RESUMEN
OBJECTIVE: The bone morphogenetic protein (BMP) signaling pathway comprises the largest subdivision of the transforming growth factor (TGFß) superfamily. BMP signaling plays essential roles in both embryonic development and postnatal tissue homeostasis. Dysregulated BMP signaling underlies human pathologies ranging from pulmonary arterial hypertension to heterotopic ossification. Thus, understanding the basic mechanisms and regulation of BMP signaling may yield translational opportunities. Unfortunately, limited tools are available to evaluate this pathway, and genetic approaches are frequently confounded by developmental requirements or ability of pathway components to compensate for one another. Specific inhibitors for type 2 receptors are poorly represented. Thus, we sought to identify and validate an antibody that neutralizes the ligand-binding function of BMP receptor type 2 (BMPR2) extracellular domain (ECD). RESULTS: Using a modified, cell-free immunoprecipitation assay, we examined the neutralizing ability of the mouse monoclonal antibody 3F6 and found a dose-dependent inhibition of BMPR2-ECD ligand-binding. Consistent with this, 3F6 blocks endogenous BMPR2 function in the BMP-responsive cell line HEK293T. The specificity of 3F6 action was confirmed by demonstrating that this antibody has no effect on BMP-responsiveness in HEK293T cells in which BMPR2 expression is knocked-down. Our results provide important proof-of-concept data for future studies interrogating BMPR2 function.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos/inmunología , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Neutralizantes/farmacología , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/antagonistas & inhibidores , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Células HEK293 , Humanos , Ratones , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
The taste receptor type 1 (TAS1R) family of heterotrimeric G protein-coupled receptors participates in monitoring energy and nutrient status. TAS1R member 3 (TAS1R3) is a bi-functional protein that recognizes amino acids such as L-glycine and L-glutamate or sweet molecules such as sucrose and fructose when dimerized with TAS1R member 1 (TAS1R1) or TAS1R member 2 (TAS1R2), respectively. It was recently reported that deletion of TAS1R3 expression in Tas1R3 mutant mice leads to increased cortical bone mass but the underlying cellular mechanism leading to this phenotype remains unclear. Here, we independently corroborate the increased thickness of cortical bone in femurs of 20-week-old male Tas1R3 mutant mice and confirm that Tas1R3 is expressed in the bone environment. Tas1R3 is expressed in undifferentiated bone marrow stromal cells (BMSCs) in vitro and its expression is maintained during BMP2-induced osteogenic differentiation. However, levels of the bone formation marker procollagen type I N-terminal propeptide (PINP) are unchanged in the serum of 20-week-old Tas1R3 mutant mice as compared to controls. In contrast, levels of the bone resorption marker collagen type I C-telopeptide are reduced greater than 60% in Tas1R3 mutant mice. Consistent with this, Tas1R3 and its putative signaling partner Tas1R2 are expressed in primary osteoclasts and their expression levels positively correlate with differentiation status. Collectively, these findings suggest that high bone mass in Tas1R3 mutant mice is due to uncoupled bone remodeling with reduced osteoclast function and provide rationale for future experiments examining the cell-type-dependent role for TAS1R family members in nutrient sensing in postnatal bone remodeling
Asunto(s)
Animales , Masculino , Resorción Ósea/metabolismo , Hueso Cortical/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Osteoblastos , Osteoclastos/metabolismo , Osteogénesis , Receptores Acoplados a Proteínas G/metabolismo , Biomarcadores/metabolismo , Resorción Ósea/inmunología , Resorción Ósea/patología , Catepsina K , Línea Celular , Hueso Cortical , Células Madre Mesenquimatosas/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones MutantesRESUMEN
The taste receptor type 1 (TAS1R) family of heterotrimeric G protein-coupled receptors participates in monitoring energy and nutrient status. TAS1R member 3 (TAS1R3) is a bi-functional protein that recognizes amino acids such as L-glycine and L-glutamate or sweet molecules such as sucrose and fructose when dimerized with TAS1R member 1 (TAS1R1) or TAS1R member 2 (TAS1R2), respectively. It was recently reported that deletion of TAS1R3 expression in Tas1R3 mutant mice leads to increased cortical bone mass but the underlying cellular mechanism leading to this phenotype remains unclear. Here, we independently corroborate the increased thickness of cortical bone in femurs of 20-week-old male Tas1R3 mutant mice and confirm that Tas1R3 is expressed in the bone environment. Tas1R3 is expressed in undifferentiated bone marrow stromal cells (BMSCs) in vitro and its expression is maintained during BMP2-induced osteogenic differentiation. However, levels of the bone formation marker procollagen type I N-terminal propeptide (PINP) are unchanged in the serum of 20-week-old Tas1R3 mutant mice as compared to controls. In contrast, levels of the bone resorption marker collagen type I C-telopeptide are reduced greater than 60% in Tas1R3 mutant mice. Consistent with this, Tas1R3 and its putative signaling partner Tas1R2 are expressed in primary osteoclasts and their expression levels positively correlate with differentiation status. Collectively, these findings suggest that high bone mass in Tas1R3 mutant mice is due to uncoupled bone remodeling with reduced osteoclast function and provide rationale for future experiments examining the cell-type-dependent role for TAS1R family members in nutrient sensing in postnatal bone remodeling.