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1.
Redox Biol ; 62: 102670, 2023 06.
Article in English | MEDLINE | ID: mdl-36958249

ABSTRACT

Keratinocytes of the mammalian skin provide not only mechanical protection for the tissues, but also transmit mechanical, chemical, and thermal stimuli from the external environment to the sensory nerve terminals. Sensory nerve fibers penetrate the epidermal basement membrane and function in the tight intercellular space among keratinocytes. Here we show that epidermal keratinocytes produce hydrogen peroxide upon the activation of the NADPH oxidase dual oxidase 1 (DUOX1). This enzyme can be activated by increasing cytosolic calcium levels. Using DUOX1 knockout animals as a model system we found an increased sensitivity towards certain noxious stimuli in DUOX1-deficient animals, which is not due to structural changes in the skin as evidenced by detailed immunohistochemical and electron-microscopic analysis of epidermal tissue. We show that DUOX1 is expressed in keratinocytes but not in the neural sensory pathway. The release of hydrogen peroxide by activated DUOX1 alters both the activity of neuronal TRPA1 and redox-sensitive potassium channels expressed in dorsal root ganglia primary sensory neurons. We describe hydrogen peroxide, produced by DUOX1 as a paracrine mediator of nociceptive signal transmission. Our results indicate that a novel, hitherto unknown redox mechanism modulates noxious sensory signals.


Subject(s)
Hydrogen Peroxide , NADPH Oxidases , Animals , Dual Oxidases/genetics , Hydrogen Peroxide/metabolism , NADPH Oxidases/metabolism , Peroxides , Nociception , NADPH Oxidase 1 , Mammals/metabolism
2.
Sci Rep ; 9(1): 6020, 2019 04 15.
Article in English | MEDLINE | ID: mdl-30988425

ABSTRACT

Phenotypic changes in lung fibroblasts are believed to contribute to the development of Idiopathic Pulmonary Fibrosis (IPF), a progressive and fatal lung disease. Long intergenic non-coding RNAs (lincRNAs) have been identified as novel regulators of gene expression and protein activity. In non-stimulated cells, we observed reduced proliferation and inflammation but no difference in the fibrotic response of IPF fibroblasts. These functional changes in non-stimulated cells were associated with changes in the expression of the histone marks, H3K4me1, H3K4me3 and H3K27ac indicating a possible involvement of epigenetics. Following activation with TGF-ß1 and IL-1ß, we demonstrated an increased fibrotic but reduced inflammatory response in IPF fibroblasts. There was no significant difference in proliferation following PDGF exposure. The lincRNAs, LINC00960 and LINC01140 were upregulated in IPF fibroblasts. Knockdown studies showed that LINC00960 and LINC01140 were positive regulators of proliferation in both control and IPF fibroblasts but had no effect upon the fibrotic response. Knockdown of LINC01140 but not LINC00960 increased the inflammatory response, which was greater in IPF compared to control fibroblasts. Overall, these studies demonstrate for the first time that lincRNAs are important regulators of proliferation and inflammation in human lung fibroblasts and that these might mediate the reduced inflammatory response observed in IPF-derived fibroblasts.


Subject(s)
Fibroblasts/pathology , Idiopathic Pulmonary Fibrosis/genetics , Lung/pathology , RNA, Long Noncoding/genetics , Cells, Cultured , Epigenesis, Genetic , Female , Fibroblasts/metabolism , Humans , Idiopathic Pulmonary Fibrosis/pathology , Lung/metabolism , Male , Middle Aged , Transcriptome
3.
Front Immunol ; 9: 2906, 2018.
Article in English | MEDLINE | ID: mdl-30619270

ABSTRACT

There is accumulating evidence to indicate that long non-coding RNAs (lncRNAs) are important regulators of the inflammatory response. In this report, we have employed next generation sequencing to identify 14 lncRNAs that are differentially expressed in human lung fibroblasts following the induction of inflammation using interleukin-1ß (IL-1ß). Knockdown of the two most highly expressed lncRNAs, IL7AS, and MIR3142HG, showed that IL7AS negatively regulated IL-6 release whilst MIR3142HG was a positive regulator of IL-8 and CCL2 release. Parallel studies in fibroblasts derived from patients with idiopathic pulmonary fibrosis showed similar increases in IL7AS levels, that also negatively regulate IL-6 release. In contrast, IL-1ß-induced MIR3142HG expression, and its metabolism to miR-146a, was reduced by 4- and 9-fold in IPF fibroblasts, respectively. This correlated with a reduced expression of inflammatory mediators whilst MIR3142HG knockdown showed no effect upon IL-8 and CCL2 release. Pharmacological studies showed that IL-1ß-induced IL7AS and MIR3142HG production and release of IL-6, IL-8, and CCL2 in both control and IPF fibroblasts were mediated via an NF-κB-mediated pathway. In summary, we have cataloged those lncRNAs that are differentially expressed following IL-1ß-activation of human lung fibroblasts, shown that IL7AS and MIR3142HG regulate the inflammatory response and demonstrated that the reduced inflammatory response in IPF fibroblast is correlated with attenuated expression of MIR3142HG/miR-146a.


Subject(s)
Fibroblasts/metabolism , Gene Expression Regulation/drug effects , Idiopathic Pulmonary Fibrosis/genetics , Inflammation/genetics , Interleukin-1beta/pharmacology , RNA, Long Noncoding/genetics , Cells, Cultured , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Female , Gene Expression Profiling/methods , Humans , Idiopathic Pulmonary Fibrosis/pathology , Inflammation/metabolism , Inflammation/pathology , Interleukin-6/genetics , Interleukin-6/metabolism , Interleukin-8/genetics , Interleukin-8/metabolism , Male , Middle Aged
4.
Front Immunol ; 8: 1038, 2017.
Article in English | MEDLINE | ID: mdl-28900427

ABSTRACT

Despite increasing evidence to indicate that long non-coding RNAs (lncRNAs) are novel regulators of immunity, there has been no systematic attempt to identify and characterize the lncRNAs whose expression is changed following the induction of the innate immune response. To address this issue, we have employed next-generation sequencing data to determine the changes in the lncRNA profile in four human (monocytes, macrophages, epithelium, and chondrocytes) and four mouse cell types (RAW 264.7 macrophages, bone marrow-derived macrophages, peritoneal macrophages, and splenic dendritic cells) following exposure to the pro-inflammatory mediators, lipopolysaccharides (LPS), or interleukin-1ß. We show differential expression of 204 human and 210 mouse lncRNAs, with positional analysis demonstrating correlation with immune-related genes. These lncRNAs are predominantly cell-type specific, composed of large regions of repeat sequences, and show poor evolutionary conservation. Comparison within the human and mouse sequences showed less than 1% sequence conservation, although we identified multiple conserved motifs. Of the 204 human lncRNAs, 21 overlapped with syntenic mouse lncRNAs, of which five were differentially expressed in both species. Among these syntenic lncRNA was IL7-AS (antisense), which was induced in multiple cell types and shown to regulate the production of the pro-inflammatory mediator interleukin-6 in both human and mouse cells. In summary, we have identified and characterized those lncRNAs that are differentially expressed following activation of the human and mouse innate immune responses and believe that these catalogs will provide the foundation for the future analysis of the role of lncRNAs in immune and inflammatory responses.

5.
Sci Rep ; 7(1): 12389, 2017 09 28.
Article in English | MEDLINE | ID: mdl-28959041

ABSTRACT

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 2 (RAMP2) comprise a receptor for adrenomedullin (AM). Although it is known that AM induces internalization of CLR•RAMP2, little is known about the molecular mechanisms that regulate the trafficking of CLR•RAMP2. Using HEK and HMEC-1 cells, we observed that AM-induced activation of CLR•RAMP2 promoted ubiquitination of CLR. A mutant (CLRΔ9KR), lacking all intracellular lysine residues was functional and trafficked similar to the wild-type receptor, but was not ubiquitinated. Degradation of CLR•RAMP2 and CLRΔ9KR•RAMP2 was not dependent on the duration of AM stimulation or ubiquitination and occurred via a mechanism that was partially prevented by peptidase inhibitors. Degradation of CLR•RAMP2 was sensitive to overexpression of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), but not to HRS knockdown, whereas CLRΔ9KR•RAMP2 degradation was unaffected. Overexpression, but not knockdown of HRS, promoted hyperubiquitination of CLR under basal conditions. Thus, we propose a role for ubiquitin and HRS in the regulation of AM-induced degradation of CLR•RAMP2.


Subject(s)
Adrenomedullin/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Phosphoproteins/metabolism , Receptors, Adrenomedullin/metabolism , Ubiquitination/physiology , Calcitonin Receptor-Like Protein/genetics , Calcitonin Receptor-Like Protein/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Gene Knockdown Techniques , HEK293 Cells , Humans , Phosphoproteins/genetics , Protein Transport , Proteolysis , RNA, Small Interfering/metabolism , Receptor Activity-Modifying Protein 2/genetics , Receptor Activity-Modifying Protein 2/metabolism , Ubiquitin/metabolism
6.
Sci Rep ; 7(1): 8024, 2017 08 14.
Article in English | MEDLINE | ID: mdl-28808260

ABSTRACT

Myositis is characterised by muscle inflammation and weakness. Although generally thought to be driven by a systemic autoimmune response, increasing evidence suggests that intrinsic changes in the muscle might also contribute to the pathogenesis. Long non-coding RNAs (lncRNAs) are a family of novel genes that regulate gene transcription and translation. To determine the potential role of lncRNAs, we employed next generation sequencing to examine the transcriptome in muscle biopsies obtained from two histologically distinct patient populations, inclusion body myositis (IBM) and anti-Jo-1-associated myositis (Jo-1). 1287 mRNAs and 1068 mRNAs were differentially expressed in the muscle from Jo-1 and IBM patients, respectively. Pathway analysis showed the top canonical pathway in both Jo-1 and IBM was oxidative phosphorylation and mitochondrial dysfunction. We identified 731 known and 325 novel lncRNAs in the muscles biopsies. Comparison with controls showed 55 and 46 lncRNAs were differentially expressed in IBM and Jo-1 myositis, respectively. Of these, 16 lncRNAs were differentially expressed in both IBM and Jo-1 myositis and included upregulated H19, lncMyoD and MALAT1. Given that these are known to regulate muscle proliferation and differentiation, we speculate that changes in lncRNAs might contribute to the phenotypic changes in Jo-1 and IBM myositis.


Subject(s)
Myositis, Inclusion Body/genetics , RNA, Long Noncoding/genetics , Transcriptome , Adult , Aged , Antibodies, Antinuclear/immunology , Humans , Middle Aged , Myositis, Inclusion Body/immunology , Myositis, Inclusion Body/metabolism , RNA, Long Noncoding/metabolism , Up-Regulation
7.
Methods Mol Biol ; 1468: 11-8, 2017.
Article in English | MEDLINE | ID: mdl-27662866

ABSTRACT

The human genome is widely transcribed outside of protein-coding genes, producing thousands of noncoding RNAs from different subfamilies including enhancer RNAs. Functional studies to determine the role of individual genes are challenging with noncoding RNAs appearing to be more difficult to knockdown than mRNAs. One factor that may have hindered progress is that the majority of noncoding RNAs are thought to be located within the nucleus, where the efficiency of traditional RNA interference techniques is debatable. Here we present an alternative RNA interference technique utilizing Locked Nucleic Acids, which is able to efficiently knockdown noncoding RNAs irrespective of intracellular location.


Subject(s)
Gene Knockdown Techniques/methods , Oligonucleotides/genetics , RNA, Long Noncoding/genetics , Cell Line , Cell Nucleus/genetics , Cells, Cultured , Enhancer Elements, Genetic , Humans , Monocytes/cytology
8.
Arthritis Rheumatol ; 68(4): 845-56, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27023358

ABSTRACT

OBJECTIVE: To identify long noncoding RNAs (lncRNAs), including long intergenic noncoding RNAs (lincRNAs), antisense RNAs, and pseudogenes, associated with the inflammatory response in human primary osteoarthritis (OA) chondrocytes and to explore their expression and function in OA. METHODS: OA cartilage was obtained from patients with hip or knee OA following joint replacement surgery. Non-OA cartilage was obtained from postmortem donors and patients with fracture of the neck of the femur. Primary OA chondrocytes were isolated by collagenase digestion. LncRNA expression analysis was performed by RNA sequencing (RNAseq) and quantitative reverse transcriptase-polymerase chain reaction. Modulation of lncRNA chondrocyte expression was achieved using LNA longRNA GapmeRs (Exiqon). Cytokine production was measured with Luminex. RESULTS: RNAseq identified 983 lncRNAs in primary human hip OA chondrocytes, 183 of which had not previously been identified. Following interleukin-1ß (IL-1ß) stimulation, we identified 125 lincRNAs that were differentially expressed. The lincRNA p50-associated cyclooxygenase 2-extragenic RNA (PACER) and 2 novel chondrocyte inflammation-associated lincRNAs (CILinc01 and CILinc02) were differentially expressed in both knee and hip OA cartilage compared to non-OA cartilage. In primary OA chondrocytes, these lincRNAs were rapidly and transiently induced in response to multiple proinflammatory cytokines. Knockdown of CILinc01 and CILinc02 expression in human chondrocytes significantly enhanced the IL-1-stimulated secretion of proinflammatory cytokines. CONCLUSION: The inflammatory response in human OA chondrocytes is associated with widespread changes in the profile of lncRNAs, including PACER, CILinc01, and CILinc02. Differential expression of CILinc01 and CIinc02 in hip and knee OA cartilage, and their role in modulating cytokine production during the chondrocyte inflammatory response, suggest that they may play an important role in mediating inflammation-driven cartilage degeneration in OA.


Subject(s)
Cartilage, Articular/metabolism , Chondrocytes/metabolism , Osteoarthritis, Hip/metabolism , Osteoarthritis, Knee/metabolism , RNA, Long Noncoding/metabolism , Aged , Arthroplasty, Replacement, Hip , Arthroplasty, Replacement, Knee , Case-Control Studies , Chondrocytes/immunology , Cytokines/immunology , Female , Femoral Neck Fractures/immunology , Femoral Neck Fractures/metabolism , Femoral Neck Fractures/surgery , Gene Expression Profiling , Humans , In Vitro Techniques , Inflammation , Interleukin-1 , Male , Osteoarthritis, Hip/immunology , Osteoarthritis, Hip/surgery , Osteoarthritis, Knee/immunology , Osteoarthritis, Knee/surgery , RNA, Long Noncoding/immunology , Reverse Transcriptase Polymerase Chain Reaction
10.
FEBS Lett ; 589(3): 396-406, 2015 Jan 30.
Article in English | MEDLINE | ID: mdl-25554418

ABSTRACT

Recent studies have indicated that non-coding RNAs transcribed from enhancer regions are important regulators of enhancer function and gene expression. In this report, we have characterised the expression of six enhancer RNAs (eRNAs) induced in human monocytic THP1 cells following activation of the innate immune response by lipopolysaccharide (LPS). Specifically, we have demonstrated that LPS-induced expression of individual eRNAs is mediated through divergent intracellular signalling pathways that includes NF-κB and the mitogen activated protein kinases, extracellular regulated kinase-1/2 and p38.


Subject(s)
Enhancer Elements, Genetic , Gene Expression Regulation/drug effects , RNA, Long Noncoding/genetics , Signal Transduction/drug effects , Cell Line , Humans , Lipopolysaccharides/pharmacology , MAP Kinase Signaling System/drug effects , Monocytes/drug effects , Monocytes/metabolism , NF-kappa B/biosynthesis , NF-kappa B/genetics , RNA, Long Noncoding/biosynthesis , p38 Mitogen-Activated Protein Kinases/biosynthesis , p38 Mitogen-Activated Protein Kinases/genetics
11.
Int J Mol Sci ; 15(1): 1112-42, 2014 Jan 16.
Article in English | MEDLINE | ID: mdl-24441568

ABSTRACT

G protein-coupled receptors (GPCRs) are important cell signaling mediators, involved in essential physiological processes. GPCRs respond to a wide variety of ligands from light to large macromolecules, including hormones and small peptides. Unfortunately, mutations and dysregulation of GPCRs that induce a loss of function or alter expression can lead to disorders that are sometimes lethal. Therefore, the expression, trafficking, signaling and desensitization of GPCRs must be tightly regulated by different cellular systems to prevent disease. Although there is substantial knowledge regarding the mechanisms that regulate the desensitization and down-regulation of GPCRs, less is known about the mechanisms that regulate the trafficking and cell-surface expression of newly synthesized GPCRs. More recently, there is accumulating evidence that suggests certain GPCRs are able to interact with specific proteins that can completely change their fate and function. These interactions add on another level of regulation and flexibility between different tissue/cell-types. Here, we review some of the main interacting proteins of GPCRs. A greater understanding of the mechanisms regulating their interactions may lead to the discovery of new drug targets for therapy.


Subject(s)
Receptors, G-Protein-Coupled/metabolism , Animals , Cell Membrane/metabolism , Humans , Molecular Chaperones/metabolism , Protein Binding , Protein Transport , Vesicular Transport Proteins/metabolism
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