ABSTRACT
The roles of long non-coding RNA (LncRNA) have been highlighted in various development processes including congenital heart defects (CHD). Here, we characterized the molecular function of LncRNA, Moshe (1010001N08ik-203), one of the Gata6 antisense transcripts located upstream of Gata6, which is involved in both heart development and the most common type of congenital heart defect, atrial septal defect (ASD). During mouse embryonic development, Moshe was first detected during the cardiac mesoderm stage (E8.5 to E9.5) where Gata6 is expressed and continues to increase at the atrioventricular septum (E12.5), which is involved in ASD. Functionally, the knock-down of Moshe during cardiogenesis caused significant repression of Nkx2.5 in cardiac progenitor stages and resulted in the increase in major SHF lineage genes, such as cardiac transcriptional factors (Isl1, Hand2, Tbx2), endothelial-specific genes (Cd31, Flk1, Tie1, vWF), a smooth muscle actin (a-Sma) and sinoatrial node-specific genes (Shox2, Tbx18). Chromatin Isolation by RNA Purification showed Moshe activates Nkx2.5 gene expression via direct binding to its promoter region. Of note, Moshe was conserved across species, including human, pig and mouse. Altogether, this study suggests that Moshe is a heart-enriched lncRNA that controls a sophisticated network of cardiogenesis by repressing genes in SHF via Nkx2.5 during cardiac development and may play an important role in ASD.
Subject(s)
Cell Differentiation/genetics , Cell Lineage/genetics , Myocytes, Cardiac/metabolism , RNA, Long Noncoding/genetics , Animals , Cell Line , Enhancer Elements, Genetic , GATA6 Transcription Factor/genetics , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Humans , Mesoderm/embryology , Mesoderm/metabolism , Mice , Myoblasts, Cardiac/cytology , Myoblasts, Cardiac/metabolism , Myocytes, Cardiac/cytology , Organogenesis/genetics , Promoter Regions, Genetic , RNA Interference , RNA, AntisenseABSTRACT
INTRODUCTION: High plasma homocysteine (Hcy) levels have been associated with increased risk of fracture. Since Hcy has been shown to induce apoptosis in many cell types, including vascular endothelial cells, we hypothesized that Hcy would have a similar apoptotic effect on osteoblasts, leading to osteoporosis by reducing bone formation. MATERIALS AND METHODS: Using primary human bone marrow stromal cells (hBMSC) and HS-5 cell line (human bone marrow stromal cell line), we investigated the effects of Hcy on these cells by cell viability assay and analysis of cytoplasmic histone-associated DNA fragments. Caspase activity assay, Western blots, and electrophoresis mobility shift assay (EMSA) were performed to find the mechanism of apoptosis. Intracellular reactive oxygen species (ROS) were measured by spectrometry using dichlorofluorescein diacetate, and cellular total glutathione level was determined by a commercially available kit. N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) were used as tools for investigating the role of ROS and nuclear factor-kappaB (NF-kappaB), respectively. RESULTS: Hcy induced apoptosis in primary human bone marrow stromal cells and the HS-5 cell line, and this apoptotic effect was caspase-dependent. In addition, Hcy increased cytochrome c release into the cytosol, and activated caspase-9 and caspase-3, but not caspase-8, indicating that Hcy induces apoptosis via the mitochondria pathway. Hcy increased ROS, and NAC inhibited the apoptotic effect of Hcy. Western blot and EMSA showed that Hcy activated the NF-kappaB pathway. PDTC blocked Hcy-induced caspase-3 activation and apoptosis. CONCLUSION: These results suggest that Hcy induces apoptosis via the ROS-mediated mitochondrial pathway and NF-kappaB activation in hBMSCs, and that Hcy may contribute to the development of osteoporosis by reducing bone formation. Antioxidants may have a role in preventing bone loss in individuals with hyperhomocysteinemia.
Subject(s)
Apoptosis/drug effects , Bone Marrow/drug effects , Homocysteine/pharmacology , Stromal Cells/cytology , Stromal Cells/drug effects , Bone Marrow/metabolism , Caspases/metabolism , Cell Differentiation/drug effects , Cell Line , Cytochromes c/metabolism , Cytosol/drug effects , Cytosol/metabolism , Humans , Mitochondria/drug effects , Mitochondria/metabolism , NF-kappa B/metabolism , Osteoblasts/cytology , Osteoblasts/drug effects , Reactive Oxygen Species/metabolism , Stromal Cells/metabolismABSTRACT
The AICD (APP intracellular Domain) and C31, caspase-cleaved C-terminal fragment of APP, have been found in Alzheimer's disease (AD) patients' brains and have been reported to induce apoptosis in neuronal cells. In recent, the C-terminal fragments of amyloid precursor protein (APP-CTs) have been reported to form a complex with Fe65 and the histone acetyltransferase Tip60 and are thought to be involved in gene transcription. In this study, based on the hypothesis that APP-CTs might exert neurotoxicity by inducing some gene transcription, we investigated the effects of APP-CTs on histone acetylation which indicates that transcription is actively going on and also on the relationship between histone acetylation and the cytotoxicity induced by APP-CTs in nerve growth factor (NGF)-differentiated PC12 cells and rat primary cortical neurons. Here we demonstrate that the expression of APP-CTs [C31, AICD (C59) and C99] induces increases in acetylation of histone 3 and histone 4 and that treatment with sodium butyrate, an inhibitor of histone deacetylase, significantly enhances the cytotoxicity induced by APP-CTs. The acetylation of histone plays an important role in allowing regulatory proteins to access DNA and is likely to be a major factor in the regulation of gene transcription. Taken together, our results suggest that APP-CTs exert neurotoxicity by transcription-dependent mechanisms and this might contribute to the pathogenesis of AD.
Subject(s)
Amyloid beta-Protein Precursor/toxicity , Histones/metabolism , Acetylation/drug effects , Amyloid beta-Protein Precursor/chemistry , Animals , Apoptosis/drug effects , Blotting, Western/methods , Butyrates/pharmacology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Survival/drug effects , Cells, Cultured , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Drug Interactions , Embryo, Mammalian , Enzyme Inhibitors/pharmacology , Immunohistochemistry , In Situ Nick-End Labeling/methods , Indoles/metabolism , L-Lactate Dehydrogenase/metabolism , Molecular Structure , Nerve Growth Factor/pharmacology , PC12 Cells , Rats , Rats, Sprague-Dawley , TransfectionABSTRACT
Many environmental factors during the pre- or postnatal period can affect an individual's cognitive function and neural development throughout life. Little is known, however, about the combined effects of the pre- and postnatal environments on cognitive function of adult offspring and structural alterations in the adult brain. In this study, we confirmed that pre- or postnatal stress impaired learning and memory performance of rats. Conversely, pre- or postnatal enriched housing improved behavioral performance. These experience-dependent behavioral alterations were consistent with changes in 5-bromo-2'-deoxyuridine-labeled cell number in the granule cell layer of the hippocampus and in the expression level of synaptic markers such as neuronal cell adhesion molecule and synaptophysin, and expression of a neurotrophic factor, brain-derived neurotrophic factor. Postnatal stress appeared to have no influence on cell proliferation, however. We did find that postnatal environment could attenuate prenatal effects partly via a longitudinal cross-housing study, in which pups born to mothers housed under enriched conditions were reared under stressful conditions and vice versa. These results suggest that postnatal environmental manipulations can counteract the cognitive alterations in early adulthood and the structural changes in the young adult brain induced by prenatal experience.
