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1.
Nat Commun ; 10(1): 2429, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160553

RESUMO

The WW domain-containing protein 2 (Wwp2) gene, the host gene of miR-140, codes for the Wwp2 protein, which is an HECT-type E3 ubiquitin ligases abundantly expressed in articular cartilage. However, its function remains unclear. Here, we show that mice lacking Wwp2 and mice in which the Wwp2 E3 enzyme is inactivated (Wwp2-C838A) exhibit aggravated spontaneous and surgically induced osteoarthritis (OA). Consistent with this phenotype, WWP2 expression level is downregulated in human OA cartilage. We also identify Runx2 as a Wwp2 substrate and Adamts5 as a target gene, as similar as miR-140. Analysis of Wwp2-C838A mice shows that loss of Wwp2 E3 ligase activity results in upregulation of Runx2-Adamts5 signaling in articular cartilage. Furthermore, in vitro transcribed Wwp2 mRNA injection into mouse joints reduces the severity of experimental OA. We propose that Wwp2 has a role in protecting cartilage from OA by suppressing Runx2-induced Adamts5 via Runx2 poly-ubiquitination and degradation.


Assuntos
Proteína ADAMTS5/metabolismo , Cartilagem Articular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoartrite/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Artrite Experimental/genética , Artrite Experimental/metabolismo , Cartilagem Articular/diagnóstico por imagem , Modelos Animais de Doenças , Humanos , Articulação do Joelho/diagnóstico por imagem , Meniscos Tibiais/cirurgia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Osteoartrite/metabolismo , RNA Mensageiro/farmacologia , Transdução de Sinais , Crânio/diagnóstico por imagem , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Microtomografia por Raio-X , Adulto Jovem
2.
Dev Cell ; 46(6): 794-806.e6, 2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30146478

RESUMO

SRY-box 9 (SOX9) is a master transcription factor that regulates cartilage development. SOX9 haploinsufficiency resulting from breakpoints in a ∼1-Mb region upstream of SOX9 was reported in acampomelic campomelic dysplasia (ACD) patients, suggesting that essential enhancer regions of SOX9 for cartilage development are located in this long non-coding sequence. However, the cis-acting enhancer region regulating cartilage-specific SOX9 expression remains to be identified. To identify distant cartilage Sox9 enhancers, we utilized the combination of multiple CRISPR/Cas9 technologies including enrichment of the promoter-enhancer complex followed by next-generation sequencing and mass spectrometry (MS), SIN3A-dCas9-mediated epigenetic silencing, and generation of enhancer deletion mice. As a result, we could identify a critical far-upstream cis-element and Stat3 as a trans-acting factor, regulating cartilage-specific Sox9 expression and subsequent skeletal development. Our strategy could facilitate definitive ACD diagnosis and should be useful to reveal the detailed chromatin conformation and regulation.


Assuntos
Sistemas CRISPR-Cas , Cartilagem/metabolismo , Condrócitos/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Fatores de Transcrição SOX9/metabolismo , Animais , Cartilagem/citologia , Células Cultivadas , Condrócitos/citologia , Cromatina/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Fatores de Transcrição SOX9/genética , Fator de Transcrição STAT3/metabolismo , Deleção de Sequência
4.
J Bone Miner Metab ; 36(1): 64-72, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28324176

RESUMO

Split hand/foot malformation (SHFM) and SHFM combined with long-bone deficiency (SHFLD) are congenital dysgeneses of the limb. Although six different loci/mutations (SHFM1-SHFM6) have been found from studies on families with SHFM, the causes and associated pathogenic mechanisms for a large number of patients remain unidentified. On the basis of the identification of a duplicated gene region involving BHLHA9 in some affected families, BHLHA9 was identified as a novel SHFM/SHFLD-related gene. Although Bhlha9 is predicted to participate in limb development as a transcription factor, its precise function is unclear. Therefore, to study its physiological function, we generated a Bhlha9-knockout mouse and investigated gene expression and the associated phenotype in the limb bud. Bhlha9-knockout mice showed syndactyly and poliosis in the limb. Moreover, some apical ectodermal ridge (AER) formation related genes, including Trp63, exhibited an aberrant expression pattern in the limb bud of Bhlha9-knockout mice; TP63 (Trp63) was regulated by Bhlha9 on the basis of in vitro analysis. These observations suggest that Bhlha9 regulates AER formation during limb/finger development by regulating the expression of some AER-formation-related genes and abnormal expression of Bhlha9 leads to SHFM and SHFLD via dysregulation of AER formation and associated gene expression.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ectoderma/embriologia , Ectoderma/metabolismo , Extremidades/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Fosfoproteínas/metabolismo , Transativadores/metabolismo
5.
Neurobiol Dis ; 106: 158-170, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28688852

RESUMO

Mutations in the Cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders accompanied by intractable epilepsies, i.e. West syndrome or atypical Rett syndrome. Here we report generation of the Cdkl5 knockout mouse and show that CDKL5 controls postsynaptic localization of GluN2B-containing N-methyl-d-aspartate (NMDA) receptors in the hippocampus and regulates seizure susceptibility. Cdkl5 -/Y mice showed normal sensitivity to kainic acid; however, they displayed significant hyperexcitability to NMDA. In concordance with this result, electrophysiological analysis in the hippocampal CA1 region disclosed an increased ratio of NMDA/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs) and a significantly larger decay time constant of NMDA receptor-mediated EPSCs (NMDA-EPSCs) as well as a stronger inhibition of the NMDA-EPSCs by the GluN2B-selective antagonist ifenprodil in Cdkl5 -/Y mice. Subcellular fractionation of the hippocampus from Cdkl5 -/Y mice revealed a significant increase of GluN2B and SAP102 in the PSD (postsynaptic density)-1T fraction, without changes in the S1 (post-nuclear) fraction or mRNA transcripts, indicating an intracellular distribution shift of these proteins to the PSD. Immunoelectron microscopic analysis of the hippocampal CA1 region further confirmed postsynaptic overaccumulation of GluN2B and SAP102 in Cdkl5 -/Y mice. Furthermore, ifenprodil abrogated the NMDA-induced hyperexcitability in Cdkl5 -/Y mice, suggesting that upregulation of GluN2B accounts for the enhanced seizure susceptibility. These data indicate that CDKL5 plays an important role in controlling postsynaptic localization of the GluN2B-SAP102 complex in the hippocampus and thereby regulates seizure susceptibility, and that aberrant NMDA receptor-mediated synaptic transmission underlies the pathological mechanisms of the CDKL5 loss-of-function.


Assuntos
Hipocampo/metabolismo , Densidade Pós-Sináptica/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsões/metabolismo , Animais , Modelos Animais de Doenças , Suscetibilidade a Doenças/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Guanilato Quinases/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Ácido Caínico , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Metilaspartato , Piperidinas/farmacologia , Densidade Pós-Sináptica/efeitos dos fármacos , Densidade Pós-Sináptica/patologia , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Convulsões/patologia , Técnicas de Cultura de Tecidos
6.
PLoS One ; 12(5): e0175673, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28467430

RESUMO

Limb bud patterning, outgrowth, and differentiation are precisely regulated in a spatio-temporal manner through integrated networks of transcription factors, signaling molecules, and downstream genes. However, the exact mechanisms that orchestrate morphogenesis of the limb remain to be elucidated. Previously, we have established EMBRYS, a whole-mount in situ hybridization database of transcription factors. Based on the findings from EMBRYS, we focused our expression pattern analysis on a selection of transcription factor genes that exhibit spatially localized and temporally dynamic expression patterns with respect to the anterior-posterior axis in the E9.5-E11.5 limb bud. Among these genes, Irx3 showed a posteriorly expanded expression domain in Shh-/- limb buds and an anteriorly reduced expression domain in Gli3-/- limb buds, suggesting their importance in anterior-posterior patterning. To assess the stepwise EMBRYS-based screening system for anterior regulators, we generated Irx3 transgenic mice in which Irx3 was expressed in the entire limb mesenchyme under the Prrx1 regulatory element. The Irx3 gain-of-function model displayed complex phenotypes in the autopods, including digit loss, radial flexion, and fusion of the metacarpal bones, suggesting that Irx3 may contribute to the regulation of limb patterning, especially in the autopods. Our results demonstrate that gene expression analysis based on EMBRYS could contribute to the identification of genes that play a role in patterning of the limb mesenchyme.


Assuntos
Botões de Extremidades/metabolismo , Fatores de Transcrição/metabolismo , Animais , Perfilação da Expressão Gênica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real
7.
J Neurosci ; 37(12): 3181-3191, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28213441

RESUMO

The secreted glycoprotein Reelin regulates embryonic brain development and adult brain functions. It has been suggested that reduced Reelin activity contributes to the pathogenesis of several neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease; however, noninvasive methods that can upregulate Reelin activity in vivo have yet to be developed. We previously found that the proteolytic cleavage of Reelin within Reelin repeat 3 (N-t site) abolishes Reelin activity in vitro, but it remains controversial as to whether this effect occurs in vivo Here we partially purified the enzyme that mediates the N-t cleavage of Reelin from the culture supernatant of cerebral cortical neurons. This enzyme was identified as a disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3). Recombinant ADAMTS-3 cleaved Reelin at the N-t site. ADAMTS-3 was expressed in excitatory neurons in the cerebral cortex and hippocampus. N-t cleavage of Reelin was markedly decreased in the embryonic cerebral cortex of ADAMTS-3 knock-out (KO) mice. Importantly, the amount of Dab1 and the phosphorylation level of Tau, which inversely correlate with Reelin activity, were significantly decreased in the cerebral cortex of ADAMTS-3 KO mice. Conditional KO mice, in which ADAMTS-3 was deficient only in the excitatory neurons of the forebrain, showed increased dendritic branching and elongation in the postnatal cerebral cortex. Our study shows that ADAMTS-3 is the major enzyme that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. Therefore, inhibition of ADAMTS-3 may be an effective treatment for neuropsychiatric and neurodegenerative disorders.SIGNIFICANCE STATEMENT ADAMTS-3 was identified as the protease that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. ADAMTS-3 was expressed in the excitatory neurons of the embryonic and postnatal cerebral cortex and hippocampus. Cleavage by ADAMTS-3 is the major contributor of Reelin inactivation in vivo Tau phosphorylation was decreased and dendritic branching and elongation was increased in ADAMTS-3-deficient mice. Therefore, inhibition of ADAMTS-3 upregulates Reelin activity and may be a potential therapeutic strategy for the prevention or treatment of neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease.


Assuntos
Proteínas ADAMTS/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Ativação Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Ligação Proteica
8.
Development ; 144(2): 313-320, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27993989

RESUMO

The periodontal ligament (PDL), which connects the teeth to the alveolar bone, is essential for periodontal tissue homeostasis. Although the significance of the PDL is recognized, molecular mechanisms underlying PDL function are not well known. We report that mohawk homeobox (Mkx), a tendon-specific transcription factor, regulates PDL homeostasis by preventing its degeneration. Mkx is expressed in the mouse PDL at the age of 10 weeks and expression remained at similar levels at 12 months. In Mkx-/- mice, age-dependent expansion of the PDL at the maxillary first molar (M1) furcation area was observed. Transmission electron microscopy (TEM) revealed that Mkx-/- mice presented collagen fibril degeneration in PDL with age, while the collagen fibril diameter gradually increased in Mkx+/+ mice. PDL cells lost their shape in Mkx-/- mice, suggesting changes in PDL properties. Microarray and quantitative polymerase chain reaction (qPCR) analyses of Mkx-/- PDL revealed an increase in osteogenic gene expression and no change in PDL- and inflammatory-related gene expression. Additionally, COL1A1 and COL1A2 were upregulated in Mkx-overexpressing human PDL fibroblasts, whereas osteogenic genes were downregulated. Our results indicate that Mkx prevents PDL degeneration by regulating osteogenesis.


Assuntos
Proteínas de Homeodomínio/fisiologia , Homeostase/genética , Ligamento Periodontal/fisiologia , Perda do Osso Alveolar/genética , Perda do Osso Alveolar/patologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteogênese/genética
9.
PLoS One ; 8(10): e76004, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24146809

RESUMO

Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.


Assuntos
Proteínas de Ligação a DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Deleção de Genes , Marcação de Genes/métodos , MicroRNAs/genética , Proteínas Recombinantes de Fusão/genética , Animais , Sequência de Bases , DNA Intergênico/genética , DNA Intergênico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Embrião de Mamíferos , Feminino , Engenharia Genética , Camundongos , Camundongos Endogâmicos ICR , MicroRNAs/metabolismo , Microinjeções , Dados de Sequência Molecular , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo
10.
Biol Reprod ; 88(6): 143, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23616593

RESUMO

MicroRNAs (miRNAs) have been shown to play key regulatory roles in a range of biological processes, including cell differentiation and development. To identify miRNAs that participate in gonad differentiation, a fundamental and tightly regulated developmental process, we examined miRNA expression profiles at the time of sex determination and during the early fetal differentiation of mouse testes and ovaries using high-throughput sequencing. We identified several miRNAs that were expressed in a sexually dimorphic pattern, including several members of the let-7 family, miR-378, and miR-140-3p. We focused our analysis on the most highly expressed, sexually dimorphic miRNA, miR-140-3p, and found that both miR-140-3p and its more lowly expressed counterpart, the previously annotated guide strand, miR-140-5p, are testis enriched and expressed in testis cords. Analysis of the miR-140-5p/miR-140-3p-null mouse revealed a significant increase in the number of Leydig cells in the developing XY gonad, strongly suggesting an important role for miR-140-5p/miR-140-3p in testis differentiation in mouse.


Assuntos
Diferenciação Celular/genética , Células Intersticiais do Testículo/citologia , MicroRNAs/metabolismo , Testículo/citologia , Animais , Contagem de Células , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , Testículo/embriologia , Testículo/metabolismo
11.
J Biol Chem ; 287(26): 22206-15, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22547066

RESUMO

Sox9 plays a critical role in early chondrocyte initiation and promotion as well as repression of later maturation. Fellow Sox family members L-Sox5 and Sox6 also function as regulators of cartilage development by boosting Sox9 activation of chondrocyte-specific genes such as Col2a1 and Agc1; however, the regulatory mechanism and other target genes are largely unknown. MicroRNAs are a class of short, non-coding RNAs that act as negative regulators of gene expression by promoting target mRNA degradation and/or repressing translation. Analysis of genetically modified mice identified miR-140 as a cartilage-specific microRNA that could be a critical regulator of cartilage development and homeostasis. Recent findings suggest Sox9 promotes miR-140 expression, although the detailed mechanisms are not fully understood. In this study we demonstrate that the proximal upstream region of pri-miR-140 has chondrogenic promoter activity in vivo. We found an L-Sox5/Sox6/Sox9 (Sox trio) response element and detailed binding site in the promoter region. Furthermore, detailed analysis suggests the DNA binding and/or transactivation ability of Sox9 as a homodimer is boosted by L-Sox5 and Sox6. These findings provide new insight into cartilage-specific gene regulation by the Sox trio.


Assuntos
Cartilagem/metabolismo , MicroRNAs/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXD/metabolismo , Animais , Condrócitos/citologia , Imunoprecipitação da Cromatina , Dimerização , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase/métodos , Isoformas de Proteínas , Ativação Transcricional , Transgenes
12.
Genes Dev ; 24(11): 1173-85, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20466812

RESUMO

Osteoarthritis (OA), the most prevalent aging-related joint disease, is characterized by insufficient extracellular matrix synthesis and articular cartilage degradation, mediated by several proteinases, including Adamts-5. miR-140 is one of a very limited number of noncoding microRNAs (miRNAs) specifically expressed in cartilage; however, its role in development and/or tissue maintenance is largely uncharacterized. To examine miR-140 function in tissue development and homeostasis, we generated a mouse line through a targeted deletion of miR-140. miR-140(-/-) mice manifested a mild skeletal phenotype with a short stature, although the structure of the articular joint cartilage appeared grossly normal in 1-mo-old miR-140(-/-) mice. Interestingly, miR-140(-/-) mice showed age-related OA-like changes characterized by proteoglycan loss and fibrillation of articular cartilage. Conversely, transgenic (TG) mice overexpressing miR-140 in cartilage were resistant to antigen-induced arthritis. OA-like changes in miR-140-deficient mice can be attributed, in part, to elevated Adamts-5 expression, regulated directly by miR-140. We show that miR-140 regulates cartilage development and homeostasis, and its loss contributes to the development of age-related OA-like changes.


Assuntos
Cartilagem/crescimento & desenvolvimento , Homeostase/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAMTS5 , Animais , Desenvolvimento Ósseo/genética , Homeostase/genética , Articulação do Joelho/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Osteoartrite/patologia
13.
Proc Natl Acad Sci U S A ; 107(23): 10538-42, 2010 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-20498044

RESUMO

Mohawk (Mkx) is a member of the Three Amino acid Loop Extension superclass of atypical homeobox genes that is expressed in developing tendons. To investigate the in vivo functions of Mkx, we generated Mkx(-/-) mice. These mice had hypoplastic tendons throughout the body. Despite the reduction in tendon mass, the cell number in tail tendon fiber bundles was similar between wild-type and Mkx(-/-) mice. We also observed small collagen fibril diameters and a down-regulation of type I collagen in Mkx(-/-) tendons. These data indicate that Mkx plays a critical role in tendon differentiation by regulating type I collagen production in tendon cells.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Tendões/crescimento & desenvolvimento , Tendões/metabolismo , Animais , Colágeno Tipo I/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Tendões/citologia , Tendões/embriologia , Resistência à Tração
14.
Arthritis Rheum ; 60(9): 2723-30, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19714579

RESUMO

OBJECTIVE: MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue-specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA-140 (miR-140). METHODS: To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR-140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin-1beta (IL-1beta) on miR-140 expression. Double-stranded miR-140 (ds-miR-140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA. RESULTS: Microarray analysis showed that miR-140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR-140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR-140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1beta suppressed miR-140 expression. Transfection of chondrocytes with ds-miR-140 down-regulated IL-1beta-induced ADAMTS5 expression and rescued the IL-1beta-dependent repression of AGGRECAN gene expression. CONCLUSION: This study shows that miR-140 has a chondrocyte differentiation-related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1beta may contribute to the abnormal gene expression pattern characteristic of OA.


Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Interleucina-1beta/metabolismo , MicroRNAs/metabolismo , Osteoartrite do Joelho/metabolismo , RNA de Cadeia Dupla/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAMTS5 , Adulto , Idoso , Idoso de 80 Anos ou mais , Agrecanas/metabolismo , Cartilagem Articular/patologia , Estudos de Casos e Controles , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Colágeno Tipo II/metabolismo , Humanos , Interleucina-1beta/farmacologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Osteoartrite do Joelho/patologia , Fatores de Transcrição SOX9/metabolismo , Transfecção
15.
Exp Cell Res ; 315(13): 2231-40, 2009 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-19306868

RESUMO

The transcription factor, Sry-related High Mobility Group (HMG) box containing gene 9 (Sox9), plays a critical role in cartilage development by initiating chondrogenesis and preventing the subsequent maturation process called chondrocyte hypertrophy. This suppression mechanism by Sox9 on late-stage chondrogenesis partially results from the inhibition of Runt-related transcription factor 2 (Runx2), the main activator of hypertrophic chondrocyte differentiation. However, the precise mechanism by which Sox9 regulates late chondrogenesis is poorly understood. In the present study, the transcriptional repressor vertebrate homolog of Drosophila bagpipe (Bapx1) was found to be a direct target of Sox9 for repression of Runx2 expression in chondrocytes. We identified a critical Sox9 responsive region in the Bapx1 promoter via a luciferase reporter assay. Analysis by chromatin immunoprecipitation and electrophoretic mobility shift assays indicated that Sox9 physically bound to this region of the Bapx1 promoter. Consistent with the notion that Bapx1 and Sox9 act as negative regulators of chondrocyte hypertrophy by regulating Runx2 expression, transient knockdown of Sox9 or Bapx1 expression by shRNA in chondrocytes increased Runx2 expression, as well as expression of the late chondrogenesis marker, Col10a1. Furthermore, while over-expression of Sox9 decreased Runx2 and Col10a1 expressions, simultaneous transient knockdown of Bapx1 diminished that Sox9 over-expressing effect. Our findings reveal that the molecular pathway modulated by Bapx1 links two major regulators in chondrogenesis, Sox9 and Runx2, to coordinate skeletal formation.


Assuntos
Condrócitos/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Condrócitos/citologia , Condrogênese/fisiologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Fatores de Transcrição SOX9/genética , Fatores de Transcrição/genética
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