RESUMEN
Calcitriol (1alpha,25(OH)(2)D(3)) plays a key role in the differentiation of osteoblasts, the cells responsible for the formation and maintenance of healthy bone matrix. Recently it has emerged that calcitriol influences the trafficking or stability of epidermal growth factor (EGF) receptors. However, how these agents might work together in regulating growth and differentiation has not been examined. Using the human osteoblast cell line, MG63, we were able to induce a profound differentiation response by treating these cells with a combination of calcitriol (100 nM) and EGF (10 ng/ml). Co-stimulation of MG63 osteoblasts with calcitriol and EGF led to synergistic increases in osteocalcin and alkaline phosphatase (ALP), proteins expressed by differentiating cells. Inhibition of differentiation was accomplished by MEK and protein kinase C (PKC) inhibitors. Other ligands known to signal via receptor tyrosine kinases could not substitute for EGF in the maturation response. These novel findings may help identify new processes that drive osteoblast differentiation.
Asunto(s)
Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Línea Celular , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor 1 de Crecimiento de Fibroblastos/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Osteoblastos/enzimología , Osteoblastos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Biosíntesis de Proteínas/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Considerable attention has been paid to the role of sex steroids during periods of major skeletal turnover, but the interaction of the gonadotropic hormones, which include LH, FSH, and human chorionic gonadotropin (hCG), within bone tissue have been overlooked. The question is pertinent due to the recent detection of extragonadal expression of gonadotropin receptors. Western blotting, immunolocalization, and RT-PCR supported the presence of osteoblast LH receptors. However, osteoblast cells failed to bind [(125)I]hCG and treatment with hCG failed to generate either cAMP or phosphorylated ERK 1/2. Bone mineral density (BMD) and bone histomorphometry were examined in the following models: 1) LH receptor null mutant (LuRKO) mice; 2) transgenic mice overexpressing hCG (hCG alphabeta+); and 3) ovariectomized (OVX) hCG alphabeta+ model. Male LuRKO mice showed a decrease in BMD after 5 months, apparently secondary to suppressed gonadal steroid production. Similarly, 9- to 10-wk-old female LuRKO mice exhibited decreases in histomorphometric parameters tested. The data indicate that loss of LH signaling results in a reduction in bone formation or an increase in bone resorption. By contrast, there were significant increases in BMD and histomorphometric indices for female, but not male, hCG alphabeta+ mice, indicating that chronic exposure to hCG results in bone formation or a decrease in bone resorption. However, OVX of the hCG alphabeta+ mice resulted in a significant reduction in BMD comparable to OVX WT controls. Although gonadotropin levels are tightly linked to sex steroid titers, it appears that their effects on the skeleton are indirect.
Asunto(s)
Huesos/fisiología , Gonadotropina Coriónica/genética , Fenotipo , Receptores de HL/deficiencia , Adulto , Animales , Densidad Ósea/fisiología , Línea Celular , Células Cultivadas , Gonadotropina Coriónica/farmacología , Gonadotropina Coriónica/fisiología , AMP Cíclico/biosíntesis , Femenino , Humanos , Tumor de Células de Leydig , Hormona Luteinizante/metabolismo , Masculino , Ratones , Ratones Transgénicos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Osteoblastos/química , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Ovariectomía , Ovario/química , Fosforilación , ARN Mensajero/análisis , Ratas , Receptores de HL/análisis , Receptores de HL/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Mammalian palatogenesis depends on palatal shelf elevation, medial edge epithelium (MEE) breakdown, and mesenchyme flow. These all require matrix remodeling, which is controlled in part by the family of matrix metalloproteinases (MMPs). We used an organ culture system to examine the effect of a general MMP inhibitor (BB3103) on mouse palatogenesis. Palates cultured in 20 micro M BB3103 contained no active MMP-2, and only one palate fused from a sample size of 15. In this single palate, MMP-3 was present at higher levels than in palates that failed to fuse. MMP-3 is known to be involved in epithelial mesenchymal transformation (EMT), and its persistence may explain why this palate fused. This implies a role for MMPs in normal palatogenesis, and disruption of their activity may result in cleft palate.
Asunto(s)
Fisura del Paladar/embriología , Metaloproteinasa 2 de la Matriz/fisiología , Metaloproteinasa 3 de la Matriz/fisiología , Hueso Paladar/embriología , Hueso Paladar/enzimología , Animales , Electroforesis en Gel de Poliacrilamida , Epitelio/embriología , Matriz Extracelular/enzimología , Ácidos Hidroxámicos/farmacología , Técnicas para Inmunoenzimas , Inhibidores de la Metaloproteinasa de la Matriz , Mesodermo , Ratones , Ratones Endogámicos , Técnicas de Cultivo de ÓrganosRESUMEN
Recent studies have indicated that maternal skeletal metabolism undergoes significant changes during gestation. The agents that are responsible for eliciting these changes in bone turnover during pregnancy have yet to be defined. We therefore sought to investigate whether chaperonin 10 (Cpn10), a homolog of early-pregnancy factor, or human placental lactogen (PL) were capable of influencing the synthesis of type I collagen by human osteoblasts in vitro. Both Cpn10 and PL are major components of the maternal circulation during pregnancy, but how they might contribute to bone metabolism has not been determined. Type I collagen represents the most abundant component of bone tissue, accounting for approximately 90% of the organic compartment. Both Cpn10 and PL were capable of stimulating the synthesis of type I collagen by human osteoblasts in culture. The inclusion of 17 beta-estradiol or prolactin, however, failed to influence the ability of cells to mobilize type I collagen. These novel findings support a role for PL and Cpn10 in the metabolism of bone tissue during pregnancy. Maternal bone collagen metabolism is clearly an important event during pregnancy, and the identification of the factors responsible will aid our understanding of the regulation of skeletal metabolism during gestation.