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1.
PLoS One ; 15(12): e0242640, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33373386

RESUMO

To examine the differential mechanobiological responses of specific resident tendon cells, we developed an in vivo model of whole-body irradiation followed by injection of either tendon stem/progenitor cells (TSCs) expressing green fluorescent protein (GFP-TSCs) or mature tenocytes expressing GFP (GFP-TNCs) into the patellar tendons of wild type C57 mice. Injected mice were subjected to short term (3 weeks) treadmill running, specifically moderate treadmill running (MTR) and intensive treadmill running (ITR). In MTR mice, both GFP-TSC and GFP-TNC injected tendons maintained normal cell morphology with elevated expression of tendon related markers collagen I and tenomodulin. In ITR mice injected with GFP-TNCs, cells also maintained an elongated shape similar to the shape found in normal/untreated control mice, as well as elevated expression of tendon related markers. However, ITR mice injected with GFP-TSCs showed abnormal changes, such as cell morphology transitioning to a round shape, elevated chondrogenic differentiation, and increased gene expression of non-tenocyte related genes LPL, Runx-2, and SOX-9. Increased gene expression data was supported by immunostaining showing elevated expression of SOX-9, Runx-2, and PPARγ. This study provides evidence that while MTR maintains tendon homeostasis by promoting the differentiation of TSCs into TNCs, ITR causes the onset of tendinopathy development by inducing non-tenocyte differentiation of TSCs, which may eventually lead to the formation of non-tendinous tissues in tendon tissue after long term mechanical overloading conditions on the tendon.


Assuntos
Condrócitos/citologia , Células-Tronco/citologia , Tendinopatia/patologia , Tendões/patologia , Tenócitos/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Forma Celular , Rastreamento de Células , Condrócitos/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Teste de Esforço , Feminino , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/genética , PPAR gama/metabolismo , Condicionamento Físico Animal/efeitos adversos , Corrida , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Células-Tronco/metabolismo , Tendinopatia/etiologia , Tendinopatia/genética , Tendinopatia/metabolismo , Tendões/metabolismo , Tenócitos/metabolismo
2.
J Biomed Nanotechnol ; 16(6): 899-909, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33187585

RESUMO

A well-studied subject of epigenetics, the histone methylation located at lysine and arginine is overseen via methyltransferases and demethylases. Lysine-specific demethylase 4A (KDM4A) comprises a lysine demethylase and possesses specificity for H3K9me3 and H3K36me3, which is capable of being used in order to activate histone transcription. Our team examined the expression of KDM4A within Sprague Dawley (SD) rats and further investigated the mechanism via which this phenomena regulates osteogenic variation within the present study. The overexpression of KDM4A facilitated the process of osteoblast differentiation in bone mesenchymal stem cells (BMSC), while the knocking down differentiation via osteoblast was restrained via the suppression of the expression of Runx2, Osterix, alkaline phosphatase (ALP), and osteocalcin (OCN). Knocking down KDM4A lowered levels of the promoter expression of Runx2, osterix, and OCN, and raised levels of H3K27me3 expression. The results demonstrated that KDM4A possesses a crucial role within the differentiation of osteoblasts and furthermore regulates the expression of Runx2, Osterix, and OCN via H3K9me3. The present research may provide new insights into the treatment of bone healing.


Assuntos
Histona Desmetilases , Lisina , Osteogênese , Animais , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Histona Desmetilases/fisiologia , Osteoblastos/metabolismo , Osteocalcina/genética , Osteogênese/genética , Ratos , Ratos Sprague-Dawley , Receptores de Ocitocina , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Ecotoxicol Environ Saf ; 206: 111194, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32866894

RESUMO

Tibial Dyschondroplasia (TD) is a prevailing skeletal disorder that mainly affects rapidly growing avian species. It results in reduced bone strength, lameness and an increase risk of fragility fractures. Total flavonoids of Rhizoma drynariae (TFRD) have been used as an effective treatment of different bone diseases in humans. The current in vitro study was conducted to explore the therapeutic effect of TFRD on thiram-induced cytotoxicity in avian growth plate cells via bone morphogenetic protein-2/runt related transcription factor-2 (BMP-2/Runx2) and Indian hedgehog/Parathyroid hormone-related peptide (IHH/PTHrP) expressions. Chondrocytes were isolated, cultured and refined from chicken's tibial growth plates in a special medium. Then chondrocytes were treated with sublethal thiram having less concentration (2.5 µg/mL) to induce cytotoxicity of chondrocyte, and then treated with providential doses (100 µg/mL) of TFRD. Thiram caused distorted morphology of chondrocytes, nuclei appeared disintegration or lysed along with decreased expressions of BMP-2/Runx2 and IHH/PTHrP. TFRD administration not only enhanced the viability of chondrocytes by itself, but also well restored the damage caused by thiram on growth plate chondrocytes by significantly up-regulating the expressions of BMP-2/Runx2 and IHH/PTHrP. Therefore, this study provides a novel insight into the further treatment of TD and other skeletal ailments and lays the foundation for prevention and treatment.


Assuntos
Proteína Morfogenética Óssea 2/genética , Condrócitos/efeitos dos fármacos , Flavonoides/farmacologia , Expressão Gênica/efeitos dos fármacos , Polypodiaceae/química , Tiram/toxicidade , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Galinhas , Condrócitos/metabolismo , Condrócitos/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Flavonoides/isolamento & purificação , Lâmina de Crescimento/citologia , Lâmina de Crescimento/efeitos dos fármacos , Proteínas Hedgehog/genética , Proteína Relacionada ao Hormônio Paratireóideo/genética , Cultura Primária de Células , Rizoma , Regulação para Cima
4.
Ecotoxicol Environ Saf ; 203: 111031, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888610

RESUMO

Bone mineral density (BMD) changes were reported to be associated with excessive fluoride exposure and abnormal expression of RUNX2. However, whether the alteration of methylation status, a most commonly used marker for the alteration of gene expression in epidemiological investigation, of RUNX2 is associated with low-to-moderate fluoride exposure and BMD changes has not been reported. Our study aims to explore the role of RUNX2 promoter methylation in BMD changes induced by low-to-moderate fluoride exposure. A total of 1124 adults (413 men and 711 women) were recruited from Kaifeng City in 2017. We measured BMD using ultrasound bone densitometer. Concentrations of urinary fluoride (UF) were measured using ion-selective electrode, and the participants were grouped into control group (CG) and excessive fluoride group (EFG) according to the concentration of UF. We extracted DNA from fasting peripheral blood samples and then detected the promoter methylation levels of RUNX2 using quantitative methylation-specific PCR. Relationships between UF concentration, RUNX2 promoter methylation and BMD changes were analyzed using generalized linear model and logistic regression. Results showed in EFG (UF concentration > 1.6 mg/L), BMD was negatively correlated with UF concentration (ß: -0.14; 95%CI: -0.26, -0.01) and RUNX2 promoter methylation (ß: -0.13; 95%CI: -0.22, -0.03) in women. The methylation rate of RUNX2 promoter increased by 2.16% for each 1 mg/L increment in UF concentration of women in EFG (95%CI: 0.37, 3.96). No any significant associations between UF concentration, RUNX2 promoter methylation, and BMD were observed in the individuals in CG. Mediation analysis showed that RUNX2 promoter methylation mediated 18.2% (95% CI: 4.2%, 53.2%) of the association between UF concentration and BMD of women in EFG. In conclusion, excessive fluoride exposure (>1.6 mg/L) is associated with changes of BMD in women, and this association is mediated by RUNX2 promoter methylation.


Assuntos
Densidade Óssea/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Exposição Ambiental/análise , Fluoretos/toxicidade , Poluentes Químicos da Água/toxicidade , Absorciometria de Fóton , Adulto , Idoso , Densidade Óssea/genética , China , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Estudos Transversais , Metilação de DNA/efeitos dos fármacos , Feminino , Fluoretos/urina , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Inquéritos e Questionários , Poluentes Químicos da Água/urina
5.
J Biol Regul Homeost Agents ; 34(3): 825-835, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32495614

RESUMO

Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer cases. MicroRNAs (miRNAs/miRs) have been reported to play significant roles in the progression of human tumors, however, the expression and biological role of miR-23b in NSCLC remains elusive. Underexpression of miR-23b was detected in NSCLC tissues in comparison with the matched para-carcinoma tissues. The clinical value of miR-23b was analyzed, and the findings showed that miR-23b expression was negatively correlated with poor overall survival and malignant clinicopathologic characteristics of NSCLC patients. Furthermore, functional assays demonstrated that overexpression of miR-23b inhibited NSCLC cell viability, invasion and migration. Luciferase reporter assay and qRT-PCR revealed that RUNX2 was a functional target of miR-23b. The elevated expression of RUNX2 was positively correlated with overall survival of NSCLC patients. Additionally, Western blot analysis indicated that EMT and Wnt/ß-catenin pathways were blocked by the upregulation of miR-23b. Taken together, these data demonstrated that dysregulation of miR-23b/RUNX2 signal may be a novel therapeutic target for the treatment of NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Neoplasias Pulmonares , MicroRNAs/genética , Via de Sinalização Wnt , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia
6.
Oncogene ; 39(28): 5152-5164, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32535615

RESUMO

Epithelial-mesenchymal transition (EMT) program, which facilitates tumor metastasis, stemness and therapy resistance, is a reversible biological process that is largely orchestrated at the epigenetic level under the regulation of different cell signaling pathways. EMT state is often heterogeneous within individual tumors, though the epigenetic drivers underlying such heterogeneity remain elusive. In colon cancer, hyperactivation of the Wnt/ß-catenin signaling not only drives tumor initiation, but also promotes metastasis in late stage by promoting EMT program. However, it is unknown whether the intratumorally heterogeneous Wnt activity could directly drive EMT heterogeneity, and, if so, what are the underlying epigenetic driver(s). Here, by analyzing a phenotypically and molecularly heterogeneous colon cancer cell line using single-cell RNA sequencing, we identified two distinct cell populations with positively correlated Wnt activity and EMT state. Integrative multi-omics analysis of these two cell populations revealed RUNX2 as a critical transcription factor epigenetically driving the EMT heterogeneity. Both in vitro and in vivo genetic perturbation assays validated the EMT-enhancing effect of RUNX2, which remodeled chromatin landscape and activated a panel of EMT-associated genes through binding to their promoters and/or potential enhancers. Finally, by exploring the clinical data, we showed that RUNX2 expression is positively correlated with metastasis development and poor survival of colon cancer patients, as well as patients afflicted with other types of cancer. Taken together, our work revealed RUNX2 as a new EMT-promoting epigenetic regulator in colon cancer, which may potentially serve as a prognostic marker for tumor metastasis.


Assuntos
Neoplasias do Colo/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Epigenômica/métodos , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica/métodos , Via de Sinalização Wnt/genética , beta Catenina/genética , Animais , Células CACO-2 , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Células HeLa , Xenoenxertos , Humanos , Estimativa de Kaplan-Meier , Células MCF-7 , Camundongos
7.
J Biol Regul Homeost Agents ; 34(2): 345-355, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32548991

RESUMO

Osteoporosis is defined as an aging-related skeletal disorder involving deterioration of bone mass and bone structure, and consequently, increased risk of fractures. Emerging evidence indicates the dysregulation of microRNAs (miRNAs) in the progression of osteoporosis. However, whether such associated miRNAs control osteoblast differentiation or constitute therapeutic targets remains elusive. In the present study, we found elevated circulating miR-374b-5p level associated with postmenopausal osteoporosis. miR-374b-5p served as a critical suppressor of osteoblast differentiation. We further identified that miR-374b-5p directly targeted Wnt family member 3 (Wnt3) and Runt-related transcription factor 2 (Runx2) through its 3'-untranslated regions (3'UTRs). Moreover, the antagonist of miR-374b-5p could promote bone formation in ovariectomy (OVX)-induced mice. Together, our results revealed that miR-374b-5p directly targeted Wnt3 and Runx2, negatively regulating osteoblast differentiation and bone formation. Collectively, circulating miR-374b-5p in the serum might serve as a potential diagnostic and therapeutic strategy for osteoporosis.


Assuntos
Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , MicroRNAs/sangue , Osteoblastos/citologia , Osteoporose/genética , Proteína Wnt3/genética , Animais , Células Cultivadas , Feminino , Humanos , Camundongos , Osteogênese
8.
Gene ; 757: 144852, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32599019

RESUMO

Until now, various methods have been introduced to fabricate 3D scaffolds to provide a suitable substrate for cell growth and proliferation and subsequent use in tissue engineering to repair damaged tissues. The 3D scaffolds can simulate the natural cellular microenvironment well. Herein, the decellularized leaf spinach has been used which not only have no problems associated with artificial scaffolds, but they also do not cost significantly. Decellularized scaffolds surface properties were characterized by the investigation of scaffolds surface roughness, hydrophilicity, mechanical properties, size and shape of porosities and specific surface area. In the next step, osteogenic differentiation potential of bone marrow derived mesenchymal stem cells cultured on the scaffold and culture plate (as a control) was evaluated using alizarin staining and calcium content, alkaline phosphatase activity and bone related genes expression assays. The results indicated that the surface properties and shape of scaffold pores were effective in the stem cells binding, growth and proliferation. This higher biocompatibility due to the ideal surface hydrophilicity as well as high specific surface area due to the presence of a rough grid surface ultimately increased the efficiency of stem cell's bone differentiation. Taken together, it can be concluded that the decellularized spinach leaf scaffold, due to its easy availability, low prices and high efficiency, can be considered as a promising potential candidate for use as a proper substrate for stem cell growth and differentiation in bone tissue engineering.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteocalcina/genética , Folhas de Planta/química , Tecidos Suporte/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Biomineralização , Cálcio/metabolismo , Linhagem Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteocalcina/metabolismo , Spinacia oleracea/química
9.
Arterioscler Thromb Vasc Biol ; 40(7): 1664-1679, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32434409

RESUMO

OBJECTIVE: Cardiovascular disease is the primary cause of mortality in patients with chronic kidney disease. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced chronic kidney disease and is now recognized as an important predictor and independent risk factor for cardiovascular and all-cause mortality in these patients. VC in chronic kidney disease is triggered by the transformation of vascular smooth muscle cells (VSMCs) into osteoblasts as a consequence of elevated circulating inorganic phosphate (Pi) levels, due to poor kidney function. The objective of our study was to investigate the role of TDAG51 (T-cell death-associated gene 51) in the development of medial VC. METHODS AND RESULTS: Using primary mouse and human VSMCs, we found that TDAG51 is induced in VSMCs by Pi and is expressed in the medial layer of calcified human vessels. Furthermore, the transcriptional activity of RUNX2 (Runt-related transcription factor 2), a well-established driver of Pi-mediated VC, is reduced in TDAG51-/- VSMCs. To explain these observations, we identified that TDAG51-/- VSMCs express reduced levels of the type III sodium-dependent Pi transporter, Pit-1, a solute transporter, a solute transporter, a solute transporter responsible for cellular Pi uptake. Significantly, in response to hyperphosphatemia induced by vitamin D3, medial VC was attenuated in TDAG51-/- mice. CONCLUSIONS: Our studies highlight TDAG51 as an important mediator of Pi-induced VC in VSMCs through the downregulation of Pit-1. As such, TDAG51 may represent a therapeutic target for the prevention of VC and cardiovascular disease in patients with chronic kidney disease.


Assuntos
Transdiferenciação Celular , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Osteogênese , Fatores de Transcrição/metabolismo , Calcificação Vascular/metabolismo , Idoso , Animais , Células Cultivadas , Colecalciferol , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Hiperfosfatemia/induzido quimicamente , Hiperfosfatemia/metabolismo , Hiperfosfatemia/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fosfatos/metabolismo , Transdução de Sinais , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Calcificação Vascular/genética , Calcificação Vascular/patologia , Calcificação Vascular/prevenção & controle
10.
BMC Mol Cell Biol ; 21(1): 29, 2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-32299362

RESUMO

BACKGROUND: Low concentrations of tumor necrosis factor-alpha (TNF-α) and its receptor TNFR2 are both reported to promote osteogenic differentiation of osteoblast precursor cells. Moreover, low concentrations of TNF-α up-regulate the expression of EphB4. However, the molecular mechanisms underlying TNF-α-induced osteogenic differentiation and the roles of TNFR2 and EphB4 have not been fully elucidated. RESULTS: The ALP activity, as well as the mRNA and protein levels of RUNX2, BSP, EphB4 and TNFR2, was significantly elevated in MC3T3-E1 murine osteoblast precursor cells when stimulated with 0.5 ng/ml TNF-α. After TNFR2 was inhibited by gene knockdown with lentivirus-mediated shRNA interference or by a neutralizing antibody against TNFR2, the pro-osteogenic effect of TNF-α was partly reversed, while the up-regulation of EphB4 by TNF-α remained unchanged. With EphB4 forward signaling suppressed by a potent inhibitor of EphB4 auto-phosphorylation, NVP-BHG712, TNF-α-enhanced expressions of TNFR2, BSP and Runx2 were significantly decreased. Further investigation into the signaling pathways revealed that TNF-α significantly increased levels of p-JNK, p-ERK and p-p38. However, only the p-ERK level was significantly inhibited in TNFR2-knockdown cells. In addition, the ERK pathway inhibitor, U0126 (10 µM), significantly reversed the positive effect of TNF-α on the protein levels of RUNX2 and BSP. CONCLUSIONS: The EphB4, TNFR2 and ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation.


Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Receptor EphB4/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Butadienos/farmacologia , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Nitrilos/farmacologia , Osteoblastos/efeitos dos fármacos , Fosforilação , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno , Receptor EphB4/antagonistas & inibidores , Receptor EphB4/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
Oncogene ; 39(19): 3893-3909, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32203164

RESUMO

The transcription factor TCF7L2 is indispensable for intestinal tissue homeostasis where it transmits mitogenic Wnt/ß-Catenin signals in stem and progenitor cells, from which intestinal tumors arise. Yet, TCF7L2 belongs to the most frequently mutated genes in colorectal cancer (CRC), and tumor-suppressive functions of TCF7L2 were proposed. This apparent paradox warrants to clarify the role of TCF7L2 in colorectal carcinogenesis. Here, we investigated TCF7L2 dependence/independence of CRC cells and the cellular and molecular consequences of TCF7L2 loss-of-function. By genome editing we achieved complete TCF7L2 inactivation in several CRC cell lines without loss of viability, showing that CRC cells have widely lost the strict requirement for TCF7L2. TCF7L2 deficiency impaired G1/S progression, reminiscent of the physiological role of TCF7L2. In addition, TCF7L2-negative cells exhibited morphological changes, enhanced migration, invasion, and collagen adhesion, albeit the severity of the phenotypic alterations manifested in a cell-line-specific fashion. To provide a molecular framework for the observed cellular changes, we performed global transcriptome profiling and identified gene-regulatory networks in which TCF7L2 positively regulates the proto-oncogene MYC, while repressing the cell cycle inhibitors CDKN2C/CDKN2D. Consistent with its function in curbing cell motility and invasion, TCF7L2 directly suppresses the pro-metastatic transcription factor RUNX2 and impinges on the expression of cell adhesion molecules. Altogether, we conclude that the proliferation-stimulating activity of TCF7L2 persists in CRC cells. In addition, TCF7L2 acts as invasion suppressor. Despite its negative impact on cell cycle progression, TCF7L2 loss-of-function may thereby increase malignancy, which could explain why TCF7L2 is mutated in a sizeable fraction of colorectal tumors.


Assuntos
Proliferação de Células/genética , Neoplasias Colorretais/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Carcinogênese/genética , Movimento Celular/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Via de Sinalização Wnt/genética , beta Catenina/genética
12.
Nat Commun ; 11(1): 1141, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111827

RESUMO

Osteosarcoma, an aggressive malignant cancer, has a high lung metastasis rate and lacks therapeutic target. Here, we reported that chromobox homolog 4 (CBX4) was overexpressed in osteosarcoma cell lines and tissues. CBX4 promoted metastasis by transcriptionally up-regulating Runx2 via the recruitment of GCN5 to the Runx2 promoter. The phosphorylation of CBX4 at T437 by casein kinase 1α (CK1α) facilitated its ubiquitination at both K178 and K280 and subsequent degradation by CHIP, and this phosphorylation of CBX4 could be reduced by TNFα. Consistently, CK1α suppressed cell migration and invasion through inhibition of CBX4. There was a reverse correlation between CK1α and CBX4 in osteosarcoma tissues, and CK1α was a valuable marker to predict clinical outcomes in osteosarcoma patients with metastasis. Pyrvinium pamoate (PP) as a selective activator of CK1α could inhibit osteosarcoma metastasis via the CK1α/CBX4 axis. Our findings indicate that targeting the CK1α/CBX4 axis may benefit osteosarcoma patients with metastasis.


Assuntos
Caseína Quinase Ialfa/metabolismo , Ligases/antagonistas & inibidores , Ligases/metabolismo , Osteossarcoma/patologia , Proteínas do Grupo Polycomb/antagonistas & inibidores , Proteínas do Grupo Polycomb/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caseína Quinase Ialfa/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Ligases/genética , Camundongos , Mutação , Metástase Neoplásica , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas do Grupo Polycomb/genética , Regiões Promotoras Genéticas , Compostos de Pirvínio/farmacologia , Compostos de Pirvínio/uso terapêutico , Análise de Sobrevida , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismo
13.
Arterioscler Thromb Vasc Biol ; 40(5): 1352-1369, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32212850

RESUMO

OBJECTIVE: Abdominal aortic aneurysms (AAAs) are highly lethal diseases without effective clinical predictors and therapeutic targets. Vascular microcalcification, as detected by fluorine-18-sodium fluoride, has recently been recognized as a valuable indicator in predicting atherosclerotic plaque rupture and AAA expansion. However, whether vascular microcalcification involved in the pathogenesis of AAA remains elusive. Approach and Results: Microcalcification was analyzed in human aneurysmal aortas histologically and in AngII (angiotensin II)-infused ApoE-/- mouse aortas by fluorine-18-sodium fluoride positron emission tomography and X-ray computed tomography scanning in chronological order in live animals. AAA patients' aortic tissue showed markedly enhanced microcalcification in the aortic media within the area proximal to elastic fiber degradation, compared with non-AAA patients. Enhanced fluorine-18-sodium fluoride uptake preceded significant aortic expansion in mice. Microcalcification-positive mice on day 7 of AngII infusion showed dramatic aortic expansion on subsequent days 14 to 28, whereas microcalcification-negative AngII-infused mice and saline-induced mice did not develop AAA. The application of hydroxyapatite, the main component of microcalcification, aggravated AngII-induced AAA formation in vivo. RNA-sequencing analysis of the suprarenal aortas of 4-day-AngII-infused ApoE-/- mice and bioinformatics analysis with ChIP-Atlas database identified the potential involvement of the osteogenic transcriptional factor Runx2 (runt-related transcription factor 2) in AAA. Consistently, vascular smooth muscle cell-specific Runx2 deficiency markedly repressed AngII-induced AAA formation in the ApoE-/- mice compared with the control littermates. CONCLUSIONS: Our studies have revealed microcalcification as a novel pathological characteristic and potential mediator of AAA, and targeting microcalcification may represent a promising strategy for AAA prevention and treatment.


Assuntos
Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Calcificação Vascular/metabolismo , Adulto , Angiotensina II , Animais , Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/genética , Estudos de Casos e Controles , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Dilatação Patológica , Modelos Animais de Doenças , Durapatita , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Transdução de Sinais , Calcificação Vascular/induzido quimicamente , Calcificação Vascular/diagnóstico por imagem , Calcificação Vascular/genética , Remodelação Vascular
14.
Chem Biol Interact ; 323: 109057, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32198086

RESUMO

Runx2 (Runt-related transcription factor 2) is a key transcription factor which is associated with osteoblast differentiation and expressed in ER+ (estrogen receptor positive) human breast cancer cell lines. Runx2 also participates in mammary gland development. Deregulation of RNA Pol III genes (polymerase III-dependent genes) is tightly linked to tumor development, while Brf1 (TFIIB-related factor 1) specifically regulates these gene transcription. However, nothing is known about the effect of Runx2 on Brf1 expression and Pol III gene transcription. Expression of Runx2, Brf1 and Pol III genes from the samples of human breast cancer and cell culture model were determined by the assays of RT-qPCR, immunoblot, luciferase reporter activity, immunohistochemistry, chromatin immunoprecipitation and Immunofluorescence. High expression of Runx2 is observed in the cases of breast cancer. The patients of high Runx2 expression at early stages display longer survival period, whereas the cases of high Runx2 at advanced stages reveal faster recurrence. The identification of signaling pathway indicates that JNK1 and c-Jun mediate Runx2 transcription. Repression of Runx2 reduces Brf1 expression and Pol III gene transcription. Further analysis indicates that Runx2 is colocalized with Brf1 in nucleus of breast cancer tissue. Both Runx2 and Brf1 synergistically modulate Pol III gene transcription. These studies indicate that Brf1 overexpression is able to be used as an early diagnosis biomarker of breast cancer, while high Runx2 expression indicates long survival period and faster recurrence. Runx2 mediates the deregulation of Brf1 and Pol III genes and its abnormal expression predicts the worse prognosis of breast cancer.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Etanol/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , RNA Polimerase III/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Transcrição Genética/efeitos dos fármacos , Adulto , Idoso , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Invasividade Neoplásica , Transdução de Sinais , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Ensaio Tumoral de Célula-Tronco , Regulação para Cima/genética , Adulto Jovem
15.
Orthop Surg ; 12(2): 668-678, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32154660

RESUMO

OBJECTIVES: This research aimed to investigate the relative level of Runt-related transcription factor 2 (RUNX2) in giant cell tumor of bone (GCTB). Through the histopathological similarities between osteoporosis and GCTB, the biological functions of exogenous RUNXS were demonstrated in GCTB cell lines. This generated awareness of the molecular mechanism of the biogenesis and metastasis of GCTB, as well as showing the pathways and processes involved in this study. This research also expected to provide hints for the clinical treatment of patients with GCTB, to release the tumor burden and reduce the recurrence rate and metastasis of patients with this condition. METHODS: The expression of RUNX2 in the tumors was verified by Western Blot, qRT-PCR and immunohistochemistry, compared with the normal tissues' adjacent tumors. Subsequently, the plasmids expressing RUNX2 were constructed, amplified and transfected into the 0404 cell line through transfection kits (0.4, 0.8, 1.6, 2.4 ng/µl). After that, the proliferation, migration, invasion, cellular viability and apoptosis of 0404 cell lines were examined by EDU assay, wound healing assay, transwell assay, annexin v staining, and CCK8 assay, respectively. RESULTS: The messenger RNA (mRNA) level of RUNX2 in tumors was over 100 folds more than the normal tissues. The protein level of tumors upregulated 8.32(±4.41) folds relatively. After the transfection of RUNX2 overexpressed plasmids into the 0404 cell line, the mRNA level of RUNX2 increased approximately 530.11(±24.87), 1117.96(±77.68), 2835.09(±45.22) and 4781.51(±79.37) folds respectively, and the protein level was upregulated about 4.12(±1.15), 16.73(±1.63), 21.53(±2.41) and 23.39(±0.85) folds respectively. The proliferation of 0404 cells was inhibited by 2.13(±1.02)% of 1.6 ng/µl group and 3.03(±1.76)% of 2.4 ng/µl group. And the migration was inhibited about 45.56(±6.13)%, 50.79(±5.27)%, 63.15(±8.62)% and 93.90(±3.65)% respectively. The invasion was decreased about 14.49(±5.4)%, 37.02(±6.52)%, 42.24(±2.59)% and 48.97(±10.61)% respectively. Meanwhile, FITC Annexin V/PI apoptosis assay demonstrated that RUNX2 plasmids could promote apoptosis rate around 4.15(±0.27)%, 5.07(±0.27)%, 7.61(±0.45)% and 11.32(±1.02)% respectively, and CCK8 proved these plasmids could weaken cellular viability in a concentration-dependent manner with the time passing. CONCLUSIONS: RUNX2 is highly expressed in giant cell tumors of bone. The RUNX2 overexpressed plasmids we constructed could be successfully transfected into 0404 cell line. Far more importantly, the exogenous RUNX2 can seriously block the biological functions of 0404 cell line in a concentration-dependent manner, including proliferation, translocation, invasion, cellular viability, and apoptosis. Meanwhile, the mechanism was hypothesized and discussed in the article.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Tumor de Células Gigantes do Osso/metabolismo , Adolescente , Adulto , Apoptose , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Plasmídeos , Transfecção , Adulto Jovem
16.
Int J Mol Sci ; 21(4)2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-32075221

RESUMO

Human cementum protein 1 (CEMP1) is known to induce cementoblast and osteoblast differentiation and alkaline phosphatase (ALP) activity in human periodontal ligament-derived cells in vitro and promotes bone regeneration in vivo. CEMP1's secondary structure analysis shows that it has a random-coiled structure and is considered an Intrinsic Disordered Protein (IDP). CEMP1's short peptide sequences mimic the biological capabilities of CEMP1. However, the role and mechanisms of CEMP1's C-terminal-derived synthetic peptide (CEMP1-p4) in the canonical Wnt/ß-catenin signaling pathway are yet to be described. Here we report that CEMP1-p4 promotes proliferation and differentiation of Human Oral Mucosa Stem Cells (HOMSCs) by activating the Wnt/ß-catenin pathway. CEMP1-p4 stimulation upregulated the expression of ß-catenin and glycogen synthase kinase 3 beta (GSK-3B) and activated the transcription factors TCF1/7 and Lymphoid Enhancer binding Factor 1 (LEF1) at the mRNA and protein levels. We found translocation of ß-catenin to the nucleus in CEMP1-p4-treated cultures. The peptide also penetrates the cell membrane and aggregates around the cell nucleus. Analysis of CEMP1-p4 secondary structure revealed that it has a random-coiled structure. Its biological activities included the induction to nucleate hydroxyapatite crystals. In CEMP1-p4-treated HOMSCs, ALP activity and calcium deposits increased. Expression of Osterix (OSX), Runt-related transcription factor 2 (RUNX2), Integrin binding sialoproptein (IBSP) and osteocalcin (OCN) were upregulated. Altogether, these data show that CEMP1-p4 plays a direct role in the differentiation of HOMSCs to a "mineralizing-like" phenotype by activating the ß-catenin signaling cascade.


Assuntos
Mucosa Bucal/crescimento & desenvolvimento , Osteogênese/genética , Ligamento Periodontal/crescimento & desenvolvimento , Proteínas/química , Células-Tronco/citologia , Regeneração Óssea/genética , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Cemento Dentário/metabolismo , Durapatita/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Sialoproteína de Ligação à Integrina/genética , Mucosa Bucal/citologia , Mucosa Bucal/metabolismo , Osteoblastos/metabolismo , Osteocalcina/genética , Peptídeos/química , Peptídeos/genética , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Estrutura Secundária de Proteína , Proteínas/genética , Proteínas/ultraestrutura , Fator de Transcrição Sp7/genética , Células-Tronco/metabolismo , Via de Sinalização Wnt/genética
17.
Arthritis Rheumatol ; 72(7): 1123-1133, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32067417

RESUMO

OBJECTIVE: To investigate the effects of a young systemic environment and growth differentiation factor 11 (GDF-11) on aging cartilage. METHODS: A heterochronic parabiosis model (2-month-old mouse and 12-month-old mouse [Y/O]), an isochronic parabiosis model (12-month-old mouse and 12-month-old mouse [O/O]), and 12-month-old mice alone (O) were evaluated. Knee joints and chondrocytes from old mice were examined by radiography, histology, cell proliferation assays, immunohistochemistry, Western blotting, and quantitative reverse transcriptase-polymerase chain reaction 16 weeks after parabiosis surgery. GDF-11 was injected into 12-month-old mouse joints daily for 16 weeks. Cartilage degeneration, cell proliferation, and osteoarthritis-related gene expression were evaluated. RESULTS: Osteoarthritis Research Society International scores in old mice were significantly lower in the Y/O group than in the O/O and O groups (both P < 0.05). The percentage of 5-ethynyl-2'-deoxyuridine-positive chondrocytes in old mice was significantly higher in the Y/O group than in the other groups (P < 0.05). Type II collagen (CII) and SOX9 messenger RNA levels differed in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). RUNX-2, CX, and matrix metalloproteinase 13 levels were significantly lower in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). Similar results were obtained for protein expression levels and after GDF-11 treatment in vitro and in vivo. Phosphorylated Smad2/3 (pSmad2/3) levels were higher in the recombinant GDF-11-treated group than in the control group. CONCLUSION: A young systemic environment promotes chondrocyte proliferation and cartilage matrix synthesis in old mice. GDF-11, a "young factor," contributes to these effects through the up-regulation of pSmad2/3.


Assuntos
Envelhecimento/genética , Proteínas Morfogenéticas Ósseas/farmacologia , Cartilagem Articular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Fatores de Diferenciação de Crescimento/farmacologia , Osteoartrite do Joelho/genética , Parabiose , Adolescente , Idoso , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Artroplastia do Joelho , Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Colágeno Tipo II/efeitos dos fármacos , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo X/efeitos dos fármacos , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Fatores de Diferenciação de Crescimento/metabolismo , Humanos , Técnicas In Vitro , Articulação do Joelho , Masculino , Metaloproteinase 13 da Matriz/efeitos dos fármacos , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Osteoartrite do Joelho/metabolismo , Fosforilação , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/efeitos dos fármacos , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Proteína Smad2/efeitos dos fármacos , Proteína Smad2/metabolismo , Proteína Smad3/efeitos dos fármacos , Proteína Smad3/metabolismo , Joelho de Quadrúpedes , Adulto Jovem
18.
Sci Rep ; 10(1): 3078, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080264

RESUMO

The highly selective magnesium transporter non-imprinted in Prader-Willi/Angelman syndrome region protein 2 (NIPA2) has recently been associated with the development and progression of type 2 diabetes osteoporosis, but the mechanisms involved are still poorly understood. Because mitophagy is involved in the pathology of type 2 diabetes osteoporosis, the present study aimed to explore the relationship among NIPA2, mitophagy and osteoblast osteogenic capacity. NIPA2 expression was reduced in C57BKS background db/db mice and in vitro models of type 2 diabetes osteoporosis, and the activation of mitophagy in primary culture osteoblast-derived from db/db mice and in high glucose-treated human fetal osteoblastic cells (hFOB1.19) was observed. Knockdown, overexpression of NIPA2 and pharmacological inhibition of peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) showed that NIPA2 increased osteoblast function, which was likely regulated by PTEN induced kinase 1 (PINK1)/E3 ubiquitin ligase PARK2 (Parkin)-mediated mitophagy via the PGC-1α/forkhead box O3a(FoxO3a)/mitochondrial membrane potential (MMP) pathway. Furthermore, the negative effect of mitophagy on osteoblast function was confirmed by pharmacological regulation of mitophagy and knockdown of Parkin. Taken together, these results suggest that NIPA2 positively regulates the osteogenic capacity of osteoblasts via the mitophagy pathway in type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Mitofagia , Osteoblastos/metabolismo , Osteoporose/metabolismo , Osteoporose/patologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Osso e Ossos/patologia , Proteínas de Transporte de Cátions/metabolismo , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Diabetes Mellitus Tipo 2/complicações , Regulação para Baixo/efeitos dos fármacos , Proteína Forkhead Box O3/metabolismo , Glucose/toxicidade , Humanos , Magnésio/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mitofagia/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Osteoblastos/ultraestrutura , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Osteoporose/complicações , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos
19.
Cancer Genomics Proteomics ; 17(2): 161-168, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32108038

RESUMO

BACKGROUND/AIM: Osteoblastoma is a rare benign tumor of the bones in which recurrent rearrangements of FOS have been found. Our aim was to investigate two osteoblastomas for possible genetic aberrations. MATERIALS AND METHODS: Cytogenetic, RNA sequencing, and molecular analyses were performed. RESULTS: A FOS-ANKH transcript was found in the first tumor, whereas a FOS-RUNX2 was detected in the second. Exon 4 of FOS fused with sequences either from intron 1 of ANKH or intron 5 of RUNX2. The fusion events introduced a stop codon and removed sequences involved in the regulation of FOS. CONCLUSION: Rearrangements and fusions of FOS show similarities with those of HMGA2 (a feature of leiomyomas and lipomas) and CSF1 (tenosynovial giant cell tumors). The replacement of a 3'-untranslated region, controlling the gene's expression, by a new sequence is thus a common pathogenetic theme shared by FOS, HMGA2, and CSF1 in many benign connective tissue tumors.


Assuntos
Neoplasias Ósseas/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Osteoblastoma/genética , Proteínas de Transporte de Fosfato/genética , Sequência de Bases , Neoplasias Ósseas/metabolismo , Criança , Subunidade alfa 1 de Fator de Ligação ao Core/deficiência , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Expressão Gênica , Humanos , Cariótipo , Masculino , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Osteoblastoma/metabolismo , Proteínas de Transporte de Fosfato/metabolismo
20.
Vascular ; 28(4): 465-474, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32089109

RESUMO

OBJECTIVE: Calcification serves as a surrogate for atherosclerosis-associated vascular diseases, and coronary artery calcification is mediated by multiple pathogenic factors. Estrogen is a known factor that protects the arterial wall against atherosclerosis, but its role in the coronary artery calcification development remains largely unclear. This study tested the hypothesis that estrogen inhibits coronary artery calcification via the hypoxia-induced factor-1α pathway. METHODS: Eight-week-old healthy female Sprague-Dawley rats were castrated, and vitamin D3 was administered orally to establish. Hypoxia-induced factor-1 inhibitor was administered to test its effect on vascular calcification and expression of bone morphogenetic protein 2 and runt-related transcription factor-2. Vascular smooth muscle cell calcification was induced with CaCl2 in rat aortic smooth muscle cells in the presence or absence of E2(17ß-estradiol) and bone morphogenetic protein 2 siRNA intervention. RESULTS: The estrogen levels in ovariectomized rats were significantly decreased, as determined by ELISA. Expression of hypoxia-induced factor-1α mRNA and protein was significantly increased in vascular cells with calcification as compared to those without calcification (p < 0.01). E2 treatment decreased the calcium concentration in vascular cell calcification and cell calcium nodules in vitro (p < 0.05). E2 also lowered the levels of hypoxia-induced factor-1α mRNA and protein (p < 0.01). Oral administration of the hypoxia-induced factor-1α inhibitor dimethyloxetane in castrated rats alleviated vascular calcification and expression of osteogenesis-related transcription factors, bone morphogenetic protein 2 and RUNX2 (p < 0.01). Finally, bone morphogenetic protein 2 siRNA treatment decreased the levels of p-Smad1/5/8 in A7r5 calcification cells (p < 0.01). CONCLUSION: Estrogen deficiency enhances vascular calcification. Treatment with estrogen reduces the expression of hypoxia-induced factor-1α as well as vascular calcification in rats. The estrogen effects occur in a fashion dependent on hypoxia-induced factor-1α regulation of bone morphogenetic protein-2 and downstream Smad1/5/8.


Assuntos
Doenças da Aorta/prevenção & controle , Estradiol/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Calcificação Vascular/prevenção & controle , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Modelos Animais de Doenças , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Ovariectomia , Fosforilação , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas Smad Reguladas por Receptor/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia
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