RESUMEN
The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor κB signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor α-toxin. Naturally elicited antibodies against α-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to α-toxin in nonleukocytic cells.
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Toxinas Bacterianas , Síndrome del Maullido del Gato , Endopeptidasas , Haploinsuficiencia , Proteínas Hemolisinas , Infecciones Estafilocócicas , Staphylococcus aureus , Toxinas Bacterianas/inmunología , Síndrome del Maullido del Gato/genética , Síndrome del Maullido del Gato/inmunología , Endopeptidasas/genética , Haploinsuficiencia/genética , Haploinsuficiencia/inmunología , Proteínas Hemolisinas/inmunología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Celular/genética , Necrosis , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/patologíaRESUMEN
Autosomal dominant (AD) NFKB1 deficiency is thought to be the most common genetic etiology of common variable immunodeficiency (CVID). However, the causal link between NFKB1 variants and CVID has not been demonstrated experimentally and genetically, and there has been insufficient biochemical characterization and enrichment analysis. We show that the cotransfection of NFKB1-deficient HEK293T cells (lacking both p105 and its cleaved form p50) with a κB reporter, NFKB1/p105, and a homodimerization-defective RELA/p65 mutant results in p50:p65 heterodimer-dependent and p65:p65 homodimer-independent transcriptional activation. We found that 59 of the 90 variants in patients with CVID or related conditions were loss of function or hypomorphic. By contrast, 258 of 260 variants in the general population or patients with unrelated conditions were neutral. None of the deleterious variants displayed negative dominance. The enrichment in deleterious NFKB1 variants of patients with CVID was selective and highly significant (P = 2.78 × 10-15). NFKB1 variants disrupting NFKB1/p50 transcriptional activity thus underlie AD CVID by haploinsufficiency, whereas neutral variants in this assay should not be considered causal.
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Inmunodeficiencia Variable Común/genética , Subunidad p50 de NF-kappa B/genética , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Células HEK293 , Haploinsuficiencia/genética , Humanos , FN-kappa B/genética , Fenotipo , Activación Transcripcional/genéticaRESUMEN
Most patients with autosomal dominant hyper-IgE syndrome (AD-HIES) carry rare heterozygous STAT3 variants. Only six of the 135 in-frame variants reported have been experimentally shown to be dominant negative (DN), and it has been recently suggested that eight out-of-frame variants operate by haploinsufficiency. We experimentally tested these 143 variants, 7 novel out-of-frame variants found in HIES patients, and other STAT3 variants from the general population. Strikingly, all 15 out-of-frame variants were DN via their encoded (1) truncated proteins, (2) neoproteins generated from a translation reinitiation codon, and (3) isoforms from alternative transcripts or a combination thereof. Moreover, 128 of the 135 in-frame variants (95%) were also DN. The patients carrying the seven non-DN STAT3 in-frame variants have not been studied for other genetic etiologies. Finally, none of the variants from the general population tested, including an out-of-frame variant, were DN. Overall, our findings show that heterozygous STAT3 variants, whether in or out of frame, underlie AD-HIES through negative dominance rather than haploinsufficiency.
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Genes Dominantes , Síndrome de Job/genética , Mutación/genética , Factor de Transcripción STAT3/genética , Adolescente , Adulto , Alelos , Empalme Alternativo/genética , Niño , Preescolar , Codón sin Sentido/genética , Evolución Molecular , Familia , Femenino , Mutación del Sistema de Lectura/genética , Genética de Población , Células HEK293 , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Linaje , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Cytomegalovirus (CMV) can cause severe disease in children and adults with a variety of inherited or acquired T-cell immunodeficiencies, who are prone to multiple infections. It can also rarely cause disease in otherwise healthy persons. The pathogenesis of idiopathic CMV disease is unknown. Inbred mice that lack the gene encoding nitric oxide synthase 2 (Nos2) are susceptible to the related murine CMV infection. METHODS: We studied a previously healthy 51-year-old man from Iran who after acute CMV infection had an onset of progressive CMV disease that led to his death 29 months later. We hypothesized that the patient may have had a novel type of inborn error of immunity. Thus, we performed whole-exome sequencing and tested candidate mutant alleles experimentally. RESULTS: We found a homozygous frameshift mutation in NOS2 encoding a truncated NOS2 protein that did not produce nitric oxide, which determined that the patient had autosomal recessive NOS2 deficiency. Moreover, all NOS2 variants that we found in homozygosity in public databases encoded functional proteins, as did all other variants with an allele frequency greater than 0.001. CONCLUSIONS: These findings suggest that inherited NOS2 deficiency was clinically silent in this patient until lethal infection with CMV. Moreover, NOS2 appeared to be redundant for control of other pathogens in this patient. (Funded by the National Center for Advancing Translational Sciences and others.).
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Infecciones por Citomegalovirus , Mutación del Sistema de Lectura , Óxido Nítrico Sintasa de Tipo II/deficiencia , Resultado Fatal , Femenino , Genotipo , Homocigoto , Humanos , Mutación con Pérdida de Función , Masculino , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Linaje , Secuenciación del ExomaRESUMEN
Juvenile-onset recurrent respiratory papillomatosis (JRRP) is a rare and debilitating childhood disease that presents with recurrent growth of papillomas in the upper airway. Two common human papillomaviruses (HPVs), HPV-6 and -11, are implicated in most cases, but it is still not understood why only a small proportion of children develop JRRP following exposure to these common viruses. We report 2 siblings with a syndromic form of JRRP associated with mild dermatologic abnormalities. Whole-exome sequencing of the patients revealed a private homozygous mutation in NLRP1, encoding Nucleotide-Binding Domain Leucine-Rich Repeat Family Pyrin Domain-Containing 1. We find the NLRP1 mutant allele to be gain of function (GOF) for inflammasome activation, as demonstrated by the induction of inflammasome complex oligomerization and IL-1ß secretion in an overexpression system. Moreover, patient-derived keratinocytes secrete elevated levels of IL-1ß at baseline. Finally, both patients displayed elevated levels of inflammasome-induced cytokines in the serum. Six NLRP1 GOF mutations have previously been described to underlie 3 allelic Mendelian diseases with differing phenotypes and modes of inheritance. Our results demonstrate that an autosomal recessive, syndromic form of JRRP can be associated with an NLRP1 GOF mutation.
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Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Mutación con Ganancia de Función , Homocigoto , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/patología , Infecciones del Sistema Respiratorio/genética , Infecciones del Sistema Respiratorio/patología , Preescolar , Citocinas/metabolismo , Femenino , Humanos , Lactante , Inflamasomas , Queratinocitos/citología , Queratinocitos/inmunología , Queratinocitos/metabolismo , Masculino , Proteínas NLR , Linaje , Hermanos , SíndromeRESUMEN
Patients with epidermodysplasia verruciformis (EV) and biallelic null mutations of TMC6 (encoding EVER1) or TMC8 (EVER2) are selectively prone to disseminated skin lesions due to keratinocyte-tropic human ß-papillomaviruses (ß-HPVs), which lack E5 and E8. We describe EV patients homozygous for null mutations of the CIB1 gene encoding calcium- and integrin-binding protein-1 (CIB1). CIB1 is strongly expressed in the skin and cultured keratinocytes of controls but not in those of patients. CIB1 forms a complex with EVER1 and EVER2, and CIB1 proteins are not expressed in EVER1- or EVER2-deficient cells. The known functions of EVER1 and EVER2 in human keratinocytes are not dependent on CIB1, and CIB1 deficiency does not impair keratinocyte adhesion or migration. In keratinocytes, the CIB1 protein interacts with the HPV E5 and E8 proteins encoded by α-HPV16 and γ-HPV4, respectively, suggesting that this protein acts as a restriction factor against HPVs. Collectively, these findings suggest that the disruption of CIB1-EVER1-EVER2-dependent keratinocyte-intrinsic immunity underlies the selective susceptibility to ß-HPVs of EV patients.
Asunto(s)
Betapapillomavirus/inmunología , Proteínas de Unión al Calcio/inmunología , Epidermodisplasia Verruciforme/inmunología , Inmunidad Innata , Queratinocitos/inmunología , Proteínas de la Membrana/inmunología , Complejos Multiproteicos/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Adhesión Celular/inmunología , Movimiento Celular/inmunología , Epidermodisplasia Verruciforme/patología , Femenino , Papillomavirus Humano 16/inmunología , Humanos , Queratinocitos/patología , Masculino , Persona de Mediana Edad , Proteínas Oncogénicas Virales/inmunologíaRESUMEN
BACKGROUND: The unfolded protein response (UPR) is activated following an endoplasmic reticulum (ER) stress. The aim of this study was to investigate the global expression of UPR genes in human OA chondrocytes in induced (I)-UPR conditions, and to explore the regulation and role of the UPR genes in homeostatic (H)-UPR conditions in human normal and OA chondrocytes. METHODS: Gene expression was determined by PCR array and qPCR. Protein production in cartilage was determined by immunohistochemistry, gene silencing by specific siRNAs, and gene regulation by treating chondrocytes with cytokines and growth factors associated with cartilage pathobiology. RESULTS: Several UPR genes, among them ERN1, PERK, and CREB3L2 were downregulated in OA compared to normal chondrocytes at both the mRNA and protein levels, but the ER stress response triggered by thapsigargin or tunicamycin treatment was similar in normal and OA chondrocytes. The activation of ER stress sensors (phosphorylated PERK, cleavage of ATF6B, and the spliced mRNA forms of XBP1) was not significantly increased in OA chondrocytes/cartilage. PDGF-BB and IL-6 significantly downregulated the expression of ERN1, PERK, and CREB3L2, but not that of ATF6B. Silencing experiments done under conditions of no ER stress (physiological conditions) revealed that decreasing ERN1 expression led to decreased COL2a1, MMP-13, ADAMTS4 and ADAMTS5 expression, while decreasing CREB3L2 and ATF6B led to decreased ADAMTS5 and ADAMTS4 expression, respectively. Importantly, the downregulation of PERK expression increased COL1a1 and suppressed COL2a1 expression. CONCLUSIONS: Although the level of ER stress is not significantly increased in OA chondrocytes, these cells respond strongly to an acute ER stress despite the decreased expression of ERN1, PERK, and CREB3L2. Emerging findings revealed for the first time that these genes play a role in cartilage biology in conditions where an acute ER stress response is not triggered and OA is not characterized by an overall basal activation of the ER stress response. Importantly, these findings identify PERK as a potential target for new OA treatment avenues.
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Condrocitos/metabolismo , Osteoartritis/genética , Respuesta de Proteína Desplegada/genética , eIF-2 Quinasa/metabolismo , Anciano , Western Blotting , Condrocitos/patología , Estrés del Retículo Endoplásmico/genética , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Osteoartritis/metabolismo , Reacción en Cadena de la Polimerasa , TranscriptomaRESUMEN
BACKGROUND: Ephrins and their related receptors have been implicated in some developmental events. We have demonstrated that ephrin-B2 (EFNB2) could play a role in knee joint pathology associated with osteoarthritis (OA). Here, we delineate the in vivo role of EFNB2 in musculoskeletal growth, development, and in OA using a cartilage-specific EFNB2 knockout (EFNB2(Col2)KO) mouse model. METHODS: EFNB2(Col2)KO was generated with Col2a1-Cre transgenic mice. The skeletal development was evaluated using macroscopy, immunohistochemistry, histomorphometry, radiology, densitometry, and micro-computed tomography. Analyses were performed at P0 (birth) and on postnatal days P15, P21, and on 8-week- and 1-year-old mice. RESULTS: EFNB2(Col2)KO mice exhibited significant reduction in size, weight, length, and in long bones. At P0, the growth plates of EFNB2(Col2)KO mice displayed increased type X collagen, disorganized hyphertrophic zone, and decreased mineralization. At P15, mutant mice demonstrated a significant reduction in VEGF and TRAP at the chondro-osseous junction and a delay in the secondary ossification, including a decrease in bone volume and trabecular thickness. At P21 and 8 weeks old, EFNB2(Col2)KO mice exhibited reduced bone mineral density in the total skeleton, femur and spine. One-year-old EFNB2(Col2)KO mice demonstrated OA phenotypic features in both the knee and hip. By P15, 27 % of the EFNB2(Col2)KO mice developed a hip locomotor phenotype, which further experiments demonstrated reflected the neurological midline abnormality involving the corticospinal tract. CONCLUSION: This in vivo study demonstrated, for the first time, that EFNB2 is essential for normal long bone growth and development and its absence leads to a knee and hip OA phenotype in aged mice.
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Desarrollo Óseo/fisiología , Cartílago Articular/patología , Efrina-B2/deficiencia , Osteoartritis/etiología , Envejecimiento , Animales , Artritis Experimental/etiología , Artritis Experimental/metabolismo , Artritis Experimental/patología , Cartílago Articular/metabolismo , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos , Osteoartritis/metabolismo , Osteoartritis/patología , Fenotipo , Microtomografía por Rayos XRESUMEN
Osteoarthritis (OA) is characterized by progressive joint destruction, including synovial membrane alteration. EphB4 and its ligand ephrin-B2 were found in vitro to positively affect OA subchondral bone and cartilage. In vivo in an experimental mouse model overexpressing bone-specific Ephb4 (TgEphB4), a protective effect was found on both the subchondral bone and cartilage during OA. We investigated in the TgEphB4 mouse model the in vivo effect on synovial membrane during OA. Knee OA was surgically induced by destabilization of the medial meniscus (DMM). Synovial membrane was evaluated using histology, histomorphometry, IHC, and real-time PCR. Compared to DMM-wild-type (WT) mice, DMM-TgEphB4 mice had a significant decrease in synovial membrane thickness, vascular endothelial growth factor, and the profibrotic markers fibrin, type 1 procollagen, type 3 collagen, connective tissue growth factor, smooth muscle actin-α, cartilage oligomeric matrix protein, and procollagen-lysine, and 2-oxoglutarate 5-dioxygenase 2. Moreover, factors known to modulate transforming growth factor-ß signaling, transforming growth factor receptor 1/ALK1, phosphorylated Smad-1, and heat shock protein 90ß were significantly decreased in DMM-TgEphB4 compared with DMM-WT mice. Ephb4 overexpression also exhibited a protective effect on synovial membrane thickness of aged (24-month-old) mice. Overexpression of bone-specific Ephb4 clearly demonstrated prevention of the development and/or progression of fibrosis in OA synovial membrane, reinforcing the hypothesis that protecting the subchondral bone prophylactically and during OA reduces the pathologic changes in other articular tissues.
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Regulación de la Expresión Génica , Osteoartritis , Receptor EphB4 , Membrana Sinovial , Animales , Fibrosis , Ratones , Ratones Transgénicos , Especificidad de Órganos/genética , Osteoartritis/genética , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoartritis/prevención & control , Receptor EphB4/biosíntesis , Receptor EphB4/genética , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
INTRODUCTION: MicroRNAs (miRNAs) down-regulate their target genes. The intronic miR-140, present in the WW domain containing E3 ubiquitin protein ligase 2 (WWP2) gene, decreases the expression of genes that play detrimental roles in osteoarthritis (OA). As the expression level of miR-140 is significantly decreased in human OA chondrocytes, we investigated its regulation in those cells. METHODS: Gene expression in human chondrocytes was determined by quantitative polymerase chain reaction (qPCR) and gene silencing was done in OA chondrocytes by transient transfection with specific small interfering RNAs (siRNAs). Binding sites of the miR-140 regulatory sequence (rsmiR-140) were identified by mutagenesis and chromatin immunoprecipitation (ChIP) in OA chondrocytes. The effects of translocation on OA chondrocytes were determined by immunocytochemistry and qPCR. RESULTS: In contrast to miR-140, the expression of WWP2 was similar in both normal and OA cells, suggesting that miR-140 has an additional level of regulation. rsmiR-140 showed activity and predicted binding sites for nuclear matrix transcription factor 4 (NMP4), myc-associated zinc (MAZ), nuclear factor of activated T-cells (NFAT), and mothers against decapentaplegic homolog 3 (SMAD3). Silencing NFAT3 (P ≤0.01) and SMAD3 (P ≤0.05) differentially regulated miR-140 independently of WWP2. Silencing NFAT5 decreased both miR-140 and WWP2 (P ≤0.003 and P ≤0.05, respectively). NFAT3 activation increased and transforming growth factor-ß (TGF-ß) decreased rsmiR-140 activity. Mutagenesis of rsmiR-140 and ChIP assays identified binding sites at which NFAT3 (activator) and SMAD3 (repressor) directly regulated miR-140. TGF-ß interfered with NFAT3 translocation, and subsequently with miR-140 expression. CONCLUSIONS: This is the first study to provide evidence of a regulatory mechanism of miR-140 independent of WWP2, and new and differential roles for NFAT3 and SMAD3 in the OA process in the regulation of miR-140 transcription. Such knowledge could advance therapeutic strategies targeting OA.
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Regulación de la Expresión Génica/fisiología , MicroARNs/biosíntesis , Factores de Transcripción NFATC/metabolismo , Osteoartritis/genética , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Osteoartritis/metabolismo , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección , Ubiquitina-Proteína Ligasas/biosíntesisRESUMEN
OBJECTIVE: In vitro activation of the receptor EphB4 positively affects human osteoarthritis (OA) articular cell metabolism. However, the specific in vivo role of this ephrin receptor in OA remains unknown. We investigated in mice the in vivo effect of bone-specific EphB4 overexpression on OA pathophysiology. METHODS: Morphometric, morphologic, and radiologic evaluations were performed on postnatal day 5 (P5) mice and on 10-week-old mice. Knee OA was induced surgically by destabilization of the medial meniscus (DMM) in 10-week-old male EphB4 homozygous transgenic (EphB4-Tg) and wild-type (WT) mice. Medial compartment evaluations of cartilage were performed using histology and immunohistochemistry, and evaluations of subchondral bone using histomorphometry, osteoclast staining, and micro-computed tomography. RESULTS: There was no obvious phenotype difference in skeletal development between EphB4-Tg mice and WT mice at P5 or at 10 weeks. At 8 and 12 weeks post-DMM, the EphB4-Tg mice demonstrated significantly less cartilage alteration in the medial tibial plateau and the femoral condyle than did the WT mice. This was associated with a significant reduction of aggrecan and type II collagen degradation products, type X collagen, and collagen fibril disorganization in the operated EphB4-Tg mice. The medial tibial subchondral bone demonstrated a significant reduction in sclerosis, bone volume, trabecular thickness, and number of tartrate-resistant acid phosphatase-positive osteoclasts at both times assessed post-DMM in the EphB4-Tg mice than in the WT mice. CONCLUSION: This is the first in vivo evidence that bone-specific EphB4 overexpression exerts a protective effect on OA joint structural changes. The findings of this study stress the in vivo importance of subchondral bone biology in cartilage integrity.
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Cartílago Articular/patología , Fémur/patología , Osteoartritis de la Rodilla , Receptor EphB4/genética , Tibia/patología , Animales , Biomarcadores/metabolismo , Cartílago Articular/inmunología , Cartílago Articular/metabolismo , Modelos Animales de Enfermedad , Edema/inmunología , Edema/patología , Femenino , Fémur/inmunología , Fémur/metabolismo , Expresión Génica/inmunología , Genotipo , Articulación de la Rodilla/inmunología , Articulación de la Rodilla/patología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteoartritis de la Rodilla/genética , Osteoartritis de la Rodilla/inmunología , Osteoartritis de la Rodilla/patología , Fenotipo , Receptor EphB4/inmunología , Tibia/inmunología , Tibia/metabolismoRESUMEN
BACKGROUND: MMP-13 and IGFBP-5 are important factors involved in osteoarthritis (OA). We investigated whether two highly predicted microRNAs (miRNAs), miR-140 and miR-27a, regulate these two genes in human OA chondrocytes. METHODS: Gene expression was determined by real-time PCR. The effect of each miRNA on IGFBP-5 and MMP-13 expression/production was evaluated by transiently transfecting their precursors (pre-miRNAs) and inhibitors (anti-miRNAs) into human OA chondrocytes. Modulation of IGFBP-5, miR-140 and miR-27a expression was determined upon treatment of OA chondrocytes with cytokines and growth factors. RESULTS: IGFBP-5 was expressed in human chondrocytes with its level significantly lower (p < 0.04) in OA. Five computational algorithms identified miR-140 and miR-27a as possible regulators of MMP-13 and IGFBP-5 expression. Data showed that both miRNAs were expressed in chondrocytes. There was a significant reduction (77%, p < 0.01) in miR-140 expression in OA compared to the normal chondrocytes, whereas miR-27a expression was only slightly decreased (23%). Transfection with pre-miR-140 significantly decreased (p = 0.0002) and with anti-miR-140 significantly increased (p = 0.05) IGFBP-5 expression at 24 hours, while pre-miR-27a did not affect either MMP-13 or IGFBP-5. Treatment with anti-miR-27a, but not with anti-miR-140, significantly increased the expression of both MMP-13 (p < 0.05) and IGFBP-5 (p < 0.01) after 72 hours of incubation. MMP-13 and IGFBP-5 protein production followed the same pattern as their expression profile. These data suggest that IGFBP-5 is a direct target of miR-140, whereas miR-27a down-regulates, likely indirectly, both MMP-13 and IGFBP-5. CONCLUSION: This study is the first to show the regulation of these miRNAs in human OA chondrocytes. Their effect on two genes involved in OA pathophysiology adds another level of complexity to gene regulation, which could open up novel avenues in OA therapeutic strategies.
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Condrocitos/metabolismo , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Metaloproteinasa 13 de la Matriz/genética , MicroARNs/metabolismo , Osteoartritis de la Rodilla/genética , Regiones no Traducidas 3' , Anciano , Artroplastia de Reemplazo de Rodilla , Sitios de Unión , Estudios de Casos y Controles , Células Cultivadas , Biología Computacional , Citocinas/metabolismo , Bases de Datos Genéticas , Regulación de la Expresión Génica , Humanos , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Persona de Mediana Edad , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/cirugía , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Índice de Severidad de la Enfermedad , Factores de Tiempo , TransfecciónRESUMEN
We generate ultrabroadband biphotons via the process of spontaneous parametric down-conversion (SPDC) in quasi-phase-matched nonlinear gratings that have a linearly chirped wave vector. By using these ultrabroadband biphotons (300-nm bandwidth), we measure the narrowest Hong-Ou-Mandel dip to date, having a full width at half maximum of 7.1 fs. This enables the generation of a high flux of nonoverlapping biphotons with ultrabroad bandwidth, thereby promoting the use of SPDC light in many nonclassical applications.
RESUMEN
A high-power linearly polarized Yb-doped silica fiber master oscillator power amplifier at 1150 nm is reported. It produced 3.35 W cw and 2.33 W of average power in 1 micros pulses at a 100 kHz repetition rate, both with 8 pm linewidth. This is the first report, to the best of our knowledge, of a high-power Yb-doped fiber amplifier at a wavelength longer than 1135 nm. The pulsed output was frequency doubled in a bulk periodically poled near-stoichiometric LiTaO(3) chip to generate 976 mW of average power at 575 nm with an overall system optical-to-optical efficiency of 9.8% with respect to launched pump power.
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Óptica y Fotónica , Diseño de Equipo , Rayos Láser , Litio/química , Oscilometría , Espectrofotometría , Tantalio/química , Iterbio/químicaRESUMEN
OBJECTIVE: Matrix metalloprotease 13 (MMP-13) plays a major role in osteoarthritic (OA) processes. We previously identified the AG-rich element (AGRE) regulatory site (GAAAAGAAAAAG) in the proximal promoter of this gene. Electrophoretic mobility shift assays (EMSAs) done with nuclear extracts from OA chondrocytes showed the presence of 2 AGRE protein-binding complexes, the formation of which depended on the pathophysiologic state (high or low) of the cells; the low OA (L-OA) chondrocytes have low MMP-13 basal levels and high interleukin-1beta (IL-1beta) inducibility, and the high OA (H-OA) chondrocytes have high MMP-13 basal levels and low IL-1beta inducibility. In this study, we sought to determine the importance of individual AGRE bases in promoter activity and to identify AGRE binding proteins from L-OA and H-OA chondrocyte complexes. METHODS: Promoter activity was determined following transient transfection into human OA chondrocytes. AGRE binding proteins were identified by mass spectroscopy. RESULTS: Individual mutations of the AGRE site differentially modulated promoter activity, indicating that the intact AGRE site is required for optimal MMP-13 expression. Damage-specific DNA binding protein 1 (DDB-1) was identified in the L-OA chondrocyte-binding complex. EMSA experiments performed with the mutation of the left AGRE site (GTGCTGAAAAAG) and nuclear extracts of L-OA chondrocytes reproduced the pattern seen in the H-OA chondrocytes. Mass spectroscopy identified p130cas as one of the proteins in this complex. Supershift experiments showed the presence of p130cas and nuclear matrix transcription factor 4 (NMP-4) in the wild-type AGRE/H-OA chondrocyte complex. CONCLUSION: These data suggest that the binding of p130(cas) and NMP-4 to the AGRE site regulates MMP-13 expression and may trigger the change in human chondrocytes from the L-OA state to the H-OA state.
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Cartílago Articular/enzimología , Condrocitos/enzimología , Colagenasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Osteoartritis de la Rodilla/metabolismo , Regiones Promotoras Genéticas , Secuencia de Bases , Cartílago Articular/química , Cartílago Articular/patología , Células Cultivadas , Condrocitos/patología , Colagenasas/análisis , Proteínas de Unión al ADN/análisis , Fibroblastos/enzimología , Fibroblastos/patología , Humanos , Metaloproteinasa 13 de la Matriz , Datos de Secuencia Molecular , Osteoartritis de la Rodilla/patología , Osteoartritis de la Rodilla/cirugía , Regiones Promotoras Genéticas/fisiología , Membrana Sinovial/química , Membrana Sinovial/enzimología , Membrana Sinovial/patología , Transactivadores/análisis , Transactivadores/metabolismoRESUMEN
OBJECTIVE: To compare gene expression in normal and osteoarthritic (OA) human chondrocytes using microarray technology. Of the novel genes identified, we selected follistatin, a bone morphogenetic protein (BMP) antagonist, and investigated its expression/regulation as well as that of 3 other antagonists, gremlin, chordin, and noggin, in normal and OA chondrocytes and synovial fibroblasts. METHODS: Basal and induced gene expression were determined using real-time polymerase chain reaction. Gene regulation was monitored following treatment with inflammatory, antiinflammatory, growth, and developmental factors. Follistatin protein production was measured using a specific enzyme-linked immunosorbent assay, and localization of follistatin and gremlin in cartilage was determined by immunohistochemical analysis. RESULTS: All BMP antagonists except noggin were expressed in chondrocytes and synovial fibroblasts. Follistatin and gremlin were significantly up-regulated in OA chondrocytes but not in OA synovial fibroblasts. Chordin was weakly expressed in normal and OA cells. Production of follistatin protein paralleled the gene expression pattern. Follistatin and gremlin were expressed preferentially by the chondrocytes at the superficial layers of cartilage. Tumor necrosis factor alpha and interferon-gamma significantly stimulated follistatin expression but down-regulated expression of gremlin. Interleukin-1beta (IL-1beta) had no effect on follistatin but reduced gremlin expression. Conversely, BMP-2 and BMP-4 significantly stimulated expression of gremlin but down-regulated that of follistatin. IL-13, dexamethasone, transforming growth factor beta1, basic fibroblast growth factor, platelet-derived growth factor type BB, and endothelial cell growth factor down-regulated the expression of both antagonists. CONCLUSION: This study is the first to show the possible involvement of follistatin and gremlin in OA pathophysiology. The increased activin/BMP-binding activities of these antagonists could affect tissue remodeling. The data suggest that follistatin and gremlin might appear at different stages during the OA process, making them interesting targets for the treatment of this disease.
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Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Condrocitos/metabolismo , Fibroblastos/metabolismo , Folistatina/análisis , Regulación de la Expresión Génica/fisiología , Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intercelular/análisis , Osteoartritis/fisiopatología , Membrana Sinovial/citología , Factor de Crecimiento Transformador beta , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/farmacología , Proteínas Portadoras , Células Cultivadas , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Folistatina/fisiología , Glicoproteínas/análisis , Glicoproteínas/fisiología , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/fisiología , Interferón gamma/farmacología , Interleucina-1/farmacología , Osteoartritis/metabolismo , Análisis por Matrices de Proteínas , Proteínas/análisis , Proteínas/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia ArribaRESUMEN
Previous studies have shown that atrial natriuretic peptide (ANP) can inhibit transcription of its receptor, guanylyl cyclase A, by a mechanism dependent on cGMP and have suggested the presence of a putative cGMP-response element (cGMP-RE) in the Npr1 gene promoter. To localize and characterize the putative cis-acting element, we have subcloned a 1520-bp fragment of the rat Npr1 promoter in an expression vector containing the luciferase reporter gene. Several fragments, generated by exonuclease III-directed deletions, were transiently transfected into cells to measure their promoter activity. Deletion from -1520 to -1396 of a 1520-bp-long Npr1 promoter led to a 5-fold increase in luciferase activity. Subsequent deletion to the position -1307 resulted in a decrease of luciferase activity by 90%. Preincubation of cells with 100 nM of ANP or 100 microM 8-bromo-cGMP inhibited luciferase activity of the 1520-bp and 1396-bp-long fragments, but not the activity of the 1307-bp fragment, suggesting that the cGMP-RE is localized between positions -1396 and -1307. The cGMP regulatory region was narrowed by gel shift assays and footprinting to position -1372 to -1354 from the transcription start site of Npr1 and indicated its interaction with transcriptional factor(s). Cross-competition experiments with mutated oligonucleotides led to the definition of a consensus sequence (-1372 AaAtRKaNTTCaAcAKTY -1354) for the novel cGMP-RE, which is conserved in the human (75% identity) and mouse (95% identity) Npr1 promoters.
Asunto(s)
GMP Cíclico/fisiología , Guanilato Ciclasa/genética , Receptores del Factor Natriurético Atrial/genética , Elementos de Respuesta/genética , Animales , Factor Natriurético Atrial/farmacología , Clonación Molecular , Secuencia de Consenso , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Elementos de Respuesta/efectos de los fármacosRESUMEN
The activity of the atrial natriuretic peptide receptor (Npr1) is altered in spontaneously hypertensive rats (SHR) in relation to its mRNA levels, suggesting abnormal transcriptional control in hypertension. A single-stranded conformational polymorphism caused by a repetitive dinucleotide segment of 10 TA in BN-Lx and of 40 TA in SHR was localized at position -943 relative to the transcription start site of the Npr1 gene, downstream of a putative cGMP-regulatory region, and was the only sequence difference noted between the two strains. Transient transfections of -1520 to -920 Npr1 promoter-SV40-luciferase fusion vector showed that the construct from BN-Lx stimulated the SV40 promoter, whereas that from SHR slightly inhibited it. In contrast to the BN-Lx construct, the activity of the SHR fragment was refractory to downregulation by atrial natriuretic peptide. Genotype-phenotype correlation studies in recombinant inbred strains (RIS) derived from BN-Lx and SHR crosses revealed significant correlations of the TA repeat with basal guanylyl cyclase activity and Npr1 mRNA levels. The correlations were heightened by a locus on chromosome 10 containing the Ace gene. The highest basal guanylyl cyclase activity and Npr1 mRNA values were found in RIS with both genes (Npr1/Ace) of BN genotypes, whereas the lowest were recorded in RIS, with the SHR genotypes at both loci. This was inversely correlated with diastolic blood pressure in these strains. In conclusion, the longer TA repeat unit in the promoter of Npr1 of SHR, in tandem with a putative cGMP responsive element, regulates the transcription of the Npr1 gene with consequences on diastolic blood pressure.
Asunto(s)
Presión Sanguínea/genética , Guanilato Ciclasa/genética , Hipertensión/genética , Peptidil-Dipeptidasa A/genética , Polimorfismo Conformacional Retorcido-Simple , Receptores del Factor Natriurético Atrial/genética , Células 3T3 , Animales , Repeticiones de Dinucleótido , Regulación de la Expresión Génica , Genotipo , Guanilato Ciclasa/biosíntesis , Guanilato Ciclasa/metabolismo , Hipertensión/metabolismo , Hipertensión/fisiopatología , Ratones , Fenotipo , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas , Receptores del Factor Natriurético Atrial/biosíntesis , Transcripción GenéticaRESUMEN
Guanylyl cyclases (GC) exist as soluble and particulate, membrane-associated enzymes which catalyse the conversion of GTP to cGMP, an intracellular signalling molecule. Several membrane forms of the enzyme have been identified up to now. Some of them serve as receptors for the natriuretic peptides, a family of peptides which includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), three peptides known to play important roles in renal and cardiovascular physiology. These are transmembrane proteins composed of a single transmembrane domain, a variable extracellular natriuretic peptide-binding domain, and a more conserved intracellular kinase homology domain (KHD) and catalytic domain. GC-A, the receptor for ANP and BNP, also named natriuretic peptide receptor-A or -1 (NPR-A or NPR- 1), has been studied widely. Its mode of activation by peptide ligands and mechanisms of regulation serve as prototypes for understanding the function of other particulate GC. Activation of this enzyme by its ligand is a complex process requiring oligomerization, ligand binding, KHD phosphorylation and ATP binding. Gene knockout and genetic segregation studies have provided strong evidence for the importance of GC-A in the regulation of blood pressure and heart and renal functions. GC-B is the main receptor for CNP, the latter having a more paracrine role at the vascular and venous levels. The structure and regulation of GC-B is similar to that of GC-A. This chapter reviews the structure and roles of GC-A and GC-B in blood pressure regulation and cardiac and renal pathophysiology.