RESUMO
Knowledge of the molecular mechanisms of bone formation has been advanced by novel findings related to genetic control. Odd-skipped related 1 (Osr1) is known to play important roles in embryonic, heart, and urogenital development. To elucidate the in vivo function of Osr1 in bone formation, we generated transgenic mice overexpressing full-length Osr1 under control of its 2.8-kb promoter, which were smaller than their wild-type littermates. Notably, abnormalities in the skull of Osr1 transgenic mice were revealed by analysis of X-ray, skeletal preparation, and morphological findings, including round skull and cranial dysraphism. Furthermore, primary calvarial cells obtained from these mice showed increased proliferation and expression of chondrocyte markers, while expression of osteoblast markers was decreased. BMP2 reduced Osr1 expression and Osr1 knockdown by siRNA-induced alkaline phosphatase and osteocalcin expression in mesenchymal and osteoblastic cells. Together, our results suggest that Osr1 plays a coordinating role in appropriate skull closure and cranial bone formation by negative regulation.
Assuntos
Osteogênese , Crânio/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Dedos de Zinco , Animais , Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos Transgênicos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Fenótipo , Crânio/metabolismoRESUMO
Osteoblasts secrete matrix vesicles and proteins to bone surfaces, but the molecular mechanisms of this secretion system remain unclear. The present findings reveal the roles of important genes in osteoblasts involved in regulation of extracellular matrix secretion. We especially focused on "soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor" (SNARE) genes and identified notable Syntaxin 4a (Stx4a) expression on the basolateral side of the plasma membrane of osteoblasts. Furthermore, Stx4a overexpression was found to increase mineralization by osteoblastic cells, whereas Stx4a knockdown reduced levels of mineralization. Also, BMP-4 and IGF-1 induced the localization of Stx4a to the basolateral side of the cells. To examine the function of Stx4a in osteoblasts, we generated osteoblast-specific Stx4a conditional knockout mice, which demonstrated an osteopenic phenotype due to reduced matrix secretion. Bone mineral density, shown by peripheral quantitative computed tomography (pQCT), was reduced in the femur metaphyseal and diaphyseal regions of Stx4a osteoblast-specific deficient mice, whereas bone parameters, shown by micro-computed tomography (µCT) and bone histomorphometric analysis, were also decreased in trabecular bone. In addition, primary calvarial cells from those mice showed decreased mineralization and lower secretion of matrix vesicles. Our findings indicate that Stx4a plays a critical role in bone matrix production by osteoblasts. © 2016 American Society for Bone and Mineral Research.
Assuntos
Matriz Óssea/metabolismo , Vesículas Citoplasmáticas/metabolismo , Osteoblastos/metabolismo , Proteínas Qa-SNARE/metabolismo , Animais , Animais Recém-Nascidos , Densidade Óssea , Calcificação Fisiológica , Camundongos Knockout , Proteínas Qa-SNARE/genética , Crânio/citologia , Tíbia/diagnóstico por imagem , Microtomografia por Raio-XRESUMO
Periodontitis is an inflammatory disease causing loss of tooth-supporting periodontal tissue. Disease susceptibility to the rapidly progressive form of periodontitis, aggressive periodontitis (AgP), appears to be influenced by genetic risk factors. To identify these in a Japanese population, we performed whole exome sequencing of 41 unrelated generalized or localized AgP patients. We found that AgP is putatively associated with single nucleotide polymorphism (SNP) rs536714306 in the G-protein coupled receptor 126 gene, GPR126 [c.3086 G>A (p.Arg1029Gln)]. Since GPR126 activates the cAMP/PKA signaling pathway, we performed cAMP ELISA analysis of cAMP concentrations, and found that rs536714306 impaired the signal transactivation of GPR126. Moreover, transfection of human periodontal ligament (HPDL) cells with wild-type or mutant GPR126 containing rs536714306 showed that wild-type GPR126 significantly increased the mRNA expression of bone sialoprotein, osteopontin, and Runx2 genes, while mutant GPR126 had no effect on the expression of these calcification-related genes. The increase in expression of these genes was through the GPR126-induced increase of bone morphogenic protein-2, inhibitor of DNA binding (ID) 2, and ID4 expression. These data indicate that GPR126 might be important in maintaining the homeostasis of periodontal ligament tissues through regulating the cytodifferentiation of HPDL cells. The GPR126 SNP rs536714306 negatively influences this homeostasis, leading to the development of AgP, suggesting that it is a candidate genetic risk factor for AgP in the Japanese population.
Assuntos
Periodontite Agressiva/genética , Polimorfismo de Nucleotídeo Único , Receptores Acoplados a Proteínas G/genética , Adolescente , Adulto , Periodontite Agressiva/metabolismo , Povo Asiático/genética , Diferenciação Celular/genética , Exoma , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Ligamento Periodontal/citologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Adulto JovemRESUMO
The Odd-skipped gene, first identified as a Drosophila pair-rule zinc-finger transcription factor, plays an important role in Drosophila development. The mammalian homolog, Odd-skipped related 2 (Osr2), regulates limb, tooth, and kidney development in mouse embryos. However, the detailed expression pattern of Osr2 during neonatal development remains unclear. In this study, we investigated Osr2 expression patterns in mouse neonatal and embryo tissues using qPCR and in situ hybridization methods. First, we examined the tissue distribution of Osr2 by qPCR, and found it to be highly expressed in the uterus and moderately in the testes, small intestine, and prostate. That expression was also found in eye, kidney, placenta, lung, thymus, lymph node, stomach, and skeletal muscle tissues, and in all embryonic stages. On the other hand, Osr2 was not expressed in brain, heart, liver, or spleen samples. Next, we examined the tissue localization of Osr2 using in situ hybridization. Osr2 was found in the craniofacial region on E13.5, with notable expression in dental germ mesenchyme as well as the renal corpuscle on E17.5. As for neonatal tissues, Osr2 was expressed in the dental papilla, dental follicle, Harderian gland, nasal bone, eyelid dermis, synovial joint, and tibial subcutis. Our findings suggest that Osr2 functions in reproductive system organs, such as the uterus, testes, prostate, placenta, and ovaries. Furthermore, based on its expression in kidney, Harderian gland, eyelid dermis, and tibial subcutis tissues, this transcription factor may be involved in hormone synthesis and function. Together, our results demonstrate the role of Osr2 in postnatal development and embryogenesis.
Assuntos
Expressão Gênica , Fatores de Transcrição/metabolismo , Animais , Osso e Ossos/metabolismo , Papila Dentária/metabolismo , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Intestino Delgado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mucosa Nasal/metabolismo , Especificidade de Órgãos , Placenta/metabolismo , Gravidez , Próstata/metabolismo , Testículo/metabolismo , Fatores de Transcrição/genética , Útero/metabolismoRESUMO
The transcription factor Odd-skipped related 2 (Osr2) functions in craniofacial and limb developments in mammals. We previously found that Osr2 gene expression is regulated by intracellular transcription factors such as Runx2, and C/EBP, whereas it remains unclear if extracellular factors would functionally regulate the Osr2 expression. In this study, we showed that TGF-ß down-regulated the Osr2 expression, which is involved in regulation of cellular migration and cell cycle. Furthermore, the down-regulation was found to be mediated by Smad3/Smad4 and p38/ATF2 signaling molecules. The Osr2 promoter was shown to possess Smad3/4 binding element and ATF2 binding element between -647 and -64 of promoter. TGF-ß induced cellular migration in C3H10T1/2 cells and arrested cell cycle at G1 phase in NMuMG-Fucci cells. In contrast, the Osr2 reduced the migration and also stimulated the cell-cycle progression. These results suggest that Osr2 is involved in TGF-ß regulating cell migration and cell cycle via a Smad3-ATF2 transcriptional complex mediating pathway.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Movimento Celular/genética , Proteína Smad3/metabolismo , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
MicroRNAs (miRNAs) are noncoding RNAs that function as key posttranscriptional regulators of gene expression. miRNA maturation is controlled by the DROSHA microprocessor complex. However, the detailed mechanism of miRNA biogenesis remains unclear. We show that the tumor suppressor breast cancer 1 (BRCA1) accelerates the processing of miRNA primary transcripts. BRCA1 increased the expressions of both precursor and mature forms of let-7a-1, miR-16-1, miR-145, and miR-34a. In addition, this tumor suppressor was shown to be directly associated with DROSHA and DDX5 of the DROSHA microprocessor complex, and it interacted with Smad3, p53, and DHX9 RNA helicase. We also found that BRCA1 recognizes the RNA secondary structure and directly binds with primary transcripts of miRNAs via a DNA-binding domain. Together, these results suggest that BRCA1 regulates miRNA biogenesis via the DROSHA microprocessor complex and Smad3/p53/DHX9. Our findings also indicate novel functions of BRCA1 in miRNA biogenesis, which may be linked to its tumor suppressor mechanism and maintenance of genomic stability.
Assuntos
Proteína BRCA1/metabolismo , MicroRNAs/biossíntese , Ribonuclease III/metabolismo , Linhagem Celular Tumoral , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , MicroRNAs/metabolismo , Proteínas de Neoplasias/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Proteína Smad3/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Disturbed endochondral ossification in X-linked hypophosphatemia indicates an involvement of P(i) in chondrogenesis. We studied the role of the sodium-dependent P(i) cotransporters (NPT), which are a widely recognized regulator of cellular P(i) homeostasis, and the downstream events in chondrogenesis using Hyp mice, the murine homolog of human X-linked hypophosphatemia. Hyp mice showed reduced apoptosis and mineralization in hypertrophic cartilage. Hyp chondrocytes in culture displayed decreased apoptosis and mineralization compared with WT chondrocytes, whereas glycosaminoglycan synthesis, an early event in chondrogenesis, was not altered. Expression of the type III NPT Pit-1 and P(i) uptake were diminished, and intracellular ATP levels were also reduced in parallel with decreased caspase-9 and caspase-3 activity in Hyp chondrocytes. The competitive NPT inhibitor phosphonoformic acid and ATP synthesis inhibitor 3-bromopyruvate disturbed endochondral ossification with reduced apoptosis in vivo and suppressed apoptosis and mineralization in conjunction with reduced P(i) uptake and ATP synthesis in WT chondrocytes. Overexpression of Pit-1 in Hyp chondrocytes reversed P(i) uptake and ATP synthesis and restored apoptosis and mineralization. Our results suggest that cellular ATP synthesis consequent to P(i) uptake via Pit-1 plays an important role in chondrocyte apoptosis and mineralization, and that chondrogenesis is ATP-dependent.
Assuntos
Trifosfato de Adenosina/biossíntese , Condrócitos/metabolismo , Condrogênese/fisiologia , Osteogênese/fisiologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 3/metabolismo , Caspase 9/metabolismo , Condrogênese/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Raquitismo Hipofosfatêmico Familiar/genética , Raquitismo Hipofosfatêmico Familiar/metabolismo , Feminino , Doenças Genéticas Ligadas ao Cromossomo X , Humanos , Masculino , Camundongos , Camundongos Mutantes , Osteogênese/efeitos dos fármacos , Fosfatos/metabolismo , Piruvatos/farmacologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genéticaRESUMO
Cell proliferation is a biological process in which chromosomes replicate in one cell and equally divide into two daughter cells. Our previous findings suggested that Odd-skipped related 2 (Osr2) plays an important role in cellular quiescence and proliferation under epigenetic regulation. However, the mechanism used by Osr2 to establish and maintain proliferation is unknown. To examine the functional role of Osr2 in cell proliferation, we analyzed its downstream target genes using microarray analysis following adenovirus-induced overexpression of Osr2 as well as knockdown with Osr2 siRNA, which showed that Osr2 regulates a multitude of genes involved in proliferation and the cell cycle, as well as development. Additional proliferation assays also indicated that Osr2 likely functions to elicit cell proliferation. Together, these results suggest that Osr2 plays important roles in proliferation and development.
Assuntos
Ciclo Celular/genética , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/fisiologia , Animais , Linhagem Celular , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/genética , Fatores de Transcrição/genéticaRESUMO
Cellular behavior and development are extensively altered during the transition from cell cycle into quiescence, though the mechanism involved in establishing and maintaining quiescence is largely unknown. We found that Odd-skipped related 2 (Osr2) was up-regulated during cellular quiescence by serum starvation as well as culturing to confluence. To investigate the regulatory mechanism of Osr2 under these conditions, we characterized the mouse Osr2 promoter. CpG islands in the flanking region of the transcription start site were predominantly methylated in exponentially growing cells, resulting in silencing of Osr2 expression. In addition, CpG demethylation in quiescence caused activation of Osr2 expression, while acetylation of the H3 and H4 histones during quiescence also led to an increase in Osr2 expression. These results suggest that epigenetically regulated Osr2 plays an important role in cellular quiescence and proliferation.
Assuntos
Ciclo Celular/genética , Epigênese Genética , Fatores de Transcrição/genética , Acetilação , Animais , Linhagem Celular , Proliferação de Células , Ilhas de CpG , Metilação de DNA , Histonas/metabolismo , Camundongos , Regiões Promotoras Genéticas , Regulação para CimaRESUMO
Odd-skipped related 1 (Osr1) gene encodes a zinc-finger transcription factor that plays important roles in embryonic, heart, and urogenital development, however, it is unknown how its expression is regulated. In this study, we analyzed the promoter region of Osr1 to elucidate its regulation mechanism. The mouse Osr1 promoter region was cloned and characterized, and found to have two repressor elements in the -4504/-2766 and -1616/-109 regions, and two enhancer elements in the -2766/-1616 and -109/+199 regions. Several Runx2 and Ikzf1 binding sites were found in both mouse and human Osr1 promoters. Osr1 promoter activity was suppressed by cotransfection with Runx2- and Ikzf1-expressing vectors in a dose-dependent manner. Electrophoresis mobility shift assays showed that purified Runx2 bound to proximal (-611/-606) Runx2 binding motifs and that Ikzf1 bound to proximal (-1652/-1644) Ikzf1 binding motifs. Chrosmatin immunoprecipitation demonstrated that Runx2 bound to both the distal (-3047/-3042) and proximal regions, and that Ikzf1 bound to both the far-distal (-3036/-3028) and proximal elements. These findings indicate that Osr1 expression is regulated by Runx2 and Ikzf1, which are known as master-gene of osteogenesis and hematopoiesis, respectively.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica/genética , Fator de Transcrição Ikaros/genética , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , Subunidade alfa 1 de Fator de Ligação ao Core/isolamento & purificação , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Elementos Facilitadores Genéticos , Fator de Transcrição Ikaros/metabolismo , Camundongos , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Severe periodontal breakdown is often associated with Down syndrome (DS); however, the etiology of this condition is not understood fully. Cellular motility of gingival fibroblasts is a critical event for wound healing and regeneration of periodontal tissues. Porphyromonas gingivalis is known to be a periodontal pathogen that invades host cells, contributing to periodontal destruction. In this study, we examined the influence of P. gingivalis infection on the motility of DS gingival fibroblasts (DGFs). METHODS: DGFs and normal gingival fibroblasts (NGFs) were infected with P. gingivalis with type II fimbriae, and cellular motility was evaluated using an in vitro wounding assay. Protein degradation of alpha5beta1-integrin subunits and a migration-regulating signaling molecule, paxillin, were investigated using specific antibodies. The adhesion to and invasion of fibroblasts by P. gingivalis were determined with a colony forming assay. The gene expressions of alpha5beta1-integrin subunits were also quantified using a reverse transcription-polymerase chain reaction method. RESULTS: The cellular motility of DGFs was impaired significantly by P. gingivalis compared to NGFs, and the former were invaded readily by P. gingivalis. Further, cellular paxillin from DGFs was degraded markedly by the pathogen. Although protein degradation of alpha5beta1 integrin was induced, its mRNA expression was not affected significantly. CONCLUSIONS: P. gingivalis readily invades DGFs and subsequently degrades paxillin, which impairs cellular motility and likely prevents wound healing and the regeneration of periodontal tissues. These characteristics may be involved in the etiology of DS periodontitis.
Assuntos
Síndrome de Down/patologia , Fibroblastos/patologia , Gengiva/patologia , Porphyromonas gingivalis/fisiologia , Adolescente , Aderência Bacteriana/fisiologia , Estudos de Casos e Controles , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Feminino , Fibroblastos/microbiologia , Fímbrias Bacterianas/classificação , Gengiva/microbiologia , Humanos , Integrina alfa5beta1/análise , Paxilina/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Porphyromonas gingivalis, a periodontal pathogen, was previously suggested to exploit alpha5beta1 integrin and lipid rafts to invade host cells. However, it is unknown if the functional roles of these host components are distinct from one another during bacterial invasion. In the present study, we analyzed the mechanisms underlying P. gingivalis invasion, using fluorescent beads coated with bacterial membrane vesicles (MV beads). Cholesterol depletion reagents including methyl-beta-cyclodextrin (MbetaCD) drastically inhibited the entry of MV beads into epithelial cells, while they were less effective on bead adhesion to the cells. Bead entry was also abolished in CHO cells deficient in sphingolipids, components of lipid rafts, whereas adhesion was negligibly influenced. Following MbetaCD treatment, downstream events leading to actin polymerization were abolished; however, alpha5beta1 integrin was recruited to beads attached to the cell surface. Dominant-negative Rho GTPase Rac1 abolished cellular engulfment of the beads, whereas dominant-negative Cdc42 did not. Following cellular interaction with the beads, Rac1 was found to be translocated to the lipid rafts fraction, which was inhibited by MbetaCD. These results suggest that alpha5beta1 integrin, independent of lipid rafts, promotes P. gingivalis adhesion to epithelial cells, while the subsequent uptake process requires lipid raft components for actin organization, with Rho GTPase Rac1.
Assuntos
Aderência Bacteriana/fisiologia , Células Epiteliais/fisiologia , Corantes Fluorescentes/química , Integrina alfa5beta1/metabolismo , Microdomínios da Membrana/metabolismo , Porphyromonas gingivalis/fisiologia , Actinas/efeitos dos fármacos , Actinas/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Células Cultivadas , Colesterol/metabolismo , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Microesferas , Porphyromonas gingivalis/citologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Esfingolipídeos/metabolismo , Propriedades de Superfície , beta-Ciclodextrinas/farmacologia , Proteína cdc42 de Ligação ao GTP/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/fisiologiaRESUMO
UNLABELLED: We report that Osr2 is one of the regulators of osteoblast function, because dominant-negative Osr2 transgenic mice exhibited decreased osteoblast activity and delayed mineralization in calvarial and tibial bone tissues. Our results indicate that Osr2 functions in regulation of osteoblast proliferation. INTRODUCTION: Molecular mechanisms that control bone formation have received attention with increasing knowledge related to genetic control of osteoblast differentiation. The odd-skipped related (Osr) gene is a zinc-finger transcription factor recently suggested to be involved in bone formation, although little is known about its role. MATERIALS AND METHODS: To elucidate the in vivo function of Osr2, we generated transgenic mice overexpressing dominant-negative Osr2. RESULTS: In this study, N-terminal-deleted Osr2 was found to act as a dominant-negative mutant toward both Osr1 and Osr2. Dominant-negative Osr2 (Osr2DeltaN) transgenic mice showed delayed mineralization in calvarial and cortical bone tissues. Furthermore, soft X-ray analysis of transgenic mice bones revealed distinctly increased radiolucency. Examinations of newborn Osr2DeltaN transgenic mice skeletons stained with alcian blue and alizarin red showed reduced intensities in the skull and skeletal elements. Morphologically, calvariae and tibias of Osr2DeltaN transgenic mice were composed of markedly thinner parietal and cortical bones and lower numbers of osteoblastic cells on bone surfaces, indicating a reduced proliferation of osteoblasts. Furthermore, calvarial osteoblasts obtained from Osr2DeltaN transgenic mice showed highly attenuated osteoblast differentiation and proliferation, confirming that Osr2 is needed for osteogenesis. Finally, results of Runx2-deficient cell assays suggested that Osr2 induces alkaline phosphatase (ALP) expression, but to a lesser degree than Runx2-expressing cells. CONCLUSIONS: Our genetic observations showed that the Osr2 gene plays a key role in osteoblastic cell proliferation.
Assuntos
Desenvolvimento Ósseo/fisiologia , Proliferação de Células , Osteoblastos/citologia , Fatores de Transcrição/fisiologia , Células 3T3 , Animais , Sequência de Bases , Diferenciação Celular/fisiologia , Linhagem Celular , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genéticaRESUMO
Severe marginal periodontitis often coexists with diabetes and is considered to be the sixth complication of the disease. Several factors associated with diabetes have been shown to be related to the development and deterioration of diabetic periodontitis, i.e., glycated hemoglobin (HbA(1C)), advanced glycation endproducts (AGEs), C-reactive protein (CRP), and glucose tolerance, as well as inflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6. A periodontal pathogen, Porphyromonas gingivalis with the type II fimbria gene, is also considered to be a related infectious factor. Among them, AGEs seem to be closely associated with the periodontitis deterioration seen in diabetic subjects. Osteoporosis and diabetes are also tightly related, and AGEs have been reported to accelerate the progression of osteoporosis. Collectively, AGEs are a critical diabetic factor to deteriorate oral osteoporosis.
Assuntos
Complicações do Diabetes , Osteoporose/etiologia , Doenças Periodontais/etiologia , Produtos Finais de Glicação Avançada , Humanos , Resistência à Insulina , Interleucina-1beta , Porphyromonas gingivalis , Fator de Necrose Tumoral alfaRESUMO
Porphyromonas gingivalis is a periodontal pathogen whose fimbriae are classified into six genotypes based on the diversity of the fimA genes encoding each fimbria subunit. It was suggested that P. gingivalis strains with type II fimbriae were more virulent than type I strains. For the present study, we generated the mutants in which fimA was substituted with different genotypes to study virulence of type II fimbriae. Using plasmid vectors, fimA of ATCC33277 (type I strain) was substituted with type II fimA, and that of OMZ314 (type II strain) with type I fimA. The substitution of type I fimA with type II enhanced bacterial adhesion/invasion to epithelial cells, whereas substitution with type I fimA resulted in diminished efficiency. Following bacterial invasion, type II clones swiftly degraded cellular paxillin and focal adhesion kinase, and inhibited cellular migration, whereas type I clones and DeltafimA mutants did not. BIAcore analysis demonstrated that type II fimbriae possess greater adhesive abilities for their receptor alpha5beta1-integrin than those of type I. In a mouse abscess model, the type II clones significantly induced serum IL-1beta and IL-6, as well as other infectious symptoms. These results suggest that type II fimbriae are a critical determinant of P. gingivalis virulence.
Assuntos
Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Porphyromonas gingivalis/genética , Abscesso/sangue , Abscesso/microbiologia , Animais , Aderência Bacteriana/genética , Infecções por Bacteroidaceae/sangue , Infecções por Bacteroidaceae/metabolismo , Infecções por Bacteroidaceae/microbiologia , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Genótipo , Células HeLa , Humanos , Immunoblotting , Integrina alfa5beta1/metabolismo , Interleucina-1beta/sangue , Interleucina-6/sangue , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mutação , Paxilina/metabolismo , Porphyromonas gingivalis/patogenicidade , Porphyromonas gingivalis/ultraestrutura , Virulência/genéticaRESUMO
Porphyromonas gingivalis fimbriae are classified into six types (types I to V and Ib) based on the fimA genes encoding FimA (a subunit of fimbriae), and they play a critical role in bacterial interactions with host tissues. In this study, we compared the efficiencies of P. gingivalis strains with distinct types of fimbriae for invasion of epithelial cells and for degradation of cellular focal adhesion components, paxillin, and focal adhesion kinase (FAK). Six representative strains with the different types of fimbriae were tested, and P. gingivalis with type II fimbriae (type II P. gingivalis) adhered to and invaded epithelial cells at significantly greater levels than the other strains. There were negligible differences in gingipain activities among the six strains; however, type II P. gingivalis apparently degraded intracellular paxillin in association with a loss of phosphorylation 30 min after infection. Degradation was blocked with cytochalasin D or in mutants with fimA disrupted. Paxillin was degraded by the mutant with Lys-gingipain disrupted, and this degradation was prevented by inhibition of Arg-gingipain activity by Nalpha-p-tosyl-l-lysine chloromethyl ketone. FAK was also degraded by type II P. gingivalis. Cellular focal adhesions with green fluorescent protein-paxillin macroaggregates were clearly destroyed, and this was associated with cellular morphological changes and microtubule disassembly. In an in vitro wound closure assay, type II P. gingivalis significantly inhibited cellular migration and proliferation compared to the cellular migration and proliferation observed with the other types. These results suggest that type II P. gingivalis efficiently invades epithelial cells and degrades focal adhesion components with Arg-gingipain, which results in cellular impairment during wound healing and periodontal tissue regeneration.
Assuntos
Adesinas Bacterianas/metabolismo , Cisteína Endopeptidases/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Fímbrias Bacterianas/fisiologia , Adesões Focais/metabolismo , Porphyromonas gingivalis/patogenicidade , Fímbrias Bacterianas/enzimologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Cisteína Endopeptidases Gingipaínas , Células HeLa , Humanos , Hidrólise , Paxilina/metabolismo , Porphyromonas gingivalis/enzimologiaRESUMO
BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is associated with chronic gingival inflammation and is suspected to influence periodontal destruction. However, the exact roles of TNF-alpha in wound healing and periodontal tissue regeneration are largely unknown. In the present study, we examined the effects of TNF-alpha on migration and proliferation of human periodontal ligament (PDL) cells. METHODS: PDL cells were cultured in the presence of TNF-alpha to determine its effects on cellular migration and proliferation. The protein expression profiles of alpha5 and beta1 integrin subunits and their related molecules, paxillin and focal adhesion kinases (FAK), were investigated. Gene expression of fibronectin also was assayed. Further, the activation of Rho-family small guanosine triphosphate (GTP)-binding protein (RhoA) was evaluated using a GTP-loading pull-down assay, and focal adhesion formation by PDL cells after transfection with the expression vector of paxillin-fused green fluorescent protein (GFP) also was observed with confocal microscopy. RESULTS: Cellular migration was impaired by TNF-alpha and recovered following the addition of anti-TNF-alpha antibodies. In contrast, PDL cell proliferation was not affected by TNF-alpha. TNF-alpha upregulated the expression of the alpha5 and beta1 integrin subunits, whereas fibronectin was not overexpressed. Phosphorylation of paxillin and FAK by PDL cells was induced, and RhoA activation also was induced. Confocal microscopic analysis revealed that TNF-alpha induced focal adhesion and stress fiber formation in all parts of the cells. CONCLUSION: Our results suggested that TNF-alpha impairs cellular migration by enhancing cellular adhesive ability following significant focal adhesion and stress fiber formation.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ligamento Periodontal/citologia , Fator de Necrose Tumoral alfa/farmacologia , Adolescente , Feminino , Fibronectinas/genética , Proteína-Tirosina Quinases de Adesão Focal/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina alfa5beta1/efeitos dos fármacos , Integrina alfa5beta1/metabolismo , Paxilina/efeitos dos fármacos , Paxilina/metabolismo , Proteína rhoA de Ligação ao GTP/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Growth and differentiation factor 5 (GDF-5) is a homodimeric protein stabilized by a single disulfide bridge between cysteine 465 in the respective monomers, as well as by three intramolecular cysteine bridges within each subunit. A mature recombinant human GDF-5 variant with cysteine 465 replaced by alanine (rhGDF-5 C465A) was expressed in E. coli, purified to homogeneity, and chemically renatured. Biochemical analysis showed that this procedure eliminated the sole interchain disulfide bond. Surprisingly, the monomeric variant of rhGDF-5 is as potent in vitro as the dimeric form. This could be confirmed by alkaline phosphatase assays and Smad reporter gene activation. Furthermore, dimeric and monomeric rhGDF-5 show comparable binding to their specific type I receptor, BRIb. Studies on living cells showed that both the dimeric and monomeric rhGDF-5 induce homomeric BRIb and heteromeric BRIb/BRII oligomers. Our results suggest that rhGDF-5 C465A has the same biological activity as rhGDF-5 with respect to binding to, oligomerization of and signaling through the BMP receptor type Ib.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Morfogenéticas Ósseas/genética , Células COS , Células Cultivadas , Chlorocebus aethiops/metabolismo , Dimerização , Imunofluorescência , Fator 5 de Diferenciação de Crescimento , Humanos , Focalização Isoelétrica , Dados de Sequência Molecular , Mapeamento de Peptídeos , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , SolubilidadeRESUMO
Porphyromonas gingivalis is a predominant periodontal pathogen, whose infection causes inflammatory responses in periodontal tissue and alveolar bone resorption. Various virulence factors of this pathogen modulate host innate immune responses. It has been reported that gingipains degrade a wide variety of host cell proteins, and fimbriae are involved in bacterial adhesion to and invasion of host cells. In the present study, we profiled ST2 stromal cell gene expression following infection with the viable P. gingivalis strain ATCC33277 as well as with its gingipain- and fimbriae-deficient mutants, using microarray technology and quantitative real-time polymerase chain reaction. Using a mouse array of about 20,000 genes, we found that infection with the wild strain elicited a significant upregulation (greater than 2-fold) of expression of about 360 genes in ST2 cells, which included the chemokines CCL2, CCL5, and CXCL10, and other proinflammatory proteins such as interleukin-6 (IL-6) and matrix metalloproteinase-13 (MMP-13). Further, infection with the gingipain-deficient mutant elicited a reduced expression of the CXCL10, IL-6 and MMP-13 genes, suggesting that gingipains play an important role in inducing the expression of those genes following P. gingivalis infection. On the other hand, the pattern of global gene expression induced by the fimbriae-deficient mutant was similar to that by the wild strain. These results suggest that P. gingivalis infection induces gene expression of a wide variety of proinflammatory proteins in stromal cells/osteoblasts, and gingipains may be involved in inducing several of the proinflammatory factors.
Assuntos
Infecções por Bacteroidaceae/metabolismo , Perfilação da Expressão Gênica , Genes/genética , Análise de Sequência com Séries de Oligonucleotídeos , Porphyromonas gingivalis , Adesinas Bacterianas/genética , Animais , Infecções por Bacteroidaceae/microbiologia , Linhagem Celular , Colagenases/genética , Colagenases/metabolismo , Cisteína Endopeptidases/deficiência , Cisteína Endopeptidases/genética , Citocinas/genética , Citocinas/metabolismo , Fímbrias Bacterianas/genética , Deleção de Genes , Cisteína Endopeptidases Gingipaínas , Hemaglutininas/genética , Metaloproteinase 13 da Matriz , Camundongos , Porphyromonas gingivalis/genética , RNA Mensageiro/genética , Células Estromais/metabolismo , Células Estromais/microbiologia , Regulação para CimaRESUMO
Odd-skipped related 2 (Osr2) gene is mouse homolog of Drosophila Odd-skipped gene involved with the pair-rule segmentation phenotype in Drosophila mutant embryos. In this study, to examine Osr2 expression regulation, the mouse Osr2 promoter region was cloned and characterized, and found to have two enhancer elements in the -1463/-1031 (distal) and -581/+3 (proximal) regions, and a repressor region (-4845/-1463, far distal). CCAAT/enhancer binding protein (C/EBP) binding sites were found in both the distal and proximal enhancer elements. Osr2 promoter activity was enhanced by C/EBPdelta, a member of the C/EBP family, in a dose-dependent manner. Electrophoresis mobility shift assays showed that purified GST-C/EBPdelta bound to distal (-1295/-1261) and proximal (-89/-55) C/EBP binding motifs. Chromatin immunoprecipitation demonstrated that acetylated histones H3, H4, and C/EBPdelta in the proximal region (-280/-43), but not the distal region (-1438/-1196), indicating that the Osr2 promoter proximal region was transcriptionally activated in C3H10T1/2 cells. Our results suggest that Osr2 expression is regulated by C/EBP regulatory elements.