RESUMEN
Bronchoalveolar stem cells (BASCs) are mobilized during injury and identified as lung progenitor cells, but the molecular regulation of this population of cells has not been elucidated. Secreted frizzled-related protein 1 (SFRP1) is a critical molecule involved in alveolar duct formation in the lung and here we demonstrate its importance in controlling cell differentiation during lung injury. Mice lacking SFRP1 exhibited a rapid repair response leading to aberrant proliferation of differentiated cells. Furthermore, SFRP1 treatment of BASCs maintained these cells in a quiescent state. In vivo overexpression of SFRP1 after injury suppressed differentiation and resulted in the accumulation of BASCs correlating with in vitro studies. These findings suggest that SFRP1 expression in the adult maintains progenitor cells within their undifferentiated state and suggests that manipulation of this pathway is a potential target to augment the lung repair process during disease.
Asunto(s)
Bronquios/citología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Proteínas de la Membrana/fisiología , Alveolos Pulmonares/citología , Células Madre/citología , Animales , Diferenciación Celular , Células Cultivadas , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Vía de Señalización WntRESUMEN
Src-null mice have higher bone mass because of decreased bone resorption and increased bone formation, whereas Abl-null mice are osteopenic, because of decreased bone formation. Compound I, a potent inhibitor of Src in an isolated enzyme assay (IC(50) 0.55 nM) and a Src-dependent cell growth assay, with lower activity on equivalent Abl-based assays, potently, but biphasically, accelerated differentiation of human mesenchymal stem cells to an osteoblast phenotype (1-10 nM). Compound I (≥0.1 nM) also activated osteoblasts and induced bone formation in isolated neonatal mouse calvariae. Compound I required higher concentrations (100 nM) to inhibit differentiation and activity of osteoclasts. Transcriptional profiling (TxP) of calvaria treated with 1 µM compound I revealed down-regulation of osteoclastic genes and up-regulation of matrix genes and genes associated with the osteoblast phenotype, confirming compound I's dual effects on bone resorption and formation. In addition, calvarial TxP implicated calcitonin-related polypeptide, ß (ß-CGRP) as a potential mediator of compound I's osteogenic effect. In vivo, compound I (1 mg/kg s.c.) increased vertebral trabecular bone volume 21% (microcomputed tomography) in intact female mice. Increased trabecular volume was also detected histologically in a separate bone, the femur, particularly in the secondary spongiosa (100% increase), which underwent a 171% increase in bone formation rate, a 73% increase in mineralizing surface, and a 59% increase in mineral apposition rate. Similar effects were observed in ovariectomized mice with established osteopenia. We conclude that the Src inhibitor compound I is osteogenic, presumably because of its potent stimulation of osteoblast differentiation and activation, possibly mediated by ß-CGRP.
Asunto(s)
Osteogénesis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Familia-src Quinasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoclastos/citología , Osteoclastos/efectos de los fármacosRESUMEN
Developmentally expressed genes are believed to play a central role in tissue repair after injury; however, in lung disease their role has not been established. This study demonstrates that SFRP1, an inhibitor of Wnt signaling normally expressed during lung embryogenesis, is induced in the lungs of emphysema patients and in two murine models of the disease. SFRP1 was found to be essential for alveolar formation as Sfrp1(-/-) mice exhibited aberrant Wnt signaling, mesenchymal proliferation, and impaired alveoli formation. In contrast, SFRP1 activated ERK and up-regulated MMP1 and MMP9 without altering TIMP1 production when expressed in human lung epithelial cells. These findings demonstrate that SFRP1 promotes normal alveolar formation in lung development, although its expression in the adult up-regulates proteins that can cause tissue destruction. Thus, SFRP1 induction during tissue injury is unlikely to contribute to the repair response but rather is a participatory factor in the pathogenesis of emphysema and tissue destruction.
Asunto(s)
Enfisema/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Pulmón , Proteínas de la Membrana/metabolismo , Organogénesis/fisiología , Animales , Línea Celular , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Pulmón/embriología , Pulmón/crecimiento & desarrollo , Pulmón/patología , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/fisiología , Humo , Nicotiana/efectos adversos , Proteínas Wnt/metabolismo , Proteína Wnt-5a , beta Catenina/metabolismoRESUMEN
Wnt proteins initiate signaling by binding to seven transmembrane spanning receptors of the frizzled (Fz) family together with the members of the low-density lipoprotein receptor-related protein (LRP) 5 and 6. A chimera of human Wnt3 and Fz1 receptor was developed that efficiently activated the TCF-luciferase reporter. Deletion of the cytoplasmic tail and point mutations in the PDZ binding region in the chimera resulted in the loss of Wnt signaling, suggesting a critical role for the Fz cytoplasmic region in Wnt signaling. The Fz CRD is also critical for Wnt signaling, as a deletion of 29 amino acids in the 2nd cysteine loop resulted in the total loss of TCF-luciferase activation. DKK-1 protein blocks upregulation of the TCF-luciferase reporter by the Wnt3-Fz1 chimera, suggesting involvement of LRP in Wnt3-Fz1 signaling. Expression of a Wnt3-Fz1 chimera in C3H10T1/2 cells resulted in the upregulation of alkaline phosphatase activity and inhibition of adipocyte formation, demonstrating that the Wnt3-Fz1 chimera is a potent activator of differentiation of C3H10T1/2 cells into osteoblasts and an inhibitor of their differentiation into the adipocyte lineage. In summary, the Wnt-Fz chimera approach has the potential to better our understanding of the mechanism of Wnt action and its role, particularly in stem cell differentiation. In addition, this methodology can be utilized to identify inhibitors of either Wnt, Fz or interactors of the canonical pathway, which may have potential therapeutic value in the treatment of cancers and other diseases.
Asunto(s)
Receptores Frizzled/metabolismo , Modelos Biológicos , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Línea Celular , Receptores Frizzled/química , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Wnt/química , Proteína Wnt3RESUMEN
The bioactive phospholipid, lysophosphatidic acid (LPA), acting through at least five distinct receptors LPA1-LPA5, plays important roles in numerous biological processes. Here we report that LPA induces osteoblastic differentiation of human mesenchymal stem cells hMSC-TERT. We find that hMSC-TERT mostly express two LPA receptors, LPA1 and LPA4, and undergo osteoblastic differentiation in serum-containing medium. Inhibition of LPA1 with Ki16425 completely abrogates osteogenesis, indicating that this process is mediated by LPA in the serum through activation of LPA1. In contrast to LPA1, down-regulation of LPA4 expression with shRNA significantly increases osteogenesis, suggesting that this receptor normally exerts negative effects on differentiation. Mechanistically, we find that in hMSC-TERT, LPA induces a rise in both cAMP and Ca(2+). The rise in Ca(2+) is completely abolished by Ki16425, whereas LPA-mediated cAMP increase is not sensitive to Ki16425. To test if LPA signaling pathways controlling osteogenesis in vitro translate into animal physiology, we evaluated the bones of LPA4-deficient mice. Consistent with the ability of LPA4 to inhibit osteoblastic differentiation of stem cells, LPA4-deficient mice have increased trabecular bone volume, number, and thickness.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Lisofosfolípidos/farmacología , Osteoblastos/citología , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores Purinérgicos/metabolismo , Animales , Huesos , Calcio/análisis , Células Cultivadas , AMP Cíclico/análisis , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Ratones Noqueados , Osteoblastos/efectos de los fármacos , Osteogénesis , Receptores Purinérgicos P2RESUMEN
Piperidinyl diphenylsulfonyl sulfonamides are a novel class of molecules that have inhibitory binding affinity for sFRP-1. As a secreted protein sFRP-1 inhibits the function of the secreted Wnt glycoprotein. Therefore, as inhibitors of sFRP-1 these small molecules facilitate the Wnt/beta-catenin canonical signaling pathway. Details of the structure-activity relationships and biological activity of this structural class of compounds will be discussed.
Asunto(s)
Glicoproteínas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Sulfonamidas/química , Sulfonamidas/farmacología , Proteínas Wnt/metabolismo , Animales , Línea Celular Tumoral , Glicoproteínas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Microsomas Hepáticos/metabolismo , Técnicas de Cultivo de Órganos , Osteogénesis/efectos de los fármacos , Ratas , Cráneo/citología , Cráneo/efectos de los fármacos , Relación Estructura-Actividad , beta Catenina/metabolismoRESUMEN
Genetic studies have identified a high bone mass of phenotype in both human and mouse when canonical Wnt signaling is increased. Secreted frizzled related protein 1 (sFRP1) is one of several Wnt antagonists and among the loss-of-function mouse models in which 32-week-old mice exhibit a high bone mass phenotype. Here we show that impact fracture healing is enhanced in this mouse model of increased Wnt signaling at a physiologic level in young (8 weeks) sFRP1(-/-) mice which do not yet exhibit significant increases in BMD. In vivo deletion of sFRP1 function improves fracture repair by promoting early bone union without adverse effects on the quality of bone tissue reflected by increased mechanical strength. We observe a dramatic reduction of the cartilage callous, increased intramembranous bone formation with bone bridging by 14 days, and early bone remodeling during the 28-day fracture repair process in the sFRP1(-/-) mice. Our molecular analyses of gene markers indicate that the effect of sFRP1 loss-of-function during fracture repair is to accelerate bone healing after formation of the initial hematoma by directing mesenchymal stem cells into the osteoblast lineage via the canonical pathway. Further evidence to support this conclusion is the observation of maximal sFRP1 levels in the cartilaginous callus of a WT mouse. Hence sFRP1(-/-) mouse progenitor cells are shifted directly into the osteoblast lineage. Thus, developing an antagonist to specifically inhibit sFRP1 represents a safe target for stimulating fracture repair and bone formation in metabolic bone disorders, osteoporosis and aging.
Asunto(s)
Remodelación Ósea , Curación de Fractura , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Tibia/metabolismo , Fracturas de la Tibia/metabolismo , Animales , Remodelación Ósea/genética , Callo Óseo/metabolismo , Callo Óseo/fisiopatología , Cartílago/metabolismo , Cartílago/fisiopatología , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Modelos Animales de Enfermedad , Curación de Fractura/genética , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Noqueados , Osteoblastos/metabolismo , Radiografía , Transducción de Señal , Tibia/diagnóstico por imagen , Tibia/fisiopatología , Fracturas de la Tibia/patología , Fracturas de la Tibia/fisiopatología , Factores de Tiempo , Proteínas Wnt/metabolismoRESUMEN
Parathyroid hormone (PTH) activates multiple signaling pathways following binding to the PTH1 receptor in osteoblasts. Previous work revealed a discrepancy between cAMP stimulation and CRE reporter activation of truncated PTH peptides, suggesting that additional signaling pathways contribute to activation of the CRE. Using a CRE-Luciferase reporter containing multiple copies of the CRE stably transfected into the osteoblastic cell line Saos-2, we tested the ability of modulators of alternative pathways to activate the CRE or block the PTH-induced activation of the CRE. Activators of non-cyclic AMP pathways, that is, EGF (Akt, MAPK, JAK/STAT pathways); thapsigargin (intracellular calcium pathway); phorbol myristate acetate (protein kinase C, PKC pathway) induced minor increases in CRE-luciferase activity alone but induced dramatic synergistic effects in combination with PTH. The protein kinase A (PKA) inhibitor H-89 (10 microM) almost completely blocked PTH-induced activation of the CRE-reporter. Adenylate cyclase inhibitors SQ 22536 and DDA had profound and time-dependent biphasic effects on the CRE response. The MAPK inhibitor PD 98059 partially inhibited basal and PTH-induced CRE activity to the same degree, while the PKC inhibitor bisindolylmaleimide (BIS) had variable effects. The calmodulin kinase II inhibitor KN-93 had no significant effect on the response to PTH. We conclude that non-cAMP pathways (EGF pathway, calcium pathway, PKC pathway) converge on, and have synergistic effects on, the response of a CRE reporter to PTH.
Asunto(s)
Adenosina Monofosfato/metabolismo , AMP Cíclico/metabolismo , Hormona Paratiroidea/farmacología , Elementos de Respuesta/efectos de los fármacos , Calcio/metabolismo , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/metabolismo , Humanos , Proteína Quinasa C/metabolismo , Transducción de SeñalRESUMEN
Dkk1 is a secreted antagonist of the LRP5-mediated Wnt signaling pathway that plays a pivotal role in bone biology. Because there are no well-documented LRP5-based assays of Dkk1 binding, we developed a cell-based assay of Dkk1/LRP5 binding using radioactive (125)I-Dkk1. In contrast to LRP6, transfection of LRP5 alone into 293A cells resulted in a low level of specific binding that was unsuitable for routine assay. However, co-transfection of LRP5 with the chaperone protein MesD (which itself does not bind Dkk1) or Kremen-2 (a known Dkk1 receptor), or both, resulted in a marked enhancement of specific binding that was sufficient for evaluation of Dkk1 antagonists. LRP5 fragments comprising the third and fourth beta-propellers plus the ligand binding domain, or the first beta-propeller, each inhibited Dkk1 binding, with mean IC(50)s of 10 and 196 nM, respectively. The extracellular domain of Kremen-2 ("soluble Kremen") was a weaker antagonist (mean IC(50) 806 nM). We also found that cells transfected with a high bone mass mutation LRP5(G171V) had a subtly reduced level of Dkk1 binding, compared to wild type LRP5-transfected cells, and no enhancement of binding by MesD. We conclude that (1) LRP5-transfected cells do not offer a suitable cell-based Dkk1 binding assay, unless co-transfected with either MesD, Kremen-2, or both; (2) soluble fragments of LRP5 containing either the third and fourth beta-propellers plus the ligand binding domain, or the first beta-propeller, antagonize Dkk1 binding; and (3) a high bone mass mutant LRP5(G171V), has subtly reduced Dkk1 binding, and, in contrast to LRP5, no enhancement of binding with MesD.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Chaperonas Moleculares/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de LDL/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Bioensayo , Huesos/metabolismo , Línea Celular , Humanos , Proteínas Relacionadas con Receptor de LDL/química , Proteínas Relacionadas con Receptor de LDL/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Chaperonas Moleculares/genética , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores de Superficie Celular/genéticaRESUMEN
Secreted frizzled related protein-1 (sFRP-1) inhibitors have the potential to be used for the treatment of osteoporosis or other bone related disorders, since the level of sFRP-1 affects osteoblast apoptosis and proliferation. From high throughput screening, we have identified a class of iminooxothiazolidines as sFRP-1 inhibitors. Structure-activity relationships were established for various regions of the scaffold along with the biochemical characterization of this class to probe selectivity, binding and ex vivo activity.
Asunto(s)
Osteogénesis/fisiología , Proteínas/aislamiento & purificación , Calcificación Fisiológica , Diferenciación Celular/fisiología , Células Cultivadas , Receptores Frizzled/antagonistas & inhibidores , Receptores Frizzled/aislamiento & purificación , Péptidos y Proteínas de Señalización Intracelular , Estructura Molecular , Proteínas/antagonistas & inhibidores , Ligando RANKRESUMEN
Secreted frizzled related protein-1 (sFRP1), an antagonist of Wnt signaling, regulates cell proliferation, differentiation and apoptosis and negatively regulates bone formation. The spatial and temporal pattern of endogenous sFRP1 expression and loss-of-function were examined in the sFRP1-LacZ knock-in mouse (sFRP1-/-) during embryonic development and post-natal growth. beta-gal activity representing sFRP1 expression is robust in brain, skeleton, kidney, eye, spleen, abdomen, heart and somites in early embryos, but sFRP1 gene inactivation in these tissues did not compromise normal embryonic and post-natal development. Kidney histology revealed increased numbers of glomeruli in KO mice, observed after 5 years of breeding. In the skeleton, we show sFRP1 expression is found in relation to the mineralizing front of bone tissue during skeletal development from E15.5 to birth. Trabecular bone volume and bone mineral density in the sFRP1-/- mouse compared to WT was slightly increased during post-natal growth. Calvarial osteoblasts from newborn sFRP1-/- mice exhibited a 20% increase in cell proliferation and differentiation at the early stages of osteoblast maturation. sFRP1 expression was observed in osteoclasts, but this did not affect osteoclast number or activity. These findings have identified functions for sFRP1 in kidney and bone that are not redundant with other sFRPs. In summary, the absence of major organ abnormalities, the enhanced bone formation and a normal life span with no detection of spontaneous tumors suggests that targeting sFRP1 can be used as a therapeutic strategy for increasing bone mass in metabolic bone disorders or promoting fracture healing by modulating Wnt signaling.
Asunto(s)
Huesos/embriología , Encéfalo/embriología , Desarrollo Embrionario , Riñón/embriología , Proteínas/metabolismo , Animales , Northern Blotting , Densidad Ósea , Remodelación Ósea/fisiología , Huesos/citología , Diferenciación Celular , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Osteogénesis/fisiologíaRESUMEN
The menopausal transition is associated with decreased ovarian function and concomitant decline in estrogen production, which may result in physiological effects such as hot flashes, reduced bone mass, and altered lipid profile. It is well established that these unfavorable changes are effectively offset with estrogen therapy (ET) or, in women with a uterus, estrogens in combination with a progestin (hormone therapy). Selective estrogen receptor (ER) modulators (SERMs), which exhibit both ER agonist and antagonist activities depending on the target tissue, have been regarded as offering the potential to provide the benefits of ET and hormone therapy with an improved safety and tolerability profile. To date, no SERM alone has demonstrated an ideal benefit-risk profile for menopausal therapy. The tissue-selective estrogen complex, or the pairing of a SERM with estrogens, may provide an optimal blend of ER agonist and antagonist activities. We evaluated the physiological profile of this novel therapeutic paradigm by using various in vivo models to assess uterine, vasomotor, lipid, and skeletal responses to a tissue-selective estrogen complex partnering bazedoxifene with conjugated estrogens (CE). Bazedoxifene at 3.0 mg/kg effectively antagonized CE-induced uterine stimulation without reversing the positive effects of CE on vasomotor instability. When paired with CE, bazedoxifene at 3.0 mg/kg reduced total cholesterol levels by up to 20% compared with CE alone and significantly increased total bone density relative to control. These preclinical findings showed that the appropriate dose combination of bazedoxifene/CE exhibits positive vasomotor, lipid, and skeletal responses with minimal uterine stimulation.
Asunto(s)
Estrógenos Conjugados (USP)/farmacología , Indoles/farmacología , Osteoporosis/prevención & control , Animales , Densidad Ósea/efectos de los fármacos , Colesterol/sangre , Estrógenos Conjugados (USP)/administración & dosificación , Femenino , Indoles/administración & dosificación , Metabolismo de los Lípidos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Moduladores Selectivos de los Receptores de Estrógeno/administración & dosificación , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Útero/efectos de los fármacos , Útero/metabolismo , Sistema Vasomotor/efectos de los fármacosRESUMEN
Wnts are secreted glycoproteins that control vital biological processes, including embryogenesis, organogenesis and tumorigenesis. Wnts are classified into several subfamilies depending on the signaling pathways they activate, with the canonical subfamily activating the Wnt/beta-catenin pathway and the non-canonical subfamily activating a variety of other pathways, including the Wnt/calcium signaling and the small GTPase/c-Jun NH2-terminal kinase pathway. Wnts bind to a membrane receptor Frizzled and a co-receptor, the low-density lipoprotein receptor related protein. More recently, both canonical and non-canonical Wnts were shown to bind the Ror2 receptor tyrosine kinase. Ror2 is an orphan receptor that plays crucial roles in skeletal morphogenesis and promotes osteoblast differentiation and bone formation. Here we examine the effects of a canonical Wnt3a and a non-canonical Wnt5a on the signaling of the Ror2 receptor. We demonstrate that even though both Wnt5a and Wnt3a bound Ror2, only Wnt5a induced Ror2 homo-dimerization and tyrosine phosphorylation in U2OS human osteoblastic cells. Furthermore, Wnt5a treatment also resulted in increased phosphorylation of the Ror2 substrate, 14-3-3beta scaffold protein, indicating that Wnt5a binding causes activation of the Ror2 signaling cascade. Functionally, Wnt5a recapitulated the Ror2 activation phenotype, enhancing bone formation in the mouse calvarial bone explant cultures and potentiating osteoblastic differentiation of human mesenchymal stem cells. The effect of Wnt5a on osteoblastic differentiation was largely abolished upon Ror2 down-regulation. Thus we show that Wnt5a activates the classical receptor tyrosine kinase signaling cascade through the Ror2 receptor in cells of osteoblastic origin.
Asunto(s)
Proteínas Proto-Oncogénicas/fisiología , Receptores de Superficie Celular/metabolismo , Proteínas Wnt/metabolismo , Proteínas Wnt/fisiología , Animales , Diferenciación Celular , Línea Celular , Dimerización , Activación Enzimática , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Osteoblastos/citología , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Transducción de Señal , Proteína Wnt-5a , Proteína Wnt3 , Proteína Wnt3ARESUMEN
Ovariectomy-induced osteopenia in the rat produces skeletal responses similar to that in a post-menopausal woman. In the ovariectomized (ovx) rat, high bone turnover, and subsequent bone loss, like in the human post-menopausal condition, can be prevented by estrogen replacement. Because of the striking resemblance of skeletal responses in humans and rats in the state of estrogen deficiency, the ovx rat is considered to be a gold standard model for evaluating drugs for prevention and reversal of osteoporosis. This chapter describes the procedure for performing ovariectomy on the rat and the utility of the ovx rat model we have utilized over the last two decades in our laboratory.
Asunto(s)
Evaluación Preclínica de Medicamentos , Osteoporosis , Ovariectomía , Animales , Densidad Ósea , Conservadores de la Densidad Ósea/uso terapéutico , Fuerza Compresiva , Modelos Animales de Enfermedad , Estrógenos/deficiencia , Femenino , Humanos , Osteoporosis/tratamiento farmacológico , Osteoporosis/fisiopatología , Ratas , Columna Vertebral/anatomía & histología , Columna Vertebral/metabolismo , Tibia/anatomía & histología , Tibia/químicaRESUMEN
Ror2 receptor plays a key role in bone formation, but its signaling pathway is not completely understood. We demonstrate that Ror2 homodimerizes at the cell surface, and that dimerization can be induced by a bivalent antibody. Antibody-mediated dimerization causes receptor autophosphorylation and induces functional consequences of its signaling, including osteogenesis in mesenchymal stem cells and bone formation in organ culture. We further show that Ror2 associates with and phosphorylates 14-3-3beta scaffold protein. Endogenous Ror2 binds 14-3-3beta in U2OS osteosarcoma cells, and purified intracellular domain of Ror2 interacts with 14-3-3beta in vitro. 14-3-3beta Is tyrosine phosphorylated in U2OS cells, and this phosphorylation is inhibited by down-regulating Ror2 and enhanced by overexpressing the kinase. Purified Ror2 phosphorylates 14-3-3beta in vitro, confirming 14-3-3beta as the first identified Ror2 substrate. Down-regulating 14-3-3beta potentiates osteoblastogenesis in mesenchymal stem cells and increases bone formation in calvarial cultures, indicating that 14-3-3beta exerts a negative effect on osteogenesis. This raises a possibility that Ror2 induces osteogenic differentiation, at least in part, through a release of the 14-3-3beta-mediated inhibition. Our research forms a foundation for several new areas of investigation, including the molecular regulation of 14-3-3 by tyrosine phosphorylation and the role of this scaffold in osteogenesis.
Asunto(s)
Proteínas 14-3-3/metabolismo , Diferenciación Celular , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis , Receptores de Superficie Celular/metabolismo , Proteínas 14-3-3/genética , Línea Celular , Dimerización , Regulación hacia Abajo , Humanos , Fosforilación , Unión Proteica , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Receptores de Superficie Celular/genéticaRESUMEN
Ror2 is a receptor tyrosine kinase, the expression of which increases during differentiation of pluripotent stem cells to osteoblasts and then declines as cells progress to osteocytes. To test whether Ror2 plays a role in osteoblastogenesis, we investigated the effects of Ror2 overexpression and down-regulation on osteoblastic lineage commitment and differentiation. Expression of Ror2 in pluripotent human mesenchymal stem cells (hMSCs) by adenoviral infection caused formation of mineralized extracellular matrix, which is the ultimate phenotype of an osteogenic tissue. Concomitantly, Ror2 over-expression inhibited adipogenic differentiation of hMSCs as monitored by lipid formation. Ror2 shifted hMSC fate toward osteoblastogenesis by inducing osteogenic transcription factor osterix and suppressing adipogenic transcription factors CCAAT/enhancer-binding protein alpha and peroxisome proliferator activated receptor gamma. Infection with Ror2 virus also strongly promoted matrix mineralization in committed osteoblast-like MC3T3-E1 cells. Expression of Ror2 in a human preosteocytic cell line by stable transfection also promoted further differentiation, as judged by inhibited alkaline phosphatase activity, potentiated osteocalcin secretion, and increased cellular apoptosis. In contrast, down-regulation of Ror2 expression by short hairpin RNA essentially abrogated dexamethasone-induced mineralization of hMSCs. Furthermore, down-regulation of Ror2 expression in fully differentiated SaOS-2 osteosarcoma cells inhibited alkaline phosphatase activity. We conclude that Ror2 initiates commitment of MSCs to osteoblastic lineage and promotes differentiation at early and late stages of osteoblastogenesis. Finally, using a mouse calvariae ex vivo organ culture model, we demonstrate that these effects of Ror2 result in increased bone formation, suggesting that it may also activate mature osteoblasts.
Asunto(s)
Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Osteogénesis , Células Madre Pluripotentes/citología , Receptores de Superficie Celular/metabolismo , Animales , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Calcificación Fisiológica/fisiología , Diferenciación Celular , Línea Celular , Dexametasona/farmacología , Regulación hacia Abajo , Glucocorticoides/farmacología , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Técnicas de Cultivo de Órganos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , PPAR gamma/metabolismo , Células Madre Pluripotentes/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Cráneo/citología , Factor de Transcripción Sp7 , Factores de Transcripción/metabolismoRESUMEN
Ror2 is an orphan receptor tyrosine kinase that plays crucial roles in developmental morphogenesis, particularly of the skeleton. We have identified human Ror2 as a novel regulator of canonical Wnt signaling in osteoblastic (bone-forming) cells with selective activities, enhancing Wnt1 but antagonizing Wnt3. Immunoprecipitation studies demonstrated physical interactions between human Ror2 and mammalian Wnt1 and Wnt3. Functionally, Ror2 antagonized Wnt1- and Wnt3-mediated stabilization of cytosolic beta-catenin in osteoblastic cells. However, Ror2 had opposing effects on a more distal step of canonical Wnt signaling: it potentiated Wnt1 activity but inhibited Wnt3 function as assessed by changes in Wnt-responsive reporter gene activity. Despite binding to Ror2, neither Wnt1 nor Wnt3 altered receptor activity as assessed by levels of Ror2 autophosphorylation. The ability of Ror2 to regulate canonical Wnt signaling in osteoblastic cells should have physiological consequences in bone, because Wnt signaling is known to modulate osteoblast survival and differentiation. Expression of Ror2 mRNA was highly regulated in a biphasic manner during human osteoblast differentiation, being virtually undetectable in pluripotent stem cells, increasing 300-fold in committed preosteoblasts, and disappearing again in osteocytes. Furthermore, Ror2 expression in osteoblasts was suppressed by the Wnt antagonist, secreted frizzled-related protein 1. The regulated expression of Ror2 during osteoblast differentiation, its inverse expression pattern with secreted frizzled-related protein 1, and its ability to modulate Wnt signaling in osteoblastic cells suggest that Ror2 may regulate bone formation.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Osteoblastos/metabolismo , Proteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Animales , Línea Celular , Proteínas del Citoesqueleto/metabolismo , Regulación de la Expresión Génica , Glicoproteínas/deficiencia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Fosforilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas/genética , ARN Mensajero/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Receptores de Superficie Celular/genética , Transactivadores/metabolismo , Proteínas Wnt , Proteína Wnt1 , Proteína Wnt3 , beta CateninaRESUMEN
We assessed the preclinical characteristics of a novel, stringently screened selective estrogen receptor modulator, bazedoxifene acetate, including its ability to bind to and activate estrogen receptors and promote increased bone mineral density and bone strength in rats, and the effects impacting the uterine endometrium, breast cancer cell proliferation, and central nervous system-associated vasomotor responses in an animal model. Bazedoxifene bound to estrogen receptor-alpha with an IC50 of 26 nm, an affinity similar to that of raloxifene. Bazedoxifene did not stimulate proliferation of MCF-7 cells but did inhibit 17beta-estradiol-induced proliferation with an IC50 of 0.19 nm. In an immature rat uterine model, bazedoxifene (0.5 and 5.0 mg/kg) was associated with less increase in uterine wet weight than either ethinyl estradiol (10 microg/kg) or raloxifene (0.5 and 5.0 mg/kg). Histological analysis revealed that coadministration of bazedoxifene also appeared to reduce raloxifene-stimulated endometrial luminal epithelial cell and myometrial cell hypertrophy. In ovariectomized rats, bazedoxifene was associated with significant increases in bone mineral density at 6 wk, compared with control, and better compressive strength of bone samples from the L4 vertebrae, compared with samples from ovariectomized animals. In the morphine-addicted rat model of vasomotor activity, bone-sparing doses of bazedoxifene alone were not associated with 17beta-estradiol inhibition of increased vasomotor activity. Bazedoxifene acetate represents a promising new treatment for osteoporosis, with a potential for less uterine and vasomotor effects than selective estrogen receptor modulators currently used in clinical practice. Controlled clinical trial data will be needed to confirm these effects.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Estrógenos/farmacología , Indoles/farmacología , Animales , Regulación de la Temperatura Corporal/efectos de los fármacos , Densidad Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Huesos/fisiología , Neoplasias de la Mama , Células CHO , Carcinoma Hepatocelular , División Celular/efectos de los fármacos , Línea Celular Tumoral , Fuerza Compresiva , Cricetinae , Endometrio/efectos de los fármacos , Estrógenos/química , Estrógenos/metabolismo , Femenino , Humanos , Indoles/química , Indoles/metabolismo , Ligandos , Lípidos/sangre , Neoplasias Hepáticas , Neuronas/citología , Osteoblastos/citología , Ratas , Ratas Sprague-DawleyRESUMEN
Estrogens exert their physiological effects on target tissues by interacting with the estrogen receptors, ERalpha and ERbeta. Estrogen replacement is one the most common and effective strategies used to prevent osteoporosis in postmenopausal women. Whereas it was thought that estrogens work exclusively by inhibiting bone resorption, our previous results show that 17beta-estradiol (E2) increases mouse bone morphogenetic protein (BMP)-2 mRNA, suggesting that estrogens may also enhance bone formation. In this study, we used quantitative real-time RT-PCR analysis to demonstrate that estrogens increase BMP-2 mRNA in mouse mesenchymal stem cells. The selective ER modulators, tamoxifen, raloxifene, and ICI-182,780 (ICI), failed to enhance BMP-2 mRNA, whereas ICI inhibited E2 stimulation of expression. To investigate if estrogens increase BMP-2 expression by transcriptional mechanisms and if the response is mediated by ERalpha and/or ERbeta, we studied the effects of estrogens on BMP-2 promoter activity in transient transfected C3H10T1/2 cells. E2 produced a dose-dependent induction of the mouse -2712 BMP-2 promoter activity in cells cotransfected with ERalpha and ERbeta. At a dose of 10 nM E2, ERalpha induced mouse BMP-2 promoter activity 9-fold, whereas a 3-fold increase was observed in cells cotransfected with ERbeta. Tamoxifen and raloxifene were weak activators of the mouse BMP-2 promoter via ERalpha, but not via ERbeta. ICI blocked the activation of BMP-2 promoter activity by E2 acting via both ERalpha and ERbeta, indicating that mouse BMP-2 promoter activation is ER dependent. In contrast to E2 and selective ER modulators, the phytoestrogen, genistein was more effective at activating the mouse BMP-2 promoter with ERbeta, compared with ERalpha. Using a deletion series of the BMP-2 promoter, we determined that AP-1 or Sp1 sites are not required for E2 activation. A mutation in a sequence at -415 to -402 (5'-GGGCCActcTGACCC-3') that resembles the classical estrogen-responsive element abolished the activation of the BMP-2 promoter in response to E2. Our studies demonstrate that E2 activation of mouse BMP-2 gene transcription requires ERalpha or ERbeta acting via a variant estrogen-responsive element binding site in the promoter, with ERalpha being the more efficacious regulator. Estrogenic compounds may enhance bone formation by increasing the transcription of the BMP-2 gene.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Estradiol/fisiología , Células Madre/metabolismo , Factor de Crecimiento Transformador beta , Animales , Células de la Médula Ósea/metabolismo , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/genética , Células Cultivadas , Relación Dosis-Respuesta a Droga , Estradiol/genética , Femenino , Regulación de la Expresión Génica , Genisteína/farmacología , Mesodermo/citología , Mesodermo/fisiología , Ratones , ARN Mensajero/metabolismo , Receptores de Estrógenos/clasificación , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Elementos de Respuesta/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Transcripción Genética , Activación TranscripcionalRESUMEN
Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults.