RESUMEN
Developing novel approaches to treat skeletal disorders requires an understanding of how critical molecular factors regulate osteoblast differentiation and bone remodeling. We have reported that (1) retinoic acid receptor-related orphan receptor beta (Rorß) is upregulated in bone samples isolated from aged mice and humans in vivo; (2) Rorß expression is inhibited during osteoblastic differentiation in vitro; and (3) genetic deletion of Rorß in mice results in preservation of bone mass during aging. These data establish that Rorß inhibits osteogenesis and that strict control of Rorß expression is essential for bone homeostasis. Because microRNAs (miRNAs) are known to play important roles in the regulation of gene expression in bone, we explored whether a predicted subset of nine miRNAs regulates Rorß expression during both osteoblast differentiation and aging. Mouse osteoblastic cells were differentiated in vitro and assayed for Rorß and miRNA expression. As Rorß levels declined with differentiation, the expression of many of these miRNAs, including miR-219a-5p, was increased. We further demonstrated that miR-219a-5p was decreased in bone samples from old (24-month) mice, as compared with young (6-month) mice, concomitant with increased Rorß expression. Importantly, we also found that miR-219a-5p expression was decreased in aged human bone biopsies compared with young controls, demonstrating that this phenomenon also occurs in aging bone in humans. Inhibition of miR-219a-5p in mouse calvarial osteoblasts led to increased Rorß expression and decreased alkaline phosphatase expression and activity, whereas a miR-219a-5p mimic decreased Rorß expression and increased osteogenic activity. Finally, we demonstrated that miR-219a-5p physically interacts with Rorß mRNA in osteoblasts, defining Rorß as a true molecular target of miR-219a-5p. Overall, our findings demonstrate that miR-219a-5p is involved in the regulation of Rorß in both mouse and human bone. © 2018 American Society for Bone and Mineral Research.
Asunto(s)
Envejecimiento , Diferenciación Celular , Regulación de la Expresión Génica , MicroARNs/metabolismo , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/biosíntesis , Osteoblastos/metabolismo , Osteoporosis/metabolismo , Animales , Humanos , Ratones , MicroARNs/genética , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Osteoblastos/patología , Osteoporosis/genética , Osteoporosis/patologíaRESUMEN
There is a clinical need to identify new molecular targets for the treatment of osteoporosis, particularly those that simultaneously inhibit bone resorption while stimulating bone formation. We have previously shown in overexpression studies that retinoic acid receptor-related orphan receptor ß (Rorß) suppresses in vitro osteoblast differentiation. In addition, the expression of Rorß is markedly increased in bone marrow-derived mesenchymal stromal cells with aging in both mice and humans. Here we establish a critical role for Rorß in regulating bone metabolism using a combination of in vitro and in vivo studies. We used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing to demonstrate that loss of Rorß in osteoblasts enhances Wnt signaling, specifically through increased recruitment of ß-catenin to T-cell factor/lymphoid enhancer factor (Tcf/Lef) DNA binding sites in the promoters of the Wnt target genes Tcf7 and Opg. This resulted in increased osteogenic gene expression and suppressed osteoclast formation through increased osteoprotegerin (OPG) secretion in Rorß-deficient cells. Consistent with our in vitro data, genetic deletion of Rorß in both female and male mice resulted in preserved bone mass and microarchitecture with advancing age due to increased bone formation with a concomitant decrease in resorption. The improved skeletal phenotype in the Rorß-/- mice was also associated with increased bone protein levels of TCF7 and OPG. These data demonstrate that loss of Rorß has beneficial skeletal effects by increasing bone formation and decreasing bone resorption, at least in part through ß-catenin-dependent activation of the Wnt pathway. Thus, inhibition of Rorß represents a novel approach to potentially prevent or reverse osteoporosis. © 2017 American Society for Bone and Mineral Research.
Asunto(s)
Resorción Ósea/metabolismo , Diferenciación Celular , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/deficiencia , Osteoblastos/metabolismo , Osteogénesis , Vía de Señalización Wnt , Animales , Resorción Ósea/genética , Resorción Ósea/patología , Resorción Ósea/prevención & control , Línea Celular , Ratones , Ratones Noqueados , Osteoblastos/patologíaRESUMEN
Aging is associated with increased cellular senescence, which is hypothesized to drive the eventual development of multiple comorbidities. Here we investigate a role for senescent cells in age-related bone loss through multiple approaches. In particular, we used either genetic (i.e., the INK-ATTAC 'suicide' transgene encoding an inducible caspase 8 expressed specifically in senescent cells) or pharmacological (i.e., 'senolytic' compounds) means to eliminate senescent cells. We also inhibited the production of the proinflammatory secretome of senescent cells using a JAK inhibitor (JAKi). In aged (20- to 22-month-old) mice with established bone loss, activation of the INK-ATTAC caspase 8 in senescent cells or treatment with senolytics or the JAKi for 2-4 months resulted in higher bone mass and strength and better bone microarchitecture than in vehicle-treated mice. The beneficial effects of targeting senescent cells were due to lower bone resorption with either maintained (trabecular) or higher (cortical) bone formation as compared to vehicle-treated mice. In vitro studies demonstrated that senescent-cell conditioned medium impaired osteoblast mineralization and enhanced osteoclast-progenitor survival, leading to increased osteoclastogenesis. Collectively, these data establish a causal role for senescent cells in bone loss with aging, and demonstrate that targeting these cells has both anti-resorptive and anabolic effects on bone. Given that eliminating senescent cells and/or inhibiting their proinflammatory secretome also improves cardiovascular function, enhances insulin sensitivity, and reduces frailty, targeting this fundamental mechanism to prevent age-related bone loss suggests a novel treatment strategy not only for osteoporosis, but also for multiple age-related comorbidities.
Asunto(s)
Huesos/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Quinasas Janus/antagonistas & inhibidores , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Osteocitos/efectos de los fármacos , Osteoporosis/metabolismo , Pirazoles/farmacología , Absorciometría de Fotón , Animales , Apoptosis/genética , Huesos/metabolismo , Hueso Esponjoso/efectos de los fármacos , Hueso Esponjoso/metabolismo , Caspasa 8/genética , Diferenciación Celular , Senescencia Celular/genética , Hueso Cortical/efectos de los fármacos , Hueso Cortical/metabolismo , Medios de Cultivo Condicionados , Citometría de Flujo , Perfilación de la Expresión Génica , Técnicas In Vitro , Ratones , Ratones Transgénicos , Nitrilos , Osteoblastos/citología , Osteoclastos/citología , Osteoporosis/genética , Pirimidinas , Reacción en Cadena en Tiempo Real de la Polimerasa , Soporte de Peso , beta-GalactosidasaRESUMEN
This corrects the article DOI: 10.1038/nm.4385.