RESUMEN
The maintenance of bone homeostasis requires tight coupling between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the precise molecular mechanism(s) underlying the differentiation and activities of these specialized cells are still largely unknown. Here, we identify choline kinase ß (CHKB), a kinase involved in the biosynthesis of phosphatidylcholine, as a novel regulator of bone homeostasis. Choline kinase ß mutant mice (flp/flp) exhibit a systemic low bone mass phenotype. Consistently, osteoclast numbers and activity are elevated in flp/flp mice. Interestingly, osteoclasts derived from flp/flp mice exhibit reduced sensitivity to excessive levels of extracellular calcium, which could account for the increased bone resorption. Conversely, supplementation of cytidine 5'-diphosphocholine in vivo and in vitro, a regimen that bypasses CHKB deficiency, restores osteoclast numbers to physiological levels. Finally, we demonstrate that, in addition to modulating osteoclast formation and function, loss of CHKB corresponds with a reduction in bone formation by osteoblasts. Taken together, these data posit CHKB as a new modulator of bone homeostasis.
Asunto(s)
Colina Quinasa/genética , Mutación , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Fosforilcolina/metabolismo , Animales , Densidad Ósea , Resorción Ósea , Huesos/metabolismo , Calcio/metabolismo , Proliferación Celular , Homeostasis , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Mutagénesis , Osteoblastos/citología , Osteoclastos/citología , Fenotipo , Microtomografía por Rayos XRESUMEN
Angiogenesis plays a pivotal role in bone formation, remodeling, and fracture healing. The regulation of angiogenesis in the bone microenvironment is highly complex and orchestrated by intercellular communication between bone cells and endothelial cells. Here, we report that EGF-like domain 7 (EGFL7), a member of the epidermal growth factor (EGF) repeat protein superfamily is expressed in both the osteoclast and osteoblast lineages, and promotes endothelial cell activities. Addition of exogenous recombinant EGFL7 potentiates SVEC (simian virus 40-transformed mouse microvascular endothelial cell line) cell migration and tube-like structure formation in vitro. Moreover, recombinant EGFL7 promotes angiogenesis featuring web-like structures in ex vivo fetal mouse metatarsal angiogenesis assay. We show that recombinant EGFL7 induces phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription 3 (STAT3), and focal adhesion kinase (FAK) in SVEC cells. Inhibition of ERK1/2 and STAT3 signaling impairs EGFL7-induced endothelial cell migration, and angiogenesis in fetal mouse metatarsal explants. Bioinformatic analyses indicate that EGFL7 contains a conserved RGD/QGD motif and EGFL7-induced endothelial cell migration is significantly reduced in the presence of RGD peptides. Moreover, EGFL7 gene expression is significantly upregulated during growth plate injury repair. Together, these results demonstrate that EGFL7 expressed by bone cells regulates endothelial cell activities through integrin-mediated signaling. This study highlights the important role that EGFL7, like EGFL6, expressed in bone microenvironment plays in the regulation of angiogenesis in bone.
Asunto(s)
Células Endoteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Integrinas/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Proteínas/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Huesos/irrigación sanguínea , Huesos/citología , Proteínas de Unión al Calcio , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Familia de Proteínas EGF , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Curación de Fractura/fisiología , Placa de Crecimiento/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteocalcina/biosíntesis , Osteoclastos/metabolismo , Osteogénesis/fisiología , Fosforilación/efectos de los fármacos , Proteínas/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Fracturas de Salter-Harris , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
Bone remodeling is regulated by secreted factors in the bone microenvironment. However, data regarding osteoclast-derived factors that influence osteoblast differentiation are lacking. Here, we show that HtrA1 is produced as a secreted protein during osteoclastogenesis, and negatively regulates osteoblast differentiation. Exogenous addition of recombinant HtrA1 attenuates osteoblast differentiation and BMP2-induced Smad1/5/8, ERK1/2 and p38 phosphorylation in pre-osteoblasts. Our studies imply a unique mode of crosstalk in which HtrA1 is produced by both osteoclasts and osteoblasts and negatively regulates osteoblast differentiation, suggesting that HtrA1 may mediate the fine tuning of paracrine and autocrine regulations during bone remodeling processes.