletion in later osteoblast stages does not replicate this response. In this study we queried whether loss of Nmp4 improves Scl-mAb potency. Experimental cohorts included global Nmp4-/- and Nmp4+/+ littermates and three conditional knockout models. Nmp4-floxed (Nmp4fl/fl) mice were crossed with mice harboring one of three Cre-drivers (i) Prx1Cre+ targeting MSPCs, (ii) BglapCre+ (mature osteocalcin-expressing osteoblasts), and (iii) Dmp1Cre+ (osteocytes). Female mice were treated with Scl-mAb or 0.9 % saline vehicle for 4 or 7 weeks from 10 weeks of age. Skeletal response was assessed using micro-computed tomography, dual-energy X-ray absorptiometry, bone histomorphometry, and serum analysis. Global Nmp4-/- mice exhibited enhanced Scl-mAb-induced increases in trabecular bone in the femur and spine and a heightened increase in whole body areal bone mineral density compared to global Nmp4+/+ controls. This improved Scl-mAb potency was primarily driven by enhanced increases in bone formation. Nmp4fl/fl;PrxCre+ mice showed an exaggerated Scl-mAb-induced increase in femoral bone but not in the spine since Prrx1 is not expressed in vertebra. The Nmp4fl/fl;BglapCre+ and Nmp4fl/fl;Dmp1Cre+ mice did not exhibit an improved Scl-mAb response. We conclude that Nmp4 expression in MSPCs interferes with the bone anabolic response to anti-sclerostin therapy.
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Introduction: Due to a lack of spatial-temporal resolution at the single cell level, the etiologies of the bone dysfunction caused by diseases such as normal aging, osteoporosis, and the metabolic bone disease associated with chronic kidney disease (CKD) remain largely unknown. Methods: To this end, flow cytometry and scRNAseq were performed on long bone cells from Sost-cre/Ai9+ mice, and pure osteolineage transcriptomes were identified, including novel osteocyte-specific gene sets. Results: Clustering analysis isolated osteoblast precursors that expressed Tnc, Mmp13, and Spp1, and a mature osteoblast population defined by Smpd3, Col1a1, and Col11a1. Osteocytes were demarcated by Cd109, Ptprz1, Ramp1, Bambi, Adamts14, Spns2, Bmp2, WasI, and Phex. We validated our in vivo scRNAseq using integrative in vitro promoter occupancy via ATACseq coupled with transcriptomic analyses of a conditional, temporally differentiated MSC cell line. Further, trajectory analyses predicted osteoblast-to-osteocyte transitions via defined pathways associated with a distinct metabolic shift as determined by single-cell flux estimation analysis (scFEA). Using the adenine mouse model of CKD, at a time point prior to major skeletal alterations, we found that gene expression within all stages of the osteolineage was disturbed. Conclusion: In sum, distinct populations of osteoblasts/osteocytes were defined at the single cell level. Using this roadmap of gene assembly, we demonstrated unrealized molecular defects across multiple bone cell populations in a mouse model of CKD, and our collective results suggest a potentially earlier and more broad bone pathology in this disease than previously recognized.
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Insuficiencia Renal Crónica , Transcriptoma , Ratones , Animales , Huesos/metabolismo , Osteoblastos/metabolismo , Hueso Cortical/metabolismo , Insuficiencia Renal Crónica/patología , Proteínas de la Membrana/metabolismo , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Serotonin (5HT) is a chemical messenger with biologic activities affecting multiple organs. Within the skeletal system, studies in mice and humans suggest blood 5HT levels affect bone, with elevations impairing and reductions enhancing bone accrual. Other studies, however, have not supported this hypothesis. Recently, administering 5HT to a Piezo1 mutant mouse strain with hyposerotonemia, intestinal dysmotility, and a doubling of femoral trabecular bone mass at 2 months of age, was reported to return the animals' intestinal motility and bone mass to normal. However, whether the 5HT directly affected bone metabolism or indirectly affected metabolism by improving intestinal function was not determined. Therefore, we administered 5HT to mice with normal intestinal function. We randomized female C57BL6/J mice and male and female mice that have increased bone mass due to a missense mutation in the WNT co-receptor LRP5 (Lrp5A214V) to receive 5HT or vehicle via daily IP injection from 4 weeks to 8 weeks of age. We did not observe consistent significant changes for distal femur trabecular, midshaft femur cortical, or vertebral body trabecular bone mass between 5HT treated and vehicle treated mice of either genotype. These data are compatible with other studies that have not observed a correlation between blood 5HT level and bone mass.
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Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Serotonina , Animales , Femenino , Masculino , Ratones , Huesos/metabolismo , Densidad Ósea/genética , Canales Iónicos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones Endogámicos C57BLRESUMEN
Age-associated low bone mass disease is a growing problem in the US. Development of osteoanabolic therapies for treating skeletal fragility has lagged behind anti-catabolic therapies, but several bone-building molecules are clinically available. We reported previously that antibody-based neutralization of the Lrp5/Lrp6 inhibitor Dkk1 has minimal effects on bone gain, but can potentiate the already potent osteoanabolic effects of sclerostin inhibition (another Lrp5/Lrp6 inhibitor highly expressed by osteocytes). In this communication, we test whether an optimized ratio of sclerostin and Dkk1 antibodies (Scl-mAb and Dkk1-mAb, respectively), administered at low doses, can maintain the same bone-building effects as higher dose Scl-mAb, in adult (6 months of age) and aged (20 months of age) wild-type mice. A 3:1 dose of Scl-mAb:Dkk1-mAb at 12.5 mg/kg was equally efficacious as 25 mg/kg of Scl-mAb in both age groups, using radiographic (DXA, µCT), biomechanical, (3-point bending tests), and histological (fluorochrome-based bone formation parameters) outcome measures. For some bone properties, including trabecular thickness and bone mineral density in the spine, and endocortical bone formation rates in the femur, the 3:1 treatment was associated with significantly improved skeletal properties compared to twice the dose of Scl-mAb. Cortical porosity in aged mice was also reduced by both Scl-mAb and low-dose 3:1 treatment. Overall, both treatments were efficacious in the mature adult (6 mo.) and aged (20 mo.) skeletons, suggesting Wnt targeting is a viable strategy for improving skeletal fragility in the very old. Further, the data suggest that low dose of combination therapy can be at least equally efficacious as higher doses of Scl-mAb monotherapy.
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Micafungin alone and combined with liposomal amphotericin B was evaluated against two strains of Histoplasma capsulatum. Micafungin was active in vitro against the mold but not the yeast form but was ineffective in vivo. Micafungin appears to be ineffective in treatment of histoplasmosis.
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Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Equinocandinas/farmacología , Equinocandinas/uso terapéutico , Histoplasma/efectos de los fármacos , Histoplasmosis/tratamiento farmacológico , Lipopéptidos/farmacología , Lipopéptidos/uso terapéutico , Animales , Histoplasma/patogenicidad , Micafungina , Ratones , Ratones Endogámicos C57BLRESUMEN
The cysteine knot protein sclerostin is an osteocyte-derived secreted inhibitor of the Wnt co-receptors LRP5 and LRP6. LRP5 plays a dominant role in bone homeostasis, but we previously reported that Sost/sclerostin suppression significantly increased osteogenesis regardless of Lrp5 presence or absence. Those observations suggested that the bone forming effects of sclerostin inhibition can occur through Lrp6 (when Lrp5 is suppressed), or through other yet undiscovered mechanisms independent of Lrp5/6. To distinguish between these two possibilities, we generated mice with compound deletion of Lrp5 and Lrp6 selectively in bone, and treated them with sclerostin monoclonal antibody (Scl-mAb). All mice were homozygous flox for both Lrp5 and Lrp6 (Lrp5f/f; Lrp6f/f), and varied only in whether or not they carried the Dmp1-Cre transgene. Positive (Cre+) and negative (Cre-) mice were injected with Scl-mAb or vehicle from 4.5 to 14 weeks of age. Vehicle-treated Cre+ mice exhibited significantly reduced skeletal properties compared to vehicle-treated Cre- mice, as assessed by DXA, µCT, pQCT, and histology, indicating that Lrp5/6 deletions were effective and efficient. Scl-mAb treatment improved nearly every bone-related parameter among Cre- mice, but the same treatment in Cre+ mice resulted in little to no improvement in skeletal properties. For the few endpoints where Cre+ mice responded to Scl-mAb, it is likely that antibody-induced promotion of Wnt signaling occurred in cell types earlier in the mesenchymal/osteoblast differentiation pathway than the Dmp1-expressing stage. This latter conclusion was supported by changes in some histomorphometric parameters. In conclusion, unlike with the deletion of Lrp5 alone, the bone-selective late-stage co-deletion of Lrp5 and Lrp6 significantly impairs or completely nullifies the osteogenic action of Scl-mAb, and highlights a major role for both Lrp5 and Lrp6 in the mechanism of action for the bone-building effects of sclerostin antibody.
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Glicoproteínas , Péptidos y Proteínas de Señalización Intercelular , Proteínas Adaptadoras Transductoras de Señales , Animales , Huesos/metabolismo , Glicoproteínas/metabolismo , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Osteoblastos/metabolismoRESUMEN
Wnt signaling plays a vital role in the cell biology of skeletal patterning, differentiation, and maintenance. Notum is a secreted member of the α/ß-hydrolase superfamily that hydrolyzes the palmitoleoylate modification on Wnt proteins, thereby disrupting Wnt signaling. As a secreted inhibitor of Wnt, Notum presents an attractive molecular target for improving skeletal health. To determine the cell type of action for Notum's effect on the skeleton, we generated mice with Notum deficiency globally (Notum-/- ) and selectively (Notumf/f ) in limb bud mesenchyme (Prx1-Cre) and late osteoblasts/osteocytes (Dmp1-Cre). Late-stage deletion induced increased cortical bone properties, similar to global mutants. Notum expression was enhanced in response to sclerostin inhibition, so dual inhibition (Notum/sclerostin) was also investigated using a combined genetic and pharmacologic approach. Co-suppression increased cortical properties beyond either factor alone. Notum suppressed Wnt signaling in cell reporter assays, but surprisingly also enhanced Shh signaling independent of effects on Wnt. Notum is an osteocyte-active suppressor of cortical bone formation that is likely involved in multiple signaling pathways important for bone homeostasis © 2021 American Society for Bone and Mineral Research (ASBMR).
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Hueso Cortical , Esterasas/genética , Osteogénesis , Vía de Señalización Wnt , Animales , Hueso Cortical/metabolismo , Ratones , Ratones Noqueados , Osteoblastos/metabolismo , Osteocitos/metabolismoRESUMEN
Bone fragility fractures are caused by low bone mass or impaired bone quality. Osteoblast/osteoclast coordination determines bone mass, but the factors that control bone quality are poorly understood. Osteocytes regulate osteoblast and osteoclast activity on bone surfaces but can also directly reorganize the bone matrix to improve bone quality through perilacunar/canalicular remodeling; however, the molecular mechanisms remain unclear. We previously found that deleting the transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-motif (TAZ) from osteoblast-lineage cells caused lethality in mice due to skeletal fragility. Here, we tested the hypothesis that YAP and TAZ regulate osteocyte-mediated bone remodeling by conditional ablation of both YAP and TAZ from mouse osteocytes using 8 kb-DMP1-Cre. Osteocyte-conditional YAP/TAZ deletion reduced bone mass and dysregulated matrix collagen content and organization, which together decreased bone mechanical properties. Further, YAP/TAZ deletion impaired osteocyte perilacunar/canalicular remodeling by reducing canalicular network density, length, and branching, as well as perilacunar flourochrome-labeled mineral deposition. Consistent with recent studies identifying TGF-ß as a key inducer of osteocyte expression of matrix-remodeling enzymes, YAP/TAZ deletion in vivo decreased osteocyte expression of matrix proteases MMP13, MMP14, and CTSK. In vitro, pharmacologic inhibition of YAP/TAZ transcriptional activity in osteocyte-like cells abrogated TGF-ß-induced matrix protease gene expression. Together, these data show that YAP and TAZ control bone matrix accrual, organization, and mechanical properties by regulating osteocyte-mediated bone remodeling. Elucidating the signaling pathways that control perilacunar/canalicular remodeling may enable future therapeutic targeting of bone quality to reverse skeletal fragility. © 2019 American Society for Bone and Mineral Research.
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Remodelación Ósea , Osteocitos , Animales , Matriz Ósea , Ratones , Osteoblastos , OsteoclastosRESUMEN
High-bone-mass (HBM)-causing missense mutations in the low density lipoprotein receptor-related protein-5 (Lrp5) are associated with increased osteoanabolic action and protection from disuse- and ovariectomy-induced osteopenia. These mutations (e.g., A214V and G171V) confer resistance to endogenous secreted Lrp5/6 inhibitors, such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). Cells in the osteoblast lineage are responsive to canonical Wnt stimulation, but recent work has indicated that osteoclasts exhibit both indirect and direct responsiveness to canonical Wnt. Whether Lrp5-HBM receptors, expressed in osteoclasts, might alter osteoclast differentiation, activity, and consequent net bone balance in the skeleton, is not known. To address this, we bred mice harboring heterozygous Lrp5 HBM-causing conditional knock-in alleles to Ctsk-Cre transgenic mice and studied the phenotype using DXA, µCT, histomorphometry, serum assays, and primary cell culture. Mice with HBM alleles induced in Ctsk-expressing cells (TG) exhibited higher bone mass and architectural properties compared to non-transgenic (NTG) counterparts. In vivo and in vitro measurements of osteoclast activity, population density, and differentiation yielded significant reductions in osteoclast-related parameters in female but not male TG mice. Droplet digital PCR performed on osteocyte enriched cortical bone tubes from TG and NTG mice revealed that ~8-17% of the osteocyte population (depending on sex) underwent recombination of the conditional Lrp5 allele in the presence of Ctsk-Cre. Further, bone formation parameters in the midshaft femur cortex show a small but significant increase in anabolic action on the endocortical but not periosteal surface. These findings suggest that Wnt/Lrp5 signaling in osteoclasts affects osteoclastogenesis and activity in female mice, but also that some of the changes in bone mass in TG mice might be due to Cre expression in the osteocyte population.
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Huesos/metabolismo , Catepsina K/metabolismo , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Mutación/genética , Absorciometría de Fotón , Alelos , Animales , Biomarcadores/sangre , Células de la Médula Ósea/metabolismo , Resorción Ósea/sangre , Resorción Ósea/patología , Huesos/diagnóstico por imagen , Diferenciación Celular , Femenino , Integrasas/metabolismo , Masculino , Ratones Transgénicos , Tamaño de los Órganos/genética , Osteoclastos/metabolismo , Osteoclastos/patología , Osteogénesis/genética , Periostio/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recombinación Genética/genética , Transgenes , Microtomografía por Rayos XRESUMEN
Mechanical 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 ß-catenin-an intracellular signaling node in the canonical Wnt pathway-in disuse mechanotransduction is not defined. Using the ß-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 ß-catenin on bone properties during disuse. We hypothesized that if ß-catenin plays an important role in Wnt-mediated osteoprotection, then artificial stabilization of ß-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 ßcatCA 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 ßcatCA 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 ß-catenin stabilized mice and tail-suspended nonstabilized mice. Our findings indicate that selectively targeting/blocking of ß-catenin degradation in bone cells could have therapeutic implications in mechanically induced bone disease. © 2019 American Society for Bone and Mineral Research.
Asunto(s)
Mecanotransducción Celular , Osteocitos/metabolismo , Osteogénesis , Tibia/metabolismo , beta Catenina/metabolismo , Animales , Densidad Ósea , Ratones , Ratones Transgénicos , Osteocitos/patología , Tibia/diagnóstico por imagen , Tibia/patología , Microtomografía por Rayos X , beta Catenina/genéticaRESUMEN
Wnt 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.
RESUMEN
Gravity 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.