RESUMEN
Teriparatide is a peptide derived from a parathyroid hormone (PTH) and an osteoporosis therapeutic drug with potent bone formation-promoting activity. To identify novel druggable genes that act downstream of PTH signaling and are potentially involved in bone formation, we screened PTH target genes in mouse osteoblast-like MC3T3-E1 cells. Here we show that Gprc5a, encoding an orphan G protein-coupled receptor, is a novel PTH-inducible gene and negatively regulates osteoblast proliferation and differentiation. PTH treatment induced Gprc5a expression in MC3T3-E1 cells, rat osteosarcoma ROS17/2.8 cells, and mouse femurs. Induction of Gprc5a expression by PTH occurred in the absence of protein synthesis and was mediated primarily via the cAMP pathway, suggesting that Gprc5a is a direct target of PTH signaling. Interestingly, Gprc5a expression was induced additively by co-treatment with PTH and 1α, 25-dihydroxyvitamin D3 (calcitriol), or retinoic acid in MC3T3-E1 cells. Reporter analysis of a 1 kb fragment of human GPRC5A promoter revealed that the promoter fragment showed responsiveness to PTH via the cAMP response element, suggesting that GPRC5A is also a PTH-inducible gene in humans. Gprc5a knockdown promoted cell viability and proliferation, as demonstrated by MTT and BrdU assays. Gprc5a knockdown also promoted osteoblast differentiation, as indicated by gene expression analysis and mineralization assay. Mechanistic studies showed that Gprc5a interacted with BMPR1A and suppressed BMP signaling induced by BMP-2 and constitutively active BMP receptors, ALK2 (ACVR1) Q207D and ALK3 (BMPR1A) Q233D. Thus, our results suggest that Gprc5a is a novel gene induced by PTH that acts in an inhibitory manner on both cell proliferation and osteoblast differentiation and is a candidate for drug targets for osteoporosis.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Osteoblastos , Hormona Paratiroidea , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Hormona Paratiroidea/farmacología , Ratones , Ratas , Humanos , Transducción de Señal/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Regiones Promotoras Genéticas/genética , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , AMP Cíclico/metabolismo , Tretinoina/farmacología , Calcitriol/farmacologíaRESUMEN
BACKGROUND: Profilin-1 (Pfn1), an evolutionarily conserved actin-binding protein, is an important regulator of the cytoskeleton. We previously reported the osteoclast-specific Pfn1-conditional knockout (cKO) mice had postnatal osteolytic phenotype with craniofacial and long-bone deformities associated with increased migration of cultured osteoclasts. We hypothesized the increased cellular processes structured with branched actin filaments may underlies the mechanism of increased bone resorption in these mutant mice. MATERIALS AND METHODS: The morphological structure and cell migration of the cultured osteoclasts were analyzed using fluorescent microscopy and time-lapse image capturing. Fractional migration distances, as well as the index of protrusive structures (%-PB) that evaluates relative border length of the protrusion were compared between the cells from control and Pfn1-cKO mice. RESULTS: Time-lapse image analysis showed that %-PB was significantly larger in Pfn1-cKO osteoclasts. In addition, the fractional migration distance was positively correlated with the index. When the branched actin filament organization was suppressed by chemical inhibitors, the osteoclast migration was declined. Importantly, the suppression was more extensive in Pfn1-cKO than in control osteoclasts. CONCLUSION: Our results indicated the causative involvement of the increased branched actin filament formation at least in part for their excessive migration. Our findings provide a mechanistic rationale for testing novel therapeutic approaches targeting branched actin filaments in osteolytic disorders.
Asunto(s)
Osteoclastos , Profilinas , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Huesos/metabolismo , Movimiento Celular , Ratones , Osteoclastos/metabolismo , Profilinas/genética , Profilinas/metabolismoRESUMEN
Sarcopenia is among the most common medical problems of the aging population worldwide and a major social concern. Here, we explored the therapeutic potential of TM5484, a novel orally available PAI-1 inhibitor, to prevent sarcopenia. The sarcopenic phenotypes of the calf muscle of 12- and 6-month-old middle-aged mice were compared. Although significant decline of isometric gastrocnemius muscle force was detected in the older untreated mice, those administered TM5484 had significantly greater calf muscle force, as determined using isometric measurements by electrical stimulation. Histological analysis indicated that cross-sectional gastrocnemius muscle fibers in untreated older mice were thinner than those in younger mice; however, TM5484-treated group showed thicker fibers than younger mice. Treatment with TM5484 for 6 months enhanced Igf1, Atrogin-1, Mt-Co1, and Chrna1 mRNA expression in the mice gastrocnemius muscle, with increased serum IGF-1 concentration. TM5484 induced dose-dependent Igf1, Atrogin-1, and Chrna1 expression in C2C12 myoblastic cells, confirming cell autonomous effect. Further, the presence of plasmin for 72 h caused significantly increased Igf1 expression in C2C12 cells. These findings suggest that oral PAI-1 inhibitors represent a promising therapeutic candidate for preventing sarcopenia progression in humans.
Asunto(s)
Fibras Musculares Esqueléticas/efectos de los fármacos , Atrofia Muscular/prevención & control , Inhibidor 1 de Activador Plasminogénico/uso terapéutico , Inhibidores de Serina Proteinasa/uso terapéutico , Envejecimiento/efectos de los fármacos , Animales , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Atrofia Muscular/etiología , Atrofia Muscular/fisiopatología , Inhibidor 1 de Activador Plasminogénico/química , Sarcopenia/etiología , Sarcopenia/patología , Sarcopenia/prevención & control , Inhibidores de Serina Proteinasa/químicaRESUMEN
Cytoplasmic polyadenylation element binding (CPEB) proteins are RNA-binding proteins involved in translational regulation of the specific target mRNAs and control function of various organs including brain, liver and hematopoietic system. However, the role of CPEB proteins during osteoclast differentiation remains unclear. Here we show that Cpeb4 is required for RANKL-induced osteoclast differentiation in mouse macrophage-derived RAW264.7 cell line. Cpeb4 mRNA and protein levels are upregulated at the late stage of osteoclast differentiation. Immunofluorescence analysis revealed that Cpeb4 is translocated from cytoplasm to nuclear bodies in response to RANKL stimulation. Inhibition of PI3K-Akt signaling or calcium-NFAT pathways using chemical inhibitors suppressed nuclear localization of Cpeb4. Loss-of-function analysis showed that shRNA-mediated Cpeb4 depletion strongly impaired TRAP-positive osteoclast formation and expression of key differentiation markers including Acp5, Ctsk, Nfatc1 and Dcstamp. These results suggest that Cpeb4 is a positive regulator in osteoclastogenesis downstream of RANKL signaling.
Asunto(s)
Osteoclastos/citología , Osteogénesis , Proteínas de Unión al ARN/metabolismo , Animales , Ratones , Osteoclastos/metabolismo , Ligando RANK/metabolismo , Células RAW 264.7 , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Transducción de Señal , Regulación hacia ArribaRESUMEN
In mammals, the ovarian follicles are regulated at least in part by bone morphogenetic protein (BMP) family members. Dullard (also known as Ctdnep1) gene encodes a phosphatase that suppresses BMP signaling by inactivating or degrading BMP receptors. Here we report that the Col1a1-Cre-induced Dullard mutant mice displayed hemorrhagic ovarian cysts, with red blood cells accumulated in the follicles, resulting in infertility. Cells expressing Cre driven by Col1a1 2.3-kb promoter and their descendants were found in granulosa cells in the ovary and in Sertoli cells in the testis. DullardmRNA was localized to granulosa cells in the ovary. Genes involved in steroid hormone genesis including Cyp11a1, Hsd3b1 and Star were reduced, whereas expression of Smad6 and Smad7, BMP-inducible inhibitory Smads, was up-regulated in the Dullard mutant ovaries. Tamoxifen-inducible Dullard deletion in the whole body using Rosa26-CreER mice also resulted in hemorrhagic ovarian cysts in 2 weeks, which was rescued by administration of LDN-193189, a chemical inhibitor of BMP receptor kinase, suggesting that the hemorrhage in the Dullard-deficient ovarian follicles might be caused by increased BMP signaling. Thus, we conclude that Dullard is essential for ovarian homeostasis at least in part via suppression of BMP signaling.
Asunto(s)
Colágeno Tipo I/metabolismo , Hemorragia/patología , Infertilidad Femenina/patología , Quistes Ováricos/patología , Folículo Ovárico/patología , Fosfoproteínas Fosfatasas/deficiencia , Animales , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Huesos/metabolismo , Huesos/patología , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hemorragia/metabolismo , Infertilidad Femenina/metabolismo , Masculino , Ratones , Ratones Noqueados , Quistes Ováricos/metabolismo , Folículo Ovárico/metabolismo , Fosfoproteínas Fosfatasas/fisiología , Fosforilación , Pirazoles/farmacología , Pirimidinas/farmacología , Transducción de Señal , Proteínas Smad/metabolismo , Testículo/metabolismo , Testículo/patologíaRESUMEN
The bone is the main storage site for Ca2+ and Mg2+ ions in the mammalian body. Although investigations into Ca2+ signaling have progressed rapidly and led to better understanding of bone biology, the Mg2+ signaling pathway and associated molecules remain to be elucidated. Here, we investigated the role of a potential Mg2+ signaling-related lysosomal molecule, two-pore channel subtype 2 (TPC2), in osteoclast differentiation and bone remodeling. Previously, we found that under normal Mg2+ conditions, TPC2 promotes osteoclastogenesis. We observed that under low-Mg2+ conditions, TPC2 inhibited, rather than promoted, the osteoclast differentiation and that the phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) signaling pathway played a role in the TPC2 activation under low-Mg2+ conditions. Furthermore, PI(3,5)P2 depolarized the membrane potential by increasing the intracellular Na+ levels. To investigate how membrane depolarization affects osteoclast differentiation, we generated a light-sensitive cell line and developed a system for the light-stimulated depolarization of the membrane potential. The light-induced depolarization inhibited the osteoclast differentiation. We then tested the effect of myo-inositol supplementation, which increased the PI(3,5)P2 levels in mice fed a low-Mg2+ diet. The myo-inositol supplementation rescued the low-Mg2+ diet-induced trabecular bone loss, which was accompanied by the inhibition of osteoclastogenesis. These results indicate that low-Mg2+-induced osteoclastogenesis involves changes in the role of TPC2, which are mediated through the PI(3,5)P2 pathway. Our findings also suggest that myo-inositol consumption might provide beneficial effects in Mg2+ deficiency-induced skeletal diseases.
Asunto(s)
Canales de Calcio/metabolismo , Magnesio/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Animales , Remodelación Ósea/efectos de los fármacos , Remodelación Ósea/fisiología , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/metabolismo , Resorción Ósea/patología , Señalización del Calcio , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Inositol/farmacología , Lisosomas/metabolismo , Deficiencia de Magnesio/tratamiento farmacológico , Deficiencia de Magnesio/metabolismo , Deficiencia de Magnesio/patología , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Fosfatos de Fosfatidilinositol/metabolismo , Células RAW 264.7 , Sodio/metabolismoRESUMEN
Osteocytes are the most abundant cells in bone and regulate bone metabolism in coordination with osteoblasts and osteoclasts. However, the molecules that control osteocytes are still incompletely understood. Profilin1 is an actin-binding protein that is involved in actin polymerization. Osteocytes possess characteristic dendritic process formed based on actin cytoskeleton. Here, we examined the expression of profilin1 and its function in osteocytes. Profilin1 mRNA was expressed in osteocytic MLO-Y4 cells and its levels were gradually increased along with the time in culture. With regard to functional aspect, knockdown of profilin1 by siRNA enhanced BMP-induced increase in alkaline phosphatase expression levels in MLO-Y4 cells. Profilin1 knockdown suppressed the levels of dendritic processes and migration of MLO-Y4 cells. Since aging causes an increase in ROS in the body, we further examined the effects of hydrogen peroxide on the expression of profilin1. Hydrogen peroxide treatment increased the levels of profilin1 mRNA in MLO-Y4 cells in contrast to the decline in alkaline phosphatase. Profilin1 was expressed not only in MLO-Y4cells but also in the primary cultures of osteocytes. Importantly, profilin1 mRNA levels in primary cultures of osteocytes were higher than those in primary cultures of osteoblasts. To examine in vivo role of profilin1 in osteocytes, profilin1 was conditionally knocked out by using DMP1-cre and profilin1 floxed mice. This conditional deletion of profilin1 specifically in osteocytes resulted in reduction in the levels of bone volume and bone mineral density. These data indicate that profilin1 is expressed in osteocytes and regulates cell shape, migration and bone mass.
Asunto(s)
Movimiento Celular , Forma de la Célula , Fémur/metabolismo , Osteocitos/metabolismo , Profilinas/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Densidad Ósea , Remodelación Ósea , Línea Celular , Movimiento Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Fémur/diagnóstico por imagen , Fémur/efectos de los fármacos , Regulación de la Expresión Génica , Genotipo , Peróxido de Hidrógeno/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Osteocitos/efectos de los fármacos , Fenotipo , Cultivo Primario de Células , Profilinas/deficiencia , Profilinas/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Microtomografía por Rayos XRESUMEN
Bone mass is determined by coordinated acts of osteoblasts and osteoclasts, which control bone formation and resorption, respectively. Osteoclasts are multinucleated, macrophage/monocyte lineage cells from bone marrow. The Dok-family adaptors Dok-1, Dok-2 and Dok-3 are expressed in the macrophage/monocyte lineage and negatively regulate many signaling pathways, implying roles in osteoclastogenesis. Indeed, mice lacking Dok-1 and Dok-2, the closest homologues with redundant functions, develop osteopenia with increased osteoclast counts compared to the wild-type controls. Here, we demonstrate that Dok-3 knockout (KO) mice also develop osteopenia. However, Dok-3 KO, but not Dok-1/-2 double-KO (DKO), mice develop larger osteoclasts within the normal cell-count range, suggesting a distinctive role for Dok-3. Indeed, Dok-3 KO, but not Dok-1/-2 DKO, bone marrow-derived cells (BMDCs) generated larger osteoclasts with more nuclei due to augmented cell-to-cell fusion in vitro. In addition, while Dok-1/-2 DKO BMDCs generated more osteoclasts, Dok-1/-2/-3 triple-KO (TKO) BMDCs generated osteoclasts increased in both number and size. Furthermore, Dok-1/-2/-3 TKO mice showed the combined effects of Dok-3 and Dok-1/-2 deficiency: severe osteopenia with more and larger osteoclasts. Together, our findings demonstrate that Dok-3 and Dok-1/-2 play distinctive but cooperative roles in osteoclastogenesis and protect mice from osteopenia, providing physiological and pathophysiological insight into bone homeostasis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Enfermedades Óseas Metabólicas/prevención & control , Proteínas de Unión al ADN/fisiología , Osteoclastos/citología , Osteogénesis , Fosfoproteínas/fisiología , Proteínas de Unión al ARN/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células de la Médula Ósea/citología , Recuento de Células , Técnicas de Cultivo de Célula , Fusión Celular , Proliferación Celular , Tamaño de la Célula , Proteínas de Unión al ADN/genética , Ratones , Ratones Noqueados , Fosfoproteínas/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Migration of the cells in osteoblastic lineage, including preosteoblasts and osteoblasts, has been postulated to influence bone formation. However, the molecular bases that link preosteoblastic/osteoblastic cell migration and bone formation are incompletely understood. Nck (noncatalytic region of tyrosine kinase; collectively referred to Nck1 and Nck2) is a member of the signaling adaptors that regulate cell migration and cytoskeletal structures, but its function in cells in the osteoblastic lineage is not known. Therefore, we examined the role of Nck in migration of these cells. Nck is expressed in preosteoblasts/osteoblasts, and its knockdown suppresses migration as well as cell spreading and attachment to substrates. In contrast, Nck1 overexpression enhances spreading and increases migration and attachment. As for signaling, Nck double knockdown suppresses migration toward IGF1 (insulin-like growth factor 1). In these cells, Nck1 binds to IRS-1 (insulin receptor substrate 1) based on immunoprecipitation experiments using anti-Nck and anti-IRS-1 antibodies. In vivo, Nck knockdown suppresses enlargement of the pellet of DiI-labeled preosteoblasts/osteoblasts placed in the calvarial defects. Genetic experiments indicate that conditional double deletion of both Nck1 and Nck2 specifically in osteoblasts causes osteopenia. In these mice, Nck double deficiency suppresses the levels of bone-formation parameters such as bone formation rate in vivo. Interestingly, bone-resorption parameters are not affected. Finally, Nck deficiency suppresses repair of bone injury after bone marrow ablation. These results reveal that Nck regulates preosteoblastic/osteoblastic migration and bone mass.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Huesos/citología , Movimiento Celular , Proteínas Oncogénicas/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Resorción Ósea/metabolismo , Resorción Ósea/patología , Huesos/diagnóstico por imagen , Huesos/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Técnicas de Silenciamiento del Gen , Proteínas Sustrato del Receptor de Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones Noqueados , Proteínas Oncogénicas/deficiencia , Tamaño de los Órganos , Osteoblastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Radiografía , Cráneo/efectos de los fármacos , Cráneo/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
LGR4 is expressed in bone and has been shown to be involved in bone metabolism. Oxidative stress is one of the key issues in pathophysiology of osteoporosis. However, the link between Lgr4 and oxidative stress has not been known. Therefore, effects of hydrogen peroxide on Lgr4 expression in osteoblasts were examined. Hydrogen peroxide treatment suppressed the levels of Lgr4 mRNA expression in an osteoblastic cell line, MC3T3-E1. The suppressive effects were not obvious at 0.1 mM, while 1 mM hydrogen peroxide suppressed Lgr4 expression by more than 50%. Hydrogen peroxide treatment suppressed Lgr4 expression within 12 h and this suppression lasted at least up to 48 h. Hydrogen peroxide suppression of Lgr4 expression was still observed in the presence of a transcription inhibitor but was no longer observed in the presence of a protein synthesis inhibitor. Although Lgr4 expression in osteoblasts is enhanced by BMP2 treatment as reported before, hydrogen peroxide treatment suppressed Lgr4 even in the presence of BMP2. Finally, hydrogen peroxide suppressed Lgr4 expression in primary cultures of osteoblasts similarly to MC3T3-E1 cells. These date indicate that hydrogen peroxide suppresses Lgr4 expression in osteoblastic cells. J. Cell. Physiol. 232: 1761-1766, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Peróxido de Hidrógeno/toxicidad , Osteoblastos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Línea Celular , Citocinas/farmacología , Regulación hacia Abajo/efectos de los fármacos , Ratones , Osteoblastos/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/genética , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
Osteoporosis is one of the most prevalent ageing-associated diseases that are soaring in the modern world. Although various aspects of the disease have been investigated to understand the bases of osteoporosis, the pathophysiological mechanisms underlying bone loss is still incompletely understood. Poldip2 is a molecule that has been shown to be involved in cell migration of vascular cells and angiogenesis. However, expression of Poldip2 and its regulation in bone cells were not known. Therefore, we examined the Poldip2 mRNA expression and the effects of bone regulators on the Poldip2 expression in osteoblasts. We found that Poldip2 mRNA is expressed in osteoblastic MC3T3-E1 cells. As FGF controls osteoblasts and angiogenesis, FGF regulation was investigated in these cells. FGF suppressed the expression of Poldip2 in MC3T3-E1 cells in a time dependent manner. Protein synthesis inhibitor but not transcription inhibitor reduced the FGF effects on Poldip2 gene expression in MC3T3-E1 cells. As for bone-related hormones, dexamethasone was found to enhance the expression of Poldip2 in osteoblastic MC3T3-E1 cells whereas FGF still suppressed such dexamethasone effects. With respect to function, knockdown of Poldip2 by siRNA suppressed the migration of MC3T3-E1 cells. Poldip2 was also expressed in the primary cultures of osteoblast-enriched cells and FGF also suppressed its expression. Finally, Poldip2 was expressed in femoral bone in vivo and its levels were increased in aged mice compared to young adult mice. These data indicate that Poldip2 is expressed in osteoblastic cells and is one of the targets of FGF. J. Cell. Biochem. 118: 1670-1677, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Factores de Crecimiento de Fibroblastos/farmacología , Proteínas Mitocondriales/metabolismo , Proteínas Nucleares/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/genética , Proteínas Nucleares/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Osteoclastogenesis is under the control of posttranscriptional and transcriptional events. However, posttranscriptional regulation of osteoclastogenesis is incompletely understood. CNOT3 is a component of the CCR4 family that regulates mRNA stability, but its function in bone is not known. Here, we show that Cnot3 deficiency by deletion of a single allele induces osteoporosis. Cnot3 deficiency causes an enhancement in bone resorption in association with an elevation in bone formation, resulting in high-turnover type bone loss. At the cellular level, Cnot3 deficiency enhances receptor activator of NF-κB ligand (RANKL) effects on osteoclastogenesis in a cell-autonomous manner. Conversely, Cnot3 deficiency does not affect osteoblasts directly. Cnot3 deficiency does not alter RANKL expression but enhances receptor activator of NF-κB (RANK) mRNA expression in bone in vivo. Cnot3 deficiency promotes RANK mRNA stability about twofold in bone marrow cells of mice. Cnot3 knockdown also increases RANK mRNA expression in the precursor cell line for osteoclasts. Anti-CNOT3 antibody immunoprecipitates RANK mRNA. Cnot3 deficiency stabilizes luciferase reporter expression linked to the 3'-UTR fragment of RANK mRNA. In contrast, Cnot3 overexpression destabilizes the luciferase reporter linked to RANK 3'-UTR. In aged mice that exhibit severe osteoporosis, Cnot3 expression levels in bone are reduced about threefold in vivo. Surprisingly, Cnot3 deficiency in these aged mice further exacerbates osteoporosis, which also occurs via enhancement of osteoclastic activity. Our results reveal that CNOT3 is a critical regulator of bone mass acting on bone resorption through posttranscriptional down-regulation of RANK mRNA stability, at least in part, even in aging-induced osteoporosis.
Asunto(s)
Resorción Ósea/fisiopatología , Regulación de la Expresión Génica/fisiología , Osteoporosis/fisiopatología , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Absorciometría de Fotón , Factores de Edad , Animales , Densidad Ósea , Cartilla de ADN/genética , Imagenología Tridimensional , Luciferasas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estabilidad del ARN/genética , ARN Interferente Pequeño/genética , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Microtomografía por Rayos XRESUMEN
Osteoporosis affects over 20 million patients in the United States. Among those, disuse osteoporosis is serious as it is induced by bed-ridden conditions in patients suffering from aging-associated diseases including cardiovascular, neurological, and malignant neoplastic diseases. Although the phenomenon that loss of mechanical stress such as bed-ridden condition reduces bone mass is clear, molecular bases for the disuse osteoporosis are still incompletely understood. In disuse osteoporosis model, bone loss is interfered by inhibitors of sympathetic tone and adrenergic receptors that suppress bone formation. However, how beta adrenergic stimulation affects osteoblastic migration and associated proliferation is not known. Here we introduced a live imaging system, fluorescent ubiquitination-based cell cycle indicator (FUCCI), in osteoblast biology and examined isoproterenol regulation of cell cycle transition and cell migration in osteoblasts. Isoproterenol treatment suppresses the levels of first entry peak of quiescent osteoblastic cells into cell cycle phase by shifting from G1 /G0 to S/G2 /M and also suppresses the levels of second major peak population that enters into S/G2 /M. The isoproterenol regulation of osteoblastic cell cycle transition is associated with isoproterenol suppression on the velocity of migration. This isoproterenol regulation of migration velocity is cell cycle phase specific as it suppresses migration velocity of osteoblasts in G1 phase but not in G1 /S nor in G2 /M phase. Finally, these observations on isoproterenol regulation of osteoblastic migration and cell cycle transition are opposite to the PTH actions in osteoblasts. In summary, we discovered that sympathetic tone regulates osteoblastic migration in association with cell cycle transition by using FUCCI system.
Asunto(s)
Osteoblastos/citología , Osteoblastos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Puntos de Control del Ciclo Celular , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Isoproterenol/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Osteoblastos/efectos de los fármacos , Hormona Paratiroidea/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de la Célula IndividualRESUMEN
Osteoporosis is one of the most prevalent diseases and the number of patients suffering from this disease is soaring due to the increase in the aged population in the world. The severity of bone loss in osteoporosis is based on the levels of impairment in the balance between bone formation and bone resorption, two arms of the bone metabolism, and bone remodeling. However, determination of bone formation levels is under many layers of control that are as yet fully defined. Bone morphogenetic protein (BMP) plays a key role in regulation of bone formation while its downstream targets are still incompletely understood. Lgr4 gene encodes an orphan receptor and has been identified as a genetic determinant for bone mass in osteoporotic patients. Here, we examine the effects of BMP on the expression of Lgr4 in osteoblastic cells. Lgr4 gene is expressed in an osteoblastic cell line, MC3T3E1 in a time dependent manner during the culture. BMP treatment enhances Lgr4 mRNA expression at least in part via transcriptional event. When Lgr4 mRNA is knocked down, the levels of BMP-induced increase in alkaline phosphatase (Alp) activity and Alp mRNA are suppressed. BMP enhancement of Lgr4 gene expression is suppressed by FGF and reversed by dexamethasone. BMP also enhances Lgr4 expression in primary cultures of calvarial osteoblasts. These data indicate that Lgr4 gene is regulated by BMP and is required for BMP effects on osteoblastic differentiation. J. Cell. Physiol. 231: 887-895, 2016. © 2015 Wiley Periodicals, Inc.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Osteoblastos/metabolismo , Receptores Acoplados a Proteínas G/genética , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Huesos/efectos de los fármacos , Huesos/metabolismo , Línea Celular , Técnicas de Silenciamiento del Gen , Ratones , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
Unloading induces bone loss and causes disuse osteoporosis. However, the mechanism underlying disuse osteoporosis is still incompletely understood. Here, we examined the effects of cathepsin K (CatK) deficiency on disuse osteoporosis induced by using sciatic neurectomy (Nx) model. After 4 weeks of surgery, CatK KO and WT mice were sacrificed and subjected to analyses. For cancellous bone rich region, Nx reduced the bone mineral density (BMD) compared to the BMD in the sham operated side in wild type mice. In contrast, CatK deficiency suppressed such Nx-induced reduction of BMD in cancellous bone. Nx also reduced BMD in the mid shaft cortical bone compared to the BMD in the corresponding region on the sham operated side in wild type mice. In contrast, CatK deficiency suppressed such Nx-induced reduction of BMD in the mid shaft cortical bone. Bone volume (BV/TV) was reduced by Nx in WT mice. In contrast, Cat-K deficiency suppressed such reduction in bone volume. Interestingly, CatK deficiency suppressed osteoclast number and osteoclast surface in the Nx side compared to sham side. When bone marrow cells obtained from Nx side femur of CatK-KO mice were cultured, the levels of the calcified area in culture were increased. Further examination of gene expression indicated that Nx suppressed the expression of genes encoding osteoblast-phenotype-related molecules such as Runx2 and alkaline phosphatase in WT mice. In contrast, CatK deficiency suppressed such reduction. These data indicate that CatK is involved in the disuse-induced bone mass reduction.
Asunto(s)
Resorción Ósea/enzimología , Resorción Ósea/etiología , Catepsina K/deficiencia , Trastornos Musculares Atróficos/complicaciones , Trastornos Musculares Atróficos/enzimología , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Densidad Ósea , Células de la Médula Ósea/metabolismo , Resorción Ósea/diagnóstico por imagen , Resorción Ósea/fisiopatología , Huesos/diagnóstico por imagen , Huesos/enzimología , Huesos/patología , Calcificación Fisiológica/genética , Catepsina K/metabolismo , Células Cultivadas , Imagenología Tridimensional , Ratones Endogámicos C57BL , Trastornos Musculares Atróficos/diagnóstico por imagen , Trastornos Musculares Atróficos/patología , Tamaño de los Órganos , Osteoclastos/patología , Osteogénesis/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Microtomografía por Rayos XRESUMEN
CIZ/NMP4 (Cas interacting zinc finger protein, Nmp4, Zfp384) is a transcription factor that is known to regulate matrix related-proteins. To explore the possible pathophysiological role of CIZ/NMP4 in arthritis, we examined CIZ/NMP4 expression in articular cartilage in arthritis model. CIZ/NMP4 was expressed in the articular chondrocytes of mice at low levels while its expression was enhanced when arthritis was induced. Arthritis induction increased clinical score in wild type mice. In contrast, CIZ/NMP4 deficiency suppressed such rise in the levels of arthritis score and swelling of soft tissue. CIZ/NMP4 deficiency also reduced invasion of inflammatory cells in joint tissue. Quantitative PCR analyses of mRNA from joints revealed that arthritis-induced increase in expressions of IL-1ß was suppressed by CIZ/NMP4 deficiency. CIZ/NMP4 bound to IL-1ß promoter and activated its transcription. The increase in CIZ/NMP4 in arthritis was also associated with enhancement in bone resorption and cartilage matrix degradation. In fact, RANKL, a signaling molecule prerequisite for osteoclastogenesis and, MMP-3, a clinical marker for arthritis were increased in joints upon arthritis induction. In contrast, CIZ/NMP4 deficiency suppressed the arthritis-induced increase in bone resorption, expression of RANKL and MMP-3 mRNA. Thus, CIZ/NMP4 plays a role in the development of arthritis at least in part through regulation of key molecules related to the arthritis.
Asunto(s)
Artritis Experimental/genética , Cartílago Articular/inmunología , Metaloproteinasa 3 de la Matriz/inmunología , Proteínas Asociadas a Matriz Nuclear/inmunología , Ligando RANK/inmunología , Factores de Transcripción/inmunología , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/inmunología , Artritis Experimental/patología , Autoanticuerpos/biosíntesis , Resorción Ósea , Cartílago Articular/patología , Condrocitos/inmunología , Condrocitos/patología , Femenino , Regulación de la Expresión Génica , Glucosa-6-Fosfato Isomerasa/antagonistas & inhibidores , Glucosa-6-Fosfato Isomerasa/genética , Glucosa-6-Fosfato Isomerasa/inmunología , Sueros Inmunes/administración & dosificación , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Articulaciones/inmunología , Articulaciones/patología , Masculino , Metaloproteinasa 3 de la Matriz/genética , Ratones , Ratones Noqueados , Proteínas Asociadas a Matriz Nuclear/deficiencia , Proteínas Asociadas a Matriz Nuclear/genética , Regiones Promotoras Genéticas , Ligando RANK/genética , Índice de Severidad de la Enfermedad , Transducción de Señal , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Profilin 1 (Pfn1) regulates cytoskeletal reorganization and migration, but its role in osteoblasts is not known. BMP (bone morphogenetic protein) is a multifunctional cytokine involved in osteoblastic differentiation and promotes bone regeneration and repair. Although several molecules are known to modulate BMP signaling, mechanisms that determine the levels of BMP action in osteoblastic function are still incompletely understood. We therefore examine the expression of Pfn1 in osteoblasts and its role in BMP-induced differentiation in osteoblasts. In osteoblastic MC3T3-E1(MC) cells, Pfn1 mRNA is expressed constitutively and its expression levels are declined during the culture in a time dependent manner in contrast to the increase in alkaline phosphatase activity revealing that Pfn1 expression is down regulated along with differentiation. To test the effects of osteoblastic differentiation on Pfn1expression further, MC cells are treated with BMP. BMP treatment suppresses the levels of Pfn1 mRNA. This suppressive effect of BMP is time dependent and further down regulation of Pfn1 mRNA levels is observed when the BMP treatment is continued for a longer period of time. Pfn1mRNA knock down (KD) by siRNAs enhances BMP-induced increase in alkaline phosphatase (Alp) activity in MC cells. To analyze the regulatory mechanism, Alp mRNA levels are examined and Pfn1 KD enhances the BMP-induced increase in the levels of Alp mRNA expression. Furthermore, Pfn1 KD enhances BMP-induced transcriptional expression of luciferase reporter activity via BMP response element in osteoblasts. These data indicate that Pfn1 is a novel target of BMP and suppresses BMP-induced differentiation of osteoblasts at least in part via transcriptional event.
Asunto(s)
Osteoblastos/metabolismo , Profilinas/metabolismo , Células 3T3 , Fosfatasa Alcalina/metabolismo , Animales , Proteína Morfogenética Ósea 2/fisiología , Inducción Enzimática , Silenciador del Gen , Ratones , Profilinas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Respuesta , Transcripción GenéticaRESUMEN
Bone formation is precisely regulated by cell-cell communication in osteoblasts. We have previously demonstrated that genetic deletion of Col6a1 or Col12a1 impairs osteoblast connections and/or communication in mice, resulting in bone mass reduction and bone fragility. Mutations of the genes encoding collagen VI cause Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM), which have overlapping phenotypes involving connective tissue and muscle. Recent studies have identified COL12A1 gene mutations in patients with UCMD- and BM-like disorders harboring no COL6 mutations, indicating the shared functions of these collagens in connective tissue homeostasis. The purpose of this investigation has been to test the hypothesis that collagens VI and XII have coordinate regulatory role(s) during bone formation. We analyzed the localization of collagens VI and XII relative to primary osteoblasts during osteogenesis. Immunofluorescence analysis demonstrated that collagens VI and XII colocalized in matrix bridges between adjacent cells during periods when osteoblasts were establishing cell-cell connections. Quantification of cells harboring collagen bridges demonstrated that matrix bridges were composed of collagens VI and XII but not collagen I. Interestingly, matrix bridge formation was impaired in osteoblasts deficient in either Col6a1 or Col12a1, suggesting that both collagens were indispensable for matrix bridge formation. These data demonstrate, for the first time, a functional relationship between collagens VI and XII during osteogenesis and indicate that a complex containing collagens VI and XII is essential for the formation of a communicating cellular network during bone formation.
Asunto(s)
Comunicación Celular , Colágeno Tipo VI/metabolismo , Colágeno Tipo XII/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis , Animales , Células Cultivadas , Colágeno Tipo I/metabolismo , Medios de Cultivo/farmacología , Matriz Extracelular/metabolismo , Ratones , Unión ProteicaRESUMEN
Heterotopic ossification (HO) in various tissues evokes clinical problems. Inflammatory responses of the stromal progenitor cells may be involved in its etiology. Previous report indicated that pro-inflammatory cytokines including IL-1ß enhanced the in vitro calcification of human mesenchymal stem cells (MSCs), by suppressing the expression of ectonucleotide pyrophosphatase/phosphodiesterase-1 gene (ENPP1). However, possible contribution of other related factors had not been investigated. Here, we investigated the expression of regulators of extracellular pyrophosphate and nucleosides including Enpp1, Nt5e, Ank, Enptds, and Ent1, examining various connective tissue stromal progenitor cells, including bone marrow stromal cells and synovium derived cells from mouse, or bone marrow MSCs from human. Consistent with previous studies, we observed characteristic suppression of the osteoblastic marker genes by IL-1ß during the osteogenic culture for 20 days. In addition, we observed a reduced expression of the important transporter genes, Ank and Ent1, whereas the alteration in Enpp1 and Nt5e levels was not always consistent among the cell types. Our results suggest that IL-1ß suppresses not only the osteoblastic but also the negative regulators of soft-tissue calcification, including Ank and Ent1 in stromal progenitor cells, which may contribute to the mechanisms of HO in various disorders.