RESUMEN

Bone remodeling is sophisticatedly regulated by two different cell types: bone-resorbing osteoclasts and bone-forming osteoblasts. Hochu-Ekki-To, a Japanese traditional herbal medicine, is commonly used for the treatment of chronic diseases or frailty after an illness; however, its effects on metabolic bone diseases such as osteoporosis are not well known. We herein report that daily oral Hochu-Ekki-To administration significantly inhibits osteoclast activation as well as the reduction in bone volume in ovariectomized mice. Our results suggest that supplementation with Hochu-Ekki-To might be beneficial for the prophylaxis and treatment of metabolic bone diseases associated with abnormal osteoclast activation.

Asunto(s)

Conservadores de la Densidad Ósea , Resorción Ósea/etiología , Resorción Ósea/prevención & control , Medicamentos Herbarios Chinos/administración & dosificación , Medicamentos Herbarios Chinos/farmacología , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Osteoporosis Posmenopáusica/etiología , Osteoporosis Posmenopáusica/prevención & control , Ovariectomía/efectos adversos , Fitoterapia , Administración Oral , Animales , Femenino , Humanos , Ratones Endogámicos

RESUMEN

The mechanistic/mammalian target of rapamycin (mTOR) is widely implicated in the pathogenesis of various diseases, including cancer, obesity, and cardiovascular disease. Bone homeostasis is maintained by the actions of bone-resorbing osteoclasts and bone-forming osteoblasts. An imbalance in the sophisticated regulation of osteoclasts and osteoblasts leads to the pathogenesis as well as etiology of certain metabolic bone diseases, including osteoporosis and osteopetrosis. Here, we identified mTOR complex 1 (mTORC1) as a pivotal mediator in the regulation of bone resorption and bone homeostasis under pathological conditions through its expression in osteoclasts. The activity of mTORC1, which was indicated by the phosphorylation level of its downstream target p70S6 kinase, was reduced during osteoclast differentiation, in accordance with the upregulation of Hamartin (encoded by tuberous sclerosis complex 1 [Tsc1]), a negative regulator of mTORC1. Receptor activator of nuclear factor-κB ligand (RANKL)-dependent osteoclastogenesis was impaired in Tsc1-deficient bone marrow macrophages. By contrast, osteoclastogenesis was markedly enhanced by Raptor deficiency but was unaffected by Rictor deficiency. The deletion of Tsc1 in osteoclast lineage cells in mice prevented bone resorption and bone loss in a RANKL-induced mouse model of osteoporosis, although neither bone volume nor osteoclastic parameter was markedly altered in these knockout mice under physiological conditions. Therefore, these findings suggest that mTORC1 is a key potential target for the treatment of bone diseases.

RESUMEN

An imbalance in the sophisticated regulation between bone-resorbing osteoclasts and bone-forming osteoblasts leads to the pathogenesis and etiology of certain metabolic bone diseases including osteoporosis. Certain polyamines are related to the pathophysiology of some disorders, including Alzheimer's disease, infectious disease, cancer, and aging. Recently, we demonstrated that oral intake of polyamines (spermidine and spermine) prevented bone loss through preferential disturbance of osteoclastic activation in ovariectomy-induced mouse model of postmenopausal osteoporosis. Here, we showed that daily oral supplementation of a diet containing polyamine-rich Saccharomyces cerevisiae S631 significantly inhibited osteoclastic activation as well as reduction of bone volume in the cancellous bone without affecting uterine weight in ovariectomized mice. Our findings recommend that daily oral supplementation with polyamine-rich yeast diet would be beneficial for prophylaxis of metabolic bone diseases associated with abnormal osteoclast activation.

RESUMEN

L-type amino acid transporter 1 (LAT1), which is encoded by solute carrier transporter 7a5 (Slc7a5), plays a crucial role in amino acid sensing and signaling in specific cell types, contributing to the pathogenesis of cancer and neurological disorders. Amino acid substrates of LAT1 have a beneficial effect on bone health directly and indirectly, suggesting a potential role for LAT1 in bone homeostasis. Here, we identified LAT1 in osteoclasts as important for bone homeostasis. Slc7a5 expression was substantially reduced in osteoclasts in a mouse model of ovariectomy-induced osteoporosis. The osteoclast-specific deletion of Slc7a5 in mice led to osteoclast activation and bone loss in vivo, and Slc7a5 deficiency increased osteoclastogenesis in vitro. Loss of Slc7a5 impaired activation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway in osteoclasts, whereas genetic activation of mTORC1 corrected the enhanced osteoclastogenesis and bone loss in Slc7a5-deficient mice. Last, Slc7a5 deficiency increased the expression of nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1) and the nuclear accumulation of NFATc1, a master regulator of osteoclast function, possibly through the canonical nuclear factor κB pathway and the Akt-glycogen synthase kinase 3ß signaling axis, respectively. These findings suggest that the LAT1-mTORC1 axis plays a pivotal role in bone resorption and bone homeostasis by modulating NFATc1 in osteoclasts, thereby providing a molecular connection between amino acid intake and skeletal integrity.

Asunto(s)

Sistema de Transporte de Aminoácidos y+L/genética , Huesos/metabolismo , Homeostasis/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Osteoclastos/metabolismo , Osteogénesis/genética , Sistema de Transporte de Aminoácidos y+L/deficiencia , Animales , Resorción Ósea/genética , Resorción Ósea/metabolismo , Huesos/citología , Células Cultivadas , Femenino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , FN-kappa B/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos/citología , Ovariectomía , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/genética

RESUMEN

The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) regulates cellular function in various cell types. Although the role of mTORC1 in skeletogenesis has been investigated previously, here we show a critical role of mTORC1/4E-BPs/SOX9 axis in regulating skeletogenesis through its expression in undifferentiated mesenchymal cells. Inactivation of Raptor, a component of mTORC1, in limb buds before mesenchymal condensations resulted in a marked loss of both cartilage and bone. Mechanistically, we demonstrated that mTORC1 selectively controls the RNA translation of Sox9, which harbors a 5' terminal oligopyrimidine tract motif, via inhibition of the 4E-BPs. Indeed, introduction of Sox9 or a knockdown of 4E-BP1/2 in undifferentiated mesenchymal cells markedly rescued the deficiency of the condensation observed in Raptor-deficient mice. Furthermore, introduction of the Sox9 transgene rescued phenotypes of deficient skeletal growth in Raptor-deficient mice. These findings highlight a critical role of mTORC1 in mammalian skeletogenesis, at least in part, through translational control of Sox9 RNA.

Asunto(s)

Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Osteogénesis/genética , Biosíntesis de Proteínas , Factor de Transcripción SOX9/genética , Esqueleto/metabolismo , Animales , Diferenciación Celular/genética , Expresión Génica , Ratones , Ratones Transgénicos , Fenotipo , Factor de Transcripción SOX9/metabolismo , Esqueleto/embriología

RESUMEN

Erk5 belongs to the mitogen-activated protein kinase (MAPK) family. Following its phosphorylation by Mek5, Erk5 modulates several signaling pathways in a number of cell types. In this study, we demonstrated that Erk5 inactivation in mesenchymal cells causes abnormalities in skeletal development by inducing Sox9, an important transcription factor of skeletogenesis. We further demonstrate that Erk5 directly phosphorylates and activates Smurf2 (a ubiquitin E3 ligase) at Thr249, which promotes the proteasomal degradation of Smad proteins and phosphorylates Smad1 at Ser206 in the linker region known to trigger its proteasomal degradation by Smurf1. Smads transcriptionally activated the expression of Sox9 in mesenchymal cells. Accordingly, removal of one Sox9 allele in mesenchymal cells from Erk5-deficient mice rescued some abnormalities of skeletogenesis. These findings highlight the importance of the Mek5-Erk5-Smurf-Smad-Sox9 axis in mammalian skeletogenesis.

Asunto(s)

Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Osteogénesis , Factor de Transcripción SOX9/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Diferenciación Celular , Condrogénesis , Humanos , Mesodermo/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteolisis , Cráneo/anomalías , Ubiquitina/metabolismo , Ubiquitinación

RESUMEN

The availability of amino acid in the brown adipose tissue (BAT) has been shown to be altered under various conditions; however, little is known about the possible expression and pivotal role of amino acid transporters in BAT under physiological and pathological conditions. The present study comprehensively investigated whether amino acid transporters are regulated by obesogenic conditions in BAT in vivo. Moreover, we investigated the mechanism underlying the regulation of the expression of amino acid transporters by various stressors in brown adipocytes in vitro. The expression of solute carrier family 38 member 1 (Slc38a1; gene encoding sodium-coupled neutral amino acid transporter 1) was preferentially upregulated in the BAT of both genetic and acquired obesity mice in vivo. Moreover, the expression of Slc38a1 was induced by hypoxic stress through hypoxia-inducible factor-1α, which is a master transcription factor of the adaptive response to hypoxic stress, in brown adipocytes in vitro. These results indicate that Slc38a1 is an obesity-associated gene in BAT and a hypoxia-responsive gene in brown adipocytes.

Asunto(s)

Adipocitos Marrones/metabolismo , Sistema de Transporte de Aminoácidos A/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Hipoxia/genética , Obesidad/genética , Animales , Línea Celular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , ARN Interferente Pequeño/genética

RESUMEN

ß-Cryptoxanthin, which is primarily obtained from citrus fruits such as Satsuma mandarins, is a major carotenoid routinely found in human serum. Recently, we demonstrated that daily oral intake of ß-cryptoxanthin prevented ovariectomy-induced bone loss and ameliorated neuropathic pain in mice. Although ß-cryptoxanthin exerts preventive effects on various lifestyle-related diseases, there have been no studies on the effect of ß-cryptoxanthin on the development of osteoarthritis, the most common degenerative joint disease, which frequently leads to loss of ability and stiffness in the elderly. Here we showed that daily oral administration of ß-cryptoxanthin significantly prevented the development of osteoarthritis developed by surgically inducing knee joint instability in mice in vivo. Furthermore, in vitro experiments revealed that ß-cryptoxanthin markedly inhibited the expression of inflammatory cytokines and enzymes critical for the degradation of the extracellular matrix in primary chondrocytes. Our results suggest that oral supplementation of ß-cryptoxanthin would be beneficial for the maintenance of joint health and as prophylaxis against osteoarthritis.

Asunto(s)

beta-Criptoxantina/uso terapéutico , Osteoartritis/prevención & control , Animales , beta-Criptoxantina/administración & dosificación , Condrocitos/metabolismo , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Ratones , Osteoartritis/tratamiento farmacológico

RESUMEN

Sympathetic tone activates the function of classical brown adipocytes, which constitutively exist in the brown adipose tissue (BAT), and inducible brown adipocytes (so-called beige adipocytes), which sporadically reside within the white adipose tissue (WAT). Here we identified the transcriptional modulator interferon-related developmental regulator 1 (Ifrd1) as a negative regulator of thermogenic and mitochondrial gene expression in brown adipocytes. Ifrd1 expression was markedly induced by cold exposure and administration of CL-316243 (a ß3 adrenergic agonist) in interscapular brown adipose and inguinal subcutaneous WATs, but not in epididymal visceral WAT, in vivo. Adrenergic stimulation also induced Ifrd1 expression in brown adipocytes in a cAMP responsive element binding protein-dependent manner in vitro. CL-316243 injection markedly elevated thermogenic and mitochondrial gene expression, including peroxisome proliferator-activated receptor γ coactivator 1α (Pgc1a) in the subcutaneous WAT of Ifrd1 knockout mice compared with gene expression in wild-type mice. Pgc1a promoter activity enhanced by the transcription factor specificity protein 1 (Sp1) was markedly repressed by co-introduction of Ifrd1 in brown adipocytes, whereas the repression was markedly prevented by the addition of trichostatin A, a histone deacetylase inhibitor. Moreover, adrenergic stimulation induced complex formation between Ifrd1, Sp1 and mSIN3B, which is a component of the SIN complex containing histone deacetylase, in brown adipocytes. These findings, therefore, suggest that Ifrd1 could be a pivotal negative regulator of sympathetic regulation of thermogenic and mitochondrial gene expression in brown adipocytes by interacting with Sp1 and the mSIN3 complex.

Asunto(s)

Adipocitos Marrones/metabolismo , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas de la Membrana/biosíntesis , Proteínas Represoras/metabolismo , Factor de Transcripción Sp1/metabolismo , Termogénesis/genética , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Agonistas Adrenérgicos/administración & dosificación , Animales , Frío , Dioxoles/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Proteínas Represoras/genética , Factor de Transcripción Sp1/genética

RESUMEN

ß-cryptoxanthin, a xanthophyll carotenoid, exerts preventive effects on various lifestyle-related diseases. Here, we found that daily oral administration of ß-cryptoxanthin significantly ameliorated the development of tactile allodynia following spinal nerve injury but was ineffective in mechanical allodynia in an inflammatory pain model in mice. Our results suggest that ß-cryptoxanthin supplementation would be beneficial for the prophylaxis of neuropathic pain.

Asunto(s)

beta-Criptoxantina/administración & dosificación , beta-Criptoxantina/farmacología , Neuralgia/tratamiento farmacológico , Administración Oral , Animales , beta-Criptoxantina/uso terapéutico , Suplementos Dietéticos , Masculino , Ratones

RESUMEN

We previously demonstrated that the transcriptional coactivator/repressor interferon-related developmental regulator 1 (Ifrd1) was expressed in osteoblasts and participated in the regulation of bone homeostasis. However, it remains unclear how Ifrd1 expression itself is regulated in osteoblasts. In the present study, we investigated the upstream regulatory mechanisms of Ifrd1 in osteoblasts during osteoblastogenesis. Ifrd1 protein expression and runt-related transcription factor 2, the master regulator of osteoblastogenesis, were markedly upregulated by bone morphogenetic protein 2 (BMP-2) stimulation in primary osteoblasts. Moreover, BMP-2 stimulation significantly induced Ifrd1 mRNA expression and promoter activity in osteoblasts. LDN193189, an inhibitor of activin-like kinase 2/3, almost completely inhibited the BMP-2-induced increase in Ifrd1 protein expression. There were at least two putative Smad-binding elements in the 5'-flanking region, which was highly conserved between mouse and human Ifrd1 genes. Co-introduction of both Smad4 and Smad1 significantly increased Ifrd1 promoter activity in osteoblasts. In addition, BMP-2 induced the recruitment of Smad1 to the Ifrd1 promoter in osteoblasts. Moreover, BMP-2-dependent osteoblastogenesis was further enhanced in Ifrd1 knocked-down osteoblasts, as determined by the intensity of Alizarin red stain and marker gene expression. These results suggest that BMP-2 directly induces Ifrd1 expression at the transcriptional level in osteoblasts via the Smad pathway, and Ifrd1 negatively regulates BMP-2-dependent osteoblastogenesis.

Asunto(s)

Proteína Morfogenética Ósea 2/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de la Membrana/metabolismo , Osteoblastos/citología , Osteoblastos/fisiología , Osteogénesis/fisiología , Activación Transcripcional/fisiología , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Regulación hacia Abajo/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Ratones

RESUMEN

We have previously shown that endochondral ossification is finely regulated by the Clock system expressed in chondrocytes during postnatal skeletogenesis. Here we show a sophisticated modulation of bone resorption and bone mass by the Clock system through its expression in bone-forming osteoblasts. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and Period1 (Per1) were expressed with oscillatory rhythmicity in the bone in vivo, and circadian rhythm was also observed in cultured osteoblasts of Per1::luciferase transgenic mice. Global deletion of murine Bmal1, a core component of the Clock system, led to a low bone mass, associated with increased bone resorption. This phenotype was recapitulated by the deletion of Bmal1 in osteoblasts alone. Co-culture experiments revealed that Bmal1-deficient osteoblasts have a higher ability to support osteoclastogenesis. Moreover, 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ]-induced receptor activator of nuclear factor κB ligand (Rankl) expression was more strongly enhanced in both Bmal1-deficient bone and cultured osteoblasts, whereas overexpression of Bmal1/Clock conversely inhibited it in osteoblasts. These results suggest that bone resorption and bone mass are regulated at a sophisticated level by osteoblastic Clock system through a mechanism relevant to the modulation of 1,25(OH)2 D3 -induced Rankl expression in osteoblasts. © 2017 American Society for Bone and Mineral Research.

Asunto(s)

Factores de Transcripción ARNTL/metabolismo , Resorción Ósea/metabolismo , Proteínas CLOCK/metabolismo , Osteoblastos/metabolismo , Proteínas Circadianas Period/metabolismo , Ligando RANK/metabolismo , Factores de Transcripción ARNTL/genética , Animales , Resorción Ósea/genética , Proteínas CLOCK/genética , Células Cultivadas , Ratones , Ratones Noqueados , Proteínas Circadianas Period/genética , Ligando RANK/genética

RESUMEN

Bone homeostasis is maintained by the sophisticated coupled actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Here we identify activating transcription factor 3 (ATF3) as a pivotal transcription factor for the regulation of bone resorption and bone remodeling under a pathological condition through modulating the proliferation of osteoclast precursors. The osteoclast precursor-specific deletion of ATF3 in mice led to the prevention of receptor activator of nuclear factor-κB (RANK) ligand (RANKL)-induced bone resorption and bone loss, although neither bone volume nor osteoclastic parameter were markedly altered in these knockout mice under the physiological condition. RANKL-dependent osteoclastogenesis was impaired in vitro in ATF3-deleted bone marrow macrophages (BMM). Mechanistically, the deficiency of ATF3 impaired the RANKL-induced transient increase in cell proliferation of osteoclast precursors in bone marrow in vivo as well as of BMM in vitro. Moreover, ATF3 regulated cyclin D1 mRNA expression though modulating activator protein-1-dependent transcription in the osteoclast precursor, and the introduction of cyclin D1 significantly rescued the impairment of osteoclastogenesis in ATF3-deleted BMM. Therefore, these findings suggest that ATF3 could have a pivotal role in osteoclastogenesis and bone homeostasis though modulating cell proliferation under pathological conditions, thereby providing a target for bone diseases.

Asunto(s)

Factor de Transcripción Activador 3/fisiología , Remodelación Ósea , Resorción Ósea/prevención & control , Osteoclastos/citología , Ligando RANK/efectos adversos , Animales , Células de la Médula Ósea/metabolismo , Resorción Ósea/etiología , Resorción Ósea/metabolismo , Resorción Ósea/patología , Diferenciación Celular , Proliferación Celular , Regulación de la Expresión Génica , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoclastos/metabolismo

RESUMEN

Bone homeostasis is maintained by the synergistic actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Here, we show that the transcriptional coactivator/repressor interferon-related developmental regulator 1 (Ifrd1) is expressed in osteoclast lineages and represents a component of the machinery that regulates bone homeostasis. Ifrd1 expression was transcriptionally regulated in preosteoclasts by receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) through activator protein 1. Global deletion of murine Ifrd1 increased bone formation and decreased bone resorption, leading to a higher bone mass. Deletion of Ifrd1 in osteoclast precursors prevented RANKL-induced bone loss, although no bone loss was observed under normal physiological conditions. RANKL-dependent osteoclastogenesis was impaired in vitro in Ifrd1-deleted bone marrow macrophages (BMMs). Ifrd1 deficiency increased the acetylation of p65 at residues K122 and K123 via the inhibition of histone deacetylase-dependent deacetylation in BMMs. This repressed the NF-κB-dependent transcription of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), an essential regulator of osteoclastogenesis. These findings suggest that an Ifrd1/NF-κB/NFATc1 axis plays a pivotal role in bone remodeling in vivo and represents a therapeutic target for bone diseases.

Asunto(s)

Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Osteoclastos/citología , Ligando RANK/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Eliminación de Gen , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/metabolismo , Regulación hacia Arriba

RESUMEN

Activating transcription factor 3 (Atf3) has been implicated in the pathogenesis of various diseases, including cancer and inflammation, as well as in the regulation of cell proliferation and differentiation. However, the involvement of Atf3 in developmental skeletogenesis and joint disease has not been well studied to date. Here, we show that Atf3 is a critical mediator of osteoarthritis (OA) development through its expression in chondrocytes. ATF3 expression was markedly up-regulated in the OA cartilage of both mice and humans. Conditional deletion of Atf3 in chondrocytes did not result in skeletal abnormalities or affect the chondrogenesis, but alleviated the development of OA generated by surgically inducing knee joint instability in mice. Inflammatory cytokines significantly up-regulated Atf3 expression through the nuclear factor-kB (NF-kB) pathway, while cytokine-induced interleukin-6 (Il6) expression was repressed, in ATF3-deleted murine and human chondrocytes. Mechanistically, Atf3 deficiency decreased cytokine-induced Il6 transcription in chondrocytes through repressing NF-kB signalling by the attenuation of the phosphorylation status of IkB and p65. These findings suggest that Atf3 is implicated in the pathogenesis of OA through modulation of inflammatory cytokine expression in chondrocytes, and the feed-forward loop of inflammatory cytokines/NF-kB/Atf3 in chondrocytes may be a novel therapeutic target for the treatment for OA. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Asunto(s)

Factor de Transcripción Activador 3/genética , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Osteoartritis/metabolismo , Factor de Transcripción Activador 3/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Cartílago Articular/efectos de los fármacos , Cartílago Articular/patología , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/patología , Femenino , Humanos , Interleucina-1beta/farmacología , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Transgénicos , Osteoartritis/genética , Osteoartritis/patología , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

We previously demonstrated a marked upregulation in the bone morphogenic protein (BMP)/growth differentiation factor (GDF) family member, GDF5, which is capable of promoting brown adipogenesis, in brown adipose tissue (BAT) of obese mice. In this study, we identified other GDF family members, besides GDF5 that are responsive to different obesogenic signals in BAT using inborn and acquired obesity animal models. In BAT from leptin-deficient ob/ob mice, GDF1 expression was preferentially downregulated, whereas the expression of several other genes in the BMP/GDF family, including GDF5, was upregulated. Moreover, in cultured brown adipocytes exposed to tunicamycin and hydrogen peroxide, at concentrations not affecting cellular viability, GDF1 expression was significantly downregulated. Recombinant GDF1 failed to significantly alter brown adipogenesis, despite the promoted phosphorylation of Smad1/5/8 in cultured brown adipocytes, but accelerated Smad1/5/8 phosphorylation with a concomitant increase in the number of migrating cells during exposure in a manner sensitive to activin-like kinase inhibitors in macrophagic RAW264.7 cells. Similarly, accelerated migration was observed in murine peritoneal macrophages exposed to GDF1. These results indicate that obesity could lead to predominant downregulation of GDF1 expression in BAT, which can modulate cellular migration through a mechanism relevant to activation of the downstream Smad signaling pathway in adjacent macrophages.

RESUMEN

Bone homeostasis is maintained by the synergistic actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Although interferon-related developmental regulator 1 (Ifrd1) has been identified as a transcriptional coactivator/repressor in various cells, little attention has been paid to its role in osteoblastogenesis and bone homeostasis thus far. Here, we show that Ifrd1 is a critical mediator of both the cell-autonomous regulation of osteoblastogenesis and osteoblast-dependent regulation of osteoclastogenesis. Osteoblast-specific deletion of murine Ifrd1 increased bone formation and decreased bone resorption, causing high bone mass. Ifrd1 deficiency enhanced osteoblast differentiation and maturation along with increased expression of Runx2 and osterix (Osx). Mechanistically, Ifrd1 deficiency increased the acetylation status of p65, a component of NF-κB, at residues K122 and K123 via the attenuation of the interaction between p65 and histone deacetylase (HDAC). This led to the nuclear export of p65 and a decrease in NF-κB-dependent Smad7 expression and the subsequent enhancement of Smad1/Smad5/Smad8-dependent transcription. Moreover, a high bone mass phenotype in the osteoblast-specific deletion of Ifrd1 was markedly rescued by the introduction of one Osx-floxed allele but not of Runx2-floxed allele. Coculture experiments revealed that Ifrd1-deficient osteoblasts have a higher osteoprotegerin (OPG) expression and a lower ability to support osteoclastogenesis. Ifrd1 deficiency attenuated the interaction between ß-catenin and HDAC, subsequently increasing the acetylation of ß-catenin at K49, leading to its nuclear accumulation and the activation of the ß-catenin-dependent transcription of OPG. Collectively, the expression of Ifrd1 in osteoblasts repressed osteoblastogenesis and activated osteoclastogenesis through modulating the NF-κB/Smad/Osx and ß-catenin/OPG pathways, respectively. These findings suggest that Ifrd1 has a pivotal role in bone homeostasis through its expression in osteoblasts in vivo and represents a therapeutic target for bone diseases.

Asunto(s)

Resorción Ósea/metabolismo , Resorción Ósea/patología , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de la Membrana/metabolismo , Osteoblastos/metabolismo , Osteogénesis , Transcripción Genética , Acetilación , Animales , Huesos/patología , Diferenciación Celular , Núcleo Celular/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Eliminación de Gen , Histona Desacetilasas/metabolismo , Homeostasis , Proteínas Inmediatas-Precoces/deficiencia , Proteínas de la Membrana/deficiencia , Ratones Noqueados , FN-kappa B/metabolismo , Tamaño de los Órganos , Osteoblastos/patología , Osteoprotegerina/metabolismo , Fenotipo , Proteínas Smad/metabolismo , Factor de Transcripción Sp7 , Factores de Transcripción/metabolismo , beta Catenina/metabolismo

RESUMEN

Although ß-cryptoxanthin, a xanthophyll carotenoid, has been shown to exert an anabolic effect on bone calcification, little attention has been paid thus far to the precise mechanism of bone remodeling. Daily oral administration of ß-cryptoxanthin significantly inhibited osteoclastic activation as well as reduction of bone volume in ovariectomized mice. In vitro studies revealed that ß-cryptoxanthin inhibited differentiation and maturation of osteoclasts by repression of the nuclear factor-κB-dependent transcriptional pathway. Our results suggest that supplementation with ß-cryptoxanthin would be beneficial for prophylaxis and for therapy of metabolic bone diseases associated with abnormal osteoclast activation.

Asunto(s)

Remodelación Ósea/efectos de los fármacos , Resorción Ósea/prevención & control , Diferenciación Celular/efectos de los fármacos , Criptoxantinas/administración & dosificación , Criptoxantinas/farmacología , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Ovariectomía , Ingesta Diaria Recomendada , Administración Oral , Animales , Enfermedades Óseas Metabólicas/tratamiento farmacológico , Enfermedades Óseas Metabólicas/etiología , Enfermedades Óseas Metabólicas/prevención & control , Citrus , Humanos , Ratones , FN-kappa B/fisiología , Transducción de Señal/efectos de los fármacos

RESUMEN

We have previously demonstrated that genetic and acquired obesity similarly led to drastic upregulation in brown adipose tissue (BAT), rather than white adipose tissue, of expression of both mRNA and corresponding protein for the bone morphogenic protein/growth differentiation factor (GDF) member GDF5 capable of promoting brown adipogenesis. In this study, we evaluated expression profiles of GDF5 in cultured murine brown pre-adipocytes exposed to pro-inflammatory cytokines and free fatty acids (FFAs), which are all shown to play a role in the pathogenesis of obesity. Both interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were effective in up-regulating GDF5 expression in a concentration-dependent manner, while similar upregulation was seen in cells exposed to the saturated FFA palmitate, but not to the unsaturated FFA oleate. In silico analysis revealed existence of the putative nuclear factor-κB (NF-κB) binding site in the 5'-flanking region of mouse GDF5, whereas introduction of NF-κB subunits drastically facilitated both promoter activity and expression of GDF5 in brown pre-adipocytes. Chromatin immunoprecipitation analysis confirmed significant facilitation of the recruitment of NF-κB to the GDF5 promoter in lysed extracts of BAT from leptin-deficient ob/ob obese mice. Upregulation o GDF5 expression was invariably inhibited by an NF-κB inhibitor in cultured brown pre-adipocytes exposed to IL-1ß, TNF-α and palmitate. These results suggest that obesity leads to upregulation of GDF5 expression responsible for the promotion of brown adipogenesis through a mechanism relevant to activation of the NF-κB pathway in response to particular pro-inflammatory cytokines and/or saturated FFAs in BAT.

Asunto(s)

Adipocitos Marrones/metabolismo , Citocinas , Factor 5 de Diferenciación de Crecimiento/metabolismo , Inflamación/metabolismo , FN-kappa B/metabolismo , Obesidad/metabolismo , Ácido Palmítico , Adipocitos Marrones/efectos de los fármacos , Animales , Células Cultivadas , Inflamación/inducido químicamente , Inflamación/complicaciones , Ratones , Ratones Endogámicos C57BL , Obesidad/complicaciones

RESUMEN

We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylation of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5(Rgsc451) mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway.

Asunto(s)

Adipogénesis/fisiología , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/fisiología , Factor 5 de Diferenciación de Crecimiento/metabolismo , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Smad/metabolismo , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Ratones , Ratones Transgénicos , Transducción de Señal/fisiología