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The original article has been corrected.
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To elucidate mutation spectrum and genotype-phenotype correlations in Japanese patients with OI, we conducted comprehensive genetic analyses using NGS, as this had not been analyzed comprehensively in this patient population. Most mutations were located on COL1A1 and COL1A2. Glycine substitutions in COL1A1 resulted in the severe phenotype. INTRODUCTION: Most cases of osteogenesis imperfecta (OI) are caused by mutations in COL1A1 or COL1A2, which encode α chains of type I collagen. However, mutations in at least 16 other genes also cause OI. The mutation spectrum in Japanese patients with OI has not been comprehensively analyzed, as it is difficult to identify using classical Sanger sequencing. In this study, we aimed to reveal the mutation spectrum and genotype-phenotype correlations in Japanese patients with OI using next-generation sequencing (NGS). METHODS: We designed a capture panel for sequencing 15 candidate OI genes and 19 candidate genes that are associated with bone fragility or Wnt signaling. Using NGS, we examined 53 Japanese patients with OI from unrelated families. RESULTS: Pathogenic mutations were detected in 43 out of 53 individuals. All mutations were heterozygous. Among the 43 individuals, 40 variants were identified including 15 novel mutations. We found these mutations in COL1A1 (n = 30, 69.8%), COL1A2 (n = 12, 27.9%), and IFITM5 (n = 1, 2.3%). Patients with glycine substitution on COL1A1 had a higher frequency of fractures and were more severely short-statured. Although no significant genotype-phenotype correlation was observed for bone mineral density, the trabecular bone score was significantly lower in patients with glycine substitutions. CONCLUSION: We identified pathogenic mutations in 81% of our Japanese patients with OI. Most mutations were located on COL1A1 and COL1A2. This study revealed that glycine substitutions on COL1A1 resulted in the severe phenotype among Japanese patients with OI.
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Osteogênese Imperfeita/genética , Adolescente , Adulto , Densidade Óssea/genética , Criança , Pré-Escolar , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Feminino , Estudos de Associação Genética , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Japão , Masculino , Mutação , Análise de Sequência de DNA , Adulto JovemRESUMO
The clarification of public concerns regarding heart transplantation is important for improving low organ donation rates in Japan. In the present study, we used the Twitter data of 4986 tweets (between August 2015 and January 2016) and 1429 tweets (between April 2016 and May 2016) to analyze public discourse on heart transplantation in Japan and identify the reasons for low organ donation rates. We manually categorized all tweets relevant to heart transplantation into nine categories and counted the number of tweets in each category per month. During the study period, the most popular category of tweets was related to the media, followed by money (tweets questioning or even criticizing the high price of fundraising goals to go overseas for heart transplantations), while some tweets were misconceptions. We also conducted a sentiment analysis, which revealed that the most popular negative tweets were related to money, while the most positive tweets were related to reports on the favorable outcomes of recipients. Our results suggest that listening to concerns, providing correct information (particularly for some misconceptions), and emphasizing the outcomes of recipients will facilitate an increase in the number of people contemplating heart transplantation and organ donation.
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Conhecimentos, Atitudes e Prática em Saúde , Transplante de Coração , Opinião Pública , Mídias Sociais , Rede Social , Obtenção de Tecidos e Órgãos/estatística & dados numéricos , Humanos , Japão , Estudos RetrospectivosRESUMO
OBJECTIVE: FGFR3 chondrodysplasia is caused by a gain-of-function mutation of the FGFR3 gene. The disease causes abnormal growth plate cartilage and lacks effective drug treatment. We sought to establish an in vivo model for the study of FGFR3 chondrodysplasia pathology and drug testing. DESIGN: We created cartilage from human induced pluripotent stem cells (hiPSCs) and transplanted the cartilage into the subcutaneous spaces of immunodeficient mice. We then created cartilage from the hiPSCs of patients with FGFR3 chondrodysplasia and transplanted them into immunodeficient mice. We treated some mice with a FGFR inhibitor after the transplantation. RESULTS: Xenografting the hiPSC-derived cartilage reproduced human growth plate cartilage consisting of zones of resting, proliferating, prehypertrophic and hypertrophic chondrocytes and bone in immunodeficient mice. Immunohistochemistry of xenografts using anti-human nuclear antigen antibody indicated that all chondrocytes in growth plate cartilage were human, whereas bone was composed of human and mouse cells. The pathology of small hypertrophic chondrocytes due to up-regulated FGFR3 signaling in FGFR3 skeletal dysplasia was recapitulated in growth plate cartilage formed in the xenografts of patient-specific hiPSC-derived cartilage. The mean diameters of hypertrophic chondrocytes between wild type and thanatophoric dysplasia were significantly different (95% CI: 13.2-26.9; n = 4 mice, one-way analysis of variance (ANOVA)). The pathology was corrected by systemic administration of a FGFR inhibitor to the mice. CONCLUSION: The patient-specific growth plate cartilage xenograft model for FGFR3 skeletal dysplasia indicated recapitulation of pathology and effectiveness of a FGFR inhibitor for treatment and warrants more study for its usefulness to study disease pathology and drug testing.
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Cartilagem/patologia , Lâmina de Crescimento/patologia , Mutação , Osteocondrodisplasias/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Animais , Cartilagem/metabolismo , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Modelos Animais de Doenças , Lâmina de Crescimento/metabolismo , Xenoenxertos , Camundongos , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de SinaisRESUMO
BACKGROUND AND OBJECTIVE: Hypophosphatasia is a rare inherited skeletal disorder characterized by defective bone mineralization and deficiency of tissue non-specific alkaline phosphatase (TNSALP) activity. The disease is caused by mutations in the liver/bone/kidney alkaline phosphatase gene (ALPL) encoding TNSALP. Early exfoliation of primary teeth owing to disturbed cementum formation, periodontal ligament weakness and alveolar bone resorption are major complications encountered in oral findings, and discovery of early loss of primary teeth in a dental examination often leads to early diagnosis of hypophosphatasia. Although there are no known fundamental treatments or effective dental approaches to prevent early exfoliation of primary teeth in affected patients, several possible treatments have recently been described, including gene therapy. Gene therapy has also been applied to TNSALP knockout mice (Alpl-/- ), which phenocopy the infantile form of hypophosphatasia, and improved their systemic condition. In the present study, we investigated whether gene therapy improved the dental condition of Alpl-/- mice. MATERIAL AND METHODS: Following sublethal irradiation (4 Gy) at the age of 2 d, Alpl-/- mice underwent gene therapy using bone marrow cells transduced with a lentiviral vector expressing a bone-targeted form of TNSALP injected into the jugular vein (n = 3). Wild-type (Alpl+/+ ), heterozygous mice (Alpl+/- ) and Alpl-/- mice were analyzed at 9 d of age (n = 3 of each), while Alpl+/+ mice and treated or untreated Alpl-/- mice were analyzed at 1 mo of age (n = 3 of each), and Alpl+/- mice and Alpl-/- mice with gene therapy were analyzed at 3 mo of age (n = 3 of each). A single mandibular hemi-section obtained at 1 mo of age was analyzed using a small animal computed tomography machine to assess alveolar bone formation. Other mandibular hemi-sections obtained at 9 d, 1 mo and 3 mo of age were subjected to hematoxylin and eosin staining and immunohistochemical analysis of osteopontin, a marker of cementum. RESULTS: Immunohistochemical analysis of osteopontin, a marker of acellular cementum, revealed that Alpl-/- mice displayed impaired formation of cementum and alveolar bone, similar to the human dental phenotype. Cementum formation was clearly present in Alpl-/- mice that underwent gene therapy, but did not recover to the same level as that in wild-type (Alpl+/+ ) mice. Micro-computed tomography examination showed that gene therapy improved alveolar bone mineral density in Alpl-/- mice to a similar level to that in Alpl+/+ mice. CONCLUSIONS: Our results suggest that gene therapy can improve the general condition of Alpl-/- mice, and induce significant alveolar bone formation and moderate improvement of cementum formation, which may contribute to inhibition of early spontaneous tooth exfoliation.
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Terapia Genética/métodos , Hipofosfatemia/terapia , Esfoliação de Dente/etiologia , Fosfatase Alcalina/genética , Processo Alveolar/patologia , Animais , Densidade Óssea , Cemento Dentário/patologia , Modelos Animais de Doenças , Hipofosfatemia/complicações , Camundongos , Camundongos Knockout , Esfoliação de Dente/terapia , Resultado do TratamentoRESUMO
Semiconducting single-walled carbon nanotubes (s-SWNTs) were extracted from SWNT mixtures using a flavin derivative (FC12). We evaluated the thermoelectric properties of the s-SWNT sheets. Electrical conductivity, power factor and figure of merit values of the sheets were increased by two orders of magnitude after removing FC12 simply by dipping in dichloromethane.
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Mandibuloacral dysplasia (MAD) is a rare autosomal recessive progeroid syndrome, characterized by mandibular hypoplasia, acroosteolysis affecting distal phalanges and clavicles, delayed closure of the cranial sutures, atrophic skin, and lipodystrophy. Recently, mutations in lamin A/C (LMNA) and zinc metalloprotease (ZMPSTE24), involved in post-translational processing of prelamin A to mature lamin A, have been identified in MAD kindreds. We now report novel compound heterozygous mutations in exon 1 (c.121C>T; p.Q41X) and exon 6 (c.743C>T; p.P248L) in ZMPSTE24 in two Japanese sisters, 7- and 3-year old, with severe MAD and characteristic facies and atrophic skin. The older sister had lipodystrophy affecting the chest and thighs but sparing abdomen. Their parents and a brother, who were healthy, had heterozygous mutations. The missense mutation, P248L, was not found in 100 normal subjects of Japanese origin. The mutant Q41X was inactive in a yeast halo assay; however, the mutant P248L retained near normal ZMPSTE24 activity. Immunoblots demonstrated accumulation of prelamin A in the patients' cell lysates from lymphoblasts. The lymphoblasts from the patients also revealed less intense staining for lamin A/C on immunofluorescence. We conclude that ZMPSTE24 deficiency results in accumulation of farnesylated prelamin A, which may be responsible for cellular toxicity and the MAD phenotype.
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Anormalidades Múltiplas/genética , Mandíbula/anormalidades , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Mutação de Sentido Incorreto , Povo Asiático , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Genótipo , Humanos , Lamina Tipo A , Lipodistrofia/genética , Proteínas de Membrana/deficiência , Metaloendopeptidases/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Prenilação , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , IrmãosRESUMO
Craniometaphyseal dysplasia (CMD) is a rare craniotubular bone dysplasia transmitted in autosomal dominant or recessive form. This disease is characterized by cranial bone hyperostosis and deformity of the metaphyses of the long bones. Using osteoclast-like cells formed from patient bone marrow cells, we investigated the pathophysiology of CMD in a 3-yr-old patient. Untreated bone marrow cells from the patient differentiated into osteoclast-like cells in vitro. These cells were shown to have vitronectin beta-receptors using a specific monoclonal antibody, i.e., 23C6 (CD51), which reacts with osteoclasts in human bone biopsy samples. However, the number of these osteoclast-like cells formed from the patient's bone marrow was only 40% of the normal controls. 1,25-dihydroxyvitamin-D3, bovine 1-34 parathyroid hormone, recombinant human interleukin-1 beta, recombinant human interleukin-6, or recombinant human macrophage colony-stimulating factor significantly increased, while salmon calcitonin significantly inhibited, the number of osteoclast-like cells. However, these cells could not resorb sperm whale dentin slices and lacked the osteoclast-reactive vacuolar proton pump as evidenced by a monoclonal antibody (E11). Western blot analysis using a monoclonal antibody to pp60c-src (327) revealed that protooncogene c-src expression by the platelets of the CMD patient was comparable to the normal control. These data suggest that: (a) the hyperostosis and the metaphyseal long bone deformity in the present CMD patient might be explained by osteoclast dysfunction due to impaired expression of the osteoclast-reactive vacuolar proton pump; and (b) a protooncogene c-src was not associated with the pathogenesis of the present CMD patient.
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Doenças do Desenvolvimento Ósseo/fisiopatologia , Medula Óssea/patologia , Osteoclastos/patologia , Bombas de Próton/genética , Crânio/patologia , Plaquetas/fisiologia , Western Blotting , Doenças do Desenvolvimento Ósseo/diagnóstico por imagem , Doenças do Desenvolvimento Ósseo/genética , Doenças do Desenvolvimento Ósseo/patologia , Medula Óssea/metabolismo , Reabsorção Óssea , Osso e Ossos/metabolismo , Calcitonina/farmacologia , Calcitriol/farmacologia , Pré-Escolar , Genes src , Humanos , Interleucina-1/farmacologia , Interleucina-6/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Masculino , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas pp60(c-src)/sangue , Radiografia , Proteínas Recombinantes/farmacologia , Crânio/diagnóstico por imagem , Teriparatida , Vacúolos/metabolismoRESUMO
McCune-Albright syndrome (MAS) is characterized by café-au-lait spot, multiple endocrine hyperfunction, and polyostotic fibrous dysplasia. A somatic point mutation of Gsalpha protein was reported to decrease GTPase activity, leading to increase in the GSalpha-associated hormone actions via cAMP. IL-6 is known to stimulate osteoclast formation and in the IL-6 promoter, a cAMP responsive element has been identified. In this paper, we investigated the role of IL-6 in the bone lesions of MAS, using the isolated fibrous cells from the polyostotic fibrous dysplasia tissues in bones of the two patients with MAS. Bone biopsy specimen revealed the increased osteoclast in number. In both patients, a GSalpha mutation (Arg201 -> His) was identified in the cultured fibrous cells. Intracellular cAMP content and IL-6 secretion by the patient cells were increased. Rp-8Br-cAMP significantly inhibited IL-6 production in the patient cells, while it had no effect on normal control. The addition of dibutyryl cAMP significantly increased the synthesis of IL-6 in normal control cells. In contrast, no effect of dibutyryl cAMP on IL-6 synthesis was observed in the cells from one of the MAS patients. These data suggest that IL-6 is, at least, one of the downstream effectors of cAMP and that the increased IL-6 synthesis has a pathogenic role in the bone lesions of MAS patients via increasing the number of osteoclasts. These results may provide a new strategy for the therapy of MAS patients.
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Osso e Ossos/metabolismo , Displasia Fibrosa Poliostótica/metabolismo , Proteínas de Ligação ao GTP/genética , Interleucina-6/biossíntese , Mutação Puntual , Sequência de Bases , Osso e Ossos/anatomia & histologia , Células Cultivadas , Criança , AMP Cíclico/análise , Feminino , Displasia Fibrosa Poliostótica/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Humanos , Lactente , Interleucina-11/biossíntese , Masculino , Dados de Sequência MolecularRESUMO
This report describes a male patient who presented with symptoms suggestive of spinocerebellar degeneration and who died of respiratory failure at the age of 7 years but was diagnosed, at autopsy, as having neuronal intranuclear hyaline inclusion disease. Neuronal intranuclear hyaline inclusion disease is a progressive and degenerative disease; diagnosis is possible only by neuropathological analysis. This is a rare disorder; few cases with early childhood onset and rapidly progressive neurologic symptoms have been documented. According to previous reports, most neurons in the central nervous system exhibited intranuclear eosinophilic inclusion bodies; neuronal depletion appeared to be restricted to the cerebellar cortex and the medullary inferior olivary nuclei, consistent with the fact that clinical deficit appears to correspond to the site of neuronal depletion and not to where eosinophilic bodies are detected. Immunohistochemical analysis revealed that these inclusions were positive for ubiquitin. The case presented herein clearly indicates that neuronal intranuclear hyaline inclusion disease should be considered as a differential diagnosis of cases involving spinocerebellar degeneration with childhood onset.
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Hialina/metabolismo , Corpos de Inclusão Intranuclear/metabolismo , Corpos de Inclusão Intranuclear/patologia , Degenerações Espinocerebelares/metabolismo , Criança , Progressão da Doença , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Humanos , Corpos de Inclusão Intranuclear/ultraestrutura , Imageamento por Ressonância Magnética/métodos , Masculino , Microscopia Eletrônica de Transmissão , Degenerações Espinocerebelares/patologia , Degenerações Espinocerebelares/fisiopatologiaRESUMO
Neuroblastoma originates from neural crest cells and is the most common extracranial solid tumor in childhood. Bone metastasis in neuroblastoma is an unfavorable prognostic factor even with intensive therapy. In the present study, we screened four cell lines of human neuroblastoma (NB-1, NB-16, NB-19, and NH-6) for tumorigenicity and metastatic capacity in nude mice and found that NB-19 cells caused osteolytic lesions after s.c. injection into mice. To detect micrometastases in the host tissue, we performed two kinds of PCR-based metastasis assays: (a) genomic PCR assay using the primers for human genome-specific Alu sequence; and (b) reverse transcription-nested PCR assay that detects the expression of tyrosine hydroxylase, a marker specific for neuroblastoma. The results of these PCR assays revealed the colonization of human neuroblastoma cells in the bone marrow of the mice that had received the s.c. injection of NB-19 cells. Because osteoclastic bone resorption has been reported to play important roles in osteolysis in some cancers such as breast cancer, we next examined the osteoclast (OC)-inducing activity of NB-19 cells using a coculture system in which NB-19 cells were cultured with murine bone marrow cells containing OC precursors and stromal cells. NB-19 cells induced tartrate-resistant acid phosphatase-positive multinucleated OC-like cells without requirement of 1,25-dihydroxyvitamin D3 or other osteoclastogenic stimulators. To investigate the factors involved in the osteoclastogenesis in the coculture of mouse marrow cells and NB-19 cells, we performed reverse transcription-PCR analysis and revealed the increased expression of receptor activator of nuclear factor kappaB ligand (RANKL) in the coculture compared with the culture of bone marrow cells alone. Interleukin-1alpha and cyclooxygenase-2 expression in the murine marrow cells was also increased in the presence of NB-19 cells. To further study the role of RANKL in the OC-like cell formation in the coculture of NB-19 cells and murine marrow cells, an expression vector encoding the active portion of the murine osteoprotegerin, which is the native inhibitor of RANKL action, was constructed and introduced into COS-7 cells. The conditioned media of the COS-7 cells transfected with the osteoprotegerin expression vector effectively blocked OC-like cell formation in the coculture of the bone marrow cells and NB-19 cells. These results suggested that in the bone microenvironment of NB-19-bearing mice, the stimulated expression of RANKL plays an important role in OC formation, leading to osteolytic bone metastasis.
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Neoplasias Ósseas/secundário , Proteínas de Transporte/biossíntese , Glicoproteínas de Membrana/biossíntese , Neuroblastoma/secundário , Osteoclastos/patologia , Fosfatase Ácida/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Neoplasias Ósseas/metabolismo , Células COS , Proteínas de Transporte/farmacologia , Comunicação Celular/fisiologia , Técnicas de Cocultura , Ciclo-Oxigenase 2 , Feminino , Glicoproteínas/farmacologia , Humanos , Interleucina-1/biossíntese , Isoenzimas/biossíntese , Isoenzimas/metabolismo , Glicoproteínas de Membrana/farmacologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoprotegerina , Prostaglandina-Endoperóxido Sintases/biossíntese , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Receptores Citoplasmáticos e Nucleares , Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fosfatase Ácida Resistente a Tartarato , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Loss of fibronectin (FN) from the cell surface has been shown to be closely associated with malignant transformation of cells. To elucidate the role of the FN matrix in the modulation of malignant phenotypes, we overexpressed a full-length cDNA encoding plasma-type FN in HT1080 human fibrosarcoma cells. The cells overexpressing FN adopted a more flattened morphology and deposited a moderately developed FN matrix both in vitro and in vivo, although the level of expression of integrin alpha5beta1 remained unchanged. FN-overexpressing cells exhibited a reduced cell motility on the substratum and grew poorly when injected s.c. into nude mice. Overexpression of FN also suppressed the ability of the tumor cells to proliferate in soft agar, whereas the suppression was reversed by inclusion in soft agar of the Arg-Gly-Asp (RGD)-containing peptide and adhesion-blocking antibodies against the central cell-binding domain of FN. Neither cell motility nor growth potential was altered by overexpression of a truncated form of FN lacking the central cell-binding domain. These results, taken together, indicate that increased deposition of FN in the pericellular matrix per se can suppress the motility and growth potential of tumor cells through interaction with RGD-recognizing integrins, most likely alpha5beta1.
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Fibronectinas/fisiologia , Fibrossarcoma/patologia , Animais , Adesão Celular , Divisão Celular , DNA Complementar/genética , Feminino , Fibronectinas/biossíntese , Fibronectinas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Fenótipo , Receptores de Fibronectina/biossíntese , Receptores de Fibronectina/genética , Proteínas Recombinantes de Fusão/biossíntese , Transfecção , Ensaio Tumoral de Célula-TroncoRESUMO
Galactocerebrosidase (GALC; EC 3.2.1.46) is a lysosomal enzyme which hydrolyzes several galactolipids and the deficiency of GALC is responsible for Krabbe disease. Recently, we cloned cDNAs for human and murine GALC. In this study we characterized the genomic organization and the promoter of the human gene. The gene was about 60 kb in length and consisted of 17 exons as reported by Luzi et al. DNA sequence analysis showed that the 5'-flanking region of the first exon was GC-rich and had not typical TATA-box but ten GC-box-like sequences within a 200 bp sequence upstream from the initiation codon. Another inframe ATG, which has better Kozak consensus sequence, was found at 48 bp upstream to the first ATG reported]. Promoter analysis using a luciferase assay in COS 7 cells showed that the -149 to -112 nucleotide (from the initiation codon A) region has dominant promoter activity. In this region three GC-box-like sequence and one YY1 binding site were detected. Primer extension revealed several transcription start sites within the region of -146 to -103 nucleotide. In this study we firstly demonstrated that the YY1 binding site and subsequent GC-box-like sequences could be a promoter in a housekeeping gene.
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Galactosilceramidase/genética , Genes/genética , Regiões Promotoras Genéticas/genética , Animais , Sequência de Bases , Sítios de Ligação , Células COS , Clonagem Molecular , Proteínas de Ligação a DNA , Fatores de Ligação de DNA Eritroide Específicos , Éxons/genética , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão , Análise de Sequência de DNA , Fatores de Transcrição , Transcrição Gênica/genética , Fator de Transcrição YY1Assuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Craniofaciais/diagnóstico , Cardiopatias Congênitas/diagnóstico , Anormalidades da Pele/diagnóstico , Adolescente , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/enzimologia , Humanos , MAP Quinase Quinase 2/genética , Masculino , Mutação , Fosforilação , SíndromeRESUMO
BACKGROUND: 1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) has been shown to modulate not only proliferation and differentiation, but also apoptosis in malignant cells, indicating that it could be useful for treating cancer. Little information is available concerning the structural motifs of the 1alpha, 25(OH)(2)D(3) molecule responsible for modulation of differentiation and apoptosis, however. We set out to synthesize singly dehydroxylated A-ring analogs of 19-nor-1alpha,25(OH)(2)D(3) in a catalytic asymmetric fashion, and to investigate their biological activities in leukemia HL-60 cells. RESULTS: A series of singly dehydroxylated 19-nor-1alpha,25-dihydroxyvitamin D(3) A-ring analogs were synthesized using a combinatiorial sequence of regioselective propiolate-ene reaction and catalytic asymmetric carbonyl-ene cyclization. Surprisingly, the analogs could be clearly divided into two categories; one group, bearing 1alpha-hydroxy or 3beta-hydroxy groups in the A-ring, were potent differentiators and the second group, bearing 1beta-hydroxy or 3alpha-hydroxy groups, were potent stimulators of apoptosis. CONCLUSIONS: We have clearly identified the structural motifs of 19-nor-1alpha,25(OH)(2)D(3) analogs responsible for differentiation and apoptosis in HL-60 cells. These findings will provide useful information not only for development of therapeutic agents for treatment of leukemia and other cancers, but also for structure-function studies of 1alpha,25(OH)(2)D(3).
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Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Calcitriol/análogos & derivados , Diferenciação Celular/efeitos dos fármacos , Antineoplásicos/química , Calcitriol/síntese química , Calcitriol/química , Calcitriol/farmacologia , Ciclo Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Células HL-60 , Humanos , Estereoisomerismo , Relação Estrutura-Atividade , Timidina/metabolismoRESUMO
The interaction of the vitamin D receptor with a vitamin D-responsive element (VDRE) derived from the human osteocalcin promoter in vitro has been shown to require a nuclear accessory factor (NAF) derived from monkey kidney cells. In this report we show that this factor is widely distributed in cells and tissues, including those that do not express the vitamin D receptor (VDR). NAF is required for VDR binding to a variety of known VDREs. VDR and NAF independently bind the VDRE weakly, as assessed by elution profiles generated during VDRE affinity chromatography. Together, however, both proteins coelute from this column with a profile that indicates a tighter strength of interaction. Analogous chromatography of the VDR derived from ROS 17/2.8 cells treated with 1,25-dihydroxyvitamin D3 in culture also reveals a dual profile of weak and strong binding, suggesting that in vivo modifications are unlikely to alter receptor DNA binding. NAF is a protein of 55 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and cross-linking experiments suggest that the VDR and NAF together form a heterodimer on a single VDRE with a mol wt of 103 kDa. These data demonstrate that NAF is required for VDR binding to specific DNA in vitro and suggest the possibility that NAF may be required for the transactivation capability of the VDR in vivo.
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Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Animais , Sequência de Bases , Calcitriol/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Cromatografia de Afinidade , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Humanos , Camundongos , Dados de Sequência Molecular , Peso Molecular , Proteínas Nucleares/isolamento & purificação , Oligodesoxirribonucleotídeos , Osteocalcina/genética , Regiões Promotoras Genéticas , Receptores de Calcitriol , Receptores de Esteroides/genética , Receptores de Esteroides/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TransfecçãoRESUMO
The purpose of this article has been to describe recent evidence that supports the idea(62) that 1,25-(OH)2D3 acts mechanistically like that of other steroid hormones. This evidence includes the finding that a clear structural interrelationship exists between the VDR and other members of the steroid receptor gene family, the observation that the VDR is required for gene promoter transactivation, and the identification of VDREs that act in cis to mediate 1,25-(OH)2D3 response. The VDR has been found to bind in vitro specifically to these functional DNA sites. Current evidence, however, indicates that the receptor may interact at these sites not as a monomer or homodimer but rather as a heterodimer with a protein whose identity remains unknown. Future studies with regard to the mechanism of vitamin D action must be aimed at gaining additional insight into the nature of VDREs, acquiring further detail about the interaction of the VDR with these elements, identifying factors that facilitate VDR DNA binding, and determining the biochemical mechanism by which the binding of receptor to these elements leads to modulation of common transcriptional machinery. In addition, 1,25-(OH)2D3 acts to suppress a number of genes, for example collagen, calcitonin, and parathyroid hormone. Efforts to elucidate these actions are currently underway, but the mechanism by which attenuation of response occurs remains largely uncharacterized. Finally, it is possible that additional mechanisms of vitamin D action may exist. Each of these areas offers a considerable challenge to future research.
Assuntos
Calcitriol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Esteroides/metabolismo , Sequências Reguladoras de Ácido Nucleico , Animais , Sequência de Bases , Sítios de Ligação , Calcitriol/genética , Calcitriol/metabolismo , DNA/metabolismo , Humanos , Dados de Sequência Molecular , Osteocalcina/química , Osteocalcina/genética , Receptores de Calcitriol , Receptores de Esteroides/química , Receptores de Esteroides/genética , Transcrição GênicaRESUMO
1 alpha-25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], together with vitamin D receptor (VDR), directly activates human osteocalcin (hOC) gene expression through a vitamin D-responsive element (VDRE) located in the promoter of the hOC gene. We investigated the effect of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] on the regulation of the hOC gene promoter and compared it with that of 1 alpha,25(OH)2D3. 24R,25(OH)2D3 did not activate the natural promoter in VDR-negative CV-1 cells. 24R,25(OH)2D3, however, induced the activation of this promoter following cotransfection with an hVDR expression vector. In VDR-positive MC3T3-E1 cells, 24R,25(OH)2D3 activated not only the natural hOC promoter but also a chimeric promoter composed of a synthetic hOC VDRE sequence linked to the thymidine kinase promoter. In combination with 1 alpha-25(OH)2D3, 24R,25(OH)2D3 did not exhibit any antagonist activity on the hOC promoter. These results suggest that under conditions of high 24R,25(OH)2D3 levels in vivo, this metabolite of vitamin D3 may activate hOC gene expression through receptor mechanisms identical to that for 1 alpha,25(OH)2D3.
Assuntos
Colecalciferol/farmacologia , Osteoblastos/metabolismo , Osteocalcina/biossíntese , Regiões Promotoras Genéticas/genética , Receptores de Calcitriol/genética , Animais , Sequência de Bases , Calcitriol/farmacologia , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Osteoblastos/efeitos dos fármacos , Osteocalcina/efeitos dos fármacos , Osteocalcina/genética , Plasmídeos , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Calcitriol/efeitos dos fármacos , TransfecçãoRESUMO
Chondromodulin-1 (ChM-1) is a cartilage-specific glycoprotein that stimulates the growth of chondrocytes and inhibits the tube formation of endothelial cells. To clarify the tissue-specific expression and the role of ChM-1 in pathophysiological conditions, we analyzed the structure of the human ChM-1 gene and its promoter. On the screening of a human genomic cosmid library using the human ChM-1 complimentary DNA (cDNA) as a probe, two clones were obtained that contained ChM-1 cDNA. The restriction enzyme map and nucleotide sequence revealed the human ChM-1 gene consisting of seven exons and exon-intron boundaries. The human ChM-1 gene was assigned to chromosome 13q14-21 by fluorescence in situ hybridization (FISH) using the clone as a probe. A primer extension analysis using total RNA extracted from human cartilage revealed a major transcription start site with the sequence CGCT+1GG. The region approximately 3-kilobase (kb) nucleotides upstream of the translation start site was then sequenced and analyzed in terms of promoter activity. We found that a region 446 base pairs (bp) upstream of the start site had promoter activity in COS7, HeLa, and ATDC5 cells. In structure the promoter is a TATA-less type without a GC-rich region. The transcription factors Sox9, Og12, and Cart-1 did not affect the promoter activity. The transcription factor Ying-Yang1 suppressed the promoter activity but GABP protein did not change the promoter activity. The construct containing -446/+87 fused to the SV40 enhancer and green fluorescent protein (GFP) exhibited expression of GFP corresponding to the differentiation of ATDC5 cells to mature chondrocytes. These results suggest that the element -446/+87 confers the cartilage-specific expression of this gene by some factor(s) other than Sox9, Og12, and Cart-1.
Assuntos
Cartilagem/metabolismo , Substâncias de Crescimento/genética , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Membrana , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Células COS , Cartilagem/citologia , Linhagem Celular , Chlorocebus aethiops , Cromossomos Humanos Par 13/genética , Cosmídeos , DNA Complementar/genética , Éxons/genética , Regulação da Expressão Gênica , Biblioteca Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Íntrons/genética , Luciferases/biossíntese , Luciferases/genética , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Polimorfismo de Fragmento de Restrição , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição/fisiologia , Transcrição Gênica , TransfecçãoRESUMO
To elucidate the regulatory mechanism of vitamin D action on insulin biosynthesis and secretion, we examined preproinsulin (ppI) mRNA levels in the pancreas of normal rats, vitamin D-deficient rats, and rats supplemented with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or calcium (Ca) for 3 days. The ppI mRNA levels determined by dot blot analysis in vitamin D-deficient, 1,25-(OH)2D3-replete, and Ca-replete rats were 39.1%, 68.7%, and 66.7%, respectively, of values in normal rats. These results concur with previously reported levels of insulin secretion in the perfused rat pancreas. The reduced level of ppI mRNA should lead to a decrease in insulin biosynthesis and, thus, impair insulin secretion in vitamin D-deficient rats. The observed partial recovery of ppI mRNA levels through supplementation of 1,25-(OH)2D3 or Ca may be one mechanism by which insulin secretion is restored in rats after 1,25-(OH)2D3 or Ca repletion. We examined further the time course of ppI mRNA accumulation in rats after a single administration of 1,25-(OH)2D3. When fasting was continued for an additional 24-h period after an overnight fast, ppI mRNA levels were not changed significantly in either vitamin D-deficient or replete rats. However, in the rats that were pair-fed after overnight fasting, ppI mRNA levels in 1,25-(OH)2D3-replete rats increased at 8 and 24 h, whereas ppI mRNA in vitamin D-deficient rats increased only at 24 h. Moreover, the increment at 24 h was significantly larger in 1,25-(OH)2D3-replete rats than in vitamin D-deficient rats. We conclude that 1,25-(OH)2D3 enhances steady state levels of ppI mRNA only under conditions of refeeding and during feeding.