RESUMO
Growth and differentiation factor 11 (GDF11) and myostatin (MSTN) are closely related transforming growth factor ß (TGF-ß) family members, but their biological functions are quite distinct. While MSTN has been widely shown to inhibit muscle growth, GDF11 regulates skeletal patterning and organ development during embryogenesis. Postnatal functions of GDF11, however, remain less clear and controversial. Due to the perinatal lethality of Gdf11 null mice, previous studies used recombinant GDF11 protein to prove its postnatal function. However, recombinant GDF11 and MSTN proteins share nearly identical biochemical properties, and most GDF11-binding molecules have also been shown to bind MSTN, generating the possibility that the effects mediated by recombinant GDF11 protein actually reproduce the endogenous functions of MSTN. To clarify the endogenous functions of GDF11, here, we focus on genetic studies and show that Gdf11 null mice, despite significantly down-regulating Mstn expression, exhibit reduced bone mass through impaired osteoblast (OB) and chondrocyte (CH) maturations and increased osteoclastogenesis, while the opposite is observed in Mstn null mice that display enhanced bone mass. Mechanistically, Mstn deletion up-regulates Gdf11 expression, which activates bone morphogenetic protein (BMP) signaling pathway to enhance osteogenesis. Also, mice overexpressing follistatin (FST), a MSTN/GDF11 inhibitor, exhibit increased muscle mass accompanied by bone fractures, unlike Mstn null mice that display increased muscle mass without fractures, indicating that inhibition of GDF11 impairs bone strength. Together, our findings suggest that GDF11 promotes osteogenesis in contrast to MSTN, and these opposing roles of GDF11 and MSTN must be considered to avoid the detrimental effect of GDF11 inhibition when developing MSTN/GDF11 inhibitors for therapeutic purposes.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Desenvolvimento Muscular/fisiologia , Miostatina/metabolismo , Osteogênese/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/genética , Osso e Ossos/patologia , Condrócitos/metabolismo , Regulação para Baixo , Folistatina , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Diferenciação de Crescimento/genética , Camundongos , Camundongos Knockout , Músculos/patologia , Osteoblastos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVE: This study aimed to investigate the effect of small molecules incorporated into the engineered nanofibrous scaffold to enhance the osteoblast differentiation MATERIALS AND METHODS: Poly-ε-caprolactone (PCL) nanofiber matrices with lithium chloride (LiCl) were fabricated using the electrospinning technique. Scaffolds were characterized using scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX). Scaffolds were seeded with MC3T3-E1 cells and assessed using Western blots (ß-catenin), alamarBlue assay (proliferation), qPCR (osteoblast differentiation), and mineralization (Alizarin Red staining). RESULTS: We observed LiCl nanofiber scaffolds induced concentration-dependent cell proliferation that correlated with an increased ß-catenin expression indicating sustained Wnt signaling. Next, we examined osteoblast differentiation markers such as osteocalcin (OCN) and Runt-related transcription factor 2 (Runx2) and noted increased expression in LiCl nanofiber scaffolds. We also noted increased bone morphogenetic protein (BMP-2, 4, and 7) expressions suggesting activated Wnt can promote cures to further osteogenic differentiation. Finally, Alizarin Red staining demonstrated increased mineral deposition in LiCl-incorporated nanofiber scaffolds. CONCLUSIONS: Together, these results indicated that LiCl-incorporated nanofiber scaffolds enhance osteoblast differentiation. CLINICAL RELEVANCE: Small molecule-incorporated nanofibrous scaffolds are an innovative clinical tool for bone tissue engineering.
Assuntos
Nanofibras , Osteogênese , Diferenciação Celular , Proliferação de Células , Osteoblastos , Poliésteres/farmacologia , Engenharia Tecidual/métodos , Alicerces TeciduaisRESUMO
Growth and differentiation factor 11 (GDF11) is a transforming growth factor ß family member that has been identified as the central player of anterior-posterior (A-P) axial skeletal patterning. Mice homozygous for Gdf11 deletion exhibit severe anterior homeotic transformations of the vertebrae and craniofacial defects. During early embryogenesis, Gdf11 is expressed predominantly in the primitive streak and tail bud regions, where new mesodermal cells arise. On the basis of this expression pattern of Gdf11 and the phenotype of Gdf11 mutant mice, it has been suggested that GDF11 acts to specify positional identity along the A-P axis either by local changes in levels of signaling as development proceeds or by acting as a morphogen. To further investigate the mechanism of action of GDF11 in the vertebral specification, we used a Cdx2-Cre transgene to generate mosaic mice in which Gdf11 expression is removed in posterior regions including the tail bud, but not in anterior regions. The skeletal analysis revealed that these mosaic mice display patterning defects limited to posterior regions where Gdf11 expression is deficient, whereas displaying normal skeletal phenotype in anterior regions where Gdf11 is normally expressed. Specifically, the mosaic mice exhibited seven true ribs, a pattern observed in wild-type (wt) mice (vs. 10 true ribs in Gdf11-/- mice), in the anterior axis and nine lumbar vertebrae, a pattern observed in Gdf11 null mice (vs. six lumbar vertebrae in wt mice), in the posterior axis. Our findings suggest that GDF11, rather than globally acting as a morphogen secreted from the tail bud, locally regulates axial vertebral patterning.
Assuntos
Padronização Corporal , Proteínas Morfogenéticas Ósseas/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Osteogênese , Coluna Vertebral/metabolismo , Animais , Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/deficiência , Proteínas Morfogenéticas Ósseas/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Diferenciação de Crescimento/deficiência , Fatores de Diferenciação de Crescimento/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mosaicismo , Osteogênese/genética , Transdução de Sinais , Coluna Vertebral/embriologiaRESUMO
Distal-less homeobox 3 (Dlx3), a member of the Dlx family of homeobox proteins, is a transcriptional activator of runt-related transcription factor 2 (Runx2) during osteogenic differentiation. It has been demonstrated that forced expression of Runx2 induces an osteogenic program and ectopic calcification in muscles. Therefore, it would be reasonable to predict that Dlx3 also affects myogenic differentiation. The relationship between Dlx3 and myogenesis, however, remains poorly understood. Therefore, in this study, the role and regulation of Dlx3 during myogenic differentiation were investigated. Expression level of Dlx3 was downregulated in human mesenchymal stem cells (MSCs), mouse MSCs, and C2C12 cells cultured in myogenic medium. Dlx3 level was inversely correlated with myogenic differentiation 1 and the muscle-specific microRNA, microRNA-133 (miR-133). The expression level of Runx2 was closely regulated by Dlx3 even under myogenic conditions. Overexpression of Dlx3 markedly downregulated expression levels of myogenic transcription factors and myotube formation in C2C12 cells, whereas Dlx3 knockdown enhanced myogenic differentiation. The Dlx3 3'-untranslated region (3'-UTR) has two potential binding sites for miR-133. Luciferase reporter assays demonstrated that Dlx3 is a direct target of miR-133a and miR-133b, and that the two target sites are redundantly active. Taken together, these results suggest that Dlx3 is a negative regulator of myogenic differentiation and that miR-133a and miR-133b enhance myogenic differentiation, partly through inhibition of Dlx3 expression via direct targeting of the Dlx3 3'-UTR.
RESUMO
Myoblast fusion is critical for muscle growth, regeneration, and repair. We previously reported that the enzyme peptidyl-prolyl cis-trans isomerase NIMA interacting 1 (Pin1) is involved in osteoclast fusion. The objective of this study was to investigate the possibility that Pin1 also inhibits myoblast fusion. Here, we show the increased number of nuclei in the Pin1+/- mice muscle fiber compared to that in wild-type mice. Moreover, we show that low dose of the Pin1 inhibitor dipentamethylene thiuram monosulfide treatment caused enhanced fusion in C2C12 cells. The R-Smads are well-known mediators of muscle hypertrophy and hyperplasia as well as being substrates of Pin1. We found that Pin1 is crucial for maintaining the stability of Smad3 (homologues of the Drosophila protein, mothers against decapentaplegic (Mad) and the Caenorhabditis elegans protein Sma). Our results show that serine 204 within Smad3 is the key Pin1-binding site during inhibition of myoblast fusion and that both the transforming growth factor-ß receptor and extracellular signal-regulated kinase (ERK)-mediated phosphorylation are required for the interaction of Pin1 with Smad3. These findings suggest that a precise level of Pin1 activity is essential for regulating myoblast fusion during myogenesis and muscle regeneration.
Assuntos
Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Proteína Smad3/metabolismo , Animais , Fusão Celular , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Atrofia Muscular/genética , Mioblastos/citologia , Mioblastos/metabolismo , Miostatina/metabolismo , Fosforilação , Ligação Proteica , Serina/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
The fibrin matrix is fundamentally formed by the polymerization of fibrinogen and thrombin in blood plasma. It is a natural biopolymeric material to widely investigate for various tissue regenerations due to good biocompatibility, rapid biodegradability, and easy fabrication. In particular, the conjugated bioactive molecules with fibrinogen can promote tissue morphogenesis or maturation after cell adhesion on the matrices, migration, proliferation, or differentiation. Using these physiological properties with cell-material interactions, the fibrin matrices have been utilized in tissue engineering applications such as skin tissue, cardiovascular tissue, musculoskeletal tissue, or nerve tissue in preclinical and clinical situations. This chapter demonstrates the fibrin material and its tissue engineering applications as the therapeutic strategies.
Assuntos
Materiais Biocompatíveis/química , Fibrina/química , Medicina Regenerativa , Engenharia Tecidual , Fibrinogênio , HumanosRESUMO
Hyperglycemic conditions in diabetic patients can affect various cellular functions, including the modulation of osteogenic differentiation. However, the molecular mechanisms by which hyperglycemia affects osteogenic differentiation are yet to be clarified. This study aimed to investigate whether the aberrant increase in protein O-linked-ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) contributes to the suppression of osteogenic differentiation due to hyperglycemia. To induce osteogenic differentiation, C2C12 cells were cultured in the presence of recombinant human bone morphogenetic protein 2 (BMP2). Excessive protein O-GlcNAcylation was induced by treating C2C12 cells with high glucose, glucosamine, or N-acetylglucosamine concentrations or by O-GlcNAc transferase (OGT) overexpression. The effect of O-GlcNAcylation on osteoblast differentiation was then confirmed by examining the expression levels of osteogenic marker gene mRNAs, activity of alkaline phosphatase, and transcriptional activity of Runx2, a critical transcription factor for osteoblast differentiation and bone formation. Cell treatment with high glucose, glucosamine or N-acetylglucosamine increased O-GlcNAcylation of Runx2 and the total levels of O-GlcNAcylated proteins, which led to a decrease in the transcriptional activity of Runx2, expression levels of osteogenic marker genes (Runx2, osterix, alkaline phosphatase, and type I collagen), and activity of alkaline phosphatase. These inhibitory effects were rescued by lowering protein O-GlcNAcylation levels by adding STO45849, an OGT inhibitor, or by overexpressing ß-N-acetylglucosaminidase. Our findings suggest that excessive protein O-GlcNAcylation contributes to high glucose-suppressed osteogenic differentiation.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , 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 , Inibidores Enzimáticos/farmacologia , Glucosamina/farmacologia , Glucose/farmacologia , Glicosilação/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Ligamento Periodontal/metabolismo , Proteínas Recombinantes/farmacologia , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Distal-less homeobox 5 (Dlx5) is a negative regulator of adipogenesis. Dlx5 expression is decreased by adipogenic stimuli, but the mechanisms of Dlx5 downregulation by adipogenic stimuli have not yet been determined. Here, we tested the impact of cAMP/PKA (protein kinase A) signaling induced by 3-isobutyl-1 methyl xanthine (IBMX), forskolin, and 8-CPT-cAMP on the expression of Dlx5 in 3T3-L1 preadipocytes. Significant downregulation of Dlx5 mRNA expression and protein production levels were observed via cAMP/PKA-dependent signaling. Forced expression of cAMP-responsive element-binding protein (CREB) and CCAAT/enhancer-binding protein ß (C/EBPß) was sufficient for downregulation of Dlx5 expression and revealed that CREB functions upstream of C/EBPß. In addition, C/EBPß knockdown by siRNA rescued Dlx5 expression in IBMX-treated 3T3-L1 preadipocytes. Luciferase assays using a Dlx5-luc-2935 reporter construct demonstrated the requirement of the Dlx5 promoter region, ranging from -774 to -95 bp that contains two putative C/EBPß binding elements (site-1: -517 to -510 bp and site-2: -164 to -157 bp), in the suppression of Dlx5 transcription. Consequently, chromatin immunoprecipitation analysis confirmed the importance of site-1, but not site-2, in C/EBPß binding and transcriptional suppression of Dlx5. In conclusion, we elucidated the underling mechanism of Dlx5 downregulation in IBMX-induced adipogenesis. IBMX activated cAMP/PKA/CREB signaling and subsequently upregulated C/EBPß, which binds to the Dlx5 promoter to suppress Dlx5 transcription.
Assuntos
Adipócitos/citologia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Homeodomínio/genética , 1-Metil-3-Isobutilxantina/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/genética , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacos , Tionucleotídeos/farmacologiaRESUMO
The canonical Wnt signaling pathway, in which ß-catenin nuclear localization is a crucial step, plays an important role in osteoblast differentiation. Pin1, a prolyl isomerase, is also known as a key enzyme in osteogenesis. However, the role of Pin1 in canonical Wnt signal-induced osteoblast differentiation is poorly understood. We found that Pin1 deficiency caused osteopenia and reduction of ß-catenin in bone lining cells. Similarly, Pin1 knockdown or treatment with Pin1 inhibitors strongly decreased the nuclear ß-catenin level, TOP flash activity, and expression of bone marker genes induced by canonical Wnt activation and vice versa in Pin1 overexpression. Pin1 interacts directly with and isomerizes ß-catenin in the nucleus. The isomerized ß-catenin could not bind to nuclear adenomatous polyposis coli, which drives ß-catenin out of the nucleus for proteasomal degradation, which consequently increases the retention of ß-catenin in the nucleus and might explain the decrease of ß-catenin ubiquitination. These results indicate that Pin1 could be a critical target to modulate ß-catenin-mediated osteogenesis.
Assuntos
Osteoblastos/citologia , Peptidilprolil Isomerase/metabolismo , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Peptidilprolil Isomerase de Interação com NIMA , Osteoblastos/metabolismo , Osteogênese , Peptidilprolil Isomerase/genética , ProteóliseRESUMO
Pin1 is a peptidyl prolyl cis-trans isomerase that specifically binds to the phosphoserine-proline or phosphothreonine-proline motifs of several proteins. We reported that Pin1 plays a critical role in the fate determination of Smad1/5, Runx2, and ß-catenin that are indispensable nuclear proteins for osteoblast differentiation. Though several chemical inhibitors has been discovered for Pin1, no activator has been reported as of yet. In this study, we directly introduced recombinant Pin1 protein successfully into the cytoplasm via fibroin nanoparticle encapsulated in cationic lipid. This nanoparticle-lipid complex delivered its cargo with a high efficiency and a low cytotoxicity. Direct delivery of Pin1 leads to increased Runx2 and Smad signaling and resulted in recovery of the osteogenic marker genes expression and the deposition of mineral in Pin1-deficient cells. These result indicated that a direct Pin1 protein delivery method could be a potential therapeutics for the osteopenic diseases. J. Cell. Physiol. 232: 2798-2805, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Peptidilprolil Isomerase de Interação com NIMA/deficiência , Peptidilprolil Isomerase de Interação com NIMA/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Osteogênese/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Células 3T3 , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Preparações de Ação Retardada , Relação Dose-Resposta a Droga , Portadores de Fármacos , Composição de Medicamentos , Fibroínas/química , Lipídeos/química , Masculino , Camundongos , Camundongos Knockout , Peptidilprolil Isomerase de Interação com NIMA/genética , Nanopartículas , Fenótipo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Fatores de Tempo , beta Catenina/metabolismoRESUMO
Cementum is a mineralized layer on the tooth's root surface and facilitates the biomechanical anchoring of fibrous connective tissues as a part of tooth-supportive complexes. Previously, we observed that OCCM30 cementoblasts cultured on fibrin matrices underwent apoptosis due to fibrin degradation through the expression of proteases. Here, we demonstrated that OCCM30 on fibrin matrices (OCCM30-fibrin) enhanced canonical Wnt signaling, which directed to plasminogen expression. The OCCM30-fibrin showed higher levels of Wnt3a expression, nuclear translocation of ß-catenin, and T-cell factor (TCF) optimal motif (TOP) reporter activity than the cells on tissue culture dishes (OCCM30-TCD), indicating that the OCCM30-fibrin enhanced canonical Wnt/ß-catenin signaling. Also, OCCM30-fibrin expressed biomineralization-associated markers at higher levels than OCCM30-TCD, of which levels were further increased with LiCl, a Wnt signaling activator. The OCCM30 cementoblasts simultaneously showed that high levels of plasminogen, a critical component of fibrinolysis, were expressed in the OCCM30-fibrin. Activation of canonical Wnt signaling with LiCl treatment or with forced lymphoid enhancer factor 1 (LEF1)-expression increased the expression of plasminogen. On the contrary, the inhibition of canonical Wnt signaling with siRNAs against Wnt3a or ß-catenin abrogated fibrin-enhanced plasminogen expression. Furthermore, there are three conserved putative response elements for the LEF1/ß-catenin complex in the plasminogen proximal promoter regions (-900 to +54). Site-directed mutations and chromatin immunoprecipitation indicated that canonical Wnt signaling directed plasminogen expression. Taken together, this study suggests that fibrin-based materials can modulate functional periodontal formations in controlling cementoblast differentiation and fibrin degradation.
Assuntos
Cemento Dentário/metabolismo , Fibrina/metabolismo , Plasminogênio/metabolismo , Via de Sinalização Wnt , Animais , Biomarcadores/análise , Linhagem Celular , Fibrina/genética , Fibrinólise/efeitos dos fármacos , Cloreto de Lítio/farmacologia , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Plasminogênio/genética , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The bone marrow of healthy individuals is primarily composed of osteoblasts and hematopoietic cells, while that of osteoporosis patients has a larger portion of adipocytes. There is evidence that the epigenetic landscape can strongly influence cell differentiation. We have shown that it is possible to direct the trans-differentiation of adipocytes to osteoblasts by modifying the epigenetic landscape with a DNA methyltransferase inhibitor (DNMTi), 5'-aza-dC, followed by Wnt3a treatment to signal osteogenesis. Treating 3T3-L1 adipocytes with 5'-aza-dC induced demethylation in the hypermethylated CpG regions of bone marker genes; subsequent Wnt3a treatment drove the cells to osteogenic differentiation. When old mice with predominantly adipose marrow were treated with both 5'-aza-dC and Wnt3a, decreased fatty tissue and increased bone volume were observed. Together, our results indicate that epigenetic modification permits direct programming of adipocytes into osteoblasts in a mouse model of osteoporosis, suggesting that this approach could be useful in bone tissue-engineering applications.
Assuntos
Transdiferenciação Celular/genética , DNA (Citosina-5-)-Metiltransferases/genética , Epigênese Genética/genética , Osteogênese/genética , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Transdiferenciação Celular/efeitos dos fármacos , Monofosfato de Citidina/administração & dosagem , Monofosfato de Citidina/análogos & derivados , DNA (Citosina-5-)-Metiltransferases/metabolismo , Epigênese Genética/efeitos dos fármacos , Humanos , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Proteína Wnt3A/genéticaRESUMO
Mesenchymal cells alter and retain their phenotype during skeletal development through activation or suppression of signaling pathways. For example, we have shown that Wnt3a only stimulates osteoblast differentiation in cells with intrinsic osteogenic potential (e.g. MC3T3-E1 pre-osteoblasts) and not in fat cell precursors or fibroblasts (3T3-L1 pre-adipocytes or NIH3T3 fibroblasts, respectively). Wnt3a promotes osteogenesis in part by stimulating autocrine production of the osteoinductive ligand Bmp2. Here, we show that the promoter regions of the genes for Bmp2 and the osteoblast marker Alp are epigenetically locked to prevent their expression in nonosteogenic cells. Both genes have conserved CpG islands that exhibit increased CpG methylation, as well as decreased acetylation and increased methylation of histone H3 lysine 9 (H3-K9) specifically in nonosteogenic cells. Treatment of pre-adipocytes or fibroblasts with the CpG-demethylating agent 5'-aza-2'-deoxycytidine or the histone deacetylase inhibitor trichostatin-A renders Bmp2 and Alp responsive to Wnt3a. Hence, drug-induced epigenetic activation of Bmp2 gene expression contributes to Wnt3a-mediated direct trans-differentiation of pre-adipocytes or fibroblasts into osteoblasts. We propose that direct conversion of nonosteogenic cells into osteoblastic cell types without inducing pluripotency may improve prospects for novel epigenetic therapies to treat skeletal afflictions.
Assuntos
Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Transdiferenciação Celular/genética , Transdiferenciação Celular/fisiologia , Epigênese Genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Via de Sinalização Wnt , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo , Células 3T3 , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Ilhas de CpG , Metilação de DNA , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Histonas/metabolismo , Camundongos , Células NIH 3T3 , Osteogênese/genética , Osteogênese/fisiologiaRESUMO
Fibroblast growth factor 2 (FGF2) signaling plays a pivotal role in bone growth/differentiation through the activation of osteogenic master transcription factor Runx2, which is mediated by the ERK/MAPK-dependent phosphorylation and the p300-dependent acetylation of Runx2. In this study, we found that Pin1-dependent isomerization of Runx2 is the critical step for FGF2-induced Runx2 transactivation function. We identified four serine or threonine residues in the C-terminal domain of Runx2 that are responsible for Pin1 binding and structural modification. Confocal imaging studies indicated that FGF2 treatment strongly stimulated the focal accumulation of Pin1 in the subnuclear area, which recruited Runx2. In addition, active forms of RNA polymerase-II also colocalized in the same subnuclear compartment. Dipentamethylene thiuram monosulfide, a Pin1 inhibitor, strongly attenuated their focal accumulation as well as Runx2 transactivation activity. The Pin1-mediated structural modification of Runx2 is an indispensable step connecting phosphorylation and acetylation and, consequently, transcriptional activation of Runx2 by FGF signaling. Thus, the modulation of Pin1 activity may be a target for the regulation of bone formation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/química , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Osteoblastos/citologia , Peptidilprolil Isomerase/metabolismo , Acetilação/efeitos dos fármacos , Animais , Sítios de Ligação , Núcleo Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células HEK293 , Humanos , Isomerismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Peptidilprolil Isomerase de Interação com NIMA , Osteoblastos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Pin1 is a peptidyl prolyl cis-trans isomerase that specifically binds to the phosphoserine-proline or phosphothreonine-proline motifs of numerous proteins. Previously, we reported that Pin1 deficiency resulted in defects in osteoblast differentiation during early bone development. In this study, we found that adult Pin1-deficient mice developed osteoporotic phenotypes compared to age-matched controls. Since BMP2 stored in the bone matrix plays a critical role in adult bone maintenance, we suspected that BMP R-Smads (Smad1 and Smad5) could be critical targets for Pin1 action. Pin1 specifically binds to the phosphorylated linker region of Smad1, which leads to structural modification and stabilization of the Smad1 protein. In this process, Pin1-mediated conformational modification of Smad1 directly suppresses the Smurf1 interaction with Smad1, thereby promoting sustained activation of the Smad1 molecule. Our data demonstrate that post-phosphorylational prolyl isomerization of Smad1 is a converging signal to stabilize the Smad1 molecule against the ubiquitination process mediated by Smurf1. Therefore, Pin1 is a critical molecular switch in the determination of Smad1 fate, opposing the death signal transmitted to the Smad1 linker region by phosphorylation cascades after its nuclear localization and transcriptional activation. Thus, Pin1 could be developed as a major therapeutic target in many skeletal diseases.
Assuntos
Proteína Morfogenética Óssea 2/genética , Diferenciação Celular/genética , Peptidilprolil Isomerase/genética , Transdução de Sinais/genética , Animais , Proteína Morfogenética Óssea 2/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/metabolismo , Fosforilação , Ligação Proteica , Proteína Smad1/metabolismo , Ativação TranscricionalRESUMO
Bone morphogenetic protein (BMP) and canonical Wnts are representative developmental signals that enhance osteoblast differentiation and bone formation. Previously, we demonstrated that epidermal growth factor (EGF) inhibits BMP2-induced osteoblast differentiation by inducing Smurf1 expression. However, the regulatory role of EGF in Wnt/ß-catenin-induced osteoblast differentiation has not been elucidated. In this study, we investigated the effect of EGF on Wnt/ß-catenin signaling-induced osteoblast differentiation using the C2C12 cell line. EGF significantly suppressed the expression of osteoblast marker genes, which were induced by Wnt3a and a GSK-3ß inhibitor. EGF increased the expression levels of Smurf1 mRNA and protein. Smurf1 knockdown rescued Wnt/ß-catenin-induced osteogenic marker gene expression in the presence of EGF. EGF treatment or Smurf1 overexpression did not affect ß-catenin mRNA expression levels, but reduced ß-catenin protein levels and TOP-Flash activity. EGF and Smurf1 promoted ß-catenin ubiquitination. Co-immunoprecipitation and GST pull-down assays showed that Smurf1 associates with ß-catenin. These results suggest that EGF/Smurf1 inhibits Wnt/ß-catenin-induced osteogenic differentiation and that Smurf1 downregulates Wnt/ß-catenin signaling by enhancing proteasomal degradation of ß-catenin.
Assuntos
Diferenciação Celular/genética , Fator de Crescimento Epidérmico/biossíntese , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , beta Catenina/metabolismo , Fator de Crescimento Epidérmico/genética , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genética , RNA Mensageiro/biossíntese , Ubiquitina-Proteína Ligases/genética , Via de Sinalização Wnt/genética , Proteína Wnt3A/antagonistas & inibidores , beta Catenina/genéticaRESUMO
MicroRNAs are novel key regulators of cellular differentiation. Dlx transcription factors play an important role in osteoblast differentiation, and Dlx5 and Dlx2 are known targets of miR-124. Therefore, in the present study, we investigated the regulatory effects of miR-124 on the osteogenic differentiation and in vivo bone formation of mesenchymal stem cells (MSCs). During osteogenic induction by BMP2, the expression levels of miR-124 were inversely correlated with those of osteogenic differentiation marker genes in human and mouse bone marrow-derived MSCs, MC3T3-E1 cells and C2C12 cells. The overexpression of a miR-124 mimic significantly decreased the expression levels of Dlx5, Dlx3, and Dlx2, whereas the silencing of miR-124 with hairpin inhibitors significantly increased the expression of these Dlx genes. Luciferase reporter assays demonstrated that miR-124 directly targets the 3'UTRs of Dlx3, Dlx5, and Dlx2. The overexpression of a miR-124 mimic suppressed the osteogenic marker gene expression levels, alkaline phosphatase activity and matrix mineralization, which were all significantly increased by the overexpression of a miR-124 inhibitor. When ectopic bone formation was induced by the subcutaneous transplantation of human bone marrow-derived MSCs in nude mice, MSCs overexpressing a miR-124 inhibitor significantly enhanced woven bone formation compared with control MSCs. However, MSCs overexpressing a miR-124 mimic exhibited increased adipocyte differentiation at the expense of ectopic bone formation. These results suggest that miR-124 is a negative regulator of osteogenic differentiation and in vivo bone formation and that the targeting of Dlx5, Dlx3, and Dlx2 genes partly contributes to this inhibitory effect exerted by miR-124.
Assuntos
Osso e Ossos/metabolismo , Diferenciação Celular , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoblastos/citologia , Regiões 3' não Traduzidas , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Osso e Ossos/citologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Nus , Osteoblastos/metabolismo , Osteogênese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression, which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation.
Assuntos
Diferenciação Celular , Proteína Básica Maior de Eosinófilos/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Osteoblastos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteína Básica Maior de Eosinófilos/genética , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteogênese , Inibidores de Proteínas Quinases/farmacologia , Proteína Smad1/genética , Proteína Smad1/metabolismo , Transcrição Gênica , Ubiquitina-Proteína Ligases/genéticaRESUMO
PURPOSE: The aim of this study was to evaluate and compare the osseointegration of zirconia implants in rabbit tibiae with and without 10-methacryloyloxydecyl dihydrogen phosphate (MDP) treatment. MATERIALS AND METHODS: Twenty-eight rabbits received a total of 112 external hex-type implants made by the powder injection molding technique with and without MDP treatment before installation in the tibiae. The contact angles on the zirconia implants and zirconia discs before and after MDP application were evaluated. Removal torque (RT) and bone-implant contact (BIC) ratios were measured. RESULTS: The MDP treatment markedly enhanced the hydrophilicity and seemed to alter the topography and chemical composition of the implant and disc surface. As the healing time increases, the BIC and RT were increased in both groups. The MDP-treated implants exhibited higher BIC values than the control implants after 2 and 4 weeks of healing. The RT was higher in MDP-treated implants after 2 weeks of healing but not after 4 weeks of healing. CONCLUSION: The 10-MDP treatments made the surface more hydrophilic and enhanced the osseointegration of the implants in the early healing phase.
Assuntos
Implantação Dentária Endóssea/métodos , Implantes Dentários , Metacrilatos/uso terapêutico , Osseointegração , Tíbia/cirurgia , Animais , Análise do Estresse Dentário , Masculino , Microscopia Eletrônica de Varredura , Coelhos , Fatores de Tempo , ZircônioRESUMO
Regulation of the hematopoietic transcription factor PU.1, a member of the ETS family, plays a critical role in the development of blood cells and in leukemia. The dosage of PU.1 has been shown to cause a shift in myelomonocytic progenitor fate. Pin1 is a unique substrate-specific enzyme that can isomerize phospho-Ser/Thr-Pro peptide bonds, accelerating the conformational change in its substrates between a cis and a trans form. Such activity has been demonstrated to be a tightly controlled mechanism regulating a wide variety of protein functions under both normal physiological and pathological conditions. We have previously reported that a conformational change in Runx2 induced by Pin1 is essential for its function in osteogenesis in vitro and in vivo. In this study, we show that the Pin1-mediated conformational change in Runx1 enhances its acetylation and stabilization and, consequently, enhances its transacting activity. The increased acetylation of Runx1 represses PU.1 transcription in pre-monocytes. Conversely, the lack of (or the inhibition of) Pin1 increases PU.1 transcription in vitro and in vivo in pre-monocytes and in the spleen tissue. Pin1 KO mice have an increased CD11b(+) /F4/80(+) cell population and F4/80 protein expression in spleen. From our data, we can conclude that the conformational change in Runx1 induced by Pin1 represses PU.1 transcription in pre-monocytes and influences the commitment to the monocyte lineage. The dosage of PU.1 is a crucial factor in acute myeloid leukemia (AML), and Pin1 may thus be a useful target for controlling PU.1-dependent hematopoiesis, as well as leukemogenesis.