Osterix and RUNX2 are Transcriptional Regulators of Sclerostin in Human Bone

Sclerostin, encoded by the SOST gene, works as an inhibitor of the Wnt pathway and therefore is an important regulator of bone homeostasis. Due to its potent action as an inhibitor of bone formation, blocking sclerostin activity is the purpose of recently developed antiosteoporotic treatments. Two bone-specific transcription factors, RUNX2 and OSX, have been shown to interact and co-ordinately regulate the expression of bone-specific genes. Although it has been recently shown that sclerostin is targeted by OSX in mice, there is currently no information of whether this is also the case in human cells. We have identified SP-protein family and AML1 consensus binding sequences at the human SOST promoter and have shown that OSX, together with RUNX2, binds to a specific region close to the transcription start site. Furthermore, we show that OSX and RUNX2 activate SOST expression in a co-ordinated manner in vitro and that SOST expression levels show a significant positive correlation with OSX/ RUNX2 expression levels in human bone. We also confirmed previous results showing an association of several SOST/RUNX2 polymorphisms with bone mineral density

Identification of an aromatase haplotype that is associated with gene expression and postmenopausal osteoporosis

Context: Osteoporosis has a significant genetic component. The aromatase-dependent conversion of androgenic precursors is the main source of estrogens in postmenopausal women. Objective: The objective of the investigation was to study the relationship of a set of single nucleotide polymorphisms (SNPs) of the aromatase gene with osteoporosis and determine their functional influence on gene transcription. Design, Participants, and Methods: This was a case-control study including 135 women with vertebral fractures due to postmenopausal osteoporosis and 312 controls. Alleles at four SNPs situated between exons I.2 and 3 were determined by Taqman assays. Total aromatase RNA and differential allelic-specific expression were studied by RT-real time PCR in adipose tissue samples taken from 50 individuals. Results: The SNPs studied were in strong linkage disequilibrium. A common haplotype, present in about half of the population, was identified as being associated with an increased risk of fractures (odds ratio 1.8, 95% confidence interval 1.2–2.8, P = 0.006). There was evidence of differential allelic expression. In heterozygous individuals, transcripts bearing T alleles at rs700518 SNP (which were included in the risk haplotype) were less abundant than those with the alternative C alleles (P < 0.001). Total aromatase expression was four times lower in fat samples from individuals who were homozygotes for the unfavorable alleles than in the opposite homozygotes (P = 0.007). Conclusions: A common haplotype of aromatase associated with gene expression is also associated with the risk of osteoporotic vertebral fractures in postmenopausal women. These data are in line with the hypothesis that the aromatase-dependent synthesis of estrogens plays an important role in bone homeostasis in postmenopausal women

Analysis of serum proteome after treatment of osteoporosis with anabolic or antiresorptive drugs

The aim of the study was to explore new markers in serum proteome associated with the response to antiosteoporosis drugs, namely teriparatide and denosumab. We obtained serum samples from 14 patients with osteoporosis, both at baseline and after 6 months of treatment with teriparatide (n = 10) or denosumab (n = 4). Samples were analyzed by nanoliquid chromatography coupled to high-resolution mass spectrometry on a QTOF 5600 (SCIEX) apparatus. The spectrometry data were analyzed with Mascot against the UniProtKB base and then several quality-control filters were applied for the identification of peptides (false discovery rate, FDR q < 0.02) and their quantification (FDR q < 0.05). In the group treated with teriparatide, 28 proteins were identified with significant differences before and after treatment. A pathway analysis by using the Reactome database revealed significant enrichment in the Insulin Like Growth Factor 1 (IGF-I) (FDR q 4 × 10-²) and innate immune system (FDR q 2 × 10-³) pathways. Among patients treated with denosumab, we observed significant differences in the levels of 10 proteins, which were also enriched in the pathways related to the innate immune system (FDR q 3 × 10-²). These results suggest that the innate immune system may be involved in the response to antiosteoporosis drugs.Funding: Supported by grants from FEIOMM (Grant No.17/0025) and ProteoRed-ISCIII. Acknowledgments: Alvaro del Real received support by the postdoctoral grant Augusto Gonzalez de Linares of the University of Cantabria

Hyperbaric Oxygen Therapy Does Not Have a Negative Impact on Bone Signaling Pathways in Humans

Introduction: Oxygen is emerging as an important factor in the local regulation of bone remodeling. Some preclinical data suggest that hyperoxia may have deleterious effects on bone cells. However, its clinical relevance is unclear. Hence, we studied the effect of hyperbaric oxygen therapy (HBOT) on serum biomarkers reflecting the status of the Wnt and receptor activator of NF-?B ligand (RANKL) pathways, two core pathways for bone homeostasis. Materials and methods: This was a prospective study of 20 patients undergoing HBOT (mean age 58 yrs., range 35?82 yrs.) because of complications of radiotherapy or chronic anal fissure. Patients were subjected to HBOT (100% oxygen; 2.4 atmospheres absolute for 90 min). The average number of HBOT sessions was 20 ± 5 (range 8?31). Serum hypoxia-inducible factor 1-? (HIF1-?), osteoprotegerin (OPG), RANKL, and the Wnt inhibitors sclerostin and dickkopf-1 (DKK1) were measured at baseline and after HBOT by using specific immunoassays. Results: HIF-1? in eight patients with measurable serum levels increased from 0.084 (0.098) ng/mL at baseline to 0.146 (0.130) ng/mL after HBOT (p = 0.028). However, HBOT did not induce any significant changes in the serum levels of OPG, RANKL, sclerostin or DKK1. This was independent of the patients? diagnosis, either neoplasia or benign. Conclusion: Despite the potential concerns about hyperoxia, we found no evidence that HBOT has any detrimental effect on bone homeostasis

Differential analysis of genome-wide methylation and gene expression in mesenchymal stemcells of patients with fractures and osteoarthritis

Insufficient activity of the bone-forming osteoblasts leads to low bone mass and predisposes to fragility fractures. The functional capacity of human mesenchymal stem cells (hMSCs), the precursors of osteoblasts, may be compromised in elderly individuals, in relation with the epigenetic changes associated with aging. However, the role of hMSCs in the pathogenesis of osteoporosis is still unclear. Therefore, we aimed to characterize the genome-wide methylation and gene expression signatures and the differentiation capacity of hMSCs from patients with hip fractures. We obtained hMSCs from the femoral heads of women undergoing hip replacement due to hip fractures and controls with hip osteoarthritis. DNA methylation was explored with the Infinium 450K bead array. Transcriptome analysis was done by RNA sequencing. The genomic analyses revealed that most differentially methylated loci were situated in genomic regions with enhancer activity, distant from gene bodies and promoters. These regions were associated with differentially expressed genes enriched in pathways related to hMSC growth and osteoblast differentiation. hMSCs from patients with fractures showed enhanced proliferation and upregulation of the osteogenic drivers RUNX2/OSX. Also, they showed some signs of accelerated methylation aging. When cultured in osteogenic medium, hMSCs from patients with fractures showed an impaired differentiation capacity, with reduced alkaline phosphatase activity and poor accumulation of a mineralized matrix. Our results point to 2 areas of potential interest for discovering new therapeutic targets for low bone mass disorders and bone regeneration: the mechanisms stimulating MSCs proliferation after fracture and those impairing their terminal differentiation

Effects of systemic or local administration of mesenchymal stem cells from patients with osteoporosis or osteoarthritis on femoral fracture healing in a mouse model

The purpose of this study was to analyze the regenerative capacity of mesenchymal stem cells (MSCs) in the treatment of fractures. MSCs extracted from patients with osteoporotic hip fractures or hip osteoarthritis undergoing hip replacement surgeries were cultured and injected into mice with femoral fracture. Two experimental models were established, one for the systemic administration of MSCs (n = 29) and another one for local administration (n = 30). Fracture consolidation was assessed by micro-CT and histology. The degree of radiological consolidation and corticalization was better with MSCs from osteoporosis than from osteoarthritis, being significant after systemic administration (p = 0.0302 consolidation; p = 0.0243 corticalization). The histological degree of consolidation was also better with MSCs from osteoporosis than from osteoarthritis. Differences in histological scores after systemic infusion were as follows: Allen, p = 0.0278; Huo, p = 0.3471; and Bone Bridge, p = 0.0935. After local administration at the fracture site, differences in histological scores were as follows: Allen, p = 0.0764; Huo, p = 0.0256; and Bone Bridge, p = 0.0012. As osteoporosis and control groups were similar, those differences depended on an inhibitory influence by MSCs from patients with osteoarthritis. In conclusion, we found an unexpected impairment of consolidation induced by MSCs from patients with osteoarthritis. However, MSCs from patients with osteoporosis compared favorably with cells from patients with osteoarthritis. In other words, based on this study and previous studies, MSCs from patients with osteoporosis do not appear to have worse bone-regenerating capabilities than MSCs from non-osteoporotic individuals of similar age.Funding: This work was supported by a grant from Instituto de Salud Carlos III [PI16-915], which can be cofounded by EU Feder funds. Acknowledgments: Álvaro del Real received support from the postdoctoral grant “Augusto González de Linares” of the University of Cantabria

Influencia del oxígeno a alta concentración en cámara hiperbárica sobre el metabolismo óseo

RESUMEN: Objetivos: Conocer las acciones del oxígeno a alta concentración en cámara hiperbárica (CH) sobre la expresión de genes relacionados con el metabolismo óseo en líneas celulares osteoblasticas y hueso trabecular humano. Material y métodos: Se analizó la expresión diferencial de varios genes relacionados con el metabolismo óseo (SOST, RUNX2, MMP14, OPG, HIF‐1α y SIRT1) en dos líneas celulares osteoblasticas humanas (Saos y Super‐Saos) y en fragmentos de hueso trabecular humano sometidos a una, tres o cinco sesiones de CH (90 minutos, oxigeno 100%; 2,3 atmosferas). En cada experimento se utilizó un control que no recibió CH. Resultados: No encontramos diferencias significativas tras la CH en la expresión de los genes estudiados, ni en las células ni en hueso trabecular. Solo en la línea celular Super‐Saos la expresión de OPG tras 5 sesiones de CH descendió 6 veces con respecto a la del grupo control (2‐ΔCt de 72; p=0,01). Conclusiones: El oxígeno a alta concentración en cámara hiperbárica no parece tener influencia en la expresión de genes relacionados con el metabolismo óseo

Efectos de la administración sistémica y local de MSCs de pacientes con osteoporosis o artrosis en un modelo animal de fractura femoral

Introducción: El objetivo fue analizar la capacidad regenerativa de las MSCs en el tratamiento de fracturas. Material y métodos: Las MSCs son extraídas de pacientes con fractura de cadera osteoporótica o artrosis de cadera, sometidos a cirugía de sustitución protésica. Las MSCs cultivadas se inyectaron en un modelo animal con fractura femoral, estableciendo dos modelos experimentales en función de la vía de administración, sistémica (n=29) o local (n=30). La consolidación de la fractura se evaluó mediante micro-TC e histología. En el estudio radiológico se analizaron los parámetros Bone Volume/Tissue Volume, grado de consolidación (0-4) y número de corticales corticalizadas (0-4). El estudio histológico se valoró según la escala de Allen (1-6), Huo (1-10) y Puente Óseo (0-2). Para el análisis de los datos se emplearon los test estadísticos de Kruskal-Wallis y U de Mann Whitney. Resultados: El grado de consolidación radiológica y corticalización fue mejor con las MSCs cultivadas de pacientes con osteoporosis que de artrosis, siendo la diferencia significativa después de la administración sistémica (p=0,0302 consolidación; p=0,0243 corticalización). El grado histológico de consolidación también fue mejor con las MSCs cultivadas de pacientes con osteoporosis que de artrosis. Cuando se realiza la inyección sistémica, las diferencias fueron las siguientes: Allen, p=0,0278; Huo, p=0,0347; y Puente Óseo, p=0,0935. Después de la administración local en el foco de fractura: Allen, p=0,0764; Huo, p=0,0256; y Puente Óseo, p=0,0012. Conclusiones: En este estudio no hemos podido demostrar una mejoría de la terapia con MSCs, independientemente de su origen (osteoporosis o artrosis). Además, encontramos peores datos de consolidación en los animales que fueron tratados con MSCs cultivadas de pacientes con artrosis, particularmente si las células se inyectaron por vía intravenosa. Estas diferencias dependieron de una influencia inhibitoria que podría estar relacionada con los procesos inmunes, en el caso de las MSCs de artrosis, o el atrapamiento de las células en los tejidos viscerales, en el caso de la administración sistémica. Sin embargo, la consolidación de las fracturas con MSCs de pacientes con osteoporosis se comparó favorablemente con las MSCs de pacientes con artrosis. Por todo ello, podemos concluir que las MSCs de pacientes con osteoporosis no tienen peores capacidades de regeneración ósea que las MSCs de individuos no osteoporóticos de edad similar

Método sensible para monitorizar la migración de las células madre mesenquimales de la médula ósea en modelos murinos

Resumen: Objetivo: Las células madre mesenquimales (MSCs) son atractivas en la terapia regenerativa de patologías humanas. En los modelos murinos, en los que se trasplantan MSCs humanas, es muy importante poder distinguir el origen de las MSCs identificadas en los órganos de ratones. El objetivo de este estudio fue determinar el rendimiento del análisis basado en PCR de secuencias Alu humanas para detectar ADN humano después de la infusión de células madre de médula ósea humana (hBMSCs) en ratones inmunodeficientes. Material y método: Las hBMSCs se obtuvieron de la cabeza femoral de pacientes sometidos a cirugía de reemplazo de cadera. Se infundieron 106 hBMSCs por vía intravenosa mediante inyección en el seno retro‐orbitario de ratones NOD/SCID. Después se evaluó la presencia de ADN humano en pulmón, hígado y hueso. Resultados: En mezclas de ADN in vitro, el ADN humano se detectó fácilmente con una buena relación logarítmica‐lineal. De manera similar, cuando se mezclaron osteoblastos humanos y de ratón, se detectaron fácilmente 1‐10 células humanas entre 105 células de ratón. Asimismo, se detectó el ADN humano en los pulmones 1 y 7 días después de las infusiones celulares en ratones NOD/SCID. Sin embargo, el ADN humano se detectó de manera inconsistente en el hígado y los huesos. Conclusión: La detección de secuencias Alu es un procedimiento eficaz para detectar ADN humano. Los resultados confirman que la mayoría de las hBMSCs inyectadas por vía intravenosa quedan atrapadas en los pulmones. Por lo tanto, de cara al tratamiento de trastornos esqueléticos, se necesitan procedimientos para aumentar la migración de dichas células al hueso

Generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies

Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D3 treatment and the immortalized cells formed a mineralized matrix, as detected by Alizarin Red staining. Moreover, these human cell lines responded by upregulating ALPL gene expression after treatment with the demethylating agent 5-aza-2 Œ-deoxycytidine (AzadC), as shown before for primary osteoblasts. We further demonstrate that these cell lines can differentiate in vivo, using a hydroxyapatite/tricalcium phosphate composite as a scaffold, to produce bone matrix. More importantly, we show that these cells respond to demethylating treatment, as shown by the increase in SOST mRNA levels, the gene encoding sclerostin, upon treatment of the recipient mice with AzadC. This also confirms, in vivo, the role of DNA methylation in the regulation of SOST expression previously shown in vitro. Altogether our results show that these immortalized cell lines constitute a particularly useful model system to obtain further insight into bone homeostasis, and particularly into the epigenetic mechanisms regulating sclerostin production