RESUMO
To explore the role of YAP, a key effector of the Hippo pathway, in temporomandibular joint (TMJ) ankylosis. The temporal and spatial expression of YAP was detected via immunohistochemistry and multiplex immunohistochemistry on postoperative Days 1, 4, 7, 9, 11, 14 and 28 in a sheep model. Isolated mesenchymal stem cells (MSCs) from samples of the Day 14. The relative mRNA expression of YAP was examined before and after the osteogenic induction of MSCs. A YAP-silenced MSC model was constructed, and the effect of YAP knockdown on MSC function was examined. YAP is expressed in the nucleus of the key sites that determine the ankylosis formation, indicating that YAP is activated in a physiological state. The expression of YAP increased gradually over time. Moreover, the number of cells coexpressing of RUNX2 and YAP-with the osteogenic active zone labelled by RUNX2-tended to increase after Day 9. After the osteogenic induction of MSCs, the expression of YAP increased. After silencing YAP, the osteogenic, proliferative and migratory abilities of the MSCs were inhibited. YAP is involved in the early development of TMJ bony ankylosis. Inhibition of YAP using shRNA might be a promising way to prevent or treat TMJ ankylosis.
Assuntos
Anquilose , Células-Tronco Mesenquimais , Osteogênese , Transtornos da Articulação Temporomandibular , Animais , Células-Tronco Mesenquimais/metabolismo , Transtornos da Articulação Temporomandibular/metabolismo , Transtornos da Articulação Temporomandibular/patologia , Transtornos da Articulação Temporomandibular/genética , Anquilose/metabolismo , Anquilose/patologia , Anquilose/genética , Proteínas de Sinalização YAP/metabolismo , Articulação Temporomandibular/metabolismo , Articulação Temporomandibular/patologia , Ovinos , Proliferação de Células , Modelos Animais de Doenças , Diferenciação Celular , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Movimento Celular , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Investigating the molecular biology underpinning the early-stage of traumatic temporomandibular joint (TMJ) ankylosis is crucial for discovering new ways to prevent the disease. This study aimed to explore the dynamic changes of transcriptome from the intra-articular hematoma or the newly generated ankylosed callus during the onset and early progression of TMJ ankylosis. METHODS: Based on a well-established sheep model of TMJ bony ankylosis, the genome-wide microarray data were obtained from samples at postoperative Days 1, 4, 7, 9, 11, 14 and 28, with intra-articular hematoma at Day 1 serving as controls. Fold changes in gene expression values were measured, and genes were identified via clustering based on time series analysis and further categorised into three major temporal classes: increased, variable and decreased expression groups. The genes in these three temporal groups were further analysed to reveal pathways and establish their biological significance. RESULTS: Osteoblastic and angiogenetic genes were found to be significantly expressed in the increased expression group. Genes linked to inflammation and osteoclasts were found in the decreased expression group. The various biological processes and pathways related to each temporal expression group were identified, and the increased expression group comprised genes exclusively involved in the following pathways: Hippo signaling pathway, Wnt signaling pathway and Rap 1 signaling pathway. The decreased expression group comprised genes exclusively involved in immune-related pathways and osteoclast differentiation. The variable expression group consisted of genes associated with DNA replication, DNA repair and DNA recombination. Significant biological pathways and transcription factors expressed at each time point postoperatively were also identified. CONCLUSIONS: These data, for the first time, presented the temporal gene expression profiling and reveal the important process of molecular biology in the early-stage of traumatic TMJ bony ankylosis. The findings might contributed to identifying potential targets for the treatment of TMJ ankylosis.
Assuntos
Anquilose , Transtornos da Articulação Temporomandibular , Articulação Temporomandibular , Animais , Ovinos/genética , Côndilo Mandibular , Anquilose/genética , Perfilação da Expressão Gênica , HematomaRESUMO
OBJECTIVE: This study aimed to investigate genetic variations in the osteoprotegerin-encoding gene (TNFRSF11B) in patients with temporomandibular joint ankylosis (TMJA). STUDY DESIGN: The sample comprised 17 patients diagnosed with TMJA, of both sexes with ages ranging from 6 to 57 years old. TNFRSF11B mutational analysis was performed using the Sanger sequencing method with DNA extracted from oral cells, and the functional impact prediction of the variants was assessed using bioinformatic analysis. RESULTS: Sequencing analysis identified 15 (88.23%) patients that presented at least 1 genetic variant in TNFRSF11B. The mutation rs202090603 (p.E33K) was found in 6 individuals, and rs140782326 (p.V281M), rs11573942 (p.L295), and rs1375250340 (p.I389T) were identified in 1 subject each. According to the pathogenicity potential of mutations, 3 variants were considered of low impact (rs2073618, rs202090603, and rs2228568) and 3 as disease causing (rs140782326, rs11573942, and rs1375250340). The variant rs202090603 (p.E33K) was found in the first cysteine domain with differences in the loop positions of p.E33K mutated the 3D structure of osteoprotegerin. CONCLUSION: Two polymorphisms (rs2073618 and rs2228568) and the mutations rs202090603 (p.E33K), rs140782326 (p.V281M), rs11573942 (p.L295), and rs1375250340 (p.I389T) in the TNFRSF11B gene may be associated with TMJA.
Assuntos
Anquilose , Transtornos da Articulação Temporomandibular , Adolescente , Adulto , Anquilose/genética , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Osteoprotegerina/genética , Articulação Temporomandibular , Transtornos da Articulação Temporomandibular/complicações , Transtornos da Articulação Temporomandibular/genética , Adulto JovemRESUMO
BACKGROUND: Traumatic haemarthrosis was hypothesized to be the etiology of temporomandibular (TMJ) ankylosis. Here, taking haematoma absorbance as a control, we aimed to reveal the molecular mechanisms involved in haematoma organizing into ankylosis using transcriptome microarray profiles. MATERIAL/METHODS: Disk removal was performed to building haematoma absorbance (HA) in one side of TMJ, while removal of disk and articular fibrous layers was performed to induced TMJ ankylosis through haematoma organization (HO) in the contralateral side in a sheep model. Haematoma tissues harvested at days 1, 4 and 7 postoperatively were examined by histology, and analyzed by Affymetrix OviGene-1_0-ST microarrays. The DAVID were recruited to perform the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis for the different expression genes (DEGs). The DEGs were also typed into protein-protein interaction (PPI) networks to get the interaction data. Six significant genes screened from PPI analysis, were confirmed by real-time PCR. RESULTS: We found 268, 223 and 17 DEGs at least twofold at days 1, 4 and 7, respectively. At day 1, genes promoting collagen ossification (POSTN, BGN, LUM, SPARC), cell proliferation (TGF-ß), and osteogenic differentiation of mesenchymal stem cells (BMP-2) were up-regulated in the HO side. At day 4, several genes involved in angiogenesis (KDR, FIT1, TEK) shower higher expression in the HO side. While HA was characterized by a continuous immune and inflammatory reaction. CONCLUSIONS: Our results provide a comprehensive understanding of the role of haematoma in the onset and progress of TMJ ankylosis. The study will contribute to explaining why few injured TMJs ankylose and most do not from the molecular level.
Assuntos
Anquilose , Hemartrose , Animais , Anquilose/genética , Côndilo Mandibular , Análise em Microsséries , Osteogênese , Ovinos , Articulação TemporomandibularRESUMO
BACKGROUND The type of traumatic temporomandibular joint (TMJ) ankylosis depends on the degree of severity of TMJ trauma. Here, we performed comprehensive differential molecular profiling between TMJ fibrous and bony ankylosis. MATERIAL AND METHODS Six sheep were used and a bilateral different degree of TMJ trauma was performed to induce fibrous ankylosis in one side and bony ankylosis in the other side. The ankylosed calluses were harvested at days 14 and 28 postoperatively and analyzed by Affymetrix OviGene-1_0-ST microarrays. DAVID was used to perform the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis for the different expression genes (DEGs). The DEGs were also typed into protein-protein interaction (PPI) networks to get the interaction data. Ten DEGs, including 7 hub genes from PPI analysis, were confirmed by real-time PCR. RESULTS We found 90 and 323 DEGs at least 2-fold at days 14 and 28, respectively. At day 14, bony ankylosis showed upregulated DEGs, such as TLR8, SYK, NFKBIA, PTPRC, CD86, ITGAM, and ITGAL, indicating a stronger immune and inflammatory response and cell adhesion, while genes associated with anti-adhesion (PRG4) and inhibition of osteoblast differentiation (SFRP1) had higher expression in fibrous ankylosis. At day 28, bony ankylosis showed increased biological process related to new bone formation, while fibrous ankylosis was characterized by a prolonged immune and inflammatory reaction. CONCLUSIONS This study provides a differential gene expression profile between TMJ fibrous and bony ankylosis. Further study of these key genes may provide new ideas for future treatment of TMJ bony ankylosis.
Assuntos
Anquilose/genética , Fibrose/genética , Transtornos da Articulação Temporomandibular/genética , Traumatismos do Nervo Trigêmeo/genética , Animais , Anquilose/patologia , Modelos Animais de Doenças , Expressão Gênica/genética , Fraturas Mandibulares/genética , Análise em Microsséries , Ovinos/genética , Articulação Temporomandibular/metabolismo , Articulação Temporomandibular/patologia , Transtornos da Articulação Temporomandibular/patologia , Transcriptoma , Traumatismos do Nervo Trigêmeo/patologiaRESUMO
Multiple synostoses syndrome (SYNS1; OMIM# 186500) is a rare autosomal dominant disorder reported in a few cases worldwide. We report a Chinese pedigree characterized by proximal symphalangism, conductive hearing loss, and distinctive facies. We examined the genetic cause and reviewed the literature to discuss the pathogeny, treatment, and prevention of SYNS1. Audiological, ophthalmological, and radiological examinations were evaluated. Whole-exome sequencing (WES) was performed to identify mutations in the proband and her parents. Sanger sequencing was used to verify the results for the proband, parents, and grandmother. The literature on the genotype-phenotype correlation was reviewed. The patient was diagnosed with multiple synostoses syndrome clinically. WES and bioinformatic analysis revealed a novel missense mutation in the NOG gene, c.554C>G (p.Ser185Cys), cosegregated in this family. The literature review showed that the phenotype varies widely, but the typical facies, conductive hearing loss, and proximal symphalangism occurred frequently. All reported mutations are highly conserved in mammals based on conservation analysis, and there are regional hot spots for these mutations. However, no distinct genotype-phenotype correlations have been identified for mutations in NOG in different races. Regular systematic examinations and hearing aids are beneficial for this syndrome. However, the outcomes of otomicrosurgery are not encouraging owing to the regrowth of bone. This study expanded the mutation spectrum of NOG and is the first report of SYNS1 in a Chinese family. Genetic testing is recommended as part of the diagnosis of syndromic deafness. A clinical genetic evaluation is essential to guide prevention, such as preimplantation genetic diagnosis.
Assuntos
Anquilose/genética , Ossos do Carpo/anormalidades , Proteínas de Transporte/genética , Deformidades Congênitas do Pé/genética , Deformidades Congênitas da Mão/genética , Perda Auditiva Condutiva/genética , Estribo/anormalidades , Sinostose/genética , Ossos do Tarso/anormalidades , Falanges dos Dedos do Pé/anormalidades , Anquilose/complicações , Anquilose/epidemiologia , Anquilose/patologia , Ossos do Carpo/patologia , Criança , Pré-Escolar , China/epidemiologia , Feminino , Deformidades Congênitas do Pé/complicações , Deformidades Congênitas do Pé/epidemiologia , Deformidades Congênitas do Pé/patologia , Estudos de Associação Genética , Predisposição Genética para Doença , Deformidades Congênitas da Mão/complicações , Deformidades Congênitas da Mão/epidemiologia , Deformidades Congênitas da Mão/patologia , Perda Auditiva Condutiva/complicações , Perda Auditiva Condutiva/epidemiologia , Perda Auditiva Condutiva/patologia , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Linhagem , Fenótipo , Estribo/patologia , Sinostose/complicações , Sinostose/epidemiologia , Sinostose/patologia , Ossos do Tarso/patologia , Falanges dos Dedos do Pé/patologia , Dedos do Pé/anormalidades , Dedos do Pé/patologia , Sequenciamento do ExomaRESUMO
BACKGROUND: Little is known about the mechanisms underlying the clinical overlap between gut inflammation and joint ankylosis, as exemplified by the concurrence of inflammatory bowel diseases (IBD) and ankylosing spondylitis (AS). As dysbiosis may serve as a common contributor, the anti-microbial pleiotropic factor lipocalin 2 could be a potential mediator due to its roles in inflammation and bone homeostasis. METHODS: Baseline colonic pathology was conducted in the ank/ank mouse model. Serum lipocalin 2 was analyzed by ELISA, in ank/ank mutants versus C3FeB6-A/Aw-jwt/wt, in patients with concurrent AS-IBD, AS alone, IBD alone, or mechanical back pain, and in healthy controls. In the ank/ank mouse model, the expression of nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) was examined by real-time PCR. Intraperitoneal injection was done with the PPARγ agonist rosiglitazone or antagonist bisphenol A diglycidyl ether for four consecutive days. Serum levels of lipocalin 2 were examined on the sixth day. RESULTS: This study showed that the ank/ank mice with fully fused spines had concurrent colonic inflammation. By first using the ank/ank mouse model with progressive ankylosis and subclinical colonic inflammation, confirmed in patients with concurrent AS and IBD, elevated circulating lipocalin 2 levels were associated with the coexisting ankylosis and gut inflammation. The intracellular pathway of lipocalin 2 was further investigated with the ank/ank mouse model involving PPARγ. Colonic expression of PPARγ was negatively associated with the degree of gut inflammation. The PPARγ agonist rosiglitazone treatment significantly upregulated the serum levels of lipocalin 2, suggesting a potential regulatory role of PPARγ in the aberrant expression of lipocalin 2. CONCLUSIONS: In summary, lipocalin 2 modulated by PPARγ could be a potential pathway involved in concurrent inflammation and ankylosis in AS and IBD.
Assuntos
Anquilose/metabolismo , Modelos Animais de Doenças , Inflamação/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Lipocalina-2/metabolismo , Espondilite Anquilosante/metabolismo , Animais , Anquilose/sangue , Anquilose/genética , Feminino , Humanos , Inflamação/sangue , Inflamação/genética , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/genética , Lipocalina-2/sangue , Lipocalina-2/genética , Masculino , Camundongos Knockout , PPAR gama/agonistas , PPAR gama/genética , PPAR gama/metabolismo , Rosiglitazona/farmacologia , Espondilite Anquilosante/sangue , Espondilite Anquilosante/genética , Regulação para Cima/efeitos dos fármacosAssuntos
Anquilose/metabolismo , Anquilose/patologia , Proteínas de Transporte/metabolismo , Perda Auditiva Condutiva/metabolismo , Perda Auditiva Condutiva/patologia , Estribo/anormalidades , Adolescente , Adulto , Anquilose/genética , Proteínas de Transporte/genética , Criança , Cromossomos Humanos Par 17/genética , Feminino , Perda Auditiva Condutiva/genética , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
PURPOSE: To evaluate the association between polymorphisms in genes that regulate bone metabolism, such as OPG, RANK, RANKL, and HIF1A, in patients with temporomandibular joint (TMJ) ankylosis. METHODS: The sample consisted of 181 individuals, the study included 17 individuals with TMJ ankylosis and 164 controls. DNA was extracted from buccal epithelial cells. The genotyping of genetic polymorphisms in OPG (rs2073618), RANK (rs3826620), RANKL (rs9594738), and HIF1A (rs2301113 and rs2057482) was performed by real-time PCR using TaqMan™ technology (Applied Biosystems). The data were subjected to statistical analysis with a level of significance of 0.05. RESULTS: The OPG (rs2073618) polymorphism was associated with TMJ ankylosis, both in the additive model and in the dominant model (p < 0.05). In the additive model, when the individuals carried the CC genotype, they presented as 10.80 times more likely to develop the condition (p = 0.03). In the dominant model, individuals that carried at least one C allele were 5.76 times more likely to have TMJ ankylosis, than those with the G allele (p = 0.01). CONCLUSION: The polymorphism rs2073618 of OPG is a possible marker that is associated with the risk of manifestation of TMJ ankylosis.
Assuntos
Anquilose/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Osteoprotegerina/genética , Ligante RANK/genética , Receptor Ativador de Fator Nuclear kappa-B/genética , Transtornos da Articulação Temporomandibular/genética , Humanos , Pacientes , Polimorfismo de Nucleotídeo Único , Articulação TemporomandibularRESUMO
Dendritic cell immunoreceptor (DCIR) is a C-type lectin receptor mainly expressed in DCs. Dcir (-/-) mice spontaneously develop autoimmune enthesitis and ankylosis accompanied by fibrocartilage proliferation and ectopic ossification. However, the mechanisms of new bone/cartilage formation in Dcir (-/-) mice remain to be elucidated. In this study, we show that DCIR maintains bone homeostasis by regulating IFN-γ production under pathophysiological conditions. DCIR deficiency increased bone volume in femurs and caused aberrant ossification in joints, whereas these symptoms were abolished in Rag2(-/-)Dcir(-/-) mice. IFN-γ-producing T cells accumulated in lymph nodes and joints of Dcir(-/-) mice, and purified Dcir(-/-) DCs enhanced IFN-γ(+) T cell differentiation. The ankylotic changes and bone volume increase were suppressed in the absence of IFN-γ. Thus, IFN-γ is a positive chondrogenic and osteoblastogenic factor, and DCIR is a crucial regulator of bone metabolism; consequently, both factors are potential targets for therapies directed against bone metabolic diseases.
Assuntos
Osso e Ossos/metabolismo , Homeostase/genética , Homeostase/imunologia , Interferon gama/biossíntese , Lectinas Tipo C/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fatores Etários , Animais , Anquilose/diagnóstico por imagem , Anquilose/genética , Anquilose/imunologia , Anquilose/patologia , Densidade Óssea/genética , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Condrócitos/citologia , Condrócitos/metabolismo , Proteínas de Ligação a DNA/deficiência , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/metabolismo , Subpopulações de Linfócitos T/citologia , Microtomografia por Raio-XRESUMO
Ankylosing spondylitis (AS) is the most common form of spondyloarthritis and is characterized by both inflammation and new bone formation. Despite many years of arduous efforts, we still do not clearly understand the pathogenesis of AS. The mechanisms behind new bone formation have been especially challenging to decipher due to the difficulty in obtaining tissue from spinal joints. The link between inflammation and bone formation looks obvious, but how inflammation drives spinal fusion is not evident. There are now two genes linked to prostaglandins (PG) that could be involved in AS pathogenesis. The first one is PTGER4 that codes for the EP4 receptor for PGE2 and the other one is PTGS1 that codes for prostaglandin-endoperoxide syntase 1 or cyclogenase 1. The bone morphogenic protein and Wnt signaling pathways could be important in signaling increased bone formation in AS. The disease-modifying potential of anti-inflammatories and tumor necrosis factor alpha inhibitors is discussed.
Assuntos
Ciclo-Oxigenase 1/genética , Osteogênese/genética , Receptores de Prostaglandina E Subtipo EP4/genética , Espondilite Anquilosante/genética , Animais , Anquilose/genética , Anquilose/fisiopatologia , Proteínas Morfogenéticas Ósseas/genética , Humanos , Espondilite Anquilosante/fisiopatologia , Via de Sinalização Wnt/genéticaRESUMO
In this study, we describe three unrelated Japanese patients with hearing loss and symphalangism who were diagnosed with proximal symphalangism (SYM1), atypical multiple synostosis syndrome (atypical SYNS1) and stapes ankylosis with broad thumb and toes (SABTT), respectively, based on the clinical features. Surgical findings in the middle ear were similar among the patients. By next-generation and Sanger sequencing analyses, we identified two novel mutations, c.559C>G (p.P178A) and c.682T>A (p.C228S), in the SYM1 and atypical SYNS1 families, respectively. No pathogenic changes were found in the protein-coding regions, exon-intron boundaries or promoter regions of the NOG, GDF5 or FGF9 genes in the SABTT family. Such negative molecular data suggest there may be further genetic heterogeneity underlying SYNS1, with the involvement of at least one additional gene. Stapedotomy resulted in good hearing in all patients over the long term, indicating no correlation between genotype and surgical outcome. Given the overlap of the clinical features of these syndromes in our patients and the molecular findings, the diagnostic term 'NOG-related-symphalangism spectrum disorder (NOG-SSD)' is advocated and an unidentified gene may be responsible for this disorder.
Assuntos
Anquilose/genética , Ossos do Carpo/anormalidades , Proteínas de Transporte/genética , Anormalidades Congênitas/genética , Deformidades Congênitas do Pé/genética , Estudos de Associação Genética , Deformidades Congênitas da Mão/genética , Mutação , Estribo/anormalidades , Sinostose/genética , Ossos do Tarso/anormalidades , Adulto , Feminino , Fator 9 de Crescimento de Fibroblastos/genética , Fator 5 de Diferenciação de Crescimento/genética , Perda Auditiva/genética , Humanos , Japão , Masculino , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
We recently have reported on a novel ankylosis gene that is closely linked to the Enpp1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) gene on chromosome 10. Here, we have discovered novel mutant mice in a Jcl:ICR closed colony with ankylosis in the toes of the forelimbs at about 3 weeks of age. The mutant mice exhibited rigidity in almost all joints, including the vertebral column, which increased with age. These mice also showed hypogrowth with age after 16 weeks due to a loss of visceral fat, which may have been caused by poor nutrition. Histological examination and soft X-ray imaging demonstrated the ectopic ossification of various joints in the mutant mice. In particular, increased calcium deposits were observed in the joints of the toes, the carpal bones and the vertebral column. We sequenced all exons and exon/intron boundaries of Enpp1 in the normal and mutant mice, and identified a G-to-T substitution (c.259+1G>T) in the 5' splice donor site of intron 2 in the Enpp1 gene of the mutant mice. This substitution led to the skipping of exon 2 (73 bp), which generated a stop codon at position 354 bp (amino acid 62) of the cDNA (p.V63Xfs). Nucleotide pyrophosphohydrolase (NPPH) activity of ENPP1 in the mutant mice was also decreased, suggesting that Enpp1 gene function is disrupted in this novel mutant. The mutant mice reported in this study will be a valuable animal model for future studies of human osteochondral diseases and malnutrition.
Assuntos
Alelos , Camundongos Mutantes , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/isolamento & purificação , Pirofosfatases/genética , Pirofosfatases/isolamento & purificação , Animais , Anquilose/genética , Sequência de Bases , Cromossomos de Mamíferos/genética , Modelos Animais de Doenças , Feminino , Transferência Genética Horizontal , Humanos , Masculino , Desnutrição/genética , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Mutação , Osteocondrodisplasias/genéticaRESUMO
OBJECTIVE: To preliminarily investigate the temporal patterns of the endogenous mRNA expression for members of the Wnt signaling and a series of genes regulating bone formation during the development of traumatic temporomandibular joint (TMJ) bony ankylosis in a sheep model. METHODS: Six sheep were used for the induction of bony ankylosis of TMJ. We performed a condylar fracture, excision of the lateral 2/3 disc and serious injury to the glenoid fossa to induce bony ankylosis on the right TMJ. An isolated condylar fracture was performed on the left side. Two sheep were sacrificed at 1 month, 3 months, and 6 months after surgery, respectively. The specimens from the ankylosed joint and the condylar fracture were harvested for RNA extraction respectively. In this report (Part I), only the bony ankylosed samples were used for analysis of gene expressions. The specimens 1 month postoperatively were taken as the control, and the changes of expression of target genes over time were examined by real-time PCR. RESULTS: mRNA expression of Wnt1, Wnt2b, Wnt3a, ß-catenin, Sfrp1, Lrp6, Lef1, CyclinD1, and Runx2 was up-regulated at 3 and 6 months compared with 1 month. The expression of Wnt5a, Sox9, and Osterix was up-regulated with a peak at 3 months, and then fell back to the basal levels at 6 months. The expression of Ocn began to up-regulate until 6 month postoperatively. CONCLUSION: Our findings suggested that Wnt signaling was involved in the formation of traumatic TMJ bony ankylosis and thus may be a potential therapeutic target for the treatment of the disease in the future.
Assuntos
Anquilose/fisiopatologia , Transtornos da Articulação Temporomandibular/fisiopatologia , Articulação Temporomandibular/lesões , Via de Sinalização Wnt/fisiologia , Animais , Anquilose/genética , Subunidade alfa 1 de Fator de Ligação ao Core/análise , Ciclina D1/análise , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Glicoproteínas/análise , Peptídeos e Proteínas de Sinalização Intercelular/análise , Peptídeos e Proteínas de Sinalização Intracelular , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/análise , Fator 1 de Ligação ao Facilitador Linfoide/análise , Côndilo Mandibular/lesões , Fraturas Mandibulares/fisiopatologia , Osteocalcina/análise , Osteogênese/genética , Projetos Piloto , Proteínas Proto-Oncogênicas/análise , Fatores de Transcrição SOX9/análise , Ovinos , Osso Temporal/lesões , Disco da Articulação Temporomandibular/lesões , Transtornos da Articulação Temporomandibular/genética , Fatores de Transcrição/análise , Proteínas Wnt/análise , Via de Sinalização Wnt/genética , Proteína Wnt1/análise , Proteína Wnt3A/análise , beta Catenina/análiseRESUMO
OBJECTIVE: The purpose of the study was to preliminarily explore the differential expressions of a series of genes regulating bone formation in temporomandibular joint (TMJ) fibrous ankylosis, bony ankylosis and condylar fracture healing. METHODS: The cDNA from either the bony ankylosed callus or fracture callus of the 6 sheep, as described in the part I, were both used in the study. The differences of gene expressions between bony ankylosis and condylar fracture at 1, 3, and 6 months postoperatively were measured by real-time PCR, with 2 samples at each time point. In addition, another 2 sheep were added to have fibrous ankylosis induced on the right TMJ, and 1 sheep was sacrificed at 3 and 6 months after surgery, respectively. The differences of gene expressions between fibrous and bony ankylosis at 3 and 6 months postoperatively were measured by real-time PCR. RESULTS: Bony ankylosis showed higher mRNA expression trends in Wnt2b, Wnt5a, ß-Catenin, Lef1, CyclinD1, Runx2, Osterix, Sox9, Col10a1, Alp, Ocn, Bmp2, and Bmp7 compared to fibrous ankylosis, although no statistical analysis was performed due to the very small sample size. Whereas bony ankylosis showed a significant lower expression of Wnt5a, ß-Catenin, Lef1, Runx2, Osterix, Sox9, Col10a1, Alp, Ocn and Bmp4 compared to condylar fracture at several time points (P < 0.05). CONCLUSION: Our data provided a preliminary molecular evidence for the hypothesis that the development of traumatic TMJ bony ankylosis was the course of delayed bone healing or hypertrophic nonunion, and deserved to be further studied.
Assuntos
Anquilose/fisiopatologia , Consolidação da Fratura/fisiologia , Côndilo Mandibular/lesões , Fraturas Mandibulares/fisiopatologia , Transtornos da Articulação Temporomandibular/fisiopatologia , Articulação Temporomandibular/lesões , Fosfatase Alcalina/análise , Animais , Anquilose/genética , Proteína Morfogenética Óssea 2/análise , Proteína Morfogenética Óssea 7/análise , Calo Ósseo/fisiopatologia , Colágeno Tipo X/análise , Subunidade alfa 1 de Fator de Ligação ao Core/análise , Ciclina D1/análise , Modelos Animais de Doenças , Fibrose , Consolidação da Fratura/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Fator 1 de Ligação ao Facilitador Linfoide/análise , Fraturas Mandibulares/genética , Osteocalcina/análise , Projetos Piloto , Proteínas Proto-Oncogênicas/análise , Fatores de Transcrição SOX9/análise , Ovinos , Transtornos da Articulação Temporomandibular/genética , Fatores de Transcrição/análise , Proteínas Wnt/análise , beta Catenina/análiseRESUMO
INTRODUCTION: Extracellular ATP (eATP) is released by articular chondrocytes under physiological and pathological conditions. High eATP levels cause pathologic calcification, damage cartilage, and mediate pain. We recently showed that stable over-expression of the progressive ankylosis gene product, ANK, increased chondrocyte eATP levels, but the mechanisms of this effect remained unexplored. The purpose of this work was to further investigate mechanisms of eATP efflux in primary articular chondrocytes and to better define the role of ANK in this process. METHODS: We measured eATP levels using a bioluminescence-based assay in adult porcine articular chondrocyte media with or without a 10 minute exposure to hypotonic stress. siRNAs for known ATP membrane transporters and pharmacologic inhibitors of ATP egress pathways were used to identify participants involved in chondrocyte eATP release. RESULTS: eATP levels increased after exposure to hypotonic media in a calcium-dependent manner in monolayer and 3-dimensional agarose gel cultures (p < 0.001). A potent transient receptor potential vanilloid 4 (TRPV4) agonist mimicked the effects of hypotonic media. ANK siRNA suppressed basal (p < 0.01) and hypotonically-stressed (p < 0.001) ATP levels. This effect was not mediated by altered extracellular pyrophosphate (ePPi) levels, and was mimicked by the ANK inhibitor, probenecid (p < 0.001). The P2X7/4 receptor inhibitor Brilliant Blue G also suppressed eATP efflux induced by hypotonic media (p < 0.001), while ivermectin, a P2X4 receptor stimulant, increased eATP levels (p < 0.001). Pharmacologic inhibitors of hemichannels, maxianion channels and other volume-sensitive eATP efflux pathways did not suppress eATP levels. CONCLUSIONS: These findings implicate ANK and P2X7/4 receptors in chondrocyte eATP efflux. Understanding the mechanisms of eATP efflux may result in novel therapies for calcium crystal arthritis and osteoarthritis.
Assuntos
Trifosfato de Adenosina/metabolismo , Condrócitos/metabolismo , Espaço Extracelular/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Animais , Anquilose/genética , Anquilose/metabolismo , Anquilose/patologia , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Leucina/análogos & derivados , Leucina/farmacologia , Proteínas de Transporte de Fosfato/antagonistas & inibidores , Proteínas de Transporte de Fosfato/genética , Probenecid/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X4/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Sulfonamidas/farmacologia , Suínos , Canais de Cátion TRPV/agonistasRESUMO
OBJECTIVE: We assessed the role of Ank in the maintenance of postnatal articular cartilage using the ank/ank mouse (mice homozygous for progressive ankylosis). METHODS: We analyzed ank/ank mice and wild-type littermates (8, 12, and 18 weeks old). Sections from decalcified, paraffin-embedded joints were stained with hematoxylin and eosin. Articular chondrocyte size and cartilage thickness were determined using morphometric methods. Immuno-histochemical staining was performed with anticollagen X, antitissue nonspecific alkaline phosphatase (TNAP), and anti-ß-catenin antibodies on fixed joint sections. Axin2 expression in paw joint lysates in wild-type versus ank/ank mice were compared using Western blot analysis. RESULTS: In all age groups of normal mice studied, calcified cartilage (CC) chondrocyte areas were significantly larger than those of uncalcified cartilage (UC) chondrocytes. However, similar chondrocyte areas (UC vs CC) were found in 12-week and 18-week-old ank/ank mice, indicating that hypertrophic chondrocytes were present in the UC of these mutant mice. The ank/ank mice showed an increase in CC thickness. The ank/ank UC hypertrophic chondrocytes showed diffuse immuno-reactivity for collagen X and TNAP. Increased ß-catenin activation was demonstrated by nuclear localization of ß-catenin staining in ank/ank chondrocytes. Axin2 expression from paw lysates was downregulated in ank/ank mice. CONCLUSION: We identified a previously unrecognized phenotype in the articular cartilage of ank/ank mice: collagen X-positive hypertrophic chondrocytes in the UC. It is possible that consequent to downregulation of axin2 expression, ß-catenin signaling was activated, leading to accelerated chondrocyte maturation and eventual ankylosis in ank/ank joints. Our studies shed new light on the contribution of a key signaling pathway in this model of joint ankylosis.
Assuntos
Anquilose/genética , Anquilose/metabolismo , Condrócitos/metabolismo , Condrócitos/patologia , Fenótipo , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Anquilose/fisiopatologia , Proteína Axina/metabolismo , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Colágeno Tipo X/metabolismo , Feminino , Heterozigoto , Hipertrofia , Articulações/metabolismo , Articulações/patologia , Camundongos , Camundongos Mutantes , Modelos AnimaisRESUMO
Human noggin (NOG) is a responsible gene for multiple synostosis syndrome (SYNS1) and proximal symphalangism (SYM1), two conditions that are recently known to be within a wider range of clinical manifestations of stapes ankylosis with symphalangism. This study was performed to determine the range of phenotype caused by NOG mutations, using Japanese patients with various phenotypes including sporadic inherited SYM1, dominantly inherited SYM1, stapes ankylosis with broad thumb and toes (Teunissen and Cremer syndrome). In addition, 33 patients with typical otosclerosis (without symphalangism) were studied. Direct sequencing analysis disclosed three novel mutations of the NOG gene in three SYM1 families. None of the otosclerosis patients without symphalangism had NOG mutations, indicating that NOG mutations may be restrictively found within patients with various skeletal abnormalities. These results together with the literature review indicated that there are no clear genotype-phenotype correlations for NOG mutations. With regard to surgical outcome, most of the patients in these three families with NOG mutations showed remarkable air-bone gap recovery after stapes surgery. Molecular genetic testing is useful to differentiate syndromic stapes ankylosis from otosclerosis, and even mild skeletal anomalies can be a diagnostic indicator of NOG-associated disease.
Assuntos
Anquilose/genética , Proteínas de Transporte/genética , Predisposição Genética para Doença/genética , Hiperopia/genética , Artropatias/congênito , Fenótipo , Sindactilia/genética , Adulto , Idoso , Anquilose/patologia , Povo Asiático/genética , Ossos do Carpo/anormalidades , Primers do DNA/genética , Feminino , Articulações dos Dedos/anormalidades , Articulações dos Dedos/patologia , Deformidades Congênitas do Pé , Deformidades Congênitas da Mão , Perda Auditiva Condutiva/genética , Perda Auditiva Condutiva/patologia , Humanos , Hiperopia/patologia , Artropatias/genética , Artropatias/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Otosclerose/genética , Linhagem , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Estribo/anormalidades , Estribo/patologia , Sindactilia/patologia , Sinostose , Ossos do Tarso/anormalidades , Polegar/anormalidades , Polegar/patologia , Dedos do Pé/anormalidades , Dedos do Pé/patologiaRESUMO
Otosclerosis is a complex bone remodeling disorder of the human otic capsule that might be associated with various mutations of A1 and A2 alleles of type-I collagen. The study herein presented, investigates the possibilty of the genetic involvement of type-I collagen in the pathogenesis of histologically confirmed otosclerosis. A total of 55 ankylotic stapes footplates were analyzed. Cortical bone fragments (n = 30), incus (n = 3) and malleus (n = 2) specimens were employed as negative controls. Specimens were divided into two groups. The first group was processed using conventional H.E. hematoxylin-eosin (H.E.) staining and type-I collagen-specific immunofluorescent assay (IFA), while the second group was examined by COL1A1 and A2-specific RT-PCR. Otosclerotic- (n = 31) and non-otosclerotic stapes footplates (n = 9) as well as cortical bones (n = 20), incus (n = 2) and malleus specimens (n = 1) showed normal and quite similar A1 and A2 allele expression confirmed by IFA. RT-PCR analysis revealed normal and consistent mRNA expression of both alleles in each specimen. Expression levels and patterns of COL1A1/A2 alleles did not show significant correlation with the histological diagnosis of otosclerosis. Type-I collagen is a highly conserved structure protein, which plays a fundamental role in the integritiy of various connective tissues. Mutations of A1 and A2 alleles result in serious systemic disorders of the skeleton, tendons and skin. Since otosclerosis is an organ-specific disease, it is difficult to explain its genetic association with type-I collagen. In conclusion, we found no evidence supporting the putative link of COL1A1 and COL1A2 alleles with otosclerosis.
Assuntos
Colágeno Tipo I/genética , Otosclerose/genética , RNA Mensageiro/análise , Estribo/metabolismo , Adulto , Idoso , Anquilose/genética , Estudos de Casos e Controles , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mutação , Otosclerose/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVES: To investigate p38 mitogen activated protein kinase (MAPK) signalling in an in vitro model of bone morphogenetic protein (BMP) and transforming growth factor ß (TGFß)-induced chondrogenesis and in vivo, with specific attention to its potential role in ankylosing enthesitis. METHODS: Human periosteum-derived cells (hPDCs) were cultured in pellets and stimulated with BMP2 or TGFß1 in the presence or absence of a p38 inhibitor SB203580 or proinflammatory cytokines. Chondrogenic differentiation was evaluated using quantitative PCR. Male DBA/1 mice from different litters were caged together at the age of 8 weeks and treated with SB203580 in both a preventive and therapeutic strategy. The mice were evaluated for prospective signs of arthritis and the toe joints were analysed histologically to assess disease severity. RESULTS: p38 inhibition by SB203580 and proinflammatory cytokines downregulated chondrogenic markers in pellet cultures stimulated by BMP2 or TGFß1. In contrast, the in vivo experiments resulted in an increased clinical incidence of arthritis and pathology severity score, reflecting progression towards ankylosis in mice given SB203580. CONCLUSION: Inhibition of p38 inhibited chondrogenic differentiation of progenitor cells, showing that not only the SMAD signalling pathways and also alternative activation of MAPKs including p38 contribute to chondrogenesis. Such an inhibitory effect is not found in an in vivo model of joint ankylosis and spondyloarthritis. Increased incidence and severity of disease in preventive experiments and shifts in disease stages in a therapeutic experimental set-up suggest that specific inhibition of p38 may have deleterious rather than beneficial effects.