Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros








Base de dados
Intervalo de ano de publicação
1.
Bone ; 183: 117085, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38522809

RESUMO

Overgrowth and intellectual disability disorders in humans are typified by length/height and/or head circumference ≥ 2 standard deviations above the mean as well as intellectual disability and behavioral comorbidities, including autism and anxiety. Tatton-Brown-Rahman Syndrome is one type of overgrowth and intellectual disability disorder caused by heterozygous missense mutations in the DNA methyltransferase 3A (DNMT3A) gene. Numerous DNMT3A mutations have been identified in Tatton-Brown-Rahman Syndrome patients and may be associated with varying phenotype severities of clinical presentation. Two such mutations are the R882H and P904L mutations which result in severe and mild phenotypes, respectively. Mice with paralogous mutations (Dnmt3aP900L/+ and Dnmt3aR878H/+) exhibit overgrowth in their long bones (e.g., femur, humerus), but the mechanisms responsible for their skeletal overgrowth remain unknown. The goal of this study is to characterize skeletal phenotypes in mouse models of Tatton-Brown-Rahman Syndrome and identify potential cellular mechanisms involved in the skeletal overgrowth phenotype. We report that mature mice with the Dnmt3aP900L/+ or Dnmt3aR878H/+ mutation exhibit tibial overgrowth, cortical bone thinning, and weakened bone mechanical properties. Dnmt3aR878H/+ mutants also contain larger bone marrow adipocytes while Dnmt3aP900L/+ mutants show no adipocyte phenotype compared to control animals. To understand the potential cellular mechanisms regulating these phenotypes, growth plate chondrocytes, osteoblasts, and osteoclasts were assessed in juvenile mutant mice using quantitative static histomorphometry and dynamic histomorphometry. Tibial growth plates appeared thicker in mutant juvenile mice, but no changes were observed in osteoblast activity or osteoclast number in the femoral mid-diaphysis. These studies reveal new skeletal phenotypes associated with Tatton-Brown-Rahman Syndrome in mice and provide a rationale to extend clinical assessments of patients with this condition to include bone density and quality testing. These findings may be also informative for skeletal characterization of other mouse models presenting with overgrowth and intellectual disability phenotypes.


Assuntos
Anormalidades Múltiplas , Deficiência Intelectual , Anormalidades Musculoesqueléticas , Humanos , Animais , Camundongos , DNA (Citosina-5-)-Metiltransferases/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , DNA Metiltransferase 3A , Anormalidades Múltiplas/genética , Mutação
2.
Cell Rep ; 42(11): 113411, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-37952155

RESUMO

Phenotypic heterogeneity in monogenic neurodevelopmental disorders can arise from differential severity of variants underlying disease, but how distinct alleles drive variable disease presentation is not well understood. Here, we investigate missense mutations in DNA methyltransferase 3A (DNMT3A), a DNA methyltransferase associated with overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity. We generate a Dnmt3aP900L/+ mouse mimicking a mutation with mild to moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. P900L mutants exhibit core growth and behavioral phenotypes shared across models but show subtle epigenomic changes, while R878H mutants display extensive disruptions. We identify mutation-specific dysregulated genes that may contribute to variable disease severity. Shared transcriptomic disruption identified across mutations overlaps dysregulation observed in other developmental disorder models and likely drives common phenotypes. Together, our findings define central drivers of DNMT3A disorders and illustrate how variable epigenomic disruption contributes to phenotypic heterogeneity in neurodevelopmental disease.


Assuntos
DNA (Citosina-5-)-Metiltransferases , DNA Metiltransferase 3A , Animais , Camundongos , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Epigênese Genética , Epigenômica , Mutação/genética
3.
Am J Physiol Cell Physiol ; 325(6): C1387-C1400, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37842749

RESUMO

Noncoding microRNAs are powerful epigenetic regulators of cellular processes by their ability to target and suppress expression of numerous protein-coding mRNAs. This multitargeting function is a unique and complex feature of microRNAs. It is now well-described that microRNAs play important roles in regulating the development and homeostasis of many cell/tissue types, including those that make up the skeletal system. In this review, we focus on microRNA-138 (miR-138) and its effects on regulating bone and cartilage cell differentiation and function. In addition to its reported role as a tumor suppressor, miR-138 appears to function as an inhibitor of osteoblast differentiation. This review provides additional information on studies that have attempted to alter miR-138 expression in vivo as a means to dampen ectopic calcification or alter bone mass. However, a review of the published literature on miR-138 in cartilage reveals a number of contradictory and inconclusive findings with respect to regulating chondrogenesis and chondrocyte catabolism. This highlights the need for more research in understanding the role of miR-138 in cartilage biology and disease. Interestingly, a number of studies in other systems have reported miR-138-mediated effects in dampening inflammation and pain responses. Future studies will reveal if a multifunctional role of miR-138 involving suppression of ectopic bone, inflammation, and pain will be beneficial in skeletal conditions such as osteoarthritis and heterotopic ossification.


Assuntos
MicroRNAs , Humanos , MicroRNAs/metabolismo , Cartilagem/metabolismo , Condrócitos/metabolismo , Diferenciação Celular/genética , Homeostase/genética , Inflamação/metabolismo , Dor/metabolismo
4.
bioRxiv ; 2023 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-36909558

RESUMO

Phenotypic heterogeneity is a common feature of monogenic neurodevelopmental disorders that can arise from differential severity of missense variants underlying disease, but how distinct alleles impact molecular mechanisms to drive variable disease presentation is not well understood. Here, we investigate missense mutations in the DNA methyltransferase DNMT3A associated with variable overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity in neurodevelopmental disease. We generate a DNMT3A P900L/+ mouse model mimicking a disease mutation with mild-to-moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. We show that the P900L mutation leads to disease-relevant overgrowth, obesity, and social deficits shared across DNMT3A disorder models, while the R878H mutation causes more extensive epigenomic disruption leading to differential dysregulation of enhancers elements. We identify distinct gene sets disrupted in each mutant which may contribute to mild or severe disease, and detect shared transcriptomic disruption that likely drives common phenotypes across affected individuals. Finally, we demonstrate that core gene dysregulation detected in DNMT3A mutant mice overlaps effects in other developmental disorder models, highlighting the importance of DNMT3A-deposited methylation in neurodevelopment. Together, these findings define central drivers of DNMT3A disorders and illustrate how variable disruption of transcriptional mechanisms can drive the spectrum of phenotypes in neurodevelopmental disease.

5.
J Cell Physiol ; 238(4): 698-713, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36780342

RESUMO

MicroRNAs (miRNAs) are epigenetic regulators that can target and inhibit translation of multiple mRNAs within a given cell type. As such, a number of different pathways and networks may be modulated as a result. In fact, miRNAs are known to regulate many cellular processes including differentiation, proliferation, inflammation, and metabolism. This review focuses on the miR-181 family and provides information from the published literature on the role of miR-181 homologs in regulating a range of activities in different cell types and tissues. Of note, we have not included details on miR-181 expression and function in the context of cancer since this is a broad topic area requiring independent review. Instead, we have focused on describing the function and mechanism of miR-181 family members on differentiation toward a number of cell lineages in various non-neoplastic conditions (e.g., immune/hematopoietic cells, osteoblasts, osteoclasts, chondrocytes, adipocytes). We have also provided information on how modulation of miR-181 homologs can have positive effects on disease states such as cardiac abnormalities, pulmonary arterial hypertension, thrombosis, osteoarthritis, and vascular inflammation. In this context, we have used some examples of FDA-approved drugs that modulate miR-181 expression. We conclude by discussing some common mechanisms by which miR-181 homologs appear to regulate a number of different cellular processes and how targeting specific miR-181 family members may lead to attractive therapeutic approaches to treat a number of human disease or repair conditions, including those associated with the aging process.


Assuntos
Diferenciação Celular , MicroRNAs , Humanos , Linhagem da Célula , Inflamação/metabolismo , Inflamação/patologia , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo
6.
Methods Mol Biol ; 2598: 197-215, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36355294

RESUMO

Since their discovery in 1993, microRNAs (miRNAs) are now recognized as important epigenetic regulators of many mammalian cellular processes including proliferation, apoptosis, metabolism, and differentiation. These small non-coding RNAs function by interacting with specific regions in the 3'-untranslated region of mRNAs, thereby resulting in mRNA degradation or suppression of translation. Since miRNAs have the ability to target many mRNAs within a given cell type, a number of cellular pathways and networks may be regulated as a result. To study the function of miRNAs, a number of methods can be used to modulate their activity in cells such as synthetic mimics or antagomirs for short-term assays or viral-based approaches for longer-term experiments such as cell differentiation assays. In this chapter, we provide our methodology to constitutively overexpress a desired miRNA during in vitro chondrogenesis of human cartilage progenitor cells (CPCs). Specifically, we describe how we obtain CPCs from human articular cartilage specimens, how we generate and titrate lentivirus engineered to overexpress a precursor miRNA, how we transduce CPCs with lentivirus and differentiate them toward the chondrocyte lineage, and how we extract RNA and measure expression levels of the miRNA of interest during in vitro chondrogenesis. We also provide some data from our laboratory demonstrating that we can achieve and maintain miRNA overexpression for up to 14 days in cartilage pellet cultures. We predict that these lentiviral-based approaches will also be useful to study how miRNA modulation of progenitor cells affects cell differentiation and extracellular matrix production within three-dimensional biomaterial scaffolds.


Assuntos
Cartilagem Articular , MicroRNAs , Animais , Humanos , Condrogênese/genética , Condrócitos/metabolismo , Diferenciação Celular/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , RNA Mensageiro/metabolismo , Mamíferos/genética
7.
J Bone Miner Res ; 36(11): 2243-2257, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34405443

RESUMO

Nonunion is defined as the permanent failure of a fractured bone to heal, often necessitating surgical intervention. Atrophic nonunions are a subtype that are particularly difficult to treat. Animal models of atrophic nonunion are available; however, these require surgical or radiation-induced trauma to disrupt periosteal healing. These methods are invasive and not representative of many clinical nonunions where osseous regeneration has been arrested by a "failure of biology". We hypothesized that arresting osteoblast cell proliferation after fracture would lead to atrophic nonunion in mice. Using mice that express a thymidine kinase (tk) "suicide gene" driven by the 3.6Col1a1 promoter (Col1-tk), proliferating osteoblast lineage cells can be ablated upon exposure to the nucleoside analog ganciclovir (GCV). Wild-type (WT; control) and Col1-tk littermates were subjected to a full femur fracture and intramedullary fixation at 12 weeks age. We confirmed abundant tk+ cells in fracture callus of Col-tk mice dosed with water or GCV, specifically many osteoblasts, osteocytes, and chondrocytes at the cartilage-bone interface. Histologically, we observed altered callus composition in Col1-tk mice at 2 and 3 weeks postfracture, with significantly less bone and more fibrous tissue. Col1-tk mice, monitored for 12 weeks with in vivo radiographs and micro-computed tomography (µCT) scans, had delayed bone bridging and reduced callus size. After euthanasia, ex vivo µCT and histology showed failed union with residual bone fragments and fibrous tissue in Col1-tk mice. Biomechanical testing showed a failure to recover torsional strength in Col1-tk mice, in contrast to WT. Our data indicates that suppression of proliferating osteoblast-lineage cells for at least 2 weeks after fracture blunts the formation and remodeling of a mineralized callus leading to a functional nonunion. We propose this as a new murine model of atrophic nonunion. © 2021 American Society for Bone and Mineral Research (ASBMR).


Assuntos
Fraturas do Fêmur , Consolidação da Fratura , Animais , Calo Ósseo/diagnóstico por imagem , Modelos Animais de Doenças , Fraturas do Fêmur/diagnóstico por imagem , Camundongos , Osteoblastos , Microtomografia por Raio-X
8.
Development ; 148(12)2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34143204

RESUMO

During retinal development, a large subset of progenitors upregulates the transcription factor Otx2, which is required for photoreceptor and bipolar cell formation. How these retinal progenitor cells initially activate Otx2 expression is unclear. To address this, we investigated the cis-regulatory network that controls Otx2 expression in mice. We identified a minimal enhancer element, DHS-4D, that drove expression in newly formed OTX2+ cells. CRISPR/Cas9-mediated deletion of DHS-4D reduced OTX2 expression, but this effect was diminished in postnatal development. Systematic mutagenesis of the enhancer revealed that three basic helix-loop-helix (bHLH) transcription factor-binding sites were required for its activity. Single cell RNA-sequencing of nascent Otx2+ cells identified the bHLH factors Ascl1 and Neurog2 as candidate regulators. CRISPR/Cas9 targeting of these factors showed that only the simultaneous loss of Ascl1 and Neurog2 prevented OTX2 expression. Our findings suggest that Ascl1 and Neurog2 act either redundantly or in a compensatory fashion to activate the DHS-4D enhancer and Otx2 expression. We observed redundancy or compensation at both the transcriptional and enhancer utilization levels, suggesting that the mechanisms governing Otx2 regulation in the retina are flexible and robust.


Assuntos
Sistema y+ de Transporte de Aminoácidos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Organogênese/genética , Fatores de Transcrição Otx/genética , Retina/metabolismo , Animais , Sequência de Bases , Elementos E-Box , Imunofluorescência , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Motivos de Nucleotídeos , Fatores de Transcrição Otx/metabolismo , Retina/embriologia
9.
Bone ; 143: 115760, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33220505

RESUMO

Epigenetic regulation is critical for proper bone development. Evidence from a large body of published literature informs us that microRNAs (miRNAs) are important epigenetic factors that control many aspects of bone development, homeostasis, and repair processes. These small non-coding RNAs function at the post-transcriptional level to suppress expression of specific target genes. Many target genes may be affected by one miRNA resulting in alteration in cellular pathways and networks. Therefore, changes in levels or activity of a specific miRNA (e.g. via genetic mutations, disease scenarios, or by over-expression or inhibition strategies in vitro or in vivo) can lead to substantial changes in cell processes including proliferation, metabolism, apoptosis and differentiation. In this review, Section 1 briefly covers general background information on processes that control bone development as well as the biogenesis and function of miRNAs. In Section 2, we discuss the importance of miRNAs in skeletal development based on findings from in vivo mouse models and human clinical reports. Section 3 focuses on describing more recent data from the last three years related to miRNA regulation of osteoblast differentiation in vitro. Some of these studies also involve utilization of an in vivo rodent model to study the effects of miRNA modulation in scenarios of osteoporosis, bone repair or ectopic bone formation. In Section 4, we provide some recent information from studies analyzing the potential of miRNA-mediated crosstalk in bone and how exosomes containing miRNAs from one bone cell may affect the differentiation or function of another bone cell type. We then conclude by summarizing where the field currently stands with respect to miRNA-mediated regulation of osteogenesis and how information gained from developmental processes can be instructive in identifying potential therapeutic miRNA targets for the treatment of certain bone conditions.


Assuntos
MicroRNAs , Animais , Desenvolvimento Ósseo/genética , Diferenciação Celular/genética , Epigênese Genética , Camundongos , MicroRNAs/genética , Osteogênese/genética
10.
Anal Chem ; 90(14): 8642-8650, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29932641

RESUMO

Melatonin is an important hormone whose functions span from regulating circadian rhythm in the brain to providing anti-inflammatory properties in the immune system. Melatonin secretion from the pineal gland is known; however, the mechanism of melatonin signaling in the immune system is not well understood. The lymph node is the hub of the immune system, and melatonin secretion from lymphocytes was proposed to be an important source specifically for regulating cytokine secretion. Methods exist to quantify the concentration of melatonin within biological samples; however, they often suffer from either a lack of selectivity for melatonin over common biological interferences or temporal resolution, which is not amenable to measuring real-time signaling dynamics. Here, we have characterized an electrochemical method for optimal melatonin detection with subsecond resolution using fast-scan cyclic voltammetry at carbon-fiber microelectrodes. The oxidation peaks detected for melatonin were at 1.0, 1.1, and 0.6 V. Evidence for electrode fouling of the tertiary peak was present; therefore, an optimized waveform was developed scanning from 0.2 to 1.3 V at 600 V/s. The optimized waveform eliminated the detection of fouling products on the electrode with a 24 ± 10 nM limit of detection. Melatonin was distinguished between biological interferences, and codetection with the major synthetic precursor, serotonin, was possible. This method was used to detect melatonin in live lymph node slices and provides the first real-time measurements within the lymph node using FSCV. Real-time detection of melatonin dynamics could provide useful information on the mechanism of immunomodulation during inflammatory disease.

11.
J Biomed Mater Res A ; 106(9): 2412-2423, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29673061

RESUMO

Intervertebral disk (IVD) degeneration is a multifactor process that results in the physical destruction of the nucleus pulposus (NP) and annulus fibrosus (AF). This compromises IVD function and causes significant disability and economic burden. Strategies to replace the entire composite structure of the IVD are limited and most approaches do not recapitulate the heterogenous biochemical composition, microarchitecture or mechanical properties of the native tissue. Our central hypothesis was that donor IVDs which resemble the size and biochemistry of human lumbar IVDs could be successfully decellularized while retaining the tissue's structure and function with the long-term goal of creating a composite scaffold for tissue engineering the human IVD. Accordingly, we optimized a procedure to decellularize bovine tail IVDs using a combination of detergents, ultrasonication, freeze-thaw cycles, and nucleases. The resultant decellularized whole IVD xenografts retained distinct AF and NP regions which contained no visible intact cell nuclei and minimal residual bovine deoxyribose nucleic acid (DNA; 65.98 ± 4.07 and 47.12 ± 13.22 ng/mg, respectively). Moreover, the NP region of decellularized IVDs contained 313.40 ± 50.67 µg/mg glycosaminoglycan. The presence of collagen type II was confirmed via immunohistochemistry. Additionally, histological analysis of the AF region of decellularized IVDs demonstrated retention of the native angle-ply collagen microarchitecture. Unconfined compression testing demonstrated no significant differences in swelling pressure and toe-region modulus between fresh and decellularized IVDs. However, linear region moduli, peak stress and equilibrium moduli were all significantly reduced. Together, this research demonstrates a successful initial step in developing a biomimetic acellular whole IVD xenograft scaffold for use in IVD tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2412-2423, 2018.


Assuntos
Xenoenxertos/fisiologia , Disco Intervertebral/fisiologia , Alicerces Teciduais/química , Animais , Anel Fibroso/fisiologia , Bovinos , Núcleo Celular/metabolismo , Colágeno/metabolismo , Força Compressiva , DNA/metabolismo , Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Processamento de Imagem Assistida por Computador , Núcleo Pulposo/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA