RESUMO
BACKGROUND: Current preclinical osteosarcoma (OS) models largely focus on quantifying primary tumor burden. However, most fatalities from OS are caused by metastatic disease. The quantification of metastatic OS currently relies on CT, which is limited by motion artifact, requires intravenous contrast, and can be technically demanding in the preclinical setting. We describe the ability for indocyanine green (ICG) fluorescence angiography to quantify primary and metastatic OS in a previously validated orthotopic, immunocompetent mouse model. QUESTIONS/PURPOSES: (1) Can near-infrared ICG fluorescence be used to attach a comparable, quantitative value to the primary OS tumor in our experimental mouse model? (2) Will primary tumor fluorescence differ in mice that go on to develop metastatic lung disease? (3) Does primary tumor fluorescence correlate with tumor volume measured with CT? METHODS: Six groups of 4- to 6-week-old immunocompetent Balb/c mice (n = 6 per group) received paraphyseal injections into their left hindlimb proximal tibia consisting of variable numbers of K7M2 mouse OS cells. A hindlimb transfemoral amputation was performed 4 weeks after injection with euthanasia and lung extraction performed 10 weeks after injection. Histologic examination of lung and primary tumor specimens confirmed ICG localization only within the tumor bed. RESULTS: Mice with visible or palpable tumor growth had greater hindlimb fluorescence (3.5 ± 2.3 arbitrary perfusion units [APU], defined as the fluorescence pixel return normalized by the detector) compared with those with a negative examination (0.71 ± 0.38 APU, -2.7 ± 0.5 mean difference, 95% confidence interval -3.7 to -1.8, p < 0.001). A strong linear trend (r = 0.81, p < 0.01) was observed between primary tumor and lung fluorescence, suggesting that quantitative ICG tumor fluorescence is directly related to eventual metastatic burden. We did not find a correlation (r = 0.04, p = 0.45) between normalized primary tumor fluorescence and CT volumetric measurements. CONCLUSIONS: We demonstrate a novel methodology for quantifying primary and metastatic OS in a previously validated, immunocompetent, orthotopic mouse model. Quantitative fluorescence of the primary tumor with ICG angiography is linearly related to metastatic burden, a relationship that does not exist with respect to clinical tumor size. This highlights the potential utility of ICG near-infrared fluorescence imaging as a valuable preclinical proof-of-concept modality. Future experimental work will use this model to evaluate the efficacy of novel OS small molecule inhibitors. CLINICAL RELEVANCE: Given the histologic localization of ICG to only the tumor bed, we envision the clinical use of ICG angiography as an intraoperative margin and tumor detector. Such a tool may be used as an alternative to intraoperative histology to confirm negative primary tumor margins or as a valuable tool for debulking surgeries in vulnerable anatomic locations. Because we have demonstrated the successful preservation of ICG in frozen tumor samples, future work will focus on blinded validation of this modality in observational human trials, comparing the ICG fluorescence of harvested tissue samples with fresh frozen pathology.
Assuntos
Angiografia/métodos , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/patologia , Corantes Fluorescentes/administração & dosagem , Verde de Indocianina/administração & dosagem , Neoplasias Pulmonares/secundário , Osteossarcoma/diagnóstico por imagem , Osteossarcoma/secundário , Animais , Linhagem Celular Tumoral , Angiografia por Tomografia Computadorizada , Medições Luminescentes , Camundongos Endogâmicos BALB C , Valor Preditivo dos Testes , Carga TumoralRESUMO
Idiopathic pulmonary fibrosis (IPF) is a disease characterized by the accumulation of apoptosis-resistant fibroblasts in the lung. We have previously shown that high expression of the transcription factor Twist1 may explain this prosurvival phenotype in vitro. However, this observation has never been tested in vivo. We found that loss of Twist1 in COL1A2+ cells led to increased fibrosis characterized by very significant accumulation of T cells and bone marrow-derived matrix-producing cells. We found that Twist1-null cells expressed high levels of the T cell chemoattractant CXCL12. In vitro, we found that the loss of Twist1 in IPF lung fibroblasts increased expression of CXCL12 downstream of increased expression of the noncanonical NF-κB transcription factor RelB. Finally, blockade of CXCL12 with AMD3100 attenuated the exaggerated fibrosis observed in Twist1-null mice. Transcriptomic analysis of 134 IPF patients revealed that low expression of Twist1 was characterized by enrichment of T cell pathways. In conclusion, loss of Twist1 in collagen-producing cells led to increased bleomycin-induced pulmonary fibrosis, which is mediated by increased expression of CXCL12. Twist1 expression is associated with dysregulation of T cells in IPF patients. Twist1 may shape the IPF phenotype and regulate inflammation in fibrotic lung injury.
Assuntos
Quimiocina CXCL12/metabolismo , Fibroblastos/fisiologia , Fibrose Pulmonar Idiopática/imunologia , Pulmão/patologia , Células-Tronco Mesenquimais/patologia , Linfócitos T/imunologia , Proteína 1 Relacionada a Twist/metabolismo , Idoso , Animais , Bleomicina , Células Cultivadas , Quimiocina CXCL12/genética , Colágeno Tipo I/metabolismo , Feminino , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , NF-kappa B/metabolismo , RNA Interferente Pequeno/genética , Proteína 1 Relacionada a Twist/genética , Regulação para CimaRESUMO
RATIONALE: Genetic association studies in chronic obstructive pulmonary disease have primarily tested for association with common variants, the results of which explain only a portion of disease heritability. Because rare variation is also likely to contribute to susceptibility, we used whole-genome sequencing of subjects with clinically extreme phenotypes to identify genomic regions enriched for rare variation contributing to chronic obstructive pulmonary disease susceptibility. OBJECTIVES: To identify regions of rare genetic variation contributing to emphysema with severe airflow obstruction. METHODS: We identified heavy smokers that were resistant (n = 65) or susceptible (n = 64) to emphysema with severe airflow obstruction in the Pittsburgh Specialized Center of Clinically Oriented Research cohort. We filtered whole-genome sequencing results to include only rare variants and conducted single variant tests, region-based tests across the genome, gene-based tests, and exome-wide tests. MEASUREMENTS AND MAIN RESULTS: We identified several suggestive associations with emphysema with severe airflow obstruction, including a suggestive association of all rare variation in a region within the gene ZNF816 (19q13.41; P = 4.5 × 10-6), and a suggestive association of nonsynonymous coding rare variation in the gene PTPRO (P = 4.0 × 10-5). Association of rs61754411, a rare nonsynonymous variant in PTPRO, with emphysema and obstruction was demonstrated in all non-Hispanic white individuals in the Pittsburgh Specialized Center of Clinically Oriented Research cohort. We found that cells containing this variant have decreased signaling in cellular pathways necessary for survival and proliferation. CONCLUSIONS: PTPRO is a novel candidate gene in emphysema with severe airflow obstruction, and rs61754411 is a previously unreported rare variant contributing to emphysema susceptibility. Other suggestive candidate genes, such as ZNF816, are of interest for future studies.
Assuntos
Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Enfisema Pulmonar/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/genética , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Osteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined. METHODS: Three human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment. RESULTS: Lung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p <0.05). Lung cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline phosphatase staining. CONCLUSIONS: Lung endothelial HULEC-5a cells are attractants for OS cell migration, proliferation, and survival. The SJSA-1 osteosarcoma cell line demonstrated greater metastatic potential than Saos-2 and U-2 cells. ALDH appears to be involved in the interaction between lung and OS cells, and ALP may be a valuable biomarker for monitoring functional OS changes during metastasis.
Assuntos
Fosfatase Alcalina/genética , Biomarcadores Tumorais/genética , Isoenzimas/genética , Osteossarcoma/genética , Retinal Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Técnicas de Cocultura , Dissulfiram/administração & dosagem , Humanos , Pulmão/citologia , Pulmão/metabolismo , Camundongos , Metástase Neoplásica , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologiaRESUMO
Transcription factor activating protein 2 (AP2) plays an important role in cellular differentiation. Although profound craniofacial and long bone developmental abnormalities have been observed in AP2-knockout mice, the molecular effects of AP2 on osteoblasts are poorly defined. We demonstrated that AP2 regulates the expression of human Frizzled 1 (FZD1), a co-receptor for the Wnt signalling pathway, in human osteoblast cell lines and primary bone marrow stromal cells (BMSCs). We also identified a putative AP2-binding site in the FZD1 proximal promoter in silico and characterized this binding element further in Saos2 in vitro by ChIP, electrophoretic mobility shift and promoter reporter assays. The transcriptional repression of the FZD1 promoter by AP2 was confirmed in normal human fetal osteoblasts (hFOB). Furthermore, overexpression of AP2 resulted in a significant reduction in both differentiation and mineralization of Saos2 cells. Knockdown of FZD1 expression before AP2 up-regulation diminished the AP2-dependent inhibition of Saos2 cell differentiation and mineralization. Similarly, overexpressing FZD1 before AP2 treatment in both Saos2 and BMSCs diminished the inhibitory effect of AP2 on osteoblast differentiation and mineralization. Taken together, these results demonstrate that AP2 is a negative regulator of osteoblast differentiation and mineralization, and its inhibitory effect may be mediated in part through down-regulation of FZD1 expression.
Assuntos
Calcificação Fisiológica/fisiologia , Diferenciação Celular/fisiologia , Regulação para Baixo/fisiologia , Proteínas de Ligação a Ácido Graxo/biossíntese , Receptores Frizzled/biossíntese , Osteoblastos/metabolismo , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Receptores Frizzled/antagonistas & inibidores , HumanosRESUMO
Frizzled homolog 1 (FZD1) is a transmembrane receptor that mediates Wnt signaling. The transcriptional regulation of FZD1 and the role of FZD1 in osteoblast biology are not well understood. We examined the role of E2F1 in FZD1 promoter activation and osteoblast differentiation and mineralization. A putative E2F1 binding site in the FZD1 promoter region was initially identified in silico and characterized further in Saos2 cells in vitro by chromatin immunoprecipitation (ChIP), electrophoretic mobility shift (EMSA) and promoter reporter assays. Over-expression of E2F1 transactivated the FZD1 promoter and increased endogenous FZD1 mRNA and protein levels in Saos2 cells. Over-expression of E2F1 in Saos2 cells up-regulated osteoblast differentiation markers alkaline phosphatase (ALP), type I collagen α (COL1A), and osteocalcin (OCN). Furthermore, E2F1 over-expression enhanced mineralization of differentiated Saos2 cells, whereas siRNA knockdown of FZD1 diminished the effects of E2F1 on osteoblast mineralization. The effects of E2F1 on FZD1 expression and osteoblast mineralization were further confirmed in normal human FOB osteoblasts. Taken together, our experiments demonstrate a role of E2F1 in osteoblast differentiation and mineralization and suggest that FZD1 is required, in part, for E2F1 regulation of osteoblast mineralization.
Assuntos
Diferenciação Celular/fisiologia , Fator de Transcrição E2F1/metabolismo , Receptores Frizzled/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Western Blotting , Diferenciação Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Fator de Transcrição E2F1/genética , Ensaio de Desvio de Mobilidade Eletroforética , Receptores Frizzled/genética , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Bone marrow mesenchymal stem cells (MSCs) can differentiate into multiple cell types including osteoblasts. How this differentiation process is controlled, however, is not completely understood. Here we show that activating transcription factor 4 (ATF4) plays a critical role in promoting bone marrow MSC differentiation towards the osteoblast lineage. Ablation of the Atf4 gene blocked the formation of osteoprogenitors and inhibited osteoblast differentiation without affecting the expansion and formation of MSCs in bone marrow cultures. Loss of ATF4 dramatically reduced the level of ß-catenin protein in MSCs in vitro and in osteoblasts/osteoprogenitors located on trabecular and calvarial surfaces. Loss of ATF4 did not decrease the expression of major canonical Wnt/ß-catenin signaling components such as Wnt3a, Wnt7b, Wnt10b, Lrp5, and Lrp6 in MSCs. Furthermore, shRNA knockdown of ATF4 expression decreased the level of ß-catenin protein in MC-4 preosteoblasts. In contrast, overexpression of ATF4 increased ß-catenin protein levels in MC-4 cells. Finally, ATF4 and ß-catenin formed a protein-protein complex in COS-7 cells coexpressing both factors or in MC-4 preosteoblastic cells. This study establishes a new role of ATF4 in controlling the ß-catenin protein levels and MSC differentiation towards the osteoblast lineage.
Assuntos
Fator 4 Ativador da Transcrição/fisiologia , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , beta Catenina/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Células da Medula Óssea/metabolismo , Células COS , Diferenciação Celular/genética , Linhagem da Célula , Chlorocebus aethiops , Feminino , Técnicas de Silenciamento de Genes , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteoblastos/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
Bone mass is controlled through a delicate balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We show here that RNA editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) is critical for proper control of bone mass. Postnatal conditional knockout of Adar1 (the gene encoding ADAR1) resulted in a severe osteopenic phenotype. Ablation of the Adar1 gene significantly suppressed osteoblast differentiation without affecting osteoclast differentiation in bone. In vitro deletion of the Adar1 gene decreased expression of osteoblast-specific osteocalcin and bone sialoprotein genes, alkaline phosphatase activity, and mineralization, suggesting a direct intrinsic role of ADAR1 in osteoblasts. ADAR1 regulates osteoblast differentiation by, at least in part, modulation of osterix expression, which is essential for bone formation. Further, ablation of the Adar1 gene decreased the proliferation and survival of bone marrow stromal cells and inhibited the differentiation of mesenchymal stem cells towards osteoblast lineage. Finally, shRNA knockdown of the Adar1 gene in MC-4 pre-osteoblasts reduced cyclin D1 and cyclin A1 expression and cell growth. Our results identify ADAR1 as a new key regulator of bone mass and suggest that ADAR1 functions in this process mainly through modulation of the intrinsic properties of osteoblasts (i.e., proliferation, survival and differentiation).
Assuntos
Adenosina Desaminase/metabolismo , Osso e Ossos/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , Adenosina Desaminase/genética , Fosfatase Alcalina/metabolismo , Animais , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/metabolismo , Proliferação de Células , Sobrevivência Celular/genética , Células Cultivadas , Ciclina A1/biossíntese , Ciclina D1/biossíntese , Inativação Gênica , Sialoproteína de Ligação à Integrina/biossíntese , Masculino , Camundongos , Camundongos Transgênicos , Osteocalcina/biossíntese , Osteogênese/genética , Proteínas de Ligação a RNA , Fator de Transcrição Sp7 , Fatores de Transcrição/biossínteseRESUMO
Bone remodeling is a complex process that must be precisely controlled to maintain a healthy life. We show here that filamin-binding LIM protein 1 (FBLP-1, also known as migfilin), a kindlin- and filamin-binding focal adhesion protein, is essential for proper control of bone remodeling. Genetic inactivation of FBLIM1 (the gene encoding FBLP-1) in mice resulted in a severe osteopenic phenotype. Primary FBLP-1 null bone marrow stromal cells (BMSCs) exhibited significantly reduced extracellular matrix adhesion and migration compared with wild type BMSCs. Loss of FBLP-1 significantly impaired the growth and survival of BMSCs in vitro and decreased the number of osteoblast (OB) progenitors in bone marrow and OB differentiation in vivo. Furthermore, the loss of FBLP-1 caused a dramatic increase of osteoclast (OCL) differentiation in vivo. The level of receptor activator of nuclear factor κB ligand (RANKL), a key regulator of OCL differentiation, was markedly increased in FBLP-1 null BMSCs. The capacity of FBLP-1 null bone marrow monocytes (BMMs) to differentiate into multinucleated OCLs in response to exogenously supplied RANKL, however, was not different from that of WT BMMs. Finally, we show that a loss of FBLP-1 promotes activating phosphorylation of ERK1/2. Inhibition of ERK1/2 activation substantially suppressed the increase of RANKL induced by the loss of FBLP-1. Our results identify FBLP-1 as a key regulator of bone homeostasis and suggest that FBLP-1 functions in this process through modulating both the intrinsic properties of OB/BMSCs (i.e., BMSC-extracellular matrix adhesion and migration, cell growth, survival, and differentiation) and the communication between OB/BMSCs and BMMs (i.e., RANKL expression) that controls osteoclastogenesis.
Assuntos
Remodelação Óssea/fisiologia , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Adesão Celular/fisiologia , Moléculas de Adesão Celular/genética , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Osteoblastos/citologia , Osteoclastos/citologia , Fosforilação/fisiologia , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Protracted inhibition of osteoblast (OB) differentiation characterizes multiple myeloma (MM) bone disease and persists even when patients are in long-term remission. However, the underlying pathophysiology for this prolonged OB suppression is unknown. Therefore, we developed a mouse MM model in which the bone marrow stromal cells (BMSCs) remained unresponsive to OB differentiation signals after removal of MM cells. We found that BMSCs from both MM-bearing mice and MM patients had increased levels of the transcriptional repressor Gfi1 compared with controls and that Gfi1 was a novel transcriptional repressor of the critical OB transcription factor Runx2. Trichostatin-A blocked the effects of Gfi1, suggesting that it induces epigenetic changes in the Runx2 promoter. MM-BMSC cell-cell contact was not required for MM cells to increase Gfi1 and repress Runx2 levels in MC-4 before OBs or naive primary BMSCs, and Gfi1 induction was blocked by anti-TNF-α and anti-IL-7 antibodies. Importantly, BMSCs isolated from Gfi1(-/-) mice were significantly resistant to MM-induced OB suppression. Strikingly, siRNA knockdown of Gfi1 in BMSCs from MM patients significantly restored expression of Runx2 and OB differentiation markers. Thus, Gfi1 may have an important role in prolonged MM-induced OB suppression and provide a new therapeutic target for MM bone disease.
Assuntos
Neoplasias Ósseas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mieloma Múltiplo/metabolismo , Osteoblastos/metabolismo , Células Estromais/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Western Blotting , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Expressão Gênica , Humanos , Interleucina-7/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Osteoblastos/patologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/patologia , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In this study, we determined the molecular mechanisms whereby forkhead transcription factor Foxo1, a key downstream signaling molecule of insulin-like growth factor 1 (IGF1)/insulin actions, regulates Runx2 activity and expression of the mouse osteocalcin gene 2 (Bglap2) in osteoblasts in vitro. We showed that Foxo1 inhibited Runx2-dependent transcriptional activity and osteocalcin mRNA expression and Bglap2 promoter activity in MC-4 preosteoblasts. Co-immunoprecipitation assay showed that Foxo1 physically interacted with Runx2 via its C-terminal region in osteoblasts or when co-expressed in COS-7 cells. Electrophoretic mobility shift assay demonstrated that Foxo1 suppressed Runx2 binding to its cognate site within the Bglap2 promoter. IGF1 and insulin prevented Foxo1 from inhibiting Runx2 activity by promoting Foxo1 phosphorylation and nuclear exclusion. In contrast, a neutralizing anti-IGF1 antibody decreased Runx2 activity and osteocalcin expression in osteoblasts. Chromatin immunoprecipitation assay revealed that IGF1 increased Runx2 interaction with a chromatin fragment of the proximal Bglap2 promoter in a PI3K/AKT-dependent manner. Conversely, knockdown of Foxo1 increased Runx2 interaction with the promoter. This study establishes that Foxo1 is a novel negative regulator of osteoblast-specific transcription factor Runx2 and modulates IGF1/insulin-dependent regulation of osteocalcin expression in osteoblasts.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Osteoblastos/metabolismo , Osteocalcina/biossíntese , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Células COS , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Chlorocebus aethiops , Cromatina/genética , Cromatina/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Insulina/genética , Fator de Crescimento Insulin-Like I/genética , Camundongos , Osteoblastos/citologia , Osteocalcina/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Elementos de Resposta/fisiologiaRESUMO
Activating transcription factor 4 (ATF4) is a critical transcription factor for osteoblast (OBL) function and bone formation; however, a direct role in osteoclasts (OCLs) has not been established. Here, we targeted expression of ATF4 to the OCL lineage using the Trap promoter or through deletion of Atf4 in mice. OCL differentiation was drastically decreased in Atf4-/- bone marrow monocyte (BMM) cultures and bones. Coculture of Atf4-/- BMMs with WT OBLs or a high concentration of RANKL failed to restore the OCL differentiation defect. Conversely, Trap-Atf4-tg mice displayed severe osteopenia with dramatically increased osteoclastogenesis and bone resorption. We further showed that ATF4 was an upstream activator of the critical transcription factor Nfatc1 and was critical for RANKL activation of multiple MAPK pathways in OCL progenitors. Furthermore, ATF4 was crucial for M-CSF induction of RANK expression on BMMs, and lack of ATF4 caused a shift in OCL precursors to macrophages. Finally, ATF4 was largely modulated by M-CSF signaling and the PI3K/AKT pathways in BMMs. These results demonstrate that ATF4 plays a direct role in regulating OCL differentiation and suggest that it may be a therapeutic target for treating bone diseases associated with increased OCL activity.
Assuntos
Fator 4 Ativador da Transcrição/fisiologia , Diferenciação Celular , Osteoclastos/citologia , Fator 4 Ativador da Transcrição/genética , Animais , Doenças Ósseas Metabólicas/etiologia , Células da Medula Óssea/patologia , Reabsorção Óssea/etiologia , Fator Estimulador de Colônias de Macrófagos/fisiologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/fisiologia , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Ligante RANK/metabolismoRESUMO
Parathyroid hormone (PTH) is a potent anabolic agent for the treatment of osteoporosis. However, its mechanism of action in osteoblast and bone is not well understood. In this study, we show that the anabolic actions of PTH in bone are severely impaired in both growing and adult ovariectomized mice lacking bone-related activating transcription factor 4 (ATF4). Our study demonstrates that ATF4 deficiency suppresses PTH-stimulated osteoblast proliferation and survival and abolishes PTH-induced osteoblast differentiation, which, together, compromise the anabolic response. We further demonstrate that the PTH-dependent increase in osteoblast differentiation is correlated with ATF4-dependent up-regulation of Osterix. This regulation involves interactions of ATF4 with a specific enhancer sequence in the Osterix promoter. Furthermore, actions of PTH on Osterix require this same element and are associated with increased binding of ATF4 to chromatin. Taken together these experiments establish a fundamental role for ATF4 in the anabolic actions of PTH on the skeleton.
Assuntos
Fator 4 Ativador da Transcrição/fisiologia , Osso e Ossos/metabolismo , Regulação da Expressão Gênica , Hormônio Paratireóideo/metabolismo , Fatores de Transcrição/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Diferenciação Celular , AMP Cíclico/metabolismo , Feminino , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Osteoblastos/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição Sp7 , Regulação para CimaRESUMO
Activating transcription factor 4 (ATF4) is essential for bone formation. However, the mechanism of its actions in bone is poorly understood. The present study examined the role for ATF4 in the regulation of proliferation and survival of primary mouse bone marrow stromal cells (BMSCs) and osteoblasts. Results showed that Atf4(-/-) cells display a severe proliferative defect as measured by multiple cell proliferation assays. Cell cycle progression of Atf4(-/-) BMSCs was largely delayed with significant G1 arrest. Expression of cyclin D1 was decreased both at the mRNA and protein level. A similar proliferation defect was observed in Atf4(-/-) calvarial periosteal osteoblasts when compared with wt control. Knocking down Atf4 mRNA by small interfering RNA in MC3T3-E1 subclone 4 preosteoblasts markedly reduced expression of cyclin D1 and cell proliferation. In contrast, overexpression of ATF4 increased cyclin D1 expression as well as cell proliferation in Atf4(-/-) BMSCs. In addition, apoptosis was significantly increased in Atf4(-/-) BMSCs and calvarial periosteal osteoblasts relative to wt controls. Taken together, these results for the first time demonstrate that ATF4 is a critical regulator of proliferation and survival in BMSCs and osteoblasts in vitro and in vivo.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Células da Medula Óssea/citologia , Proliferação de Células , Osteoblastos/citologia , Fator 4 Ativador da Transcrição/genética , Animais , Apoptose , Células da Medula Óssea/metabolismo , Ciclo Celular , Sobrevivência Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , Crânio/citologia , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
ATF4 (activating transcription factor 4) is an osteoblast-enriched transcription factor that regulates terminal osteoblast differentiation and bone formation. ATF4 knock-out mice have reduced bone mass (severe osteoporosis) throughout life. Runx2 (runt-related transcription factor 2) is a runt domain-containing transcription factor that is essential for bone formation during embryogenesis and postnatal life. In this study, we identified general transcription factor IIA gamma (TFIIA gamma) as a Runx2-interacting factor in a yeast two-hybrid screen. Immunoprecipitation assays confirmed that TFIIA gamma interacts with Runx2 in osteoblasts and when coexpressed in COS-7 cells or using purified glutathione S-transferase fusion proteins. Chromatin immunoprecipitation assay of MC3T3-E1 (clone MC-4) preosteoblast cells showed that in intact cells TFIIA gamma is recruited to the region of the osteocalcin promoter previously shown to bind Runx2 and ATF4. A small region of Runx2 (amino acids 258-286) was found to be required for TFIIA gamma binding. Although TFIIA gamma interacts with Runx2, it does not activate Runx2. Instead, TFIIA gamma binds to and activates ATF4. Furthermore, TFIIA gamma together with ATF4 and Runx2 stimulates osteocalcin promoter activity and endogenous mRNA expression. Small interfering RNA silencing of TFIIA gamma markedly reduces levels of endogenous ATF4 protein and Ocn mRNA in osteoblastic cells. Overexpression of TFIIA gamma increases levels of ATF4 protein. Finally, TFIIA gamma significantly prevents ATF4 degradation. This study shows that a general transcription factor, TFIIA gamma, facilitates osteoblast-specific gene expression through interactions with two important bone transcription factors ATF4 and Runx2.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteocalcina/biossíntese , Fator de Transcrição TFIIA/metabolismo , Células 3T3-L1 , Fator 4 Ativador da Transcrição/genética , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Knockout , Osteoblastos , Osteocalcina/genética , Regiões Promotoras Genéticas/fisiologia , Ligação Proteica/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Fator de Transcrição TFIIA/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
PTH is an important peptide hormone regulator of calcium homeostasis and osteoblast function. However, its mechanism of action in osteoblasts is poorly understood. Our previous study demonstrated that PTH activates mouse osteocalcin (Ocn) gene 2 promoter through the osteoblast-specific element 1 site, a recently identified activating transcription factor-4 (ATF4) -binding element. In the present study, we examined effects of PTH on ATF4 expression and activity as well as the requirement for ATF4 in the regulation of Ocn by PTH. Results show that PTH elevated levels of ATF4 mRNA and protein in a dose- and time-dependent manner. This PTH regulation requires transcriptional activity but not de novo protein synthesis. PTH also increased binding of nuclear extracts to osteoblast-specific element 1 DNA. PTH stimulated ATF4-dependent transcriptional activity mainly through protein kinase A with a lesser requirement for protein kinase C and MAPK/ERK pathways. Lastly, PTH stimulation of Ocn expression was lost by small interfering RNA down-regulation of ATF4 in MC-4 cells and Atf4(-/-) bone marrow stromal cells. Collectively, these studies for the first time demonstrate that PTH increases ATF4 expression and activity and that ATF4 is required for PTH induction of Ocn expression in osteoblasts.
Assuntos
Fator 4 Ativador da Transcrição/genética , Osteoblastos/metabolismo , Osteocalcina/genética , Hormônio Paratireóideo/farmacologia , Células 3T3 , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Transdução de Sinais , Transcrição GênicaRESUMO
While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation.