RNA-seq of THP-1 and U937 exposed to enamel matrix derivative.
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PROVIDER: PRJNA1327401 | ENA |
REPOSITORIES: ENA
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Project description:Enamel matrix derivatives (EMDs) treat periodontal defects and gingival recession, a process where macrophages are believed to contribute to the clinical outcome. However, the present findings are heterogeneous, raising the need for standardized bioassays to better understand and monitor EMD activity in vitro. To this aim, we have conducted THP-1 and U937, both widely established monocytic cell lines, as bioassays in EMD research. To understand their differential response to EMD, we employed an RNA-seq approach to identify changes in the genetic signatures of THP-1 and U937 cells. When applying a threshold of 1.5 log2 fold-change and a significance of 2.0-log10, we could identify 5/37 and 30/23 up-and-down-regulated genes in THP1 and U937 cells, respectively. In THP-1, the upregulated genes included S100A8, S100A9 and CD38; downregulated gene included ADM, CD48, IL24, MMP1, and PDGFB. In U937, most striking was the increase of alpha subunit integrins ITGA1, ITGA2, ITGA6, and the decrease of genes including OLR1, CCL1, CCL4L2, CCL8, IL21R, MMP7, PDGFB and MMP25. We further show that the TGF-β receptor type I kinase inhibitor SB431542 blocked the expression changes of S100A8, S100A9, CD38, ITGA2, ITGA6, and OLR1 but failed to reverse PDGFB. These data serve as a primer for developing macrophage bioassays to measure EMD activity in the context of TGF-β signaling.
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Project description:In a systemic effort to survive environmental stress, organ systems fluctuate and adapt to overcome external pressures. The evolutionary drive back towards homeostasis, makes it difficult to determine if an organism experienced a toxic exposure to stress, especially in early prenatal and neonatal periods of development. Previous studies indicate that primary human teeth may provide historical records of experiences related to stressors during that early time window. To assess the molecular effects of early life adversity on enamel formation, we used a limited bedding and nesting (LBN) mouse model of early life adversity (ELA), to assess changes in enamel organ gene expression and enamel mineralization. Enamel of postnatal day 12 (P12) weight-matched ELA mice was more mineralized as compared control enamel. RNAseq showed 724 genes significantly upregulated in ELA enamel organs as compared to controls, and 574 significantly downregulated genes (DESeq2 p-value <= 0.05). Transcripts expressing the enamel matrix proteins amelogenin (Amelx) and enamelin (Enam) were among the top 4 most differentially expressed genes. The most significantly enriched GO and Reactome pathway was Extracellular Matrix Organization, while the most significantly enriched KEGG Pathways were Butanoate metabolism and Synthesis and Degradation of Ketone Bodies. qPCR analysis of weight-matched ELA and control enamel organs confirmed that expression of the ameloblast-specific proteins Amelx and Enam were highly upregulated in ELA mice. When evaluating molecular mechanisms for amelogenin upregulation, we found significantly increased expression of Dlx3, while transcripts for clock genes Per1 and Nrd1, which have also positively associated with amelogenin expression, were downregulated. These findings suggest that the developing enamel organs are sensitive to the pressures of early life adversity and produce structural biomarkers in tooth enamel mineral to reflect these changes. Together these results support the possibility that tooth enamel may contain biomarkers of cellular effects associated with systemic early life adversity.
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Project description:Tooth enamel forms in an ephemeral protein matrix where changes in protein abundance, composition and post-translational modifications are critical to achieve healthy enamel properties. Amelogenin (AMELX) with its splice variants is the most abundant enamel matrix protein, with only one known phosphorylation site at serine 16 shown in vitro to be critical for regulating mineralization. The phosphorylated form of AMELX stabilizes amorphous calcium phosphate, while crystalline hydroxyapatite forms in the presence of the unphosphorylated protein. While it is necessary to our understanding of how AMELX regulates mineral transitions over space and time, it is unknown whether and when un-phosphorylated amleogenin occurs during enamel mineralization. This study aims to reveal the spatiotemporal distribution of the most abundant AMLEX splice variants including the full length P173, the shorter leucine-rich amelogenin protein (LRAP), and the exon 4-containing P190 in forming enamel, all within the context of the changing enamel matrix proteome during mineralization. We microsampled permanent pig molars, capturing known stages of enamel formation from both crown surface and inner enamel. Nano-LC-MS/MS proteomic analyses after tryptic digestion rendered more than 500 unique protein identifications in enamel, dentin, and bone. We mapped collagens, keratins, and proteolytic enzymes (CTSL, MMP2, MMP10) and determined distributions of P173, LRAP and P190, the enamel proteins enamelin (ENAM) and ameloblastin (AMBN), and matrix-metalloprotease-20 (MMP20) and kallikrein-4 (KLK4). All enamel proteins and KLK4 were near-exclusive to enamel and in excellent agreement with published expression levels. Phosphorylated P173 and LRAP decreased in abundance from recently deposited matrix towards older enamel, mirrored by increasing abundances of testicular acid phosphatase (ACPT). Our results showed that hierarchical clustering analysis of secretory enamel links closely matching distributions of unphosphorylated P173 and LRAP with ACPT and nontraditional amelogenesis proteins, many associated with enamel defects. We report higher protein diversity than previously published and Gene Ontology (GO)-defined protein functions related to the regulation of mineral formation in secretory enamel (e.g. casein α-S1, CSN1S1), immune response in erupted enamel (e.g. peptidoglycan recognition protein, PGRP), and phosphorylation. This study presents a novel approach to characterize and study functional relationships through spatiotemporal mapping of the ephemeral extracellular matrix proteome.
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Project description:Methylation of CpG islands is associated with transcriptional repression and, in cancer, leads to the abnormal silencing of tumor-suppressor genes. Genome wide methylation profiling of myeloid leukemia cell lines identified a large number of genes with aberrantly methylated CpG islands. Comparative mRNA expression analysis suggests that more than half of these genes show extremely low or absent expression in normal cells, suggesting that hypermethylation in cancer may be independent of the transcriptional status of the affected gene. Experiment Overall Design: The expression profiles of the three leukemia cell lines KG-1, U937 and THP-1 was compared to normal human blood monocytes (reference). The set includes a single hybridization for each sample.
2008-06-12 | E-GEOD-3280 | biostudies-arrayexpress
Project description:The present study aimed to investigate the periodontal regenerative effect of enamel matrix derivative (EMD) in diabetes. Thirty-six rats were assigned to streptozotocin-induced diabetes or control (non-diabetic) groups. Three-wall intrabony defects were surgically generated in the bilateral maxilla molar, followed by application of EMD or saline. Primary wound closure and defect fill were evaluated via histomorphological analysis and micro-computed tomography. mRNA expression levels of inflammatory and angiogenic factors in the defects were quantified via real-time polymerase chain reaction. Gingival fibroblasts were isolated from control animals and cultured in high-glucose (HG) or control medium. The effects of EMD on insulin resistance and PI3K/Akt/VEGF signaling were evaluated. The achievement rate of primary closure and the parameters of defect fill were significantly higher at EMD-treated site than at EMD-untreated sites in both diabetic and non-diabetic rats, although defect fill in the diabetic groups was significantly lower in the control groups on two-way repeated-measures analysis of variance (for both, p<0.05). Newly formed bone and cementum were significantly increased at EMD-treated sites in diabetic rats than at EMD-untreated sites in control rats (for both, p<0.05). Vegf was significantly upregulated at EMD-treated sites in both diabetic and non-diabetic rats (for both, p<0.05). In vitro, insulin or EMD-induced Akt phosphorylation was significantly lower in cells cultured in HG medium (p<0.05). EMD-mediated Vegf upregulation was suppressed by the Akt inhibitor wortmannin, although the effect was significantly lower in HG medium (p<0.01). In conclusion, EMD might promote periodontal tissue regeneration via Akt/VEGF signaling, even in a diabetic condition.
| S-EPMC6237339 | biostudies-literature
Project description:Background: Pyroptosis is a caspase-dependent catabolic process relevant to periodontal disorders for which inflammation is central to the pathophysiology of the disease. Although enamel matrix derivative (EMD) has been applied to support periodontal regeneration, its capacity to modulate the expression of pyroptosis-related genes remains unknown. Considering EMD has anti-inflammatory properties and pyroptosis is linked to the activation of the inflammasome in chronic periodontitis, the question arises whether EMD could reduce pyroptosis signalling. Methods: To answer this question, primary macrophages obtained from murine bone marrow and RAW 264.7 macrophages were primed with EMD before being challenged by lipopolysaccharide (LPS). Cells were then analysed for pyroptosis-signalling components by gene expression analyses, interleukin-1β (IL-1β) immunoassay, and the detection of caspase-1 (CAS1). The release of mitochondrial reactive oxygen species (ROS) was also detected. Results: We report here that EMD, like the inflammasome (NLRP3) and CAS1 specific inhibitors-MCC950 and Ac-YVAD-cmk, respectively-lowered the LPS-induced expression of NLRP3 in primary macrophages (EMD: p = 0.0232; MCC950: p = 0.0426; Ac-YVAD-cmk: p = 0.0317). EMD further reduced the LPS-induced expression of NLRP3 in RAW 264.7 cells (p = 0.0043). There was also a reduction in CAS1 and IL-1β in RAW 264.7 macrophages on the transcriptional level (p = 0.0598; p = 0.0283; respectively), in IL-1β protein release (p = 0.0313), and CAS1 activity. Consistently, EMD, like MCC950 and Ac-YVAD-cmk, diminished the ROS release in activated RAW 264.7 cells. In ST2 murine mesenchymal cells, EMD could not be tested because LPS, saliva, and IL-1β + TNF-α failed to provoke pyroptosis signalling. Conclusion: These findings suggest that EMD is capable of dampening the expression of pyroptosis-related genes in macrophages.
| S-EPMC9099857 | biostudies-literature