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1.
J Oral Sci ; 62(4): 423-426, 2020 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-32863319

RESUMO

PURPOSE: Diabetes causes hyperglycemic disorders due to insufficient activity of insulin, and it also increases blood glucose level. Recent studies have reported the relationship between diabetes and periodontal disease. Periodontitis is advanced by inflammatory cytokines stimulated with LPS. The purpose of this study was to investigate the effects of hyperglycemia on the expression of inflammatory cytokines induced by LPS in osteoblasts. METHODS: Cells were cultured for 7 and 14 days in the presence or absence of LPS and glucose. The expression mRNA level of IL-6, RANKL and OCN was determined using real-time PCR. The protein expression of IL-6 and RANKL was also measured using ELISA. RESULTS: LPS and glucose increased the mRNA expression of IL-6, coupled with a decrease in the mRNA expression of OCN, which is associated with IL-6 and glucose. It also increased the protein expression of IL-6 compared to LPS. However, LPS+Glucose did not affect the mRNA and protein expression of RANKL. Furthermore, GLUT4 inhibitor, WZB117, blocked the stimulatory effect of glucose on LPS-induced IL-6 mRNA expression. WZB117 did not affect LPS-reduced OCN mRNA expression. CONCLUSION: These results suggest that high glucose levels increase LPS-induced IL-6 expression mediated by GLUT4.


Assuntos
Transportador de Glucose Tipo 4/fisiologia , Interleucina-6 , Lipopolissacarídeos , Glucose , Proteínas Facilitadoras de Transporte de Glucose , Interleucina-6/metabolismo , Osteoblastos/metabolismo
2.
PLoS One ; 15(9): e0237463, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32970688

RESUMO

Titanium is essentially absent from biological systems yet reliably integrates into bone. To achieve osseointegration, titanium must activate biological processes without entering cells, defining it as a bio-activating material. Nanostructuring bulk titanium reduces grain size, increases strength, and improves other quantifiable physical properties, including cytocompatibility. The biological processes activated by increasing grain boundary availability were detected with total RNA-sequencing in mouse pre-osteoblasts grown for 72 hours on nanometrically smooth substrates of either coarse grain or nanostructured ultrafine grain titanium. The average grain boundary length under cells on the conventional coarse grain substrates is 273.0 µm, compared to 70,881.5 µm for cells adhered to the nanostructured ultrafine grain substrates; a 260-fold difference. Cells on both substrates exhibit similar expression profiles for genes whose products are critical for mechanosensation and transduction of cues that trigger osteoconduction. Biological process Gene Ontology term enrichment analysis of differentially expressed genes reveals that cell cycle, chromatin modification, telomere maintenance, and RNA metabolism processes are upregulated on ultrafine grain titanium. Processes related to immune response, including apoptosis, are downregulated. Tumor-suppressor genes are upregulated while tumor-promoting genes are downregulated. Upregulation of genes involved in chromatin remodeling and downregulation of genes under the control of the peripheral circadian clock implicate both processes in the transduction of mechanosensory information. Non-coding RNAs may also play a role in the response. Merging transcriptomics with well-established mechanobiology principles generates a unified model to explain the bio-activating properties of titanium. The modulation of processes is accomplished through chromatin remodeling in which the nucleus responds like a rheostat to grain boundary concentration. This convergence of biological and materials science reveals a pathway toward understanding the biotic-abiotic interface and will inform the development of effective bio-activating and bio-inactivating materials.


Assuntos
Materiais Biocompatíveis/química , Regeneração Óssea , Nanoestruturas/química , Osteoblastos/citologia , Titânio/química , Animais , Linhagem Celular , Teste de Materiais , Mecanotransdução Celular , Camundongos , Osseointegração , Osteoblastos/metabolismo , Análise de Sequência de RNA , Propriedades de Superfície , Transcriptoma
3.
Acta Odontol Latinoam ; 33(2): 125, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32920615

RESUMO

Melatonin (MLT) is a potential signaling molecule in the homeostasis of bone metabolism and may be an important mediator of bone formation and stimulation. The aim of this in vitro study was to evaluate the effect of MLT on the viability, mRNA/protein expression and mineralization of pre-osteoblastic cells. The concentrations 5, 2.5, 1, 0.1 and 0.01 mM MLT were tested on pre-osteoblastic cells (MC3T3) compared to control (no MLT), evaluating proliferation and cell viability (C50), gene expression (RT-PCR) and secretion (ELISA) of COL-I and OPN at 24h, 48h and 72h, and the formation of mineral nodules (alizarin red and fast red) after 10 days of treatment. MLT at 5 and 2.5 mM proved to be cytotoxic (C50), so only 0.01, 0.1 and 1 mM were used for the subsequent analyses. OPN mRNA expression increased with MLT at 0.1 mM - 1 mM, which was followed by increased secretion of OPN both at 24h and 72h compared to the remaining groups (p <0.05). COL-I mRNA and COL-1 secretion followed the same pattern as OPN at 0.1 mM MLT at 72h of treatment (p <0.05). Regarding mineralization, all MLT doses (except 1mM) caused an increase (p <0.05) in the formation of mineral nodules compared to the control. Melatonin at 0.01mM - 1mM had a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/ secretion and mineralization, thereby fostering osteogenesis.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Metaloproteinase 2 da Matriz/metabolismo , Melatonina/farmacologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Osteopontina/metabolismo , Fragmentos de Peptídeos/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/genética , Osteoblastos/metabolismo , Osteopontina/genética , Fragmentos de Peptídeos/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real
4.
Gene ; 763: 145059, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32858177

RESUMO

Osteopontin (OPN) is not only a marker of osteoblasts but it is also related to cancer progression and inflammation. The expression of OPN increases in response to inflammatory cytokines, hormones, and mechanical stress. Among them, cyclic-AMP (cAMP) elevating agents stimulate OPN expression in the presence of 1, 25-OH vitamin D3 (VD3). We aimed to clarify the mechanism by which cAMP enhances OPN expression in osteoblastic cells. The OPN promoter (-2335 to +76, OPNp2335) exerted a cell type specific response to forskolin (FK) and VD3. Sequential deletion analysis of OPNp revealed that the OPNp (-833 to +76) contained essential responsive regions to respond to cAMP signaling. In particular, both Vitamin D response element (VDRE, -758 to -743) and osteoblast-specific cis- acting element 2 (OSE2, -695 to -690) were essential for cAMP-mediated OPNp activity. The expression of vitamin D receptor (VDR), but not runt-related transcription factor 2 (Runx2), a nuclear receptor for OSE2, was induced by the treatment of the cells with FK. Although, VD3-induced OPNp activity was slightly enhanced in VDR-overexpressing osteoblasts, it reached the same level as that of osteoblasts induced by both VD3 and FK in the presence of histone deacetylase (HDAC) inhibitor. Moreover, we identified histone acetylation on the OPN promoter region by FK treatment. These results strongly suggest that OPNp activity is controlled by the cAMP signaling via genetic and epigenetic regulations.


Assuntos
AMP Cíclico/metabolismo , Epigênese Genética , Osteoblastos/metabolismo , Osteopontina/genética , Acetilação , Animais , Células HEK293 , Código das Histonas , Humanos , Camundongos , Osteopontina/química , Osteopontina/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Vitamina D/metabolismo
5.
Life Sci ; 258: 118195, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32781073

RESUMO

AIMS: The estrogen-ERα axis participates in osteoblast maturation. This study was designed to further evaluated the roles of the estrogen-ERα axis in bone healing and the possible mechanisms. MAIN METHODS: Female ICR mice were created a metaphyseal bone defect in the left femurs and administered with methylpiperidinopyrazole (MPP), an inhibitor of ERα. Bone healing was evaluated using micro-computed tomography. Colocalization of ERα with alkaline phosphatase (ALP) and ERα translocation to mitochondria were determined. Levels of ERα, ERß, PECAM-1, VEGF, and ß-actin were immunodetected. Expression of chromosomal Runx2, ALP, and osteocalcin mRNAs and mitochondrial cytochrome c oxidase (COX) I and COXII mRNAs were quantified. Angiogenesis was measured with immunohistochemistry. KEY FINDINGS: Following surgery, the bone mass was time-dependently augmented in the bone-defect area. Simultaneously, levels of ERα were specifically upregulated and positively correlated with bone healing. Administration of MPP to mice consistently decreased levels of ERα and bone healing. As to the mechanisms, osteogenesis was enhanced in bone healing, but MPP attenuated osteoblast maturation. In parallel, expressions of osteogenesis-related ALP, Runx2, and osteocalcin mRNAs were induced in the injured zone. Treatment with MPP led to significant inhibition of the alp, runx2, and osteocalcin gene expressions. Remarkably, administration of MPP lessened translocation of ERα to mitochondria and expressions of mitochondrial energy production-related coxI and coxII genes. Furthermore, exposure to MPP decreased levels of PECAM-1 and VEGF in the bone-defect area. SIGNIFICANCE: The present study showed the contributions of the estrogen-ERα axis to bone healing through stimulation of energy production, osteoblast maturation, and angiogenesis.


Assuntos
Regeneração Óssea , Diferenciação Celular , Metabolismo Energético , Receptor alfa de Estrogênio/metabolismo , Neovascularização Fisiológica , Osteoblastos/citologia , Transdução de Sinais , Fosfatase Alcalina/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/patologia , Diferenciação Celular/efeitos dos fármacos , Cromossomos de Mamíferos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Regulação para Cima/efeitos dos fármacos , Cicatrização/efeitos dos fármacos
6.
PLoS Biol ; 18(8): e3000792, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745129

RESUMO

A ubiquitous feature of the circadian clock across life forms is its organization as a network of cellular oscillators, with individual cellular oscillators within the network often exhibiting considerable heterogeneity in their intrinsic periods. The interaction of coupling and heterogeneity in circadian clock networks is hypothesized to influence clock's entrainability, but our knowledge of mechanisms governing period heterogeneity within circadian clock networks remains largely elusive. In this study, we aimed to explore the principles that underlie intercellular period variation in circadian clock networks (clonal period heterogeneity). To this end, we employed a laboratory selection approach and derived a panel of 25 clonal cell populations exhibiting circadian periods ranging from 22 to 28 h. We report that a single parent clone can produce progeny clones with a wide distribution of circadian periods, and this heterogeneity, in addition to being stochastically driven, has a heritable component. By quantifying the expression of 20 circadian clock and clock-associated genes across our clone panel, we found that inheritance of expression patterns in at least three clock genes might govern clonal period heterogeneity in circadian clock networks. Furthermore, we provide evidence suggesting that heritable epigenetic variation in gene expression regulation might underlie period heterogeneity.


Assuntos
Proteínas CLOCK/genética , Relógios Circadianos/genética , Ritmo Circadiano/genética , Epigênese Genética , Redes Reguladoras de Genes , Animais , Proteínas CLOCK/metabolismo , Linhagem Celular Tumoral , Células Clonais , Perfilação da Expressão Gênica , Genes Reporter , Heterogeneidade Genética , Humanos , Padrões de Herança , Luciferases/genética , Luciferases/metabolismo , Camundongos , Células NIH 3T3 , Osteoblastos/citologia , Osteoblastos/metabolismo , Processos Estocásticos
7.
Mol Cell ; 79(3): 425-442.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32615088

RESUMO

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.


Assuntos
Adenosina/análogos & derivados , Neoplasias de Cabeça e Pescoço/genética , Metiltransferases/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Células HEK293 , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Hibridização de Ácido Nucleico , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Ecotoxicol Environ Saf ; 203: 110930, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32684523

RESUMO

Benzo[a]pyrene(BaP), a polycyclic aromatic hydrocarbons (PAH) of environmental pollutants, is one of the main ingredients in cigarettes and an agonist of the aryl hydrocarbon receptor (AhR). Mesenchymal stem cells (MSCs) including C3H10T1/2 and MEF cells, adult multipotent stem cells, can be differentiated toward osteoblasts during the induction of osteogenic induction factor-bone morphogenetic protein 2(BMP2). Accumulating evidence suggests that BaP decreases bone development in mammals, but the further mechanisms of BaP on BMP2-induced bone formation involved are unknown. Here, we researched the role of BaP on BMP2-induced osteoblast differentiation and bone formation. We showed that BaP significantly suppressed early and late osteogenic differentiation, and downregulated the runt-related transcription factor 2(Runx2), osteocalcin(OCN) and osteopontin (OPN) during the induction of BMP2 in MSCs. Consistent with in vitro results, administration of BaP inhibited BMP2-induced subcutaneous ectopic osteogenesis in vivo. Interestingly, blocking AhR reversed the inhibition of BaP on BMP2-induced osteogenic differentiation, which suggested that AhR played an important role in this process. Moreover, BaP significantly decreased BMP2-induced Smad1/5/8 phosphorylation. Furthermore, BaP significantly reduced bone morphogenetic protein receptor 2(BMPRII) expression and excessively activated Hey1. Thus, our data demonstrate the role of BaP in BMP2-induced bone formation and suggest that impaired BMP/Smad pathways through AhR regulating BMPRII and Hey1 may be an underlying mechanism for BaP inhibiting BMP2-induced osteogenic differentiation.


Assuntos
Benzo(a)pireno/toxicidade , Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Benzo(a)pireno/metabolismo , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células HCT116 , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Nus , Osteoblastos/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(32): 19276-19286, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719141

RESUMO

Bone homeostasis requires continuous remodeling of bone matrix to maintain structural integrity. This involves extensive communication between bone-forming osteoblasts and bone-resorbing osteoclasts to orchestrate balanced progenitor cell recruitment and activation. Only a few mediators controlling progenitor activation are known to date and have been targeted for intervention of bone disorders such as osteoporosis. To identify druggable pathways, we generated a medaka (Oryzias latipes) osteoporosis model, where inducible expression of receptor-activator of nuclear factor kappa-Β ligand (Rankl) leads to ectopic formation of osteoclasts and excessive bone resorption, which can be assessed by live imaging. Here we show that upon Rankl induction, osteoblast progenitors up-regulate expression of the chemokine ligand Cxcl9l. Ectopic expression of Cxcl9l recruits mpeg1-positive macrophages to bone matrix and triggers their differentiation into osteoclasts. We also demonstrate that the chemokine receptor Cxcr3.2 is expressed in a distinct subset of macrophages in the aorta-gonad-mesonephros (AGM). Live imaging revealed that upon Rankl induction, Cxcr3.2-positive macrophages get activated, migrate to bone matrix, and differentiate into osteoclasts. Importantly, mutations in cxcr3.2 prevent macrophage recruitment and osteoclast differentiation. Furthermore, Cxcr3.2 inhibition by the chemical antagonists AMG487 and NBI-74330 also reduced osteoclast recruitment and protected bone integrity against osteoporotic insult. Our data identify a mechanism for progenitor recruitment to bone resorption sites and Cxcl9l and Cxcr3.2 as potential druggable regulators of bone homeostasis and osteoporosis.


Assuntos
Matriz Óssea/metabolismo , Quimiocina CXCL9/metabolismo , Proteínas de Peixes/metabolismo , Oryzias/metabolismo , Osteoclastos/metabolismo , Osteoporose/metabolismo , Receptores CXCR3/metabolismo , Células-Tronco/metabolismo , Animais , Matriz Óssea/crescimento & desenvolvimento , Diferenciação Celular , Quimiocina CXCL9/genética , Modelos Animais de Doenças , Proteínas de Peixes/genética , Humanos , Macrófagos/metabolismo , Oryzias/genética , Oryzias/crescimento & desenvolvimento , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoporose/genética , Osteoporose/fisiopatologia , Ligação Proteica , Receptores CXCR3/genética , Células-Tronco/citologia
10.
Life Sci ; 257: 118044, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32622944

RESUMO

AIMS: High-dose glucocorticoid (GC) administration causes osteoporosis. Many previous studies from our group and other groups have shown that melatonin participates in the regulation of osteoblast proliferation and differentiation, especially low concentrations of melatonin, which enhance osteoblast osteogenesis. However, the role of melatonin in glucocorticoid-induced osteoblast differentiation remains unknown. MATERIALS AND METHODS: An examination of the expression of osteoblast differentiation markers (ALP, OCN, COLL-1), as well as alkaline phosphatase staining and alkaline phosphatase enzymatic activity assay to measure osteoblast differentiation and quantifying Alizarin red S staining to measure mineralization, were performed to determine the effects of dexamethasone (Dex) and melatonin on the differentiation of MC3T3-E1 cells. We used immunofluorescence staining to detect the expression of Runx2 in melatonin-treated MC3T3-E1 cells. The expression of mRNA was determined by qRT-PCR, and protein levels were measured by western blotting. KEY FINDINGS: In the present study, we found that 100 µM Dex significantly reduced osteoblast differentiation and mineralization in MC3T3-E1 cells and that 1 µM melatonin attenuated these inhibitory effects. We found that only inhibition of PI3K/AKT (MK2206) and BMP/Smad (LDN193189) signalling abolished melatonin-induced differentiation and mineralization. Meanwhile, MK2206 decreased the expression of P-AKT and P-Smad1/5/9 and LDN193189 decreased the expression of P-Smad1/5/9 but had no obvious effect on P-AKT expression in melatonin-treated and Dex-induced MC3T3-E1 cells. SIGNIFICANCE: These findings suggest that melatonin rescues Dex-induced inhibition of osteoblast differentiation in MC3T3-E1 cells via the PI3K/AKT and BMP/Smad signalling pathways and that PI3K/AKT signalling may be the upstream signal of BMP/Smad signalling.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Melatonina/metabolismo , Osteoblastos/metabolismo , Animais , Biomineralização/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Linhagem Celular , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/efeitos adversos , Glucocorticoides/farmacologia , Melatonina/farmacologia , Camundongos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(29): 17187-17194, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32636266

RESUMO

Osteoprotegerin (OPG), a secreted decoy receptor for receptor activator of nuclear factor B ligand (RANKL), plays an essential role in regulating bone resorption. While much is known about the function of the N-terminal domains of OPG, which is responsible for binding to RANKL, the exact biological functions of the three C-terminal domains of OPG remain uncertain. We have previously shown that one likely function of the C-terminal domains of OPG is to bind cell surface heparan sulfate (HS), but the in vivo evidence was lacking. To investigate the biological significance of OPG-HS interaction in bone remodeling, we created OPG knock-in mice (opg AAA ). The mutated OPG is incapable of binding to HS but binds RANKL normally. Surprisingly, opg AAA/AAA mice displayed a severe osteoporotic phenotype that is very similar to opg-null mice, suggesting that the antiresorption activity of OPG requires HS. Mechanistically, we propose that the HS immobilizes secreted OPG at the surface of osteoblasts lineage cells, which facilitates binding of OPG to membrane-anchored RANKL. To further support this model, we altered the structure of osteoblast HS genetically to make it incapable of binding to OPG. Interestingly, osteocalcin-Cre;Hs2st f/f mice also displayed osteoporotic phenotype with similar severity to opg AAA/AAA mice. Combined, our data provide strong genetic evidence that OPG-HS interaction is indispensable for normal bone homeostasis.


Assuntos
Conservadores da Densidade Óssea/metabolismo , Conservadores da Densidade Óssea/farmacologia , Heparitina Sulfato/metabolismo , Osteoprotegerina/metabolismo , Osteoprotegerina/farmacologia , Animais , Sítios de Ligação , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Osso e Ossos/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Osteogênese/fisiologia , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Osteoprotegerina/genética , Ligante RANK/metabolismo , Transcriptoma
12.
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
13.
Life Sci ; 256: 117964, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32534036

RESUMO

AIMS: Vascular smooth muscle cells (VSMCs) are important regulators of vascular functions and their conversion to osteoblasts is a key to development of vascular calcification. This study aimed to characterize in vitro effect of hepatoma-derived growth factor (HDGF) on phenotypic conversion of cultured aortic VSMCs into osteoblast-like cells. MATERIALS AND METHODS: Cell proliferation and migration assays were used to examine cell behaviors. Western blotting, alkaline phosphatase activity and calcium staining were used to evaluate osteoblastic marker expression and function, respectively. KEY FINDINGS: Recombinant HDGF treatment enhanced VSMC growth and motility. Treatment of osteogenic medium (OM) increased expression of not only HDGF but also osteoblastic markers, including Runx2 and osteopontin (OPN), while VSMC marker α-smooth muscle actin (α-SMA) declined. Coincidentally, HDGF and OM treatment alone stimulated signaling activities in both PI3K/Akt and MAPK pathways. Conversely, inhibition of Akt and p38 significantly blocked the OM-upregulated HDGF, Runx2, and OPN expression and NF-κB phosphorylation, but did not reversed the α-SMA downregulation, implicating the involvement of Akt and p38 activities in the osteoblastic transformation of VSMCs. Small interfering RNA-mediated HDGF gene silencing effectively prevented the Runx2 and OPN upregulation, alkaline phosphatase activation, and calcium deposition, but did not affect the α-SMA levels in the transformed cells, supporting the involvement of HDGF in regulation of Runx2 and OPN expression. SIGNIFICANCE: In conclusion, in synergism with other osteogenic factor, HDGF may promote the progression of osteobastic transformation of VSMCs via Akt and p38 signaling pathways and contribute to vascular calcification in arteriosclerosis. CHEMICAL COMPOUNDS STUDIED IN THIS STUDY: HDGF (PubChem CID:); LY294002 (PubChem CID: 3973); PD98059 (PubChem CID: 4713); SB203580 (PubChem CID: 176155); SB431542 (PubChem CID: 4521392); SP600125 (PubChem CID: 8515); Wortmannin (PubChem CID: 312145).


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Osteoblastos/citologia , Animais , Biomarcadores/metabolismo , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Inativação Gênica/efeitos dos fármacos , Cinética , Miócitos de Músculo Liso/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteopontina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
PLoS Genet ; 16(6): e1008805, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32497039

RESUMO

Osteoporosis is a genetic disease characterized by progressive reductions in bone mineral density (BMD) leading to an increased risk of fracture. Over the last decade, genome-wide association studies (GWASs) have identified over 1000 associations for BMD. However, as a phenotype BMD is challenging as bone is a multicellular tissue affected by both local and systemic physiology. Here, we focused on a single component of BMD, osteoblast-mediated bone formation in mice, and identified associations influencing osteoblast activity on mouse Chromosomes (Chrs) 1, 4, and 17. The locus on Chr. 4 was in an intergenic region between Wnt4 and Zbtb40, homologous to a locus for BMD in humans. We tested both Wnt4 and Zbtb40 for a role in osteoblast activity and BMD. Knockdown of Zbtb40, but not Wnt4, in osteoblasts drastically reduced mineralization. Additionally, loss-of-function mouse models for both genes exhibited reduced BMD. Our results highlight that investigating the genetic basis of in vitro osteoblast mineralization can be used to identify genes impacting bone formation and BMD.


Assuntos
Densidade Óssea/genética , Proteínas de Ligação a DNA/fisiologia , Osteoblastos/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteogênese/genética , Proteína Wnt4/genética
15.
Am J Physiol Cell Physiol ; 319(2): C345-C358, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32520608

RESUMO

The maturity of osteoblasts by proliferation and differentiation in preosteoblasts is essential for maintaining bone homeostasis. The beneficial effects of vitamin D on bone homeostasis in mammals have been demonstrated experimentally and clinically. However, the direct actions of vitamin D on preosteoblasts remain to be fully elucidated. In this study, we found that the functional activity of intermediate-conductance Ca2+-activated K+ channels (KCa3.1) positively regulated cell proliferation in MC3T3-E1 cells derived from mouse preosteoblasts by enhancing intracellular Ca2+ signaling. We examined the effects of treatment with vitamin D receptor (VDR) agonist on the expression and activity of KCa3.1 by real-time PCR examination, Western blotting, Ca2+ imaging, and patch clamp analyses in mouse MC3T3-E1 cells. Following the downregulation of KCa3.1 transcriptional modulators such as Fra-1 and HDAC2, KCa3.1 activity was suppressed in MC3T3-E1 cells treated with VDR agonists. Furthermore, application of the KCa3.1 activator DCEBIO attenuated the VDR agonist-evoked suppression of cell proliferation rate. These findings suggest that a decrease in KCa3.1 activity is involved in the suppression of cell proliferation rate in VDR agonist-treated preosteoblasts. Therefore, KCa3.1 plays an important role in bone formation by promoting osteoblastic proliferation under physiological conditions.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Osteoblastos/metabolismo , Receptores de Calcitriol/genética , Vitamina D/genética , Células 3T3 , Animais , Benzimidazóis/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/genética , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Histona Desacetilase 2/genética , Humanos , Camundongos , Osteoblastos/citologia , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Técnicas de Patch-Clamp , Proteínas Proto-Oncogênicas c-fos/genética , Receptores de Calcitriol/agonistas , Transdução de Sinais/efeitos dos fármacos
16.
J Cancer Res Clin Oncol ; 146(9): 2241-2253, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32494918

RESUMO

PURPOSE: Bone metastasis is the result of complex crosstalk between tumor cells and bone marrow cells. Bone marrow adipocytes (BMAs) are the most abundant cell type in adult bone marrow. Therefore, we explore the effects of BMAs on bone metastasis in lung cancer. METHODS: RNA-seq was used to compare the mRNA expression level of bone metastatic SBC5 cells and non-bone metastatic SBC3 cells. Rosiglitazone-induced marrow adiposity and intra-femoral injection of SBC5 cells were used to demonstrate the relationship between BMAs and SBC5 cells in vivo. Co-culture system, gene co-expression, gene ontology (GO) enrichment analysis and protein-protein interaction (PPI) network were used to explore the potential mechanism. RESULTS: BMAs specially enhance the invasion of bone metastatic SBC5 instead of non-bone metastatic SBC3 in vitro. SBC5 instead of SBC3 promoted osteoblast and osteoclast differentiation as well as de-differentiation of mature BMAs. Rosiglitazone-induced marrow adiposity significantly enhanced osteolytic lesion induced by SBC5 in vivo. RNA-seq revealed that compared with SBC3, S100A9 and S100A8 genes were the most prominent genes up-regulated in SBC5 cells. High expression of S100A8/9 in SBC5 could be responsible for the crosstalk between lung cancer cells and BMAs. More importantly, interleukin 6 receptor (IL6R), which is adjacent to S100A8/A9 in 1q21.3, was significantly up-regulated by BMAs in vitro. S100A8/A9 (1 µg/ml) could obviously enhance the osteoblastic differentiation and inhibit adipogenic differentiation, whereas TLR4 inhibitor TAK242 (10 µmol/l) significantly attenuated this effect. CONCLUSIONS: Our study suggested that bone marrow adipocyte may communicate with lung cancer cells via 1q21.3 (S100A8/A9-IL6R)-TLR4 pathway to promote osteolytic bone destruction. 1q21.3 (S100A8/A9-IL6R) is a potential target for the treatment of lung cancer bone metastasis.


Assuntos
Adipócitos/metabolismo , Medula Óssea/metabolismo , Osso e Ossos/metabolismo , Neoplasias Pulmonares/metabolismo , Osteólise/metabolismo , Receptores de Interleucina-6/metabolismo , Proteínas S100/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia
17.
Environ Toxicol ; 35(9): 991-997, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32401414

RESUMO

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease, in which the immune system attacks synovial joint tissues. Interleukin (IL)-1ß is a critical proinflammatory cytokine in RA progression. Sphingosine-1-phosphate (S1P), a platelet-derived lysophospholipid mediator, reportedly regulates osteoimmunology. Here, we investigated how S1P mediates IL-1ß expression in osteoblasts. Our analysis of records from the Gene Expression Omnibus (GEO) database demonstrate higher levels of IL-1ß in patients with RA compared with those with osteoarthritis. Stimulation of osteoblasts with S1P concentration dependently increased mRNA and protein expression of IL-1ß. Elevations in IL-1ß mRNA expression induced by S1P were reduced by the small interfering RNA (siRNA) against the S1P1 receptor. S1P also augmented JAK and STAT3 molecular cascades. We also found that JAK and STAT3 inhibitors and their siRNAs antagonized S1P-promoted IL-1ß expression. Our results indicate that S1P promotes the expression of IL-1ß in osteoblasts via the S1P1 receptor and the JAK and STAT3 signaling pathways.


Assuntos
Interleucina-1beta/genética , Janus Quinases/metabolismo , Lisofosfolipídeos/fisiologia , Osteoblastos/metabolismo , Fator de Transcrição STAT3/metabolismo , Esfingosina/análogos & derivados , Artrite Reumatoide/metabolismo , Células Cultivadas , Humanos , Lisofosfolipídeos/farmacologia , Masculino , Osteoartrite/metabolismo , Osteoblastos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Esfingosina/farmacologia , Esfingosina/fisiologia , Receptores de Esfingosina-1-Fosfato/genética
18.
PLoS Genet ; 16(5): e1008586, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463816

RESUMO

The strength of bone depends on bone quantity and quality. Osteocalcin (Ocn) is the most abundant noncollagenous protein in bone and is produced by osteoblasts. It has been previously claimed that Ocn inhibits bone formation and also functions as a hormone to regulate insulin secretion in the pancreas, testosterone synthesis in the testes, and muscle mass. We generated Ocn-deficient (Ocn-/-) mice by deleting Bglap and Bglap2. Analysis of Ocn-/-mice revealed that Ocn is not involved in the regulation of bone quantity, glucose metabolism, testosterone synthesis, or muscle mass. The orientation degree of collagen fibrils and size of biological apatite (BAp) crystallites in the c-axis were normal in the Ocn-/-bone. However, the crystallographic orientation of the BAp c-axis, which is normally parallel to collagen fibrils, was severely disrupted, resulting in reduced bone strength. These results demonstrate that Ocn is required for bone quality and strength by adjusting the alignment of BAp crystallites parallel to collagen fibrils; but it does not function as a hormone.


Assuntos
Apatitas/metabolismo , Calcificação Fisiológica/genética , Metabolismo dos Carboidratos/genética , Glucose/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Osteocalcina/fisiologia , Testosterona/biossíntese , Animais , Apatitas/química , Osso e Ossos/metabolismo , Colágeno/metabolismo , Cristalização , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Tamanho do Órgão/genética , Osteoblastos/metabolismo , Osteocalcina/genética , Osteogênese/genética , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
19.
Phytomedicine ; 71: 153225, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32464299

RESUMO

BACKGROUND: Impaired bone formation is one of the reasons behind osteoporosis. Alterations in the patterns of mesenchymal stromal cell differentiation towards adipocytes instead of osteoblasts contribute to osteoporosis progression. Natural anti-osteoporotic agents are effective and safe alternatives for osteoporosis treatment. PURPOSE: In this context, 3,5-dicaffeoyl­epi-quinic acid (DCEQA) which is a derivative of chlorogenic acid with reported bioactivities was studied for its osteogenic differentiation enhancing potential in vitro. METHODS: Anti-osteoporotic effects of DCEQA were investigated in human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) which were induced to differentiate into osteoblasts or adipocytes with or without DCEQA treatment. Changes in the osteogenic and adipogenic markers such as ALP activity and lipid accumulation, respectively, were observed along with differentiation-specific activation of mitogen activated protein kinase (MAPK) pathways. RESULTS: At 10 µM concentration, DCEQA increased the proliferation of bone marrow-derived human mesenchymal stromal cells (hBM-MSCs) during osteoblast differentiation. The expression of osteogenic markers ALP, osteocalcin, Runx2, BMP2 and Wnt 10a was upregulated by DCEQA treatment. The ALP activity and extracellular mineralization were also increased. DCEQA elevated the phosphorylation levels of p38 and JNK MAPKs as well as the activation of ß-catenin and Smad1/5. DCEQA suppressed the lipid accumulation and downregulated expression of adipogenic markers PPARγ, C/EBPα and SREBP1c in adipo-induced hBM-MSCs. DCEQA also decreased the phosphorylation of p38 and ERK MAPKs and stimulated the activation of AMPK in hBM-MSC adipocytes. CONCLUSION: DCEQA was suggested to enhance osteoblast differentiation via stimulating Wnt/BMP signaling. The adipocyte differentiation inhibitory effect of DCEQA was suggested to arise from its ability to increase AMPK phosphorylation. Overall, DCEQA was shown to possess osteogenesis enhancing and adipogenesis inhibitory properties which might facilitate its use against osteoporotic conditions.


Assuntos
Adipócitos/citologia , Atriplex/química , Ácido Clorogênico/análogos & derivados , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/efeitos dos fármacos , Células da Medula Óssea , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Ácido Clorogênico/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo
20.
Nat Commun ; 11(1): 2289, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385263

RESUMO

The osteoblast differentiation capacity of skeletal stem cells (SSCs) must be tightly regulated, as inadequate bone formation results in low bone mass and skeletal fragility, and over-exuberant osteogenesis results in heterotopic ossification (HO) of soft tissues. RUNX2 is essential for tuning this balance, but the mechanisms of posttranslational control of RUNX2 remain to be fully elucidated. Here, we identify that a CK2/HAUSP pathway is a key regulator of RUNX2 stability, as Casein kinase 2 (CK2) phosphorylates RUNX2, recruiting the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which stabilizes RUNX2 by diverting it away from ubiquitin-dependent proteasomal degradation. This pathway is important for both the commitment of SSCs to osteoprogenitors and their subsequent maturation. This CK2/HAUSP/RUNX2 pathway is also necessary for HO, as its inhibition blocked HO in multiple models. Collectively, active deubiquitination of RUNX2 is required for bone formation and this CK2/HAUSP deubiquitination pathway offers therapeutic opportunities for disorders of inappropriate mineralization.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ossificação Heterotópica/metabolismo , Osteogênese , Adulto , Idoso , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Diferenciação Celular , Displasia Cleidocraniana/genética , Displasia Cleidocraniana/patologia , Feminino , Deleção de Genes , Haploinsuficiência/genética , Membro Posterior/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Ossificação Heterotópica/genética , Ossificação Heterotópica/patologia , Osteoblastos/metabolismo , Fosforilação , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo
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