RESUMO
Runx2 (runt related transcription factor 2) is an essential transcription factor for osteoblast proliferation and differentiation. Uridine diphosphate (UDP)-N-acetylgalactosamine (GalNAc): polypeptide GalNAc-transferase 3 (Galnt3) prevents proteolytic processing of fibroblast growth factor 23 (Fgf23), which is a hormone that regulates the serum level of phosphorus. Runx2 and Galnt3 were expressed in osteoblasts and osteocytes, and Fgf23 expression was restricted to osteocytes in bone. Overexpression and knock-down of Runx2 upregulated and downregulated, respectively, the expressions of Galnt3 and Fgf23, and Runx2 directly regulated the transcriptional activity of Galnt3 in reporter assays. The expressions of Galnt3 and Fgf23 in osteoblast-specific Runx2 knockout (Runx2fl/flCre) mice were about half those in Runx2fl/fl mice. However, the serum levels of phosphorus and intact Fgf23 in Runx2fl/flCre mice were similar to those in Runx2fl/fl mice. The trabecular bone volume was increased during aging in both male and female Galnt3-/- mice, but the osteoid was reduced. The markers for bone formation and resorption in Galnt3-/- mice were similar to the control in both sexes. Galnt3-/- mice exhibited hyperphosphatemia and hypercalcemia, and the intact Fgf23 was about 40% that of wild-type mice. These findings indicated that Runx2 regulates the expressions of Galnt3 and Fgf23 and that Galnt3 decelerates the mineralization of osteoid by stabilizing Fgf23.
Assuntos
Calcificação Fisiológica , Calcinose , N-Acetilgalactosaminiltransferases , Osteoblastos , Animais , Feminino , Masculino , Camundongos , Calcinose/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fatores de Crescimento de Fibroblastos/metabolismo , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Osteoblastos/metabolismo , Fósforo , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Bcl2l1 (Bcl-XL) belongs to the Bcl-2 family, Bcl2 and Bcl2-XL are major anti-apoptotic proteins, and the apoptosis of osteoblasts is a key event for bone homeostasis. As the functions of Bcl2l1 in osteoblasts and bone homeostasis remain unclear, we generated osteoblast-specific Bcl2l1-deficient (Bcl2l1fl/flCre) mice using 2.3-kb Col1a1 Cre. Trabecular bone volume and the trabecular number were lower in Bcl2l1fl/flCre mice of both sexes than in Bcl2l1fl/fl mice. In bone histomorphometric analysis, osteoclast parameters were increased in Bcl2l1fl/flCre mice, whereas osteoblast parameters and the bone formation rate were similar to those in Bcl2l1fl/fl mice. TUNEL-positive osteoblastic cells and serum TRAP5b levels were increased in Bcl2l1fl/flCre mice. The deletion of Bcl2l1 in osteoblasts induced Tnfsf11 expression, whereas the overexpression of Bcl-XL had no effect. In a co-culture of Bcl2l1-deficient primary osteoblasts and wild-type bone-marrow-derived monocyte/macrophage lineage cells, the numbers of multinucleated TRAP-positive cells and resorption pits increased. Furthermore, serum deprivation or the deletion of Bcl2l1 in primary osteoblasts increased apoptosis and ATP levels in the medium. Therefore, the reduction in trabecular bone in Bcl2l1fl/flCre mice may be due to enhanced bone resorption through osteoblast apoptosis and the release of ATP from apoptotic osteoblasts, and Bcl2l1 may inhibit bone resorption by preventing osteoblast apoptosis.
Assuntos
Reabsorção Óssea , Osteogênese , Animais , Feminino , Masculino , Camundongos , Trifosfato de Adenosina/metabolismo , Apoptose/genética , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Osso Esponjoso/metabolismo , Diferenciação Celular , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes developmental and reproductive disorders in pups due to the attenuated luteinizing hormone (LH) production during the perinatal stage; however, the administration of α-lipoic acid (LA) to TCDD-exposed pregnant rats reversed the attenuated LH production. Therefore, reproductive disorders in pups are expected to be ameliorated with LA supplementation. To address this issue, pregnant rats orally received low dose TCDD at gestational day 15 (GD15) and proceeded to parturition. The control received a corn oil vehicle. To examine the preventive effects of LA, supplementation with LA was provided until postnatal day 21. In this study, we demonstrated that maternal administration of LA restored the sexually dimorphic behavior of male and female offspring. TCDD-induced LA insufficiency is likely a direct cause of TCDD reproductive toxicity. In the analysis to clarify the mechanism of the decrease in LA, we found evidence suggesting that TCDD inhibits the synthesis and increases the utilization of S-adenosylmethionine (SAM), a cofactor for LA synthesis, resulting in a decrease in the SAM level. Furthermore, folate metabolism, which is involved in SAM synthesis, is disrupted by TCDD, which may adversely affect infant growth. Maternal supplementation of LA restored SAM to its original level in the fetal hypothalamus; in turn, SAM ameliorated abnormal folate consumption and suppressed aryl hydrocarbon receptor activation induced by TCDD. The study demonstrates that the application of LA could prevent and recover next-generation dioxin reproductive toxicity, which provides the potential to establish effective protective measures against dioxin toxicity.
Assuntos
Ácido Fólico , Exposição Materna , Dibenzodioxinas Policloradas , Efeitos Tardios da Exposição Pré-Natal , Caracteres Sexuais , Desenvolvimento Sexual , Ácido Tióctico , Animais , Feminino , Masculino , Gravidez , Ratos , Feto/efeitos dos fármacos , Feto/metabolismo , Ácido Fólico/metabolismo , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Exposição Materna/efeitos adversos , Dibenzodioxinas Policloradas/toxicidade , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/prevenção & controle , S-Adenosilmetionina/metabolismo , Desenvolvimento Sexual/efeitos dos fármacos , Ácido Tióctico/administração & dosagem , Ácido Tióctico/farmacologia , Ácido Tióctico/uso terapêutico , Reprodução/efeitos dos fármacosRESUMO
RUNX proteins, such as RUNX2, regulate the proliferation and differentiation of chondrocytes and osteoblasts. Haploinsufficiency of RUNX2 causes cleidocranial dysplasia, but a detailed analysis of Runx2+/- mice has not been reported. Furthermore, CBFB is required for the stability and DNA binding of RUNX family proteins. CBFB has two isoforms, and CBFB2 plays a major role in skeletal development. The calvaria, femurs, vertebrae and ribs in Cbfb2-/- mice were analyzed after birth, and compared with those in Runx2+/- mice. Calvarial development was impaired in Runx2+/- mice but mildly delayed in Cbfb2-/- mice. In femurs, the cortical bone but not trabecular bone was reduced in Cbfb2-/- mice, whereas both the trabecular and cortical bone were reduced in Runx2+/- mice. The trabecular bone in vertebrae increased in Cbfb2-/- mice but not in Runx2+/- mice. Rib development was impaired in Cbfb2-/- mice but not in Runx2+/- mice. These differences were likely caused by differences in the indispensability of CBFB and RUNX2, the balance of bone formation and resorption, or the number and maturation stage of osteoblasts. Thus, different amounts of CBFB and RUNX2 were required among the bone tissues for proper bone development and maintenance.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core , Osteoblastos , Animais , Camundongos , Diferenciação Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Osteoblastos/metabolismo , Osteogênese/genética , Costelas/metabolismo , Crânio/metabolismo , Coluna Vertebral/metabolismoRESUMO
A 60-year-old man visited our hospital to treat a large cystic mass in the pelvis which had been found by abdominal ultrasonography in December 201X. Computed tomography (CT) and magnetic resonance imaging (MRI) showed a multilocular cyst with a maximum diameter of about 10 cm. CT-guided drainage and sclerotherapy with minocycline reduced the size of tumor by 40%, but symptoms such as difficulty of defecation and urinary frequency appeared a year and a half later due to re-enlargement of the cysts. Laparoscopic resection of the multilocular cysts was performed, and all cysts were removed almost completely using transrectal ultrasonography. The multilocular cyst was positive for NKX3.1 by immunohistochemical staining, and was diagnosed as a giant multilocular prostatic cystadenoma. After surgery, the symptoms such as difficulty of defecation and urinary frequency were relieved promptly. One year after the surgery, the patient was free from recurrence of the disease.
Assuntos
Cistadenoma , Cistos , Laparoscopia , Neoplasias da Próstata , Cistadenoma/diagnóstico por imagem , Cistadenoma/cirurgia , Cistectomia , Cistos/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Minociclina , Pelve/patologia , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/cirurgiaRESUMO
Immune checkpoint blockade (ICB) treatment improves the prognosis of several types of solid tumors, however, responsiveness to ICB therapy remains low in pancreatic ductal adenocarcinoma (PDACs), which has a rich tumor microenvironment (TME). The TME is composed of various stromal cells, including cancer-associated fibroblasts (CAFs), which contribute to the establishment of an immunosuppressive microenvironment. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is an innate immune pathway that results in the upregulation of immune cell recruiting-cytokines and anti-tumor efficacy. In this study, we aimed to investigate the impact of cGAS-STING expression and the presence of CAFs upon immune cell infiltration in PDACs. cGAS and STING co-expressing PDAC cases showed favorable survival, with many cytotoxic CD8 + T cell infiltrations from the stromal component adjacent to the cancer cells toward cancer cells, but not in cGAS-STING signaling defected PDAC cases. The signatures of tumor-restrain CAFs were expressed in tumors with cGAS-STING signaling. Finally, transwell co-culture experiments demonstrated that immune cell infiltration was impeded by the presence of CAFs, but not by activation of cGAS-STING signaling. In conclusion, pro-infiltration signals, such as cGAS-STING, and characterization of CAFs are crucial in defeating CAF barricades and encouraging immune cell infiltration in PDACs.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma Ductal Pancreático , Proteínas de Membrana , Nucleotidiltransferases , Neoplasias Pancreáticas , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Humanos , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Microambiente TumoralRESUMO
Runt-related transcription factor-2 (Runx2) is an essential transcription factor for osteoblast differentiation. However, its functions after the commitment into osteoblasts are controversial and remain to be clarified. We generated enhanced green fluorescent protein (EGFP)-Cre transgenic mice driven by the 2.3-kilobase (kb) Col1a1 promoter, and Runx2 was deleted in osteoblasts and odontoblasts in Runx2fl/flCre mice. The sutures and fontanelles were more widely opened in Runx2fl/flCre newborns than in Runx2fl/fl newborns. Runx2fl/flCre mice exhibited dwarfism with shorter incisors and 37% had irregularly aligned incisors. The volume of trabecular bone in femurs and vertebrae and their bone mineral density (BMD), in addition to the cortical thickness and BMD were reduced in Runx2fl/flCre mice compared with Runx2fl/fl mice in both sexes. The bone formation of both trabecular and cortical bone, osteoblast number, osteoclast surface, osteoblast proliferation, and the serum levels of procollagen type 1 N-terminal propeptide (P1NP), tartrate-resistant acid phosphatase 5b (TRAP5b), and C-terminal cross-linked telopeptide of type 1 collagen (CTX1) were reduced in Runx2fl/flCre mice. The expression of major bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and Bglap&Bglap2, and of Tnfsf11 was lower in Runx2fl/flCre mice than in Runx2fl/fl mice. The expression of Runx2 target genes, including Ihh, Fgfr1, Fgfr2, Fgfr3, Tcf7, Wnt10b, Pth1r, Sp7, and Dlx5, was also reduced. Osteoblasts in Runx2fl/fl mice were cuboidal and contained abundant type I collagen α1 (Col1a1), whereas those in Runx2fl/flCre mice were deflated and contained a small amount of Col1a1. Runx2 activated the reporter activity of the 2.3-kb Col1a1 promoter and bound the region around the Col1a1 transcription start site. The deletion of Runx2 by Cre-expressing adenovirus in Runx2fl/fl primary osteoblasts impaired osteoblast differentiation and the expression of genes encoding major bone matrix proteins, and osteoclastogenesis was inhibited due to the reduction of Tnfsf11 expression in the osteoblasts. This study demonstrated that Runx2 is required for the expression of the major bone matrix protein genes and Tnfsf11 after commitment into osteoblasts in mice. © 2021 American Society for Bone and Mineral Research (ASBMR).
Assuntos
Matriz Óssea , Subunidade alfa 1 de Fator de Ligação ao Core , Animais , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos , Osteoblastos , Fatores de TranscriçãoRESUMO
Plant activators, including acibenzolar-S-methyl (ASM), are chemical compounds that stimulate plant defense responses to pathogens. ASM treatment inhibits infection by a variety of plant viruses, however, the mechanisms of this broad-spectrum and strong effect remain poorly understood. We employed green fluorescent protein (GFP)-expressing viruses and Nicotiana benthamiana plants to identify the infection stages that are restricted by ASM. ASM suppressed infection by three viral species, plantago asiatica mosaic virus (PlAMV), potato virus X (PVX), and turnip mosaic virus (TuMV), in inoculated cells. Furthermore, ASM delayed the long-distance movement of PlAMV and PVX, and the cell-to-cell (short range) movement of TuMV. The ASM-mediated delay of long-distance movement of PlAMV was not due to the suppression of viral accumulation in the inoculated leaves, indicating that ASM restricts PlAMV infection in at least two independent steps. We used Arabidopsis thaliana mutants to show that the ASM-mediated restriction of PlAMV infection requires the NPR1 gene but was independent of the dicer-like genes essential for RNA silencing. Furthermore, experiments using protoplasts showed that ASM treatment inhibited PlAMV replication without cell death. Our approach, using GFP-expressing viruses, will be useful for the analysis of mechanisms underlying plant activator-mediated virus restriction.
Assuntos
Nicotiana , Potexvirus , Tiadiazóis , Adjuvantes Imunológicos/farmacologia , Resistência à Doença/efeitos dos fármacos , Imunidade Vegetal/efeitos dos fármacos , Potexvirus/fisiologia , Tiadiazóis/farmacologia , Nicotiana/imunologia , Nicotiana/virologiaRESUMO
Runx2 and Sp7 are essential transcription factors for osteoblast differentiation. However, the molecular mechanisms responsible for the proliferation of osteoblast progenitors remain unclear. The early onset of Runx2 expression caused limb defects through the Fgfr1-3 regulation by Runx2. To investigate the physiological role of Runx2 in the regulation of Fgfr1-3, we compared osteoblast progenitors in Sp7-/- and Runx2-/- mice. Osteoblast progenitors accumulated and actively proliferated in calvariae and mandibles of Sp7-/- but not of Runx2-/- mice, and the number of osteoblast progenitors and their proliferation were dependent on the gene dosage of Runx2 in Sp7-/- background. The expression of Fgfr2 and Fgfr3, which were responsible for the proliferation of osteoblast progenitors, was severely reduced in Runx2-/- but not in Sp7-/- calvariae. Runx2 directly regulated Fgfr2 and Fgfr3, increased the proliferation of osteoblast progenitors, and augmented the FGF2-induced proliferation. The proliferation of Sp7-/- osteoblast progenitors was enhanced and strongly augmented by FGF2, and Runx2 knockdown reduced the FGF2-induced proliferation. Fgfr inhibitor AZD4547 abrogated all of the enhanced proliferation. These results indicate that Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation, at least partly, by regulating Fgfr2 and Fgfr3 expression.
Assuntos
Proliferação de Células/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Células-Tronco/fisiologia , Animais , Benzamidas/farmacologia , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Osteoblastos/fisiologia , Osteogênese/genética , Piperazinas/farmacologia , Cultura Primária de Células , Pirazóis/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Fator de Transcrição Sp7/genéticaRESUMO
Plant viruses in the genus Carlavirus include more than 65 members. Plants infected with carlaviruses exhibit various symptoms, including leaf malformation and plant stunting. Cysteine-rich protein (CRP) encoded by carlaviruses has been reported to be a pathogenicity determinant. Carlavirus CRPs contain two motifs in their central part: a nuclear localization signal (NLS) and a zinc finger motif (ZF). In addition to these two conserved motifs, carlavirus CRPs possess highly divergent, N-terminal, 34 amino acid residues with unknown function. In this study, to analyse the role of these distinct domains, we tested six carlavirus CRPs for their RNA silencing suppressor activity, ability to enhance the pathogenicity of a heterologous virus and effects on virus accumulation levels. Although all six tested carlavirus CRPs showed RNA silencing suppressor activity at similar levels, symptoms induced by the Potato virus X (PVX) heterogeneous system exhibited two different patterns: leaf malformation and whole-plant stunting. The expression of each carlavirus CRP enhanced PVX accumulation levels, which were not correlated with symptom patterns. PVX-expressing CRP with mutations in either NLS or ZF did not induce symptoms, suggesting that both motifs play critical roles in symptom expression. Further analysis using chimeric CRPs, in which the N-terminal region was replaced with the corresponding region of another CRP, suggested that the N-terminal region of carlavirus CRPs determined the exhibited symptom types. The up-regulation of a plant gene upp-L, which has been reported in a previous study, was also observed in this study; however, the expression level was not responsible for symptom types.
Assuntos
Carlavirus/metabolismo , Doenças das Plantas/virologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Carlavirus/patogenicidade , Sinais de Localização Nuclear/metabolismo , Folhas de Planta/virologia , Potexvirus/metabolismo , Interferência de RNA , RNA Viral/metabolismo , Especificidade da Espécie , Nicotiana/virologia , Proteínas Virais/químicaRESUMO
Our previous studies demonstrated that treating pregnant rats with dioxins, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), targets the pituitary expression of luteinizing hormone (LH) to attenuate testicular steroidogenesis in fetuses, resulting in the imprinting of sexual immaturity of the offspring after reaching maturity. Furthermore, we found that although TCDD disturbs the tricarboxylic acid (TCA) cycle in the fetal hypothalamus, maternal co-treatment with α-lipoic acid (α-LA), a cofactor of the TCA cycle, restores a TCDD-produced reduction in the LH-evoked steroidogenesis as well as the TCA cycle activity in fetuses. However, the mechanism underlying the beneficial effect of α-LA remains to be fully elucidated. To address this issue, we compared the effect of α-LA with that of thiamine, another cofactor of the TCA cycle. As with α-LA, supplying thiamine to dams exposed to TCDD alleviates the reduced level of not only hypothalamic ATP but also pituitary LH and testicular steroidogenic protein in fetuses. However, thiamine had a much weaker effect than α-LA. In agreement with ATP attenuation, TCDD activated AMP-activated protein kinase (AMPK), a negative regulator of LH production, whereas the supplementation of α-LA allowed recovery from this defect. Furthermore, α-LA restored the TCDD-produced reduction in the pituitary expression of the receptor for gonadotropin-releasing hormone (GnRH), an upstream regulator of LH synthesis. These results suggest that α-LA rescues TCDD-produced attenuation during fetal steroidogenesis due not only to facilitation of energy production through the TCA cycle but also through suppression of AMPK activation, and the pituitary GnRH receptor may serve as a mediator of these effects.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Encéfalo/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , Receptores LHRH/genética , Ácido Tióctico/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/metabolismo , Feminino , Feto/efeitos dos fármacos , Feto/metabolismo , Hormônio Liberador de Gonadotropina/genética , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hormônio Luteinizante/sangue , Hormônio Luteinizante/genética , Masculino , Miocárdio/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Ratos Wistar , Testículo/efeitos dos fármacos , Testículo/metabolismoRESUMO
Synthesis and reactivity of iron-dinitrogen complexes have been extensively studied, because the iron atom plays an important role in the industrial and biological nitrogen fixation. As a result, iron-catalyzed reduction of molecular dinitrogen into ammonia has recently been achieved. Here we show that an iron-dinitrogen complex bearing an anionic PNP-pincer ligand works as an effective catalyst towards the catalytic nitrogen fixation, where a mixture of ammonia and hydrazine is produced. In the present reaction system, molecular dinitrogen is catalytically and directly converted into hydrazine by using transition metal-dinitrogen complexes as catalysts. Because hydrazine is considered as a key intermediate in the nitrogen fixation in nitrogenase, the findings described in this paper provide an opportunity to elucidate the reaction mechanism in nitrogenase.
RESUMO
Cbfb is a cotranscription factor that forms a heterodimer with Runx proteins Runx1, Runx2, and Runx3. It is required for fetal liver hematopoiesis and skeletal development. Cbfb has two functional isoforms, Cbfb1 and Cbfb2, which are formed by alternative splicing. To address the biological functions of these isoforms in skeletal development, we examined Cbfb1(-/-) and Cbfb2(-/-) mouse embryos. Intramembranous and endochondral ossification was retarded and chondrocyte and osteoblast differentiation was inhibited in Cbfb2(-/-) embryos but not in Cbfb1(-/-) embryos. Cbfb2 mRNA was upregulated in calvariae, limbs, livers, thymuses, and hearts of Cbfb1(-/-) embryos but Cbfb1 mRNA was not in those of Cbfb2(-/-) embryos, and the total amount of Cbfb1 and Cbfb2 mRNA in Cbfb1(-/-) embryos was similar to that in wild-type embryos but was severely reduced in Cbfb2(-/-) embryos. The absolute numbers of Cbfb2 mRNA in calvariae, limbs, livers, thymuses, and brains in wild-type embryos were about three times higher than those of Cbfb1 in the respective tissue. The levels of Runx proteins were reduced in calvariae, limbs, and primary osteoblasts from Cbfb2(-/-) embryos, but the reduction in Runx2 protein was very mild. Furthermore, the amounts of Runx proteins and Cbfb in Cbfb2(-/-) embryos differed similarly among skeletal tissues, livers, and thymuses, suggesting that Runx proteins and Cbfb are mutually required for their stability. Although Cbfb1(-/-) embryos developed normally, Cbfb1 induced chondrocyte and osteoblast differentiation and enhanced DNA binding of Runx2 more efficiently than Cbfb2. Our results indicate that modulations in the relative levels of the isoforms may adjust transcriptional activation by Runx2 to appropriate physiological levels. Cbfb2 was more abundant, but Cbfb1 was more potent for enhancing Runx2 activity. Although only Cbfb2 loss generated overt skeletal phenotypes, both may play major roles in skeletal development with functional redundancy. © 2016 American Society for Bone and Mineral Research.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Diferenciação Celular/fisiologia , Condrócitos/metabolismo , Embrião de Mamíferos/embriologia , Osteoblastos/metabolismo , Esqueleto/embriologia , Animais , Fator de Ligação a CCAAT/genética , Condrócitos/citologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Embrião de Mamíferos/citologia , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Esqueleto/citologiaRESUMO
Runx family proteins, Runx1, Runx2, and Runx3, play important roles in skeletal development. Runx2 is required for osteoblast differentiation and chondrocyte maturation, and haplodeficiency of RUNX2 causes cleidocranial dysplasia, which is characterized by open fontanelles and sutures and hypoplastic clavicles. Cbfb forms a heterodimer with Runx family proteins and enhances their DNA-binding capacity. Cbfb-deficient (Cbfb(-/-) ) mice die at midgestation because of the lack of fetal liver hematopoiesis. We previously reported that the partial rescue of hematopoiesis in Cbfb(-/-) mice revealed the requirement of Cbfb in skeletal development. However, the precise functions of Cbfb in skeletal development still remain to be clarified. We deleted Cbfb in mesenchymal cells giving rise to both chondrocyte and osteoblast lineages by mating Cbfb(fl/fl) mice with Dermo1 Cre knock-in mice. Cbfb(fl/fl/Cre) mice showed dwarfism, both intramembranous and endochondral ossifications were retarded, and chondrocyte maturation and proliferation and osteoblast differentiation were inhibited. The differentiation of chondrocytes and osteoblasts were severely inhibited in vitro, and the reporter activities of Ihh, Col10a1, and Bglap2 promoter constructs were reduced in Cbfb(fl/fl/Cre) chondrocytes or osteoblasts. The proteins of Runx1, Runx2, and Runx3 were reduced in the cartilaginous limb skeletons and calvariae of Cbfb(fl/fl/Cre) embryos compared with the respective protein in the respective tissue of Cbfb(fl/fl) embryos at E15.5, although the reduction of Runx2 protein in calvariae was much milder than that in cartilaginous limb skeletons. All of the Runx family proteins were severely reduced in Cbfb(fl/fl/Cre) primary osteoblasts, and Runx2 protein was less stable in Cbfb(fl/fl/Cre) osteoblasts than Cbfb(fl/fl) osteoblasts. These findings indicate that Cbfb is required for skeletal development by regulating chondrocyte differentiation and proliferation and osteoblast differentiation; that Cbfb plays an important role in the stabilization of Runx family proteins; and that Runx2 protein stability is less dependent on Cbfb in calvariae than in cartilaginous limb skeletons.
Assuntos
Desenvolvimento Ósseo/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Subunidade beta de Fator de Ligação ao Core/fisiologia , Animais , Diferenciação Celular , Condrócitos/citologia , Subunidade beta de Fator de Ligação ao Core/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/citologiaRESUMO
Runx2 is essential for osteoblast differentiation and chondrocyte maturation. The expression of Runx2 is the first requisite step for the lineage determination from mesenchymal stem cells to osteoblasts. Although the transcript from Runx2 distal promoter is majorly expressed in osteoblasts, the promoter failed to direct green fluorescent protein (GFP) expression to osteoblasts. To find the regulatory region, we generated GFP reporter mice driven by a bacterial artificial chromosome (BAC) of Runx2 locus, and succeeded in the reproduction of endogenous Runx2 expression. By serially deleting it, we identified a 343-bp enhancer, which directed GFP expression specifically to osteoblasts, about 30 kb upstream of the distal promoter. The sequence of the 343-bp enhancer was highly conserved among mouse, human, dog, horse, opossum, and chicken. Dlx5, Mef2c, Tcf7, Ctnnb1, Sp7, Smad1, and Sox6, which localized on the enhancer region in primary osteoblasts, synergistically upregulated the enhancer activity, whereas Msx2 downregulated the activity in mouse osteoblastic MC3T3-E1 cells. Msx2 was predominantly bound to the enhancer in mouse multipotent mesenchymal C3H10T1/2 cells, whereas Dlx5 was predominantly bound to the enhancer in MC3T3-E1 cells. Dlx5 and Mef2 directly bound to the enhancer, and the binding sites were required for the osteoblast-specific expression in mice, whereas the other factors bound to the enhancer by protein-protein interaction. The enhancer was characterized by the presence of the histone variant H2A.Z, the enrichment of histone H3 mono- and dimethylated at Lys4 and acetylated at Lys18 and Lys27, but the depletion of histone H3 trimethylated at Lys4 in primary osteoblasts. These findings indicated that the enhancer, which had typical histone modifications for enhancers, contains sufficient elements to direct Runx2 expression to osteoblasts, and that Dlx5 and Mef2, which formed an enhanceosome with Tcf7, Ctnnb1, Sp7, Smad1, and Sox6, play an essential role in the osteoblast-specific activation of the enhancer. © 2014 American Society for Bone and Mineral Research.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Elementos Facilitadores Genéticos/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição MEF2/metabolismo , Osteoblastos/metabolismo , Animais , Pareamento de Bases/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Cromossomos Artificiais Bacterianos/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica , Genes Reporter , Loci Gênicos , Proteínas de Fluorescência Verde/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Histonas/metabolismo , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Fatores de Transcrição SOX/metabolismo , Proteína Smad1/metabolismo , Fator de Transcrição Sp7 , Fatores de Transcrição/metabolismo , beta Catenina/metabolismoRESUMO
We present a 73-year-old female with advanced thyroid papillary carcinoma who complained of gross hematuria. We found a bladder tumor and considered it the cause of her symptom. Cystoscopic findings of the tumor were unusual, with peri-tumor vessel formation. Pathological examination of the bladder tumor was consistent with metastasis of thyroid papillary carcinoma. Therefore, we identified thyroid carcinoma metastasis to the urinary bladder as the cause of hematuria in our patient. Thyroid carcinoma metastasis to the bladder is extremely rare, but it should be included among differential diagnoses for gross hematuria in patients with a clinical history of thyroid carcinoma.
RESUMO
Amino-acid substitution at positions 70 and 91 of the hepatitis C virus (HCV)-1b core region is a factor that contributes to a non-virological response in treatment using interferon/ribavirin combination. In this study, a system was developed for detection of nucleotide sequence variation in the core region that is simpler and less expensive than the current direct sequencing method. A PCR detection method using mutation-specific primers was developed, and amino acids at positions 70 and 91 were identified. The protein type was determined based on band intensity in electrophoresis, and classified into wild (70:R, 91:L), mutant (70:Q/H, 91:M) and competitive types. The detection rate, sensitivity and reproducibility were investigated in 108 patients with HCV-1b who were treated with interferon/ribavirin combination therapy, and correlation with the results of direct sequencing was examined. The detection rate was 94.4%, the sensitivity was 10 KIU/mL, the reproducibility was high, and in detectable cases the consistency with direct sequencing was 97.1%; inconsistency was noted in two competitive-type cases and in one case with an unusual amino-acid substitution. The results suggest that the system described in this paper provides an effective, simple and low-cost approach to detection of nucleotide sequence variation in the core region of HCV-1b.
Assuntos
Variação Genética , Hepacivirus/genética , Hepatite C/virologia , Proteínas do Core Viral/química , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Antivirais/uso terapêutico , Sequência de Bases , Quimioterapia Combinada , Eletroforese , Hepacivirus/classificação , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Humanos , Interferons/uso terapêutico , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Viral/sangue , Reprodutibilidade dos Testes , Ribavirina/uso terapêutico , Sensibilidade e Especificidade , Albumina Sérica/análise , Proteínas do Core Viral/genética , Proteínas não Estruturais Virais/genéticaRESUMO
The authors describe the results of fat thickness patterning of the abdominal sites in 50 patients, all of whom required breast reconstruction with a transverse rectus abdominis musculocutaneous flap. The thickness of the abdominal fat was measured at 12 anatomic locations with an ultrasonic instrument. The highest value of the subcutaneous fat thickness was 29.0+/-10.0 mm at a site 2 cm below the umbilicus at the center of the rectus abdominis muscle. The lowest value of the subcutaneous fat thickness was 17.8+/-7.6 mm at a site 2 cm above the umbilicus on the anterior superior iliac spine. Average subcutaneous fat thickness over the abdomen of 50 patients was 24.0+/-9.4 mm. There were 13 patients (group 1) who had an abdominal fat thickness of more than 30 mm, 19 patients (group 2) with an abdominal fat thickness less than 30 mm and more than 20 mm, and 18 patients (group 3) with an abdominal fat thickness less than 20 mm. Complications occurred in 12 of 50 flaps (24%). Among groups 1, 2, and 3 there was no significant difference (p<0.01) in the overall flap complications (15.4: 36.8: 16.7). In summary, subcutaneous fat thickness showed the higher value at the center of the abdomen and the lower value at the lateral site. Abdominal fat thickness is not a risk factor for necrosis of pedicled transverse rectus abdominis musculocutaneous flaps in patients who are thin, average, or mildly obese. Preoperative examination of the abdominal subcutaneous fat thickness should provide useful information for detailed simulation of a reconstructive operation.