RESUMEN
Haptoglobin (Hp), a type of acute-phase protein, is known to have a systemic anti-inflammatory function and to modulate inflammation by directly affecting immune cells, such as T cells, dendritic cells, and macrophages. However, the effects of Hp on osteoclast differentiation are not well studied, even though osteoclast precursor cells belong to a macrophage-monocyte lineage. In this study, we found that the bone volume was reduced, and the number of osteoclasts was increased in Hp-deficient mice compared with wild-type mice. Moreover, our in vitro studies showed that Hp inhibits osteoclastogenesis by reducing the protein level of c-Fos at the early phase of osteoclast differentiation. We revealed that Hp-induced suppression of c-Fos was mediated by increased IFN-ß levels. Furthermore, Hp stimulated IFN-ß via a TLR4-dependent mechanism. These results demonstrate that Hp plays a protective role against excessive osteoclastogenesis via the Hp-TLR4-IFN-ß axis.
Asunto(s)
Haptoglobinas/metabolismo , Interferón beta/metabolismo , Osteoclastos/fisiología , Reacción de Fase Aguda , Animales , Resorción Ósea/genética , Diferenciación Celular , Células Cultivadas , Haptoglobinas/genética , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteogénesis , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de SeñalRESUMEN
Amelogenesis imperfecta (AI) is a heterogeneous group of genetic disorders affecting tooth enamel. The affected enamel can be hypoplastic and/or hypomineralized. In this study, we identified ACPT (testicular acid phosphatase) biallelic mutations causing non-syndromic, generalized hypoplastic autosomal-recessive amelogenesis imperfecta (AI) in individuals from six apparently unrelated Turkish families. Families 1, 4, and 5 were affected by the homozygous ACPT mutation c.713C>T (p.Ser238Leu), family 2 by the homozygous ACPT mutation c.331C>T (p.Arg111Cys), family 3 by the homozygous ACPT mutation c.226C>T (p.Arg76Cys), and family 6 by the compound heterozygous ACPT mutations c.382G>C (p.Ala128Pro) and 397G>A (p.Glu133Lys). Analysis of the ACPT crystal structure suggests that these mutations damaged the activity of ACPT by altering the sizes and charges of key amino acid side chains, limiting accessibility of the catalytic core, and interfering with homodimerization. Immunohistochemical analysis confirmed localization of ACPT in secretory-stage ameloblasts. The study results provide evidence for the crucial function of ACPT during amelogenesis.
Asunto(s)
Fosfatasa Ácida/genética , Amelogénesis Imperfecta/genética , Proteínas del Esmalte Dental/genética , Genes Recesivos , Mutación , Fosfatasa Ácida/metabolismo , Amelogénesis Imperfecta/diagnóstico , Niño , Esmalte Dental/anomalías , Proteínas del Esmalte Dental/metabolismo , Exones , Femenino , Homocigoto , Humanos , Masculino , Linaje , Conformación Proteica , Alineación de Secuencia , TurquíaRESUMEN
Mitofusin 2 (Mfn2) is a mitochondrial outer membrane protein that participates in tethering mitochondria to the ER. Mitochondria-ER tethering has been demonstrated to play important roles in many cellular activities by regulating homeostasis of metabolites and calcium. Intracellular calcium signaling is crucial for the differentiation of osteoclasts, the bone-resorbing cells. In this study, we investigated whether Mfn2 plays a role in osteoclastogenesis by receptor activator of nuclear factor kappa B (RANKL) in primary cells. We found that RANKL increased Mfn2 expression during osteoclast formation from mouse bone marrow-derived macrophages (BMMs). When Mfn2 expression was suppressed in BMMs by using a siRNA-mediated gene knock-down system, osteoclast differentiation and activity of mature osteoclasts were reduced. Mfn2 knock-down also decreased the RANKL-mediated induction of NFATc1, the key transcription factor for osteoclast gene expression, without affecting c-Fos level. This effect on NFATc1 was associated with decreased calcium oscillation and calcineurin activity in Mfn2-deficient osteoclasts. Taken together, our results indicate that Mfn2 positively contributes to RANKL-induced osteoclast differentiation by regulating the calcium-calcieurin-NFATc1 axis, raising the importance of a previously under-recognized role of mitochondria in osteoclastogenesis.
Asunto(s)
Calcineurina/metabolismo , Calcio/metabolismo , GTP Fosfohidrolasas/metabolismo , Factores de Transcripción NFATC/metabolismo , Osteogénesis , Transducción de Señal , Animales , Señalización del Calcio , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos ICR , Mitocondrias/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismoRESUMEN
OBJECTIVE: Amelogenesis imperfecta (AI) is a rare hereditary disorder affecting the quality and quantity of the tooth enamel. The purpose of this study was to identify the genetic etiology of hypoplastic AI families based on the candidate gene approach. MATERIALS AND METHODS: We recruited three Turkish families with hypoplastic AI and performed a candidate gene screening based on the characteristic clinical feature to find the pathogenic genetic etiology. RESULTS: The candidate gene sequencing of the LAMB3 gene for family 1 revealed a heterozygous nonsense mutation in the last exon [c.3431C > A, p.(Ser1144*)]. FAM20A gene sequencing for families 2 and 3 identified a homozygous deletion [c.34_35delCT, p.(Leu12Alafs*67)] and a homozygous deletion-insertion (c.1109 + 3_1109 + 7delinsTGGTC) mutation, respectively. CONCLUSION: The candidate gene approach can be successfully used to identify the genetic etiology of the AI in some cases with characteristic clinical features. CLINICAL RELEVANCE: Identification of the genetic etiology of the AI will help both the family members and dentist understand the nature of the disorder. Characteristic clinical feature can suggest possible genetic causes.
Asunto(s)
Amelogénesis Imperfecta/genética , Moléculas de Adhesión Celular/genética , Proteínas del Esmalte Dental/genética , Codón sin Sentido , Análisis Mutacional de ADN , Homocigoto , Humanos , Mutación INDEL , Linaje , Eliminación de Secuencia , Turquía , KalininaRESUMEN
Lipid raft microdomains have important roles in various cellular responses. Caveolae are a specialized type of lipid rafts that are stabilized by oligomers of caveolin proteins. Here, we show that caveolin-1 (Cav-1) plays a crucial role in the regulation of osteoclastogenesis. We found that caveolin-1 was dramatically up-regulated by receptor activator of nuclear factor κB ligand (RANKL), the osteoclast differentiation factor. Knockdown of Cav-1 reduced osteoclastogenesis and induction of NFATc1, the master transcription factor for osteoclastogenesis, by RANKL. Consistent with the in vitro results, injection of caveolin-1 siRNA onto mice calvariae showed reduction in RANKL-induced bone resorption and osteoclast formation. Moreover, Cav-1(-/-) female mice had higher bone volume and lower osteoclast number compared with wild type mice. However, Cav-1(-/-) male mice had both osteoclast and osteoblast numbers higher than wild type mice with no difference in bone volume. The sex dependence in the effect of Cav-1 deficiency was partly attributed to decreased receptor activator of nuclear factor κB and increased cFms expression in osteoclast precursors of female and male mice, respectively. Taken together, these data demonstrate that Cav-1 has a complicated but critical role for osteoclastogenesis.
Asunto(s)
Desarrollo Óseo/genética , Caveolina 1/genética , Diferenciación Celular/genética , Osteoclastos/metabolismo , Animales , Caveolina 1/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Masculino , Microdominios de Membrana/genética , Microdominios de Membrana/metabolismo , Ratones , Factores de Transcripción NFATC/biosíntesis , Osteoclastos/química , Osteogénesis/genética , Ligando RANK/biosíntesis , Caracteres SexualesRESUMEN
Tetraspanin family proteins regulate morphology, motility, fusion, and signaling in various cell types. We investigated the role of the tetraspanin 7 (Tspan7) isoform in the differentiation and function of osteoclasts. Tspan7 was up-regulated during osteoclastogenesis. When Tspan7 expression was reduced in primary precursor cells by siRNA-mediated gene knock-down, the generation of multinuclear osteoclasts was not affected. However, a striking cytoskeletal abnormality was observed: the formation of the podosome belt structure was inhibited and the microtubular network were disrupted by Tspan7 knock-down. Decreases in acetylated microtubules and levels of phosphorylated Src and Pyk2 in Tspan7 knock-down cells supported the involvement of Tspan7 in cytoskeletal rearrangement signaling in osteoclasts. This cytoskeletal defect interfered with sealing zone formation and subsequently the bone-resorbing activity of mature osteoclasts on dentin surfaces. Our results suggest that Tspan7 plays an important role in cytoskeletal organization required for the bone-resorbing function of osteoclasts by regulating signaling to Src, Pyk2, and microtubules.
Asunto(s)
Resorción Ósea/metabolismo , Resorción Ósea/patología , Osteoclastos/metabolismo , Osteoclastos/patología , Podosomas/metabolismo , Tetraspaninas/metabolismo , Animales , Movimiento Celular , Supervivencia Celular , Células Cultivadas , Femenino , Ratones , Osteogénesis , Podosomas/patologíaRESUMEN
Osteogenesis imperfecta (OI) is a heterogenous group of genetic disorders of bone fragility. OI type V is an autosomal-dominant disease characterized by calcification of the forearm interosseous membrane, radial head dislocation, a subphyseal metaphyseal radiodense line, and hyperplastic callus formation; the causative mutation involved in this disease has not been discovered yet. Using linkage analysis in a four-generation family and whole-exome sequencing, we identified a heterozygous mutation of c.-14C>T in the 5'-untranslated region of a gene encoding interferon-induced transmembrane protein 5 (IFITM5). It completely cosegregated with the disease in three families and occurred de novo in five simplex individuals. Transfection of wild-type and mutant IFITM5 constructs revealed that the mutation added five amino acids (Met-Ala-Leu-Glu-Pro) to the N terminus of IFITM5. Given that IFITM5 expression and protein localization is restricted to the skeletal tissue and IFITM5 involvement in bone formation, we conclude that this recurrent mutation would have a specific effect on IFITM5 function and thus cause OI type V.
Asunto(s)
Proteínas de la Membrana/genética , Osteogénesis Imperfecta/genética , Regiones no Traducidas 5'/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Exoma/genética , Femenino , Ligamiento Genético , Humanos , Masculino , Datos de Secuencia Molecular , Osteogénesis Imperfecta/diagnóstico por imagen , Mutación Puntual/genética , Radiografía , Análisis de Secuencia de ADNRESUMEN
In bone marrow, bone marrow stromal cells (BMSCs) have the capacity to differentiate into osteoblasts and adipocytes. Age-related osteoporosis is associated with a reciprocal decrease of osteogenesis and an increase of adipogenesis in bone marrow. In this study, we demonstrate that disruption of nuclear factor I-C (NFI-C) impairs osteoblast differentiation and bone formation, and increases bone marrow adipocytes. Interestingly, NFI-C controls postnatal bone formation but does not influence prenatal bone development. We also found decreased NFI-C expression in osteogenic cells from human osteoporotic patients. Notably, transplantation of Nfic-overexpressing BMSCs stimulates osteoblast differentiation and new bone formation, but inhibits adipocyte differentiation by suppressing peroxisome proliferator-activated receptor gamma expression in Nfic(-/-) mice showing an age-related osteoporosis-like phenotype. Finally, NFI-C directly regulates Osterix expression but acts downstream of the bone morphogenetic protein-2-Runx2 pathway. These results suggest that NFI-C acts as a transcriptional switch in cell fate determination between osteoblast and adipocyte differentiation in BMSCs. Therefore, regulation of NFI-C expression in BMSCs could be a novel therapeutic approach for treating age-related osteoporosis.
Asunto(s)
Factores de Transcripción NFI/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Factores de Transcripción/biosíntesis , Anciano , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Transgénicos , Factores de Transcripción NFI/genética , Osteogénesis/fisiología , Factor de Transcripción Sp7 , TransfecciónRESUMEN
5-Lipoxygenase (5-LO) catalyzes the formation of two major groups of leukotrienes, leukotriene B4 and cysteinyl leukotrienes (CysLTs), and it has been implicated as a promising drug target to treat various inflammatory diseases. However, its role in osteoclastogenesis has not been investigated. In this study, we used mouse bone marrow-derived macrophages (BMMs) to show that 5-LO inhibitor suppresses RANKL-induced osteoclast formation. Inhibition of 5-LO was associated with impaired activation of multiple signaling events downstream of RANK, including ERK and p38 phosphorylation, and IκB degradation, followed by a decrease in NFATc1 expression. Ectopic overexpression of a constitutively active form of NFATc1 partly rescued the antiosteoclastogenic effect of 5-LO inhibitor. The knockdown of 5-LO in BMMs also resulted in a significant reduction in RANKL-induced osteoclast formation, accompanied by decreased expression of NFATc1. Similar effects were shown with CysLT receptor (CysLTR)1/2 antagonist and small RNA for CysLTR1 in BMMs, indicating the involvement of CysLT and CysLTR1 in 5-LO-mediated osteoclastogenesis. Finally, 5-LO inhibitor suppressed LPS-induced osteoclast formation and bone loss in the in vivo mouse experiments, suggesting a potential therapeutic strategy for treating diseases involving bone destruction. Taken together, the results of this study demonstrate that 5-LO is a key mediator of RANKL-induced osteoclast formation and possibly a novel therapeutic target for bone-resorption diseases.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Resorción Ósea/prevención & control , Indoles/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Osteoclastos/efectos de los fármacos , Ligando RANK/antagonistas & inhibidores , Receptores de Leucotrienos/metabolismo , Animales , Araquidonato 5-Lipooxigenasa/genética , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Médula Ósea/patología , Resorción Ósea/genética , Resorción Ósea/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos ICR , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patología , Fosforilación/efectos de los fármacos , Ligando RANK/genética , Ligando RANK/metabolismo , ARN Interferente Pequeño/genética , Receptores de Leucotrienos/genética , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Apolipoprotein E (ApoE) plays a major role in the transport and metabolism of lipid. Other functions of ApoE include modulation of innate and adaptive immune responses. The expression of ApoE in osteoblasts and its relevance with bone formation have also been reported. However, the effect of ApoE on osteoclasts has not yet been examined. Here, we investigated the role of ApoE in osteoclast differentiation using bone marrow-derived macrophages (BMMs) and RAW264.7 cells. We found a down-regulation of ApoE gene expression during osteoclastic differentiation of those cells. Overexpression of ApoE in BMMs and RAW264.7 cells significantly blocked the induction of c-Fos and nuclear factor of activated T cell c1 (NFATc1), transcription factors critical for expression of osteoclast marker genes, by receptor activator of nuclear factor κB ligand (RANKL), the osteoclast differentiation factor. ApoE inhibited osteoclast differentiation, as measured by decreased number of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs). In addition, ApoE reduced the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and ATPase, H(+) transporting, lysosomal 38kDa, V0 subunit d2 (ATP6v0d2), genes involved in cell-cell fusion during osteoclastogenesis. Knock-down of ApoE using a specific siRNA promoted the RANKL-mediated induction of osteoclast differentiation. While ApoE did not affect the activation of ERK, JNK, and p38 MAPK signaling pathways by RANKL, the phosphorylation of p65 trans-activation domain on serine 536 and transcription activity of NF-κB were reduced by ApoE overexpression. These findings suggest that ApoE plays an inhibitory role in osteoclast differentiation via the suppression of RANKL-dependent activation of NF-κB and induction of c-Fos and NFATc1.
Asunto(s)
Apolipoproteínas E/fisiología , Diferenciación Celular/genética , Genes fos , FN-kappa B/genética , Factores de Transcripción NFATC/genética , Osteoclastos/fisiología , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Apolipoproteínas E/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Regulación de la Expresión Génica/efectos de los fármacos , Genes fos/efectos de los fármacos , Genes fos/genética , Genes fos/fisiología , Ratones , FN-kappa B/metabolismo , FN-kappa B/fisiología , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/fisiología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Ligando RANK/farmacología , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , TransfecciónRESUMEN
Hypodontia, i.e., missing one or more teeth, is a relatively common human disease; however, oligodontia, i.e., missing six or more teeth, excluding the third molars, is a rare congenital disorder. Many genes have been shown to cause oligodontia in non-syndromic or syndromic conditions. In this study, we identified two novel PAX9 mutations in two non-syndromic oligodontia families. A mutational analysis identified a silent mutation (NM_006194.4: c.771G>A, p.(Gln257=)) in family 1 and a frameshift mutation caused by a single nucleotide duplication (c.637dup, p.(Asp213Glyfs*104)) in family 2. A minigene splicing assay revealed that the silent mutation resulted in aberrant pre-mRNA splicing instead of normal splicing. The altered splicing products are ones with an exon 4 deletion or using a cryptic 5' splicing site in exon 4. Mutational effects were further investigated using protein expression, luciferase activity assay and immunolocalization. We believe this study will not only expand the mutational spectrum of PAX9 mutations in oligodontia but also strengthen the diagnostic power related to the identified silent mutation.
RESUMEN
The bone morphogenetic protein (BMP) ligand Glass bottom boat (Gbb) acts as a retrograde growth signal at the Drosophila neuromuscular junction (NMJ). Endocytic regulation of presynaptic BMP receptors has been proposed to attenuate retrograde BMP signaling. However, it remains unknown whether the Gbb signal is also regulated by postsynaptic mechanisms. Here, we provide evidence that Drosophila Cdc42-interacting protein 4 (dCIP4) functions postsynaptically to inhibit synaptic growth. dCIP4 is localized postsynaptically at NMJs. dcip4 mutations lead to synaptic overgrowth and increased presynaptic phosphorylated mothers against decapentaplegic (Mad) levels, and these defects are rescued by muscle-specific expression of dCIP4. Biochemical and genetic analyses demonstrate that dCIP4 acts downstream of Cdc42 to activate the postsynaptic Wsp-Arp2/3 pathway. We also show that BMP signaling is necessary for synaptic overgrowth in larvae lacking postsynaptic dcip4 or wsp. Finally, dCIP4 and Wsp inhibit Gbb secretion. Thus, we propose that dCIP4 restrains synaptic growth by inhibiting postsynaptic Gbb secretion through the Wsp-Arp2/3 pathway.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Unión Neuromuscular/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras/genética , Células Cultivadas , Drosophila , Proteínas de Drosophila/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Microscopía Confocal/métodos , Biología Molecular/métodos , Mutación/genética , Proteínas del Tejido Nervioso/genética , Unión Neuromuscular/citología , Unión Neuromuscular/efectos de los fármacos , Fosforilación , Terminales Presinápticos/metabolismo , Interferencia de ARN/fisiología , Transducción de Señal/genética , Transfección/métodos , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Amelogenesis imperfecta (AI) is a collection of diverse inherited disorders featuring dental-enamel defects in the absence of significant nondental symptoms. AI phenotypes vary and are categorized as hypoplastic, hypocalcified, and hypomaturation types. Phenotypic specificity to enamel has focused research on genes encoding enamel-matrix proteins. We studied two families with autosomal-dominant hypocalcified AI and have identified nonsense mutations (R325X and Q398X) in the FAM83H gene on chromosome 8q24.3. The mutations perfectly cosegregate with the disease phenotype and demonstrate that FAM83H is required for proper dental-enamel calcification.
Asunto(s)
Amelogénesis Imperfecta/genética , Cromosomas Humanos Par 8/genética , Fenotipo , Proteínas/genética , Amelogénesis Imperfecta/patología , Secuencia de Bases , Codón sin Sentido/genética , Cartilla de ADN/genética , Humanos , Hibridación in Situ , Escala de Lod , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
Vitamin E, an essential nutrient with powerful antioxidant activity, is the mixture of two classes of compounds, tocopherols (TPs) and tocotrienols (TTs). Although TTs exhibit better bone protective activity than α-TP, the underlying mechanism is poorly understood. In this study, we investigated whether α-TT and α-TP can modulate osteoclastic bone resorption. We found that α-TT but not α-TP inhibits osteoclastogenesis in coculture of osteoblasts and bone marrow cells induced by either IL-1 or combined treatment with 1α,25(OH)(2) vitamin D(3) and prostaglandin E(2). In accordance with this, only α-TT inhibited receptor activator of NF-κB ligand (RANKL) expression in osteoblasts. In addition, α-TT but not α-TP inhibited RANKL-induced osteoclast differentiation from precursors by suppression of c-Fos expression, possibly through inhibiting ERK and NF-κB activation. This anti-osteoclastogenic effect was reversed when c-Fos or an active form of NFATc1, a critical downstream of c-Fos during osteoclastogenesis, was overexpressed. Furthermore, only α-TT reduced bone resorbing activity of mature osteoclasts without affecting their survival. Overall, our results demonstrate that α-TT but not α-TP has anti-bone resorptive properties by inhibiting osteoclast differentiation and activation, suggesting that α-TT may have therapeutic value for treating and preventing bone diseases characterized by excessive bone destruction.
Asunto(s)
Resorción Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Ligando RANK/antagonistas & inhibidores , Vitamina E/análogos & derivados , Animales , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/patología , Supervivencia Celular , Células Cultivadas , Ratones , Osteoclastos/citología , Ligando RANK/biosíntesis , Transducción de Señal/efectos de los fármacos , Tocotrienoles , Vitamina E/farmacología , Vitamina E/uso terapéuticoRESUMEN
Amelogenesis imperfecta (AI) is a heterogeneous group of genetic disorders with regard to genetic aetiology and clinical phenotype and affects tooth enamel with no other non-oral syndromic conditions. X-linked AI is caused by mutations in the amelogenin (AMELX) gene, the only AI candidate gene located on the X chromosome. To date, 15 mutations in the AMELX gene have been found to cause AI. We identified a proband with generalized hypoplastic enamel and unusual multiple crown resorption in premolars and molars. Pedigree analysis suggested an X-linked hereditary pattern. We performed mutational analysis for the AMELX gene based on the candidate gene approach. Sequencing analysis revealed a novel mutation in exon 6 (g.4090delC, c.517delC, p.Pro173LeufsX16). This frameshift mutation produces a premature stop codon within exon 6 and is predicted to replace 33 amino acids at the C-terminus with 15 novel amino acids if the mutant mRNA escapes the nonsense-mediated decay system. Although crown resorptions occur frequently in patients with the hypoplastic type of A1, an association with the AMELX mutation has not been previously reported. We believe that these findings will broaden our understanding of the clinical phenotype and pathogenesis of X-linked AI.
Asunto(s)
Amelogénesis Imperfecta/genética , Amelogenina/genética , Resorción Dentaria/genética , Amelogénesis Imperfecta/complicaciones , Niño , Codón sin Sentido , Análisis Mutacional de ADN , Mutación del Sistema de Lectura , Humanos , Masculino , Mordida Abierta/complicaciones , Corona del Diente/patologíaRESUMEN
Hereditary dentin defects can be categorized as a syndromic form predominantly related to osteogenesis imperfecta (OI) or isolated forms without other non-oral phenotypes. Mutations in the gene encoding dentin sialophosphoprotein (DSPP) have been identified to cause dentinogenesis imperfecta (DGI) Types II and III and dentin dysplasia (DD) Type II. While DGI Type I is an OI-related syndromic phenotype caused mostly by monoallelic mutations in the genes encoding collagen type I alpha 1 chain (COL1A1) and collagen type I alpha 2 chain (COL1A2). In this study, we recruited families with non-syndromic dentin defects and performed candidate gene sequencing for DSPP exons and exon/intron boundaries. Three unrelated Korean families were further analyzed by whole-exome sequencing due to the lack of the DSPP mutation, and heterozygous COL1A2 mutations were identified: c.3233G>A, p.(Gly1078Asp) in Family 1 and c.1171G>A, p.(Gly391Ser) in Family 2 and 3. Haplotype analysis revealed different disease alleles in Families 2 and 3, suggesting a mutational hotspot. We suggest expanding the molecular genetic etiology to include COL1A2 for isolated dentin defects in addition to DSPP.
RESUMEN
Epigallocatechin-3-gallate (EGCG), the major anti-inflammatory compound in green tea, has been shown to suppress osteoclast differentiation. However, the precise molecular mechanisms underlying the inhibitory action of EGCG in osteoclastogenesis and the effect of EGCG on inflammation-mediated bone destruction remain unclear. In this study, we found that EGCG inhibited osteoclast formation induced by osteoclastogenic factors in bone marrow cell-osteoblast cocultures but did not affect the ratio of receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) to osteoprotegerin induced by osteoclastogenic factors in osteoblasts. We also found that EGCG inhibited osteoclast formation from bone marrow macrophages (BMMs) induced by macrophage colony-stimulating factor plus RANKL in a dose-dependent manner without cytotoxicity. Pretreatment with EGCG significantly inhibited RANKL-induced the gene expression of c-Fos and nuclear factor of activated T-cells (NFATc1), essential transcription factors for osteoclast development. EGCG suppressed RANKL-induced activation of c-Jun N-terminal protein kinase (JNK) pathway, among the three well known mitogen-activated protein kinases and also inhibited RANKL-induced phosphorylation of the NF-kappaB p65 subunit at Ser276 and NF-kappaB transcriptional activity without affecting the degradation of IkappaBalpha and NF-kappaB DNA-binding in BMMs. The inhibitory effect of EGCG on osteoclast formation was somewhat reversed by retroviral c-Fos overexpression, suggesting that c-Fos is a downstream target for antiosteoclastogenic action of EGCG. In addition, EGCG treatment reduced interleukin-1-induced osteoclast formation and bone destruction in mouse calvarial bone in vivo. Taken together, our data suggest that EGCG has an antiosteoclastogenic effect by inhibiting RANKL-induced the activation of JNK/c-Jun and NF-kappaB pathways, thereby suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.
Asunto(s)
Resorción Ósea/tratamiento farmacológico , Catequina/análogos & derivados , FN-kappa B/antagonistas & inhibidores , Osteoclastos/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Transducción de Señal/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Resorción Ósea/prevención & control , Catequina/farmacología , Células Cultivadas , Técnicas de Cocultivo , Regulación hacia Abajo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Factores de Transcripción NFATC , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Osteoprotegerina , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Ligando RANKRESUMEN
cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-beta. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.
Asunto(s)
Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Osteólisis/metabolismo , Animales , Neoplasias Óseas/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Interleucina-17/farmacología , Metaloproteinasas de la Matriz/genética , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Osteólisis/patología , Osteoprotegerina/genética , Proteína Relacionada con la Hormona Paratiroidea/genética , Factor de Crecimiento Transformador beta/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Metastasis to bone is a frequent occurrence in patients with breast and prostate cancers and inevitably threatens the patient's quality of life and survival. Identification of cancer-derived mediators of bone metastasis and osteolysis may lead to novel therapeutic strategies. In this study, we established highly bone-metastatic PC3 prostate and MDA-MB-231 (MDA) breast cancer cell sublines by in vivo selection in mice. In bone-metastatic cancer cells, the expression and secretion of connective tissue growth factor (CTGF) were highly upregulated. CTGF knockdown in bone-metastatic cells decreased invasion activity and MMP expression. RUNX2 overexpression in the CTGF knockdown cells restored the invasion activity and MMP expression. In addition, CTGF increased RUNX2 protein stability by inducing its acetylation via p300 acetyl transferase. The integrin αvß3 receptor mediated these effects of CTGF. Furthermore, CTGF promoted RUNX2 recruitment to the RANKL promoter, resulting in increased RANKL production from the tumor cells and subsequent stimulation of osteoclastogenesis from precursor cells. In addition, animal model with injection of CTGF knocked-down prostate cancer cells into 6-week old BALB/c male mice showed reduced osteolytic lesions. More importantly, the expression levels of CTGF and RANKL showed a strong positive correlation in human primary breast tumor tissues and were higher in bone metastases than in other site metastases. These findings indicate that CTGF plays crucial roles for osteolytic bone metastasis both by enhancing invasiveness of tumor cells and by producing RANKL for osteoclastogenesis. Targeting CTGF may lead to the development of effective preventive and therapeutic strategies for osteolytic metastasis. © 2019 American Society for Bone and Mineral Research.