RESUMO
White matter degeneration in the central nervous system (CNS) has been correlated with a decline in cognitive function during aging. Ultrastructural examination of the aging human brain shows a loss of myelin, yet little is known about molecular and biochemical changes that lead to myelin degeneration. In this study, we investigate myelination across the lifespan in C57BL/6 mice using electron microscopy and Fourier transform infrared (FTIR) spectroscopic imaging to better understand the relationship between structural and biochemical changes in CNS white matter tracts. A decrease in the number of myelinated axons was associated with altered lipid profiles in the corpus callosum of aged mice. FTIR spectroscopic imaging revealed alterations in functional groups associated with phospholipids, including the lipid acyl, lipid ester and phosphate vibrations. Biochemical changes in white matter were observed prior to structural changes and most predominant in the anterior regions of the corpus callosum. This was supported by biochemical analysis of fatty acid composition that demonstrated an overall trend towards increased monounsaturated fatty acids and decreased polyunsaturated fatty acids with age. To further explore the molecular mechanisms underlying these biochemical alterations, gene expression profiles of lipid metabolism and oxidative stress pathways were investigated. A decrease in the expression of several genes involved in glutathione metabolism suggests that oxidative damage to lipids may contribute to age-related white matter degeneration.
Assuntos
Substância Branca , Envelhecimento/fisiologia , Animais , Encéfalo/metabolismo , Corpo Caloso/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina , Espectroscopia de Infravermelho com Transformada de Fourier , Substância Branca/metabolismoRESUMO
SIRT2 is a NAD+ -dependent deacetylase that deacetylates a diverse array of protein substrates and is involved in many cellular processes, including regulation of inflammation. However, its precise role in the inflammatory process has not completely been elucidated. Here, we identify heat-shock protein 90α (Hsp90α) as novel substrate of SIRT2. Functional investigation suggests that Hsp90 is deacetylated by SIRT2, such that overexpression and knock-down of SIRT2 altered the acetylation level of Hsp90. This subsequently resulted in disassociation of Hsp90 with glucocorticoid receptor (GR), and translocation of GR to the nucleus. This observation was further confirmed by glucocorticoid response element (GRE)-driven reporter assay. Nuclear translocation of GR induced by SIRT2 overexpression repressed the expression of inflammatory cytokines, which were even more prominent under lipopolysaccharide (LPS) stimulation. Conversely, SIRT2 knock-down resulted in the up-regulation of cytokine expression. Mutation analysis indicated that deacetylation of Hsp90 at K294 is critical for SIRT2-mediated regulation of cytokine expression. These data suggest that SIRT2 reduces the extent of LPS-induced inflammation by suppressing the expression of inflammatory factors via SIRT2-Hsp90-GR axis.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Inflamação/metabolismo , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais , Sirtuína 2/metabolismo , Acetilação/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Escherichia coli/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Modelos Biológicos , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Sirtuína 2/química , SolubilidadeRESUMO
BACKGROUND/AIMS: Runt-related transcription factor 2 (Runx2) is a master regulator of osteogenic differentiation, but most of the direct downstream targets of RUNX2 during osteogenesis are unknown. Likewise, High-temperature requirement factor A1 (HTRA1) is a serine protease expressed in bone, yet the role of Htra1 during osteoblast differentiation remains elusive. We investigated the role of Htra1 in osteogenic differentiation and the transcriptional regulation of Htra1 by RUNX2 in primary mouse mesenchymal progenitor cells. METHODS: Overexpression of Htra1 was carried out in primary mouse mesenchymal progenitor cells to evaluate the extent of osteoblast differentiation. Streptavidin agarose pulldown assay, chromatin immunoprecipitation assay, and dual luciferase assay were carried out to investigate the interaction of RUNX2 protein at the Htra1 promoter during osteoblast differentiation. RESULTS: Overexpression of Htra1 increased the production of mineralized bone matrix, upregulating several osteoblast genes, such as Sp7 transcription factor (Sp7) and Alkaline phosphatase, liver/bone/kidney (Alpl). In addition, Htra1 upregulated osteogenesis-related signalling genes, such as Fibroblast growth factor 9 (Fgf9) and Vascular endothelial growth factor A (Vegfa). A series of experiments confirmed Htra1 as a direct RUNX2 transcriptional target. Overexpression of Runx2 resulted in the upregulation of Htra1 mRNA and protein. Chromatin immunoprecipitation and streptavidin agarose pull-down assays showed that RUNX2 binds a proximal -400 bp region of the Htra1 promoter during osteogenic differentiation. Dual luciferase assays confirmed that RUNX2 activates the proximal Htra1 promoter during osteogenic differentiation. Mutation of putative RUNX2 binding sites revealed that RUNX2 interacts with the Htra1 promoter at -252 bp and -84 bp to induce Htra1 expression. CONCLUSION: We demonstrate that Htra1 is a positive regulator of osteogenic differentiation, showing for the first time that Htra1 is a direct downstream target of RUNX2.
Assuntos
Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fator 9 de Crescimento de Fibroblastos/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteogênese , Regiões Promotoras Genéticas , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Myelination is controlled by timely expression of genes involved in the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (OLs). Sirtuin 2 (SIRT2), a NAD+-dependent deacetylase, plays a critical role in OL differentiation by promoting both arborization and downstream expression of myelin-specific genes. However, the mechanisms involved in regulating SIRT2 expression during OL development are largely unknown. The RNA-binding protein quaking (QKI) plays an important role in myelination by post-transcriptionally regulating the expression of several myelin specific genes. In quaking viable (qkv/qkv ) mutant mice, SIRT2 protein is severely reduced; however, it is not known whether these genes interact to regulate OL differentiation. Here, we report for the first time that QKI directly binds to Sirt2 mRNA via a common quaking response element (QRE) located in the 3' untranslated region (UTR) to control SIRT2 expression in OL lineage cells. This interaction is associated with increased stability and longer half-lives of Sirt2.1 and Sirt2.2 transcripts leading to increased accumulation of Sirt2 transcripts. Consistent with this, overexpression of qkI promoted the expression of Sirt2 mRNA and protein. However, overexpression of the nuclear isoform qkI-5 promoted the expression of Sirt2 mRNA, but not SIRT2 protein, and delayed OL differentiation. These results suggest that the balance in the subcellular distribution and temporal expression of QKI isoforms control the availability of Sirt2 mRNA for translation. Collectively, our study demonstrates that QKI directly plays a crucial role in the post-transcriptional regulation and expression of Sirt2 to facilitate OL differentiation.
Assuntos
Diferenciação Celular , Oligodendroglia/citologia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/fisiologia , Sirtuína 2/genética , Animais , Regulação da Expressão Gênica , Camundongos , Ligação Proteica , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Elementos de RespostaRESUMO
Sirtuin2 (SIRT2) is a deacetylase enzyme predominantly expressed in myelinating glia of the central nervous system (CNS). We have previously demonstrated that Sirt2 expression enhances oligodendrocyte (OL) differentiation and arborization in vitro, but the molecular targets of SIRT2 in OLs remain speculative. SIRT2 has been implicated in cholesterol biosynthesis by promoting the nuclear translocation of sterol regulatory element binding protein (SREBP)-2. We investigated this further in CNS myelination by examining the role of Sirt2 in cholesterol biosynthesis in vivo and in vitro employing Sirt2 -/- mice, primary OL cells and CG4-OL cells. Our results demonstrate that expression of cholesterol biosynthetic genes in the CNS white matter or cholesterol content in purified myelin fractions did not differ between Sirt2 -/- and age-matched wild-type mice. Cholesterol biosynthetic gene expression profiles and total cholesterol content were not altered in primary OLs from Sirt2 -/- mice and in CG4-OLs when Sirt2 was either down-regulated with RNAi or overexpressed. In addition, Sirt2 knockdown or overexpression in CG4-OLs had no effect on SREBP-2 nuclear translocation. Our results indicate that Sirt2 does not impact the expression of genes encoding enzymes involved in cholesterol biosynthesis, total cholesterol content, or nuclear translocation of SREBP-2 during OL differentiation and myelination.
Assuntos
Diferenciação Celular/fisiologia , Colesterol/biossíntese , Neurogênese/fisiologia , Oligodendroglia/metabolismo , Sirtuína 2/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Colesterol/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Experimental models of multiple sclerosis (MS) have significantly advanced our understanding of pathophysiology and therapeutic interventions. Although in vivo rodent models are considered to most closely represent the complex cellular and molecular disease states of the human central nervous system (CNS), these can be costly to maintain and require long timelines. Organotypic slice cultures maintain the cytotypic organization observed in the intact CNS, yet provide many of the experimental advantages of in vitro cell culture models. Cerebellar organotypic cultures have proven useful for studying myelination and remyelination, but this model has only been established using early postnatal tissue. This young brain tissue allows for neuro development ex vivo to mimic the 'mature' CNS; however, there are many differences between postnatal and adult organotypic cultures. This may be particularly relevant to MS, as a major barrier to myelin regeneration is age. This paper describes a modified protocol to study demyelination and remyelination in adult cerebellar tissue, which has been used to demonstrate neuroprotection with omega-3 fatty acids. Thus, adult cerebellar organotypic cultures provide a novel ex vivo platform for screening potential therapies in myelin degeneration and repair.
Assuntos
Cerebelo/metabolismo , Cerebelo/patologia , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Remielinização/fisiologia , Adulto , Fatores Etários , Animais , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Cerebelo/citologia , Humanos , Bainha de Mielina/metabolismo , Técnicas de Cultura de ÓrgãosRESUMO
PTEN is a tumour suppressor that is frequently mutated in a variety of cancers. Hence, PTEN has significant potential as a therapeutic molecule. PTEN-long is an alternative translation variant, with an additional 173 amino acids added to the N-terminal of the canonical PTEN when CUG of the mRNA is utilized as the start codon. PTEN-long is secreted into serum and can re-enter cells throughout the body. One of the major barriers for gene therapy is to efficiently and specifically deliver DNA or RNA material to target cells. As an alternative approach, if a therapeutic protein can be directly delivered to target cell of interest, it should theoretically function well within the cells, particularly for genes that are deficiently expressed in vivo. Most therapeutic proteins are incapable of efficiently permeating the cell membrane. In this study, we have employed CRISPR/Cas9 gene editing tool combined with single-stranded template to edit CTG of PTEN-long to ATG in the genome. Two guide RNAs close to CTG site were found to have similar efficiency in driving PTEN-long expression. Furthermore, we detected PTEN-long expression in transfected whole-cell lysate and in concentrated culture media in Western blot. Interestingly, the culture media of PTEN-long expression can reduce Akt phosphorylation level and repress U87 cell proliferation compared to wild-type U87 or control media. Taken together, PTEN-long driven by CRISPR/Cas9 imports and exports cells and represses nearby cell proliferation, indicating the PTEN-long generated by CRISPR/Cas9 has potential to be an alternative strategy for PTEN gene therapy.
Assuntos
Sistemas CRISPR-Cas , Neuroglia/efeitos dos fármacos , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , Proteínas Supressoras de Tumor/genética , Processamento Alternativo , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Edição de Genes , Engenharia Genética , Terapia Genética/métodos , Humanos , Neuroglia/metabolismo , Neuroglia/patologia , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/farmacologiaRESUMO
Pharmacogenomics has a significant potential to impact how we treat diseases. It involves targeting genetically identifiable populations with therapeutic interventions that promises to yield immediate positive health outcomes with lower or no side effects. The 'trial and error' method of treatment will no longer be necessary with the successful implementation of personalized medicine. The following is an overview of some new developments in pharmacogenomics of multiple sclerosis, and how it has the potential to improve future treatment.
Assuntos
Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/genética , Farmacogenética/tendências , Animais , Humanos , Pesquisa/tendênciasRESUMO
The objectives of this study are to investigate the possible ways by which the curcumin analogs 2a and 2b exert their antiproliferative properties. The analogs 2a and 2b have submicromolar IC(50) values towards human HCT-116 colon cancer cells but are far less toxic to human non-malignant CRL-1790 colon cells. Both compounds affected a number of mitochondrial functions in HCT-116 cells namely increasing the intracellular concentrations of reactive oxygen species, inhibiting oxygen consumption and decreasing the mitochondrial membrane potential. These molecules also produced swelling of isolated rat liver mitochondria, supporting a mitochondrial mechanism of cytotoxicity. Both compounds reacted with glutathione in the presence of glutathione S-transferase π and hence they may be classified as thiol alkylators.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Piperidonas/farmacologia , Animais , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Glutationa/metabolismo , Células HCT116 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Piperidonas/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Hoxa2 gene was reported to be expressed by oligodendrocytes (OLs) and down-regulated at the terminal differentiation stage during oligodendrogenesis in mice (Nicolay et al. 2004b). To further investigate the role of Hoxa2 in oligodendroglial development, a tetracycline regulated controllable expression system was utilized to establish a stable cell line (CG4-SHoxa2 [sense Hoxa2]), where the expression level of Hoxa2 gene could be up-regulated. The impact of Hoxa2 over-expression on the proliferation and differentiation of CG4-SHoxa2 cells was investigated. Up-regulation of Hoxa2 increased the proliferation of CG4-SHoxa2 cells. The mRNA levels of PDGFαR (platelet-derived growth factor [PDGF] alpha receptor), which is expressed by OL progenitor cells, were not different in CG4-SHoxa2 cells compared to wild-type CG4 cells. Semi-quantitative RT-PCR revealed that the mRNA levels of myelin basic protein (MBP) was lower in CG4-SHoxa2 cells than in wild-type CG4 cells indicating the differentiation of CG4-SHoxa2 cells was delayed when the Hoxa2 gene was up-regulated.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Proteína Básica da Mielina/metabolismo , Oligodendroglia/citologia , Tetraciclina/farmacologia , Animais , Bromodesoxiuridina/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Gangliosídeos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Camundongos , Proteína Básica da Mielina/genética , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
BACKGROUND: Development of the secondary palate (SP) is a complex event and abnormalities during SP development can lead to cleft palate, one of the most common birth disorders. Matrix metalloproteinases (MMPs) are required for proper SP development, although a functional role for any one MMP in SP development remains unknown. MMP-25 may have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the MMP-25 gene. We report on the gene expression profile of MMP-25 in the developing mouse SP and identify its functional role in mouse SP development. RESULTS: MMP-25 mRNA and protein are found at all SP developmental stages in mice, with the highest expression at embryonic day (E) 13.5. Immunohistochemistry and in situ hybridization localize MMP-25 protein and mRNA, respectively, to the apical palate shelf epithelial cells and apical mesenchyme. MMP-25 knockdown with siRNA in palatal cultures results in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium. MMP-25 mRNA and protein levels significantly decrease when cultured palate shelves are incubated in growth medium with 5 µg/mL of a TGF-ß3-neutralizing antibody. CONCLUSIONS: Our findings indicate: (i) MMP-25 gene expression is highest at E12.5 and E13.5, which corresponds with increasing palate shelf growth downward alongside the tongue; (ii) MMP-25 protein and mRNA expression predominantly localize in the apical epithelium of the palate shelves, but are also found in apical areas of the mesenchyme; (iii) knockdown of MMP-25 mRNA expression impairs palate shelf fusion and results in significant medial edge epithelium remaining in contacted areas; and (iv) bio-neutralization of TGF-ß3 significantly decreases MMP-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development and are the first to identify a role for a single MMP in mouse SP development.
Assuntos
Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Palato/embriologia , Fator de Crescimento Transformador beta3/metabolismo , Animais , Feminino , Proteínas Ligadas por GPI , Técnicas In Vitro , Metaloproteinases da Matriz Associadas à Membrana/genética , Camundongos , Palato/metabolismoRESUMO
During aging, there is a decrease both in the stability of central nervous system (CNS) myelin once formed and in the efficiency of its repair by oligodendrocytes (OLs). To study CNS remyelination during aging, we used the cuprizone (a copper chelator) mouse model. Inclusion of cuprizone in the diet kills mature OLs and demyelinates axons in the rostral corpus callosum (CC) of mice, which enabled us to characterize age-related changes (i.e., 2-16 months of age) in glial cell response during the recruitment (i.e., demyelination) and differentiation (i.e., remyelination) phases of myelin repair. We have found that the time between 12 and 16 months of age is a critical period during which there is an age-related decrease in the number of OL lineage cells (Olig2(Nuc)+ve/GFAP-ve cells) in the rostral CC of both control mice and mice recovering from cuprizone-induced demyelination. Our results also show there was an age-related impaired recruitment of progenitor cells to replace lost OLs in spite of there being no major age-related decrease in the size of the progenitor cell pool (PDGFalphaR+ve/GFAP-ve, and Olig2(Nuc) +ve/PDGFalphaR+ve cells). However, there were cuprizone-induced increased numbers of astrocyte progenitor cells (Olig2(Cyto)+ve/PDGFalphaR+ve) in these same mice; thus PDGFalphaR+ve progenitor cells in mice as old as 16 months of age retain the ability to differentiate into astrocytes, with this fate choice occurring following cytoplasmic translocation of Olig2. These data reveal for the first time age-related differences in the differentiation of PDGFalphaR+ve progenitor cells into OLs and astrocytes and lead us to suggest that during aging there must be a transcriptional switch mechanism in the progenitor cell fate choice in favor of astrocytes. This may at least partially explain the age-related decrease in efficiency of OL myelination and remyelination.
Assuntos
Envelhecimento/fisiologia , Corpo Caloso/citologia , Cuprizona/farmacologia , Expressão Gênica/efeitos dos fármacos , Bainha de Mielina , Oligodendroglia , Fatores de Transcrição , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Biomarcadores/metabolismo , Linhagem da Célula/efeitos dos fármacos , Quelantes/farmacologia , Corpo Caloso/fisiologia , Dieta , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/fisiologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The cleft palate exhibited by Hoxa2 null murine embryos has been described as being secondary to abnormalities of tongue musculature, and Hoxa2 was presumed to not play a direct role in palate development. However, we detected Hoxa2 expression in the developing palate at both the mRNA and protein levels between embryonic day (E) 12.5 and E15.5. Organ cultures of Hoxa2(-/-) palates maintained in the absence of the tongue showed decreased fusion rates than either Hoxa2(+/-) or Hoxa2(+/+) palate cultures. Knocking down Hoxa2 expression with antisense retroviral constructs resulted in decreased fusion rates than corresponding controls. An overall increase in cell proliferation was observed in Hoxa2 null palates providing a potential mechanism by which Hoxa2 may be affecting palate development. Hoxa2 also repressed the expression of its downstream targets Msx1, Bmp4, Barx1, and Ptx1 within the palate. These results demonstrate the cleft palate phenotype of Hoxa2 null embryos is not solely due to abnormal tongue musculature, and indicate a direct role of Hoxa2 in regulating murine palatogenesis.
Assuntos
Proteínas de Homeodomínio/fisiologia , Palato/embriologia , Palato/metabolismo , Animais , Western Blotting , Proteína Morfogenética Óssea 4/metabolismo , Proliferação de Células , Fissura Palatina/embriologia , Fissura Palatina/genética , Fissura Palatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Fator de Transcrição MSX1/metabolismo , Camundongos , Camundongos Mutantes , Técnicas de Cultura de Órgãos , Fatores de Transcrição Box Pareados/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismoRESUMO
We have previously found that Sirt2 enhanced the outgrowth of cellular processes and MBP expression in CG4 cells, where Sirt2 expression is suppressed by transcription factor Nkx2.2. However, the detailed mechanism of Sirt2 facilitating oligodendroglial cell differentiation remained unclear. In the present study, we observed that Sirt2 partially translocated into the nuclei when CG4 cells were induced to differentiate. Sirt2 was detected at the CpG island of PDGFRα promoter via ChIP assay during the cells differentiation process rather than during the state of growth. Sirt2 deacetylated protein(s) bound to the promoter of PDGFRα and simultaneously appeared to facilitate histone3 K27 tri-methylation, both of which are suppressive signatures on gene transcription activation. In bisulfate assay, we identified that Sirt2 significantly induced DNA methylation of PDGFRα promoter compared with the control. Consistently, Sirt2 overexpression down-regulated PDGFRα expression in CG4 cells. The knock-down of PDGFRα or Sirt2 over-expression repressed cell proliferation, but knock-down of Sirt2 promoted cell proliferation. Taken together, Sirt2 translocated into the nuclei while the cells initiated a differentiation process, facilitating CG4 cell differentiation partially through epigenetic modification and suppression of PDGFRα expression. The repression of PDGFRα expression mediated by Sirt2 appeared to facilitate a transition of cellular processes, i.e. from a proliferating progenitor state to a post-mitotic state in CG4 cells.
Assuntos
Diferenciação Celular , Epigênese Genética , Sirtuína 2/fisiologia , Acetilação , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Proliferação de Células , Ilhas de CpG , Metilação de DNA , Técnicas de Silenciamento de Genes , Células HEK293 , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio , Humanos , Camundongos , Células NIH 3T3 , Proteínas Nucleares , Regiões Promotoras Genéticas , Ratos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Sirtuína 2/análise , Sirtuína 2/genética , Fatores de TranscriçãoRESUMO
Protein-energy malnutrition (PEM) exacerbates functional impairment caused by brain ischemia. This is correlated with reactive gliosis, which suggests an increased inflammatory response. The objective of the current study was to investigate if PEM increases hippocampal activation of nuclear factor kappaB (NFkappaB), a transcription factor that amplifies the inflammatory response involved in ischemic brain injury. Mongolian gerbils (11-12 weeks old) were randomly assigned to control diet (12.5% protein) or protein-deficient diet (2%) for 4 weeks. The 2% protein group had a 15% decrease in voluntary food intake (P<.001; unpaired t test), resulting in PEM. Body weight after 4 weeks was 20% lower in the PEM group (P<.001). Gerbils were then exposed to sham surgery or global ischemia induced by 5-min bilateral common carotid artery occlusion. PEM independently increased hippocampal NFkappaB activation detected by electrophoretic mobility shift assay at 6 h after surgery (P=.014; 2-factor ANOVA). Ischemia did not significantly affect NFkappaB activation nor was there interaction between diet and ischemia. Serum glucose and cortisol concentrations at 6 h postischemia were unaltered by diet or ischemia. A second experiment using gerbils of the same age and feeding paradigm demonstrated that PEM also increases hippocampal NFkappaB activation in the absence of surgery. These findings suggest that PEM, which exists in 16% of elderly patients at admission for stroke, may worsen outcome by increasing activation of NFkappaB. Since PEM increased NFkappaB activation independent of ischemia or surgery, the data also have implications for the inflammatory response of the many individuals affected globally by PEM.
Assuntos
Isquemia Encefálica/patologia , Hipocampo/metabolismo , Isquemia , NF-kappa B/biossíntese , Desnutrição Proteico-Calórica/metabolismo , Ração Animal , Animais , Glicemia/metabolismo , Peso Corporal , Encéfalo/patologia , Gerbillinae , Hidrocortisona/sangue , Masculino , Modelos Biológicos , NF-kappa B/metabolismo , Acidente Vascular CerebralRESUMO
Cleft palate is one of the most common congenital birth defects worldwide. The homeobox (Hox) family of genes are key regulators of embryogenesis, with Hoxa2 having a direct role in secondary palate development. Hoxa2-/- mice exhibit cleft palate; however, the cellular and molecular mechanisms leading to cleft palate in Hoxa2-/- mice is largely unknown. Addressing this issue, we found that Hoxa2 regulates spatial and temporal programs of osteogenic differentiation in the developing palate by inhibiting bone morphogenetic protein (BMP) signaling dependent osteoblast markers. Expression of osteoblast markers, including Runx2, Sp7, and AlpI were increased in Hoxa2-/- palatal shelves at embryonic day (E) 13.5 and E15.5. Hoxa2-/- mouse embryonic palatal mesenchyme (MEPM) cells exhibited increased bone matrix deposition and mineralization in vitro. Moreover, loss of Hoxa2 resulted in increased osteoprogenitor cell proliferation and osteogenic commitment during early stages of palate development at E13.5. Consistent with upregulation of osteoblast markers, Hoxa2-/- palatal shelves displayed higher expression of canonical BMP signaling in vivo. Blocking BMP signaling in Hoxa2-/- primary MEPM cells using dorsomorphin restored cell proliferation and osteogenic differentiation to wild-type levels. Collectively, these data demonstrate for the first time that Hoxa2 may regulate palate development by inhibiting osteogenic differentiation of palatal mesenchyme via modulating BMP signaling.
RESUMO
Multiple sclerosis (MS) is a progressive, neurodegenerative disease with unpredictable phases of relapse and remission. The cause of MS is unknown, but the pathology is characterized by infiltration of auto-reactive immune cells into the central nervous system (CNS) resulting in widespread neuroinflammation and neurodegeneration. Immunomodulatory-based therapies emerged in the 1990s and have been a cornerstone of disease management ever since. Interferon ß (IFNß) was the first biologic approved after demonstrating decreased relapse rates, disease activity and progression of disability in clinical trials. However, frequent dosing schedules have limited patient acceptance for long-term therapy. Pegylation, the process by which molecules of polyethylene glycol are covalently linked to a compound, has been utilized to increase the half-life of IFNß and decrease the frequency of administration required. To date, there has been one clinical trial evaluating the efficacy of pegylated IFN. The purpose of this article is to provide an overview of the role of IFN in the treatment of MS and evaluate the available evidence for pegylated IFN therapy in MS.
RESUMO
Cleft palate is a common congenital abnormality that results from defective secondary palate (SP) formation. The Sine oculis-related homeobox 2 (Six2) gene has been linked to abnormalities of craniofacial and kidney development. Our current study examined, for the first time, the specific role of Six2 in embryonic mouse SP development. Six2 mRNA and protein expression were identified in the palatal shelves from embryonic days (E)12.5 to E15.5, with peak levels during early stages of palatal shelf outgrowth. Immunohistochemical staining (IHC) showed that Six2 protein is abundant throughout the mesenchyme in the oral half of each palatal shelf, whereas there is a pronounced decline in Six2 expression by mesenchyme cells in the nasal half of the palatal shelf by stages E14.5-15.5. An opposite pattern was observed in the surface epithelium of the palatal shelf. Six2 expression was prominent at all stages in the epithelial cell layer located on the nasal side of each palatal shelf but absent from the epithelium located on the oral side of the palatal shelf. Six2 is a putative downstream target of transcription factor Hoxa2 and we previously demonstrated that Hoxa2 plays an intrinsic role in embryonic palate formation. We therefore investigated whether Six2 expression was altered in the developing SP of Hoxa2 null mice. Reverse transcriptase PCR and Western blot analyses revealed that Six2 mRNA and protein levels were upregulated in Hoxa2-/- palatal shelves at stages E12.5-14.5. Moreover, the domain of Six2 protein expression in the palatal mesenchyme of Hoxa2-/- embryos was expanded to include the entire nasal half of the palatal shelf in addition to the oral half. The palatal shelves of Hoxa2-/- embryos displayed a higher density of proliferating, Ki-67 positive palatal mesenchyme cells, as well as a higher density of Six2/Ki-67 double-positive cells. Furthermore, Hoxa2-/- palatal mesenchyme cells in culture displayed both increased proliferation and elevated Cyclin D1 expression relative to wild-type cultures. Conversely, siRNA-mediated Six2 knockdown restored proliferation and Cyclin D1 expression in Hoxa2-/- palatal mesenchyme cultures to near wild-type levels. Our findings demonstrate that Six2 functions downstream of Hoxa2 as a positive regulator of mesenchymal cell proliferation during SP development.